Handbooks of Vitamins

Preface
Knowledge of vitamins in health and disease has increased markedly in recent years due
to extraordinary advances in analytical and separation methodologies (1,8,9). Although
each of the past six decades has been exciting from the standpoint of improved approaches
in the isolation, identification, and synthesis of vitamins, the past 10 years have been
particularly important. In addition, improved methodology has never been so available to
the typical researcher. With improved methodologies and sources of inexpensive vitamins,
it is now possible to study the functions of vitamins in clinical settings. Improved methods
have led, in turn, to a better understanding of the roles that vitamins play as catalysts,
cellular regulators, and co-substrates. Moreover, there is now a better appreciation of vitaminlike
accessory factors, which may be beneficial to overall health or required on a
conditional basis.
As a result of the continued interest in vitamin-related research, as might be expected,
a large body of information has accumulated in the past 10 years. One goal for
the Handbook of Vitamins: Third Edition, Revised and Expanded, is to provide basic and
fundamental background material to aid the reader in assessing the importance of new
findings regarding vitamin function. In addition to the Handbook of Vitamins, the reader
is directed to other books published by Marcel Dekker, Inc. (5–7, 10–13), to the Methods
in Enzymology series (e.g., Ref. 8), edited by two of the editors of the Handbook of
Vitamins, and to other related textbooks (2–4).
Much of the interest in vitamins stems from an appreciation that there remain sizable
populations at risk for vitamin deficiencies. In addition, the need for vitamins in some
individuals is known to be influenced by genetic polymorphisms and related genetic factors.
Some of the material in the Handbook of Vitamins addresses such points and relationships
that have significant public health impact, e.g., the relationship of B12 and folate
metabolism to homocysteine regulation and, in turn, the connections that homocysteine
may have to vascular diseases and developmental defects.
v
vi Preface
Indeed, for each of the vitamins, there now exists a broader understanding of function
and requirement. In the chapters dedicated to fat-soluble vitamins, new roles for vitamin
A, K, and D are described. An overview of vitamin E’s role as an antioxidant is
included, as well as a separate chapter on various flavinoids that play roles in oxidant
defense. The section on water-soluble vitamins begins with a brief discussion and review
of bioorganic mechanisms. Each of the subsequent chapters focusing on water-soluble
vitamins was written to provide a general understanding of chemical, physiological, and
nutritional relationships. Some sections highlight methods for evaluating deficiencies or
nutritional requirements and the interaction of vitamins with environmental factors. Also,
the efficacy and safety of vitamin use at high levels is addressed.
The chapters were written with a varied audience in mind: the clinician, the biochemist
whose physiological background may be limited, the advanced nutrition student, and
the dietician. The Handbook of Vitamins is a single source, a summary of vitamin functions,
that is complete, yet accessible. In this kind of endeavor, the challenge is to deal
with an expanding factual information base. The contributors have often chosen to develop
the material in a conceptual, rather than in a highly detailed manner. The Handbook of
Vitamins serves as an authoritative and comprehensive source.
We are indebted to the staff at Marcel Dekker, Inc., for their patience and assistance.
The Handbook of Vitamins is part of a serious and significant effort by Dekker to provide
seminal works that speak broadly to micronutrient function. In addition to those referenced
below, other offerings that complement this book may be found at http://www.dekker.com.
We also wish to acknowledge three colleagues who died before this edition was
completed. Graham Garratt, Executive Vice President and Publisher, was very helpful in
the early stages of assembling the handbook. James Olson, one of the eminent leaders in
the field of vitamin A metabolism, and Lawrence Machlin, who edited the first two editions
of Handbook of Vitamins, passed away during the past year. Each man was a dear friend,
truly distinguished in his field, and will be missed.
Robert B. Rucker
John W. Suttie
Donald B. McCormick
Lawrence J. Machlin
REFERENCES
1. G. F. M. Ball, Bioavailability and Analysis of Vitamins in Foods, Chapman & Hall, New
York, 1998, pp. 1–569.
2. J. Basu and T. Kumar, Vitamins in Human Health and Disease, Wallingford: CAB International,
New York, 1996, pp. 1–345.
3. G. F. Combs, The Vitamins: Fundamental Aspects in Nutrition and Health, Academic Press,
San Diego, CA, pp. 1–618.
4. P. J. Quinn and V. E. Kagan, eds., Fat-Soluble Vitamins, Plenum, New York, 1998, pp. 1–
533.
5. C. Rice-Evans and L. Packer, eds., Flavonoids in health and disease, Marcel Dekker, New
York, 1997.
6. L. B. Bailey, ed., Folate in Health and Disease, Marcel Dekker, New York, 1994.
7. A. Bendich, C. E. Butterworth, Micronutrients in Health and in Disease Prevention, Marcel
Dekker, New York, 1991.
Preface vii
8. D. B. McCormick, J. W. Suttie, C. Wagner, eds., Vitamins and Coenzymes, Parts I, J, K, and
L, Academic Press, San Diego, CA, Methods in Enzymology series, Vol. 279–283, 1997.
9. Sauberlich, H. E., Laboratory Tests for the Assessment of Nutritional Status, 2nd ed., CRC
Press, Boca Raton, 1999, pp. 1–486.
10. R. Blomhoff, Vitamin A in Health and Disease, Marcel Dekker, New York, 1994.
11. L. Packer, J. Fuchs, eds., Vitamin C in Health and Disease, Marcel Dekker, New York, 1997.
12. L. Packer, J. Fuchs, eds., Vitamin E in Health and Disease: Biochemistry and Clinical Applications,
Marcel Dekker, New York, 1992.
13. S. K. Gaby, A. Bendich, V. N. Singh, L. J. Machlin, eds., Vitamin Intake and Health: A
Scientific Review, Marcel Dekker, New York, 1990.
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Contents
Preface v
Contributors xi
1. Vitamin A 1
James Allen Olson
2. Vitamin D 51
Elaine D. Collins and Anthony W. Norman
3. Vitamin K 115
John W. Suttie
4. Vitamin E 165
Ching K. Chow
5. Bioorganic Mechanisms Important to Coenzyme Functions 199
Donald B. McCormick
6. Niacin 213
James B. Kirkland and Jean M. Rawling
7. Riboflavin (Vitamin B2) 255
Richard S. Rivlin and John Thomas Pinto
ix
x Contents
8. Thiamine 275
Vichai Tanphaichitr
9. Pantothenic Acid 317
Nora S. Plesofsky
10. Vitamin B6 339
James E. Leklem
11. Biotin 397
Donald M. Mock
12. Folic Acid 427
Tom Brody and Barry Shane
13. Cobalamin (Vitamin B12) 463
William S. Beck
14. Choline 513
Steven H. Zeisel and Minnie Holmes-McNary
15. Ascorbic Acid 529
Carol S. Johnston, Francene M. Steinberg, and Robert B. Rucker
16. Ascorbic Acid Regulation of Extracellular Matrix Expression 555
Jeffrey C. Geesin and Richard A. Berg
17. Nutrients and Oxidation: Actions, Transport, and Metabolism of
Dietary Antioxidants 569
J. Bruce German and Maret G. Traber
Index 589
Contributors
William S. Beck, M.D. Department of Medicine and Department of Biochemistry and
Cell Biology, Harvard University, Cambridge, Massachusetts
Richard A. Berg, Ph.D. Biomaterials Research, Collagen Corporation, Palo Alto, California
Tom Brody, Ph.D. Department of Nutritional Sciences and Toxicology, University of
California, Berkeley, California
Ching K. Chow, Ph.D. Graduate Center for Nutritional Sciences, University of Kentucky,
Lexington, Kentucky
Elaine D. Collins, Ph.D. Department of Chemistry, San Jose? State University, San Jose?,
California
Jeffrey C. Geesin, Ph.D. Wound Healing Technology Resource Center, Johnson and
Johnson, Skillman, New Jersey
J. Bruce German, Ph.D. Department of Food Science and Technology, University of
California, Davis, California
Minnie Holmes-McNary, Ph.D. Lineberger Comprehensive Cancer Center, School of
Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
xi
xii Contributors
Carol S. Johnston, Ph.D. Department of Nutrition, Arizona State University East,
Mesa, Arizona
James B. Kirkland, Ph.D. Department of Human Biology and Nutritional Sciences,
University of Guelph, Guelph, Ontario, Canada
James E. Leklem, Ph.D. Department of Nutrition and Food Management, Oregon State
University, Corvallis, Oregon
Donald B. McCormick, Ph.D. Department of Biochemistry, School of Medicine, Emory
University, Atlanta, Georgia
Donald M. Mock, M.D., Ph.D. Departments of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences, Little Rock, Arkansas
Anthony W. Norman, Ph.D. Division of Biomedical Sciences, Department of Biochemistry,
University of California, Riverside, California
James Allen Olson, Ph.D.† Department of Biochemistry, Biophysics, and Molecular
Biology, Iowa State University, Ames, Iowa
John Thomas Pinto, Ph.D. Nutrition Research Laboratory, Department of Medicine,
Memorial Sloan-Kettering Cancer Center, New York, New York
Nora S. Plesofsky, Ph.D. Department of Plant Biology, University of Minnesota, St.
Paul, Minnesota
Jean M. Rawling, M.D., Ph.D. Department of Family Medicine, Faculty of Medicine,
University of Calgary, Calgary, Alberta, Canada
Richard S. Rivlin, M.D. Weill Medical College of Cornell University, and Department
of Medicine, Clinical Nutrition Research Unit, Memorial Sloan-Kettering Cancer Center,
New York, New York
Robert B. Rucker, Ph.D. Department of Nutrition, University of California, Davis,
California
Barry Shane, Ph.D. Department of Nutritional Sciences and Toxicology, University of
California, Berkeley, California
Francene M. Steinberg, Ph.D., R.D. Department of Nutrition, University of California,
Davis, California
John W. Suttie, Ph.D. Department of Biochemistry, University of Wisconsin, Madison,
Wisconsin
† Deceased.
Contributors xiii
Vichai Tanphaichitr, M.D., Ph.D., F.A.C.P., F.R.A.C.P., F.R.I. Department of Medicine,
Ramathibodi Hospital, and Faculty of Medicine, Mahidol University, Bangkok, Thailand
Maret G. Traber, Ph.D. Linus Pauling Institute, Oregon State University, Corvallis,
Oregon
Steven H. Zeisel, M.D., Ph.D. Department of Nutrition, School of Public Health and
School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North
Carolina
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1
Vitamin A
JAMES ALLEN OLSON
Iowa State University, Ames, Iowa
I. HISTORY
Probably the first nutritional deficiency disease to be clearly recognized was night blindness.
The ancient Egyptians, as indicated in the Papyrus Ebers and later in the London
Medical Papyrus, recommended that juice squeezed from cooked liver be topically applied
to the eye to cure night blindness. These writings date from 1500 bc, but the observations
probably are of much earlier origin. The Greeks, who depended heavily on Egyptian medicine,
recommended both the ingestion of cooked liver and its topical application as a cure
for night blindness, a tradition that has persisted in many societies to this day (1).
Although interesting references to vitamin A deficiency diseases and their cure can
be found throughout history, the modern science of nutrition is only about a century old.
The observation that experimental animals lose weight and die on purified diets was noted
by many investigators toward the end of the nineteenth century. In the early part of this
century, specific factors necessary for growth and survival were beginning to be identified.
Frederick Gowland Hopkins in England, for example, during the period 1906–1912 found
that a growth-stimulating principle from milk was present in an alcoholic extract of milk
rather than in the ash.
During the same period, Stepp in Germany identified one of these ‘‘minimal qualitative
factors’’ as a lipid. Soon thereafter, E. V. McCollum and Marguerite Davis in Wisconsin
showed that butter or egg yolk, but not lard, contained a lipid-soluble factor necessary
for the growth of rats. In 1913 they coined the term ‘‘fat-soluble A’’ and thereby
attributed for the first time the growth-stimulating property of these extracts to a single
compound. Approaching the problem in a very different way, Osborne and Mendel at
Yale concomitantly found that cod liver oil or butter was an essential growth-promoting
food for rats. The year 1913, therefore, was the beginning of the modern age of vitamin
A exploration.
1
2 Olson
Many outstanding findings have been made during the past eight decades, of which
only the most notable will be mentioned here. Inasmuch as both colorless extracts of
liver as well as colored plant lipids showed biological activity, Steenbock in Wisconsin
postulated in 1919 the interconversion of two forms of the vitamin. A decade later, Moore
in England showed that ?-carotene, the plant pigment, was converted to the colorless form
of the vitamin in liver tissue. In the early 1920s, experimental vitamin A deficiency was
well characterized by Wolbach and Howe in Boston, and the existence of vitamin A defi-
ciency in children was noted by Bloch in Denmark. In 1930, Karrer and his colleagues
in Switzerland determined the structure both of ?-carotene and of vitamin A and shortly
thereafter synthesized some of its derivatives. One of these derivatives was retinaldehyde,
and in 1935 George Wald of Harvard, while working in Germany, proved that the retinene
found in visual pigments of the eye was identical to Karrer’s chemical compound retinaldehyde.
Of great importance for applied nutrition was the elegant synthesis of all-trans vitamin
A from the inexpensive precursor ?-ionone by Otto Isler and his collaborators in
Basel in the late 1940s. Within a few years the price of vitamin A, which earlier had been
painstakingly isolated from fish liver oils by molecular distillation, fell 10-fold, and the
possibility of using vitamin A more generally in foods at a reasonable cost was assured.
With the availability of radioactive isotopes in the 1950s, many studies were initiated
on the metabolism of vitamin A, and much of our present knowledge concerning it was
defined. The details of the visual cycle involving rhodopsin in the eye were in large part
worked out, and attention began to focus on the possible somatic function of vitamin A in
growth and cellular differentiation. In subsequent sections, the present state of knowledge
relative to vitamin A will be summarized. Much of the early history of vitamin A is
discussed in Moore’s highly readable monograph (2).
II. CHEMISTRY
A. Isolation
In nature vitamin A is largely found as an ester and, consequently, is highly soluble in
organic solvents but not in aqueous solutions. The major provitamin carotenoid, ?-carotene,
has similar solvent properties. One of the richest sources of vitamin A is liver tissue,
in particular the liver oils of marine fish and mammals. The esters can be directly isolated
from these oils by molecular distillation at very low pressure, a procedure that has been
used extensively for the commercial preparation of vitamin A–rich oils. Alternatively,
vitamin A might be directly extracted with chloroform or with some other solvent combination,
such as hexane together with ethanol, followed by purification of vitamin A by
chromatographic means. To hydrolyze esters, not only of vitamin A and carotenoids but
also of triglycerides and other lipids, saponification with KOH is commonly used, followed
by extraction with organic solvents. Retinol or its esters can be readily crystallized at low
temperature from a variety of organic solvents, including ethyl formate, propylene oxide,
and methanol.
B. Structure and Nomenclature
The structure and recommended names of major compounds in the vitamin A group of
substances are depicted in Fig. 1. The acknowledged reference compound is called alltrans
retinol, whose formula and numbering system are given in Fig. 1a (3).
Vitamin A 3
Fig. 1 Formulas for retinol and its derivatives. All are all-trans isomers except as noted. (a) retinol,
(b) retinal, (c) retinoic acid, (d) 3,4-didehydroretinol, (e) 11-cis retinal, (f) 9-cis retinoic acid, (g)
5,6-epoxyretinol, (h) retro anhydroretinol, (i) 4-oxoretinol, (j) retinoyl ?-glucuronide, (k) 14-
hydroxy-4,14-retro retinol, (l) retinyl palmitate.
In addition to the all-trans form of vitamin A, all 15 of the other possible isomers
have been prepared and characterized (4–6). The most interesting and important isomers
(4–6) are 11-cis retinal, depicted in Fig. 1e, which is the chromophore of the visual pigments
rhodopsin and iodopsin; 9-cis retinoic acid (Fig. 1f), which is the ligand for the
retinoid X receptor (RXR) transcription factors; and 13-cis retinal, which serves lightdependent
functions in halobacteria. As the number of cis bonds increase, both the absorption
maximum of the isomer and its absorbance tend to decrease. Thus, whereas all-trans
retinaldehyde has an absorption maximum and molecular extinction coefficient of 368 nm
and 48,000 in hexane, respectively, the 9,11,13-cis isomer shows corresponding values
of 302 nm and 15,500 (5,6). The tetra-cis isomer, 7,9,11,13-cis, of ethyl retinoate, retinal,
and retinol show similar hypsochromic and hypochromic shifts relative to their all-trans
analogs (4,5). Although the terms ‘‘cis’’ and ‘‘trans’’ have traditionally been used to
denote various isomers of vitamin A, the current chemical notation is ‘‘Z’’ for ‘‘cis’’
and ‘‘E’’ for ‘‘trans.’’ Thus, 11-cis retinal might equally well be termed 7E,9E,11Z,13Eretinal.
4 Olson
The major commercial forms of vitamin A are all-trans retinyl acetate and all-trans
retinyl palmitate (Fig. 1l). The esters are generally produced because of their increased
stability and better solubility in oils and other commercial preparations. The esters can
also be incorporated into a gelatin matrix that protects them from oxidation over reasonable
periods of time, even when subjected to cooking. These latter forms have been extensively
used in the fortification of foods and animal feeds.
Carotenoids not only serve as precursors of vitamin A but also can quench singlet
oxygen and act as both antioxidants and prooxidants. Of over 600 carotenoids that have
been isolated from nature, only about 50 possess provitamin A activity. An excellent
summary of many aspects of carotenoid isolation, structure, and synthesis is given in the
fine treatise edited by Isler (7) and in a recent comprehensive work (8). Thus, the term
provitamin A is used as a generic indicator for all carotenoids that show the biological
activity of vitamin A. Carotenoids commonly found in ingested fruits and vegetables are
depicted in Fig. 2. The most active and quantitatively the most important of these provitamins
is all-trans ?-carotene (Fig. 2:5). Generally, carotenoids must contain at least one
?-ionone ring that is not hydroxylated to show vitamin A activity.
C. Structures of Important Synthetic Analogs of Vitamin A
A very large number of analogs of vitamin A have been synthesized during the past 15
years. The procedures for synthesizing these retinoids have been comprehensively reviewed
(9). The initial impetus for this vast effort was to identify analogs of all-trans
retinoic acid with a better efficacy/toxicity ratio (‘‘therapeutic index’’) in the prevention
of chemically induced cancers in experimental animals (10). With the discovery of the
retinoid receptors, RAR and RXR, the focus has shifted to the synthesis of analogs that
bind strongly and specifically to various forms of these receptors, thereby either activating
them or inhibiting their activation by retinoic acid (9). Some retinoids with interesting
therapeutic or binding properties are given in Fig. 3. In brief, 13-cis retinoic acid (Accutane)
(Fig. 3a) has been used effectively in the treatment of acne, etretinate (Fig. 3b) and
acitretin (Fig. 3c) in the treatment of psoriasis, and 4-hydroxyphenylretinamide (fenretinide)
(Fig. 3d) in preventive studies of breast cancer. TTNPB (Fig. 3e) is one of the most
toxic retinoids as well as one of the most physiologically active (11). TTNPB interacts
well with RARs but poorly with RXRs. Its 3-methyl derivative (Fig. 3f), in contrast, reacts
well with RXRs but not with RARs. The naphthylamide derivative Am-580 (Fig. 3g) is
specific for RAR?, CD-417 (Fig. 3h) for RAR?, CD-666 (Fig. 3i) for RAR?, and SR-
11237 (Fig. 3j) for RXR (12,13). The latter compound has a fixed cisoid configuration at
the position equivalent to 9-cis retinoic acid.
D. Synthesis
Since the complete elucidation of the structure of vitamin A by Karrer in 1930, intense
effort has been expended in synthesizing vitamin A from a variety of precursors. Of many
possibilities, the two major synthetic procedures used commercially are those of Hoffmann-
La Roche and of Badische Anilin- und Sod-Fabrik (BASF). The Roche procedure
involves as a key intermediate a C14 aldehyde and further requires the efficient reduction
of acetylenic to olefinic bonds near the end of the synthesis. The BASF procedure, on the
other hand, depends heavily on the Wittig reaction, by which a phosphonium ylid reacts
with an aldehyde or ketone to give an olefin and phosphine oxide. The major steps in
Fig. 2 Polyenes and carotenoids in foods that may also be found in animal tissues. 1, phytoene;
2, phytofluene; 3, lycopene; 4, ?-carotene; 5, ?-carotene; 6, ?-cryptoxanthin; 7, zeaxanthin; 8, lutein;
9, canthaxanthin; 10, violaxanthin; 11, neoxanthin; 12, astaxanthin. (From Ref. 139.)
6 Olson
Fig. 3 Some synthetic retinoids with specific therapeutic or protein-binding properties. (a) 13-
cis retinoic acid, (b) etretinate, (c) acitretin, (d) 4-hydroxyphenylretinamide (HPR, Fenretinide), (e)
tetrahydrotetramethylnaphthalenylpropenylbenzoic acid (TTNPB), (f) 3-methyl TTNPB, (g) AM-
580, (h) CD-417, (i) CD-666, (j) SR-11237.
Fig. 4 Major commercial routes to the synthesis of retinyl acetate (B) from ?-ionone (A). The
Isler (Roche) procedure is on the left, the Wittig (BASF) method on the right.
Vitamin A 7
Table 1 Physical Properties of All-trans Retinol and Its Esters
Property Retinol Retinyl acetate Retinyl palmitate
Formula C20H30O C22H32O2 C36H60O2
Formula weight 286.46 328.50 524.88
Melting point (°C) 63–64 57–59 28–29
UV absorptiona
?max 325 326 326
E1%
1cm 1820 1530 960

 52,140 50,260 50,390
Fluorescence
Excitation ?max 325 325 325
Emission ?max 470 470 470
aIn isopropanol. Values are similar in ethanol but differ in chloroform and other solvents. Absorbance values
for retinyl esters in hexane are about 3% higher than for retinol in hexane.
these two procedures are presented in Fig. 4. Major synthetic routes to vitamin A and the
retinoids have been exhaustively reviewed (7,9,14).
E. Chemical and Physical Properties
In concentrated solution, retinol and its esters are light yellow to reddish hued oils that
solidify when cooled and have a mild pleasant odor. As already mentioned, they are insoluble
in water or glycerol but are readily miscible with most organic solvents. The formula,
formula weights, melting points, and characteristics of the absorption and fluorescent spectra
are summarized in Table 1. Indeed, many other physicochemical properties, including
infrared spectra, polarographic characteristics, proton magnetic resonance, and the like,
have also been studied (7–9,14). Nuclear magnetic resonance (NMR) spectra are particularly
useful in characterizing various isomers of vitamin A (5,14). Vitamin A in crystalline
form or in oil, if kept under a dry nitrogen atmosphere in a dark cool place, is stable for
long periods. In contrast, vitamin A and its analogs are particularly sensitive to oxidation
by air in the presence of light, particularly when spread as a thin surface film. In its natural
form, whether present in liver or bound to protein in the plasma, vitamin A also is quite
stable when stored in the frozen state, preferably at 70°C, in a hermetically closed container
in the dark (15). On the other hand, upon extraction from biological materials, care
must be taken to prevent both oxidation and isomerization of vitamin A (15).
Commercial firms have been successful in preparing several stabilized forms of
vitamin A. For example, retinyl acetate, propionate, or palmitate can be coated in the
presence of antioxidants into a gelatin-carbohydrate matrix. The beadlets thus formed,
upon being mixed in animal feeds or in human food, retain 90% or more of their activity
for at least 6-months if kept under good storage conditions. However, in the presence of
high humidity, heat, and oxygen, the loss is considerably greater.
III. ANALYTICAL PROCEDURES
The most common method for analyzing vitamin A and its analogs in pharmaceutical
preparations, feedstuffs, and tissues is high-performance liquid chromatography (HPLC)
combined with a UV detector, usually set at 325 nm (15). Two major types of chromato8
Olson
graphic columns are employed, so-called straight-phase and reverse-phase supports. In
the former, hydrophobic compounds are eluted first and more polar compounds later,
whereas in the latter the reverse order of elution occurs. Isomers of a given retinoid are
best separated on the former (16), whereas different retinyl esters are best resolved on
the latter. Carotenoids can also be well resolved by HPLC, usually in conjunction with a
UV-visible (UV-Vis) detector set at 450 nm (17–19). Other retinoids are usually measured
at their maximal absorption wavelengths.
One of the most sensitive methods for measuring vitamin A is by its intensive greenish
yellow fluorescence (Table 1). Because many other natural compounds fluoresce, such
as the polyene pigment phytofluene, chromatographic separation by HPLC or by other
procedures is usually necessary to ensure adequate specificity in the response. The intense
fluorescence of vitamin A has recently been used as well in separating liver cells containing
large amounts of retinyl ester (stellate cells) from those containing smaller amounts by
means of laser-activated flow cytometry (20).
Mass spectroscopy (MS) is also being increasingly used in the analysis of retinoids
and carotenoids (15). The combination of gas chromatography on selected capillary columns
with MS has been used to study the in vivo kinetics and the equilibration of deuterated
retinol with endogenous reserves of vitamin A in humans (21,22). By using ?-carotene
richly labeled with carbon 13, isotope ratio mass spectrometry has provided important
new information about its metabolism in humans (23,24).
The older conventional methods, such as the colorimetric measurement of the transient
blue complex formed at 620 nm when vitamin A is dehydrated in the presence of
Lewis acids and the decrease in absorbance at 325 nm when vitamin A in serum extracts
is inactivated by UV light, remain of value. Currently, however, these procedures are the
methods of choice only in field surveys in which more sophisticated instrumentation is
not available (25).
IV. BIOASSAY METHODS
Biological tests have a utility that cannot be replaced by specific chemical or physical
methods. The physiological response of a species to a given provitamin, a mixture of
provitamins, or a vitamin A preparation can only be assessed by biological procedures.
Indeed, the complexities of absorption, metabolism, storage, transport, and uptake by tissues
is integrated into a meaningful whole only by biological tests.
Biological methods for vitamin A include the classical growth response tests in
vitamin A–deficient rats, liver storage assays in rats and chicks, and the vaginal smear
technique. The requisite design and pitfalls of using the classical rat growth test have been
well reviewed (26). These classical procedures are rarely used now.
Cell culture systems are now commonly employed for measuring the biological
activity, including the toxicity, of retinoids (27–29). A major focus has been the effects
of retinoids in inducing the differentiation of transformed cells in vitro (28–30).
V. NUTRITIONAL ASPECTS
A. Foods
Common dietary sources of preformed vitamin A are various dairy products, such as milk,
cheese, butter, ice cream, and eggs; liver; other internal organs, such as kidney and heart;
Vitamin A 9
and many fish, such as herring, sardines, and tuna. The very richest sources are liver oils
of shark; of marine fish, such as halibut; and of marine mammals, such as polar bear.
With respect to carotenoids, carrots and green leafy vegetables, such as spinach and
amaranth, generally contain large amounts. Although tomato contains some vitamin A–
active carotenoids, the major pigment is lycopene, which has no nutritional activity. Fruits
like papaya and orange have appreciable quantities of carotenoids. The cereal grains generally
contain very little vitamin A, particularly when milled (an important consideration
in dealing with the problem of vitamin A deficiency among very young children throughout
the world). Yellow maize does have vitamin A–active carotenoids, whereas white
maize, which is popular in many parts of Africa, does not. The richest source of carotenoids
is red palm oil, which contains about 0.5 mg of mixed ?- and ?-carotene per
milliliter. Thus, about 7 mL of red palm oil per day should meet the nutritional needs of
a preschool child.
Food composition tables are commonly used to determine the content of vitamin
A, including carotenoid sources, in the diet. Although the adequacy of vitamin A and
carotenoid intake can usually be estimated by their use, such tables are much less helpful
for quantifying mean intakes of vitamin A.
B. Assessment of Vitamin A Status
Vitamin A status can be classified into five categories: deficient, marginal, satisfactory,
excessive, and toxic. The deficient and toxic states are characterized by clinical signs,
whereas the other three states are not. The most commonly used indicator of clinical
deficiency in surveys is the presence of xerophthalmia (25). Of various eye signs that
appear, Bitot’s spots (XlB) in preschool children have proven to be the most useful. Corneal
involvement, although quite specific, is a relatively rare sign, and conjunctival xerosis
(XlA) is fairly nonspecific. Of course, the disappearance of any reversible sign in response
to vitamin A treatment strengthens its validity. Night blindness, which is difficult to evaluate
in young children by direct measurement, can often be satisfactorily assessed by interviewing
the mother (25).
Other methods of assessing vitamin A status include dietary, physiological, histological,
and biochemical procedures. Dietary methods provide useful information about food
habits but, as already indicated, are less useful for quantifying mean intakes. The current
availability of better values for various carotenoids in foods (31), however, enhances the
utility of this approach. Several physiological techniques exist for measuring the impaired
sensitivity of rod cells in the eye (25). The major histological method is conjunctival
impression cytology. In vitamin A–depleted individuals, goblet cells in the conjunctiva
of the eye tend to disappear, and epithelial cells often assume abnormal shapes (25). However,
the subjectivity of the technique and the existence of confounding variables, e.g.,
trachoma, limit its specificity somewhat. Biochemical methods include the measurement
of concentrations of retinol and retinol-binding protein (RBP) in plasma and tears (25).
Because plasma retinol is maintained homeostatically and is affected by other nutrients
and infections, its measurement is most useful when individuals are depleted of the vitamin.
Two response tests, the relative dose response (RDR) and modified relative dose
response (MRDR) methods, provide more specific information about the vitamin A statuses
of individuals and populations from deficiency through marginal status (25). These
latter tests are increasingly being used to assess vitamin A status. Finally, isotope dilution
10 Olson
techniques, employing vitamin A labeled with deuterium or 13C, are starting to be used
to determine total body stores of vitamin A in populations at risk (21–23,25,32).
C. Requirements and Recommendations
A nutritional requirement, whether for animals or humans, must be defined operationally
in terms of nutrient-specific indicators. If a different indicator is selected, the value of the
requirement may well change. Thus, the presumed requirement will progressively increase
as survival, prevention of clinical signs of deficiency, reproductive performance, and longevity
are used as end points. As a consequence, several tiers of requirements might be
defined, based on the physiological level of satisfactory performance that is desired. Many
vitamins, including vitamin A, also show biological actions, some beneficial and some
adverse at yet higher levels, e.g., the adjuvant effect of large doses of vitamin A in stimulating
the immune system. These effects, which are pharmacological in nature, should not
be considered in setting the nutritional requirement. However, some responses to nutrients
that fall between clearly nutritional and clearly pharmacological effects can be difficult
to classify.
Strictly speaking, the nutritional requirement for a population, as defined by a given
selected indicator, should be expressed as the mean requirement, i.e., when 50% of those
in the population show satisfactory values and 50% show unsatisfactory values of a given
indicator. However, because the objective of nutritional analysis is the maintenance of a
satisfactory status in most individuals in a population, the recommended dietary intake
(RDI), a term used worldwide (33), or recommended dietary allowance (RDA), as used
in the United States and in a few other countries, has been defined as the average intake
over time that theoretically would meet or exceed the nutritional needs of 97.7% (mean
 2 SD) of a selected healthy population group, or, more pragmatically, of ‘‘nearly all’’
members of the group. Although reliable data on the frequency distribution of requirements
for many species are not available, a coefficient of variation of 20% seems to apply
to several vitamins in humans and other species.
Often, the RDI of a nutrient is set at a level generously above that which provides
for any possible physiological need but significantly lower than that which causes toxic
effects. In such instances, a ‘‘safety factor,’’ usually selected somewhat arbitrarily, is
included in the calculation of the desired intake level.
Because all of these procedures have been used in setting recommended intakes
of vitamin A, a range of recommended values exists. In selecting an appropriate level
for a given application, the basis of various recommendations must consequently be
probed.
Three sets of recommended dietary intakes for vitamin A are given in Table 2: the
1988 RDI of the Food and Agriculture Organization and the World Health Organization
(FAO/WHO) (34); the 1989 RDAs of the Food and Nutrition Board, National Research
Council, U.S. National Academy of Sciences (35); and the 1991 dietary reference values
(DRV) for the United Kingdom (36). The age categories cited in the table (37) are similar
but not identical in the three recommendations. Three different systems have been used:
a single value in the RDA (USA), a two-tier system in the RDI (FAO/WHO), and a threetier
system in the DRV (UK). In a conceptual sense, the higher tier in the FAO/WHO
system, termed ‘‘safe’’ intake, the top tier in the UK system, termed ‘‘reference nutrient
intake’’ (RNI), and the single RDA values in the U.S. system are roughly equivalent (37).
All presume the presence of an adequate total body reserve of vitamin A for most individuVitamin
A 11
Table
2
Recommended
Dietary
Intakes
of
Vitamin
A
in
µg
Retinol
Equivalents
a,b
RDI
DRV
(UK)e
(FAO
/
WHO)
c
Lower
reference
Estimated
average
Reference
Group
Basal
Safe
RDA
(USA)d
nutrient
intake
requirement
nutrient
intake
Infants
0
–
0.5
y
180
350
375
150
250
350
0.5–1
y
180
350
375
150
250
350
Children
1
–
2
y
200
400
400
200
300
400
2
–
6
y
200
400
500
200
300
400
6
–
10
y
250
400
700
250
350
500
Males
10
–12
y
300
500
1000
250
400
600
12
–70
y
300
600
1000
300
500
700
Females
10
–70
y
270
500
800
250
400
600
Pregnancy
100
100
0
100
Lactation
0–6
mo
180
350
500
350
6mo
180
350
400
350
aA
µg
retinol
equivalent
is
defined
as
1
µg
all-trans
retinol,
which
is
considered
equal
to
6
µg
all-trans
?-carotene
or
12
µg
of
mixed
provitamin
A
carotenoids.
In
molar
terms,
1
µg
retinol
equivalent

3.5
nmol
retinol

11.2
nmol
?-carotene.
bModified
from
Ref.
37.
cRef.
34.
dRef.
35.
eRef.
36.
12 Olson
als but also include the requirements of ‘‘those few members of the community with
particularly high needs’’ (36).
The lowest tiers in the FAO/WHO system and in the DRV system show similar
values but are defined differently. The FAO/WHO defines basal requirements as the ‘‘(average
daily) amount needed to prevent clinically demonstrable impairment of function’’
(34). The lower reference nutrient intake (LRNI) is defined as the ‘‘(average daily) amount
of the nutrient that is enough for only the few people in a group who have low needs’’
(36).
The estimated average requirement (EAR) in the UK system is the (average daily)
amount of a nutrient that meets the needs of 50% of the analyzed group of people. Thus,
it is truly a mean requirement. In the UK system, the LRNI is 2 SD below the EAR and
the RNI is 2 SD above the EAR. The assigned values for the LRNI in the UK system,
which in large part is an inadequate intake for most people, and for the basal requirement
in the FAO/WHO classification, which is a minimally adequate intake for most people,
are similar. This confounding fact may in part be explained by the different reference
standards for weight that are used. The reference weights for adult men and women, respectively,
in the FAO/WHO report are 65 and 55 kg, in the UK report are 74 and 60
kg, and in the U.S. report are 76 and 62 kg. Despite these differences, the RDA in the
United States is still very generous relative to those of many other countries (37).
Recommended nutrient intakes are established for healthy individuals. Febrile conditions
and lipid malabsorption can markedly increase needs. Genetic defects in the handling
of vitamin A also can significantly affect requirements. Homozygous defects have not been
identified for vitamin A, however, probably because they are inconsistent with survival.
Nonetheless, cases of vitamin A intolerance, in which toxic signs appear in some individuals
who ingest moderate amounts of vitamin A, have been reported (38). These instances,
though rare, seem to have a genetic basis (37).
Another aspect of nutritional requirements that merits comment is the concept of a
single ‘‘optimal’’ intake. Little evidence favors such a view. Rather, most studies indicate
that a broad range of satisfactory intakes exists between a low level that prevents defi-
ciency and a high level that causes toxicity. The ‘‘satisfactory range’’ viewpoint is inherent
in the establishment of a ‘‘safe and adequate range’’ for several nutrients (35).
The most suitable indicator of vitamin A nutriture, as indicated earlier, is its total
body reserve. The status of many other nutrients, such as iron, folic acid, vitamin B12, and
vitamin C, can also be related to their total body reserves. The relation of various indicators
of nutritional status to the total body reserve and the selection of a total body reserve that
meets specified operational criteria of adequacy are essential for the future definition of
more scientifically based nutritional requirements and recommendations.
VI. PHYSIOLOGIC RELATIONSHIPS
A. Metabolism
1. Absorption
After foods are ingested, preformed vitamin A of animal tissues and the provitamin carotenoids
of vegetables and fruits are released from proteins by the action of pepsin in the
stomach and of proteolytic enzymes in the small intestine. In the stomach the free carotenoids
and retinyl esters tend to aggregate in fatty globules, which then enter the duodenum.
In the presence of bile salts, the globules are broken up into smaller lipid aggregates,
Vitamin A 13
which can be more easily digested by pancreatic lipase, retinyl ester hydrolase, cholesteryl
ester hydrolase, and the like. The resultant mixed micelles, which contain retinol, the
carotenoids, sterols, some phospholipids, mono- and diglycerides, and fatty acids, then
diffuse into the glycoprotein layer surrounding the microvillus and make contact with the
cell membranes. Various components of the micelles, except for the bile salts, are then
readily absorbed into mucosal cells, mainly in the upper half of the intestine (39). A
specific transporter exists for vitamin A (40) but evidently not for carotenoids. Nonetheless,
carotenoids do interact with each other during absorption (41,42).
The bioavailability and the digestion of vitamin A and carotenoids, needless to say,
are affected by the overall nutritional status of the individual and the integrity of the
intestinal mucosa. Nutritional factors of importance are protein, fat, vitamin E, zinc, and
probably iron. Some types of fiber, e.g., highly methoxylated pectins, markedly reduce
carotenoid absorption (43).
Bile salts, which are detergents, promote the rapid cleavage of retinyl and carotenoid
esters and assist in the transfer of these lipids into mucosal cells. Interestingly, carotenoid
absorption has an absolute requirement for bile salts, independent of its dispersion in a
suitable micelle, whereas vitamin A in any properly solubilized form is readily absorbed
(24,39).
The overall absorption of dietary vitamin A is approximately 80–90%, with somewhat
less efficient absorption at very high doses. The efficiency of absorption of carotenoids
from foods is 50–60%, depending on their bioavailability. The absorption effi-
ciency of carotenoids decreases markedly at high intakes or doses (24,39,44).
2. Transport
Retinol in intestinal mucosal cells, whether derived directly from the diet or formed from
carotenoids, is largely converted to retinyl esters in these cells. Retinyl esters, together
with some unesterified retinol, phospholipids, triglycerides, and apolipoproteins, are incorporated
into chylomicra, which are released into the lymph.
During the transport of chylomicra from the lymph into the general circulation, the
triglycerides are degraded by lipoprotein lipase of the plasma. The latter is activated by
divalent metal ions and requires the presence of albumin as a fatty acid acceptor. After
much of the triglyceride is digested and removed from the chylomicron, the resultant
chylomicron remnant, which contains retinyl ester, cholesteryl ester, some other lipids,
and several apolipoproteins, is taken up primarily by the liver but also to some extent by
other tissues (39). Uptake by liver involves interaction between apolipoproteins E and
B48 in the chylomicron remnant and high-affinity receptors on the cell surface of parenchymal
cells (39).
Whereas dietary retinol is transported to the liver largely as an ester in lipoproteins
formed in the intestine, the mobilization of vitamin A from the liver stores and its delivery
to peripheral tissues is a highly regulated process. A precursor (pre-RBP) of a specific
retinol-binding protein is synthesized in liver parenchymal cells (45). After the removal
of a 3500-d polypeptide, the resultant apo-RBP binds all-trans retinol, and the complex
(holo-RBP), together with transthyretin, is secreted into the plasma. Human RBP, first
isolated in the late 1960s, is a single polypeptide chain, has a molecular weight of 21,000,
and possesses a single binding site for retinol. Under normal conditions of vitamin A
nutriture, approximately 90% of RBP is saturated with retinol. In children the usual level
of RBP in plasma is approximately 1–1.5 µmol/L (20–30 µg/mL), which rises at puberty
to adult levels of 2–2.5 µmol/L (40–50 µg/mL). In human plasma RBP is largely com14
Olson
plexed with transthyretin, a tetramer that also binds thyroid hormones, in a 1:1 molar
complex. The association constant (Ka) between transthyretin and RBP is approximately
106 M/L. The amino acid sequence and three-dimensional structure of RBP have been
determined, and a cDNA clone for it has been identified and studied (45).
Within RBP, retinol resides in a hydrophobic ? barrel (46), which protects it from
oxidation or destruction during transport (Fig. 5). RBP has been isolated from the plasma
of many other species, including rat, monkey, rabbit, cow, dog, and chicken. In all of
these species, RBP is similar in size to that found in the human, and in most instances it
forms a complex with transthyretin.
The formation of the larger transthyretin–RBP complex may minimize the loss of
RBP in the urine during its passage through the kidney (45). In chronic renal disease, the
levels of RBP and of plasma retinol are greatly elevated, whereas in severe protein calorie
malnutrition, the amounts of both are reduced to approximately 50%. Thus, the steadystate
concentrations of both vitamin A and RBP in the plasma are dependent on factors
other than vitamin A status alone.
Under normal physiological conditions, the turnover of holo-RBP in the plasma is
quite rapid. When associated with transthyretin, its half-life is approximately 11 to 16 h,
Fig. 5 The structure of human holo-RBP, depicted as a ribbon. The loop that changes conformation
between the holo and apo forms of RBP is depicted in black. The N-terminal end is at the
upper left, the C-terminal end at the lower right. Retinol is also depicted in black within a eightstranded
? barrel. (Graciously provided by Dr. Marcia Newcomer, Vanderbilt University, Nashville,
TN. See also Ref. 46.)
Vitamin A 15
whereas in the apo form it is removed much more rapidly. These half-life values increase,
i.e., turnover decreases, about 50% in severe protein calorie malnutrition.
The retinol ligand of holo-RBP in the plasma not only is taken up by peripheral
target tissues but also is recycled back to the liver. By use of in vivo kinetic analysis,
Green and Green (47) found that an average retinol molecule in the plasma recycles 7–
13 times before being irreversibly lost. As another expression of the efficiency of recycling,
the turnover rate of plasma retinol is 10 times the irreversible disposal rate of wholebody
vitamin A (47). The kidney is the major organ involved in recycling. If its function
is adversely affected by disease, infection, and high fever, large amounts of holo-RBP
are excreted in the urine (48).
Most of the retinol recycled from tissues back to the liver, on kinetic grounds, is
in the form of holo-RBP. However, other transport agents, e.g., retinyl ester on lipoproteins
and retinyl ?-glucuronide, may play some role in recycling. The key point is that retinol
is carefully conserved by the body and is not indiscriminately lost through excretion.
Indeed, the irreversible daily loss of vitamin A in the feces and urine of vitamin A–
depleted animals, and presumably of humans, is much reduced relative to the rate of loss
in vitamin A–sufficient animals (47).
Of all processes relative to the uptake, storage, and transport of vitamin A, the
combination of retinol with RBP in the liver seems to be one of the most specific. Thus,
the only ligand found under normal physiological conditions is all-trans retinol. Other
vitamin A analogs will combine with apo-RBP in vitro, however, and 3-dehydroretinol,
15-methylretinol, and 15-dimethylretinol form holo-RBP analogs in vivo (49,50). Indeed,
the abilities of specific analogs to saturate RBP in vivo can be closely related to their
biological activities (49).
Retinoic acid is primarily transported on plasma albumin and retinyl esters in
low-density lipoproteins (39). The ?-glucuronides of retinol and retinoic acid, although
water-soluble, are bound to plasma albumin and possibly to other plasma proteins in vivo
(51).
3. Uptake, Storage, and Release by Cells
The major circulating form of vitamin A in plasma is holo-RBP. Two mechanisms have
been explored for its uptake by cells, one of which is dependent on cell surface receptors
and the other of which is not (45,52).
Cell surface receptors for holo-RBP have been best characterized from the retinal
pigment epithelium of the vertebrate eye. The receptor on the external surface of these
cells, i.e., that in contact with plasma, binds holo-RBP strongly, i.e., with a Ka of 1–3 
107 M/L (45). The estimated size of the receptor is 63 kd. Transthyretin is not bound to
the receptor, nor is an RBP–transthyretin complex necessary for binding. Each cell of the
retinal pigment epithelium has about 50,000 cell surface receptors for RBP. Cells of other
tissues, such as kidney, lung, and muscle, do not possess this receptor, although other
proteins that facilitate retinol uptake may be present on them (45,52). Receptors for holo-
RBP may also be present on cells of the liver, skin, placenta, and the barriers between
the blood and testes and the blood and brain (52). After complexing with the cell surface
receptor, holo-RBP, in all likelihood, is internalized within target cells by receptor-mediated
endocytosis. Thereafter, retinol is released into the cytosol, and the apo-RBP is either
degraded or secreted from cells in a modified form (45,52). Apo-RBP is bound to surface
receptors less tightly than holo-RBP.
16 Olson
Receptor-independent uptake of retinol has been demonstrated in keratinocytes and
in some liver cell lines cultured in vitro (45). Retinol can dissociate from RBP at a rate
sufficient to explain its direct uptake by lipid membranes (52). In all probability, both
mechanisms operate with different cells and under different conditions.
Under normal conditions, more than 90% of the vitamin A in the animal body is
stored in the liver, although small quantities are also found in all other tissues. Thus, most
of the attention has been given to elucidating the mechanism of its storage and release
from the liver.
Endocytosed chylomicron remnants are initially located in low-density endosomes
of parenchymal cells (52,53), where retinyl esters are hydrolyzed. Retinol is then translocated,
presumably in a complex with CRBP-I (cellular retinol-binding protein I), to the
endoplastic reticulum, which is rich in apo-RBP and contains both lecithin : retinol acyltransferase
(LRAT) and acyl-CoA: retinol acyltransferase (ARAT). Retinol might continue
to circulate as holo-CRBP-I, be transferred to apo-RBP for release into the plasma or to
stellate cells, be transferred to stellate cells by some other mechanism, or be esterified
to retinyl ester. Retinyl esters, which predominantly consist of palmitate with smaller
amounts of stearate, oleate, and linoleate, are then stored in vitamin A–containing globules
(VAGs) within the parenchymal cells. When liver reserves of vitamin A are low, parenchymal
cells are the major storage site of vitamin A (39,52,53).
When liver reserves are adequate (20 µg retinol per gram), approximately 80%
of the newly absorbed vitamin A is transferred from parenchymal cells to a specialized
type of perisinusoidal cell, termed the stellate cell (fat-storing cell, lipocyte, Ito cell) (52–
54). The mechanism of intercellular transport is not clear, although complexes of retinol
with CRBP and RBP have been postulated to serve as possible carriers. Within stellate
cells, retinol is rapidly esterified to fatty acids in a pattern similar to that found in parenchymal
cells. The resultant retinyl esters are stored in vitamin A–containing globules, which
contain up to 60% retinyl esters, a significant amount of triglyceride, less phospholipid,
and small amounts of cholesterol, cholesteryl esters, and ?-tocopherol. Several proteins,
including retinyl ester synthase and hydrolase, are associated with these globules. Stellate
cells also contain appreciable amounts of CRBP-I and cellular retinoic acid–binding protein
(CRABP). Whether stellate cells can synthesize RBP is controversial (52,53). Circulating
radiolabeled RBP, however, can be taken up by stellate cells. All other cells of the
liver, including Kupffer’s cells, endothelial cells, and other nonparenchymal cells, contain
very small amounts (5%) of the total liver reserve of vitamin A.
In the storage of dietary vitamin A, the hydrolysis and formation of retinyl esters
is important. Similarly, in the release of vitamin A from the liver as holo-RBP, hydrolysis
of retinyl esters is a key step. In regard to the control of these processes, it is interesting
that several forms of retinyl ester hydrolase exist in the liver (53) and that their activity
is enhanced by a poor vitamin A status and by vitamin E deficiency. On the other hand,
the esterifying enzymes are enhanced when large amounts of vitamin A are ingested. Thus,
these enzymes should be included among factors that control vitamin A metabolism in
vivo. Another important factor may be the ratio of apo-CRBP-I to holo-CRBP-I (55).
When apo-CRBP-I predominates, the hydrolysis of vitamin A is favored, but when holo-
CRBP-I is the major form, the storage of retinyl esters is enhanced.
Stellate cells are found not only in rat liver but in many other tissues and in many
other species (54). Besides storing vitamin A, stellate cells are highly active in synthesizing
collagen and other structural proteins. The factors underlying the association of these two
important processes in stellate cells have not been elucidated.
Vitamin A 17
As already mentioned, retinol is released from parenchymal cells of the liver as a
1:1 complex of holo-RBP and transthyretin. In mice that cannot make transthyretin, holo-
RBP concentrations in the plasma are very low (56). Stellate cells of the liver may also
release holo-RBP directly or may transfer retinol to circulating apo-RBP (52). Cells of
other tissues, and particularly of kidney, seem to release holo-RBP for transport back to
the liver. No structural differences have been noted between RBP synthesized in the liver
and that formed in peripheral tissues (45). Retinyl esters are released from the intestine as
chylomicra, as already noted, and from the liver as very low-density lipoproteins (VLDLs)
(52,53). Although retinoic acid, retinyl ?-glucuronide, and retinoyl ?-glucuronide are present
in plasma in low concentrations, the mode of their release from tissues has not been
defined.
4. Transformations
Dietary forms of vitamin A are retinyl esters, derived entirely from animals sources, and
provitamin A carotenoids, derived largely from plant foods. Of the total carotenoids present
in nature, however, less than 10% serve as precursors of vitamin A. Retinol is released
from its dietary esters by the action of pancreatic hydrolases, which act in the presence
of bile salts within the intestinal lumen. Within intestinal mucosal cells, retinol combines
with CRBP-II (cellular retinol-binding protein II) to give a complex that can undergo
either esterification to retinyl ester or oxidation to retinal (39,40,53,55). The major source
of the acyl moiety is the usually saturated fatty acid in the ? position of phosphatidylcholine,
but a transfer from acyl-CoA can also occur (39,40,53,55).
The major reactions of vitamin A metabolism, which are summarized in Fig. 6,
are esterification, oxidation at C-15, oxidation at C-4, conjugation, isomerization, other
miscellaneous oxidative reactions, and chain cleavage (53,55). Retinol and retinal, as well
Fig. 6 Major metabolic transformations of vitamin A. ROL, retinol; RE, retinyl ester; RAL, retinal;
RA, retinoic acid; RG, retinyl ?-glucuronide; RAG, retinoyl ?-glucuronide; 5,6-EROL, 5,6-
epoxyretinol; 5,6-ERE, 5,6-epoxyretinyl ester; 14-HrROL, 14-hydroxy-4,14-retro retinol; 4-HRA,
4-hydroxyretinoic acid; 4-ORA, 4-oxoretinoic acid; 5,6-ERA, 5,6-epoxyretinoic acid; C-19 and C-
16, chain-shortened, oxidized products with the indicated number of carbon atoms. Double arrows
indicate reversible reactions; single arrows irreversible changes. (From Ref. 39.)
18 Olson
as other metabolites reversibly converted to them, all possess significant biological activity.
Retinoic acid and its glucuronide are active in growth but not in vision or, in most
species, in reproduction. Except for 14-hydroxy-4,14-retro retinol, more oxidized products,
such as 4-hydroxyretinoic acid, 5,6-epoxyretinoic acid, and C-19 metabolites, are
largely devoid of biological activity. Retinoyl ?-glucuronide, retinyl ?-glucuronide, and
retinoic acid are normally present in small amounts (3–11 nmol/L, or 1–5 µg/L) in human
plasma. Retinoyl ?-glucuronide is not hydrolyzed in some cells and slowly hydrolyzed
in vivo. Retinoic acid can also be covalently bound to proteins, possibly by means of a
coenzyme A intermediate (39,57).
The enzymology of these reactions in most instances is conventional. Retinol is
reversibly oxidized to retinal by a nicotinamide adenine dinucleotide (NAD)–dependent
pathway in many tissues. Although alcohol dehydrogenase can catalyze this reaction, specific
retinol dehydrogenases (retinal reductases) also exist.
Retinal is then irreversibly converted to retinoic acid in many tissues by aldehyde
dehydrogenases and oxidases (53). The rate of conversion of retinal to retinoic
acid is several fold faster than the oxidation of retinol to retinal. Retinoic acid is inactivated
biologically either by hydroxylation at the C-4 position or by epoxidation at
the C-5/6 positions. In animal tissues, both of these oxidative reactions are irreversible.
4-Hydroxyretinoic acid can be further oxidized to the 4-keto derivative, followed by
a variety of oxidative, chain-cleaving, and conjugative reactions. One of the major
conjugated cleavage products is retinotaurine, which appears in the bile in significant
amounts (58).
Vitamin A also forms several ?-glucuronides by interacting with UDP-glucuronic
acid in the presence of glucuronyltransferases. The most interesting of these conjugates
are retinyl ?-glucuronide and retinoyl ?-glucuronide, both of which retain high biological
activity (53). Both are synthesized in the intestine, in the liver, and probably in other
tissues. In addition, both are endogenous components of human blood and, together with
the glucuronides of 4-ketoretinoic acid and of other oxidized metabolites, appear in the
bile (53). The rate of formation of ?-glucuronides of 9-cis and of 13-cis retinoic acid by
rat liver microsomes in vitro is significantly faster than that of the all-trans isomer (59).
When retinyl acetate or retinyl ?-glucuronide is administered to rats, 5,6-epoxyretinyl
ester is found in discrete amounts in the liver (60). Thus, epoxidation occurs at the
level of retinol as well as of retinoic acid.
?-Carotene and several other provitamin A carotenoids are converted by oxidative
cleavage at the 15,15? double bond to yield two molecules of retinal. The enzyme catalyzing
this reaction, 15,15?-carotenoid dioxygenase, is found in the intestine, liver, kidney,
and some other tissues. This enzyme will also convert ?-apocarotenoids found in plants
and in some animal tissues to retinal (53). 9-cis ?-Carotene is also cleaved by the enzyme
to yield 9-cis retinal, which can be oxidized to 9-cis retinoic acid (61,62), the ligand for
RXR receptors. ?-Carotene can also be cleaved eccentrically to ?-apocarotenals, which
can be further degraded oxidatively to retinal (63). Although the relative importance of
the two pathways in vivo in mammals has not been determined, cell-free preparations of
pig and guinea pig intestines yield retinal as the major, if not sole, product of ?-carotene
cleavage (64,65).
5. Retinoid-Binding Proteins
Retinoid-binding proteins play key roles in the metabolism and function of their ligands
(39,45,53,55,66,67). Major well-characterized binding proteins for retinoids are listed in
Vitamin A 19
Table 3 Properties of Major Retinoid-Binding Proteinsa
Molecular
Name Abbreviation weight (kd) Major ligand
Retinol-binding protein RBP 21.2 All-trans retinol
Cellular retinol-binding protein, CRBP-I 15.7 All-trans retinol
type I
Cellular retinol-binding protein, CRBP-II 15.6 All-trans retinol
type II
Cellular retinoic acid–binding CRABP-I 15.5 All-trans retinoic acid
protein, type I
Cellular retinoic acid–binding CRABP-II 15.0 All-trans retinoic acid
protein, type II
Cellular retinaldehyde-binding CRALBP 36.0 11-cis retinol and retinal
protein
Interphotoceptor retinol-binding IRBP 135.0 11-cis retinal and all-trans
protein retinol
Epididymal retinoic acid–bind- E-RABP 18.5 All-trans and 9-cis retinoic
ing protein acid
aBased on Refs. 46, 55, and 68.
Table 3. Several are coligands for enzymatic transformations of retinoids, namely, CRBPII,
CRBP-I, CRABP-I, and possibly CRABP-II (66,67). RBP (as already mentioned), interphotoreceptor
retinal-binding protein (IRBP) and epididymal retinoic acid–binding protein
(E-RABP) primarily serve transport functions, the first in the plasma, and the second
in the eye, and the third in the testes (39,45,68). Cellular retinaldehyde-binding protein
(CRALBP) probably also serves a transport function in retinal pigment epithelial cells and
possibly in Mu?ller cells of the eye, but it may also have some role during morphogenesis of
the eye (68). The ratio of apo to holo forms of some of these proteins, as already mentioned,
may regulate metabolic processes (66,67). Apart from these major functions, all
binding proteins sequester retinoids in hydrophobic cavities, thereby protecting the ligand
from degradation and membrane systems of the cell from unwanted interaction with these
amphiphilic ligands. Finally, the RBPs render the hydrophobic ligands water-soluble,
much like glucuronidation does. Some redundancy in their functions may exist in that
transgenic mice without CRABP-II and/or CRABP-I developed normally in one study
(69) but showed polydactyly in another (70).
Other proteins, such as several fatty acid–binding proteins, serum albumin, and ?-
lactoglobulin, also bind retinoids. These interactions, although relatively nonspecific,
might play some physiological role.
6. Excretion
In a quantitative sense, ingested vitamin A is generally metabolized in the following way:
(a) 10–20% is not absorbed and, hence, is excreted within 1–2 days into the feces; (b)
of the 80–90% absorbed, 20–60% is either conjugated or oxidized to products that are
excreted within approximately 1 week in the feces or urine, with a small amount in expired
CO2; and (c) the remainder (30–60%) of the absorbed vitamin A is stored, primarily in
the liver. However, when the initial liver reserves are depleted, the relative amount stored
in the liver is much lower (39).
20 Olson
Stored vitamin A is metabolized much more slowly in the liver and peripheral tissues
to conjugated and oxidized forms of vitamin A, which then are excreted. The half-life for
the overall depletion rate in humans is 128–156 days (71). As a general rule, derivatives
of vitamin A with an intact carbon chain are excreted in the feces, whereas acidic chain–
shortened products tend to be excreted in the urine. In the steady state, approximately
equal amounts of metabolites are excreted in the feces and the urine. During periods of
severe infection, however, large amounts of holo-RBP, often exceeding the daily requirement
for vitamin A, are excreted in the urine (48).
B. Functions
1. Vision
When a photon of light strikes the dark-adapted retina of the eye, 11-cis retinal, present
as a protonated Schiff base of lysine residue 296 in rhodopsin, is converted to a highly
strained transoid form in bathorhodopsin. The more stable all-trans form of bathorhodopsin
is then converted to metarhodopsin I, and subsequently by deprotonation to metarhodopsin
II (68).
Rhodopsin contains both hydrophilic and hydrophobic regions; has a molecular
weight of approximately 38,000 in the cow; is asymmetrical, with a folded length of
approximately 70A° ; and absorbs maximally at 498–500 nm. Like several other transmembrane
proteins, rhodopsin contains seven helical segments that extend back and forth
across the disk membrane. Approximately 60% of the total amino acid structure is found
in these helical proteins. In addition to loops between helical portions, less structured Cterminal
and N-terminal portions extend into the cytosol and the intradisk space, respectively.
The chromophore resides in a hydrophobic pocket formed by several transmembrane
segments near the cytosolic side of the disk membrane. Residues close to the
chromophore include Phe115, Ala117, Glu122, Trp126, Ser127, Trp265, and Tyr268 (68).
Rhodopsin contains an acetylated N terminus and two oligosaccharides on asparagines 2
and 15. The complete amino acid sequence of rhodopsin is known.
Three color pigments exist in the cone cells of the human eye, with maximal absorption
at 420 nm (blue cones), 534–540 nm (green cones), and 563–570 nm (red
cones). The amino acid sequences of these iodopsins are similar, but not identical, to
each other and to that of rhodopsin. All adopt a similar conformation within the disk
membrane, being composed of seven transmembrane segments. Only seven amino acids
differ among the transmembrane segments of the cone pigments. Thus, the differences in
absorption maxima must reside in subtle changes in the ambience surrounding the chromophore
(68).
Metarhodopsin II, the penultimate conformational state of the light-activated visual
pigment, reacts with transducin, a G protein attached to the disk membrane that contains
three subunits. In response, the ? subunit of transducin binds GTP in place of endogenously
associated GDP, thereby activating cGMP phosphodiesterase, which hydrolyzes
cGMP to GMP. Because cGMP specifically maintains in an open state the sodium pore
in the plasmalemma of the rod cell, a decrease in its concentration causes a marked reduction
in the influx of sodium ions into the rod outer segment. The membrane consequently
becomes hyperpolarized, which triggers the nerve impulse to other cells of the retina
through the synaptic terminal of the rod cell (68). This sequence of events is summarized
in Fig. 7.
Vitamin A 21
Fig. 7 Possible sequence of steps between the light-induced activation of rhodopsin and hyperpolarization
of the rod cell membrane. R., Rhodopsin; R: light-activated metarhodopsin II; R-PO4,
phosphorylated rhodopsin; ATP, adenosine triphosphate; GTP, guanosine triphosphate; GDP, guanosine
diphosphate; cGMP, cyclic 3?–5?-guanosine monophosphate; GMP, guanosine monophosphate;
T., transducin; T.-GDP, a complex of transducin with GDP; T.-GTP, a complex of transducin
with GTP; PDE, phosphodiesterase. (From Ref. 39.)
Recovery from this activated state occurs in three ways: (a) The ? subunit of transducin,
which also shows GTPase activity, hydrolyzes bound GTP to GDP, thereby leading
to subunit reassociation to inactive transducin. As a consequence, cGMP phosphodiesterase
activity falls and the cGMP level increases back to a normal level. (b) Metarhodopsin
II is phosphorylated at sites in the C-terminal portion by ATP, which then reacts with
arrestin to form a complex that no longer activates transducin. (c) Metarhodopsin II dissociates
through metarhodopsin III to yield all-trans retinal and opsin, which also does not
activate transducin. These events are also summarized in Fig. 7.
Retinol is very much involved in the process of vision, as shown in Fig. 8 (68).
All-trans retinol bound to RBP interacts with a cell surface receptor and is internalized
into retinal pigment epithelial cells (RPEs) (reaction 8). Thereafter, probably as a CRBP
complex, retinol is transacylated from phosphatidylcholine to yield retinyl ester (reaction
3), which in turn undergoes a concerted hydrolysis and isomerization to 11-cis retinol
(reaction 4) (72). The latter, probably as a CRALBP complex, can either be esterified by
transacylation (reaction 3) or be oxidized to 11-cis retinal (reaction 5). The latter compound
is transferred to IRBP, which ferries it to the rod outer segment in the neural retina.
11-cis Retinal, probably in association with a protein but not with CRALBP, then combines
with opsin in the disk membranes to form rhodopsin (reaction 7). Another source
of both all-trans and 11-cis retinal in the RPEs is the discarded tips of the constantly
regenerating rod outer segments, which are phagocytized by the RPE (not shown in Fig. 8).
Light activation of rhodopsin, as already noted, ultimately gives rise to all-trans
retinal (reaction 1), which is reduced in the rod outer segment to all-trans retinol (reaction
2). The latter is transported by IRBP back to the RPE, where it is stored as all-trans retinyl
ester (reaction 3). Thus, the cycle is complete.
During continued exposure of the retina to strong light, rhodopsin is largely converted
to opsin plus all-trans retinal, which tends to accumulate as all-trans retinyl ester
in the RPE. During dark adaptation, the reverse process occurs. As the rhodopsin concentration
increases in the rod outer segment, the retina becomes increasingly sensitive to
light of very low intensity.
22 Olson
Fig. 8 Reactions of the mammalian visual cycle. Horizontal lines separate cellular and extracellular
compartments in the retina involved in retinoid metabolism. Most of the enzymatic reactions of
the cycle occur in RPE, including the generation of 11-cis retinol. The reactions shown below the
double line occur within the photoreceptor cell. Retinoid-binding proteins found within the indicated
compartments are shown on the left. CRALBP, cellular retinaldehyde-binding protein; CRBP, cellular
retinol-binding protein; IRBP, interphotoreceptor retinoid-binding protein; RBP, retinol-binding
protein; at-, all-trans-; 11-, 11-cis-; Rol, retinol; Ral, retinaldehyde; RE, retinyl ester; gNa, sodium
conductance; RPE, retinal pigment epithelium. Reactions shown are: 1, photoisomerization; 2, alltrans
retinol dehydrogenase; 3, lecithin:retinol acyltransferase (LRAT); 4, retinyl ester isomerohydrolase;
5, 11-cis retinol dehydrogenase; 6, retinyl ester hydrolase; 7, regeneration of rhodopsin
(nonenzymatic); 8, uptake of plasma retinol. (From Ref. 68.)
Most of the processes involved in the visual cycle have been extensively studied,
including the enzymes that are involved (68).
2. Growth
The earliest assays for vitamin A were based on the growth response of rats fed a purified
diet. The fact that maintenance of normal vision and enhancement of growth are two
separate properties of the vitamin A molecule was dramatically demonstrated by the observation
that retinoic acid stimulated growth but could not maintain vision (73). In nutritional
studies, the onset of vitamin A deficiency has often been detected by a so-called growth
plateau, which after several days is followed by a rapid loss of weight and, ultimately,
death. However, in animals cycled on retinoic acid, i.e., given retinoic acid for 18 days
followed by 10 days of deprivation, rats become exquisitely sensitive to the removal of
Vitamin A 23
retinoic acid from the diet (74). In these animals loss of appetite occurs within 1–2 days
after the withdrawal of retinoic acid, which is closely followed by a depression in growth.
Loss of appetite is therefore one of the first symptoms noted in all vitamin A–deficient
animals. In humans, however, night blindness and mild xerophthalmia seem to be the
earliest signs of clinical deficiency.
Decreased food intake is not due in this case to impaired taste function or to the
poor palatability of the deficient diet. Because many factors affect appetite, it has not been
possible to define specifically the molecular effect of vitamin A on this process. Inasmuch
as distortions in nitrogen metabolism and in amino acid balances within tissues and in
the plasma occur concomitantly, the effect of vitamin A deficiency on appetite may be
related to these latter abnormalities as well as to disturbances in cell differentiation.
3. Cellular Differentiation
In vitamin A deficiency, mucus-secreting cells are replaced by keratin-producing cells in
many tissues of the body. Conversely, the addition of vitamin A to vitamin A–deficient
keratinizing cells in tissue culture induces a shift to mucus-producing cells. Retinoids also
rapidly induce F-9 teratocarcinoma cells, as well as many other cell lines, to differentiate.
In this process, many new proteins appear in the newly differentiated cells. Thus, vitamin
A and its analogs, both in vivo and in vitro, markedly influence the way in which cells
differentiate (30,39).
The mechanism by which retinoids induce cellular differentiation is becoming clear
(Fig. 9). Within tissue cells, all-trans retinol, in association with CRBP, can be oxidized
to all-trans retinoic acid and presumably can also be isomerized to 9-cis retinol, which
in turn can be oxidized to 9-cis retinoic acid. The latter can also arise by isomerization
of all-trans retinoic acid or by central cleavage of 9-cis ?-carotene (61,62). All-trans or
9-cis retinoic acid is transported on CRABP or on other RBPs to the nucleus, where it is
tightly bound to one or more of the three (?, ?, ?) retinoic acid receptors (RAR) or to
one or more of the three (?, ?, ?) retinoid X receptors (RXR), respectively (39,75–79).
The RAR and RXR receptors, like other nuclear hormone receptors, possess six
protein domains with specific functions. At the N-terminal end, domains A and B serve
as physiological activators of the receptor; domain C, which is highly conserved, contains
zinc–sulfhydryl interactions (‘‘zinc fingers’’) that bind to DNA; domain D is a hinge
region that provides the necessary conformation of the receptor; domain E binds the ligand;
and domain F, at the C-terminal end, enhances dimerization. All nuclear retinoid receptors
contain 410–467 amino acids and have molecular weights of 45–51 kd (39,75–79). RAR?
and RXR? show very limited homology to each other (only 61% in the DNA binding
domain and 27% in the ligand binding domain). Each of the six human retinoid receptors
shows different chromosomal locations, developmental expression, and organ localization.
Thus, RAR? and RXR? are widespread in their occurrence; RAR? and RXR? are both
present in adult muscle and heart but differ in their embryonic appearance and localization;
RAR? is found in adult skin and lung but has a variable embryonic pattern; and RXR?
is found in liver, skin, and kidney of both adults and embryos. Thus, each receptor seems
to show its own pattern of expression and function. Furthermore, no functional relationship
exists between the respective ?, ?, and ? forms of the two receptors (75–79).
Of the two retinoid receptor families, RXR shows the broadest actions. In the absence
of 9-cis retinoic acid, RXR forms heterodimers with the vitamin D receptor (VDR),
the thyroid receptor (TR), and RAR but only when the latter are charged with their respective
ligands, namely, 1?, 25-calcitriol, triiodothyronine, and all-trans or 9-cis retinoic
24 Olson
Fig. 9 Roles of retinoids in cellular differentiation at, All-trans; 9c, 9-cis, ROL, retinol; HrROL,
14-hydroxy-4,14-retro retinol; RAL, retinal; RA, retinoic acid; RAG, retinoyl ?-glucuronide; RR,
cell surface receptor for holo-RBP; RBP, plasma retinol-binding protein; CRBP, cellular retinolbinding
protein; CRABP, cellular retinoic acid–binding protein; RAR?, ?, ?, nuclear retinoic acid
receptors, forms ?, ?, and ?; RXR?, ?, ?, nuclear 9-cis retinoic acid receptors, forms ?, ?, ?; TR,
triiodothyronine bound to the nuclear thyroid receptor; VDR, calcitriol bound to the nuclear vitamin
D receptor; X, unknown nuclear receptors; solid arrows, known transformations or effects; dashed
arrows, postulated transformations or effects; ?, unknown occupancy. (From Ref. 39.)
acid (75–79). In the presence of 9-cis retinoic acid, RXR will bind its ligand and form a
homodimer. Thus, heterodimer and homodimer formation are competitive actions that are
dependent on the ratio of all-trans retinoic acid to 9-cis retinoic acid and the latter’s
concentration in the nucleus.
The hormone response element (HRE) in DNA for both RXR and RAR is the
consensus sequence AGGTCA. For gene activation, a direct repeat, i.e., AGGTCA–
other bases–AGGTCA, rather than a palindromic arrangement, is most common. The
spacing between the two sequences and the number of tandem repeats largely defines
the specificity of the interaction. This observation has given rise to the DR 1-2-3-4-5
rule, in which the numbers refer to the number of intervening deoxynucleotide base pairs
between the directly repeating (DR) consensus sequences (75–79). The RXR response
element usually employs DR-1 spacing, the RAR response element uses DR-2 or DR-5
spacing, and the VDR and TR response elements use DR-3 and DR-4 spacing, respectively.
The sequence of intervening bases often is important as well, but not always. The
RXR homodimer is demonstrably formed in the presence of 9-cis retinoic acid, whereas
the RAR homodimer only seems to form under special circumstances within cells
(75–79).
In addition to the interaction with RAR, VDR, and TR, RXR also forms heterodimers
with two orphan receptors, the chicken ovalbumin upstream promoter transcription
Vitamin A 25
factor (COUP-TF) and the peroxisomal proliferator activated receptor (PPAR). The ligands
for these two receptors are not known. Although most heterodimers of RXR, including
that with PPAR, activate gene expression, RXR-COUP-TF, either as such or as a
COUP-TF homodimer, inhibits the expression mediated by the RXR homodimer of the
CRABP-II gene. Similarly, RAR, probably by combining with cJun in a nonproductive
complex, also inhibits the activation of the AP-1 site, which is important for cell proliferation.
The AP-1 site normally is activated by the cJun-cFos heterodimer. Thus, the retinoid
receptors can show both activation and suppression of gene expression, depending on the
nature of the heterodimers formed. Genes are activated by retinoids as a result of the
binding of an appropriate homo- or heterodimer to a hormone response element in DNA,
whereas gene expression seems to be suppressed by the competition between a retinoid
receptor and some other transcription factor for the latter’s activating partner protein
(75–79).
Many genes contain response elements for the retinoid receptors, as shown in Table
4 (75–80). Although all of these effects cannot be placed in a cohesive physiological
framework, some deserve special mention, namely: (a) the stimulation of both certain
cytosolic and nuclear binding proteins for retinoids by retinoic acid via retinoic acid response
elements; (b) the stimulation of Hox a-1 (Hox 1.6) and Hox b-1 (Hox 2.9), initiating
genes of embryonic development in several cells and species and (c) enhancement of class
I alcohol dehydrogenase type 3, which may well induce the synthesis of more retinoic
acid from retinol.
Some retinoids may stimulate differentiation by a different pathway; for example,
retinoyl ?-glucuronide does not bind to CRBP, CRABP, or nuclear RAR but nonetheless
is highly active biologically (39,53). Similarly, B lymphocytes differentiate in response
to 14-hydroxy-4,14-retro retinol but not to all-trans retinoic acid (81). Furthermore, the
binding to RAR of various acidic retinoids, both natural and synthetic, relates closely to
their invoked cellular responses but not to their binding affinities for CRABP, at least in
some cellular systems (39).
4. Morphogenesis
Both a deficiency and an excess of vitamin A and of most other retinoids adversely affect
embryogenesis (79,82). Studies on the role of vitamin A in embryogenesis were greatly
stimulated by the demonstration that an implant containing all-trans retinoic acid, when
placed in the anterior part of the developing chick limb bud, mimics the activity of the
naturally occurring zone of polarizing activity (ZPA) (82,83). Thus, the hypothesis arose
Table 4 Some Proteins Whose Genes Contain Response
Elements for Retinoid Receptors
RAR?2 Hox a-1 (Hox 1.6)
RAR?2 Hox b-1 (Hox 2.9)
RAR?2 Phosphoenolpyruvate carboxykinase
CRBP-I Apolipoprotein A-I
CRBP-II Ovalbumin
CRABP-II Complement factor H
Laminin B1 Alcohol dehydrogenase (class I, type 3)
Acyl coenzyme A oxidase
26 Olson
that all-trans retinoic acid might well be one of a presumed host of morphogens that
control embryologic development (39).
The initial hypothesis was refined to postulate that a posterior-to-anterior gradient
of all-trans retinoic acid, but not of retinol, was primarily responsible for pattern formation
in the chick limb bud (82,83). In a historical context, pattern formation in the skin was
postulated to depend on gradients of vitamin A more than 20 years ago (84). A significant
body of experimental data accord with the gradient hypothesis. On the other hand, many
other observations do not, including the observation that the gene product of Sonic hedgehog
induces the polarizing activity of the ZPA, independent of the presence of RA (85).
Thus, the gradient hypothesis is not as attractive today as it was when first formulated
(80,82,84). Nonetheless, retinoic acid, some of its metabolites, and its receptors clearly
play crucial roles in development (79,80,82,83).
Other retinoids also induce limb bud duplication: tetrahydrotetramethylnaphthalenylpropenylbenzoic
acid (TTNPB), CD-367, a structurally related compound, and 9-cis
retinoic acid are much more active than all-trans retinoic acid; all-trans 3,4-didehydroretinoic
acid and Am-580 are as active; and 13-cis retinoic acid and many other retinoids are
less active (82).
Retinoid receptors and cellular retinoid-binding proteins are expressed in a temporally
dependent manner in various parts of the developing embryo. In 9.5- to 10-d mouse
embryos, for example, RAR? and RAR? are uniformly expressed throughout the mesenchyme,
whereas RAR? is restricted to the proximal part of the limb bud. Concomitantly,
CRABP-I is expressed uniformly in the mesenchyme as well as in the apical ectodermal
ridge. As the embryo develops, the distribution and concentrations of both retinoid-binding
proteins and retinoid receptors change.
To define the possible roles of specific retinoid receptors in development, mice lacking
one allele of various isoforms of the RAR family, of all RAR? isoforms, and of all
RAR? isoforms were created (79). Null mice homozygous for single RAR isoforms
showed no deformities, whereas mice homozygous for either all RAR? or all RAR? isoforms
showed an altered phenotype. Although indistinguishable at birth from their wildtype
littermates, mice null for RAR? were rejected by their mothers, grew slowly, and
eventually died, even though no malformations could be detected (79). In contrast, most
RAR? null mice showed abnormalities of the axial skeleton but not of the limbs. Null
mice for both RAR? and RAR? showed more severe abnormalities and died soon after
birth (79). The RXR? null mutation was embryolethal. These interesting findings strongly
implicate the retinoid receptors in embryogenesis but also indicate that considerable redundancy
exists in the functions of specific isoforms. The observation that limbs develop
normally in the absence of RAR? or RAR? is particularly interesting in view of the marked
actions of retinoic acid on that process (79).
Genes that clearly play direct roles in development are the four Hox gene clusters
(80,82). As already indicated, Hox a-1 (Hox 1.6) contains a retinoic acid response element
(RARE) at its 3? end. Activated Hox a-1 could set in motion sequential activation of other
5?-located genes in its cluster (Hox a-2 to a-13). Hox b-1 (Hox 2.9) of the Hox b family,
which is involved in hindbrain development, contains an enhancing RARE at its 3? end
and a suppressing RARE at its 5? end (86). Genes of the Hox d family, which are known
to be involved in limb development, are also activated by retinoic acid (80,82). Other
genes are also activated or suppressed by treatment with retinoic acid (80). Their interplay
in development is slowly being clarified.
Vitamin A 27
Retinoids not only influence the normal physiological development of the embryo
but also, in larger amounts, induce fetal abnormalities. Thus, the regulation of retinoic
acid metabolism in the developing embryo is a crucial aspect of the overall complex
process.
5. The Immune Response
Vitamin A was early termed the ‘‘antiinfective’’ vitamin, based on the increased number
of infections noted in vitamin A–deficient animals and humans (81). In vitamin A defi-
ciency, both specific and nonspecific protective mechanisms are impaired: namely, the
humoral response to bacterial, parasitic, and viral infections; cell-mediated immunity; mucosal
immunity; natural killer cell activity; and phagocytosis (39). Large doses of vitamin
A can also serve as an adjuvant. When vitamin A–deficient animals are supplemented
with vitamin A, immune responses generally improve. The immune responses to certain
antigens in vitamin A–depleted children are also enhanced by vitamin A supplementation
(87). However, increased responsiveness in children is less marked than in animals, probably
because of the presence of multiple nutritional deficiencies in malnourished populations
and a poor, but not acutely deficient, vitamin A status (39).
The primary immune response to protein antigens, such as tetanus toxoid, is markedly
reduced in vitamin A deficiency (81). On the other hand, the process of immunological
memory, essential for a marked secondary response, does not seem to be adversely
affected. In a similar manner, the primary immune response to membrane polysaccharides
of bacteria is severely depressed by vitamin A depletion but quickly recovers with vitamin
A supplementation (81). In contrast, the immune response to bacterial lipopolysaccharides
is unaffected by vitamin A status. Interestingly, the injection of lipopolysaccharide into
vitamin A–deficient rats greatly enhances their immune response to tetanus toxoid and
to bacterial polysaccharides (81). The nature of this stimulatory effect of lipopolysaccharide
has not been clarified. Nonetheless, the observation shows that the antibody-forming
process in vitamin A–deficient animals is functionally intact (81).
The humoral response to many viruses, including measles, herpes simplex, and the
Newcastle disease virus, is impaired in vitamin A deficiency and improved by repletion.
Similarly, the host response to some parasitic infections is reduced by vitamin A depletion
(81).
In most instances, except as noted below, retinoic acid was more potent than retinol
in restoring the humoral response in vitamin A–depleted animals (88). At least one
mechanism of action for retinoic acid in the stimulation of antibody formation is now
becoming clear (88). In vitamin A–sufficient animals, an antigen is phagocytized by an
antigen-presenting cell (APC), often a macrophage, which metabolizes the antigen to a
fragment that is presented on the cell surface bound to class II molecules of the major
histocompatibility complex (MHC). T-helper lymphocytes are stimulated by contact with
APC to form interleukin-2 (IL-2), which in turn enhances T-cell proliferation as well
as B-cell growth and differentiation. Activated B cells (plasma cells) initially produce
IgM antibodies but switch to the production of high-affinity IgG antibodies upon maturation
(81).
Two types of T-helper cells exist, TH1 and TH2. TH1 cells secrete cytokines that
stimulate cell-mediated immunity (CMI) and TH2 cells secrete cytokines that enhance
antibody production (88). Thus, the balance between TH1 and TH2 will determine the
nature of the response to a given antigen. TH1 cells produce interferon ? (IFN-?) and TH2
28 Olson
cells produce IL-10, IL-5, and IL-4. Cross-regulation exists, inasmuch as IFN-? inhibits
TH2 cell proliferation and IL-10 and IL-4, via different mechanisms, inhibit both TH1 cell
development and IFN-? production (88).
In mesenteric lymph node cells of vitamin A–deficient mice, IFN-? is overproduced
and IL-10 and IL-5 are underproduced (88). Thus, the ratio of TH1 to TH2 cells is increased,
presumably leading to an enhancement of the CMI response and to a decrease in the
antibody response (88). These interesting observations explain the decrease in humoral
immunity in vitamin A deficiency but do not accord with the general observation that
CMI responses are usually depressed in vitamin A deficiency (81). Nonetheless, these
valuable studies pinpoint the T-helper cell as a major site of vitamin A action in the
immune response.
Much interest is now being shown in ?-14-hydroxy-retro retinol (HRR). B-Lymphoblastoid
cells transformed with Epstein–Barr virus die unless HRR, which is derived
from retinol, is present in the medium (81). Retinoic acid is inactive in this system. Retinol,
presumably via HRR, also is involved in the proliferation of normal B cells and T cells.
Various cytokines can modulate the process but cannot replace HRR in it. HRR has also
been identified in many other types of cells. These studies have stimulated much interest
for two reason: (a) retinoic acid, which in most instances is presumed to be the active
form of vitamin A, is inactive in these systems, and (b) other retro derivatives of vitamin
A, such as anhydroretinol and retroretinol, do not enhance animal growth. As yet, however,
the enzyme responsible for the conversion of retinol to HRR has not been studied. These
observations indicate that another signaling pathway for retinoids, albeit specialized in
given cells, may exist.
Phagocytosis, particularly the ‘‘oxygen burst’’ following the ingestion of a foreign
body, and IgA secretion are also depressed in vitamin A deficiency (81). The synthesis
of goblet cell mucins is reduced as well by vitamin A depletion, both in the intestinal
mucosa and in the conjunctiva of the eye. Because protein malnutrition also adversely
affects the immune response, children afflicted with both deficiencies are clearly at increased
risk of developing severe infections (39).
In vitamin A–sufficient animals, carotenoids also enhance the immune response.
Both nutritionally inactive (canthaxanthin) and nutritionally active (?-carotene) carotenoids
have similar effects (39). Their mechanism of action is not known.
6. Transmembrane Proton Transfer
The past generalization that vitamin A was found only in the animal kingdom was upset
in 1971 by the dramatic discovery of a new retinaldehyde-containing pigment, bacteriorhodopsin,
in the membrane of the purple bacterium Halobacterium halobium (89). Soon
thereafter, its basic function was discovered, namely, that under the influence of light,
protons are pumped from the inside to the outside of the bacterial cell. The chemiosmotic
gradient thereby established could be used for the active transport of nutrients into the
cell and for the formation of ATP and other energy storage compounds.
Bacteriorhodopsin and rhodopsin are similar in many ways: Bacteriorhodopsin contains
248 amino acids and has a molecular weight of 26,000, 70% of which is in the form
of ? helices. Seven helical segments stretch back and forth across the membrane, each
of which contains approximately 23 amino acid residues (90). The two chromophores of
bacteriorhodopsin are 13-cis and all-trans retinaldehyde, both of which are bound to the
protein as a Schiff base at Lys216 in approximately a 1:1 ratio at thermal equilibrium.
The chromophore lies in the middle of the membrane at a tilted angle of 27 degrees from
Vitamin A 29
the plane of the membrane. In the light, the 13-cis isomer is converted to the all-trans
form. In the dark-adapted pigment, as in the case of rhodopsin, the Schiff base is protected
from reagents like hydroxylamine; however, after exposure to light, the reaction sites are
exposed. After the absorption of a photon, a sequence of conformational changes occur
in bacteriorhodopsin that are analogous to those seen with rhodopsin.
In the photocycle of bacteriorhodopsin, the Schiff base is deprotonated and then
reprotonated. Because only one proton is transferred across the membrane in one photocycle,
the Schiff base proton released probably plays a role in proton transfer.
In addition to bacteriorhodopsin, halobacteria contain four other related pigments:
halorhodopsin, involved in chloride transport; sensory photosystems SR-I and SR-II,
which control phototaxis; and slow-cycling rhodopsin (SCR), the function of which is
unclear (91). SR-I (?max 587 nm) provides a light-attractant signal and SR-II (?max 373) a
light-repellant signal. SR-I forms a molecular complex with a membrane-bound transducer
protein, Htrl (92). The phototactic action of the SR-I–Htrl complex does not involve deprotonation
and reprotonation. However, SR-I, when separated from Htrl, pumps protons
across the membrane just like bacteriorhodopsin. Thus, the phototactic function of SR-I, as
contrasted with the proton-pumping, energy-yielding action of bacteriorhodopsin, depends
crucially on its interaction with the transducer protein Htrl (92). The actions of SR-I in
photosynthetic bacteria and of rhodopsin in mammalian physiology clearly are highly
analogous.
In animals, retinoids do not seem to play similar roles, although they can inhibit
some processes involving active transport.
7. Gap Junction Communication
All-trans retinoic acid and some carotenoids elevate mRNA in cells in vitro for connexin
43, a gap junctional protein involved in intercellular communication (93). Interestingly,
the action of canthaxanthin in enhancing connexin 43 synthesis is mediated by its cleavage
product, 4-oxoretinoic acid (94). A transcription factor for 4-oxoretinoic acid, however,
has not as yet been identified.
VII. PHARMACOLOGY
Vitamin A and various retinoids have been used to treat nutritional inadequacy, some skin
disorders, and certain forms of cancer.
A. Nutrition
Oral doses of vitamin A of 200,000 IUa (60 mg or 2.1 mmol) in oil have been used as a
prophylactic measure in many less industrialized countries, but particularly in Asia
(95,96). Such doses are usually given one to three times a year to children 2–6 years of
age. Smaller doses (25,000–100,000 IUa) have been used in younger children. Because
vitamin A is generally well absorbed and is stored in the liver and other tissues, the procedure
has been effective in reducing the incidence of acute vitamin A deficiency in children.
Although vitamin A is relatively inexpensive, the logistics of maintaining a public health
measure involving the individual dosing of children has not always proved easy.
Lactating women in less industrialized countries are also at risk. Thus, the administration
of 200,000 IUa of vitamin A to a mother soon after the birth of her child is helpful
in enhancing her vitamin A reserves as well as in increasing the concentration of vitamin
30 Olson
A in her milk. The only danger is that a lactating woman may again become pregnant
and, as a consequence, the dose may have teratogenic effects.
Other intervention strategies for improving the vitamin A status of groups at risk
include the fortification of foods with vitamin A, horticultural programs featuring carotenoid-
rich foods, and nutrition education. All of these interventions have been applied
successfully in various countries. Needless to say, each approach has its own set of advantages
and drawbacks (95,96).
In general, the vitamin A status of children in most less industrialized countries is
improving. This encouraging positive development may be attributed to a variety of factors:
the above-cited intervention strategies, other government-sponsored programs in nutrition
and health, improved communication, a better standard of living, and political stability
(95,96).
B. Skin Disorders
Various retinoids, and particularly 13-cis retinoic acid (Fig. 3a), etretinate (Fig. 3b), and
acitretin (Fig. 3c), are used to treat acne, psoriasis, and other skin disorders (97,98). Although
highly effective, retinoic acid and etretinate are teratogenic at high doses when
given orally, and retinoic acid is a skin irritant when applied topically. Although most
efficacious retinoids are also toxic, some conjugated forms, such as retinoyl ?-glucuronide
and hydroxyphenylretinamide, retain their therapeutic actions with less, if any, toxicity
(99). Topical all-trans retinoic acid can also reduce wrinkling and hyperpigmentation
caused by photo-induced aging (39,97,98).
The mechanism of these effects is probably multifactorial. On the physiological
level, retinoic acid can alter epidermal differentiation pathways, induce epidermal hyperplasia,
inhibit transglutaminase, and, as a result, reduce the cross-linking of proteins in the
cornified envelope, change the pattern of keratins formed by keratinocytes, alter membrane
viscosity, stimulate gap junction formation, influence various facets of the immune system
(including the inflammatory response), inhibit collagenase formation, and reduce sebum
production (97,98).
On the molecular level, skin cells contain the usual cytosolic binding proteins and
some nuclear receptors for retinoids (98,100). The distribution of the latter, however,
seems unique for skin: RXRs predominate over RARs by a ratio of 5: 1. Of the RARs,
RAR? represents 87% and RAR? 13% of the total. RAR? is not detected. RXR? is the
only detected receptor of the RXR family. The major heterodimer present, as expected,
is RXR?/RAR? (100). Topically applied all-trans retinoic acid induces CRABP-II
strongly, but not CRABP-I, in human skin. Thus, all of the elements for the control of
differentiation by retinoids are present in skin cells, but the key factors involved in the
therapeutic effects of RA on abnormal skin are only being identified slowly (98,101).
C. Cancer
Retinoids have been used both as therapeutic and as chemopreventive agents against a
large variety of tumors in both experimental animals (102) and humans (103).
In experimental animals, retinoids have been tested, in most instances with positive
results, as chemopreventive agents against chemically induced cancers of the mammary
gland, skin, lung, bladder, pancreas, liver, digestive tract, and prostate gland (102). Combination
therapy, e.g., chemopreventive use of hydroxyphenylretinamide and tamoxifen
against N-methyl-N-nitrosourea-induced mammary cancers, has often proved more effecVitamin
A 31
tive, and sometimes synergistically so, than the use of only one agent (102). Retinoids
alone show the best chemopreventive effects with tumors that follow discrete promotional
stages of carcinogenesis, e.g., mammary and skin tumors.
In humans, trials with retinoids or carotenoids, often together with other nutrients,
have been conducted to prevent oral premalignancies, bronchial metaplasia, laryngeal papillomatosis,
Barrett’s esophagus, actinic keratosis, and cervical dysplasia—all considered
to be premalignant lesions (103). Chemoprevention trials against secondary primary tumors
of the head and neck, skin, breast, lung, and bladder have also been initiated or
conducted, as have several primary prevention trials for epithelial cancer. Therapeutic
trials have been conducted with a variety of hematological and epithelial malignancies
(103).
The most promising results have been found with all-trans retinoic acid for acute
promyelocytic leukemia, but drug resistance developed rapidly. Other positive responses
were obtained with 13-cis retinoic acid for squamous cell cancers of the skin; with 13-
cis retinoic acid for mycosis fungoides; with 13-cis retinoic acid, etretinate or ?-carotene
for oral leukoplakia; with 13-cis retinoic acid for laryngeal papillomatosis; with etretinate
for actinic keratosis; with all-trans retinoic acid for cervical dysplasia; and with 13-cis
retinoic acid for secondary primary tumors in patients with head and neck cancer (103).
Responses to retinoids were poor or unconvincing in a large number of other cancers.
Responses to retinoids tended understandably to be better in diseases that were not too
far advanced. The major problem encountered was the toxicity caused by the most efficacious
doses (103).
In only one disease, acute promyelocytic leukemia (APL), is the mechanism of action
of retinoic acid fairly well defined (30,75,103). In essence, the RAR? gene and the
PML gene form a hybrid because of a chromosomal translocation. Presumably higher
concentrations of retinoic acid are therefore required to activate the modified RAR? transcription
factor and to induce cellular differentiation. After several months, resistance to
retinoic acid treatment develops, which may be due to an increased rate of retinoic acid
metabolism, increased sequestration of retinoic acid by induced CRABP, or reduced permeability
of the cell to retinoic acid. Thus, the treatment of APL by retinoic acid, which
initially was heralded with enthusiasm, is clearly less effective as long-term therapy than
initially hoped.
Cancer is such a complex and variegated group of diseases that generalizations about
possible mechanisms of action of retinoic acid have not been very fruitful. Nonetheless,
retinoids probably act by stimulating the differentiation of precancerous stem cells,
whereas carotenoids probably are involved in a network of antioxidants that include vitamin
E, vitamin C, sulfhydryl groups, and a variety of other enzymatic and nonenzymatic
processes.
D. Other Diseases
Retinoids and carotenoids have been implicated as protective agents in a variety of other
diseases, including heart disease, cortical cataract, and age-related macular degeneration
(104,105). Epidemiological and intervention studies with carotenoids are considered later.
VIII. TOXICITY
Three types of toxicity to retinoids exist: acute, chronic, and teratogenic (106–111). Acute
toxicity is due to a single dose or a limited number of large doses taken during a short
32 Olson
period, whereas chronic toxicity is caused by moderately high doses taken frequently,
usually daily, over a span of months or years. Teratogenicity is caused by the ingestion
of moderate to high doses during the first trimester of pregnancy in humans and at selected
‘‘sensitive’’ periods during gestation in animals.
A. Acute Vitamin A Toxicity
When a single dose of more than 0.7 mmol of vitamin A (200 mg, or 660,000 IUa)
is ingested by adults or when a dose larger than 0.35 mmol (100 mg or 330,000 IUa)
is ingested by children, nausea, vomiting, headache, increased cerebrospinal fluid pressure,
vertigo, blurred (double) vision, muscular incoordination, and (in infants) bulging of the
fontanelle may occur (39,106). Some infants can be adversely affected by single doses
of only 0.1 mmol. These signs are generally transient and subside within 1 to 2 days.
When the dose is extremely large, drowsiness, malaise, inappetence, reduced physical
activity, skin exfoliation, itching around the eyes, and recurrent vomiting soon follow.
Finally, when lethal doses are given to young monkeys, an excellent model for vitamin
A toxicity in the human, the animals have deepening coma, convulsions, and respiratory
irregularities, and they finally die of either respiratory failure or convulsions (107). The
median lethal dose (LD50 value) of vitamin A injected intramuscularly in a water-miscible
form in the young monkey is 0.6 mmol (168 mg) retinol per kilogram body weight. Extrapolated
to a 3-kg child and a 70-kg adult, the total LD50 dose would be 1.8 mmol (500
mg) and 41 mmol (11.8 g), respectively. A newborn child, who mistakenly was given
0.09 mmol (25 mg) daily, or 28 µmol/kg for 11 days, died of vitamin A toxicity (108).
The total dose received was 0.31 mmol/kg, 50% of the LD50 value for young monkeys.
Such enormous amounts of vitamin A are present only in high-potency preparations of
vitamin A or in large amounts (
500 g) of livers particularly rich in vitamin A (0.035
mmol/g or 10 mg/g) (39).
The LD50 values of all-trans retinyl ester, 13-cis retinoic acid, and all-trans retinoic
acid in the adult rat are 27.7 mmol/kg body weight, 13.3 mmol/kg body weight, and 6.7
mmol/kg body weight, respectively (109). These LD50 values reflect the general observation
that the relative toxicities of these retinoids are all-trans retinoic acid 13-cis retinoic
acid  all-trans retinol and its esters (109,110). Retinoids injected intraperitoneally are
much more toxic than those administered orally, which in turn are more toxic than those
applied topically to the skin. Species differences in toxicity also exist.
B. Chronic Vitamin A Toxicity
Chronic toxicity is induced in humans by the recurrent intake of vitamin A in amounts
at least 10 times the RDA, i.e., 13 µmol (3.75 mg retinol equivalents or 12,500 IUa) for
an infant or 35 µmol (10 mg retinol equivalents or 33,300 IUa) for an adult. A healthfood
enthusiast who ingested 26 µmol (25,000 IUa) of vitamin A as a supplement daily
plus a similar amount in food showed severe signs of toxicity (39,109,110). Approximately
50 signs of chronic toxicity have been reported, of which the most common are alopecia,
ataxia, bone and muscle pain, cheilitis, conjunctivitis, headache, hepatotoxicity, hyperlipemia,
hyperostosis, membrane dryness, pruritis, pseudotumor cerebri, various skin disorders,
and visual impairment (39,109,110). Particular attention has recently been paid to
serious adverse effects on the liver caused by daily doses of 26–52 µmol (25,000–50,000
IUa) of vitamin A (111,112). When the supplemental intake of vitamin A is eliminated,
these signs usually, but not always, disappear over a period of weeks to months (39).
Vitamin A 33
In chronic hypervitaminosis A, holo-RBP in the plasma is not much elevated,
whereas retinyl esters are usually increased markedly. Factors that enhance toxicity include
alcohol ingestion, low protein intake, viral hepatitis, other diseases of the liver and kidney,
and possibly tetracycline use. Elderly individuals may be more sensitive because of a
slower rate of storage in the liver and a reduced plasma clearance of administered vitamin
A. Tocopherol, taurine, and zinc are protective in tissue culture cells, but they may or
may not be effective in vivo (39,110).
Some individuals seem to suffer from vitamin A intolerance, i.e., the appearance of
signs of toxicity upon routinely ingesting moderate amounts of vitamin A. This relatively
rare condition, which seems to be genetic, mainly affects males (38,39,110).
C. Teratogenicity
Vitamin A and other retinoids are powerful teratogens both in experimental animals and
in women (39,109,110,113). A single extremely large dose, exposure for as short as a
week on high daily doses (0.1–0.3 mmol, or 30–90 mg), or long-term daily intakes of
26 µmol (25,000 IUa or 7500 retinol equivalents) of vitamin A during early pregnancy
can induce spontaneous abortions or major fetal malformations. Common defects are craniofacial
abnormalities, including microcephaly, microtia, and harelip; congenital heart
disease; kidney defects; thymic abnormalities; and central nervous system disorders
(39,109,110,113).
Permanent learning disabilities have been noted in otherwise normal rat pups whose
dams received nonteratogenic doses of vitamin A (114), and similar effects have been
noted in children whose mothers received large doses of 13-cis retinoic acid (115,116).
Most of the children with severely impaired intelligence quotients in this study, however,
also had major physical malformations. In general, the doses that induce demonstrable
learning disabilities in rats are approximately 40% (range 10–70%) of those that cause
terata (114). Many procedures have been employed to measure behavior, and usually the
measurement has been conducted within weeks of birth. Whether the observed effects
become worse or are ameliorated at a later time is still unclear.
Synergism between vitamin A and other teratogens, such as alcohol and drugs, at
nonteratogenic doses of each is probable. Thus, women who are pregnant, or who might
become so, should carefully control their intake of vitamin A, both in regard to rich food
sources, such as liver, and to vitamin A supplements (39).
D. Relative Toxic Effects of Different Retinoids
Most attention has focused on the relative teratogenic effects of natural and synthetic
retinoids that have been, or might be, used therapeutically. As shown in Table 5, a 10,000-
fold range exists, with TTNPB as the most teratogenic and retinoyl ?-glucuronide as the
least (117). The reasons for this wide range of teratogenic activities are only partly understood.
All-trans retinoyl ?-glucuronide, for example, when given in large doses, seems
to be absorbed from the intestinal tract less well than all-trans retinoic acid, is transferred
across the placenta much less well than all-trans retinoic acid, is converted to a significant
extent to the less toxic 13-cis isomer, is taken up by the embryo less well than all-trans
retinoic acid, and is inherently much less teratogenic than all-trans retinoic acid (118).
Indeed, the formation of glucuronides of retinoids may well be a protective metabolic
process to prevent toxic effects (113). Isomerization may also serve a protective action,
e.g., 9-cis retinoic acid is rapidly converted to the much less active 9,13-cis retinoic acid
34 Olson
Table 5 Relative Teratogenicity of Retinoids in Hamstersa
Retinoid TDb
50 (mg/kg)
TTNPB 0.02
Etretinate 6
All-trans retinoic acid 11
13-cis retinoic acid 22
All-trans retinal 23
All-trans retinol 23
All-trans hydroxyphenylretinamide 139
All-trans retinoyl ?-glucuronide 200c
aSelected data from Refs. 117 and 118.
bTeratogenic dose to the dam that induced malformations in 50% of the
offspring.
cExtrapolated value.
during pregnancy (119,120). Nonetheless, 9,13-cis retinoic acid might possibly play some
functional role in pregnancy (120).
TTNPB, as a stable aromatic molecule, might be metabolized less rapidly than retinoic
acid. Thus, its high activity both physiologically and as a teratogen might be attributed
to a lack of its conversion to less active products. Inasmuch as the rate of glucuronidation
of TTNPB and of all-trans retinoic acid by rat liver microsomes is similar (G. Genchi
and J. A. Olson, unpublished observations), some other metabolic step might be affected.
E. Mechanisms of Toxic Action
Acute toxicity is probably caused by the presence of significant amounts of ‘‘free’’ retinol
and retinyl ester in blood and tissues. Retinol bound to RBP is much less cytotoxic than
unbound retinol. Similarly, the activity of all-trans retinoic acid in cell differentiation is
reduced by the presence of large amounts of CRABP in the cells (30). When large doses
of retinol are given to monkeys, the amount of retinol in the brain markedly increases
(107), consistent with the many signs of central nervous system involvement in acute
toxicity.
Chronic toxicity and teratogenicity seem to involve primarily the nuclear retinoid
receptors (80,109,113). The teratogenic effects of AM-580 (Fig. 3g), a RAR?-specific
agonist, in pregnant mice are offset by a sulfur-containing analog of TTNBP that serves
as a RAR? antagonist (109). As already mentioned, retinoic acid activates via its receptors
several families of Hox genes that are closely implicated with specific developmental
structures. Thus, the concentration of retinoic acid at various locations in the embryo must
be very carefully regulated so that a proper spacial and temporal sequence of activations
can occur. By flooding the embryo with retinoic acid, regulation will be lost. Thus, in
early mouse embryos treated with all-trans retinoic acid, Hox a-1 (Hox 1.6) expression
at 7.5 d is much more extensive and less localized than in control embryos (109).
Other possible actions of retinoids include effects on cell proliferation, cell differentiation,
cell migration, programmed cell death (apoptosis), and membrane integrity. Many
of these effects also involve known retinoid receptors, but others do not (109,113).
Vitamin A 35
The structural characteristics of highly teratogenic retinoids comprise the following
(117):
1. A polar terminus with an acidic pKa
2. A lipophilic polyene side chain with good ? electron delocalization, e.g., cis
isomers are less active than the all-trans isomer
3. A fairly lipophilic ring with no set nature or dimensions opposite the polar
terminus, and
4. Conformational restriction (for acidic retinoids only)
Interestingly, the two least teratogenic compounds, 4-hydroxyphenylretinamide
(Fig. 3d) and retinoyl ?-glucuronide (Fig. 1j) are conjugated, more polar molecules.
F. Safe Levels of Vitamin A Intake in Humans
This issue of crucial public health importance can be approached in two ways: (a) by
assessing the no-effect level of vitamin A intake experimentally in various populations
at risk and (b) by reviewing the formal recommendations made by expert groups. The
major groups at risk are infants, young children, pregnant women, and lactating women.
The issue is complicated in part by whether or not a given population will clearly benefit
as well as be put at-risk by the ingestion of a large dose of vitamin A. In the following
discussion, however, only the issue of toxicity will be considered.
Statistical issues are also crucial. It clearly is easier to define an appropriate maximal
intake for a population in which an allowable percentage of that group, e.g., 1%, 3%, or
5%, is adversely affected than to specify an intake that presumably will affect nobody.
As already mentioned, genetic vitamin A intolerance is known to exist in a handful of
persons (38,110,121). However, recommendations for a healthy population certainly
should not be based on the adverse reactions of a few hypersensitive individuals. Finally,
some relatively nonspecific reactions to moderate doses might not be causally related to
the vitamin A in the dose.
Nonetheless, despite these caveats, some useful guidelines can be defined for vitamin
A intake. RDIs of vitamin A are completely safe insofar as we know. RDA values for
infants, children 1–6 years, and pregnant women are 375, 400–500, and 800 µg retinol
equivalents, respectively (35); (Table 2). These values, however, assume that intakes are a
mixture of preformed vitamin A and corotenoids. Thus, the estimated amount of preformed
vitamin A in the diet of infants, children 1–6 years, and pregnant women are approximately
340 µg (1133 IUa), 300–375 µg (1000–1250 IUa), and 600 µg (2000 IUa), respectively.
1. Pregnant Women
Although additional vitamin A is not usually needed by healthy pregnant women, daily
multivitamin tablets containing 5000 IUa (1500 µg) of vitamin A are ingested by large
numbers of women. Toxic reactions to their ingestion have not been reported.
Toxic reactions have been noted, however, in women, both pregnant and nonpregnant,
ingesting daily doses of 18,000 IUa (5400 µg) (106,110–113), whereas only
sporadic claims, often poorly documented, have been made for toxic effects at lower daily
doses. In a study of retinoid embryopathy in pregnant women, the lowest dose of 13-cis
retinoic acid that was associated with birth defects was 10 mg/day (122). Thus, 1500 µg
of vitamin A seems safe for essentially all pregnant women, whereas 5400 µg probably
is not.
36 Olson
Responding to concerns about the teratogenicity of vitamin A, the Teratology Society
recommended that women who might become pregnant limit their daily intake of
preformed vitamin A to 8000 IUa (2400 µg) and ingest provitamin A carotenoids as a
primary source of dietary vitamin A (123). Furthermore, they recommended that the unit
dose of commercially available vitamin A be limited to 5000–8000 IUa and that the hazards
of excessive intakes of vitamin A be indicated on the labels of such products. Similarly,
the American Institute of Nutrition, the American Society for Clinical Nutrition,
and the American Dietetic Association issued a joint formal statement that supplements of
vitamins and minerals were not needed by well-nourished, healthy individuals, including
pregnant women, except in some specific instances (124). The Council for Responsible
Nutrition, a group sponsored by industry, has also advised that pregnant women, while
needing to ensure an adequate intake of vitamin A, should prudently limit their intake
of nutritional supplements of vitamin A to 5000–10,000 IUa (125). Subsequently, they
recommended that the unit dosage of retinol in commercial vitamin A preparations be
limited to 10,000 IUa.
In recognition of the fact that a deficiency of vitamin A in the mother can also
cause abortion and fetal abnormalities, the International Vitamin A Consultative Group
(IVACG) has recommended that the average daily diet of pregnant women should supply
620 µg retinol equivalents, in keeping with FAO/WHO recommendations (34). However,
in areas of the world where this level of intake does not occur and little opportunity exists
for dietary improvement, or in emergency situations in which food supplies are disrupted,
IVACG recommends that daily supplements of 3000 µg retinol equivalents (10,000 IUa)
can be given safely anytime during pregnancy. They do not suggest, by the way, that
well-nourished women take supplements.
To summarize, well-nourished healthy women of reproductive potential should include
carotenoid-rich fruits and vegetables in their diet. They should also avoid taking
supplements of preformed vitamin A during the first trimester of pregnancy, during which
increased nutritional demands are small and the risk of fetal abnormalities is high. If
supplements of vitamin A are subsequently taken, the daily dose should be carefully limited
to 5000–10,000 IUa.
2. Infants and Young Children
As already indicated, the probable daily amounts of preformed vitamin A in recommended
diets of infants and young children are 1133 IUa and 1250 IUa, respectively. As a general
public health measure, oral doses (200,000 IUa) of all-trans retinyl palmitate in oil have
been administered one to three times a year to preschool children, usually 1–6 years of
age, in less industrialized countries (95). Side effects, e.g., nausea, vomiting, and bulging
of the fontanelle in infants, have usually been reported in 5% of the treated children
and have been transient (1–3 days) in nature. Because vitamin A toxicity is a function
of weight, younger children tend to be most affected.
Because of international public health interest in combining vitamin A supplementation
with the expanded program of immunization (EPI), the effects of dosing infants with
vitamin A from 6 weeks to 9 months of age has been explored. When 50,000 IUa of
vitamin A was given orally in oil to Bangladesh infants at 6, 11, and 16 weeks of age,
11% showed transient bulging of the fontanelle. In a subsequent study in which 25,000
IUa was given orally at approximately the same three times to Bangladesh infants, 8%,
corrected for the 2.5% incidence in the placebo group, showed the same effect (126). In
Vitamin A 37
this study, nearly all of the affected children had received three doses of vitamin A; none
showed any toxicity after one dose. If we assume that the infants’ daily intake from breast
milk was approximately 100 µg (333 µg IUa), the calculated mean daily increment from
the dose is only an additional 715 IUa, giving a total of 1048 IUa, essentially the same
as the recommended daily intake. A single oral dose of 50,000 IUa given to Indonesian
infants showed much less toxicity, i.e., 4.5% in the vitamin A–treated group and 2.5%
in the control group (127). Furthermore, the cerebral fluid volume, but not the pressure,
was transiently increased in these infants, and no lasting side effects were noted. Bulging
of the fontanelle, in consequence, may be more of a transient physiological response to
a dose of this magnitude than an indicator of toxicity.
As yet, the relationship between an acceptably safe dose and the age of infants has
not been defined. Quite possibly, infants suffering from inadequate intakes of protein and
calories may be more susceptible to vitamin A toxicity than better nourished infants. If
so, the dose of vitamin A that is selected for a given country might well be based on
anthropometric indices within that country.
Thus, a generally safe single dose for most infants, although currently undefined,
probably will fall in the range of 4000–5000 IUa per kilogram body weight with an interval
between doses of 5 weeks.
IX. CAROTENOIDS
Unlike retinoids, including vitamin A, carotenoids are generally nontoxic. However, individuals
who routinely ingest large amounts of carotenoids, either in tomato or carrot juice
or in commercial supplements of ?-carotene, can develop hypercarotenosis, characterized
by a yellowish coloration of the skin and a very high concentration of carotenoids in the
plasma. This benign condition, although resembling jaundice, gradually disappears upon
correcting the excessive intake of carotenoids. The only known toxic manifestation of
carotenoid intake is canthaxanthin retinopathy, which can develop in patients with erythropoietic
porphyria and related disorders who are treated with large daily doses (50–100
mg) of canthaxanthin, the 4,4?-diketo derivative of ?-carotene, for long periods (128). In
most instances, however, these deposits of canthaxanthin disappear slowly upon termination
of treatment (128). Canthaxanthin-containing supplements are not currently available
in the United States. ?-Carotene at similar doses is not known to cause retinopathy. Carotenoids,
even when ingested in large amounts, are not known to cause birth defects or
hypervitaminosis A, primarily because the efficiency of their absorption from the intestine
falls rapidly as the dose increases and because their conversion to vitamin A is not suffi-
ciently rapid to induce toxicity (39).
Quite apart from their function as precursors of vitamin A, carotenoids are distributed
widely in mammalian tissues, can quench singlet oxygen, can serve as an antioxidant
in tissues (particularly under conditions of low oxygen tension), and can stimulate the
immune response (39,104).
Thus, by using provitamin A activity as the nutritional function and singlet oxygenquenching
and antioxidant activity as the biological action, four classes of carotenoids
might be defined: those that are both nutritionally and biologically active, such as ?-
carotene; those that are nutritionally active and biologically inactive, such as 14?-?-apocarotenal;
those that are nutritionally inactive but biologically active, such as lycopene and
violaxanthin; and those that are both nutritionally and biologically inactive, such as phytoene.
38 Olson
Because over 90% of the 600 characterized carotenoids in nature are not precursors
of vitamin A, their biological effects in mammalian physiology, independent of their provitamin
A activity, are being followed with interest.
X. POTENTIAL HEALTH BENEFITS OF RETINOIDS AND
CAROTENOIDS
A. Retinoids
The requirements for vitamin A and safe levels of intake have already been discussed. A
positive response to chemopreventive treatment with large doses of vitamin A has been
shown in leukoplakia and actinic keratosis (103). The retinoids most commonly used for
therapy and chemoprevention, however, are 13-cis retinoic acid, all-trans retinoic acid,
hydroxyphenylretinamide, etretinate, and acitretin (103). As indicated earlier, these agents
have shown promising results in the prevention or treatment of some carcinomas (103).
The major drawbacks in their use are their toxicity at highly efficacious doses and, in the
case of APL, a rapidly developing resistance to the drug. Of the retinoids listed, all-trans
retinoic acid and etretinate, because of the latter’s slow turnover in the body, are the
most toxic and hydroxyphenylretinamide the least. The search consequently continues to
identify new retinoids with high efficacy but low toxicity. Retinoyl ?-glucuronide, a naturally
occurring metabolite of retinoic acid, shows these properties (99,118). It is active in
treating acne but has not been tested against other diseases. In epidemiological surveys,
the dietary intake of preformed vitamin A and the plasma concentration of retinol are
rarely associated with a reduced incidence of chronic diseases.
B. Carotenoids
1. Cancer
One of the most dramatic and consistent observations in epidemiological studies is the
inverse association between ?-carotene intake and the incidence of lung cancer (104,129).
These findings have stimulated intervention trials in two high-risk groups: asbestos workers
in Tyler, Texas and middle-aged male smokers in Finland. The results of these intervention
trials are disappointing. In asbestos workers, no differences in the prevalence of sputum
atypia was noted between treated (50 mg ?-carotene  25,000 IUa retinol every other
day) and control groups over a 5-year period. In the Finnish study, the group treated daily
with ?-carotene (20 mg) showed a significantly higher incidence of lung cancer [relative
risk (RR)  1.18, 95% confidence interval (CI)  1.03–1.36] and total mortality (RR 
1.08, 95% CI  1.01–1.16) than did the placebo group. Supplemental ?-carotene did not
affect the incidence of other major cancers found in this population (129).
The unexpected negative finding in the Finnish study has several possible explanations:
(a) Supplemental ?-carotene is interfering with the intestinal absorption of other
possible chemopreventive nutrients. In this regard, ?-carotene inhibits the absorption in
humans of lutein, which shows good antioxidant activity (42). In that same vein, ?-carotene,
which shows chemopreventive properties, might be similarly affected. (b) Supplemental
?-carotene may be serving as a pro-oxidant in the well-oxygenated ambient of the
lung (130). (c) The population of middle-aged male smokers is not representative of other
groups, who might well benefit from a higher intake of carotenoids. (d) A comparison
between treated and control subjects that fall only in the lowest quartile of initial plasma
Vitamin A 39
carotene values might yield different results. (e) Vitamin C, which is low in the plasma
of most Finns, may have played some role in the outcome. (f) Alcohol intake may also
play an important role in the outcome.
Several other trials have provided results that support the findings in the Finnish
study, namely, the Carotene and Retinal Efficacy Trial (CARET) and the Physician’s
Health Study (129).
The development of head and neck cancers, including those of the oral cavity, pharynx,
and larynx, are influenced by many factors, including smoking, other uses of tobacco,
alcohol, and diet (129). Serum carotene concentrations, adjusted for smoking, are inversely
related to the incidence of these carcinomas. Supplements of ?-carotene can markedly
reduce leukoplakia, although the lesion returns upon cessation of treatment (129). A chemoprevention
trial designed to assess the effect of daily supplements of ?-carotene (50 mg)
on the recurrence of head and neck cancer is currently under way (129).
The effects of various nutrient combinations on esophageal cancer and on stomach
cancer was evaluated in Linxian, China, where the incidence of esophageal cancer is 100-
fold higher than in the United States (129). Of four nutrient treatments, only one, involving
supplements of ?-carotene, selenium, and ?-tocopherol, showed a positive effect: the reductions
in total deaths, cancer deaths, esophageal cancer deaths, and gastric cancer deaths
were 9% (RR  0.91, 95% CI  0.84–0.99), 13% (RR  0.87, 95% CI  0.75–1.00),
4% (RR  0.96, 95% CI  0.78–1.18), and 21% (RR  0.79, 95% CI  0.64–0.99),
respectively (129). Although these results support the concept that diet influences cancer
incidence, the general nutritional status of the population was poor. Thus, whether the
mixed supplement, or one component of it, was protective as a result of generally improved
health or of a more specific anticancer effect is not clear (129).
The dietary intake and serum concentrations of carotenoids are often inversely associated
with the risk of colorectal cancer (129). However, by using adenomas as an indicator,
supplemental ?-carotene (25 mg/day) was found to be ineffective (RR  1.01, 95%
CI  0.85–1.20) in preventing the recurrence of this lesion (129).
?-Carotene intake has been associated with an improved survival rate in breast cancer
patients (129). Whether supplements of carotenoids reduce the incidence of breast
cancer in a well-designed clinical trial is not known. In an ongoing trial, the effect of
hydroxyphenylretinamide on the recurrence of breast cancer is being explored (103).
The risk of cervical cancer has been correlated with the prediagnostic serum levels
of ?-, ?-, and total carotenoids (RR  2.7–3.1, 95% CI  1.1–	8.1) (129). On the
other hand, invasive cervical cancer among white women in the United States did not
relate to any specific food group of the diet or to the use of supplements of vitamins A,
C, and E and folic acid (131). However, cervical dysplasia, considered to be a precancerous
lesion, did respond to ?-carotene supplements (30 mg/day) (129).
The recurrence of skin cancer was not affected by ?-carotene supplements (50 mg/
day) over a 5-year period (RR 1.05, 95% CI  0.91–1.22) (129).
Thus, a dichotomy exists. Most of the associations found between diseases in dietary
or plasma level studies do not agree with the results of intervention trials. The former
tend to show strong significant correlations and the latter, in large part, do not. Possible
explanations are as follows: (a) ?-Carotene, which is only one of approximately 600 known
carotenoids, might not be the most active one, or indeed, might inhibit the absorption of
other more chemopreventive carotenoids and other nutrients. (b) Carotenoids might be
only one of a group of chemopreventive agents in foods that act synergistically in preventing
carcinogenesis. The fact that the relative risk values for colored fruits and vegeta40
Olson
bles usually are less (more protective) than those for carotenoids or for any other component
of the food supports this viewpoint. (c) Carotenoids may serve solely as a useful
marker for a healthful lifestyle. (d) The preventive action of carotenoids might occur very
early in disease progression but be ineffective later. Thus, subjects in identified high-risk
groups, who often have had a primary tumor, may be resistant to nutritional supplements.
(e) The associations found in observational epidemiology are not causal and can be confounded
by a variety of unanticipated and unmeasured factors. In essence, the intervention
trials may well be providing more valid answers (129).
2. Photosensitivity Disorders
Patients with erythropoietic porphyria and similar diseases benefit by ingesting supplements
(180 mg/day) of ?-carotene. Canthaxanthin, though also protective, is no longer
used because of the reversible retinopathy that results (128). Although the concentrations
of ?-carotene and vitamin A are elevated in the livers of these patients, the side effects
of ?-carotene ingestion over a period of years are minimal (129).
3. Cardiovascular Disease
Epidemiologic studies suggest protective effects of carotenoid intake against both coronary
events and stroke (104,129). In a European study (WHO/MONICA, i.e., monitoring cardiovascular
disease), mortality from ischemic heart disease correlated inversely with serum
vitamin E concentrations (r2  0.63) but not with ?-carotene levels (r2  0.04) (132).
If the 3 Finnish sites, which were outliers, of the 16 examined were excluded, however,
the inverse correlation with ?-carotene concentrations improved markedly (r2  0.50). A
mean serum ?-carotene concentration in populations of 0.4 µmol/L or higher was associated
with good health in the European studies, whereas a concentration 0.25 µmol/L
in populations was related to an increased risk of coronary disease, stroke, and cancer
(132). The risk of myocardial infarction was inversely related to adipose ?-carotene content
in smokers (RR  2.62, 95% CI  1.79–3.83) but not in nonsmokers (RR  1.07)
(129). Furthermore, physicians with stable angina or prior coronary revascularizations,
who were supplemented with ?-carotene for 5 years, showed a 51% reduction in the risk
of major coronary events (129). ?-Carotene did not show beneficial effects, however,
in the total population enrolled in the Physicians Health Study (129). The incidence of
cardiovascular deaths in the Finnish lung cancer study also was not affected by ?-carotene
supplementation.
The overall results, therefore, are somewhat mixed. The most likely mechanism of
action of carotenoids, but by no means the only one, is a reduction in the oxidation of
low-density lipoproteins, which seem to play a key role in atherogenesis (104). Of various
antioxidants studied both in vivo and in vitro, however, ?-carotene does not seem to be
very protective, if at all. Thus, the relationship among dietary intakes of carotenoids, their
plasma and tissue concentrations, and cardiovascular disease remains unclear.
4. Age-Related Macular Degeneration
The macular of the eye predominantly contains two pigments, lutein and zeaxanthin
(133,134). Because these two pigments account for less than 25% of plasma carotenoids,
their uptake from plasma and deposition in the macula show specificity. These pigments
might consequently play a role in protecting the macula from damage caused by light and
particularly by blue light. In a recent study with patients suffering from age-related macular
degeneration (ARMD) vs. matched controls, subjects in the highest quintile of carotVitamin
A 41
enoid intake had a 43% lower risk (RR  0.57, 95% CI  0.35–0.92) of suffering from
ARMD than those in the lowest quintile (129). Of various carotenoids, the intake of spinach
and collard greens, rich in these two carotenoids, was most strongly associated with
reduced risk. However, not all studies support this finding (105). Nonetheless, higher
plasma concentrations of lutein and zeaxanthin as well as ?-carotene showed a significant
trend toward a lower risk of developing ARMD. These interesting findings are currently
being investigated.
5. Senile Cataract
Cataract consists of gradual opacification of the lens with aging, which may in part result
from oxidative stress. Carotenoid intake, as well as that of vitamins C and E, has been
associated with a reduced risk of cataract (105,129). In the main Linxian, China trial,
however, combined supplements of ?-carotene, selenium, and ?-tocopherol were not associated
with a reduction in the incidence of cataracts, and inconclusive results have been
reported by others (105,129). Thus, whereas the concept that antioxidant nutrients might
prevent oxidative damage to a fairly exposed structure, such as the lens, is highly feasible,
the data supporting a protective role of dietary components in the process are mixed.
6. HIV Infection
In HIV infection T-helper (CD4) cells are destroyed, thereby impairing the immune response.
In humans as well as in experimental animals, both ?-carotene, which is a provitamin
A carotenoid, and canthaxanthin, which is not, can enhance the immune response
(135). Indeed, in HIV-infected patients, large doses of ?-carotene increased the CD4/CD8
ratio, which is usually depressed in HIV infection, and improved the response to vaccines
(135). UV light tends both to activate human HIV expression, at least in transgenic mice,
and to reduce plasma carotenoid concentrations in humans. In phase II HIV-infected subjects,
plasma carotenoid concentrations are reduced by 50%. AIDS patients treated daily
with a combination of ?-carotene supplementation (120 mg) and whole-body hyperthermia
(42°C, 1 h) showed a better and longer lasting response than either treatment separately
(136). Thus, carotenoids seem to ameliorate the condition of AIDS patients, probably, at
least in part, by enhancing the immune response.
A quite different effect of a carotenoid has also been reported, namely, that halocynthiaxanthin
(5,6-epoxy-3,3?-dihydroxy-7?,8?-didehydro-5,6,7,8-tetrahydro-?,?-carotene-8-
one) strongly and rather specifically inhibits RNA-dependent DNA polymerase of the HIV
virus (135). The use of carotenoids in the treatment of subjects with HIV infections clearly
merits further attention.
XI. CONCLUDING REMARKS
Revising a chapter that was written approximately 9 years ago (137) is a valuable but
humbling experience. The facts cited earlier have not changed, and many of the concepts
have been modified only slightly; however, interests have shifted markedly and a whole
new body of information and hypotheses—some quite clear, some conflicting—has arisen.
Old observations are viewed in new ways, and a completely new set of research questions
are being asked. Thus, in revising the chapter, the addition of a paragraph here and a
reference there was just not feasible. As a consequence, the chapter is largely rewritten,
and the reference list is in large part new. This new chapter and the previous one are,
therefore, complementary to each other.
42 Olson
A major recent advance has been the discovery of the nuclear retinoid receptors and
their impact on embryogenesis, cell differentiation, disease, and pharmacology. In truth,
the paradigm of interpreting vitamin A actions has markedly changed. A new chemistry
has arisen, with the focus on finding compounds that serve as specific agonists and antagonists
for given retinoid receptors. The linkage between nutrition and molecular biology,
seemingly such diverse fields, has been strengthened by the observation that the RXR
receptors for vitamin A interact meaningfully with the thyroid receptor, dependent on
iodine for its activity, and with the vitamin D receptor, dependent of course on another
fat-soluble vitamin. The hint (not the demonstration) that ?-tocopherol may also have
specific nuclear effects adds further interest to this linkage.
Diet, admittedly along with a variety of other factors, is known to affect the onset
and possibly the severity of major chronic diseases. In the past several years, an explosion
of information about specific nutrients that may play roles in these processes has appeared.
Despite the great care with which most of these studies have been done, these surveys
have inherent constraints. Thus, the findings in large part have tantalized us rather than
presented a coherent picture. Carotenoids, primarily together with vitamin E, have played
a central role in these surveys.
Although these studies have great potential impact, some biases have arisen: namely,
that carotenoids, vitamin A, vitamin E, vitamin C, and selenium all act solely as antioxidants
in these processes. Indeed, the term ‘‘antioxidant vitamins’’ has become common
parlance. To stimulate a broader, less constrained view of their potential actions, these
nutrients, as well as many other naturally occurring compounds, both of endogenous as
well as of dietary origin, might better be called ‘‘physiological modulators’’ (138). By so
doing, the mechanism of action is not automatically inferred from the outset for whatever
beneficial or adverse effect that they might show (138,139).
To keep the reference list within bounds, references are largely made to reviews,
which in turn can serve as a guide to the primary research literature. Recent reviews,
monographs, and articles of particular interest are cited in references 140–162. I regret
the necessary omission of many specific research papers that have enriched our knowledge
in this dynamic field.
ACKNOWLEDGMENTS
This study has been supported in part by grants from the National Institutes of Health
(DK-39733), the USDA (NRICGP 94-37200-0490 and ISU/CDFIN/CSRS 94-34115-
2835), and the W. S. Martin Fund. This is Journal Paper J-16524 of the Iowa Agriculture
and Home Economics Experiment Station, Ames, IA (Iowa Project No. 3335). The author
is indebted to Ms. Margaret Haaland for outstanding administrative and secretarial assistance.
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Vitamin A 47
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2
Vitamin D
ELAINE D. COLLINS
San Jose? State University, San Jose?, California
ANTHONY W. NORMAN
University of California, Riverside, California
I. INTRODUCTION
Vitamin D designates a group of closely related compounds that possess antirachitic activity.
The two most prominent members of this group are ergocalciferol (vitamin D2) and
cholecalciferol (vitamin D3). Ergocalciferol is derived from a common plant steroid, ergosterol,
and is the form that was employed for vitamin D fortification of foods from the
1940s to the 1960s. Cholecalciferol is the form of vitamin D obtained when radiant energy
from the sun strikes the skin and converts the precursor 7-dehydrocholesterol into vitamin
D3. Since the body is capable of producing cholecalciferol, vitamin D does not meet the
classical definition of a vitamin. It is more accurate to call vitamin D a prohormone; thus,
vitamin D is metabolized to a biologically active form that functions as a steroid hormone
(1–4). However, since vitamin D was first recognized as an essential nutrient, it has historically
been classified among the lipid-soluble vitamins. Even today it is thought of by many
as a vitamin, although it is now known that there exists a vitamin D endocrine system
that generates the steroid hormone 1?,25-dihydroxyvitamin D3 [1?,25(OH)2D3].
Vitamin D functions to maintain calcium homeostasis together with two peptide
hormones, calcitonin and parathyroid hormone (PTH). Vitamin D is also important for
phosphorus homeostasis (5,6). Calcium and phosphorus are required for a wide variety
of biological processes (Table 1). Calcium is necessary for muscle contraction, nerve pulse
transmission, blood clotting, and membrane structure. It also serves as a cofactor for such
enzymes as lipases and ATPases and is needed for eggshell formation in birds. It is an
important intracellular signaling molecule for signal transduction pathways, such as those
involving calmodulin and protein kinase C. Phosphorus is an important component of
51
52 Collins and Norman
Table 1 Biological Calcium and Phosphorus
Calcium Phosphorus
Utilization
Body content: 70-kg man has 1200 g Ca2
 Body content: 70-kg man has 770 g P
Structural: bone has 95% of body Ca Structural: Bone has 90% of body P
Plasma [Ca2
] is 2.5 mM, 10 mg % Plasma [Pi] is 2.3 mM, 2.5–4.3 mg %
Muscle contraction Intermediary metabolism (phosphorylated intermediates)
Nerve pulse transmission
Blood clotting Genetic information (DNA and RNA)
Membrane structure Phospholipids
Enzyme cofactors (amylase, trypsinogen, Enzyme/protein components (phosphohistilipases,
ATPases) dine, phosphoserine)
Eggshell (birds) Membrane structure
Daily requirements (70-kg man)
Dietary intake: 700a Dietary intake: 1200a
Fecal excretion: 300–600a,b Fecal excretion: 350–370a,b
Urinary excretion: 100–400a,b Urinary excretion: 200–600a,b
aValues in milligrams per day.
bBased on the indicated level of dietary intake.
DNA, RNA, membrane lipids, and the intracellular energy–transferring ATP system. The
phosphorylation of proteins is important for the regulation of many metabolic pathways.
Furthermore, the maintenance of serum calcium and phosphorus levels within narrow
limits is important for normal bone mineralization. Any perturbation in these levels results
in bone calcium accretion or resorption. Disease states, such as rickets, can develop if the
serum ion product is not maintained at a level consistent with that required for normal bone
mineralization. Maintaining a homeostatic state for these two elements is of considerable
importance to a living organism.
Recently, 1?,25(OH)2D3 has been shown to act on novel target tissues not related
to calcium homeostasis. There have been reports characterizing receptors for the hormonal
form of vitamin D and activities in such diverse tissues as brain, pancreas, pituitary, skin,
muscle, immune cells, and parathyroid (Table 2). These studies suggest that vitamin D
status is important for insulin and prolactin secretion, muscle function, immune and stress
response, melanin synthesis, and cellular differentiation of skin and blood cells.
There are a number of recent books (7–9) and comprehensive reviews (10–19) that
cover many aspects of vitamin D, including its endocrinological aspects.
II. HISTORY
Rickets, a deficiency disease of vitamin D, appears to have been a problem in ancient
times. There is evidence that rickets occurred in Neanderthal man about 50,000 bc (20).
The first scientific descriptions of rickets were written by Dr. Daniel Whistler (21) in 1645
and by Professor Francis Glisson (22) in 1650. Rickets became a health problem in northern
Europe, England, and the United States during the Industrial Revolution when many
people lived in urban areas with air pollution and little sunlight. Prior to the discovery of
vitamin D, theories on the causative factors of rickets ranged from heredity to syphilis
(2).
Vitamin D 53
Table 2 Distribution of 1,25(OH)2D3 Actionsa
Tissue Distribution of Nuclear 1,25(OH)2D3 Receptor
Adipose Intestine Pituitary
Adrenal Kidney Placenta
Bone Liver (fetal) Prostrate
Bone marrow Lung Retina
Brain Muscle, cardiac Skin
Breast Muscle, embryonic Stomach
Cancer cells Muscle, smooth Testis
Cartilage Osteoblast Thymus
Colon Ovary Thyroid
Eggshell gland Pancreas ? cell Uterus
Epididymus Parathyroid Yolk sac (bird)
Hair follicle Parotid
Distribution of Nongenomic Responses
Intestine Transcaltachia
Osteoblast Ca2
 channel opening
Osteoclast Ca2
 channel opening
Liver Lipid metabolism
Muscle A variety
aSummary of the tissue location of the nuclear receptor for 1?,25(OH)2D3 (nVDR) (top) and
tissues displaying ‘‘rapid’’ or membrane-initiated biological responses (bottom).
Some of the important scientific discoveries leading to the understanding of rickets
were dependent on discoveries about bone. As reviewed by Hess (23), the first formal
descriptions of bone were made by Marchand (1842), Bibard (1844), and Friedleben
(1860). In 1885, Pommer wrote the first description of the pathological process taking
place in the rachitic skeleton. In 1849, Trousseau and Lasque recognized that osteomalacia
and rickets were different manifestations of the same disorder. In 1886 and 1890,
Hirsch and Palm did a quantitative geographical study of the worldwide distribution of
rickets and found that the incidence of rickets paralleled the incidence of lack of sunlight
(23). This was substantiated in 1919 when Huldschinsky demonstrated that ultraviolet
(UV) rays were effective in healing rickets (24).
In the early 1900s, the concept of vitamins was developed and nutrition emerged
as an experimental science, allowing for further advances in understanding rickets. In
1919, Sir Edward Mellanby (25,26) was able to experimentally produce rickets in puppies
by feeding synthetic diets to over 400 dogs. He further showed that rickets could be prevented
by the addition of cod liver oil or butterfat to the feed. He postulated that the
nutritional factor preventing rickets was vitamin A since butterfat and cod liver oil were
known to contain vitamin A (26). Similar studies were also conducted by McCollum et
al. (27).
The distinction between the antixerophthalmic factor, vitamin A, and the antirachitic
factor, vitamin D, was made in 1922 when McCollum’s laboratory showed that the antirachitic
factor in cod liver oil could survive both aeration and heating to 100°C for 14 h,
whereas the activity of vitamin A was destroyed by this treatment. McCollum named the
new substance vitamin D (28).
Although it was known that UV light and vitamin D are equally effective in preventing
and curing rickets, the close interdependence of the two factors was not immedi54
Collins and Norman
ately recognized. Then, in 1923, Goldblatt and Soames (29) discovered that food that was
irradiated and fed to rats could cure rickets; food that was not irradiated could not cure
rickets. In 1925, Hess and Weinstock demonstrated that a factor with antirachitic activity
was produced in the skin upon UV irradiation (30,31). Both groups demonstrated that the
antirachitic agent was in the lipid fraction. The action of the light appeared to produce a
permanent chemical change in some component of the diet and the skin. They postulated
that a provitamin D existed that could be converted to vitamin D by UV light absorption.
Much more work ultimately demonstrated that the antirachitic activity resulted from the
irradiation of 7-dehydrocholesterol.
The isolation and characterization of vitamin D was now possible. In 1932, the
structure of vitamin D2 was simultaneously determined by Windaus in Germany, who
named it vitamin D2 (32), and by Askew in England, who named it ergocalciferol (33).
In 1936, Windaus identified the structure of vitamin D found in cod liver oil, vitamin D3
(34). Thus, the ‘‘naturally’’ occurring vitamin is vitamin D3, or cholecalciferol. The structure
of vitamin D was determined to be that of a steroid or, more correctly, a seco-steroid.
However, the relationship between its structure and its mode of action was not realized
for an additional 30 years.
Vitamin D was believed for many years to be the active agent in preventing rickets.
It was assumed that vitamin D was a cofactor for reactions that served to maintain calcium
and phosphorus homeostasis. However, when radioisotopes became available, more precise
measurements of metabolism could be made. Using radioactive 45Ca2
, Linquist found
that there was a lag period between the administration of vitamin D and the initiation of its
biological response (35). Stimulation of intestinal calcium absorption required 36–48 h for
a maximal response. Other investigators found delays in bone calcium mobilization and
serum calcium level increases after treatment with vitamin D (36–40). The duration of the
lag and the magnitude of the response were proportional to the dose of vitamin D used (37).
One explanation for the time lag was that vitamin D had to be further metabolized
before it was active. With the development of radioactively labeled vitamin D, it became
possible to study the metabolism of vitamin D. Norman et al. were able to detect three
metabolites that possessed antirachitic activity (41). One of these metabolites was subsequently
identified as the 25-hydroxy derivative of vitamin D3 [25(OH)D3] (42). Because
25(OH)D3 was found to have 1.5 times more activity than vitamin D in curing rickets in
the rat, it was thought that this metabolite was the biologically active form of vitamin D
(43). However, in 1968, Haussler et al. reported a more polar metabolite that was found
in the nuclear fraction of the intestine from chicks given tritiated vitamin D3 (44). Biological
studies demonstrated that this new metabolite was 13–15 times more effective
than vitamin D in stimulating intestinal calcium absorption and 5–6 times more effective in
elevating serum calcium levels (45). The new metabolite was also as effective as vitamin D
in increasing growth rate and bone ash (45). In 1971, the structural identity of this metabolite
was reported to be the 1?,25-dihydroxy derivative of vitamin D[1?,25(OH)2D3] (46–
48), the biologically active metabolite of vitamin D.
In 1970, the site of production of 1?,25(OH)2D3 was demonstrated to be the kidney
(49). This discovery, together with the finding that 1?,25(OH)2D3 is found in the nuclei
of intestinal cells, suggested that vitamin D was functioning as a steroid hormone (44,50).
Subsequently, a nuclear receptor protein for 1?,25(OH)2D3 was identified and characterized
(50,51). Since the cDNA for the 1?,25(OH)2D3 nuclear receptor from several species
has now been cloned and sequenced (52–55), the relationship between vitamin D and the
other steroid hormones has been clearly established (56). The discovery that the biological
Vitamin D 55
actions of vitamin D could be explained by the classical model of steroid hormone action
marked the beginning of the modern era of vitamin D.
III. CHEMISTRY
A. Structure
As previously mentioned, vitamin D refers to a family of compounds that possess antirachitic
activity. Members of the family are derived from the cyclopentanoperhydrophenanthrene
ring system (Fig. 1), which is common to other steroids, such as cholesterol.
However, vitamin D has only three intact rings; the B ring has undergone fission of the 9,10
carbon bond, resulting in the conjugated triene system of double bonds that is possessed by
all D vitamins. The structure of vitamin D3 is shown in Fig. 1. Naturally occurring mem-
Fig. 1 Chemistry and irradiation pathway for production of vitamin D3 (a natural process) and
vitamin D2 (a commercial process). In each instance the provitamin, with a ?5,?7 conjugated doublebond
system in the B ring, is converted to the seco-B previtamin, with the 9,10 carbon–carbon bond
broken. Then the previtamin D thermally isomerizes to the ‘‘vitamin’’ form, which contains a system
of three conjugated double bonds. In solution vitamin D is capable of assuming a large number of
conformations due to rotation about the 6,7 carbon–carbon bond of the B ring. The 6-s-cis conformer
(the steroid-like shape) and the 6-s-trans conformer (the extended shape) are presented for both
vitamin D2 and vitamin D3.
56 Collins and Norman
Table 3 Side Chains of Provitamin D
bers of the vitamin D family differ from each other only in the structure of their side
chains; the side chain structures of the various members of the vitamin D family are given
in Table 3.
From the x-ray crystallographic work of Nobel laureate Crowfoot-Hodgkin et al.,
it is now known that the diene system of vitamin D that extends from C-5 to C-8 is transoid
and nearly planar (57,58). However, the C-6 to C-19 diene system is cisoid and not planar.
The C-10 to C-19 double bond is twisted out of the plane by 60°. As a result, the A ring
exists in one of two possible chair conformations. It is also known that the C and D rings
are rigid and that the side chain prefers an extended configuration. In 1974, Okamura et
al. reported that vitamin D and its metabolites have a high degree of conformational mobility
(59). Using nuclear magnetic resonance (NMR) spectroscopy, they were able to detect
that the A ring undergoes rapid interconversion between the two chair conformations, as
shown in Fig. 2. This conformational mobility is unique to the vitamin D molecule and
is not observed for other steroid hormones. It is a direct consequence of the breakage of
the 9,10 carbon bond of the B ring, which serves to ‘‘free’’ the A ring. As a result of this
mobility, substituents on the A ring are rapidly and continually alternating between the
axial and equatorial positions.
B. Nomenclature
Vitamin D is named according to the new revised rules of the International Union of Pure
and Applied Chemists (IUPAC). Since vitamin D is derived from a steroid, the structure
retains its numbering from the parent steroid compound. Vitamin D is designated ‘‘seco’’
because its B ring has undergone fission. Asymmetrical centers are named using R, S
Vitamin D 57
Fig. 2 The dynamic behavior of 1?,25(OH)2D3. The topological features of the hormone
1?,25(OH)2D3 undergo significant changes as a consequence of rapid conformational changes (due
to single-bond rotation) or, in one case, as a consequence of a hydrogen shift (resulting in the
transformation of 1?,25(OH)2D3 to pre-1?,25(OH)2D3). (top) The dynamic changes occurring
within the seco-B conjugated triene framework of the hormone (C5, 6, 7, 8, 9, 10, 19). All of the
carbon atoms of the 6-s-trans conformer of 1?,25(OH)2D3 are numbered using standard steroid
notation for the convenience of the reader. Selected carbon atoms of the 6-s-cis conformer are also
numbered as are those of pre-1?,25(OH)2D3. (middle) The rapid chair–chair inversion of the A ring
of the secosteroid. (bottom) The dynamic single-bond conformational rotation of the cholesterollike
side chain of the hormone. The C/D trans-hydrindane moiety is assumed to serve as a rigid
anchor about which the A-ring, seco-B triene, and side chain are in dynamic equilibrium.
notation and Cahn’s rules of priority. The configuration of the double bonds is notated
E, Z; E for ‘‘trans,’’ Z for ‘‘cis.’’ The formal name for vitamin D3 is 9,10-seco(5Z,7E)-
5,7,10(19)-cholestatriene-3?-ol and for vitamin D2 is 9,10-seco(5Z,7E)-5,7,10(19),21-
ergostatetraene-3?-ol.
C. Chemical Properties
1. Vitamin D3(C27H44O)
Three double bonds
Melting point, 84–85°C
UV absorption maximum at 264–265 nm with a molar extinction coefficient of
18,300 in alcohol or hexane, ?D20 
84.8° in acetone
58 Collins and Norman
Molecular weight, 384.65
Insoluble in H2O
Soluble in benzene, chloroform, ethanol, and acetone
Unstable in light
Will undergo oxidation if exposed to air at 24°C for 72 h
Best stored at 0°C
2. Vitamin D2(C28H44O)
Four double bonds
Melting point, 121°C
UV absorption maximum at 265 nm with a molar extinction coefficient of 19,400
in alcohol or hexane, ?D20 
 106° in acetone
Same solubility and stability properties as D3
D. Isolation
Many of the studies that have led to our understanding of the mode of action of vitamin
D have involved tissue localization and identification of vitamin D and its various metabolites.
Since vitamin D is a steroid, it is isolated from tissue by methods that extract total
lipids. The technique most frequently used for this extraction is that of Bligh and Dyer
(60).
Over the years, a wide variety of chromatographic techniques have been used to
separate vitamin D and its metabolites. These include paper, thin-layer, column, and gas
chromatographic methods. Paper and thin-layer chromatography usually require long development
times with unsatisfactory resolutions and have limited capacity. Column chromatography,
with alumina, Floridin, Celite, silicic acid, and Sephadex LH-20 as supports,
has been used to rapidly separate many closely related vitamin D compounds (2). However,
none of the above methods are capable of resolving and distinguishing vitamin D2
from vitamin D3. Gas chromatography can separate these two compounds, but in the process
vitamin D is thermally converted to pyrocalciferol and isopyrocalciferol, resulting in
two peaks. High-performance liquid chromatography (HPLC) has become the method of
choice for the separation of vitamin D and its metabolites (61,62). This powerful technique
is rapid and gives good recovery with high resolution.
E. Synthesis of Vitamin D
1. Photochemical Production
In the 1920s, it was recognized that provitamins D were converted to vitamins D upon
treatment with UV radiation (Fig. 1). The primary structural requirement for a provitamin
D is a sterol with a C-5 to C-7 diene double-bond system in ring B. The conjugated
double-bond system is a chromophore which upon UV irradiation initiates a series of
transformations resulting in the production of the vitamin D seco-steroid structure. The
two most abundant provitamins D are ergosterol (provitamin D2) and 7-dehydrocholesterol
(provitamin D3).
2. Chemical Synthesis
There are two basic approaches to the synthesis of vitamin D. The first involves the chemical
synthesis of a provitamin that can be converted to vitamin D by UV irradiation. The
second is a total chemical synthesis.
Vitamin D 59
Since vitamin D is derived from cholesterol, the first synthesis of vitamin D resulted
from the first chemical synthesis of cholesterol. Cholesterol was first synthesized by two
groups in the 1950s. The first method involves a 20-step conversion of 4-methoxy-2,5-
toluquinone to a progesterone derivative, which is then converted in several steps to progesterone,
testosterone, cortisone, and cholesterol (63). The other method uses the starting
material 1,6-dihydroxynaphthalene. This is converted to the B and C rings of the steroid.
A further series of chemical transformations leads to the attachment of the A ring and
then the D ring. The final product of the synthesis was epiandrosterone, which could be
converted to cholesterol (64). The cholesterol was then converted to 7-dehydrocholesterol
and UV-irradiated to give vitamin D. The yield of vitamin D from photochemical conversion
is normally 10–20%.
The first pure chemical synthesis of vitamin D, without any photochemical irradiation
steps, was accomplished in 1967 (65). This continuing area of investigation allows
for the production of many vitamin D metabolites and analogs without the necessity of
a photochemical step. Pure chemical synthesis also allows for the synthesis of radioactive
vitamin D and metabolites for the study of the metabolism of vitamin D.
Figure 3 summarizes some of the currently used synthetic strategies (14). Method
A involves the photochemical ring opening of a 1-hydroxylated side-chain-modified deriv-
Fig. 3 Summary of approaches to the chemical synthesis of 1?,25(OH)2D3. The general synthetic
approaches A–H, which are discussed in the text, represent some of the major synthetic approaches
used in recent years to synthesize the hormone 1?,25(OH)2D3 and analogs of 1?,25(OH)2D3.
60 Collins and Norman
ative of 7-dehydrocholesterol 1 producing a provitamin that is thermolyzed to vitamin D
(66,67). Method B is useful for producing side chain and other analogs. In this method,
the phosphine oxide 2 is coupled to a Grundmann’s ketone derivative 3, producing the
1?,25(OH)2D3 skeleton (68,69). In method C, dienynes such as 4 are semihydrogenated
to a previtamin structure that undergoes rearrangement to the vitamin D analog (70,71).
Method D involves the production of the vinylallene 6 from compound 5 and the subsequent
rearrangement with heat- or metal-catalyzed isomerization followed by sensitized
photoisomerization (72). Method E starts with an acyclic A ring precursor 7 that is intramolecularly
cross-coupled to bromoenyne 8, resulting in the 1,25(OH)2D3 skeleton
(73,74). Method F starts with the tosylate of 11, which is isomerized to the i-steroid 10.
This structure can be modified at C-1 and then reisomerized under sovolytic conditions
to 1?,25(OH)2D3 or analogs (75,76). In method G, vitamin D derivatives 11 are converted
to 1-oxygenated 5,6-trans vitamin D derivatives 12 (77). Finally, method H involves the
direct modification of 1?,25(OH)2D3 or an analog 13 through the use of protecting groups,
such as transition metal derivatives, or by other direct chemical transformations on 13
(78). These synthetic approaches have allowed the synthesis of more than 300 analogs
of 1?,25(OH)2D3. For an article that gives an extensive review of all the synthetic
approaches, see (14).
IV. METABOLISM
The elucidation of the metabolic pathway by which vitamin D is transformed into its
biologically active form is one of the most important advances in our understanding of
how vitamin D functions and the development of the vitamin D endocrine system. It is
now known that both vitamin D2 and vitamin D3 must be hydroxylated at the C-1 and C-
25 positions (Fig. 4) before they can produce their biological effects. The activation of
vitamin D2 occurs via the same metabolic pathway as does the activation of vitamin D3,
and the biological activities of both vitamin D2 and vitamin D3 have been shown to be
identical in all animals except birds and the New World monkey. Apparently, these animals
have the ability to discriminate against vitamin D2 (79).
A. Absorption
Vitamin D can be obtained from the diet, in which case it is absorbed in the small intestine
with the aid of bile salts (80,81). In rat, baboon, and human, the specific mode of vitamin
D absorption is via the lymphatic system and its associated chylomicrons (82,83). It has
been reported that only about 50% of a dose of vitamin D is absorbed (83,84). However,
considering that sufficient amounts of vitamin D can be produced daily by exposure to
sunlight, it is not surprising that the body has not evolved a more efficient mechanism
for vitamin D absorption from the diet.
Although the body can obtain vitamin D from the diet, the major source of this
prohormone is its production in the skin from 7-dehydrocholesterol. The 7-dehydrocholesterol
is located primarily in the malpighian layer of the skin. Upon exposure to UV
light, it is photochemically converted to previtamin D, which then isomerizes to vitamin
D over a period of several days (85). Once formed, vitamin D is preferentially removed
from the skin into the circulatory system by the blood transport protein for vitamin D,
the vitamin D–binding protein (DBP).
Vitamin D 61
Fig. 4 Overview of the vitamin D endocrine and paracrine system. Target organs and cells for
1?,25(OH)2D3 by definition contain receptors for the hormone. Biological effects are generated by
both genomic and nongenomic signaling pathways.
B. Transport
The actual site of transfer of vitamin D from the chylomicrons to its specific plasma carrier
protein, DBP, is unknown. After an oral dose of radioactive vitamin D, the radioactivity
becomes associated with the lipoprotein fraction of the plasma (86). As time passes, there
is a progressive shift from this fraction to the ?-globulin fraction (87,88). It has been
shown that the electrophoretic mobility of DBP is identical to that of ?2-globulins and
albumins (89,90).
In mammals, vitamin D, 25(OH)D3, 24R,25(OH)2D3, and 1?,25(OH)2D3 are transported
on the same protein, DBP (91,92). DBP, also known as group-specific protein (Gc
protein), is a globulin protein with a molecular weight in humans of 58,000. It is a bifunctional
protein, responsible both for the transport of vitamin D and its metabolites as well
as functioning as a scavenger for actin which may be inappropriately present in the plasma.
DBP possesses a high-affinity binding site for monomeric actin and forms a high molecular
weight complex with it (93). DBP also possesses a high-affinity binding site for 25(OH)D3
and binds other vitamin D metabolites with somewhat lower affinity (92). Sequence analysis
of the cDNA for DBP indicates that it shares homology with serum albumin and ?-
fetoprotein (94). DBP has been proposed to help in the cellular internalization of vitamin
D sterols, and levels of DBP influence the concentration of ‘‘bound’’ and ‘free’ hormone
62 Collins and Norman
in the plasma (95). The concentration of the free hormone may be important in determining
the biological activity of the hormone (95–99). Several review articles on DBP are available
(97–99).
C. Storage
Vitamin D is taken up rapidly by the liver. Since it was known that the liver serves as a
storage site for retinol, another fat-soluble vitamin, it was thought that the liver also functioned
as a storage site for vitamin D. However, it has since been shown that blood has
the highest concentration of vitamin D, in comparison with other tissues (100,101). From
studies in rats it was concluded that no rat tissue can store vitamin D or its metabolites
against a concentration gradient (82). The persistence of vitamin D in animals during
periods of vitamin D deprivation may be explained by the slow turnover rate of vitamin
D in certain tissues, such as skin and adipose tissue. During times of deprivation, the
vitamin D in these tissues is released slowly, thus meeting the vitamin D needs of the
animal over a period of time. In contrast, it was found that in pig tissue concentrations
of 1?,25(OH)2D3, especially in adipose tissue, are three- to seven-fold higher than plasma
levels (102).
Similarly, Mawer et al. carried out studies in humans on the distribution and storage
of vitamin D and its metabolites (103). In human tissue, adipose tissue and muscle were
found to be major storage sites for vitamin D. Their studies also indicated that adipose
tissue serves predominantly as the storage site for vitamin D3 and that muscle serves as
the storage site for 25(OH)D3.
D. Metabolism
The parent vitamin D is largely biologically inert; before vitamin D can exhibit any biological
activity, it must be metabolized by the body to its active forms. 1?,25(OH)2D3 is the
most active metabolite known, but there is evidence that 24R,25(OH)2D3 is required for
some of the biological responses attributed to vitamin D (16,104,105). Both of these metabolites
are produced in vivo following carbon-25 hydroxylation of the parent vitamin
D molecule.
1. 25(OH)D3
In the liver, vitamin D undergoes its initial transformation, which involves the addition
of a hydroxyl group to the 25-carbon. The metabolite thus formed is 25(OH)D3, which
is the major circulating form of vitamin D. Although there is some evidence that this
metabolite can be formed in other tissues, such as intestine and kidney, it is generally
accepted that the formation of 25(OH)D3 occurs predominantly in the liver.
The production of 25(OH)D3 is catalyzed by the enzyme vitamin D3 25-hydroxylase.
The 25-hydroxylase is found in liver microsomes and mitochondria (106–109). It is a
P450-like enzyme that is poorly regulated (110). Therefore, circulating levels of 25(OH)D3
are a good index of vitamin D status, i.e., they reflect the body content of the parent
vitamin D3 (111,112). Recent studies have suggested that the 25-hydroxylation of vitamin
D is partially regulated by 1?,25(OH)2D3. Other studies suggest that 25-hydroxylation is
dependent on intracellular calcium levels (113). However, the extent, nature, and physiological
significance of any regulatory mechanism of this step in the metabolism of vitamin
D remains uncertain. The 25-hydroxylase has been cloned (114) and expressed in yeast
cells (115).
Vitamin D 63
2. 1?,25(OH)2D3
From the liver, 25(OH)D3 is returned to the circulatory system where it is transported
via DBP to the kidney. In the kidney, a second hydroxyl group can be added at the
C-1 position. The enzyme responsible for the 1?-hydroxylation of 25(OH)D3 is the 25-
hydroxyvitamin D3-1?-hydroxylase (1-hydroxylase) (116).
1-Hydroxylase is located in the mitochondria of the proximal tubules in the kidney.
The enzyme belongs to a class of enzymes known as mitochondrial mixed-function oxidases.
Mixed-function oxidases use molecular oxygen as the oxygen source instead of
water. 1-Hydroxylase is composed of three proteins that are integral components of the
mitochondrial membrane: they are renal ferredoxin reductase, renal ferredoxin, and a cytochrome
P450.
The most important point of regulation of the vitamin D endocrine system occurs
through the stringent control of the activity of the renal 1-hydroxylase (117). In this way,
the production of the hormone 1?,25(OH)2D3 can be modulated according to the calcium
needs of the organism. Although extrarenal production of 1?,25(OH)2D3 has been demonstrated
in placenta (118,119), cultured pulmonary alveolar and bone macrophages (120–
122), cultured embryonic calvarial cells (123), and cultured keratinocytes (124,125), which
can provide the hormone to adjacent cells in a paracrine fashion,the kidney is considered
the primary source of circulating 1?,25(OH)2D3. Several regulatory factors have been
identified that modulate 1-hydroxylase activity, but some are functional only in certain
species and under certain experimental conditions. The major factors are 1?,25(OH)2D3
itself, PTH, and the serum concentrations of calcium and phosphate (126).
Probably the most important determinant of 1-hydroxylase activity is the vitamin
D status of the animal. When circulating levels of 1?,25(OH)2D3 are low, the production
of 1?,25(OH)2D3 in the kidney is high, and when circulating levels of 1?,25(OH)2D3 are
high, synthesis of 1?,25(OH)2D3 is low (117). The changes of enzyme activity induced
by 1?,25(OH)2D3 can be inhibited by cycloheximide and actinomycin D (127), which
suggests that 1?,25(OH)2D3 is acting at the level of transcription. Another modulator of
renal 1?,25(OH)2D3 production is PTH. PTH is released when plasma calcium levels are
low, and in the kidney it stimulates the activity of the 1-hydroxylase and decreases the
activity of the 24-hydroxylase. 1?,25(OH)2D3 and 24R,25(OH)2D3 also operate in a feedback
loop to modulate and/or reduce the secretion of PTH. Other modulators of renal
1?,25(OH)2D3 production are shown in Fig. 4.
3. 24R,25(OH)2D3
A second dihydroxylated metabolite of vitamin D is produced in the kidney, namely,
24R,25(OH)2D3. Also, virtually all other tissues that have receptors for 1?,25(OH)2D3
can also produce 24R,25(OH)2D3. There is some controversy concerning the possible
unique biological actions of 24R,25(OH)2D3. However, there is some evidence that
24R,25(OH)2D3 plays a role in the suppression of PTH secretion (128,129), in the mineralization
of bone (130,131), and in fracture healing (132,133). Other studies demonstrated
that the combined presence of 24R,25(OH)2D3 and 1?,25(OH)2D3 are required for normal
egg production, fertility, and hatchability in chickens (104) and quail (134). From these
studies it was apparent that only combination doses of both compounds were capable of
eliciting the same response as the parent vitamin D. Thus, it appears that both
1?,25(OH)2D3 and 24R,25(OH)2D3 may be required for some of the known biological
responses to vitamin D.
64 Collins and Norman
The enzyme responsible for the production of 24R,25(OH)2D3 is the 25-hydroxyvitamin
D3-24R-hydroxylase (24-hydroxylase). Experimental evidence suggests that this
enzyme is also a mixed-function oxidase. The activity of this enzyme is regulated so that
when 1?,25(OH)2D3 levels are low, the activity of the 24R-hydroxylase is also low, but
when 1?,25(OH)2D3 levels are high, the activity of the 24R-hydroxylase is high. Under
normal physiological conditions both 1?,25(OH)2D3 and 24R,25(OH)2D3 are secreted
from the kidney and circulated in the plasma of all classes of vertebrates.
In addition to these three metabolites, many other vitamin D3 metabolites have been
chemically characterized, and the existence of others appears likely. The chemical struc-
Fig. 5 Summary of the metabolic transformations of vitamin D3. Shown here are the structures
of all known chemically characterized vitamin D3 metabolites.
Vitamin D 65
tures of the 37 known metabolites are shown in Fig. 5. Most of these metabolites appear
to be intermediates in degradation pathways of 1?,25(OH)2D3. None of these other metabolites
have been shown to have biological activity except for the 1?,25(OH)2D3-26,23-
lactone. The lactone is produced by the kidney when the plasma levels of 1?,25(OH)2D3
are very high. The metabolite appears to be antagonistic to 1?,25(OH)2D3 because it mediates
a decrease in serum calcium levels in the rat. Other experiments suggest that lactone
inhibits bone resorption and blocks the resorptive action of 1?,25(OH)2D3 on the bone
(135), perhaps functioning as a natural antagonist of 1?,25(OH)2D3 to prevent toxic effects
from overproduction of 1?,25(OH)2D3.
E. Catabolism and Excretion
Several pathways exist in humans and animals to further metabolize 1?,25(OH)2D3. These
include oxidative cleavage of the side chain, hydroxylation of C-24 to produce
1?,24,25(OH)3D3, formation of 24-oxo-1?,25(OH)2D3, formation of 1?,25(OH)2D3-
26,23-lactone, and formation of 1?,25,26(OH)3D3 (Fig. 5). It is not known which of these
pathways are involved in the breakdown or clearance of 1?,25(OH)2D3 in humans.
The catabolic pathway for vitamin D is obscure, but it is known that the excretion
of vitamin D and its metabolites occurs primarily in the feces with the aid of bile salts.
Very little appears in the urine. Studies in which radioactively labeled 1?,25(OH)2D3 was
administered to humans have shown that 60–70% of the 1?,25(OH)2D3 was eliminated
in the feces as more polar metabolites, glucuronides, and sulfates of 1?,25(OH)2D3. The
half-life of 1?,25(OH)2D3 in plasma has two components. Within 5 min, only half of an
administered dose of radioactive 1?,25(OH)2D3 remains in the plasma. A slower component
of elimination has a half-life of about 10 h. 1?,25(OH)2D3 is catabolized by a number
of pathways that result in its rapid removal from the organism (136).
V. BIOCHEMICAL MODE OF ACTION
The major classical physiological effects of vitamin D are to increase the active absorption
of Ca2
 from the proximal intestine and to increase the mineralization of bone. This is
achieved via two major signal transduction pathways: genomic and nongenomic.
A. Genomic
Vitamin D, through its daughter metabolite 1?,25(OH)2D3, functions in a manner homologous
to that of steroid hormones. A model for steroid hormone action is shown in
Fig. 6. In the general model, the hormone is produced in an endocrine gland in response
to a physiological stimulus and then circulates in the blood, usually bound to a protein
carrier (i.e. DBP), to target tissues where the hormone interacts with specific,
high-affinity, intracellular receptors. The receptor–hormone complex localizes in the
nucleus, undergoes some type of ‘‘activation’’ perhaps involving phosphorylation
(137–140), and binds to a hormone response element (HRE) on the DNA to modulate
the expression of hormone-sensitive genes. The modulation of gene transcription results
in either the induction or repression of specific mRNAs, ultimately resulting in
changes in protein expression needed to produce the required biological response. Highaffinity
receptors for 1?,25(OH)2D3 have been identified in at least 26 target tissues
(12,16,141) and more than 50 genes are known to be regulated by 1?,25(OH)2D3 (142).
66 Collins and Norman
Fig. 6 General model for the mode of action of steroid hormones. Target tissues contain receptors
for the steroid which confer on them the ability to modulate gene transcription. S, steroid; R, receptor
protein, which may be present inside the cell in either the cytosol or nuclear compartment; SR,
steroid–receptor complex; DBP, serum vitamin D–binding protein, which functions to transport the
steroid hormone from the endocrine gland to its various target tissues.
Genes that have been shown to be transcriptionally regulated by 1?,25(OH)2D3 are listed
in Table 4.
1. Nuclear Receptor
The 1?,25(OH)2D3 receptor was originally discovered in the intestine of vitamin-D defi-
cient chicks (50,51). It has been extensively characterized and the cDNA for the nuclear
receptor has been cloned and sequenced (52–55). The 1?,25(OH)2D3 receptor is a DNAbinding
protein with a molecular weight of about 50,000 da. It binds 1?,25(OH)2D3 with
high affinity with a KD in the range of 1–50  1010 M (143–145). The ligand specificity
of the nuclear 1?,25(OH)2D3 receptor is illustrated in Table 5. The 1?,25(OH)2D3 receptor
protein belongs to a superfamily of homologous nuclear receptors (52). To date only a
single form of the receptor has been identified.
The superfamily of ligand-dependent nuclear receptors includes receptors for glucocorticoids
(GR), progesterone (PR), estrogen (ER), aldosterone, androgens, thyroid hormone
(T3R), hormonal forms of vitamins A (RAR, RXR) and D (VDR), and several
orphan receptors (141,146,147). Comparative studies of these receptors reveal that they
have the common structural organization consisting of five domains (148), shown in Fig.
7. The different domains act as distinct modules that can function independently of each
other (149–151).
The DNA binding domain, C, is the most conserved domain throughout the family.
About 70 amino acids fold into two zinc finger–like motifs. Conserved cysteines
coordinate a zinc ion in a tetrahedral arrangement. The first finger, which contains four
cysteines and several hydrophobic amino acids, determines the DNA response element
specificity. The second zinc finger, which contains five cysteines and many basic amino
Vitamin D 67
Table 4 Genes Regulated by 1?,25(OH)2D3
Gene Reg. Evidence Tissue/cell
?-Tubulin Down mRNA Chick intestine
Aldolase subunit B Up mRNA Chick kidney
Alkaline phosphatase Up mRNA Rat intestine
Chick intestine
TE-85 cells
ATP synthase Up mRNA Rat intestine
Chick intestine
Down mRNA Chick kidney
c-FMS Up mRNA HL-60 cells
c-FOS Up mRNA MG-63 cells
HL-60 cells
c-KI-RAS Up mRNA BALB-3T3 cells
c-MYB Down mRNA HL-60 cells
c-MYC Up mRNA MG-63
Down mRNA U937 cells
HL-60 cells Transcription
HL-60 cells
Calbindin28K Up mRNA Chick intestine
Mouse kidney Transcription
Chick intestine
Calbindin9K Up mRNA Mouse kidney
VDRE Rat
Carbonic anhydrase Up mRNA Marrow cells
Transcription Myelomonocytes
CD-23 Down mRNA PBMC
Collagen type I Down mRNA/VDRE Rat
Cytochrome oxidase sub- Up mRNA Rat intestine
unit I
Chick intestine
Down mRNA Chick kidney
Cytochrome oxidase sub- Up mRNA Chick intestine
unit II
Down mRNA Chick kidney
Cytochrome oxidase sub- Up mRNA Rat intestine
unit III
Chick intestine
Down mRNA Chick kidney
Cytochrome B Down mRNA Chick kidney
Fatty acid–binding pro- Down mRNA Chick intestine
tein
Ferridoxin Down mRNA Chick kidney
Fibronectin Up mRNA MG-63
TE-85
HL-60 cells
?-Interferon Down mRNA T lymphocytes
PBMC
Glyceraldehyde-3- Up mRNA BT-20 cells
phosphate dehydrogenase
68 Collins and Norman
Table 4 Continued
Gene Reg. Evidence Tissue/cell
GM-colony-stimulating Down mRNA T lymphocytes
factor
Heat shock protein 70 Up mRNA PBMC
Histone H4 Down mRNA/ HL-60 cells
Ttranscription
1-Hydroxyvitamin D-24- Up mRNA Rat kidney
hydroxylase mRNA/ Rat kidney
Transcription
Integrin???3 Up mRNA/ Avian osteoclast precursor cells
Transcription
Interleukin-6 Up mRNA U937
Interleukin-4 Up mRNA U937 cells
Interleukin-2 Down mRNA T lymphocytes
Interleukin-3 receptor Up mRNA MC3T3 cells
Matrix gla protein Up mRNA UMR106-01, ROS 25/1, 25/4 cells
Metallothionein Up mRNA Rat kertinocytes
Mouse liver/kidney/skin
Chick kidney
Monocyte-derived neutro- Up mRNA/transcription HL-60 cells
phil-activating peptide
NADH DH subunit I Down mRNA Chick kidney
NADH DH subunit III Up mRNA Chick intestine
NADH DH subunit IV Up mRNA Chick intestine
Nerve growth factor Up mRNA L-929 cells
Osteocalcin Up mRNA ROS 17/2.8
ROS 25/1
VDRE ROS 17/2.8
Rat
Osteopontin Up mRNA ROS 17/2.8
VDRE ROS 17/2.8
Plasma membrane cal- Up mRNA Chick intestine
cium pump
Pre-pro-PTH Down mRNA Rat
mRNA/transcription Bovine parathyroid
Prolactin Up mRNA GH4C1 cells
Protein kinase inhibitor Down mRNA Chick kidney
Protein kinase C Up mRNA/transcription HL-60 cells
PTH Down mRNA Rat parathyroid
PTH-related protein Down mRNA/transcription TT cells
Transferrin receptor Down mRNA PBMC
Tumor necrosis factor ? Up mRNA U937 cells
Transcription HL-60 cells
VDR Up mRNA Rat intestine
Rat pituitary
MG-60 cells
Source: Ref. 142.
Vitamin D 69
Table 5 Ligand Specificity of the Nuclear 1?,25(OH)2D3 Receptor
RCI
Ligand Structural modification (%)a
1?,25(OH)2D3 100
1?,25(OH)2-24-nor-D3 Shorten side chain by 1 carbon 67
1?,25(OH)2-3-epi-D3 Orientation of 3?-OH altered 24
1?,25(OH)2-24a-dihomo-D3 Lengthen side chain by 2 carbons 24
1?,25(OH)2D3 Orientation of 1?-OH changed 0.8
1?(OH)D3 Lacks 25-OH 0.15
25(OH)D3 Lacks 1?-OH 0.15
1?,25(OH)2-7-dehydrocholesterol Lacks a broken B ring; is not a seco steroid 0.10
Vitamin D3 Lacks 1? and 25-OH 0.0001
aThe Relative Competitive Index (RCI) is a measure of the ability of a nonradioactive ligand to compete, under
in vitro conditions, with radioactive 1?,25(OH)2D3 for binding to the nuclear 1?,25(OH)2D3 receptor (VDR)
Source: Ref. 14.
acids, is also necessary for DNA binding and is involved in receptor dimerization
(146,150,152,153).
The next conserved region is the steroid binding domain (region E). This region
contains a hydrophobic pocket for ligand binding and also contains signals for several
other functions, including dimerization (154–157), nuclear translocation, and hormonedependent
transcriptional activation (149,150,158).
The A/B domain is also known as the immuno- or transactivation domain. This
region is poorly conserved in amino acids and in size, and its function has not been clearly
defined. The VDR has the smallest A/B domain (25 amino acids) of the known receptors;
mineralocorticoid receptor has the largest (603 amino acids). An independent transcriptional
activation function is located within the A/B region (146,150,151) that is constitutive
in receptor constructs lacking the ligand binding domain (region E). The relative
importance of the transcriptional activation by this domain depends on the receptor, the
context of the target gene promoter, and the target cell type (159).
Region D is the hinge region between the DNA binding domain and the ligand
bonding domain. The hinge region in the VDR contains 156 amino acids and has immunogenic
properties. The VDR has the longest hinge region of the known receptors (160).
Human GR and PR have hinge regions of 92 and 101 amino acids, respectively.
The VDR belongs to a subgroup of the receptors designated group II, which includes
T3R, RAR, RXR, and several orphan receptors. All of the group II receptors can form
heterodimers with RXR (161,162), and other heterodimeric interactions have also been
Fig. 7 Schematic representation of the human nuclear VDR. The DNA binding domain (C) and
ligand binding domain (E) are boxed.
70 Collins and Norman
reported (163). T3R lacking the DNA binding domain can inhibit the transactivation of
RAR (156) and VDR (152), but not a chimeric receptor containing the ligand binding
domain of GR. The VDR can also form heterodimers with RAR (163,164). The ability
to form heterodimers with other receptors allows for enhanced affinity for distinct DNA
targets, generating the diverse range of physiological effects.
2. Calbindin D
One of the major effects of 1?,25(OH)2D3 in many of its target tissues is the induction
of the calcium binding protein, calbindin D. In the mammalian kidney and brain and
in avians, a larger form of the protein is expressed, calbindin D28K (165), whereas in the
mammalian intestine and placenta a smaller form is expressed, calbindin D9K (166). The
expression of calbindins in various tissues and species appears to be regulated to differing
degrees by 1?,25(OH)2D3 (167).
Early experiments showed that actinomycin D and ?-amanitin, transcriptional inhibitors,
could block the induction of calbindin D28K by 1?,25(OH)2D3 (168). Later experiments
showed that 1?,25(OH)2D3 was able to stimulate total RNA synthesis in the chick
intestine (169) in addition to specifically inducing the mRNA for calbindin D28K (170).
Nuclear transcription assays have shown that transcription of calbindin D28K mRNA is
directly induced by 1?,25(OH)2D3 in the chick intestine and is correlated to the level of
occupied 1?,25(OH)2D3 receptors (171). The gene for calbindin D28K has now been cloned
and sequenced, but there is still much to learn about how 1?,25(OH)2D3 induces this gene
(172).
B. Nongenomic Actions
Recent studies (173) suggest that not all of the actions of 1?,25(OH)2D3 can be explained
by receptor–hormone interactions with the genome. 1?,25(OH)2D3 can stimulate the intestinal
transport of calcium within 4–6 min, i.e., too quickly to involve genome activation.
The rapid transport of calcium mediated by 1?,25(OH)2D3 in the intestine has been termed
‘‘transcaltachia’’ (‘‘trans’’  across; ‘‘cal’’  calcium; ‘‘tachia’’  swiftly). Transcaltachia
is not inhibited by actinomycin D but is inhibited by colchicine, an antimicrotubule
agent, and by leupeptin, an antagonist of lysosomal cathepsin B. Transcaltachia induced
by 1?,25(OH)2D3 in the intestine appears to involve the internalization of calcium in
endocytic vesicles at the brush-border membrane, which then fuse with lysosomes and
travel along microtubules to the basal lateral membrane where exocytosis occurs. Therefore,
some of the actions of 1?,25(OH)2D3 may be mediated at the cell membrane or by
extranuclear subcellular components.
Other effects of 1?,25(OH)2D3 that do not appear to be mediated by the nuclear
receptor are phosphoinositide breakdown (174), enzymatic activity in osteoblast-derived
matrix vesicles (175), certain secretion events in osteoblasts (176), rapid changes in cytosolic
Ca2
 levels in primary cultures of osteoblasts and osteosarcoma cells (177–179),
and increases in cyclic guanosine monophosphate levels in fibroblasts (180). These rapid
effects appear to be mediated by a membrane receptor–like protein for 1?,25(OH)2D3
(181); a candidate membrane receptor for 1?,25(OH)2D3 has been proposed (182). Other
steroid hormones, i.e., estrogen (183), progesterone (184–187), testosterone (188), glucocorticoids
(189,190), corticosteroid (191), and thyroid (192,193), have been shown to have
similar membrane effects (181). A model for the nongenomic signal transduction pathway
is shown in Fig. 8.
Vitamin D 71
Fig. 8 Model describing the signal transduction pathways associated with the nongenomic response
of transcaltachia. The general model of vesicular Ca2
 transport includes formation of Ca2
-
containing endocytic vesicles at the brush-border membrane, fusion of endocytic vesicle with lysosomes,
movement of lysosomes along microtubules, and exocytotic extrusion of Ca2
 via fusion of
the lysosomes with the basal lateral membrane of the intestinal enterocyte. The binding of
1?,25(OH)2D3 to a membrane receptor results in an increase of several second messengers, including
IP3, cAMP, activation of PKC or intracellular Ca2
, which may result in the transient opening of
Ca2
 channels. The increased Ca2
 concentration may then initiate the exocytosis of the lysosomal
vesicles.
VI. SPECIFIC FUNCTIONS OF 1
,25(OH)2D3
A. 1
,25(OH)2D3 and Mineral Metabolism
The classical target tissues for 1?,25(OH)2D3 are those tissues that have been found to
be directly involved in the regulation of mineral homeostasis. In humans, serum calcium
levels are normally maintained between 9.5 and 10.5 mg/100 mL, whereas the phosphorus
concentration is between 2.5 and 4.3 mg/100 mL (2). Together with PTH and calcitonin,
1?,25(OH)2D3 maintains serum calcium and phosphate levels by its actions on the intestine,
kidney, bone, and parathyroid gland.
In the intestine, one of the best characterized effects of 1?,25(OH)2D3 is the stimulation
of intestinal lumen–to–plasma flux of calcium and phosphate (39,194,195). Although
extensive evidence exists showing that 1?,25(OH)2D3 interacting with its receptor upregulates
calbindin D in a genome-mediated fashion, the relationship between calbindin
D and calcium transport is not clear (196). In the vitamin D–deficient state, both mammals
and birds have severely decreased intestinal absorption of calcium with no detectable
72 Collins and Norman
levels of calbindin. There is a linear correlation between the increased cellular levels of
calbindin D and calcium transport. When 1?,25(OH)2D3 is given to vitamin D–deficient
chicks, the transport of calcium reaches maximal rates at 12–14 h, whereas calbindin D
does not reach its maximal levels until 48 h (173). In one study employing immunohistochemical
techniques, it was demonstrated that the cellular location of calbindin D28K
changed with the onset of calcium transport (197).
1?,25(OH)2D3 treatment also is known to alter the biochemical and morphological
characteristics of the intestinal cells (198,199). The size of the villus and the size of the
microvilli increase upon 1?,25(OH)2D3 treatment (200). The brush border undergoes noticeable
alterations of structure and composition of cell surface proteins and lipids, occurring
in a time frame corresponding to the increase in Ca2
 transport mediated by
1?,25(OH)2D3 (201). However, despite extensive work, the exact mechanisms involved
in the vitamin D–dependent intestinal absorption of calcium remain unknown (202–204).
The kidney is the major site of synthesis of 1?,25(OH)2D3 and of several other
hydroxylated vitamin D derivatives. Probably the most important effect of 1?,25(OH)2D3
on the kidney is the inhibition of 25(OH)D3-1?-hydroxylase activity, which results in a
decrease in the synthesis of 1?,25(OH)2D3 (205,206). Simultaneously, the activity of the
25(OH)D3-24-hydroxylase is stimulated. The actions of vitamin D on calcium and phosphorus
metabolism in the kidney has been controversial, and more research is needed to
clearly define the actions of 1?,25(OH)2D3 on the kidney.
Although vitamin D is a powerful antirachitic agent, its primary effect on bone is
the stimulation of bone resorption leading to an increase in serum calcium and phosphorus
levels (207). With even slight decreases in serum calcium levels, PTH is synthesized,
which then stimulates the synthesis of 1?,25(OH)2D3 in the kidney. Both of these hormones
stimulate bone resorption. Maintaining constant levels of calcium in the blood is
crucial, whether calcium is available from the diet or not. Therefore, the ability to release
calcium from its largest body store—bone—is vital. Bone is a dynamic tissue that is
constantly being remodeled. Under normal physiological conditions, bone formation and
bone resorption are tightly balanced (208). The stimulation of bone growth and mineralization
by 1?,25(OH)2D3 appears to be an indirect effect of the provision of minerals for
bone matrix incorporation through an increase of intestinal absorption of calcium and
phosphorus. In bone, nuclear receptors for 1?,25(OH)2D3 have been detected in normal
osteoblasts (209), osteoblast-like osteosarcoma cells, but not in osteoclasts. In addition,
1?,25(OH)2D3 can induce rapid changes in cytosolic Ca2
 levels in osteoblast and osteosarcoma
cells by opening voltage-gated Ca2
 channels via a nongenomic signal transduction
pathway (178,179).
Some of the actions of 1?,25(OH)2D3 in bone are related to changes in bone cell
differentiation. 1?,25(OH)2D3 is known to affect a number of osteoblast-related functions.
For example, 1?,25(OH)2D3 decreases type I collagen production (210), and increases
alkaline phosphatase production and the proliferation of cultured osteoblasts (211);
1?,25(OH)2D3 increases the production of osteocalcin (212) and matrix Gla protein (213),
and decreases the production of type I collagen by fetal rat calvaria (214).
1?,25(OH)2D3 also affects the growth and differentiation of osteoclasts and osteoclast-
like cells in vivo in rats (215) and in primate bone marrow cell cultures (216). Since
the osteoclast does not have a nuclear VDR, 1?,25(OH)2D3 must affect osteoclasts indirectly
or by nongenomic mechanisms. There is some evidence that a factor produced by
osteoblasts promotes the formation of osteoclasts (217,218). It is possible that the only
effect of 1?,25(OH)2D3 on the osteoclasts is to stimulate its generation from progenitor
cells.
Vitamin D 73
PTH is an important tropic stimulator of 1?,25(OH)2D3 synthesis by the kidney.
High circulating levels of 1?,25(OH)2D3 have been shown to decrease the levels of PTH
by two different mechanisms: an indirect mechanism due to the resulting increase in serum
calcium levels, which is an inhibitory signal for PTH production, and a direct mechanism
involving the interaction of 1?,25(OH)2D3 and its receptor, which directly suppresses the
expression of the prepro-PTH gene.
During pregnancy and lactation, large amounts of calcium are needed for the developing
fetus and for milk production. Hormonal adjustments in the vitamin D endocrine
system are critical to prevent depletion of minerals leading to serious bone damage for
the mother. Although receptors for 1?,25(OH)2D3 have been found in placental tissue and
in the mammary gland, the role of vitamin D is not clear.
B. Vitamin D in Nonclassical Systems
In the 1970s and 1980s, nuclear receptors for 1?,25(OH)2D3 were discovered in a variety
of tissues and cells not directly involved in calcium homeostasis. Thus, the role of the
vitamin D endocrine system has expanded to include general effects on cell regulation
and differentiation (12,18). Nuclear VDRs are present in muscle, hematolymphopoietic,
reproductive, and nervous tissue, as well as in other endocrine tissues and skin. More than
50 proteins are known to be regulated by 1?,25(OH)2D3, including several oncogenes
(56,142) (Table 2), which extend by far the classical limits of vitamin D actions on calcium
homeostasis. In many of these systems it is not yet clear what the effect of vitamin D is
on the tissue or its mode of action.
Skeletal muscle is a target organ for 1?,25(OH)2D3. Clinical studies have shown
the presence of muscle weakness or myopathy during metabolic bone diseases related to
vitamin D deficiency (19,22,219). These abnormalities can be reversed with vitamin D
therapy. Experimental evidence has shown that 1?,25(OH)2D3 has a direct effect on Ca2

transport in cultured myoblasts and skeletal muscle tissue. Furthermore, there is evidence
that the action of 1?,25(OH)2D3 on skeletal muscle may be important for the calcium
homeostasis of the entire organism because the hormone induces a rapid release of calcium
from muscle into the serum of hypocalcemic animals. 1?,25(OH)2D3 receptors have been
detected in myoblast cultures, and the changes in calcium uptake have been shown to be
RNA- and protein synthesis–dependent, suggesting a genomic mechanism. 1?,25(OH)2D3
has also been shown to be important for cardiac muscle function (220–223).
In the skin, 1?,25(OH)2D3 appears to exert effects on cellular growth and differentiation.
Receptors for 1?,25(OH)2D3 have been found in human (224) and mouse skin (225).
1?,25(OH)2D3 inhibits the synthesis of DNA in mouse epidermal cells (225). The hormone
induces changes in cultured keratinocytes, which are consistent for terminal differentiation
of nonadherent cornified squamous cells (226). Additional experiments have shown that
human neonatal foreskin keratinocytes produce 1?,25(OH)2D3 from 25(OH)D3 under in
vitro conditions (227), suggesting that keratinocyte-derived 1?,25(OH)2D3 may affect epidermal
differentiation locally. Psoriasis is a chronic hyperproliferative skin disease. Some
forms of psoriasis have been shown to improve significantly when treated topically with
calcipotriol, a nonhypercalcemic analog of 1?,25(OH)2D3 (228–230). In mouse skin carcinogenesis,
1?,25(OH)2D3 blocks the production of tumors induced by 12-O-tetradecanoylphorbol-
12-acetate (231).
In the pancreas, 1?,25(OH)2D3 has been found to be essential for normal insulin
secretion. Experiments with rats have shown that vitamin D increases insulin release from
the isolated perfused pancreas, in both the presence and the absence of normal serum
74 Collins and Norman
calcium levels (232–236). Human patients with vitamin D deficiency, even under conditions
of normal calcemia, exhibit impaired insulin secretion but normal glucagon secretion,
suggesting that 1?,25(OH)2D3 directly affects ?-cell function (237).
Receptors for 1?,25(OH)2D3 have been found in some sections of the brain
(238,239). However, the role of 1?,25(OH)2D3 in the brain is not well understood. Both
calbindins D have been found in the brain, but neither the expression of calbindin D28K
nor that of calbindin D9K appears to be directly modulated by vitamin D (238,239). In the
rat, 1?,25(OH)2D3 appears to increase the activity of the choline acetyltransferase in specific
regions of the brain (238). Other steroid hormones have also been shown to affect
the metabolism of specific brain regions (240,241).
Also, normal, benign, hyperplastic and malignant prostatic epithelial and fibroblastic
cells contain receptors for 1?,25(OH)2D3 (242). In hematopoietic tissue, 1?,25(OH)2D3
promotes the differentiation and inhibits proliferation of both malignant and nonmalignant
hematopoietic cells. Human promyelocytic leukemia cells, HL-60, have been shown to
have receptors for 1?,25(OH)2D3 and to differentiate toward macrophages upon treatment
with 1?,25(OH)2D3 (243,244). Other effects of 1?,25(OH)2D3 on the immune system will
be discussed in the next section.
C. Immunoregulatory Roles
In 1979, when the VDR was discovered in several neoplastic hematopoietic cell lines as
well as in normal human peripheral blood mononuclear cells, monocytes, and activated
lymphocytes (245,246), a role for 1?,25(OH)2D3 in immune function was suggested. Since
then, 1?,25(OH)2D3 has been shown to affect cells of the immune system in a variety of
ways. 1?,25(OH)2D3 reduces the proliferation of HL-60 cells and induces their differentiation
to monocytes (243) and macrophages (244,247,248). The actions of 1?,25(OH)2D3
on normal monocytes are controversial, but it appears that the molecule may enhance
monocyte function. 1?,25(OH)2D3 appears to reduce levels of HLA-DR and CD4
 class
II antigens on monocytes or macrophages with no effect on the expression of class I
antigens (249). The enhancement of class II antigen expression is a common feature of
autoimmunity and often precedes the onset of autoimmune diseases.
1?,25(OH)2D3 also promotes the differentiation of leukemic myeloid precursor cells
toward cells with the characteristics of macrophages (243). Subsequent experiments have
shown that 1?,25(OH)2D3 does not alter the clonal growth of normal myeloid precursors
but does induce the formation of macrophage colonies preferentially over the formation
of granulocyte colonies (247). In addition, macrophages derived from different tissues can
synthesize 1?,25(OH)2D3 when activated by ?-interferon (121). Also, 1?,25(OH)2D3 can
suppress immunoglobulin production by activated B lymphocytes (250) and inhibit DNA
synthesis and proliferation of both activated B and T lymphocytes (251–253). These findings
suggest that a vitamin D paracrine system exists that involves activated macrophages
and activated lymphocytes (Fig. 4).
1?,25(OH)2D3 also affects some functions of T and B lymphocytes and natural killer
(NK) cells. In T lymphocytes, the mitogen activation of lymphocyte proliferation is
blocked in the presence of 1?,25(OH)2D3 (251,254,255), apparently by interference with
cell cycle progression from early G1 to late G1 phase (254). 1?,25(OH)2D3 exhibits a
permissive or enhancing effect on T-cell suppressor activity. In an in vitro model of transplant
compatibility, the mixed lymphocyte reaction, 1?,25(OH)2D3 significantly enhanced
T-cell suppressor activity (256). 1?,25(OH)2D3 also affects the cytotoxicity of NK and
T-cytotoxic cells probably by interfering with their generation from precursor cells.
Vitamin D 75
1?,25(OH)2D3 has been shown to decrease mRNA levels for IL-2 (257), ?-interferon
(258) and granulocyte-macrophase colony-stimulating factor (GM-CSF) (259,260).
1?,25(OH)2D3 also attenuates the inducing effect of T-helper cells on IgG synthesis by
B cells (261).
Despite the wide range of actions of 1?,25(OH)2D3 on various immune cells, no
general immunomodulatory role for 1?,25(OH)2D3 has been defined. In contrast to the
serum calcium elevation observed in patients with sarcoidosis, lymphoma, and an anephric
patients with end-stage renal disease, no systemic immunosuppressive activity of
1?,25(OH)2D3 has been described in these disease states to date, suggesting that
1?,25(OH)2D3 acts in an autocrine or paracrine fashion to modulate local immune function
(261,262). 1?,25(OH)2D3 and cyclosporine, a potent immunosuppressive drug, appear to
affect the immune system in a similar fashion. They both affect T lymphocytes during
initial activation by antigen, select the generation of T-helper cells by inhibiting lymphokine
production at a genomic level, and inhibit the generation of T-cytotoxic and NK
cells. Both are involved in the enhancement of T-suppressor function, a key element in
the efficacy of cyclosporine as a drug that reduces allograft tissue rejection (263).
1?,25(OH)2D3 appears to work synergistically with cyclosporine when the two compounds
are used in combination (264,265).
The use of nonhypercalcemic 1?,25(OH)2D3 analogs can result in enhanced immunosuppressive
effects without the toxicity risks of 1?,25(OH)2D3. Because of the synergistic
effect when 1?,25(OH)2D3 is used with cyclosporine, synthetic 1?,25(OH)2D3 analogs
may be used in the treatment of autoimmune diseases (266) or for transplantation
(267) in combination with cyclosporine to reduce the toxicity of both compounds.
D. Structures of Important Analogs
In nephrectomized animals, vitamin D compounds cannot be hydroxylated at the C-1
position because the kidney is the site where this hydroxylation occurs. Researchers
found that neither vitamin D nor 25(OH)D was able to elicit a significant biological response
when administered in physiological doses to nephrectomized animals (48,268).
Also, it was noted in the 1940s and 1950s that dihydrotachysterol3 (a 5,6-trans analog
of vitamin D3) was biologically active under circumstances where the parent vitamin D
demonstrated little or no biological activity. These findings raised the question of the
functional importance of the various structural elements of the vitamin D molecule.
Studies using analogs of vitamin D have been used to address this question. The ability
of analogs to bind to the nuclear receptor for 1?,25(OH)2D3, to increase intestinal calcium
absorption (ICA) and bone calcium mobilization (BCM), and to promote cellular
differentiation are then determined. Because of recent advances in new vitamin D syntheses
described above and in Fig. 3, analogs have been synthesized with modifications
in the A ring, seco-B ring, C ring, C/D ring junction, D ring, and/or side chain (14,
269).
The importance of the configuration of the A ring has been studied by synthesizing
5,6-trans analogs. Because of the rotation of the A ring, these analogs cannot undergo 1-
hydroxylation and have been found to be only 1/1000 as biologically effective as
1?,25(OH)2D3. The relative significance of the 3?-hydroxyl group has been assessed by
preparing analogs such as 3-deoxy-1?,25(OH)2D3. Although this analog is active in vivo,
it is interesting in that it preferentially stimulates intestinal calcium absorption over bone
calcium mobilization (270). Of all the analogs synthesized, only a few show such selective
biological activity.
76 Collins and Norman
The effect of altering the length of the side chain has been studied. The 27-nor-
25(OH)D3 and 26,27-bis-nor-25(OH)D3 are reportedly able to stimulate intestinal calcium
absorption and bone calcium mobilization in both normal and anephric rats but are 10–
100 times less active than 25(OH)D3 (271). The 24-nor-25(OH)D3 was found to have no
biological activity (272), although it was able to block the biological response to vitamin
D but not to 25(OH)D3 or 1?,25(OH)2D3. This suggests that it might have anti–vitamin
D activity.
One of the most interesting side-chain analogs of 1?,25(OH)2D3 is 1?(OH)D3. This
metabolite appears to have the same biological activity in the chick as 1?,25(OH)2D3
(273) and is approximately half as active in the rat (274). In an attempt to determine if
the biological activity of 1?(OH)D3 is the result of in vivo 25-hydroxylation, the 25-fluoro-
1?(OH)D3 derivative was prepared (275). The fluorine on C-25 prevents hydroxylation of
this carbon. The fluoro compound was found to be one-fiftieth as active as 1?,25(OH)2D3,
suggesting that 1?(OH)D3 has some activity even without 25-hydroxylation.
From such studies, the particular attributes of the structure of 1?,25(OH)2D3 that
enables it to elicit its biological responses are being defined. It is now known that the 3?-
hydroxy group does not appear to be as important for biological activity as the 1?- or
25-hydroxyl groups; the cis configuration of the A ring is preferred over the trans configuration;
and the length of the side chain appears critical, as apparently there is little tolerance
for its being shortened or lengthened.
Analogs of 1?,25(OH)2D3 have been used to study the in vivo metabolism and mode
of action of vitamin D compounds. There has also been widespread interest in developing
1?,25(OH)2D3 analogs to use as therapeutic agents in the treatment of osteoporosis, renal
osteodystrophy, cancer, immunodeficiency syndromes, autoimmune diseases, and some
skin disorders. Of particular interest are analogs that separate the calcemic effects from
the proliferation and differentiation effects of 1?,25(OH)2D3.
One of the most successful analogs in terms of separating biological activities is a
cyclopropyl derivative of 1?,25(OH)2D3, 1?,24S(OH)2-22ene-26,27-dehydrovitamin D3,
designated calcipotriol; this analog has weak systemic effects on calcium metabolism but
potent effects on cell proliferation and differentiation (276,277). It is rapidly converted
to inactive metabolites in vivo (278,279) and is 200-fold less potent than 1?,25(OH)2D3
in causing hypercalciuria and hypercalcemia in rats (276). It is as effective in binding to
the nuclear receptor as 1?,25(OH)2D3 and has similar effects on the growth and differentiation
of keratinocytes (280,281). It is currently marketed as a topical treatment for psoriasis,
a proliferative disorder of the skin (228,282–285).
Another analog that has potential as a therapeutic agent is 22-oxa-1?,25(OH)2D3.
This analog has been shown to suppress the secretion of PTH and may be useful in the
treatment of secondary hyperparathyroidism (286). It is 10 times more potent in suppressing
proliferation and inducing differentiation than 1?,25(OH)2D3, with only 1/50 to
1/100 of the in vitro bone-resorbing activity of 1?,25(OH)2D3 (287).
Still another set of analogs of 1?,25(OH)2D3 with potential therapeutic applications
are the compounds with a double bond at C-16 and/or a triple bond at C-23. The best
characterized of these compounds is 1?,25(OH)2-16ene-23yne-D3 (288–290). This analog
is 300-fold less active in intestinal calcium absorption (ICA) and bone calcium mobilization
(BCM) and 10 to 15 times less active in inducing hypercalcemia in vivo in mice than
1?,25(OH)2D3. In three leukemia models, therapy with the analog resulted in a significant
increase in survival (288,289). All of the 16-ene and or 23-yne analogs that have been
tested are equivalent or more potent than 1?,25(OH)2D3 in the induction of HL-60 cell
Vitamin D 77
differentiation and inhibition of clonal proliferation (289,291), and ten to two hundred
fold less active in ICA and BCM (291,292).
Fluorinated analogs of 1?,25(OH)2D3 have been especially useful for studying the
in vivo metabolism of 1?,25(OH)2D3. Fluorine groups have been substituted for the hydroxyls
at C-25, C-1, and C-3 to study the importance of these hydroxylations for the
biological activity of 1?,25(OH)2D3. Also, fluorine groups have been substituted for hydrogens
at C-23, C-24, and C-26 to facilitate the study of 1?,25(OH)2D3 catabolism. The
analog 1?,25(OH)2-26,26,26,27,27,27-hexafluoro-D3 has been shown to be 10 times more
potent than 1?,25(OH)2D3 in calcium mobilization, with longer lasting effects due to its
slower rate of catabolism and metabolic clearance (293). This analog is also 10 times
more potent than 1?,25(OH)2D3 in suppressing proliferation and inducing differentiation
of HL-60 cells (247,294,295).
VII. BIOLOGICAL ASSAYS
With the exception of vitamin B12, vitamin D is the most potent of the vitamins (as defined
by the amount of vitamin required to elicit a biological response). Consequently, biological
samples and animal tissues usually contain very low concentrations of vitamin D. For
example, the circulating plasma level of vitamin D3 in humans is only 10–20 ng/mL, or
2–5  108 M (296). In order to detect such low concentrations of vitamin D, assays
that are specific for and sensitive to vitamin D and its biologically active metabolites are
required.
A. Rat Line Test
From 1922 to 1958, the only official assay for determination of the vitamin D content of
pharmaceutical products or food was the rat line test. The term ‘‘official’’ indicates that
the reproducibility and accuracy of the assay are high enough that the results of the test
can be accepted legally. This assay, which is capable of detecting 1–12 IU (25–300 ng)
of vitamin D, is still widely used today to determine the vitamin D content of many foods,
particularly milk (297–299). The rat line test for vitamin D employs recently weaned
rachitic rats; these rats are fed a rachitogenic diet for 19–25 days until severe rickets
develops. The rats are then divided into groups of 7–10 animals and are fed diets that
have been supplemented either with a graded series of known amounts of vitamin D3 as
standards or with the unknown test sample. (Although vitamin D oils can be directly
assayed, milk, vitamin tablets, and vitamin D–fortified foods must be saponified and the
residue taken up into a suitable oil vehicle prior to assay.) The rats are maintained on
their respective diets for 7 days. The animals are sacrificed and their radii and ulnae dissected
out and stained with a silver nitrate solution. Silver is deposited in areas of bone
where new calcium has been recently deposited. The regions turn dark when exposed to
light. Thus, the effects of the unknown sample on calcium deposition in the bone can be
determined by visual comparison with the standards. Typical results for the rat line test
are shown in Fig. 9.
B. AOAC Chick Assay
Since the rat line test is done in rats, it cannot discriminate between vitamin D2 and vitamin
D3. In the chick, vitamin D3 is 10 times more potent than vitamin D2, so it is important
to accurately determine the amount of vitamin D3 in poultry feeds. The Association of
78 Collins and Norman
Fig. 9 Rat line test chart is shown in panel A. Photographs of radii sections scored according to
the line test chart are shown in panel B.
Official Analytical Chemists (AOAC) chick test was developed to specifically measure
vitamin D3 (300,301).
Groups of 20 newly hatched chicks are placed on vitamin D–deficient diets containing
added levels of vitamin D3 (1–10 IU) or the test substance. After 3 weeks on the
diet, the birds are sacrificed and the percentage of bone ash of their tibia is determined.
A rachitic bird typically has 25–27% bone ash, whereas a vitamin D–supplemented has
40–45% bone ash. This assay is not used frequently because it is time consuming and
expensive.
C. Intestinal Calcium Absorption
Other biological assays have been developed that make use of the ability of vitamin D
to stimulate the absorption of calcium across the small intestine. Two basic types of assays
measure this phenomenon: those that measure the effect of the test substance on intestinal
calcium uptake in vivo (302) and those that employ in vitro methods (39,303). Each is
capable of detecting physiological quantities, i.e., 2–50 IU (50–1250 ng; 0.13–3.2 nmol),
of vitamin D.
1. In Vivo Technique
The in vivo technique for measuring intestinal calcium absorption uses rachitic chicks
that have been raised on a low-calcium (0.6%), rachitogenic diet for 3 weeks. The birds
are then given one dose of the test compound orally, intraperitoneally, or intracardially.
Twelve to 48 h later, the chicks are anesthetized and 4.0 mg of 40Ca2
 and approximately
6  106 dpm 45Ca2
 are placed in the duodenal loop. Thirty minutes later, the chicks are
killed by decapitation and serum is collected. Aliquots of serum are measured for 45Ca2

in a liquid scintillation counter (302).
2. In Vitro Technique
The general design of this technique is the same as that of the in vivo technique because
vitamin D activity is measured in terms of intestinal calcium transport. In these assays,
Vitamin D 79
a vitamin D standard or test compound is given orally or intraperitoneally 24–48 h before
the assay. At the time of the assay, the animals are killed and a 10-cm length of duodenum
is removed and turned inside out. A gut sac is formed by tying off the ends of the segment
so that the mucosal surface is on the outside and the serosal surface on the inside. The
everted intestinal loop is incubated with solutions of 45Ca2
. The mucosal surface of the
intestine actively transports the calcium through the tissue to the serosal side. The ratio
of calcium concentration on the serosal vs. the mucosal side of the intestine is a measure
of the ‘‘active’’ transport of calcium (39,304,305). In a vitamin D–deficient animal this
ratio is 1–2.5; in a vitamin D–dosed animal it can be as high as 6–7. The chick in vivo
assay is usually preferred because of the tedious nature of preparing the everted gut sacs.
The in vitro technique is used primarily for studies with mammals rather than birds.
D. Bone Calcium Mobilization
Another assay for vitamin D activity that often is performed simultaneously with the chick
in vivo intestinal calcium absorption assay is measurement of the vitamin D–mediated
elevation of serum calcium levels. If 3-week-old rachitic chicks are raised on a zerocalcium
diet for at least 3 days before the assay and then are given a compound containing
vitamin D, their serum calcium levels will rise in a highly characteristic manner, proportional
to the amount of steroid given (302). Since there is no dietary calcium available,
the only calcium source for elevation of serum calcium is bone. By carrying out this assay
simultaneously with the intestinal calcium absorption assay, it is possible to measure two
different aspects of the animal’s response to vitamin D at the same time.
E. Growth Rate
The administration of vitamin D to animals leads to an enhanced rate of whole-body
growth. An assay for vitamin D was developed in the chick using the growth-promoting
properties of the steroid (45,306). One-day-old chicks are placed on a rachitogenic diet
and given standard doses of vitamin D3 or the test compound three times weekly. The
birds are weighed periodically, and their weight is plotted vs. age. In the absence of vitamin
D, the rate of growth essentially plateaus by the fourth week, whereas 5–10 IU of vitamin
D3 per day is sufficient to maintain a maximal growth rate in the chick. The disadvantage
of this assay is the 3- to 4-week time period needed to accurately determine the growth
rate.
F. Radioimmunoassay and Enzyme-Linked Immunosorbent Assay
for Calbindin D28K
Additional biological assays utilize the presence of calbindin D28K protein as an indication
of vitamin D activity. Calbindin D28K is not present in the intestine of vitamin D–deficient
chicks and is only synthesized in response to the administration of vitamin D. Therefore,
it is possible to use the presence of calbindin D28K to determine vitamin D activity. A
radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA), both capable
of detecting nanogram quantities of calbindin D28K, have been developed for this
purpose (307).
A comparison of the sensitivity and working range of the biological assays for vitamin
D is given in Table 6.
80 Collins and Norman
Table 6 Comparison of Sensitivity and Working Range of Biological Assays for Vitamin D
Minimal level
detectable in
Time Usual assay
required working
Assay for assay ng nmol range
Rat line test 7 d 12 0.03 25–300 ng
AOAC chick 21 d 50 0.113 50–1250 ng
Intestinal Ca2
 absorption
In vivo
45Ca2
 1 d 125 0.33 0.125–25 g
47Ca2
 1 d 125 0.33 0.125–25 g
In vitro
Everted sacs 1 d 250 0.65 250–1000 ng
Duodenal uptake of 45Ca2
 1 d 250 0.65 250–1000 ng
Bone Ca2
 mobilization
In vivo 24 h 125 0.32 0.125–25 g
Body growth 21–28 d 50 0.06 50–1250 ng
Immunoassays for calcium-binding 1 d 1 0.0025 1 ng
protein
VIII. ANALYTICAL PROCEDURES
Although considerable progress has been made in the development of chemical or physical
means to measure vitamin D, these methods at present generally lack the sensitivity and
selectivity of the biological assays. Thus, they are not adequate for measuring samples
that contain low concentrations of vitamin D. However, these physical and chemical means
of vitamin D determination have the advantage of not being as time consuming as the
biological assays and so are frequently used on samples known to contain high levels of
vitamin D.
A. Ultraviolet Absorption
The first techniques available for quantitation of vitamin D were based on the measurement
of the UV absorption at 264 nm. The conjugated triene system of double bonds in the
vitamin D seco-steroids produces a highly characteristic absorption spectra (Fig. 10). The
absorption maxima for vitamin D occurs at 264 nm, and at this wavelength the molar
extinction coefficient for both vitamins D2 and D3 is 18,300. Thus, the concentration of
an unknown solution of vitamin D can be calculated once its absorption at 264 nm is
known. Although this technique is both quick and easy, it suffers from the disadvantage
that the sample must be scrupulously purified prior to assay in order to remove potential
UV-absorbing contaminants.
B. Colorimetric Methods
Several colorimetric methods for the quantitation of vitamin D have been developed over
the years. Among these various colorimetric assays is a method based on the isomerization
of vitamin D to isotachysterol. This procedure, which employs antimony trichloride, can
detect vitamin D in the range of 1–1000 µg. Because it can detect such large amounts
Vitamin D 81
A
B
Fig. 10 Ultraviolet spectrum of provitamin D and vitamin D. Panel A illustrates the characteristic
UV absorption spectrum of provitamin D. The wavelengths of the several absorption maxima are
262, 271, 282, and 293 nm. The molar extinction coefficient at 282 nm is 11,500. Panel B illustrates
the characteristic UV spectrum of vitamin D. The molar extinction coefficient at the 265- to 265-
nm absorption maxima is 18,300.
of vitamin D, this assay is now used primarily to determine the vitamin D content of
pharmaceutical preparations and has become the official U.S. Pharmacopoeia (USP) colorimetric
assay for vitamin D3.
In this assay, three types of tubes are normally prepared: one containing the standard
vitamin D or unknown sample plus the color reagent; one containing only the solvent,
ethylene chloride; and a third containing ethylene chloride, acetic anyhdride, and the color
reagent. The absorbance at 500 nm is measured 45 s after addition of the color reagent.
82 Collins and Norman
The concentration of vitamin D in the assay tube is proportional to its absorbance, which
is corrected for the solvent blank and for the tube containing the acetic anhydride. This
procedure has been found to follow Beer’s law for solutions containing 3.25–6.5 nmol
vitamin D per milliliter assay solution. The major disadvantage to this assay is that the
copresence of vitamin A, which is often present in pharmaceutical samples along with
vitamin D, interferes with the assay. Thus, purification procedures that include adsorption
chromatography and partition chromatography are required, making this assay procedure
rather laborious and time consuming. Another disadvantage is the necessity for careful
timing of the reaction because of the short time required for the appearance of maximal
intensity of color. However, since this is the only direct chemical method routinely available,
it has widespread industrial application (308).
C. Fluorescence Spectroscopy
There have been two reports in the literature describing assays that depend on the reaction
of vitamin D with a substance capable of fluorescing (309,310). Both of these procedures
are based on the fact that an acetic anhydride–sulfuric acid solution containing vitamin
D is capable of fluorescence if the solution is activated by light of the correct wavelength.
The lower limits of detectability of this assay are the same as those of the antimony
trichloride colorimetric assay, and the same compounds that interfere with the colorimetric
assay also interfere with this fluorescence assay. As a result, this assay is not normally
used for the analytical determination of vitamin D concentration.
D. Gas Chromatography–Mass Spectrometry
One of the most powerful modern techniques available to steroid chemists for the analytical
determination of samples containing mixtures of steroids is mass spectrometry (MS),
or mass spectrometry coupled with prior separation by gas chromatography (GC). The
gas chromatography–mass spectrometry (GC-MS) technique can be coupled to an on-line
computer that collects information on the fragmentation patterns of steroids in the mass
spectrometer. In this way a sophisticated quantitative assay can be developed with a sensitivity
and selectivity approaching that of RIAs. There have recently appeared several GCMS
procedures applicable to vitamin D seco-steroids (311), but they are not yet widely
employed.
E. High-Performance Liquid Chromatography
Several papers that describe the separation of vitamin D and its various metabolites by
HPLC have appeared (312,313). This separation process has an exceedingly high resolving
capability due to the large number of theoretical plates present in a typical column. Of
equal importance to this technique is the sensitivity of the detector used for observing the
separated compounds. All of the published procedures for the separation of vitamin D by
HPLC have used an UV detector, and so their sensitivity is limited to approximately 5
ng. The chief advantage of using HPLC is the reduction in labor and time required to
separate vitamin D and its metabolites. In the current official USP method for the determination
of vitamin D, two prepurification steps, requiring up to 8 h, are necessary before
the colorimetric analysis can be performed (314). However, with HPLC, reproducible
separation of closely related compounds can be achieved in less than 1 h. HPLC has great
Vitamin D 83
potential once a more sensitive detection method is developed. There are now available
several official AOAC procedures for the HPLC determination of vitamin D in a variety
of sample types, including vitamin preparations, multivitamin preparations, vitamin D oil
concentrates, and fortified milk and milk powder (308).
F. Competitive Binding Assays
Various competition assays that can specifically quantitate the levels of 25(OH)D3,
24,25(OH)2D3, or 1?,25(OH)2D3 in a sample are now available. Such assays were developed
as a consequence of the discovery of specific vitamin D–binding proteins in the
serum and tissues of mammals and birds, along with the availability of high specific activity
tritiated 25(OH)D3 and 1?,25(OH)2D3. Since these steroid competition assays are sensitive
(they are capable of detecting picogram quantities), they are now routinely used to
measure vitamin D metabolite levels in plasma.
Two different types of steroid competition assays have been developed for the detection
of 1?,25(OH)2D3. The first employs incubation of intestinal mucosal cytosol plus the
nuclear chromatin fractions with standardized amounts of tritiated 1?,25(OH)2D3. The
1?,25(OH)2D3 in the sample competes with the tritiated hormone for the binding sites of
the 1?,25(OH)2D3 receptors present in the cytosol and chromatin fractions. By measuring
the amount of tritiated 1?,25(OH)2D3 bound to the receptor, the amount of 1?,25(OH)2D3
in the sample can be determined. The first such assay developed that could be used to
measure 1?,25(OH)2D3 levels in plasma was that of Brumbaugh and Haussler (315). Their
technique requires a minimum of 10 mL plasma and involves a laborious three-stage
chromatographic procedure. The final 1?,25(OH)2D3 peak is then assayed. Separation of
bound from free steroid is achieved by filtration of the incubation media through glass
filters. The steroid associated with the chromatin-cytosol receptor is specifically bound to
these filters. A similar assay has been described by Procsal et al., except that separation
of the bound from free steroid is achieved by high-speed differential centrifugation (316).
The second type of competition assay involves the use of calf thymus cytosol as
the source of binding protein. Reinhardt et al. developed a radioreceptor assay for vitamin
D2, vitamin D3, and their metabolites that does not require HPLC (317). Their technique
includes the use of a stable 1?,25(OH)2D3 receptor preparation from calf thymus, nonequilibrium
assay conditions, and solid-phase extraction of vitamin D metabolites from serum
or plasma samples. This procedure requires 0.2–1.0 mL plasma. 1?,25(OH)2D3 is removed
from the plasma on a C18-silica cartridge. The cartridge is first reverse-phase-eluted and
then switched to normal-phase elution (318). The 1?,25(OH)2D3 is recovered and incubated
with [3H]1?,25(OH)2D3 and reconstituted thymus receptor. Separation of receptorbound
hormone from free hormone is achieved by the addition of dextran-coated charcoal.
Similar assays have been developed for 25(OH)D3 and 24,25(OH)2D3 (146,319,320). This
assay has a sensitivity of 0.7 pg.
IX. NUTRITIONAL REQUIREMENTS FOR VITAMIN D
A. Humans
The vitamin D requirement for healthy adults has never been precisely defined. Since
vitamin D3 is produced in the skin upon exposure to sunlight, a human being does not
84 Collins and Norman
have a requirement for vitamin D when sufficient sunlight is available. However, vitamin
D does become an important nutritional factor in the absence of sunlight. In addition to
geographical and seasonal factors, UV light from the sun may be blocked by factors such
as air pollution and sun screens. In fact, as air pollution became prevalent during the
industrial revolution, the incidence of rickets became widespread in industrial cities. It is
now known that the rickets epidemic was partly caused by lack of sunlight due to air
pollution. Thus, rickets has been called the first air pollution disease. Any condition that
blocks sunlight from skin, such as the wearing of clothes, use of sunscreens, living indoors
and in cities with tall buildings, or living in geographical regions of the world that do not
receive adequate sunlight, can contribute to the inability of the skin to biosynthesize suffi-
cient amounts of vitamin D. Under these conditions, vitamin D becomes a true vitamin
in that it must be supplied in the diet on a regular basis.
Since vitamin D can be endogenously produced by the body and since it is retained
for long periods of time by vertebrate tissue, it is difficult to determine minimum daily
requirements for this substance. The requirement for vitamin D is also dependent on the
concentration of calcium and phosphorus in the diet, the physiological stage of development,
age, gender, degree of exposure to the sun, and the amount of pigment in the
skin.
The current allowance of vitamin D recommended in 1989 by the National Research
Council is 300 IU/day [1 IU  0.025 µg vitamin D (321)] for infants from birth to 6
months in age; 400 IU/day for children, adolescents, and pregnant and lactating women;
and 200 IU/day for other adults (322). Because rickets is more prevalent in preschool
children, the Food and Agricultural Organization/World Health Organization (FAO/
WHO) committee recommended that children receive 400 IU/day until the age of 6 years,
after which the recommended daily allowance (RDA) is 100 IU/day.
In the United States, adequate amounts of vitamin D can readily be obtained from
the diet and from casual exposure to sunlight. However, in some parts of the world where
food is not routinely fortified and sunlight is often limited, obtaining adequate amounts
of vitamin D becomes more of a problem. As a result, the incidence of rickets in these
countries is higher than in the United States. Rickets was practically eradicated from the
United States in the mid-1920s, but it has reappeared in the past two decades. The increase
is predominantly due to changes in infant feeding and dietary preferences. Of 27 cases
of infant rickets reported within the first year of life, 26 were found in breast-fed infants
(323). Of 62 cases involving older children, 56 were from families following strict vegetarian
diets that included no meat products, milk products, fish, or eggs.
B. Animals
The task of assessing the minimum daily vitamin D requirement for animals is no easier
than it is for humans. Such factors as the dietary calcium/phosphorus ratio, physiological
stage of development, gender, amount of fur or hair, color, and perhaps even breed, all
affect the daily requirement for vitamin D in animals. Also, some animals, such as chicken
and turkey, do not respond as well to vitamin D2 as to vitamin D3. As with humans,
animals that are maintained in sunlight can produce their own vitamin D, so that dietary
supplementation is not really necessary. For animals that are kept indoors or that live in
climates where the sunlight is not adequate for vitamin D production, the vitamin D content
of food becomes important. Sun-cured hays are fairly good sources of vitamin D, but
Vitamin D 85
Table 7 Vitamin D Requirements of Animals
Animal Daily requirements (IU)
Chickens, growing 90a
Dairy cattle:
Calves 660b
Pregnant, lactating 5000–6000c
Dogs:
Growing puppies 22d
Adult maintenance 11d
Ducks 100d
Monkey, growing
rhesus 25d
Mouse, growing 167d
Sheep:
Lambs 300e
Adults 250e
Swine:
Breed sows 550c
Lactating sows 1210c
Young boars 690c
Adult boars 550c
Turkeys 400a
aIU required per pound of feed.
bIU required for 100 kg body weight.
cIU required per animal.
dIU required per kg body weight.
eIU required per 45 kg body weight.
Source: Published information by the Committee of Animal
Nutrition, Agricultural Board (National Research Council).
dehydrated hays, green feeds, and seeds are poor sources. A brief list of the RDAs for
animals is given in Table 7.
X. FOOD SOURCES OF VITAMIN D
For the most part, vitamin D is present in unfortified foods in only very small and variable
quantities (Table 8). The vitamin D that occurs naturally in unfortified foods is
generally derived from animal products. Saltwater fish, such as herring, salmon, and sardine,
contain substantial amounts of vitamin D, and fish liver oils are extremely rich
sources. However, eggs, veal, beef, unfortified milk, and butter supply only small quantities
of the vitamin. Plants are extremely poor sources of vitamin D; fruits and nuts contain
no vitamin D, and vegetable oils contain only negligible amounts of the provitamin. As
a consequence, in the United Stated dietary requirements for vitamin D are met by the
artificial fortification of suitable foods. Among these fortified foods are milk, both fresh
and evaporated; margarine and butter; cereals; and chocolate mixes. Milk is fortified to
supply 400 IU vitamin D per quart, and margarine usually contains 2000 IU or more per
pound. A more complete listing of the vitamin D values of food is given by Booher et
al. (324).
86 Collins and Norman
Table 8 Vitamin D Content of Unfortified Foods
Vitamin D
Food source (IU/100 g)
Beef steak 13
Beet greens 0.2
Butter 35
Cabbage 0.2
Cheese 12
Cod 85
Cod liver oil 10,000
Corn oil 9
Cream 50
Egg yolk 25
Herring (canned) 330
Herring liver oil 140,000
Liver:
Beef (raw) 8–40
Calf (raw) 0–15
Pork (raw) 40
Chicken (raw) 50–65
Lamb (raw) 20
Mackerel 120
Milk:
Cow (100 mL) 0.3–4
Human (100 mL) 0–10
Salmon (canned) 220–440
Sardines (canned) 1500
Shrimp 1150
Spinach 0.2
Source: Refs. 2 and 324.
XI. SIGNS OF VITAMIN D DEFICIENCY
A. Humans
A deficiency of vitamin D results in inadequate intestinal absorption and renal reabsorption
of calcium and phosphate. As a consequence, serum calcium and phosphate levels fall
and serum alkaline phosphatase activity increases. In response to these low serum calcium
levels, hyperparathyroidism occurs. The result of increased levels of PTH, along with
whatever 1?,25(OH)2D3 is still present at the onset of the deficiency, is the demineralization
of bone. This ultimately leads to rickets in children and osteomalacia in adults. The
classical skeletal symptoms associated with rickets, i.e., bowlegs, knock-knees, curvature
of the spine, and pelvic and thoracic deformities (Fig. 11), result from the application of
normal mechanical stress to demineralized bone. Enlargement of the bones, especially in
the knees, wrists, and ankles, and changes in the costochondral junctions also occur. Since
in children bone growth is still occurring, rickets can result in epiphysial abnormalities
not seen in adult osteomalacia. Rickets also results in inadequate mineralization of tooth
Vitamin D 87
Fig. 11 Classic appearance of rickets in a child.
enamel and dentin. If the disease occurs during the first 6 months of life, convulsions and
tetany can occur. Few adults with osteomalacia develop tetany.
Low serum calcium levels in the range of 5–7 mg per 100 mL and high serum
alkaline phosphatase activity can be used to diagnose rickets and osteomalacia. Also, a
marked reduction in circulating 1?,25(OH)2D3 levels in individuals with osteomalacia or
rickets has been reported (325–327). Radiographic changes are also evident and can be
used in diagnosis.
B. Animals
The response to vitamin D deficiency in animals closely resembles that in humans. Among
the first symptoms of the deficiency is a decline in the plasma concentration of calcium
and phosphorus. This is followed by an abnormally low growth rate and the characteristic
alteration of bones, including faulty calcification of the bone matrix. As the disease progresses,
the forelegs bend sideways and the joints become swollen. In laying birds, the
eggs are thin-shelled, egg production declines, and hatchability is markedly reduced (328);
classic symptoms of rickets develop, followed by tetany and death.
88 Collins and Norman
XII. HYPERVITAMINOSIS D
Excessive amounts of vitamin D are not available from natural sources. However, vitamin
D intoxication is a concern in patients being treated with vitamin D or vitamin D analogs
for hypoparathyroidism, vitamin D–resistant rickets, renal osteodystrophy, osteoporosis,
psoriasis, some cancers, or in those who are taking supplemental vitamins. Hypervitaminosis
D is a serious problem because it can result in irreversible calcification of the heart,
lungs, kidneys, and other soft tissues. Therefore, care should be taken to detect early
signs of vitamin D intoxication in patients receiving pharmacological doses. Symptoms
of intoxication include hypercalcemia, hypercalciuria, anorexia, nausea, vomiting, thirst,
polyuria, muscular weakness, joint pains, diffuse demineralization of bones, and disorientation.
If allowed to go unchecked, death will eventually occur.
Vitamin D intoxication is thought to occur as a result of high 25(OH)D levels rather
than high 1?,25(OH)2D levels (329,330). Patients suffering from hypervitaminosis D have
been shown to exhibit a 15-fold increase in plasma 25(OH)D concentration as compared
to normal individuals. However, their 1?,25(OH)2D levels are not substantially altered
(331). Furthermore, anephric patients can still suffer from hypervitaminosis D even though
they are for the most part incapable of producing 1?,25(OH)2D. It has also been shown
that large concentrations of 25(OH)D can mimic the actions of 1?,25(OH)2D at the level
of the receptor (315,329,332,333).
In the early stages of intoxication, the effects are usually reversible. Treatment consists
of merely withdrawing vitamin D and perhaps reducing dietary calcium intake until
serum calcium levels fall. In more severe cases, treatment with glucocorticoids, which
are thought to antagonize some of the actions of vitamin D, may be required to facilitate
the correction of hypercalcemia. Since calcitonin can bring about a decline in serum calcium
levels, it may also be used in treatment.
XIII. FACTORS THAT INFLUENCE VITAMIN D STATUS
A. Disease
In view of the complexities of the vitamin D endocrine system, it is not surprising that
many disease states are vitamin D–related. Figure 12 classifies some of the human disease
states that are believed to be associated with vitamin D metabolism according to the metabolic
step where the disorder occurs.
1. Intestinal Disorders
The intestine functions as the site of dietary vitamin D absorption and is also a primary
target tissue for the hormonally active 1?,25(OH)2D3. Impairment of intestinal absorption
of vitamin D can occur in those intestinal disorders that result in the malabsorption of
fat. Patients suffering from such disorders as tropical sprue, regional enteritis, and multiple
jejunal diverticulosis often develop osteomalacia because of what appears to be a malabsorption
of vitamin D from the diet (334). Surgical conditions, such as gastric resection
and jejunoileal bypass surgery for obesity, may also impair vitamin D absorption. Also,
patients receiving total parenteral nutrition in the treatment of the malnutrition caused by
profound gastrointestinal disease often develop bone disease (335).
On the other hand, intestinal response to vitamin D can be affected by certain disease
states. Patients suffering from idiopathic hypercalciuria exhibit an increased intestinal
Vitamin D 89
Fig. 12 Human disease states related to vitamin D. PTH, parathyroid hormone; CT, calcitonin;
VDRR, vitamin D–resistant rickets; Pi, inorganic phosphate; Ca2
, calcium.
absorption of calcium that may result from an enhanced intestinal sensitivity to
1?,25(OH)2D3 or from an overproduction of 1?,25(OH)2D3. The disease sarcoidosis also
results in enhanced sensitivity to vitamin D. Sarcoidosis is characterized by hypercalcemia
and hypercalciuria in patients receiving only modest amounts of vitamin D. Experiments
have shown that these patients have elevated levels of serum 1?,25(OH)2D3. The excess
1?,25(OH)2D3 is likely of extrarenal origin and therefore not regulated by circulating
levels of PTH (336). Other experiments have shown that macrophages from patients with
sarcoidosis can produce 1?,25(OH)2D3 (120).
Other disease states that can result in extrarenal production of 1?,25(OH)2D3 are
tuberculosis (337), leprosy (338), and some lymphomas (339).
2. Liver Disorders
The liver plays an important role in the vitamin D endocrine system; not only is it the
primary site for the production of 25(OH)D, but it is also the source of bile salts that aid
in the intestinal absorption of vitamin D. Furthermore, it is likely that the liver is the site
where binding of 25(OH)D by vitamin D-binding protein occurs, and it may even be the
site at which this binding protein is synthesized. Hence, malfunctions of the liver can
possibly interfere with the absorption, transport, and metabolism of vitamin D. Malabsorption
of calcium and the appearance of bone disease have been reported in patients suffering
from either primary biliary cirrhosis or prolonged obstructive jaundice. The disappearance
of radioactive vitamin D from the plasma of these patients is much slower than in normal
humans (340), and their plasma 25(OH)D levels are reduced (341). Although these patients
respond poorly to vitamin D treatment, they immediately respond if treated with
90 Collins and Norman
25(OH)D3. Thus, it appears that the bone disease experienced by these patients results
from their inability to produce 25(OH)D.
3. Renal Disorders
The kidney functions as the endocrine gland for 1?,25(OH)2D3. Thus, disease states that
affect the kidney can concomitantly alter the production of this calcium homeostatic hormone.
It is well known that patients suffering from renal failure also often suffer from
skeletal abnormalities. Termed renal osteodystrophy, these skeletal abnormalities include
growth retardation, osteitis fibrosa, osteomalacia, and osteosclerosis. It became apparent
with the discovery that under normal conditions 1?,25(OH)2D3 is produced in the kidney
that these skeletal abnormalities result from the failure of patients to produce
1?,25(OH)2D3. Support for this theory came from studies on the metabolism of radioactively
labeled vitamin D in normal persons vs. patients with chronic renal failure.
From these studies, anephric or uremic individuals appeared incapable of producing
1?,25(OH)2D3. Direct evidence for this came from the observation that circulating level
of 1?,25(OH)2D3 in the normal subject is in the range of 30–35 pg/mL, whereas in chronic
renal failure the levels have been reported as low as 3–6 pg/mL (342,343). However,
after a successful renal transplant, 1?,25(OH)2D3 levels return to the normal range. Also,
the administration of 1?,25(OH)2D3 to these patients results in the stimulation of intestinal
calcium absorption and an elevation of serum calcium levels (344).
4. Parathyroid Disorders
As previously outlined, PTH influences the production of 1?,25(OH)2D3, so that any disease
state that affects the secretion of PTH may, in turn, have an effect on the metabolism
of vitamin D. Hyperactivity of the parathyroid glands, as in primary hyperparathyroidism,
results in the appearance of bone disease resembling osteomalacia. Circulating
1?,25(OH)2D3 levels in these subjects have been reported to be significantly elevated
(345), as is their intestinal calcium transport (346). On the other hand, in hypoparathyroidism,
hypocalcemia occurs. In these patients, a slight reduction in circulating
1?,25(OH)2D3 levels has been reported (347). When these patients are treated with
1?,25(OH)2D3 their serum levels are increased to normal.
There are several published reviews on the role of vitamin D in disease (18,334,348).
B. Genetics
Vitamin D–resistant rickets (hypophosphatemic rickets) appears to be an X-linked, dominant
genetic disorder. Winters et al. presented evidence that this disease is almost always
inherited and is usually congenital (349). Males are usually more severely affected by this
disease than females. Associated with the disease are skeletal abnormalities, such as rickets
or osteomalacia, and a diminished renal tubular reabsorption of phosphate that results in
hypophosphatemia. Individuals with this disease do not respond to physiological doses
of vitamin D; treatments with 25(OH)D3 and 1?,25(OH)2D3 are also ineffective, although
an increase in intestinal calcium absorption does occur (350). These patients have also
been reported to have normal serum 1?,25(OH)2D3 levels. Thus, it appears that this disorder
does not result from an alteration in the metabolism of vitamin D or from an impaired
intestinal response to 1?,25(OH)2D3, but rather from a specific defect in renal tubular
reabsorption of phosphate.
Vitamin D 91
A genetic defect that interferes with vitamin D metabolism has also been suggested
in vitamin D–dependent rickets type I. This ailment differs from rickets in that it appears
in children who are receiving adequate amounts of vitamin D and requires pharmacological
doses of vitamin D or 25(OH)D3 to reverse the harmful effect of disease on bone. However,
the disease is responsive to physiological amounts of 1?,25(OH)2D3, which suggests that
the defect occurs in the metabolism of 25(OH)D3 to 1?,25(OH)2D3. This disease state
appears to be the result of an autosomal recessively inherited genetic defect (351). It is
not known how this defect affects the metabolism of 25(OH)D3.
Vitamin D–dependent rickets type II also has a genetic basis. This ailment is similar
to vitamin D–dependent rickets type I except that children do not respond to large doses
of vitamin D, 25(OH)D3, or 1?,25(OH)2D3. The combination of symptoms, i.e., defective
bone mineralization, decreased intestinal calcium absorption, hypocalcemia, and increased
serum levels of 1?,25(OH)2D3, suggest end-organ resistance to the action of
1?,25(OH)2D3. Experiments have shown that these children have a single-point mutation
in the nuclear receptor for 1?,25(OH)2D3 (352–355).
C. Drugs
Recent evidence suggests that prolonged use of anticonvulsant drugs, such as diphenylhydantoin
or phenobarbital, can result in an impaired response to vitamin D; this results in
an alteration of calcium metabolism and the appearance of rickets or osteomalacia. Serum
25(OH)D3 levels in patients receiving these drugs have been reported to be markedly
reduced (356). Also, studies in animals suggest that these drugs stimulate the hepatic
microsomal cytochrome P450 enzymes, which could lead to an increased catabolism of
25(OH)D3 (357). However, 1?,25(OH)2D3 levels have been shown to be normal or even
increased after drug treatment (358). It appears that this drug-induced osteomalacia may
not be the result of an effect of the drug on vitamin D metabolism. Studies on rat and
chick duodena in organ culture indicate that anticonvulsant drugs may act on the gastrointestinal
tract and affect the absorption of calcium (359). Anticonvulsant drugs have also
been shown to inhibit calcium reabsorption in organ culture mouse calvaria (360). Further
research is needed to determine the mechanism by which these anticonvulsant drugs affect
calcium metabolism.
D. Alcohol
Persons suffering from chronic alcoholism exhibit a decrease in plasma 25(OH)D3 levels
and in intestinal calcium absorption and bone mineral content. This is observed in patients
with and without cirrhosis of the liver. Current evidence indicates that the impairment of
intestinal calcium absorption is the result of low 25(OH)D3 levels (361,362). However,
how chronic alcoholism results in low 25(OH)D3 levels is at present not understood.
E. Age
The fact that changes in the metabolism of vitamin D may occur with aging has been
suggested by the observation that the ability to absorb dietary calcium decreases with age
(363). In addition, loss of bone increases in the elderly and an age-related hypoplasia of
bone cells occurs. 1?,25(OH)2D3 levels in the plasma decrease with age, possibly due to
an age-related reduction of epidermal concentrations of 7-dehydrocholesterol, resulting in
less photochemical production of 1?,25(OH)2D3 by the skin. Also, the 1?-hydroxylase
enzyme is less responsive to induction by PTH due to the decrease of glomerular filtration
with age (364).

Gray et al. demonstrated that men and women differ in their metabolism of vitamin D in
response to various physiological stimuli (365). In women, they observed that dietary
phosphate deprivation resulted in a decrease in serum phosphorus levels with a concomitant
increase in plasma 1?,25(OH)2D3 concentrations. However, no change of either of
these parameters was noted in men. Thus, the mechanism by which men and women
respond to dietary phosphate deprivation seems to differ.
XIV. EFFICACY OF PHARMACOLOGICAL DOSES
Several ailments are known to respond to massive doses of vitamin D. For example, the
intestinal malabsorption of calcium that results from chronic renal failure and the subsequent
development of rickets or osteomalacia can be overcome by administration of
100,000–300,000 IU vitamin D per day (364). Patients suffering from hypoparathyroidism
can usually be treated by giving 80,000–100,000 IU vitamin D per day (366). Also, children
afflicted with vitamin D–dependent rickets type I can be treated with 10,000–100,000
IU per day (2). The therapeutic effect of such massive doses can be explained by the fact
that 25(OH)D in sufficiently high concentrations will mimic the action of 1?,25(OH)2D3
at the receptor. However, as mentioned earlier, the administration of such pharmacological
doses of vitamin D to patients over a prolonged period of time carries with it the danger
of vitamin D toxicity.
Table 9 shows the drug forms of vitamin D metabolites that are currently available
for the treatment of several disease states. Experiments are in progress to develop additional
vitamin D analogs that will be useful pharmacological agents without the hypercalcemic
side effect of 1?,25(OH)2D3.
XV. RECENT DEVELOPMENTS
Recently, the x-ray crystal structure of the ligand-binding domain (LBD) of VDR (367)
and several other nuclear receptors have been determined. These LBDs are composed
primarily of 11–13 ?-helices that are folded to form three layers. The interior of the LBD
is composed primarily of hydrophobic residues designed to bind lipophilic ligands like
1,25(OH)D3. Ligand binding to its cognate receptor induces conformational changes in
the receptor, which increases its ability to activate gene transcription. The major structural
difference between unoccupied and ligand-bound nuclear receptors is the repositioning of
the C-terminal helix 12 containing the ligand-dependent transactivation domain, AF-2.
Upon ligand binding, helix 12 moves from projecting away from the LBD to a position
tightly packed against helix 3 of the LBD (368,369). Repositioning of the AF-2 domain
results in the formation of new surfaces, including a hydrophobic cleft (370), needed to
interact with other nuclear factors called coactivators. Coactivators are adapter proteins
that link transcriptional activators such as nuclear receptors to basal transcription machinery
resulting in increased gene transcription (371).
Three families of coactivator proteins are known to interact with nuclear receptors:
SRC-1 (372)/NcoA-1 (373), TIF2 (374)/GRIP-1 (375), and ACTR/pCIP (376). These
94 Collins and Norman
proteins contain a conserved leucine-containing motif (LXXLL), important for interactions
between coactivators and nuclear receptors (377). The coactivator complexes have activities
associated with them such as histone acetyltransferase activity (378) or methyl transferase
activity (379), which aid in the decondensation of chromatin to facilitate binding
of RNA polymerase II transcription complex (RNA Pol II) to DNA. Some of the proteins
in the coactivator complex can also interact with other proteins that are part of the RNA
Pol II core complex. In this way, the coactivators can link upstream activators such as
nuclear receptors to RNA Pol II and modulate gene transcription (380). Identifying these
intermediary factors involved in transcriptional control of specific target genes is essential
for understanding the biological actions of vitamin D and will continue to be an exciting
area of research for several years.
XVI. CONCLUSION
Vitamin D, through its hormonally active metabolite 1?,25(OH)2D3, is known to act on
bone and intestine to maintain calcium homeostasis. However, the actions of
1?,25(OH)2D3 are much broader than was originally thought. There is evidence that
1?,25(OH)2D3 is involved in the physiology of tissues not related to calcium homeostasis,
such as skin, pancreas, pituitary, muscle, and hematopoietic cells. Figure 4 demonstrates
the complexity of the vitamin D endocrine system as it is understood today. Although
many advances have been made in the past decade, there is still much to learn about the
detailed cellular and molecular mode of action of vitamin D and its metabolites.
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3
Vitamin K
JOHN W. SUTTIE
University of Wisconsin, Madison, Wisconsin
I. HISTORY
The discovery of vitamin K was the result of a series of experiments by Henrik Dam on
the possible essential role of cholesterol in the diet of the chick. Dam (1) noted that chicks
ingesting diets that had been extracted with nonpolar solvents to remove the sterols developed
subdural or muscular hemorrhages and that blood taken from these animals clotted
slowly. The disease was subsequently observed by McFarlene et al. (2), who described
a clotting defect seen when chicks were fed ether-extracted fish or meat meal, and also
by Holst and Halbrook (3). Studies in a number of laboratories soon demonstrated that
this disease could not be cured by the administration of any of the known vitamins or
other known physiologically active lipids. Dam continued to study the distribution and
lipid solubility of the active component in vegetable and animal sources and in 1935
proposed (4,5) that the antihemorrhagic vitamin of the chick was a new fat-soluble vitamin,
which he called vitamin K. Not only was K the first letter of the alphabet that was not
used to described an existing or postulated vitamin activity at that time, but it was also
the first letter of the German word Koagulation. Dam’s reported discovery of a new vitamin
was followed by an independent report of Almquist and Stokstad (6,7), describing
their success in curing the hemorrhagic disease with ether extracts of alfalfa and clearly
pointing out that microbial action in fish meal and bran preparations could lead to the
development of antihemorrhagic activity.
The only plasma proteins involved in blood coagulation that were clearly defined
at that time were prothrombin and fibrinogen, and Dam et al. (8) succeeded in preparing
a crude plasma prothrombin fraction and demonstrating that its activity was decreased
when it was obtained from vitamin K–deficient chick plasma. At about the same period
of time, the hemorrhagic condition resulting from obstructive jaundice or biliary problems
was shown to be due to poor utilization of vitamin K by these patients, and the bleeding
115
116 Suttie
episodes were attributed to a lack of plasma prothrombin. The prothrombin assays used
at that time were not specific for prothrombin, and it was widely believed that the defect
in the plasma of animals fed vitamin K–deficient diets was due solely to a lack of prothrombin.
A real understanding of the various factors involved in regulating the generation
of thrombin from prothrombin did not begin until the mid-1950s, and during the next 10
years, factors VII, IX, and X were discovered and shown to be dependent on vitamin K
for synthesis.
A number of groups were involved in the attempts to isolate and characterize this
new vitamin, and Dam’s collaboration with Karrer of the University of Zurich resulted
in the isolation of the vitamin from alfalfa as a yellow oil. Subsequent studies soon established
that the active principle was a quinone and vitamin K1 was characterized as 2-
methyl-3-phytyl-1,4-naphthoquinone (9) and synthesized by Doisy’s group in St. Louis.
Their identification was confirmed by independent synthesis of this compound by Karrer
et al. (10), Almquist and Klose (11), and Fieser (12). The Doisy group also isolated a
form of the vitamin from putrified fish meal, which in contrast to the oil isolated from
alfalfa was a crystalline product. Subsequent studies demonstrated that this compound
called vitamin K2, contained an unsaturated side chain at the 3-position of the naphthoquinone
ring. Early investigators recognized that sources of the vitamin, such as putrified
fish meal, contained a number of different vitamins of the K2 series with differing chainlength
polyprenyl groups at the 3-position. Early observations suggested that the alkylated
forms of vitamin K could be formed in animal tissues from the parent compound, menadione.
This was not definitely established until Martius and Esser (13) demonstrated that
they could isolate a radioactive polyprenylated form of the vitamin from tissues of rats
fed radioactive menadione. Much of the early history of the discovery of vitamin K has
been reviewed by Almquist (14), and he and others (15–17) have reviewed the literature
in this field shortly after discovery of the vitamin.
II. CHEMISTRY
A. Isolation
Vitamin K can be isolated from biological material by standard methods used to obtain
physiologically active lipids. The isolation is always complicated by the small amount of
desired product in the initial extracts. Initial extractions are usually made with the use of
some type of dehydrating conditions, such as chloroform-methanol, or by first grinding
the wet tissue with anhydrous sodium sulfate and then extracting it with acetone followed
by hexane or ether. Large samples (kilogram quantities) of tissues can be extracted with
acetone alone, and this extract is partitioned between water and hexane to obtain the crude
vitamin. Small samples, such as in vitro incubation mixtures or buffered subcellular fractions,
can be effectively extracted by shaking the aqueous suspension with a mixture of
isopropanol and hexane. The phases can be separated by centrifugation and the upper
layer analyzed directly. Methods for the efficient extraction of vitamin K from various
food matrices have been developed (18) and will be useful in developing accurate food
composition data.
Crude nonpolar solvent extracts of tissues contain large amounts of contaminating
lipid in addition to the desired vitamin. Further purification and identification of vitamin
K in this extract can be facilitated by a preliminary fractionation of the crude lipid extract
on hydrated silicic acid (19). A number of the forms of the vitamin can be separated from
each other and from other lipids by reversed-phase partition chromatography, as described
Vitamin K 117
by Matschiner and Taggart (20). These general procedures appear to extract the majority
of vitamin K from tissues. Following separation of the total vitamin K fraction from much
of the contaminated lipid, the various forms of the vitamin can be separated by the procedures
described in Sec. III.
B. Structure and Nomenclature
The nomenclature of compounds possessing vitamin K activity has been modified a number
of times since the discovery of the vitamin. The nomenclature in general use at the
present time is that most recently adopted by the IUPAC-IUB Subcommittee on Nomenclature
of Quinones (21). The term vitamin K is used as a generic descriptor of 2-
methyl-1,4-naphthoquinone (I) and all derivatives of this compound that exhibit an
antihemorrhagic activity in animals fed a vitamin K–deficient diet. The compound 2-
methyl-3-phytyl-1,4-naphthoquinone (II) is generally called vitamin K1, but is preferably
called phylloquinone. The USP nomenclature for phylloquinone is phytonadione. The
compound, first isolated from putrified fish meal and called at that time vitamin K2, is
one of a series of vitamin K compounds with unsaturated side chains, called multiprenylmenaquinones,
that are found in animal tissues and bacteria. This particular menaquinone
(2-methyl-3-farnesylgeranylgeranyl-1,4-naphthoquinone) had 7 isoprenoid units, or 35
carbons in the side chain; it was once called vitamin K2 (35) but now is called menaquinone-
7 (MK-7) (III). Vitamins of the menaquinone series with up to 13 prenyl groups
have been identified, as well as several partially saturated members of this series. The
parent compound of the vitamin K series, 2-methyl-1,4-naphthoquinone, has often been
called vitamin K3 but is more commonly and correctly designated as menadione.
C. Structures of Important Analogs, Commercial Forms, and
Antagonists
1. Analogs and Their Biological Activity
Following the discovery of vitamin K, a number of related compounds were synthesized
in various laboratories and their biological activity compared with that of the isolated
forms. A large number of compounds were synthesized by the Fieser group (22), and data
on the biological activity of these and other compounds have been reviewed and summarized
elsewhere (23,24). The data from some of the early studies are somewhat difficult
to compare because of variations in methods of assay, but a number of generalities were
apparent rather early. Although there were early suggestions that menadione (I) might be
118 Suttie
functioning as a vitamin, it is now usually assumed that the compound is alkylated to a
biologically active menaquinone either by intestinal microorganisms or by tissue alkylating
enzymes. The range of compounds that can be utilized by animals (or intestinal bacteria)
is wide, and compounds such as 2-methyl-4-amino-1-naphthol or 2-methyl-1-naphthol
have biological activity similar to that of menadione when fed to animals. Compounds
such as the diphosphate, the disulfate, the diacetate, and the dibenzoate of reduced vitamin
K series have been prepared and have been shown to have full biological activity. Most
early studies compared the activities of various compounds with that of menadione. The
2-methyl group is usually considered essential for activity, and alterations at this position,
such as the 2-ethyl derivative (IV), result in inactive compounds. This is not due to the
inability of the 2-ethyl derivative to be alkylated, as 2-ethyl-3-phytyl-1,4-naphthoquionone
is also inactive. There is some evidence from feeding experiments that the methyl group
may not be absolutely essential. 2-Phytyl-1,4-naphthoquionone (V) has biological activity,
but the available data do not establish that V functions as a biologically active desmethylphylloquinone
rather than being methylated to form phylloquinone.
Studies with substituted 2-methyl-1,4-naphthoquinones have revealed that polyisoprenoid
side chains are the most effective substituents at the 3-position. The biological
activity of phylloquinone, 2-methyl-3-phytyl-1,4-naphthoquinone (II), is reduced by saturation
of the double bond to form 2-methyl-3-(?,?-dihydrophytyl)-1,4-naphthoquinone
(VI). This compound is, however, considerably more active than 2-methyl-3-octadecyl-
1,4-naphthoquinone (VII), which has an unbranched alkyl side chain of similar size. Natural
phylloquinone is the trans isomer, and although there has been some confusion in the
past, Matschiner and Bell (25) have shown that the cis isomer of phylloquinone (VIII) is
essentially inactive. The naphthoquinone nucleus cannot be altered appreciably, as methylation
to form 2,6-dimethyl-3-phytyl-1,4-naphthoquinone (IX) results in loss of activity,
and the benzoquinone most closely corresponding to phylloquinone, 2,3,5-trimethyl-6-
phytyl-1,4-benzoquinone, has been reported (22) to have no activity.
Vitamin K 119
The activity of various structural analogs of vitamin K in whole-animal assay systems
is, of course, a summation of the relative absorption, transport, metabolism, and
effectiveness of this compound at the active site as compared with that of the reference
compound. Much of the data on biological activity of various compounds were obtained
by the use of an 18-h oral dose curative test utilizing vitamin K–deficient chickens. This
type of assay allows metabolic alterations of the administered form to an active form of
the vitamin, and significant activity of a compound in this assay may result from bioconversion
to an active form. Activity varies with length of the isoprenoid side chain, and
isoprenalogs with three to five isoprenoid groups have maximum activity (24) when administered
orally. The lack of effectiveness of higher isoprenalogs in this type of assay
may be due to the relatively poor absorptions of these compounds. Matschiner and Taggart
(26) have shown that when the intracardial injection of vitamin K in deficient rats is used
as a criterion, the very high molecular weight isoprenalogs of the menaquinone series are
the most active; maximum activity was observed with MK-9 (Table 1). Structure–function
relationships of vitamin K analogs have also been studied utilizing in vitro assays of the
vitamin K–dependent ?-glutamylcarboxylase, and these will be discussed in Sec. VII.
2. Commercial Form of Vitamin K
Only a few forms of the vitamin are commercially important. The major use of vitamin
K in the animal industry is in poultry diets. Chicks are very sensitive to vitamin K restriction,
and antibiotics that decrease intestinal vitamin synthesis are often added to poultry
diets. Supplementation is therefore required to ensure an adequate supply. Phylloquinone
is too expensive for this purpose, and different forms of menadione have been used. Menadione
itself possesses high biological activity in a deficient chick, but its effectiveness
depends on the presence of lipids in the diet to promote absorption. There are also problems
of its stability in feed products, and because of this, water-soluble forms are used.
Menadione forms a water-soluble sodium bisulfite addition product, menadione sodium
bisulfite (MSB) (X), which has been used commercially but which is also somewhat unstable
in mixed feeds. In the presence of excess sodium bisulfite, MSB crystallizes as a
complex with an additional mole of sodium bisulfite; this complex, known as menadione
Table 1 Effect of Route of Administration on Biological Activitya
Relative biological activity
Phylloquinone
series Menaquinone series
Number of C atoms
in side chain Oral (chick) Oral (chick) Intracardial (rat)
10 10 15 
2
15 30 40 —
20 100 100 13
25 80 120 15
30 50 100 170
35 — 70 1700
40 — 68 —
45 — 60 2500
50 — 25 1700
a Data are expressed on a molar basis with phylloquinone assigned a value of 100.
Source: From Refs. 24 and 26.
120 Suttie
sodium bisulfite complex (MSBC) (XI), has increased stability and it is widely used in
the poultry industry. A third water-soluble compound is a salt formed by the addition
of dimethylpyridionol to MSB; it is called menadione pyridionol bisulfite (MPB) (XII).
Comparisons of the relative biopotency of these compounds have often been made on the
basis of the weight of the salts rather than on the basis of menadione content, and this
has caused some confusion in assessing their value in animal feeds. However, a number
of studies (27–29) have indicated that MPB is somewhat more effective in chick rations
than in MSBC. This form of the vitamin has also been demonstrated to be effective in
swine rations (30).
The clinical use of vitamin K is largely limited to two forms. A water-soluble form
of menadione, menadiol sodium diphosphate, which is sold as Kappadione or Synkayvite,
is still used in some circumstances, but the danger of hyperbilirubinemia associated with
menadione usage (see Sec. XI) had led to the use of phylloquinone as the desired form of
the vitamin. Phylloquinone (USP phytonadione) is sold as AquaMEPHYTON, Konakion,
Mephyton, and Mono-Kay.
3. Antagonists of Vitamin Action
The history of the discovery of the first antagonists of vitamin K, the coumarin derivatives,
has been documented and discussed by Link (31). A hemorrhagic disease of cattle, traced
to the consumption of improperly cured sweet clover hay, was described in Canada and
the U.S. Midwest in the 1920s. If serious hemorrhages did not develop, animals could be
aided by transfusion with whole blood from healthy animals, and by the early 1930s it
was established that the cause of the prolonged clotting times was a decrease in the concentration
of prothrombin in the blood. The compound present in spoiled sweet clover that
was responsible for this disease had been studied by a number of investigators but was
finally isolated and characterized as 3,3?-methylbis-(4-hydroxycoumarin) by Link’s group
(32,33) during the period from 1933 to 1941 and was called dicumarol (XIII). Dicumarol
was successfully used as a clinical agent for anticoagulant therapy in some early studies,
and a large number of substituted 4-hydroxycoumarins were synthesized both in Link’s
laboratory and elsewhere. The most successful of these, both clinically for long-term lowering
of the vitamin K–dependent clotting factors and subsequently as a rodenticide, have
been warfarin, 3-(?-acetonylbenzyl)-4-hydroxycoumarin (XIV), or its sodium salt; phenprocoumon,
3-(1-phenylpropyl)-4-hydroxycoumarin (XV); and ethyl biscovmacetate,
3,3?-carboxymethylenebis-(4-hydroxycoumarin) ethyl ester (XVI). The various drugs that
Vitamin K 121
have been used differ in the degree to which they are absorbed from the intestine, in their
plasma half-life, and presumably in their effectiveness as a vitamin K antagonist in the
active site. Because of this, their clinical use differs. Much of the information on the
structure–activity relationships of the 4-hydroxycoumarins has been reviewed by Renk
and Stoll (34). These drugs are synthetic compounds, and although the clinically used
compound is the racemic mixture, studies of the two optical isomers of warfarin have
shown that they differ both in their effectiveness as anticoagulants and in the influence
of other drugs on their metabolism. The clinical use of these compounds and many of
their pharmacodynamic interactions have been reviewed by O’Reilly (35).
Warfarin is widely used as a rodenticide, and concern has been expressed in recent
years because of the identification of anticoagulant-resistant rat populations. These were
first observed in northern Europe (39) and subsequently in the United States (37). Resistance
is now a significant problem in both North America (38) and Europe (39), and
concern over the spread of resistance has led to the synthesis of more effective coumarin
derivatives. Two of the most promising appear to be 3-(3-p-diphenyl-1,2,3,4-tetrahydronaphth-
1-yl)-4-hydroxycoumarin, difenacoum (XVII) and 3-(3-[4?-bromobiphenyl-4-
yl]-1,2,3,4-tetrahydronaphth-1-yl)-4 hydroxycoumarin, bromodifenacoum (XVIII). The
genetics of resistance and the differences between various resistant strains are now better
understood (40), and it appears that the problems can be brought under control.
A second class of chemical compounds having anticoagulant activity that can be
reversed by vitamin K administration (41) are the 2-substituted 1,3-indandiones. Many of
122 Suttie
these compounds also have been synthesized, and two of the more commonly used members
of the series have been 2-phenyl-1,3-indandione (XIX) and 2-pivalyl-1, 3-indandione
(XX). These compounds have had some commercial use as rodenticides, but because of
the potential for hepatic toxicity (35), they are no longer used clinically. Studies on the
mechanism of action of these compounds have not been as extensive as those on the 4-
hydroxycoumarins, but the observations that warfarin-resistant rats are also resistant to
the indandiones and their effects on vitamin K metabolism (42) suggest that the mechanism
of action of the indandiones is similar to that of the 4-hydroxycoumarins.
During the course of a series of investigations into the structural requirements for
vitamin K activity, it was shown (43) that replacement of the 2-methyl group of phylloquinones
by a chlorine atom to form 2-chloro-3-phytyl-1,4-naphthoquinone (XXI) or by a
bromine atom to form 2-bromo-3-phytyl-1,4-naphthoquinone resulted in compounds that
were potent antagonists of vitamin K. The most active of these two compounds is the
chloro derivative (commonly called chloro-K. Lowenthal (44) has shown that, in contrast
to the coumarin and indandione derivatives, chloro-K acts like a true competitive inhibitor
of the vitamin at its active site(s). Because its mechanism of action is distinctly different
from that of the commonly used anticoagulants, chloro-K has been used to probe the
mechanism of action of vitamin K, and, as it is an effective anticoagulant in coumarin
anticoagulant–resistant rats (45), it has been suggested as a possible rodenticide. Lowenthal’s
studies of possible agonists and antagonists of vitamin K indicated (46) that, in
contrast to earlier reports (22), some of the para-benzoquinones do have biological activity.
The benzoquinone analog of vitamin K1, 2,5,6-trimethyl-3-phytyl-1,4-benzoquinone,
was found to have weak vitamin K activity, and 2-chloro-5,6-dimethyl-3-phytyl-1,4-
benzoquinone (XXII) is an antagonist of the vitamin. When these compounds were modi-
fied to contain shorter isoprenoid side chains at the 3-position, they were neither agonists
nor antagonists.
Two compounds that appear to be rather unrelated to either vitamin K or the coumarins
and that have anticoagulant activity have recently been described. Marshall (47) has
shown that 2,3,5,6-tetrachloro-4-pyridinol (XXIII) has anticoagulant activity, and on the
basis of its action in warfarin-resistant rats (42), it would appear that it is functioning as
a direct antagonist of the vitamin, as does chloro-K. A second series of compounds even
less structurally related to the vitamin are the 6-substituted imidazole-[4-5-b]-pyrimidines.
These compounds were described by Bang et al. (48) as antagonists of the vitamin, and
Vitamin K 123
the action of 6-chloro-2-trifluoromethylimidazo-[4,5-b]-pyrimidine (XXIV) in warfarinresistant
rats (48,49) suggests that they function in the same way as a coumarin or indandione
type of compound. As is the case with potentially active forms of the vitamin,
studies of vitamin K antagonists have more recently been studied utilizing in vitro assays
and will be discussed in Sec. VII.
A hypoprothrombinemia can also be produced in some species by feeding animals
sulfa drugs and antibiotics. There is little evidence that these compounds are doing anything
other than decreasing intestinal synthesis of the vitamin by altering the intestinal
flora. They should, therefore, not be considered antagonists of the vitamin.
D. Synthesis of Vitamin K
The methods used in the synthesis of vitamin K have remained essentially those originally
described by Doisy’s group (50), Almquist and Klose (11,51), and Fieser (12,52) in 1939.
Those procedures involved the condensation of phytol or its bromide with menadiol or
its salt to form the reduced addition compound, which is then oxidized to the quinone.
Purification of the desired product from unreacted reagents and side products occurred
either at the quinol stage or after oxidation. These reactions have been reviewed in considerable
detail, as have methods to produce the specific menaquinones rather than phylloquionone
(53,54). The major side reactions in this general scheme is the formation of the cis
rather than the trans isomer at the ?2 position and alkylation at the 2- rather than the 3-
position to form the 2-methyl-2-phytyl derivative. The use of monoesters of menadiol and
newer acid catalysts for the condensation step (55) is the basis for the general method of
industrial preparation used at the present time. Naruta and Maruyama (56) have described
a new method for the synthesis of compounds of the vitamin K series based on the coupling
of polyprenyltrimethyltins to menadione. This method is a regio- and stereocontrolled
synthesis that gives a high yield of the desired product. It is likely that this method may
have particular utility in the synthesis of radiolabeled vitamin K for metabolic studies, as
the purification of the desired product appears to be somewhat simpler than with the synthesis
currently in use.
E. Physical and Chemical Properties
Compounds with vitamin K activity are substituted 1,4-naphthoquinones and, therefore,
have the general chemical properties expected of all quinones. The chemistry of quinoids
Fig. 1 Physical properties of phylloquinone. (A) Ultraviolet absorption spectra in petroleum ether.
(B) Infrared absorption spectra. (C) Nuclear magnetic resonance spectra in CDCl3 at 60 Mc. (D)
Mass fragmentation spectrum of the parent molecular ion is seen at m/e 450.
Vitamin K 125
has been reviewed in a book edited by Patai (57), and much of the data on the spectral
and other physical characteristics of phylloquinone and the menaquinones have been summarized
by Sommer and Kofler (58) and Dunphy and Brodie (59). The oxidized form of
the K vitamins exhibits an ultraviolet (UV) spectrum that is characteristic of the naphthoquinone
nucleus, with four distinct peaks between 240 and 280 nm and a less sharp absorption
at around 320–330 nm. The extinction coefficient (E1%
1cm) decreases with chain length
and has been used as a means of determining the length of the side chain. The molar
extinction value  for both phylloquinone and the various menaquinones is about 19,000.
The absorption spectrum changes drastically upon reduction to the hydroquinone, with
an enhancement of the 245-nm peak and disappearance of the 270-nm peak. These compounds
also exhibit characteristic infrared and nuclear magnetic resonance (NMR) absorption
spectra that are again largely those of the naphthoquinone ring. NMR analysis of
phylloquinone has been used to establish firmly that natural phylloquinone is the trans
isomer and can be used to establish the cis–trans ratio in synthetic mixtures of the vitamin.
Mass spectroscopy has been useful in determining the length of the side chain and the
degree of saturation of vitamins of the menaquinone series isolated from natural sources.
The UV, infrared, NMR and mass fragmentation spectra of phylloquinone are shown in
Fig. 1. Phylloquinone is an oil at room temperature; the various menaquinones can easily
be crystallized from organic solvents and have melting points from 35 to 60°C, depending
on the length of the isoprenoid chain.
III. ANALYTICAL PROCEDURES
Vitamin K can be analyzed by a variety of color reactions or by direct spectroscopy (58–
60). Chemical reactivity of the vitamin is a function of the naphthoquinone nucleus, and
as other quinones also react with many of the colorimetric assays, they lack specificity
for the vitamin. The number of interfering substances present in crude extracts is such
that a significant amount of separation is required before UV absorption spectra can be
used to quantitate the vitamin in anything other than reagent grade preparations. These
methods are therefore of little value in the determination of the small amount of vitamin
present in natural sources. Early quantitation determinations depended on a biological
assay, and analytical methods suitable for the small amounts of vitamin K present in tissues
and most food sources have been available only recently. The separation of the extensive
mixtures of menaquinones in bacteria and animal sources was first achieved with various
thin-layer or paper chromatographic systems (53,58,59,61). All separations involving concentrated
extracts of vitamin K should be carried out in subdued light to minimize UV
decomposition of the vitamin. Compounds with vitamin K activity are also sensitive to
alkali, but they are relatively stable to an oxidizing atmosphere and to heat and can be
vacuum-distilled with little decomposition.
Interest in the quantitation of vitamin K in serum and animal tissues has led to an
increasing emphasis on the use of high-performance liquid chromatography (HPLC) as
an analytical tool to investigate vitamin K metabolism. This method was first demonstrated
to be applicable to the separation of vitamin K by Williams et al. (62). The general development
of HPLC techniques for the quantitation of phylloquinone and menaquinones in
biological materials has been reviewed (63), and specific methods will be discussed in
Secs. V and VI.
126 Suttie
IV. BIOASSAY PROCEDURES
The classical assay for the vitamin K content of an unknown source was based on determination
of the whole-blood clotting time of the chick (64) and at the present time is largely
of historical interest. A small amount of material to be assayed was placed in the crop of
vitamin K–deficient chicks, and the response at 20 h was compared with that of known
amounts of vitamin K. When larger amounts of material were available, they were fed
for 2 weeks and the degree of vitamin sufficiency compared with known amounts of
the vitamin. Utilization of more sensitive plasma clotting factor assays (65,66) improves
sensitivity, as does better standardization of the degree of deficiency (67,68). Some investigators
have modified the degree of hypoprothrombinemia of the test chicks by anticoagulant
administration (29,65,66), but this modification makes the assay somewhat insensitive
to menadione and other unalkylated forms of the vitamin.
Because of the ease in producing a vitamin K deficiency in this species, chicks have
most often been used in biological assays; however, rats have also been used (26). All
oral bioassay procedures are complicated by the effects of different rates and extents of
absorption of the desired nutrients from the various products being assayed. They have
been superseded by HPLC techniques and will be used in the future only to compare the
biological activity of different forms of the vitamin or to investigate nutrient interactions
that modify biological activity.
V. CONTENT IN FOOD
Satisfactory tables of the vitamin K content of various commonly consumed foods have
not been made available until recently. Many of the values commonly quoted have apparently
been recalculated in an unspecified way from data obtained by a chick bioassay
(69,70) that was not intended to be more than qualitative and should not in any sense be
used to give absolute values. Tables in various texts and reviews (71–73) may also contain
data from this source, as well as considerable amounts of unpublished data. A major
problem has been the lack of suitable bioassays or chemical assays for the vitamin. The
range of values reported for the same foods has therefore been wide and is illustrated in
Table 2. The significant variation observed suggests that reporting a single value for the
vitamin K content of different foods gives an erroneous impression of the confidence that
should be given such a value.
Current methodology utilizes HPLC analysis of lipid extracts, and has been reported
(18) to have a within-sample coefficient of variation for different foods in the range of
7–14% and a between-sample coefficient of variation of 9–45%. Although green leafy
vegetables have been known for some time to be the major source of vitamin K in the
diet, it is now apparent that cooking oils, particularly soybean oil and rapeseed oil (79),
are major contributors. Interest in hemorrhagic diseases of the newborn has led to a determination
of phylloquinone in cow (80) and human milk (80–82) and in infant formulas
(80). Human milk contains only 1–2 ng of phylloquinone per milliliter, which is somewhat
less than that found in cow’s milk. Infant formulas are currently supplemented with vitamin
K, providing a much higher intake than that provided by breast milk.
The data in Table 3 are taken from a survey of literature (83), which considered
most of the reported HPLC derived values for various food items and from analyses of
the FDA total diet study (83a). In general, green and/or leafy vegetables are the best
sources of the vitamin, with cooking oils being the next major sources. Due to the low
Vitamin K 127
Table 2 Determination of Vitamin K by Bioassay and Chemical Methods
Vitamin K (µg per 100 g fresh)
Bioassay Chemical assay
Food Ia IIb IIIc IVd Ve
Beans, green 14 22 46 — —
Broccoli 20 65 147 230 —
Cabbage 95 37 110 110 61
Cauliflower 136 
10 27 — —
Carrots 20 — 5 — —
Peas 19 — 39 50 —
Potatoes, white 4 — 
1 — —
Spinach 177 130 415 260 231
Tomato, ripe 11 — 6 — —
a Doisy (75), chick bioassay of fresh food with phylloquinone as a standard.
b Richardson et al. (74), chick bioassay of frozen food with menadione as a standard.
c Shearer et al. (76), HPLC assay for phylloquinone.
d Kodaka et al. (77), HPLC assay for phylloquinone.
e Seifert (78), gas chromatography assay of phylloquinone; data have been recalculated on a dry weight basis.
Table 3 Vitamin K Content of Ordinary Foodsa
µg Phylloquinone/100 g of edible portion
Vegetables Nuts, Oils, Seeds Fruits
Kale 817 Soybean oil 193 Avocado 40
Parsley 540 Rapeseed oil 141 Grapes 3
Spinach 400 Olive oil 49 Cantaloupe 1
Endive 231 Walnut oil 15 Bananas 0.5
Green onions 207 Safflower oil 11 Apples 0.1
Broccoli 205 Sunflower oil 9 Oranges 0.1
Brussels sprouts 177 Corn oil 3
Cabbage 147 Dry soybeans 47 Meat & Dairy
Lettuce 122 Dry kidney beans 19 Ground beef 0.5
Green beans 47 Sesame seeds 8 Chicken 0.1
Peas 36 Dry navy beans 2 Pork 
0.1
Cucumbers 19 Raw peanuts 0.2 Turkey 
0.1
Tomatoes 6 Tuna 
0.1
Carrots 5 Grains Butter 7
Cauliflower 5 Bread 3 Cheddar cheese 3
Beets 3 Oat meal 3 3.5% Milk 0.3
Onions 2 White rice 1 Yogurt 0.3
Potatoes 0.8 Wheat flour 0.6 Skim milk 
0.1
Sweet corn 0.5 Dry spaghetti 0.2 Mayonnaise 81
Mushrooms 
0.1 Shredded wheat 0.7 Egg yolk 2
Corn flakes 
0.1 Egg white 
0.1
a Values are taken from a provisional table (83) and are median values from a compilation of reported assays.
Only HPLC assays are included in the database.
128 Suttie
dietary requirement for vitamin K, a few servings of foods with high vitamin K content
contribute a significant amount toward satisfying this requirement, and an uncomplicated
dietary deficiency is unlikely. Utilizing current food composition data and 14-day food
diaries obtained from 2000 households, the mean adult phylloquinone intake in the United
States has been determined to be 80 µg for men and 73 µg for women (83b). Dihydrophylloquinone
formed during hydrogenation of cooking oils has lower biological activity and
is present in the U.S. diet at about 20% of the amount of phylloquinone. Because most
dietary vitamin K is present in a few foods, it is possible (84) to construct palatable research
diets that meet the recommended dietary allowances for other nutrients but that
have a phylloquinone content of less than 10 µg/day.
The vitamin K content of individual food items is probably more variable than that
of many other nutrients. Significant changes in the vitamin K content of plants during
growth and maturity and at various geographical locations have been reported (85). The
effect of food processing and cooking on vitamin K content has not been carefully considered.
Richardson et al. (74) have investigated the effect of heat processing (canning) or
of sterilization by ionizing radiation on the vitamin K content of six vegetables. The vitamin
K content of these foods, as determined by chick bioassay, did not differ consistently
from that of the fresh frozen food, nor did storage of any of the products for 15 months
have a significant effect. Phylloquinone in cooking oils has been shown to be relatively
stable to heat but rapidly destroyed by both daylight and fluorescent light (79). Other than
being affected by light, vitamin K present in food is most likely relatively stable; however,
only limited data are available.
VI. METABOLISM
A. Absorption
The absorption of nonpolar lipids, such as vitamin K, into the lymphatic system depends on
their incorporation into mixed micelles, and optimal formation of these micellar structures
requires the presence of both bile and pancreatic juice (86,87). Normal subjects have been
reported (88) to excrete less than 20% of a radiolabeled 1-mg dose of phylloquinone in
the feces, but as much as 70–80% of the ingested phylloquinone was excreted unaltered
in the feces of patients with impaired fat absorption caused by obstructive jaundice, pancreatic
insufficiency, or adult celiac disease. Much of the fecal radioactivity found in studies
such as these is due to polar biliary excretion products rather than unmetabolized phylloquinone.
Very little is known about the absorption of vitamin K from different food sources.
Vitamin K in spinach (89) was found to be absorbed 13.3% as well as from a commercial
detergent-solubilized preparation (Konakion) of phylloquinone when 25 g of butter was
included with the meal, but only 1.4% as well in the absence of butter. A second study
(89a) also indicates the phylloquinone in food sources is only 15–20% as bioavailable as
added phylloquinone. These limited data suggest that the absorption of vitamin K is probably
low but may be highly dependent on food sources and total dietary composition.
The potential for absorption of the large-bowel menaquinone pool has been reviewed
(90,91). Early, rather nonphysiological, experiments (92) demonstrated that MK-9 disappeared
from the lumen of the isolated rat ileum or large intestine but did not document
its appearance in plasma or lymph. Ichihashi et al. (93) have shown that in the presence
Vitamin K 129
of bile MK-9 is absorbed via the lymphatic pathway from rat jejunum but that in the
absence of bile no uptake of MK-9 from the colon to lymph or blood occurred within 6
h. The oral administration of 1 mg mixed long-chain menaquinones to anticoagulated
human subjects has been shown (94) to effectively decrease the extent of the acquired
hypoprothrombinemia. This demonstrates that the human digestive tract can absorb these
more hydrophobic forms of the vitamin when they are presented to the small intestine
but does not address their absorption from the large bowel. In addition, a small but nutritionally
significant portion of the intestinal content of the vitamin is located not in the
large bowel but in a region where bile acid–mediated absorption could occur (95).
Menadione is widely used in poultry, swine, and laboratory animal diets as a source
of vitamin K. It can be absorbed from both the small intestine and the colon by a passive
process (96,97), and after absorption it can be alkylated to MK-4, a biologically active
form of the vitamin.
B. Transport
The lymphatic system has been demonstrated to be the major route of transport of absorbed
phylloquinone from the intestine. There is no evidence that the vitamin in the lymph is
in any way modified. Phylloquinone is transported in chylomicrons and other low-density
lipoprotein particles (91), and its clearance is affected by apolipoprotein E polymorphism
(98). Hyperlipidemic patients have elevated phylloquinone concentrations, and this increase
is associated with the triglyceride-rich very low-density lipoprotein (VLDL) fraction
of the lipoprotein pool (91,98). The clearance of an injected dose of radioactive phylloquinone
from plasma has been investigated in humans (99,100) and shown to consist
of a two-phase exponential decline in radioactivity. The first phase had a half-life of 20–
30 min and the second a half-life of 120–165 min. Although the body pool of vitamin K
cannot be calculated from such data, it can be calculated (99) that the total body pool of
vitamin K is replaced approximately every 2.5 h.
C. Plasma Concentrations
The resolution provided by the development of HPLC system (63) and the increased sensitivity
provided by newer methods of detection have made it possible to obtain a quantitative
measure of the amount of vitamin K in serum or plasma. The clinical significance
of these measurements is not yet fully established, and little information on the relationship
between dietary intake and serum levels is available. Measurements of endogenous serum
phylloquinone concentrations (0.5–2 ng/mL) require a preliminary semipreparative column
to rid the sample of contaminating lipids followed by an analytical column. The chief
alterations and improvement in methodology in recent years have been associated with
the use of different methods of detection. Early methods utilized UV detectors, which
lack sensitivity, and electrochemical detection or fluorescence detection of the vitamin
following chemical or electrochemical reduction have replaced this methodology. These
techniques have been recently reviewed (101,102).
Initial reports of plasma or serum phylloquinone concentrations were probably too
high, and it now appears (103,104) that normal fasting values are in the region of 0.5 ng/
mL (1.1 nmol/L). This is a lower concentration than is found for the other fat-soluble
vitamins. Although concentrations of individual menaquinones were at one time thought
too low to be measured, this does not appear to be the case. The combined plasma concen130
Suttie
tration of MK-7 and MK-8 in three studies (105–107) are in the same range as the concentration
of phylloquinone (108), and substantial amounts of plasma MK-6 have been reported
by other investigators (109,110). The significance of these observations is not yet
known. Circulating concentrations of phylloquinone vary with dietary intake, and in animal
(111,112) or human (113–115) studies where vitamin K intake has been restricted
or in patients with decreased food intake (116) there is a rapid decrease in plasma vitamin
K concentrations.
D. Tissue Deposition and Storage
Early studies of the distribution of dietary vitamin K in tissues were hampered by the low
specific radioactivity of the vitamin available. These studies, which utilized milligram
quantities of vitamin K injected into rats, indicated that phylloquinone was specifically
concentrated and retained in the liver but that menadione was poorly retained in this organ.
Menadione was found to be widely distributed in all tissues and to be very rapidly excreted.
The metabolism of more physiological doses of the vitamin has also been studied, and
significant differences in the distribution of phylloquinone and menadione have been seen
(117,118). About 50% of a 10-µg dose of phylloquinone administered to a rat was found
in liver at 3 h, but only 2% of a 2-µg dose of menadiol diphosphate was found in the
liver at that time. The half-life of phylloquinone in rat liver is in the range of 10–15 h
(111,112,119), so hepatic concentrations are greatly dependent on recent intake. Vitamin
K distribution has also been studied (120) by the technique of whole-body radiography
following administration of radioactive menadione, phylloquinone, or MK-4. Essentially
equal body distribution of phylloquinone and MK-4 were found at 24 h. Radioactive menadione
was spread over the whole body much faster than the other two compounds, but
the amount retained in the tissues was low. Whole-body radiography also confirmed that
vitamin K is concentrated by organs other than liver, and the highest activity was seen
in the adrenal glands, lungs, bone marrow, kidneys, and lymph nodes.
Early studies of vitamin K distribution in the liver (121) depended on bioassay of
isolated fractions of normal liver and indicated that the vitamin was distributed in all
subcellular organelles. When 0.02 or 3µg of phylloquinone was injected (122) into vitamin
K–deficient rats, more than 50% of the liver radioactivity was recovered in the microsomal
fraction and substantial amounts were found in the mitochondria and cellular debris fractions.
The specific activity (picomoles vitamin K per milligram protein) of injected radioactive
phylloquinone has been studied (118), and only the mitochondrial and microsomal
fractions had a specific activity that was enriched over that of the entire homogenate, with
the highest activity in the microsomal fraction. The less biologically active cis isomer of
phylloquinone was found to be concentrated in liver microsomes (25), but the mitochondrial
fraction had the highest specific activity. Nyquist et al. (123) found the highest specific
activity (dpm/mg protein) of radioactive phylloquinone to be in the Golgi and smooth
microsomal membrane fractions. Only limited data on the distribution of menaquinones
are available, and MK-9 has been reported (124) to be preferentially localized in a mitochondrial
rather than a microsomal subcellular fraction. Factors influencing intracellular
distribution of the vitamin are not well understood, and only preliminary evidence of an
intracellular vitamin K–binding protein that might facilitate intraorganelle movement has
been presented (125). The vitamin does appear to be lost more rapidly from the cytosol
than from membrane fractions as a deficiency develops (126).
Vitamin K 131
Because of the small amounts of vitamin K in animal tissues, it has been difficult
to determine which of the vitamers are present in tissue from different species. Only
limited data are available, and they have been reviewed by Matschiner (127,128). These
data, obtained largely by thin-layer chromatography, indicate that phylloquinone is found
in the liver of those species ingesting plant material and that, in addition to this, menaquinones
containing 6–13 prenyl units in the alkyl chain are found in the liver of most species.
Some of these long-chain vitamers appear to be partially saturated. More recently, analysis
of a limited number of human liver specimens has shown that phylloquinone represents
only about 10% of the total vitamin K pool and that a broad mixture (Table 4) of menaquinones
is present. The predominant forms appear to be MK-7, MK-8, MK-10, and MK-
11. Kayata et al. (131) have reported that the hepatic menaquinone content of five 24-
month-old infants was approximately sixfold higher than that of three infants less than 2
weeks of age, and another study (132) failed to find menaquinones in neonatal livers.
Although the long-chain menaquinones are potential sources of vitamin K activity in liver,
the extent to which they are utilized is not known. A recent study (124) has demonstrated
that the utilization of MK-9 as a substrate for the vitamin K–dependent carboxylase is
only about 20% as extensive as phylloquinone when the two compounds are present in
the liver in equal concentrations. Recent data have also suggested that MK-4 may play a
unique role in satisfying the vitamin K requirement of some species or tissues. Most
analyses of liver from various species have not detected significant amounts of MK-4.
However, chicken liver has been shown (133,134) to contain more MK-4 than phylloquinone,
and some nonhepatic tissues of the rat have been shown (135) to contain much
more MK-4 than phylloquinone. The significance of these observations and the possibility
that MK-4 may play a unique role in some aspect of vitamin K nutrition is not yet
known.
E. Metabolism
1. Alkylation of Menadione
Animals cannot synthesize the naphthoquinone ring, and this portion of the vitamin must
be furnished in the diet. Bacteria and plants synthesize this aromatic ring system from
shikimic acid, and these pathways have been reviewed (136). It appears that bacterial
synthesis of menaquinones does not usually proceed through free menadione as an intermediate,
but rather 1,4-dihydroxy-2-naphthoic acid is prenylated, decarboxylated, and then
methylated to form the menaquinones. The transformation of menadione to MK-4 in animal
tissues was first observed by Martius and Esser (13). Radioactive menadione was
used (137) to establish that menadione could be converted to a more lipophilic compound
that, on the basis of limited characterization, appeared to be MK-4. It was then demonstrated
(138) that menadione could be converted to MK-4 by an in vitro incubation of rat
or chick liver homogenates with geranylgeranyl pyrophosphate. The activity in chick liver
was much higher than that in the rat. These studies have been extended (139), and it has
been demonstrated that other isoprenoid pyrophosphates could serve as alkyl donors for
menaquinone synthesis. Early studies (140) also demonstrated that administered phylloquinone
or other alkylated forms could be converted to MK-4. It was originally believed
that the dealkylation and subsequent realkylation with a geranylgeranyl side chain occurred
in the liver, but subsequently it was concluded that phylloquinone was not converted
to MK-4 unless it was administered orally. This suggested that intestinal bacterial action
132 Suttie
Table
4
Phylloquionone
and
Menaquinone
Content
of
Human
Liver
Vitamin
K
(pmol
/
g
liver)a
Kb
MK-5
MK-6
MK-7
MK-8
MK-9
MK-10
MK-11
MK-12
MK-13
Ref.
22

512
18
12

13
57

59
95

157
2

467
71
90

74
15

13
5

6
(129)
18

4
NR
NR
122

61
11

24

296
16
94

26
21

68

3
(130)
28

4NR
NR
34

12
9

22

175
10
99

15
14

25

1
(116)
a
Values
are
mean

SEM
for
6
or
7
subjects
in
each
study.
Values
from
Refs.
129
and
130
have
been
recalculated
from
data
presented
as
ng/
g
liver.
NR,
not
reported.
b
Phylloquinone.
Vitamin K 133
was required for the dealkylation step. More recent studies (133–135) have demonstrated
that the phylloquinone-to-MK-4 conversion is very extensive in some tissues and it has
been demonstrated that gut bacteria are not needed for this conversion (135a,135b).
2. Metabolic Degradation and Excretion
Menadione metabolism has been studied in both whole animals (142,143) and isolated
perfused livers (135). The phosphate, sulfate, and glucuronide of menadiol have been
identified in urine and bile, and studies with hepatectomized rats (145) have suggested
that extrahepatic metabolism is significant. Early studies of phylloquinone metabolism
(146) demonstrated that the major route of excretion was in the feces and that very little
unmetabolized phylloquinone was present. Wiss and Gloor (147) observed that the side
chains of phylloquinone and MK-4 were shortened by the rat to seven carbon atoms,
yielding a carboxylic acid group at the end that cyclized to form a ?-lactone. This lactone
was excreted in the urine, presumably as a glucuronic acid conjugate. The metabolism of
radioactive phylloquinone has now been studied in humans by Shearer’s group (88). They
found that about 20% of an injected dose of either 1 mg or 45 µg of vitamin K was
excreted in the urine in 3 days, and that 40–50% was excreted in the feces via the bile.
Two different aglycones of phylloquinone were tentatively identified as the 5- and 7-
carbon side-chain carboxylic acid derivatives, respectively. It was concluded that the ?-
lactone previously identified was an artifact formed by the acidic extraction conditions
used in previous studies.
More recent studies of vitamin K metabolism have been conducted following the
discovery of the significance of the 2,3-epoxide of the vitamin (XXV). This metabolite,
commonly called vitamin K oxide, was discovered by Matschiner et al. (148), who were
investigating an observation (122) that warfarin treatment caused a buildup of liver phylloquinone.
This increase in radioactive vitamin was shown to be due to the presence of a
significant amount of a metabolite more polar than phylloquinone that was isolated and
characterized as phylloquinone 2,3-epoxide. Further studies of this compound (149) revealed
that about 10% of the vitamin K in the liver of a normal rat is present as the
epoxide and that this can become the predominant form of the vitamin following treatment
with coumarin anticoagulants.
As might be expected from the effects of coumarin anticoagulants on tissue metabolism
of vitamin K, anticoagulant treatment has a profound effect on vitamin K excretion.
Disappearance of radiolabeled vitamin K from the plasma is not significantly altered by
coumarin anticoagulant administration, but the ratio of plasma vitamin K epoxide to vitamin
K does increase drastically under these conditions. Warfarin administration also
greatly increases urinary excretion and decreases fecal excretion of phylloquinone (150)
The distribution of the various urinary metabolites of phylloquinone is also substantially
altered by warfarin administration. The amounts of the two major urinary glucuronides that
have tentatively been identified as the conjugates of 2-methyl-3-(5?-carboxy-3?-methyl-
2?pentenl)-1,4-naphthoquinone (XXVI) and 2-methyl-3-(3?-carboxy-3?-methylpropyl)-
134 Suttie
1,4-naphthoquinone (XXVII) are substantially decreased, and three new metabolites appear
(151).
It is likely that there are a number of excretion products of vitamin K that have not
yet been identified. Matschiner (127) has fed double-labeled phylloquinone—a mixture
of [6,7-3H]phylloquinone and [14C]phylloquinone-(phytyl-U) with a 3H/14C ratio of 66—
to vitamin K–deficient rats and recovered radioactivity in the urine with a 3H/14C ratio of
264. This ratio is higher than would be expected for the excretion of a metabolite with
seven carbons remaining in the side chain and is more nearly that expected from the
excretion of the metabolite with five carbons remaining in the side chain. These data
suggest that some more extensively degraded metabolites of phylloquinone are also
formed. An exchange of the phytyl side chain of phylloquinone for the tetraisoprenoid
chain of MK-4 before degradative metabolism would also be expected to contribute to
an increase in the 3H/14C ratio, and current data suggest that this exchange may occur. It
is doubtful, however, that this exchange is extensive enough to account for the increased
ratio of naphthoquinone ring atoms to side-chain atoms that was observed in this study.
The available data relating to vitamin K metabolism therefore suggest that menadione
is rapidly metabolized and excreted, and that only a relatively minor portion of this
synthetic form of the vitamin gets converted to biologically active MK-4. The degradative
metabolism of phylloquinone and the menaquinones is much slower. The major products
appear to have been identified, but there may be a number of urinary and biliary products
not yet characterized. The ratio of the various metabolites formed is drastically altered
by anticoagulant administration. However, there is no evidence to indicate that the isoprenoid
forms of the vitamin must be subjected to any metabolic transformation before they
serve as a cofactor for the vitamin K–dependent carboxylases.
VII. BIOCHEMICAL FUNCTION
A. Vitamin K–Dependent Clotting Factors
Soon after Dam’s discovery of a hemorrhagic condition in chicks that could be cured by
certain plant extracts, it was demonstrated that the plasma of these chicks contained a
decreased concentration of prothrombin. At this time, the complex series of reactions
involved in the conversion of circulating fibrinogen to a fibrin clot were poorly understood.
A basis for a clear understanding of the role of the multitude of factors involved in coagulation
came with the realization that many of the proteins involved could be looked at as
zymogens that could be activated by a specific protease and that these modified proteins
could in turn activate still other zymogens. This led to the development of a generalized
‘‘cascade’’ or ‘‘waterfall’’ theory of blood coagulation, which has now been shown (152)
to be an oversimplification. As shown in Fig. 2, the key step is the activation of prothrombin
(factor II) to thrombin by the activated form of factor X (factor Xa). Factor X can be
Vitamin K 135
Fig. 2 Involvement of vitamin K–dependent clotting factors in coagulation. The vitamin K–dependent
proteins (ellipses) circulate as inactive forms of serine proteases until converted to their
active (subscript a) forms. These conversions occur in stages where an active protease, a substrate,
and a protein cofactor (triangles) form a Ca2-mediated associated with a phospholipid surface. The
protein cofactors V and VII are activated by thrombin (IIa) to achieve their full activity. The clotting
system is traditionally divided into two pathways: the extrinsic pathway, which involves a tissue
factor (TF) in addition to blood components, and an intrinsic pathway, which involves components
present in the blood. Protein C is activated by IIa in the presence of an endothelial cell protein called
thrombomodulin (TM). Protein C is not a procoagulant but rather functions in a complex with protein
S to inactivate Va and VIIIa.
activated by two pathways involving the vitamin K–dependent proteins factor VII and
factor IX. These activations are carried out in a Ca2/phospholipid-dependent manner, and
the enzymology of these conversions is now well understood. The four proteins involved in
thrombin generation were collectively called the ‘‘vitamin K–dependent clotting factors’’
for 25 years before protein C and protein S were discovered. These two proteins have an
anticoagulant role as they are able to inactivate the accessory proteins, factor Va and factor
VIIIa (153). A seventh vitamin K–dependent protein, termed protein Z, has been described
but its function is not known.
There was a period of about 40 years between the discovery of vitamin K and the
elucidation of the metabolic function of the vitamin. Theories of the participation of vitamin
K in oxidative phosphorylation somehow being related to the synthesis of specific
proteins could not be substantiated, nor could theories of the vitamin-controlled production
of specific proteins at a transcriptional level be proven. Involvement of an intracellular
precursor in the biosynthesis of prothrombin was first clearly stated by Hemker et al. (154)
who postulated that an abnormal clotting time in anticoagulant-treated patients was due
to a circulating inactive form of plasma prothrombin. Direct evidence of the presence of
a liver precursor protein was obtained when Shah and Suttie (155) demonstrated that
136 Suttie
the prothrombin produced when hypoprothrombinemic rats were given vitamin K and
cycloheximide was not radiolabeled if radioactive amino acids were administered at the
same time as the vitamin. These observations were consistent with the presence of a hepatic
precursor protein pool in the hypoprothrombinemic rat that was rapidly being synthesized
and that could be converted to prothrombin in a step that did not require protein
synthesis. This hypothesis was strengthened by direct observations (156) that the plasma
of patients treated with coumarin anticoagulants contained a protein that was antigenically
similar to prothrombin but lacked biological activity. A similar protein was first demonstrated
in bovine plasma by Stenflo (157), but it appears (158) to be present in low concentrations
or altogether absent in other species.
Studies of this ‘‘abnormal’’ prothrombin (159) demonstrated that it contained normal
thrombin, had the same molecular weight and amino acid composition, but did not
absorb to insoluble barium salts as did normal prothrombin. This difference, and the altered
calcium-dependent electrophoretic and immunochemical properties, suggested a difference
in calcium binding properties of these two proteins that was subsequently demonstrated
by direct calcium binding measurements. The critical difference in the two proteins
was the inability of the abnormal protein to bind to calcium ions, which are needed for
the phospholipid-stimulated activation of prothrombin by factor X (160). These studies
of the abnormal prothrombin clearly implicated the calcium binding region of prothrombin
as the vitamin K–dependent region. Acidic, Ca2-binding peptides could be isolated from
a tryptic digest of the fragment 1 region of normal bovine prothrombin but could not
be obtained when similar isolation procedures were applied to preparations of abnormal
prothrombin. The nature of the vitamin K–dependent modification was elucidated when
Stenflo et al. (161) succeeded in isolating an acidic tetrapeptide (residues 6–9 of prothrombin)
and demonstrating that the glutamic acid residues of this peptide were modified so that
they were present as ?-carboxyglutamic acid (3-amino-1,1,3-propanetricarboxylic acid)
residues (Fig. 3). Nelsestuen et al. (162) independently characterized ?-carboxyglutamic
acid (Gla) from a dipeptide (residues 33 and 34 of prothrombin), and these characterizations
of the modified glutamic acid residues in prothrombin were confirmed by Magnusson
Fig. 3 Structure of ?-carboxyglutamic acid (Gla) and a diagramatic representation of the prothrombin
molecule. Specific proteolysis of prothrombin by factor Xa and thrombin will cleave prothrombin
into the specific large peptides shown: fragment 1 (F-1), fragment 2 (F-2), prethrombin
1 (P-1), prethrombin 2 (P-2), and thrombin (thr). For details of the activation of prothrombin to
thrombin and for sequences of vitamin K–dependent proteins, see Ref. (146). The Gla residues in
bovine prothrombin are located at residues 7, 8, 15, 17, 20, 21, 26, 27, 30, and 33, and they occupy
homologous positions in the other vitamin K–dependent plasma proteins.
Vitamin K 137
et al. (163), who demonstrated that all 10 Glu residues in the first 33 residues of prothrombin
are modified in this fashion.
B. Vitamin K–Dependent Carboxylase
After the vitamin K–dependent step in prothrombin synthesis was shown to be the formation
of ?-carboxyglutamic acid residues, Esmon et al. (164) demonstrated that the addition
of vitamin K and H14CO3 to vitamin K–deficient rat liver microsomal preparations resulted
in the fixation of CO2 into microsomal proteins. It was possible to isolate radioactive
prothrombin from this incubation mixture and show that essentially all of the incorporated
radioactivity was present as ?-carboxyglutamic acid residues in the fragment 1 region of
prothrombin. These observations would appear to offer final proof of the biochemical role
of vitamin K as a cofactor for this microsomal glutamyl carboxylase (Fig. 4).
This enzyme activity was soon shown to be active when solubilized in a number
of detergents, and it was demonstrated (165) that the pentapeptide Phe-Leu-Glu-Glu-Val
would serve as a substrate for this enzyme. Most subsequent studies have utilized this or
a similar peptide substrate, rather than the endogenous microsomal substrates, to study
enzyme activity. The rough microsomal fraction of liver is highly enriched in carboxylase
activity, and lower but significant levels are found in smooth microsomes. Mitochondria,
nuclei, and cytosol have negligible activities. The data obtained from protease sensitivity
studies are consistent with the hypothesis that the carboxylation event occurs on the lumen
side of the rough endoplasmic reticulum (166). Details of the properties of the enzyme
can be found in a number of reviews (167–173). This carboxylation reaction does not
require ATP, and the available data are consistent with the view that the energy to drive
this carboxylation reaction is derived from the reoxidation of the reduced form of vitamin
K. This unique carboxylase requires O2, and studies ruled out the involvement of biotin
in the system. These findings and a direct study of the CO2/HCO3
 requirement indicate
that carbon dioxide rather than HCO3
 is the active species in the reaction. The vitamin
K antagonist 2-chloro-3-phytyl-1,4-naphthoquinone is an effective inhibitor of the carboxylase,
and the reduced form of this analog has been shown to be competitive with the
reduced vitamin site. Polychlorinated phenols are strong inhibitors, and substitution of a
trifluoromethyl group, a hydroxymethyl group, or a methoxymethyl group at the 2-position
also results in inhibitory compounds (174).
A review of studies of the substrate specificity at the vitamin site of the carboxylase
suggests that the only important structural features of this substrate in a detergent-solubilized
system are a 2-methyl-1,4-naphthoquinone substituted at the 3-position with a somewhat
hydrophobic group (174). Methyl substitution of the benzenoid ring has little effect
or increases the apparent Km for the reduced vitamin. A large number of low molecular
Fig. 4 The vitamin K–dependent carboxylation reaction.
138 Suttie
weight peptide substrates of the enzyme have been synthesized, and their assay has failed
to reveal any unique sequence needed as a signal for carboxylation. In general, peptides
with Glu-Glu sequences are better substrates than those with single Glu residues. Gln, DGlu,
or Homo-Glu residues have been demonstrated to be noncarboxylated residues, while
Asp residues are poorly carboxylated. Why only the first of the two adjacent Glu residues
in these synthetic, nonphysiological substrates is carboxylated by the enzyme is not yet
apparent. A consensus sequence within the Gla region that may be important for efficient
carboxylation has been identified (175), but its significance is not known. The manner by
which the enzyme carboxylates its normal physiological protein substrates is not known.
Data obtained by characterizing the partially ?-carboxylated forms of prothrombin secreted
by a dicoumarol-treated cow would suggest that carboxylation may begin at the most
amino-terminal of the 10 potential Gla sites in prothrombin and move toward the more
carboxy terminal sites (176). However, an in vitro study utilizing des-?-carboxyosteocalcin
has shown carboxylation preferentially occurring at the most carboxy terminal of the three
potential Gla sites (177), and this aspect of the carboxylation reaction is not understood.
The stereochemistry of the reactions at the glutamyl residue has also been determined
(178). The incorporation of threo- or erythro-?-fluoroglutamate into a peptide substrate
results in a stereospecific hydrogen abstraction that corresponds to the elimination of the
4-pro-S-hydrogen of the glutamyl residues. Studies with tritium-labeled substrates have
also established that the hydrogen exchange catalyzed by the enzyme in the absence of
CO2 proceeds with a stereospecific abstraction of the same 4-pro-S-hydrogen of the glutamyl
residue that is eliminated in the carboxylation reaction. The stereochemistry associated
with the addition of CO2 to the active intermediate to form Gla has been determined by
the demonstration (179) that carboxylation of 4-S-fluoroglutamate proceeds with inversion
of configuration.
The substrate Phe-Leu-Glu-Glu-Leu, tritiated at the ? carbon of each Glu residue,
was used (180) to demonstrate that the enzyme catalyzed a vitamin KH2-dependent and
O2-dependent, but CO2-independent release of tritium from this substrate, thus establishing
the role of the vitamin in removing the ? hydrogen of the Glu substrate. The 2,3-epoxide
of vitamin K is a coproduct of Gla formation, and at saturating concentrations of CO2
there is an apparent equivalent stoichiometry between epoxide formation and Gla formation
(181). At lower CO2 concentrations a large excess of vitamin K epoxide is produced.
How epoxide formation is coupled to ?-hydrogen abstraction has not yet been firmly established,
but presumably involves an oxygenated intermediate that is on the pathway to
epoxide formation. This enzyme will catalyze a vitamin KH2- and oxygen-dependent exchange
of 3H from 3H2O into the ? position of a Glu residue of a peptide substrate
(182,183). Exchange of 3H from water with the ?-carbon hydrogen is decreased as the
concentration of HCO3
 in the media is increased. Studies utilizing ?-3H-labeled Glu substrates
have also demonstrated a close association between epoxide formation, Gla formation,
and ?-CEH bond cleavage. The efficiency of the carboxylation reaction, Gla/?-CEH
bond cleaved, is independent of Glu substrate concentration, and the data suggest that this
ratio approaches unity at high CO2 concentrations (184). The available data are consistent
with the model shown in Fig. 5, which indicates that the role of vitamin K is to abstract
the ?-methyl hydrogen to leave a carbanion.
A major gap in an understanding of the mechanism of action of this enzyme has
been the assumption that abstraction of the ?-methyl hydrogen requires a strong base
(presumably formed from the vitamin) and the lack of evidence for such an intermediate.
A series of reports by Dowd and co-workers (185,186) has suggested that an initial attack
Vitamin K 139
Fig. 5 Proposed mechanism of action for the vitamin K–dependent carboxylase enzyme. The
available data support the interaction of O2 with the reduced form of vitamin K to form an oxygenated
intermediate that is sufficiently basic to abstract the ?-hydrogen of the glutamyl residue. The products
of this reaction would be vitamin K epoxide and a glutamyl carbanion. The bracketed peroxy, dioxetane,
and alkoxide intermediates have not been identified in the enzyme-catalyzed reaction but are
postulated based on model organic reactions.
of O2 at the carbonyl carbon adjacent to the methyl group results in the formation of a
dioxetane ring, which generates an alkoxide intermediate. This intermediate is hypothesized
to be the strong base that abstracts the ?-methyl hydrogen. This pathway leads to
the possibility that a second atom of molecular oxygen can be incorporated into the carbonyl
group of the epoxide product, and this activity can be followed by utilization of
either 18O2 or vitamin K with 18O incorporated into the carbonyl oxygens in the reaction.
This partial dioxygenase activity of the carboxylase has been verified by a second group
(187), and although the general scheme (188) shown in Fig. 5 is consistent with all of
the available data, the mechanism remains a hypothesis at this time.
140 Suttie
Normal functioning of the vitamin K–dependent carboxylase poses an interesting
question in terms of enzyme–substrate recognition. This microsomal enzyme recognizes
a small fraction of the total hepatic secretory protein pool and then carboxylates 9–12
Glu sites in the first 45 residues of these proteins. Cloning of the vitamin K–dependent
proteins has revealed that the primary gene products contain a very homologous ‘‘propeptide’’
between the amino terminus of the mature protein and the signal peptide (189). This
region appears from both early in vitro and in vivo lines of evidence (190) to be a ‘‘docking’’
or ‘‘recognition site’’ for the enzyme. This domain of the carboxylase substrates has
also been shown (191) to be a modulator of the activity of the enzyme by decreasing the
apparent Km of the Glu site substrate. The structural features of the propeptide domain
that are important in stimulating the activity of the enzyme have been demonstrated (192)
to be the same as those that have been shown to be involved in targeting these proteins
for carboxylation. This propeptide domain is undoubtedly of major importance in directing
the efficient carboxylation of the multiple Glu sites in these substrates.
Significant progress toward a detailed understanding of the properties of the vitamin
K–dependent carboxylase were limited for years by the lack of a purified enzyme. A
preparation that has 500–1000 times the specific activity of microsomes and can be routinely
prepared in 2 days has been available for a number of years (193). The first report
of purification of the enzyme to homogeneity (194) was of a 77-kd protein that was subsequently
acknowledged (195) to represent the purification of the endoplasmic reticulum
heat shock protein BiP or GRP78. Two microsomal proteins of 94 kd (196,197) and 98
kd (198) have been purified and claimed to be the carboxylase. The subsequent expression
of the 94-kd protein in an insect cell line that lacks any endogenous carboxylase activity
utilizing a baculovirus expression system (199) has established that this protein carries
the activity previously associated with crude preparations of the carboxylase. The role of
the 98-kd protein in the overall carboxylase mechanism, if any, is not known.
Although recombinant carboxylase is available, little information on the structure
of this enzyme is available. The affinity of the propeptide region of various vitamin K–
dependent proteins for the carboxylase has been studied (199a), as has the influence of
the Glu substrate on the epoxidase activity (199b) of the enzyme. The fate of proteins that
are not carboxylated has also been investigated, and, at least in the case of prothrombin, it
has been shown (199c) that in the rat, but not in the human or bovine, lack of carboxylation
leads to degradation in the endoplasmic reticulum rather than secretion.
The substrates for the vitamin K–dependent carboxylase contain multiple potential
Gla residues, and the available data (199d, 199e) suggest that carboxylation is an ordered
process leading to complete carboxylation once initiated. The enzyme has been shown
(199f) to be a substrate for itself, and this modification may be involved in control of the
activity of the enzyme.
C. Vitamin K–Dependent Proteins in Skeletal Tissues
Hauschka et al. (200) first reported the presence of Gla in the EDTA-soluble proteins of
chick bone and demonstrated that it was located in an abundant low molecular weight
protein that would bind tightly to BaSO4. Price et al. (201) independently discovered a
low molecular weight Gla-containing protein in bovine bone that bound tightly to hydroxyapatite
and that inhibited hydroxyapatite formation from saturated solutions of calcium
phosphate. This protein was soon sequenced and shown to contain three Gla residues in
a 49-residue sequence (MW 5700) that shows no apparent homology to the vitamin K–
Vitamin K 141
dependent plasma proteins. This protein has been called either ‘‘osteocalcin’’ or bone Gla
protein (BGP), and the terms are used interchangeably by most investigators. A second
protein, matrix Gla protein (MGP), that has been found in bone in cartilage is structurally
related to bone Gla protein (BGP) (202) and is also expressed in numerous other tissues
(203). Protein S, a vitamin K–dependent protein with an antithrombotic role in hemostasis,
has also been shown (204) to be present in bone matrix. A number of reviews of the
properties and metabolic role of these skeletal tissue components are available (205–208).
Clear evidence for the physiological role of these vitamin K–dependent bone proteins
has been difficult to obtain. Early reports tended to assume some role of osteocalcin
in bone mineralization, but no evidence to support this role has been obtained. Price and
Williamson (209) developed a vitamin K–deficient rat model in which animals were maintained
by administration of a ratio of warfarin to vitamin K that prevented hemorrhage
but resulted in bone osteocalcin levels only 2% of normal. No defect in bone size, morphology,
or mineralization was observed in these animals, and calcium homeostasis was not
impaired. Subsequent studies (210) demonstrated that when rats were maintained on this
protocol for 8 months rather than 2 months, a mineralization disorder characterized by
complete fusion of proximal tibia growth plate and cessation of longitudinal growth was
observed. Whether this response is due to a defect in osteocalcin, MGP, or protein S
synthesis is not yet known. These data do, however, suggest that a skeletal vitamin K–
dependent protein is involved in regulating the deposition of bone mineral.
Osteocalcin is synthesized in bone rather than accumulating there after synthesis in
a different organ. However, small amounts do circulate and can be measured by radioimmunoassay.
Circulating osteocalcin is four- to fivefold higher in young children than
in adults and reaches the adult level of 5–7 ng/mL at puberty. Levels are probably slightly
higher in males, and a slight increase with advanced age has been found. In males, it has
been shown that plasma osteocalcin increases in Paget’s disease, bone metastasis, renal
osteodystrophy, hyperparathyroidism, and osteopenia. In general, plasma osteocalcin increased
markedly in Paget’s disease and is also elevated in other diseases characterized
by increased resorption and increased bone formation (211,212).
A number of reports have suggested that vitamin K status might influence skeletal
health. Low concentrations of circulating vitamin K have been reported in patients with
bone fractures, and under-?-carboxylated circulating osteocalcin has been associated with
low bone density and fracture rate (208,212,214). A large multicenter study (214a) has
identified increased under-?-carboxylated osteocalcin as a predictor of hip fracture, and
low phylloquinone intake has been correlated with hip fractures (214b). If vitamin K status
is related to osteoporosis incidence, the anticoagulant-treated population should be at risk.
However, only marginal associations between warfarin treatment and lower bone mineral
density were found in a recent meta-analysis of nine studies (214c). If there is a relationship
between bone health and vitamin K status, it is unlikely to be mediated through the
rather small alterations in the amount of biologically active osteocalcin that have been
observed in these reports. Both osteocalcin and MGP have been cloned, and the phenotype
of mice lacking the osteocalcin gene was increased bone mass and stronger bones (214d)
and of MGP an increase in aortic calcification (214e).
D. Other Vitamin K–Dependent Proteins
Proteins containing Gla residues have been found in mineralized tissues other than bone
and in pathologically calcified tissues (215–217). These proteins have not been extensively
142 Suttie
characterized, and their relationship to osteocalcin is not clear. A Gla-containing protein
has been purified from kidney (218) and shown to be distinct from osteocalcin, but its
function has not been established. The reported presence of a Gla-containing protein in
liver mitochondria (219) has been questioned (220) but later confirmed by a third investigator
(221). Spermatozoa (222) and urine (223) have also been reported as sources of
specific Gla-containing proteins. Specific proteins have not been characterized following
these reports; however, two novel proline-rich proteins have been cloned (223a). The role
of these proteins has not been elucidated, but a vitamin K–dependent protein encoded by
a growth arrest–specific gene (gas-b) (224) with considerable homology to protein S appears
to be important in cellular development. These proteins are not restricted to higher
vertebrates, and proteins containing Gla residues have been found in elasmobranch species
(225), snake venoms (226), and murine gastropods (227).
These reports of purified or nearly purified proteins containing Gla residues make
it abundantly clear that the action of vitamin K–dependent carboxylase is not limited to
the hepatic modification of a few mammalian plasma proteins. With the exception of
muscle, most mammalian tissues and organs contain significant levels of carboxylase activity.
In considering the physiological role of vitamin K–dependent proteins without a
known function, the widespread use of vitamin K antagonists as clinical anticoagulants
should be considered. Patients are routinely given sufficient amounts of coumarin anticoagulants
to depress vitamin K–dependent clotting factor levels of 15–35% of normal. It
might, therefore, have been expected that a number of problems unrelated to clotting
factor synthesis would have been observed in these patients. However, widespread clinical
problems associated with an effect on synthesis of other vitamin K–dependent proteins
during routine anticoagulant therapy has not been observed. Vitamin K metabolism in
these other tissues may not be as sensitive to coumarin anticoagulants as that in liver, or
the normal level of these proteins may be in excess of that needed for their physiological
function. It may also be that these proteins are part of a secondary backup system to a
metabolic pathway that is warfarin-insensitive.
E. Metabolic Effects of 4-Hydroxycoumarins
The oral anticoagulants are not only effective antithrombotic clinical drugs (228), but
they have also been widely used as rodenticides. Vitamin K and warfarin or other 4-
hydroxycoumarins exhibit a general in vivo agonist/antagonist relationship, and early
investigators recognized that the relationship was not what would be expected for the
competition of two compounds for a single active site. When the vitamin K–dependent
carboxylase was discovered, it was shown that the enzyme was not particularly sensitive
to inhibition by these drugs and that in contrast to the situation in animals in vitro inhibition
of the carboxylase enzyme by warfarin could not be reversed by high concentrations of
vitamin. The microsomal associated activities that have been identified as being involved
in the metabolic interconversions of the liver vitamin K pool are shown in Fig. 6. In
addition to the carboxylase/epoxidase, they include a vitamin K epoxide reductase and
two or more vitamin K quinone reductases. The microsomal epoxide reductase requires
a dithiol rather than a reduced pyridine nucleotide for activity and is commonly studied
in vitro with dithiothreitol as a reductant. This membrane-associated activity has been
difficult to purify but has now been reported (228a) to be composed of a microsomal
epoxide hydrase and a second protein. The thioredoxin/thioredoxin reductase systems can
Vitamin K 143
Fig. 6 Vitamin K–related activities in rat liver microsomes. Vitamin K epoxide formed in the
carboxylation reaction is reduced by a warfarin-sensitive pathway ‘‘vitamin K epoxide reductase’’
that is driven by dithiothreitol (DTT) as a reducing agent in in vitro studies. The quinone form of
the vitamin can be reduced to the hydroquinone either by a warfarin-sensitive DTT-driven quinone
reductase or by one or more nicotinamide nucleotide–linked quinone reductases which are less
sensitive to warfarin. Warfarin and other coumarin anticoagulants do not have a significant effect
on the carboxylase/epoxidase activity.
drive the reactions in vitro, and it is possible that this is the physiologically relevant reductase
(229,230).
A real understanding of the mechanism of action of warfarin began when Matschiner
and colleagues (231,232) demonstrated that the 2,3-epoxide of vitamin K was a normal
metabolite in rat liver and that the ratio of vitamin K epoxide to vitamin K was increased
by warfarin administration. Although it was originally thought that these high levels of
vitamin K epoxide inhibited the carboxylase, this was shown to be unlikely (233). The
current general theory is that inhibition of vitamin K epoxide reduction by warfarin prevents
efficient recycling of the vitamin to its enzymatically active form and therefore limits
the action of the carboxylase.
A strain of wild rats that were resistant to the action of the common 4-hydroxycoumarin
anticoagulants were used to establish this theory. The vitamin K epoxide reductase
preparation obtained from livers of the warfarin-resistant rats were relatively insensitive
to inhibition by warfarin (234,235). These preparations were, however, strongly inhibited
by a second 4-hydroxycoumarin, difenacoum, which had been developed as an effective
rodenticide for control of the warfarin-resistant rat population. These data appeared to
provide the final proof that the inhibition of the epoxide reductase by warfarin was related
to its anticoagulant action. The dithiol-dependent vitamin K quinone reductase has also
been reported to be warfarin-sensitive, and it has been shown that this activity, like that of
the epoxide reductase, is less sensitive to warfarin when assayed in tissues of the warfarinresistant
rat (236,237). It is, therefore, likely that the effects of the 4-hydroxycoumarin
144 Suttie
anticoagulants involve not only the reduction of vitamin K epoxide to the quinone but also
the reduction of the quinone to the hydroquinone (238). The NADH-dependent quinone
reductases are less sensitive to warfarin inhibition and constitute a pathway for vitamin
K quinone reduction in the anticoagulant-treated animal. The presence of this pathway
explains the ability of administered vitamin K to counteract the hemorrhagic condition
resulting from a massive dose of warfarin (239).
The alteration of two enzyme activities by what has been assumed to be a single
mutation responsible for the development of warfarin resistance in the wild rat population
raises interesting questions in terms of the structural relationship of the proteins involved.
Whether or not the two microsomal dithiol-dependent activities, vitamin K epoxide reductase
and vitamin K quinone reductase, are catalyzed by the same active site, two active
sites on the same protein, or on two subunits of the same protein is unclear at present. A
significant number of data do, however, support the theory that the same enzyme catalyzes
both activities (240).
VIII. DEFICIENCY SIGNS AND METHODS OF NUTRITIONAL
ASSESSMENT
The classical method used to define an inadequate intake of vitamin K was to measure
the plasma concentration of one of the vitamin K–dependent clotting factors, prothrombin
(factor II), factor VII, factor IX, or factor X. The various tests of clotting function used
in clinical practice, which are based on the activity of these factors, have been summarized
by Denson and Biggs (241).
Whole-blood clotting times, which were used in early work, are notoriously inaccurate,
variable, and insensitive and should not be used. Tests used at present measure the
time it takes recalcified citrated or oxalated plasma to form a fibrin clot. The standard
‘‘one-stage prothrombin time’’ assay measures clotting time after the addition of calcium
and a lung or brain extract (thromboplastin) preparation to furnish phospholipids and tissue
factor. Variations of this assay have been developed, and commercial reagent kits are
available. Because of the presence of tissue extract, factor IX is bypassed, and the assay
responds to the level of prothrombin and factors VII and X. Of these, factor VII has the
shortest half-life, and its concentration decreases earliest when vitamin K action is impaired.
It is likely, therefore, that these one-stage prothrombin assays often measure the
level of factor VII rather than prothrombin. Specific assays for factors VII and X are also
available but are seldom used in studies of vitamin K sufficiency. The classic assay for
prothrombin is a ‘‘two-stage’’ assay in which thrombin is generated from prothrombin in
one tube and a sample of this added to fibrinogen in a second tube to measure thrombin
concentration. This is a much more tedious procedure; although capable of giving an
accurate measurement of the amount of prothrombin in a plasma sample, it has seldom
been used for routine assay of vitamin K status. A number of snake venom preparations
liberate thrombin from prothromin and have been used (158,242,243) to develop onestage
clotting assays for prothrombin. The enzymes in these preparations do not require
that prothrombin be present in a calcium-dependent phospholipid complex for activation,
and they will therefore activate the descarboxyprothrombin formed in vitamin K–deficient
animals. For this reason they cannot be used to monitor a vitamin K deficiency.
All of these methods depend on the observation of the formation of a fibrin clot as
an end point to assay and are not amenable to automation. The vitamin K–dependent
clotting factors are serine proteases, and in recent years there has been considerable interest
Vitamin K 145
in the development of chromogenic substrates for the assay of these proteins, particularly
factor X and prothrombin. These assays, when utilized to assay prothrombin activity,
actually measure the concentration of thrombin that has been generated from prothrombin
by various methods (242). Because they can be readily adapted to an automated analysis,
these substrates are receiving a great deal of attention as clinical tools to follow anticoagulant
therapy but have not yet been utilized in any attempt to monitor vitamin K adequacy.
Human vitamin K deficiency results in the secretion into the plasma of partially
carboxylated species of vitamin K–dependent proteins. Because they lack the full complement
of ?-carboxyglutamic acid residues, their calcium binding affinity is altered and they
can be separated from normal prothrombin by alterations in their ability to bind to barium
salts or by electrophoresis. Antibodies that are specific for these ‘‘abnormal’’ prothrombins
have been developed and can also be used to detect a vitamin K deficiency. These assays
or similar methods used to detect the concentration of under-?-carboxylated osteocalcin
have greatly increased the sensitivity with which a vitamin K deficiency can be detected
(244). It is also likely that vitamin K status is reflected in alterations of circulating levels
of the vitamin. The extremely low concentration of vitamin K in plasma made these measurements
very difficult at one time, but satisfactory HPLC methods for the determination
of plasma or serum phylloquinone have now been developed (see Sec. VI.C). The amount
of vitamin K found in ‘‘normal’’ plasma appears to be about 0.5 ng/mL, and limited
information on the response of circulating vitamin K to changes in dietary vitamin K is
currently available.
IX. NUTRITIONAL REQUIREMENT
A. Animals
The establishment of a dietary vitamin K requirement for various species has been difficult.
The challenge in demonstrating dietary requirement in many species presumably comes
from the varying degrees to which they utilize the large amount of vitamin K synthesized
by intestinal bacteria and the degree to which different species practice coprophagy. A
spontaneous deficiency of vitamin K was first noted in chicks, and poultry are much more
likely to develop symptoms of a dietary deficiency than any other species. This has usually
been assumed to be due to the rapid transit rate of material through the relatively short
intestinal tract of the chick or to limited synthesis of menaquinones in this species. A
more recent study (133) suggests that limited recycling of vitamin K because of low epoxide
reductase activity may be the cause of the increased requirement.
Ruminal microorganisms synthesize large amounts of vitamin K, and ruminants do
not appear to need a source of vitamin in the diet. Deficiencies have, however, been produced
in most monogastric species. Estimations of vitamin K requirements by different
workers are difficult to compare because of the different forms of the vitamin that were
used and different methods that were employed to establish the requirement. Some studies
have utilized a curative assay in which animals were first made hypoprothrombinemic by
feeding a vitamin K–deficient diet. They were then either injected with the vitamin and
prothrombin levels assayed after a few hours or a day, or fed diets containing various
amounts of the vitamin for a number of days and the response in clotting factor synthesis
noted. Preventive or prophylactic assays have also been used and an attempt made to
determine the minimum concentration of the vitamin that must be present in a diet to
maintain normal clotting factor levels.
146 Suttie
Phylloquinone has often been used in experimental nutrition studies, whereas other
forms of vitamin K are usually used in practical rations. Menadione is usually considered
to be from 20% to 40% as effective as phylloquinone on a molar basis, but this depends
a great deal on the type of assay which is used. It is rather ineffective in a curative assay,
where the rate of its alkylation to a menaquinone is probably the rate-limiting factor, but
often shows activity nearly equal to phylloquinone in a long-term preventive assay. Practical
nutritionists have often preferred to utilize a water-soluble form of menadione, such
as menadione sodium bisulfite complex (MSBC). This compound appears to be about as
active on a molar basis as phylloquinone in poultry rations, and at least in this species,
the activity of menadione, MSBC, and phylloquinone are roughly equal on a weight basis.
Detailed discussions of the vitamin K requirements of various species are available
in articles by Scott (245), Doisy and Matschiner (246), and Griminger (67). The data
indicate that the requirement for most species falls in a range of 2–200 µg vitamin K per
kilogram body weight per day. The data in Table 5, which have been adopted from a table
presented by Griminger (67), give an indication of the magnitude of the requirement for
various species. It should be remembered that this requirement can be altered by age, sex,
or strain, and that any condition influencing lipid absorption or conditions altering intestinal
flora will have an influence of these values (see Sec. X). A considerably higher level
of dietary vitamin K has been recommended for most laboratory animals by the National
Research Council (247). Recommendations for most species are in the range of 3000 µg/
kg of diet, but the rat requirement has been set at 50 µg/kg.
Although this level is sufficient in most cases, it does not prevent all signs of defi-
ciency (111), and the American Institute of Nutrition (248) has now recommended that
purified diets for laboratory rodents should have 750 µg of phylloquinone added to each
kilogram of diet.
B. Humans
The requirement of the adult human for vitamin K is extremely low, and there seems little
possibility of a simple dietary deficiency developing in the absence of complicating factors.
Until recently, the low requirement and the relatively high levels of vitamin K found
in most diets had prevented an accurate assessment of the requirement. Frick et al. (249)
Table 5 Vitamin K Requirements of Various Speciesa
Dietary
Daily intake concentration
Species (µg/kg per day) (µg/kg diet)
Dog 1.25 60
Pig 5 50
Rhesus monkey 2 60
Rat, male 11–16 100–150
Chicken 80–120 530
Turkey poult 180–270 1200
a Data have been summarized from a more extensive table (Ref. 67) and
are presented as the amount of vitamin needed to prevent the development
of a deficiency. No correction for differences in potency of equal weights
of different forms of the vitamin has been made.
Vitamin K 147
studied the vitamin K requirement of starved intravenously fed debilitated patients given
antibiotics to decrease intestinal vitamin K synthesis. They determined that 0.1 µg/kg per
day was not sufficient to maintain normal prothrombin levels and that 1.5 µg/kg per day
was sufficient to prevent any decreases in clotting factor synthesis. Their data indicate
that the requirement was of the order of 1 µg/kg per day. Two other studies (75,250) with
very limited numbers of subjects successfully decreased vitamin K intake to the extent
that clotting factor activities were lowered and also suggested that the vitamin requirement
of the human is in the range of 0.5–1.0 µg vitamin K per kilogram per day.
A major problem in determining a dietary requirement for vitamin K has been the
relative insensitivity of the commonly used prothrombin time measurement (244). A more
recent study modifying the vitamin K intake of young adults by restriction of foods with
a high phylloquinone content (251) has resulted in two symptoms of a mild deficiency:
increased circulating under-?-carboxylated prothrombin and decreased Gla excretion.
These responses were reversed by additional dietary vitamin, and the data obtained are
consistent with a requirement in the range previously suggested. A more carefully controlled
metabolic ward study (113) found alterations in the same two sensitive measures
of deficiency in subjects consuming about 10 µg phylloquinone per day, and increased
circulating under-?-carboxylated prothrombin has been observed in a second metabolic
ward controlled study (252). Based largely on these studies, the RDA for vitamin K has
been set at 1 µg phylloquinone per kilogram body weight for adults, and 5 µg phylloquinone
per day for infants from birth to 6 months and 10 µg phylloquinone per day from
6 to 12 months. The 1 µg/kg body weight was also applied to children.
This requirement may be subject to change as more data become available. Because
of the close relationship of plasma phylloquinone concentration to recent intakes, this
measurement lacks utility for assessing vitamin K status. Assessment of adequacy by use
of the somewhat insensitive one-stage prothrombin time has meant that a large decrease
in vitamin K–dependent clotting factor synthesis was needed to produce an apparent defi-
ciency. More sensitive clotting assays and the ability to immunochemically detect circulating
des-?-carboxyprothrombin (253) provide an opportunity to monitor much milder forms
of vitamin K deficiency. There are a number of reports that vitamin K status may be
important in maintaining skeletal health (214), and in recent studies (254,255,255a) the
extent of under-?-carboxylation of circulating osteocalcin has been shown to be a very
sensitive criterion of vitamin K sufficiency. It is likely that this method will become increasingly
important in future studies directed at defining the human requirement for vitamin
K.
X. FACTORS INFLUENCING VITAMIN K STATUS AND POSSIBLE
GROUPS AT RISK
A. Adult Human Considerations
The human population normally consumes a diet containing a great excess of vitamin K,
but a vitamin K–responsible hypoprothrombinemia can sometimes be a clinically signifi-
cant human problem. O’Reilly (35) has reviewed the potential problem areas and has
pointed out the basic factors needed to prevent a vitamin K deficiency: (a) a normal diet
containing the vitamin (b) the presence of bile in the intestine, (c) a normal absorptive
surface in the small intestine, and (d) a normal liver. Cases of an acquired vitamin K
deficiency do, therefore, occur in the adult population and, though relatively rare, present
148 Suttie
a significant problem for some individuals. It has usually been assumed that a general
deficiency is not possible, but Hazell and Bloch (256) have observed that a relatively high
percentage of an older adult hospital-admitted population has a hypoprothrombinemia that
responds to administration of oral vitamin K. The basis for this apparent increase in vitamin
K requirement was not determined and was probably multicausal. It has, however,
been shown (257) that it is much easier to develop a vitamin K deficiency in older than
in young rats. Whether this is related to anything other than an increased intake of
nutrients/unit of body weight in the younger animals has not been determined.
B. Hemorrhagic Disease of the Newborn
A vitamin K–responsive hemorrhagic disease of the human newborn has been a long
recognized syndrome. The classical disease is seen in infants at the age of 2–5 days, but
‘‘late hemorrhagic disease’’ is a condition also seen in infants older than 1 month (258).
The newborn infant has low levels of the vitamin K–dependent clotting factors (259), and
these normally increase with time. Infants are at risk because the gut of the newborn is
relatively sterile and provides little vitamin K, and human breast milk is low in vitamin
K. Serious problems appear to be associated almost exclusively with breast-fed infants.
Because of the possibility of hemorrhagic episodes, the American Academy of Pediatrics
recommends that phylloquinone be administered parenterally to all newborn infants at a
dose of 0.5–1.0 mg. Commercial infant formulas are routinely fortified with vitamin K.
Improvement in methods for detecting mild vitamin K deficiencies (260) has led to
a renewed interest in vitamin K nutrition in the infant, and the importance of the low
vitamin K intake of breast-fed infants to the development of signs of deficiency is now
more clearly understood. A critical factor in determining the vitamin K status of the breastfed
infant has been shown to be milk intake (261,262). There is currently increased interest
in oral rather than parenteral administration of vitamin K to infants, and efforts are being
made to develop satisfactory products.
C. Effects of Drugs
Early studies (see Ref. 257) of vitamin K action and requirement established the need to
prevent coprophagy to produce a vitamin K deficiency in the rat. It is easily demonstrated
that the vitamin K requirement of the rat is greatly increased under germ-free conditions
(263) and that feeding sulfa drugs increases the vitamin K requirement of the chick (264).
The importance of menaquinones in satisfying the human requirement for vitamin K is
unclear (108). Numerous cases of vitamin K–responsive hemorrhagic events in patients
receiving antibiotics have been reviewed by Savage and Lindenbaum (265). These have
been assumed to be due to decreased menaquinone utilization by these patients, but it is
possible that many cases may represent low dietary intake alone and that the presumed
adverse drug interaction is not always related to the hypoprothrombinemia. The secondand
third-generation cephalosporins have been implicated in a large number of hypothrombinemic
episodes; although it has been suggested that these drugs have a direct effect on
the vitamin K–dependent carboxylase (266), it is more likely that they are exerting a weak
coumarin-like response (267,268).
A series of reports have demonstrated (269,270) that dietary butylated hydroxytoluene
causes a hemorrhagic condition in rats that can be cured by vitamin K supplementation.
The mechanism by which the effect is mediated has not yet been clarified. Phenobarbital
and diphenylhydantoin administration to mothers has been reported (271) to produce a
Vitamin K 149
vitamin K–responsive hemorrhage in the newborn, and clofibrate appears to alter coumarin
responsiveness (272) through an effect on vitamin K utilization of availability. The nature
of these effects has not been extensively investigated.
The widely used coumarin anticoagulants (see Secs. II.C and VII.E) effectively antagonize
the action of vitamin K and also influence metabolism of the vitamin. Clinically,
the effect of this vitamin K antagonism has been thought to be limited to their effect on
clotting factor synthesis. Oral anticoagulant therapy during the first trimester is known to
be associated with development of the fetal ‘‘warfarin syndrome’’ (273). Whether this
outcome is related to the influence of warfarin on the action of osteocalcin, matrix Gla
protein, protein S, or an unknown vitamin K–dependent protein is not known. It is also
possible that this adverse drug effect is related not to the action of warfarin as a vitamin
K antagonist but to an unrelated action of the drug.
D. Influence of Hormones
Early studies of vitamin K requirements indicated that female rats had higher plasma
prothrombin concentrations and a lower vitamin K requirement. Plasma prothrombin levels
are also higher in pregnant rats, and this increased concentration of prothrombin in
female and pregnant rats results from an effect on rate of synthesis rather than rate of
prothrombin degradation (274). Castration of both sexes unifies that vitamin K response,
and in the castrated rat, prothrombin concentrations can be increased with estrogens and
decreased with androgens (275). There are some indications (126) from studies utilizing
[3H]vitamin K1 that the estrogen effects are related to the amount of vitamin needed in
the liver to maintain normal levels of prothrombin. The available evidence suggests that
the influence of estrogens on rate of synthesis is reflected in a higher rate of synthesis
and accumulation of prothrombin precursors in the microsomes (276,277).
The effect of other hormones on vitamin K metabolism or action is less clear. Nishino
(278) has shown that hypophysectomy prevents the estrogen stimulation of prothrombin
synthesis in castrated females, and that prolactin injections protect intact males from
the development of hypoprothrombinemia. It has also been shown that hypothyroidism
in humans results in a decrease in both the rate of synthesis and destruction of the vitamin
K–dependent clotting factors (279). It is likely that the effects of these hormones are
related to rates of synthesis of the proteins involved rather than to any effect on vitamin
K metabolism. But it is possible that there are also effects on the vitamin itself.
E. Other Dietary and Disease State Factors
Early studies of vitamin K functions (280) established that the inclusion of mineral oil in
diets prevented its absorption, and mineral oil has often been used in vitamin K–deficient
diets. High dietary vitamin A has also been recognized for some time to adversely influence
vitamin K action (246). Whether this is a general effect on nonpolar lipid absorption
or a specific vitamin K antagonism is not clear, but it can be observed at relatively low
dietary levels of retinol acetate and retinoic acid (257). Dietary oxidized squalene also
has a potent hemorrhagic effect (257), and a d-?-tocopherol hydroquinone administration
has been shown to produce a vitamin K–responsive hemorrhagic syndrome in the pregnant
rat (281). High vitamin E consumption may be of some clinical significance, as the addition
of vitamin K to the diet of a patient on coumarin anticoagulant therapy has been
shown to result in a hemorrhagic episode (282). It is possible that high vitamin E intakes
may exacerbate a borderline vitamin K deficiency. There are indications that ?-tocopherol
150 Suttie
quinone, rather than ?-tocopherol (283), may be the causative agent, and this may explain
some of the differences in the reported results.
Insufficient assimilation of vitamin K can occur in adults on protracted antibiotic
treatment or those receiving long-term parenteral hyperaliminentation without vitamin K
supplementation. Malabsorption of vitamin K has also occurred as a result of obstructive
jaundice, biliary fistula, pancreatic insufficiency, steatorrhea, or chronic diarrhea. Specific
references to observations of vitamin K–responsive hemorrhagic conditions in these various
diseases states can be found in Refs. 35 and 265.
XI. EFFICACY AND HAZARDS OF PHARMACOLOGICAL DOSES OF
VITAMIN K
No hazards attribute to the long-term ingestion of elevated amounts of the natural forms
of vitamin K have been reported (284,285). For treatment of prolonged clotting times
when hemorrhage is not a problem, vitamin K can be given orally or parenterally. If given
orally to patients with impaired biliary function, bile salts should also be administered.
Vitamin K1 is available as the pure compound or as an aqueous colloidal solution that
can be given intramuscularly or intravenously. Some adverse reactions have been noted
following intervenous administration, and unless a severe hemorrhagic episode is present,
intramuscular infection is the recommended route of therapy. Effective therapy requires
synthesis of normal clotting factors, and a couple of hours may be necessary before a
substantial decrease in clotting times is apparent.
The relative safety of phylloquinone and, presumably, menaquinones does not hold
for menadione or its water-soluble derivatives. These compounds can be safely used at
low levels to prevent the development of a deficiency but should not be used as a pharmacological
treatment for a hemorrhagic condition. Although once prescribed for treatment
of the hemorrhagic disease of the newborn, these compounds are known to react with free
sulfhydryl groups of various tissues and to cause hemolytic anemia, hyperbilirubinemia,
and kernicterus. This marked increase in conjugated bilirubin is extremely toxic to the
neonatal brain and has caused death in some instances (284).
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in Current Advances in Vitamin K Research (J. W. Suttie, ed.). Elsevier, New York, 1988,
pp. 59–64.
284. C. A. Owen, Vitamin K group. XI. Pharmacology and toxicology, in The Vitamins, Vol. 3,
2nd ed. (W. H. Sebrell and R. S. Harris, eds.). Academic Press, New York, 1971, pp. 492–
509.
285. National Research Council, Vitamin Tolerance of Animals, National Academy Press, Washington,
D.C., 1987.
4
Vitamin E
CHING K. CHOW
University of Kentucky, Lexington, Kentucky
I. INTRODUCTION
Vitamin E was discovered by Evans and Bishop more than 75 years ago as a lipid-soluble
substance in lettuce and wheat, necessary for the prevention of fetal death and resorption
in rats fed a rancid lard diet (1). This substance was designated as vitamin E following
the recognition of vitamin D (2,3). The term ‘‘tocopherol’’ is used after the Greek words
‘‘tokos’’ (childbirth), ‘‘phero’’ (to bring forth), and ‘‘ol’’ (alcohol). ?-Tocopherol was
isolated from wheat germ oil in 1936 (4). It was first synthesized by Karrer (5), and its
structure was determined by Fernholz (6) in 1938. The antioxidant properties of tocopherols
were first reported by Olcott and Emerson in 1937 (7).
In addition to fetal resorption in rats, a number of species-dependent deficiency
symptoms of vitamin E, such as liver necrosis in rats and pigs, erythrocyte hemolysis in
rats and chicken, and white muscle disease in calves, sheep, mice, and mink, were reported
during the 1940s and 1950s (8–10). However, due to the lack of a definite clinical syndrome
attributable to vitamin E deficiency and the difficulty of inducing vitamin E defi-
ciency in human adults, the need for or use of this vitamin had been questioned. In the
late 1960s, the essentiality of vitamin E for humans was recognized in connection with
studies on premature infants in which hemolytic anemia was associated with vitamin E
deficiency (9,11). Subsequent studies have shown that neurologic abnormalities do occur
in association with malabsorption syndromes of various etiologies (9,11,12). In recent
years, the involvement of free radicals in the pathogenesis of degenerative diseases and
the possible prevention or slow-down of the disease process by antioxidants have promoted
a renewed and expanded interest in vitamin E.
165
166 Chow
II. CHEMISTRY AND ANTIOXIDANT PROPERTIES
Vitamin E is the term suggested for all tocol and tocotrienol derivatives qualitatively
exhibiting the biological activity of ?-tocopherol. The term ‘‘tocopherols’’ is the generic
description for all mono-, di-, and trimethyltocols and tocotrienols, and is not synonymous
with the term ‘‘vitamin E.’’ All eight naturally occurring tocopherol compounds isolated
from plant sources have a 6-chromanol ring (head) and a phytyl side chain (tail) (8–
10,13,14). There are four tocopherols and four tocotrienols that occur naturally, differing
in the number and position of methyl groups on the phenolic ring (Fig. 1). Tocotrienols
have a structure similar to that of tocopherols, except that the side chain contains three
isolated double bonds at the 3?, 7?, and 11? positions.
The tocopherol molecule has three chiral centers (2, 4?, and 8?) in its phytyl tail.
Since a total of eight stereoisomeric forms may exist, tocopherols of unspecified configuration
are more accurately called methyl-substituted tocols (Table 1). The term ‘‘tocol’’
is the trivial designation for 2-methyl-2-(4?, 8?, 12?-trimethyltridecylchroman-6-ol. All naturally
occurring tocopherols (?-, ?-, ?-, and ?-) have the same molecular configuration
Fig. 1 Structural formula of tocopherols.
Vitamin E 167
Table 1 Vitamin E Compounds
Vitamin Trivial name Chemical name
?-Tocopherol 5,7,8-Trimethyltocol 2,5,7,8-Tetramethyl-2-(4?,8?,12?-trimethyltridecyl)-
6-chromanol
?-Tocopherol 5,8-Dimethyltocol 2,5,8-Trimethyl-2-(4?,8?,12?-trimethyltridecyl)-6-
chromanol
?-Tocopherol 7,8-Dimethyltocol 2,7,8-Trimethyl-2-(4?,8?,12?-trimethyltridecyl)-6-
chromanol
?-Tocopherol 8-Monomethyltocol 2,8-Dimethyl-2-(4?,8?,12?-trimethyltridecyl)-6-
chromanol
?-Tocotrienol 5,7,8-Trimethyl tocotrienol 2,5,7,8-Tetramethyl-2-(4?,8?,12?-trimethyltridecyl)-
6-chromanol
?-Tocotrienol 5,8-Dimethyl tocotrienol 2,5,8-Trimethyl-2-(4?,8?,12?-trimethyltridecyl)-6-
chromanol
?-Tocotrienol 7,8-Dimethyl tocotrienol 2,7,8-Trimethyl-2-(4?,8?,12?-trimethyltridecyl)-6-
chromanol
?-Tocotrienol 8-Monomethyl tocotrienol 2,8-Dimthyl-2-(4?,8?,12?-trimethyltridecyl)-6-
chromanol
[RRR, 2d, 4?d, 8?d, d-tocopherol or (
)-tocopherols] in their phytyl groups. Since tocotrienols
have only one chiral center at position 2, they can only have 2d and 2l stereoisomers.
However, the double bonds at positions 3? and 7? of the phytyl tail allow for the existence
of four cis/trans geometrical isomers (a total of eight isomers, theoretically) per tocotrienol
(13,14).
In addition to naturally occurring isomers, several types of synthetic vitamin E,
mainly in ester forms (e.g., ?-tocopheryl acetate and tocopheryl succinate), are available
commercially. The ester form is less susceptible to oxidation and is therefore more suitable
for food and pharmaceutical applications than the free form. To distinguish it from the
synthetic one, the naturally occurring stereoisomer of ?-tocopherol, formerly known as
d-?-tocopherol, has been designated as RRR-?-tocopherol. The totally synthetic ?-tocopherol,
previously known as dl-?-tocopherol or 2dl, 4?dl, 8?dl-tocopherol, which consists
of eight stereoisomers [2d, 4?d, 8?d (RRR), 2l, 4?d, 8?d (SRR), 2d, 4?d, 8?l (RRS), 2l,
4?d,?8?l (SRS), 2d, 4?l, 8?d (RSR), 2l, 4?l, 8?d (SSR), 2d, 4?l, 8?l (RSS), and 2l, 4?l, 8?l
(SSS)] (13,14), has been designated as all-rac ?-tocopherol. All-rac ?-tocopherol is usually
synthesized by the condensation of trimethylhydroquinone with isophytol. The majority
of tocopherol present in plants is not the ? form. Therefore, RRR-?-tocopherol, which
is referred as natural ?-tocopherol, is often obtained following methylation of tocopherol
mixtures isolated from vegetable oils.
Tocopherols are pale yellow or yellow-brown viscous liquids at room temperature.
At high purity they are almost odorless and colorless. Free tocopherols are practically
insoluble in water but are soluble in oils, fats, acetone, ethanol, chloroform, and other
organic solvents. The boiling point of tocopherols at 0.1 mm Hg pressure is 200–220°C,
their molecular weight ranges from 396.6 to 430.69, and the molar absorbance (E1%; 1
cm) at 292–298 nm ranges from 75.8 to 91.4 (Table 2) (14a). Tocopherols can be oxidized
by atmospheric oxygen, and the oxidation is accelerated by heat, light, alkali, and metal
ions. On the other hand, tocopherols are rather stable to heat and alkali when oxygen is
absent.
168 Chow
Table 2 Molecular Weight and UV Absorption Maxima and Molar
Absorbance of Vitamin E Compounds
Mol w ?max (nm) E%
1 cm
?-Tocopherol 430.7 292.0 75.8
?-Tocopherol 416.7 296.0 89.4
?-Tocopherol 416.7 298.0 91.4
?-Tocopherol 402.6 298.0 87.3
?-Tocotrienol 424.6 292.5 91.0
?-Tocotrienol 410.6 294.0 87.3
?-Tocotrienol 410.6 296.0 90.5
?-Tocotrienol 396.6 297.0 88.1
Source: Adapted from Ref. 14a.
Generally, the first step of tocopherol oxidation is the formation of resonance-stabilized
chromanoxyl (chroman-6-oxyl) radical, due to the donation of the phenolic hydrogen
to a lipid peroxyl radical (15,16). The chromanoxyl radicals are very reactive toward alkyl
and alkylperoxy radicals. Depending on the severity of the oxidation conditions and the
presence of other chemicals, different tocopherol oxidation products are obtained. For
example, oxidation of ?-tocopherol in polar solvents (water or alcohol) leads to the formation
of 5-formyl and hemiacetal derivatives of 5-hydroxymethylene chromanol (13,17).
In lipophilic solvents, the chromanoxy radicals tend to react mainly via radical–radical
coupling reactions and form dimers or trimers (17,18). In the presence of peroxy radicals,
?-tocopherol is primarily oxidized to 8?-peroxy-substituted tocopherone, which is then
degraded to form ?-tocopherylquinone, plus various tocopherone and quinone epoxides
and spiro-dimer and trimer (19–21). Relatively little is known about the products of and
mechanisms for the antioxidant reactions of tocotrienols. Tocotrienols, however, are expected
to yield the same type of radicals and dimeric products as their corresponding
tocopherols. Two dimers of ?-tocotrienol [5-(?-tocotrienyloxy)-?-tocotrienol and 5-(?-tocotrienyl)-
?-tocotrienol] are the principal oxidation products of ?-tocotrienol-rich Hevea
latex (22).
It has long been recognized that tocopherols possess antioxidant activity (7), and
the biological activity of vitamin E is mainly attributed to their ability to donate their
phenolic hydrogen to lipid free radicals. Tocopherols, as well as tocotrienols, can react
with peroxyl radicals more rapidly than can polyunsaturated fatty acids and, therefore,
are very effective free-radical chain-breaking antioxidants. Antioxidant activity of tocopherols
is determined by their chemical reactivity with molecular oxygen, superoxide radicals,
peroxyl radicals, or other radicals, or by their ability to inhibit autoxidation of fats
and oils. The chemical structures of the tocopherols and tocotrienols support a hydrogendonating
power in the order ?  ?  ?  ? (23). The presence of electron-releasing
substituents ortho and/or para to the hydroxy function increased the lectern density of the
active centers, facilitating the homolytic fission of the O-H bond, increasing the stability of
the phenoxy radical, and improving the reactivity with peroxy radicals (23). Thus, ?-
tocopherol is structurally expected to be a more potent hydrogen donor than ?-, ?-, and
?-tocopherols because these lack one or two ortho-methyl group. The order of ?  ? 
Vitamin E 169
?  ? is generally found in the relative antioxidant activity of the tocopherols in vivo
(see Sec. V). The relative antioxidant activity of tocopherols in vitro, however, varies
considerably depending on the experimental conditions and the assessment method employed.
?-tocopherol and some other isomers, for example, have sometimes been shown
to exhibit a higher antioxidant activity than the ? form in a number of in vitro test systems
(24). Also, tocotrienols are more effective than tocopherols in preventing oxidation under
certain conditions (25). In vitro antioxidant activities of the tocopherols depend not only
on their absolute chemical reactivities toward hydroperoxy and other free radicals, but
also on such factors as tocopherol concentrations, temperature, light, type of substrate and
solvent, and other chemical species, which may act as pro-oxidants and synergists, in the
test system (13,23).
Unlike free tocopherols, tocopheryl esters are much more stable to oxidation, and
do not function as antioxidants in vitro.
III. ABSORPTION, TRANSPORT AND METABOLISM
A. Intestinal Absorption
Similar to lipid components, intestinal absorption of vitamin E depends on pancreatic
function, biliary secretion, micelle formation, and transport across intestinal membrane
(26,27). Tocopherol is first emulsified and solubilized within bile salt micelles and transported
across the water layer to come in contact with the absorption brush-border membrane
of the enterocyte. Tocopheryl esters, such as tocopheryl palmitate, tocopheryl acetate,
and tocopheryl succinate, are hydrolyzed to free tocopherol during the absorptive
process. Under physiological conditions the hydrolysis occurs in gut lumen, although a
mucosal esterase has also been found in the endoplasmic reticulum of the enterocytes
(28). Free tocopherol is absorbed by a passive diffusion process from small intestine to
the enterocyte (29). The absorption process is nonsaturable, non-carrier-mediated, and
does not require energy. The area at the junction between the upper and middle thirds of
the small intestine appears to be the region for tocopherol uptake by enterocytes (29,30).
Within the enterocyte, tocopherol is incorporated into chylomicrons and secreted into the
intracellular spaces and lymphatics, and thus into the bloodstream. This transfer process
does not appear to require a specific protein.
The efficacy of tocopherol absorption varies considerably depending on the conditions
and methods for its estimation. Generally, the rate of absorption decreases as the
amount taken increases. Medium-chain triglycerides have been shown to enhance the absorption
process, whereas long-chain polyunsaturated fatty acids reduce the absorption of
?-tocopherol (31,32). The adverse effect of dietary long-chain polyunsaturated fatty acids
on vitamin E absorption may partly be due to an increase in oxidation of tocopherol during
digestion.
In humans, the majority of ?-tocopherol is absorbed via the lymph ducts, and a
small portion is absorbed by the portal vein (26,27). The absorption of tocopherols other
than the ? form is not as well understood. However, studies have shown that there are
no major differences in the rate of intestinal absorption between ?- and non-?-tocopherols,
such as ?-tocopherol, and that excess of the ? form does not reduce the absorption of ?-
tocopherols (33,34). Thus, a higher level of ?-tocopherols than other tocopherols in human
plasma and tissues is the result of factors other than absorption.
170 Chow
B. Plasma Transport
Due to its hydrophobicity vitamin E requires special transport mechanisms in the aqueous
milieu of the plasma, body fluid, and cells. Similar to dietary lipids, tocopherols absorbed
in the small intestine are incorporated into triglyceride-rich chylomicrons, secreted into
intestinal lymph, and then delivered to the liver. There is no discrimination between various
forms of vitamin E during chylomicron secretion (35,36).
Following delivery to the liver from small intestine, tocopherol is repackaged with
very low-density lipoproteins (VLDLs) and secreted into plasma. At this stage, RRR-?-
tocopherol is preferentially secreted with VLDL over SRR-?-tocopherol and RRR-?-
tocopherol (35–37). During the conversion of VLDLs to low-density lipoproteins (LDLs)
in the circulation, a portion of tocopherol is transferred to LDLs. Exchange of tocopherol
also occurs between LDLs and high-density lipoproteins (HDLs) (38). Therefore, tocopherol
is distributed in all lipoproteins (39,40). In humans, the highest concentration of vitamin
E is found in LDL and HDL. Vitamin E can also be exchanged between lipoproteins
and liposomes, and between plasma and red blood cells. While no specific plasma/serum
carrier protein for vitamin E has been reported, a phospholipid transfer protein that accelerates
exchange/transfer of ?-tocopherol between lipoproteins and cells has been identified
in human plasma (41). This protein may play a role in determining the concentration of
tocopherols present in the circulation and in tissues.
Normal plasma vitamin E concentration in humans ranged from 11 to 37 µmol/L
(5 to 16 mg/L), 1.6 to 5 µmol ?-tocopherol/mmol lipid (0.8 to 1.7 mg/g), or 2.5 to 8
µmol ?-tocopherol/mmol cholesterol (2.8 to 8 mg/g). Approximately 10–20% of total
tocopherols in human plasma is the ? form. In malabsorption states, such as cystic fibrosis,
plasma lipids, lipoproteins, and vitamin E concentration are frequently reduced concurrently
(10–12,26).
C. Tissue Uptake
The mechanisms involved in the uptake of tocopherols by tissue remain unclear. Lipoprotein
lipase bound to the surface of the endothelial lining of capillary walls catabolizes the
triglycerides in the core of chylomicrons and forms chylomicron remnants (26,27). The
latter is then taken up by the parenchymal cells in the liver via the remnant receptors on
the surface of hepatocytes (42,43). Along with the free fatty acids, some vitamin E is also
taken up by peripheral tissues during catabolism of lipoproteins by lipoprotein lipase.
Tocopherol is taken up by tissues via several mechanisms. This includes lipoprotein receptor
(apolipoprotein B/E)–dependent and receptor-independent pathways, independent
transport and cotransport of ?-tocopherol and LDL, and uptake from a number of lipoproteins
(44). The action of lipoprotein lipase is an important process prior to tissue uptake
of tocopherols.
Most tissues, including liver, skeletal muscle, and adipose tissue, have the capacity
to accumulate ?-tocopherol (45–47). In adipose tissue, tocopherol is located mainly in
the bulk lipid droplet and its turnover is slow. The adrenal gland has the highest concentration
of ?-tocopherol, although lung and spleen also contain relatively high concentrations
(47,48). ?-Tocopherol is primarily taken up and located in parenchymal cells (49). The
transfer of tocopherol between parenchymal and nonparenchymal cells takes place after
uptake in parenchymal cells. Parenchymal, but not nonparenchymal, cells have a large
storage capacity for surplus ?-tocopherol and are more resistant to tocopherol depletion
than nonparenchymal cells. Light mitochondria has the highest concentration of ?-tocoVitamin
E 171
pherol, whereas the concentration is low in cytosol (49,50). The majority of tocopherol
is localized in the membranes. For example, approximately three-fourths of mitochondrial
?-tocopherol is found in the outer membrane, and one-fourth is associated with the inner
membrane (49). Also, essentially all tocopherol in the red cells is found in the membranes
(51).
D. Hepatic Secretion
Vitamin E taken up by the liver is stored in the parenchymal cells or secreted into the
bloodstream within nascent VLDLs. Following the action of lipoprotein lipase, some vitamin
E in the VLDLs may end up in LDLs as these lipoproteins are transformed in plasma.
Vitamin E in LDLs may again be taken up by liver via the LDLs (apolipoprotein B/E)
receptor or by non-receptor-mediated uptake (26,42,43). Some vitamin E in association
with chylomicrons and VLDL may also be transferred to peripheral cells and HDLs during
lipolysis by lipoprotein lipase. Secreted tocopherols are either rapidly returned from
plasma to the liver during the course of lipid metabolism or excreted via bile. The secretory
pathway via nascent VLDLs from liver seems to be critical in maintaining tocopherol
concentrations in plasma. Studies have shown that discrimination between the stereoisomers
of tocopherol occurs during hepatic secretion of nascent VLDLs (36). RRR-?-
Tocopherol is preferentially incorporated over other tocopherols into VLDLs, which are
then secreted into plasma. This is evidenced by the much higher levels of RRR-?-tocopherol
than either SRR-?- or RRR-?-tocopherol found in the nascent VLDLs and plasma of
cynomolgus monkeys after the same amount of each was given (37). The preference of
a specific hepatic tocopherol-binding protein to RRR-?-tocopherol appears to be responsible
for this discrepancy.
E. Tocopherol-Binding Proteins
A cystosolic tocopherol-binding protein has been shown to selectively facilitate the incorporation
of RRR-?-tocopherols into nascent VLDLs. By preferentially binding the ? form
over the other forms of tocopherols, the binding protein determines which tocopherols
are returned to the liver from plasma via HDLs and LDLs (52–55). The discrimination
between the tocopherol isomers and homologues appears to be due to the preference of
the tocopherol-binding protein to recognize specific structural features: a fully methylated
aromatic ring, a saturated phytyl side chain, and a stereochemical RRR configuration of
the methyl group’s branching of the side chain (14,36). There is a chiral discrimination
between different isomers of ?-tocopherol, with preference retention of the RRR stereoisomer
over the SRR form (35,36). By regulating the binding and/or transfer of tocopherol,
the transfer protein plays a key role in determining plasma concentration and biological
activity of tocopherols (55). The process results in the preferential enrichment of LDL
and HDL with ?-tocopherol in plasma. The critical role of tocopherol-binding protein in
regulating plasma tocopherol concentration has been indicated in patients with familial
isolated vitamin E deficiency (56,57). Those patients have clear signs of vitamin E defi-
ciency (extremely low plasma vitamin E and neurological abnormalities) but have no fat
malabsorption or lipoprotein abnormalities. Absence of the transfer protein in those patients,
which impairs secretion of tocopherol into hepatic lipoproteins (VLDLs), has been
postulated to be responsible for their low plasma vitamin E status (26,55). The human ?-
tocopherol transfer protein gene was shown to be located at chromosome 8q13 (57a).
Studies of the gene structure and mutations of ?-tocopherol transfer protein in patients
172 Chow
with familial vitamin E deficiency have confirmed that mutations of the gene for ?-tocopherol
transfer protein are responsible for isolated vitamin E deficiency (58,58a). The degree
of functionality of the mutant ?-tocopherol transfer protein seems to be associated with
the degree of severity of the neurological damage and age of onset (59).
A tocopherol/binding protein with a molecular weight of 30–36 kd has been purified
from rat liver cytosol (54,55,60,61). The protein binds ?-tocopherol and enhances its transfer
between membranes. The purified protein has two isoforms with isoelectric points at
5.0 and 5.1, respectively (61). The binding protein exhibits a structural homology with
the cellular retinaldehyde-binding protein present only in visual tissues (62). A tocopherolbinding
protein with a molecular weight of 36.6 kd has also been identified and purified
from human liver cytosol (63). An apparently different ?-tocopherol binding protein with
a molecular mass of 14.2 kd has been isolated and purified from rabbit heart (64) and rat
liver and heart (65). The binding of this smaller binding protein to ?-tocopherol is rapid,
reversible, and saturable, and neither ?- nor ?-tocopherol can replace the binding of ?-
tocopherol. It also markedly stimulates the transfer of tocopherol from liposomes to mitochondria.
The binding protein is different from the cytosolic fatty acid–binding protein
and may be involved in intracellular transport and metabolism of ?-tocopherol (66). The
relationship between those two binding proteins, if any, is not known.
F. Metabolism
The major route of excretion of absorbed tocopherol is fecal elimination. Excess ?-tocopherol
and other forms of tocopherols are excreted first into the bile and then into the
feces. After exerting its antioxidant activity tocopherol is first converted to tocopheryl
chromanoxy radical. The chromanoxy radical is readily reverted to tocopherol, and the
process can be facilitated by such reducing agents as glutathione and ascorbate and/or a
yet to be identified enzymic system. Tocopheryl chromanoxy radicals can also form dimer
or trimer, or be further oxidized to form tocopheryl quinone (Fig. 2). A small amount of
?-tocopheryl quinone is found in liver (67). Studies on the metabolic fate of C14-labeled
?-tocopheryl quinone and ?-tocopheryl hydroquinone have showed that there is no conversion
to ?-tocopherol in vivo (68). ?-Tocopheryl quinone, however, can be reduced
likely by an enzymic process to ?-tocopheryl hydroquinone, and then further metabolized
to ?-tocopheronic acid. Higher activity of NADPH-dependent tocopheryl quinone reductase
is found in the mitochondria and microsomes than in the cytosol of rat hepatocytes
(69). Also, NADPH-cytochrome P450 reductase is capable of catalyzing tocopheryl hydroquinone
formation from tocopheryl quinone (69). Tocopheronic acid is subsequently conjugated
with glucuronic acid or other compounds, and excreted in urine (68). The urinary
metabolite, ?-tocopheronic acid, was first isolated by Simon and co-workers (70) after
administration of large doses of ?-tocopherol to rabbits and humans. A portion of bound
or conjugated ?-tocopheryl hydroquinone is secreted in the bile and eliminated in the
feces (68). A small portion of dimer and trimer of ?-tocopherol has also been found in
rat liver (71,72).
Results obtained form recent studies suggest that 2,5,7,8-tetramethyl-1(2?-carboxyethyl)-
6-hydroxy chroman, instead of ?-tocopheronic acid, may be the major urinary metabolite
of ?-tocopherol (72a). The urinary metabolite appears to be formed in liver directly
from a side chain degradation of ?-tocopherol without oxidative splitting of the
chroman ring (Fig. 2). The compound can be oxidized readily to form ?-tocopheronic
Vitamin E 173
Fig. 2 Metabolic fate of ?-tocopherol. GSH represents reduced glutathione; GSSG, oxidized glutathione;
NADPH and NADP, reduced and oxidized nicotinamide dinucleotide phosphate, respectively.
acid (or tocopheronolactone). Similarly, 2,7,8-trimethyl-2-(2?-carboxyethyl)-6-hydroxy
chroman and 2,8-dimethyl-2(2?-carboxyethyl)-6-hydroxy chroman have been identified as
the principal urinary metabolites for ?- and ?-tocopherols, respectively (72b,72c).
IV. DEFICIENCY SYMPTOMS AND TOXICITY
A. Deficiency Symptoms
A number of species-dependent and tissue-specific symptoms of vitamin E deficiency have
been reported (Table 3). The development and severity of certain vitamin E deficiency
symptoms are associated with the status of other nutrients, including polyunsaturated fatty
acids, selenium, and sulfur amino acids (8,9). For example, the most common sign of
deficiency is necrotizing myopathy, which occurs in almost all species in the skeletal
muscle, as well as in some heart and smooth muscles. However, in lambs and calves,
myopathy primarily results from selenium deficiency, and in rabbits and guinea pigs, severe
debilitating myopathy develops when fed a low vitamin E diet alone. On the other
174 Chow
Table 3 Pathology of Vitamin E Deficiency
Condition Animal Tissues affected
Reproductive failure:
Embronic degeneration Female: rat, hen, turkey, Vascular system of embryo
ewe
Male: rat, guinea pig, ham- Male gonads
ster, dog, cock
Liver, blood, brain, capillaries:
Liver necrosis Rat, pig Liver
Erythrocyte destruction Rat, chick Blood (hemolysis)
Anemia Monkey Bone marrow
Blood protein loss Chick, turkey Serum albumin
Encephalomalacia Chick Cerebellum (Purkinje cells)
Exudative diathesis Chick, turkey Vascular system
Depigmentation Rat Incisors
Kidney degeneration Rat, monkey, mink Kidney tubular epithelium
Steatitis Mink, pig, chick Depot fat
Eosinophilia Rat Blood, small intestine, liver,
stomach, and skeletal
muscle
Nutritional myopathies:
Nutritional muscular dystrophy Rabbit, guinea pig, rat, mon- Skeletal muscle
key, duck, turkey
Stiff lamb Lamb, kid Skeletal muscle
White-muscle disease Calf, sheep, mouse, mink Skeletal and heart muscle
Myopathy of gizzard and heart Turkey, poult Gizzard, heart, and skeletal
muscle
Neuromuscular disorder Rat, monkey, humans Skeletal muscle, spinal cord
hand, rats manifest a relatively benign myopathy when fed a vitamin E–deficient diet,
and chickens develop no myopathy unless the diet is depleted of both vitamin E and
sulfur amino acids. In the chicken, encephalomalacia, a disorder characterized by localized
hemorrhage and necrosis in the cerebellum, is prevented by vitamin E but not by selenium.
However, in chicken, exudative diathesis (a disorder characterized by increased capillary
permeability) can be prevented by either vitamin E or selenium (8,9). Similarly, eosinophilic
enteritis and eosinophilia in rats is preventable by vitamin E or selenium (73). The
cause of the species-dependent and tissue-specific vitamin E deficiency symptoms remains
to be delineated.
In humans, lower plasma/serum levels of vitamin E (0.5 mg/dL) are associated
with a shorter life span of the red cells and increased susceptibility to hemolytic stress.
Most of the vitamin E deficiency states in humans are associated with fat malabsorption
disorders (9,11,12,74). Low serum vitamin E levels have been observed in patients with
a variety of fat malabsorption conditions (Table 4). Recent studies of children and adults
with diseases that cause fat/vitamin E malabsorption, such as abetalipoproteinemia,
chronic cholestatic hepatobiliary disorder, and cystic fibrosis, as well as patients with the
primary form of vitamin E deficiency, familial isolated vitamin E deficiency syndrome,
have conclusively demonstrated that neurological dysfunction is associated with vitamin
E deficiency (12,26,56).
Vitamin E 175
Table 4 Tocopherol Levels in Human Vitamin E Deficiency States
Mean plasma/
serum ?-tocopherol
Condition (mg/100 mL) Range
Premature birth 0.25 0–0.56
Cystic fibrosis 0.24 0–0.97
Malabsorption syndromes other than cystic fibrosis:
Biliary atresia 0.11 —
Biliary cirrhosis 0 —
Biliary obstruction 0.08 0–0.14
Celiac disease 0.20 0–0.35
Chronic pancreatitis 0.40 0.17–0.79
Gastrectomy 0.36 0.10–0.80
Intestinal lymphangiectasia 0.28 —
Intestinal resection 0.31 —
Nontropical sprue 0.25 0.12–0.32
Regional enteritis plus intestinal resection 0.27 0.15–0.39
Tropical sprue 0.28 —
Ulcerative colitis 0.24 0.17–0.36
Whipple’s disease 0.30 0.14–0.45
Protein-calorie malnutrition 0.48 0–0.91
Normal 1.05 7.7–15.5
Source: After Ref. (11.)
B. Toxicity
Both acute and chronic studies with several species of animals have shown that high doses
of vitamin E are relatively nontoxic. However, extremely high levels of vitamin E
(16,000–64,000 IU/kg diet) have been shown to result in decreased pigmentation and a
waxy, feather like appearance (75), and abnormal mineralization and clotting abnormalities
(76) in the chick. Also, feeding pelicans with a high dose of vitamin E (5000 IU/kg
diet) results in vitamin K deficiency (77). These findings suggest that interference with
absorption or metabolic interaction with vitamins D and K is associated with extreme
dietary levels of vitamin E. In humans, supplementation of 800 IU vitamin E per day or
more for several years has consistently shown no adverse effects (78). Although increased
post-surgery bleeding and one individual case of breeding in patient receiving anticoagulant
therapy has been reported, a recent study (78a) has shown that moderate to large
doses of vitamin E (up to 1200 IU daily) can be safely used in patients receiving warfarin.
V. BIOPOTENCY AND BIOLOGICAL FUNCTIONS
A. Biopotency
The utilization or function of tocopherols in biological tissues is governed not solely by
their chemical reactivities but by the biokinetics of their distribution, transport, retention,
and localization as well. Tocopherols differ widely in their antioxidant and biological
activities. A vitamin E activity of RRR-?-tocopherol higher than that of the other forms
176 Chow
suggests that the RRR configuration of the phytyl tail must be optimal for maximum
biopotency. The biological activity of tocopherol is assessed in terms of its relative ability
to prevent such deficiency-related symptoms as fetal resorption-gestation and erythrocyte
hemolysis (Table 5), and, more recently, by a curative myopathy test in rats (8,9,79,79a).
The curative myopathy test is based on the ability of tocopherol to suppress the increase
of pyruvate kinase activity in the plasma of vitamin E–deficient rats. Increased plasma
pyruvate kinase activity resulting from myodegeneration is a sensitive and specific indicator
for assessing the extent of vitamin E deficiency in rats (80). The increase of plasma
pyruvate kinase activity in rats with vitamin E deficiency is related to the nutritional status
of selenium (81). Markedly increases in plasma pyruvate kinase activity is also found in
dystrophic chickens and hamsters (82). Similarly, the pyruvate kinase activity in the serum
of patients with neuromuscular disorders is approximately 20- to 40-fold that of the control
values (83–85).
The biological activity of various forms of tocopherol is expressed as units of activity
in relation to that of all-rec-?-tocopheryl acetate. The relative values in international units
(IU/mg) are: all-rec-?-tocopheryl acetate, 1.00; all-rec-?-tocopherol, 1.10; RRR-?-tocopheryl
acetate, 1.36; RRR-?-tocopherol, 1.49; all-rec-?-tocopheryl succinate, 0.89; and
RRR-?-tocopheryl succinate, 1.21.
B. Biological Functions
Although many biochemical abnormalities are associated with vitamin E deficiency, the
mechanism by which vitamin E prevents various metabolic and pathological lesions has
not yet been elucidated. Vitamin E is the major lipid-soluble chain-breaking antioxidant
found in plasma, red cells, and tissues (14,86,87), and it plays an essential role in maintaining
the integrity of biological membranes. Among the biological functions proposed
for vitamin E, prevention against free-radical–initiated lipid peroxidation tissue damage
is the most important one accepted by most investigators. The antioxidant role of vitamin
E in vivo is supported by the findings that synthetic antioxidants can prevent or lessen
certain vitamin E–deficient symptoms, and that increased production of peroxidation prod-
Table 5 Vitamin E Activity of Tocopherolsa
Resorption-
Structure gestation (%) Hemolysis (%)
RRR-?-tocopherol 100 100
RRR-?-tocopherol 25–50 15–27
RRR-?-tocopherol 8–19 3–20
RRR-?-tocopherol 0.1–3 0.3–2
RRR-?-tocotrienol 21–50 17–25
RRR-?-tocotrienol 4–5 1–5
RRR-?-tocotrienol — —
RRR-?-tocotrienol — —
RRR-?-tocopheryl acetate 91 —
All-rec-?-tocopherolb 74 —
All-rec-?-tocopheryl acetateb 67 —
a Bioassay methods.
b A mixture of eight steroisomers.
Vitamin E 177
ucts, such as malondialdehyde, ethane, and pentane, are found in vitamin E–depleted
animals (9,87). The suggestion that vitamin E may play a structural role in the control of
membrane permeability and stability (88) does not conflict with this view.
Several non-antioxidant functions of vitamin E have also been suggested. Vitamin
E, for example, has been reported to regulate the de novo synthesis of xanthine oxidase
(89), modulate the activities of microsomal enzymes (90,91), and down-regulate protein
kinase activity and cell proliferation (92,92a). It is interesting to note that RRR-?-tocopherol
prevents the inhibition of protein kinase activity and cell growth of smooth-muscle
cells caused by RRR-?-tocopherol (93). Also, vitamin E may regulate immune response
or cell-mediated immunity by modulating the generation of prostaglandins and lipid peroxidation
products, and the metabolism of arachidonic acid (94–96). Additionally, vitamin
E has been shown to be essential for optimally developing and maintaining the integrity
and function of nervous mechanism and skeletal muscle (12,97). Recently, vitamin E has
been shown to be capable of down-regulate mitochondrial generation of superoxide and
hydrogen peroxide (97a,97b). By reducing mitochondrial generation of superoxide and
related ROS, dietary vitamin E not only attenuates oxidative damage but also modulates
the expression and activation of signal transduction pathways and other redox-sensitive
biological modifiers, and thereby may prevent or delay the onset of degenerative tissue
changes.
VI. VITAMIN E AND FREE-RADICAL–INDUCED PEROXIDATIVE
DAMAGE
A. Free-Radical–Induced Peroxidative Damage
The harmful effects resulting from inhalation of high concentration of oxygen have been
attributed to the formation of reactive oxygen species rather than molecular oxygen per se.
Superoxide radicals, nitric oxide, lipid alkoxyl and peroxyl radicals are the most significant
reactive oxygen species generated in systems living in aerobic environments. Among
them, peroxy radical derived from polyunsaturated fatty acids has special significance due
to its involvement in lipid peroxidation, the most common indicator of free-radical processes
in living systems (14).
The process of lipid peroxidation (or autoxidation) can be divided into three phases:
initiation, propagation, and termination. In the initiation phase (reaction 1), carbon-centered
lipid radicals (R•) can be produced by proton abstraction from, or addition to, a
polyunsaturated fatty acid (RH) when a free-radical initiator (I*) is present. The initiation
reaction is generally very slow and is dependent on the type of initiator employed. However,
the reaction can be catalyzed by heat, light, trace metals, and/or certain enzymes
(e.g., lipoxygenases). During the propagation phrase, the lipid radical reacts readily with
available molecular oxygen to form a peroxy radical (ROO•) at a very high rate (reaction
2). The peroxyl radical formed can react with another polyunsaturated fatty acid (R?H),
at a slow rate, to form a hydroperoxide (ROOH) and a new carbon-centered radical (reaction
3):
RH ?>
I*
R• (1)
R• 
 O2 > ROO• (2)
ROO• 
 R?H > ROOH 
 R?• (3)
178 Chow
The propagative process can continue until all polyunsaturated fat is consumed or
the chain reaction is broken (termination phase). Free radicals can also be scavenged and
the chain reaction terminated by self-quenching to form dimers (reaction 4) or by the
action of antioxidant (AH) (reaction 5).
R• (or ROO•) 
 R?• > R-R? (or ROOR?) (4)
R• (or ROO•) 
 AH > RH (or ROOH) 
 A• (5)
Of the free-radical process inhibitors, tocopherols are among the most effective
chain breakers available. This is because tocopherols can react more rapidly with peroxy
radicals than do polyunsaturated fatty acid (14). Since lipid peroxy radicals can react with
tocopherols, several orders of magnitude faster (Ka  104–109 M1 s1) than their reaction
with acyl lipids (Ka  10–60 M1 s1) (14,23), one tocopherol molecule can protect up
to 103–108 polyunsaturated fatty acid molecules at low peroxide levels. Similarly, a small
ratio of ?-tocopherol to polyunsaturated fatty acid molecules (1:1500) in the red cell membrane
is sufficient to interrupt the free-radical chain reactions (98).
Tocopherols can also inhibit the oxidations induced by the electronically excited
singlet oxygen. The relative effectiveness of ?-, ?-, ?-, and ?-tocopherols as singlet oxygen
quenchers is 100:55:26:10, respectively (99,100). In addition, tocopherols can react with
hydroxyl, perhydroxy, and superoxide radicals (101,102), as well as nitric oxide (103,104),
and form tocopheryl quinones, epoxytocopherones, tocored, and/or other oxidation products.
The antioxidant property of various tocopherols is dependent on their stoichiometric
factors, the rate of their reaction with peroxy radicals, and their concentrations. Under
certain conditions, tocopherol may exert pro-oxidant property. The pro-oxidant effect of
?-tocopherol reported in in vitro studies appears to be related to its tocopheroxy radicals
(23). When the concentration of tocopheroxy radical is high, the radicals may react reversibly
with unperoxidized lipids and lipid hydroperoxides by chain transfer and generate
alkyl and peroxy radicals, respectively.
Reactive oxygen species may react with cellular components, with resultant degradation
and/or inactivation of essential cellular constituents (105–109). The products derived
from the reaction with reactive oxygen species may be more or less reactive or harmful.
Cell membranes, which contain a relative high proportion of polyunsaturated fatty acids,
are more susceptible to free-radical–induced lipid peroxidation. The process of free-radical–
induced lipid peroxidation has been implicated as a critical initiating event leading
to cell injury or organ degeneration. There are a large variety of conditions that are capable
of initiating or enhancing oxidative stress within the cellular environment. These conditions
include inadequate intake of antioxidants, excess intake of prooxidants, exposure to
noxious chemical or physical agents, strenuous physical activities, injuries and wounds,
and certain hereditary disorders (87).
B. Antioxidant Systems
In view of the potential adverse effects of oxygen and its reactive intermediates, it is
important that a number of enzymatic and nonenzymatic antioxidant systems are present
in the cell. Under normal conditions, the overall antioxidant activity of the cell is able to
control or prevent most of the adverse effects of oxygen and its reactive intermediates.
However, when the antioxidant potential is weakened or oxidative stress is greatly increased,
irreversible damage to the cell may occur. The susceptibility of a given organ or
Vitamin E 179
Fig. 3 Relationship between vitamin E and other antioxidant systems. LH represents membrane
or polyunsaturated lipids; LOOH, lipid hydroperoxides, LOH, hydroxy acid; LOO•, peroxy radical;
L•, alkyl radical; LO•, alkoxy radical; OH•, hydroxyl radical; •O2, superoxide radical; X•, other radicals;
1O2, singlet oxygen; H2O2, hydrogen peroxide; RCHO, aldehydes; , epoxides; Vit.E•,
vitamin E radical; Vit.C•, ascorbate radical or dehydroascorbate; 6-PG, 6-phosphogluconate; GSH,
reduced glutathione; GSSG, oxidized glutathione; NADH and NAD, reduced and oxidized nicotinamide
adenine dinucleotide, respectively; NADPH and NADP, reduced and oxidized nicotinamide
dinucleotide phosphate, respectively; Se selenium; SOD, superoxide dismutase; GP, GSH peroxidase
or phospholipid hydroperoxide GSH peroxidase; GR, GSSG reductase; G-6-PD, glucose-6-
phosphate dehydrogenase; ADH, aldehyde dehydrogenase; EH, epoxide hydrase; AR, ascorbate or
dehydroascorbate reductase.
180 Chow
Fig. 4 Possible regeneration pathways of ?-tocopherol. LOOH represents lipid hydroperoxides;
LOO•, peroxy radical; GSH, reduced glutathione; GSSG, oxidized glutathione; NADH and NAD,
reduced and oxidized nicotinamide adenine dinucleotide, respectively; NADPH and NADP, reduced
and oxidized nicotinamide dinucleotide phosphate, respectively.
organ system to oxidative damage is determined by the overall balance between the extent
of oxidative stress and antioxidant capability.
Major cellular antioxidant mechanisms include (a) direct interaction with oxidants
or oxidizing agents by ascorbic acid, glutathione (GSH), and other reducing agents; (b)
scavenging of free radicals and singlet oxygen by vitamin E, ascorbic acid, carotenoids,
superoxide dismutase, and other scavengers; (c) reduction of hydroperoxides by glutathione
peroxidases and catalase; (d) binding or removal of transition metals by ferritin, transferrin,
ceruloplasmin, albumin, and other chelators; (e) separation or prevention of reactive
oxygen species and other factors from reaching the specific site of action or reacting with
essential cellular components by membrane barriers; and (f) repair of resulting damage
by dietary nutrients and metabolic activities (Fig. 3). While various antioxidant systems
act at different stages and cellular localization, vitamin E occupies a unique position in
the overall antioxidant defense (87,110,111). The essential role of vitamin E in maintaining
the red cell integrity, for example, is supported by the findings that ascorbic acid and GSH
promote oxidative damage to the vitamin E–deficient red cells, while protecting vitamin
E–supplemented red cells (111). The contradictory role of these reducing agents appears
to be the result of (a) their participation in vitamin E regeneration when certain vitamin
E levels are maintained and (b) their involvement in free-radical generation by virtue of
their maintaining transition metal ions in a reduced state (112,113) when vitamin E is low
or depleted. Approximately 1 part of ?-tocopherol is capable of protecting 1000 parts of
Vitamin E 181
lipid molecule in biological membranes (98,114). The efficacy of tocopherol is augmented
by the presence of other antioxidant systems and the factors involved in its regeneration
(Fig. 4).
VII. FUNCTIONAL INTERRELATIONSHIP WITH OTHER NUTRIENTS
It has long been recognized that the function of vitamin E is closely interrelated with other
dietary nutrients. In addition to dietary lipids, vitamin E is functionally related to the status
of polyunsaturated fatty acids, selenium, vitamin C, iron, ?-carotene, and sulfur-containing
amino acids.
A. Lipids
Cell membranes contain a high proportion of polyunsaturated fatty acids, and vitamin E
is found predominantly in the membranes. The dietary requirement for vitamin E is related
to the degree of unsaturation of the fatty acids in tissue lipids, which can be altered by
dietary lipids (115,116). The intake of tocopherols generally parallels the intake of polyunsaturated
fatty acids. Foods that contained high polyunsaturated fatty acids generally are
rich sources of tocopherols. A typical U.S. mixed diet has a ratio of approximately 0.5
mg ?-tocopherol equivalent per gram of polyunsaturated fatty acids (114).
B. Selenium
The functional interrelationships between vitamin E and selenium have long been recognized.
Selenium has been shown to prevent or reduce the severity of several symptoms
of vitamin E deficiency, including necrotic liver degeneration and eosinophilic enteritis
in rats and exudative diathesis in chicks (8,9,73). Selenium deficiency in domestic animals
is frequently associated with an increased vitamin E requirement, and the situation is often
aggravated by the oxidation of vitamin E in feed during storage.
The finding that selenium is an integral part of the enzymes GSH peroxidase (117)
and phospholipid hydroperoxide GSH peroxidase (118) provides a reasonable explanation
of the metabolic interrelationship between vitamin E and selenium. Selenium may complement
the antioxidant function of vitamin E by reducing lipid hydroperoxide (ROOH) to
corresponding hydroxy acid (ROH) via the action of GSH peroxidases (reaction 6). This
reduction prevents the decomposition of hydroperoxides to form free radicals that may
initiate further peroxidation and may thus reduce the requirement of vitamin E.
ROOH 
 2 GSH ???????>
GSH peroxidase
ROH 
 GSSG 
 H2O (6)
C. Vitamin C (Ascorbic Acid)
In addition to being an important water-soluble free-radical scavenger and reducing agent,
vitamin C may complement the function of vitamin E and reduce vitamin E requirement.
This effect is partly attributable to the involvement of vitamin C in the regeneration or
restoration of vitamin E (Fig. 4) after exerting its antioxidant function (119–122). However,
unambiguous experimental evidence for the sparing effect of vitamin C on the requirement
of vitamin E has yet to be provided (123). In addition to ascorbic acid, GSH,
in association with an enzyme or enzyme system(s), seem to be involved in the regeneration
of vitamin E (122,124). Also, a GSH-dependent dehydroascorbate reductase and an
182 Chow
NADH-semidehydroascorbate reductase appear to be involved in the regeneration or restoration
of vitamin C (125). Furthermore, dihydrolipoic acid, NADH-cytochrome b5, and
ubiquinol have been shown to play a role in vitamin E regeneration (127–129), and dihydrolipoic
acid is also involved in the regeneration of ubiquinol, ascorbate, GSH, and thioredoxin
(129). While the nature of the vitamin E regenerative process in vivo is not entirely
clear, the process does provide a rational explanation for the fact that it is very difficult
to deplete adult animals or human subjects of the vitamin (115).
D. 
-Carotene
Of the more than 500 carotenoids that have been identified, about 50 possess some vitamin
A activity (129). In the leafy green and yellow-orange vegetables, the predominant provitamin
A activity is due to a hydrocarbon carotene, ?-carotene, which makes up over 50%
of the total carotenoids. Other carotenoids, such as lycopene and lutein, are found in the
circulation but cannot be converted to retinol (130). In addition to being a precursor of
vitamin A, ?-carotene is an effective quencher of singlet oxygen and free-radical scavenger
(131–133). As a more effective scavenger of singlet oxygen, ?-carotene may complement
vitamin E in protecting cellular components against oxidative damage. ?-Carotene may
also modulate membrane properties important to cell–cell signaling or possibly serve as
an intracellular precursor to retinoic acid (134). Vitamin E has been shown to enhance
the lymphatic transport of ?-carotene and its conversion to vitamin A in the ferret (135).
On the other hand, excessive intake of ?-carotene has been reported to decrease the plasma
level of vitamin E, and excess vitamin E lowers the levels of ?-carotene (136–138).
E. Iron
Separation and removal of iron and other transition metal ions is of critical importance
in preventing iron-catalyzed generation of hydroxyl radicals from hydrogen peroxide (reaction
8). Hydrogen peroxide is readily formed from superoxide following the action of
superoxide dismutase (SOD) (reaction 7).
•O2
 
 •O2
 
 2H
 ??>
SOD
H2O2 
 O2 (7)
H2O2 
 Fe2
 ??> OH 
 OH• 
 Fe3
 (8)
Transition metal ions, particularly Fe2
 and Cu
, can exacerbate membrane damage
by catalyzing the decomposition of lipid hydroperoxides to yield more free radicals (reaction
9) and aggravate lipid peroxidation membrane damage (112,113):
ROOH ??> R•, RO•, ROO•, etc. (9)
Dietary iron exists mainly in the ferric state but is absorbed in the ferrous form.
Iron absorbed from the diet or released from ferritin can be sequestered by transferrin,
lactoferrin, citrate, ATP, and other phosphate esters. However, excess iron absorption
occurs in certain pathological conditions. The genetic disease idiopathic hemochromatosis,
for example, causes a condition similar to iron overload, and the effects of iron overload
can be lessened by increased vitamin E intake (139).
Vitamin E 183
F. Sulfur-Containing Amino Acids
It has long been recognized that muscular dystrophy, the condition that occurs in most
animal species fed a vitamin E–deficient diet, appears in the chick only when fed a diet
that contains low-sulfur amino acids (8,9). This interrelationship of vitamin E with sulfur
amino acids appears to be due to a requirement for sulfur amino acids in the synthesis
of GSH (140). GSH is needed for the activity of GSH peroxidase and for the
restoration/regeneration of vitamin E (Fig. 4). Oxidized glutathione (GSSG), in turn, is
reduced by GSSG reductase utilizing NADPH generated in the pentose shunt pathway
(reaction 10).
NADPH 
 GSSG ??????>
GSSG reductase
2GSH 
 NADP (10)
VIII. FOOD CONTENTS AND SOURCES
Tocopherols exist mainly in the free-alcohol form and are widely distributed in a variety
of plant life. A notable exception is latex lipids, in which the vast majority of tocopherols
(mainly ?-tocotrienol) are present as esters of fatty acids (10,141). Vegetable oils are the
primary dietary sources of tocopherols in the United States.
While ?-tocopherol predominates in most animal species and is significantly more
active biologically than any other form of tocopherol, ?-tocopherol is the major form
consumed in the United States. ?-Tocopherol in soybean oil, corn oil, and other vegetable
oils, accounts for over half of the estimated total tocopherol intake (Table 6). The signifi-
cant sources of vitamin E in U.S. diets include regular margarines, regular mayonnaise
and salad dressings, vitamin E–fortified breakfast cereals, vegetable shortenings, peanut
butter, eggs, cooking oils, potato chips, whole milk, tomato products, and apples (142).
Sheppard et al. (143) have summarized the tocopherol content and vitamin E activity in
various food products based on recent information.
IX. REQUIREMENTS AND ASSESSMENTS
A. Requirements
In 1968, vitamin E was officially recognized as an essential nutrient, and 30 IU was recommended
for men by the Food and Nutrition Board. Reassessment of dietary fat and vitamin
E content led in 1974 to a revised recommendation of 15 IU. In the 1980 recommended
dietary allowance (RDA), efforts were made to take into account the dietary contribution
of non-?-tocopherols, and the requirement was expressed as tocopherol equivalent (TE).
One TE is equal to 1 mg RRR-?-tocopherol, and the other isomers were converted to TE
by multiplying the 1 mg of each isomer by the relative activity factor (e.g., ?-tocopherol,
0.5; ?-tocopherol, 0.1; ?-tocotrienol, 0.3). The tenth RDA expressed in both TE and IU
is shown in Table 7, and the values ranged from 4.5 IU (three ?-tocopherol equivalents
or 3 mg d-tocopherol) in infants, 6.0–10.4 in children (10 years or under), 14.9 in males
(11 years), 11.9 in females (11 years), 14.9 in pregnant women, 16.4 to 17.9 in lactating
women (144). The normal intake of vitamin E in U.S. diets ranges between 4 and 33
TE daily in adults not taking vitamin E supplements, with average values between 11 and
13 TE. This level of intake results in average plasma/serum levels in adults of approximately
9.5 ug ?-tocopherol/mL (9).
184 Chow
Table
6
Tocopherol
Content
of
Major
Fats
and
Oils
Used
in
Food
Products
in
the
United
States
Individual
tocopherolb
(%
of
total)
%
Total
Total
tocopherol
Type
of
fats
and
oils
fats/oilsa
(mg/100
g)
?-T
?-T-3
?-T
?-T-3
?-T ?-T-3 ?-T ?-T-3
Animal
fats:
Lard
13.3
0.6–1.3
90
5
—
—
—
—
—
—
Butter
8.2
1.0–5.0
90
—
—
—
5—
—
—
Tallow
4.9
1.5–2.4
90
—
—
—
10
—
—
—
Vegetable
oils:
Soybean
53.4
56
–
160
4
–
18
—
—
—
58
–
69
—
—
—
Cotton
seed
8.9
30
–
81
51
–
67
—
—
—
33
–
49
—
—
—
Corn
3.8
53
–
162
11
–
24
—
—
—
76
–
89
—
—
—
Coconut
3.1
1
–
4
14
–
67
14
—
3—
53
17
—
Peanut
1.4
20
–
32
48
–
61
—
—
—
39
–
52
—
—
—
Palm
1.0
33
–
73
28
–
50
16
–
19
—
4
—
34
–
39
—
—
Palm
kernel
0.7
0
—
—
—
—
—
—
—
—
Safflower
0.7
25
–
49
80
–
94
—
—
—
6
–
20
—
—
—
Olive
0.6
5
–
15
65
–
85
—
—
—
15
–
35
—
—
—
a
53.4
pounds
per
capita.
b
?-T,
?-T,
?-T,
and
?-T
are
?-,
?-,
?-,
and
?-tocopherol,
respectively;
?-T-3,
?-T-3,
?-T-3,
and
?-T-3
are
the
corresponding
tocotrienols.
After
Ref.
10.
Vitamin E 185
Table 7 U.S. Recommended Daily Dietary Allowance for
Vitamin E
International Tocopherol
Age (years) units equivalents
Infants 0–0.5 4.5 3.0
Children 0.5–1.0 6.0 4.0
1–3 8.9 6.0
4–10 10.4 7.0
Males 11 14.9 10.0
Females 11 11.9 8.0
Pregnant 14.9 10.0
Lactating, first
6 mo. 17.9 12.0
Lactating, second
6 mo. 16.4 11.0
Vitamin E deficiency is rarely seen in the population of the United States. When it
occurs, it is usually a result of lipoprotein deficiencies or lipid malabsorption syndromes
(11,12,26). A high intake of polyunsaturated fat may increase the vitamin E requirement.
Increased levels have also been recommended to provide protection against oxidants and
oxidizing agents in the environment. In addition, recent information suggests that a higher
intake of vitamin E is associated with enhanced immune response and reduced risk of
certain chronic diseases, particularly coronary heart disease (see Sec. X).
B. Assessments
The nutritional status of vitamin E is assessed based on the concentration of tocopherols
in body store and its physiological functions. Currently, several indices, such as tocopherol
concentrations in plasma/serum erythrocytes, platelets, or tissues; degree of erythrocyte
hemolysis, and amounts of lipid peroxidation products (e.g., ethane, pentane, and malondialdehyde)
generated, have been used to assess the nutritional status of vitamin E in humans.
Measurement of tocopherol concentrations in plasma/serum is the most commonly
used approach. The tocopherol concentration in erythrocytes is an indicator of longer term
status for vitamin E than that in plasma/serum. Platelets are more sensitive for measurement
of dose response to dietary vitamin E when compared with plasma, erythrocytes, or
lymphocytes (145). Also, platelet tocopherol concentrations are independent of serum lipid
levels (146), an important advantage relative to serum or plasma tocopherol concentrations.
However, none of the above accurately reflects both dietary intake and body stores
of vitamin E. Measurement of ?-tocopherol tissue store, such as adipose tissue, is a reliable
way to assess long-term vitamin E status in humans. However, tissue biopsy is highly
invasive and therefore impractical for application to the general public. Measurement of
tocopherol concentrations in the relatively noninvasive and easily collectible skin and
buccal mucosal cells has shown that the nutritional status of vitamin E, and the concentrations
of ?- and ?-tocopherol are significantly correlated with that of the plasma (147).
The significant plasma–tissue relationships suggest that plasma tocopherol concentrations,
in lieu of their target tissue concentrations, can be used as a reliable indicator of vitamin
E status.
186 Chow
Long-term intake of vitamin E is difficult to assess accurately because diets vary
substantially over time. Also, since tocopherols are widely distributed in foods and products
made from them, their consumption is difficult to quantify based on a dietary history.
Potential sources of error in the use of interviews for obtaining food/nutrient intake information
are as follows: (a) only recent intake is estimated, whereas earlier dietary intake
may be very different; (b) a limited number of foods were analyzed; and (c) errors may
have occurred in subject estimation of food intake frequency and size of portion eaten.
The accuracy of vitamin E intake information obtained through the use of frequency
questionnaires also depends on the reliability of information in the food composition data
used. The tocopherol content of foods is highly variable, depending genetic, seasonal,
processing, storage, and other factors. Also, the tocopherol content varies greatly among
foods, and the tocopherol contents of various foods were obtained using colorimetry procedure
rather than HPLC. In addition, food processing, storage, and culinary practices can
significantly influence the destruction of tocopherols present in the food. Furthermore, the
possible interaction of vitamin E and other nutrients, such as polyunsaturated lipids, ?-
carotene, selenium, sulfur amino acids, and ascorbic acid, further complicates evaluation
of vitamin E status.
X. HEALTH EFFECTS
A. Immune Response
The immune response generally declines with age, and suboptimal immune responses in
the elderly may be responsible for the increased risk of infections and immune-mediated
diseases (148). Therefore, maintaining proper immune function is important for the elderly.
Several indices of immune response, including responses on delayed-type hypersensitivity
skin tests, antibody production, lymphocyte proliferation, cytokine production, and
counts of the specific subgroups of white blood cells, are influenced by the status of essential
nutrients, including vitamin E. Vitamin E supplementation is associated with enhanced
production of the cytokine interleukin-2 (IL-2), enhanced lymphocyte proliferation, and
decreased production of the immunosuppressive prostaglandin E2 (149). The antioxidant
property is likely to play a role in the immunostimulatory effects of vitamin E for the
immune response. For example, vitamin E supplementation (60, 200, or 800 IU/day) for
a month or longer resulted in significant increases in delayed-type hypersensitivity skin test
response, and T-cell subpopulations and proliferative responses, as well as IL-2 activity in
the healthy, free-living elderly (150,151,151a). These findings suggest that current dietary
vitamin E intake levels of 10–15 IU/day are insufficient to attain optimal immune responses
in the general population. However, the 800 IU/day supplement group showed
lower immune responses on some tests than the 200 IU/day group, and was comparable
with the 60 IU/day or the placebo group, suggesting that more is not necessarily better
(151a,151b).
B. Cardiovascular Disease
Cardiovascular disease is the leading cause of morbidity and mortality in industrialized
countries. Several dietary factors, including decreased vitamin E status, have been implicated
in the incidence of coronary heart disease. In recent years, several large-scale obserVitamin
E 187
vational and experimental studies have shown that higher intake or serum/plasma levels
of ?-tocopherol are associated with a decreased risk of cardiovascular diseases. For example,
in a study that included 87,245 female nurses and 39,910 male health professionals, the
age-adjusted relative risk of coronary heart disease was found to be significantly lowered in
the highest quintile of vitamin E intake compared with the lowest quintile (132,153). The
apparent benefit was attributable mainly to subjects consuming 100 IU vitamin E or more
daily for 2 years or longer. In another study, which included 21,809 postmenopausal
women, higher food-derived vitamin E intake was associated with decreased death rate
due to coronary heart disease (154). Results obtained from intervention studies generally
corroborated with the epidemiological data. In the Cambridge Heart Antioxidant Study
(155), for example, patients with angiographically proven coronary atherosclerosis receiving
400 or 800 IU of vitamin E for 510 days had a significant reduction of the rate of
major cardiovascular events (47%) and of nonfatal myocardial infarction (77%), in comparison
with those receiving placebo. However, cardiovascular deaths were not signifi-
cantly different between groups.
While the information available suggests that increased intake of vitamin E is associated
with reduced risk of cardiovascular disease, the mechanism by which vitamin E reduces
the risk of cardiovascular disease is not clear. It is possible that the ability of vitamin
E to prevent LDL oxidation (156,157) and/or to reduce platelet adhesion (158,159) may
be responsible. Evidence indicates that enhanced uptake of oxidatively modified LDL by
macrophages through a specific receptor gives rise to lipid-laden foam cells, one of the
earliest stages in the development of atherosclerosis (160–162). Oxidized LDL is toxic
to cells and may be responsible for damage to the endothelial layer and destruction of
smooth-muscle cells (160,162).
C. Cancer
Carcinogenesis is a multiple process, and a large number of chemical and physical agents
can affect the development of cancer in numerous ways. Vitamin E may protect against
cancer development by reacting directly with mutagens/carcinogens, altering metabolic
activation, enhancing the immune system, inhibiting cell proliferation, or other mechanisms.
A large number of observational and experimental studies have been performed
to determine the relationship between vitamin E status and cancer risk (163,164). Also, a
number of well-designed large-scale chemoprevention trials for antioxidant micronutrients
have been conducted in recent years (165–167). However, only a few of those intervention
studies are designed specifically to examine the efficacy of vitamin E. Most of the studies
dealing with head/neck, lung, and colorectal cancer suggest a protective effect of vitamin
E against cancer risk, though reports dealing with the association between vitamin E status
and risk of breast, bladder, and cervical cancer are less consistent (168). Nevertheless,
the majority of studies suggest that low vitamin E status is associated with increased risk
of certain cancer.
D. Neurodegenerative Diseases
Neurodegenerative diseases, such as dementia of Alzheimer type and Parkinson’s disease,
are characterized neurochemically by a transmitter-specific loss of neurons, which progresses
and extends to several neuronal systems over the course of the disease. While the
188 Chow
pathophysiology of the heterogenous disorder is largely unknown, free radical-induced
oxidative damage may play a role in the cellular events leading to the degeneration of
neurons in the brain. In patients with Alzheimer disease, for example, amyloid beta-peptide
accumulates in plaques of the brain. The compound is neurotoxic by a mechanism involving
induction of reactive oxygen species. The concentration of ?-tocopherol is lower in
cerebrospinal fluid from patients with Alzheimer type dementia that the control (169).
Also, the concentrations of basal and iron-ascorbate stimulated lipid peroxidation products
are higher in the inferior temporal cortex of Alzheimer subjects than the controls (170).
These reports suggest that the brain of patients with Alzheimer disease is associated with
inadequate antioxidant status and/or increased oxidative stress. In a double-blind,
multicenter trial, Sano et al. (171) evaluated the effect of ?-tocopherol (2,000 IU daily)
on primary outcome in 341 patients with Alzheimer’s disease of moderate severity over
a 2 year period and found that treatment with ?-tocopherol was beneficial in delaying the
primary outcome of disease progression (death, severe dementia, loss of the ability to
perform basic activities of daily living or the need for institutionalization). The degree of
functionality of the mutant ?-tocopherol transfer protein in patients with familial vitamin
E deficiency are associated with the degree of severity of the neurological damage and
age of onset as well as with plasma vitamin E concentration (59). High doses of vitamin
E can prevent or mitigate the neurological course of this disease. Plasma vitamin E concentrations
in patients increase when they are treated with large doses of vitamin E, presumably
because of the direct transfer of tocopherol from chylomicrons to other circulating
lipoproteins.
XI. SUMMARY AND CONCLUSION
The essentiality of vitamin E for humans had been questioned after the vitamin was identified
as the fat-soluble substance necessary for reproduction in rats more than 75 years
ago. This is because a number of tissue-specific deficiency symptoms can be easily produced
in experimental animals but not in humans. The essentiality of vitamin E for humans
was only recognized in the late 1960s. Subsequent studies of patients with certain
neurological disorders have clearly demonstrated the need of the vitamin in the development
and maintenance of the integrity and function of the nervous mechanism and skeletal
muscle.
Recent studies also provide a much better understanding of the mechanisms of tocopherol
transport and selective retention of certain forms or isomers of tocopherols. The
finding that tocopherol can be regenerated or restored in association with GSH, ascorbic
acid, and other systems provides a feasible explanation as to why it is difficult to deplete
vitamin E in human adults. Information is available that supports the view that the function
of vitamin E is interrelated with other antioxidant systems and that ascorbic acid, ?-
carotene, sulfur amino acids, and selenium complement the antioxidant function of vitamin
E.
In recent years, public interest in vitamin E has increased drastically. This is mainly
promoted by experimental findings which suggest that vitamin E may protect against oxidative
stress resulting from exposure to environmental agents, and that increased intake
of the vitamin is associated with a reduced risk of cardiovascular disease, cancer, and
other degenerative diseases, as well as with enhanced immune response. However, more
research is needed to establish more definitively the relationship between vitamin E intake
and the risk of degenerative diseases, and to elucidate further the mechanisms by which
Vitamin E 189
vitamin E is involved in altering disease processes. In addition, the optimal requirement
of various population groups for vitamin E against oxidative stress has yet to be established.
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5
Bioorganic Mechanisms Important to
Coenzyme Functions
DONALD B. McCORMICK
Emory University, Atlanta, Georgia
I. INTRODUCTION
A. Definition
With the use (and misuse) of terms over significant periods of time, there is sufficient
uncertainty regarding a specific word that it is best to avoid confusion by defining it. This
is the case for the word ‘‘coenzyme,’’ which together with ‘‘cofactor’’ and ‘‘prosthetic
group’’ have to some become ambiguous biochemical jargon (1). As defined succinctly,
a coenzyme is a natural, organic molecule that functions in a catalytic enzyme system
(2). Coenzymes bind specifically within protein apoenzymes to constitute catalytically
competent holoenzymes. Hence, coenzymes are organic cofactors that augment the diversity
of reactions that otherwise would be limited to chemical properties, principally simple
acid–base catalysis, of side-chain substituents from amino acid residues within enzymes.
With tight binding, coenzymes may be referred to as prosthetic groups; with loose binding,
they may be called cosubstrates.
The focus of this chapter will be on the molecular means or mechanisms by which
coenzymes participate as the loci of bond making and breaking steps in holoenzymecatalyzed
reactions. Most coenzymes are more complex metabolic derivatives of watersoluble
vitamins. Since the subject of this volume is vitamins, coverage of coenzyme
mechanisms will bear emphasis on those coenzymes derived from such vitamins covered
in other regards in subsequent chapters. References are sparsely used for typical or recent
material that otherwise has largely become the domain of mechanistically inclined textbooks
of biochemistry (3,4), especially when set for the more advanced student or professional
(5).
199
200 McCormick
B. Groupings
Attempts to place each coenzyme in a singular mechanistic group are sometimes fraught
with difficulty because some can arguably fit into more than one category. For example,
the lipoyl residues of transacylases undergo oxidation–reduction as well as participate in
acyl transfers, but it is the latter that distinguishes the biological function. In at least a
few instances, the tetrahydro forms of pterin coenzymes are oxidized to the dihydro level
during operations of the enzymic systems, e.g., 5,10-methylene tetrahydrofolate in thymidylate
synthase and tetrahydrobiopterin in phenylalanine hydroxylase. More conventionally,
folyl coenzymes are pulled together in a broad mechanistic view under one-carbon
transfers.
The six groups that are subdivisions of this chapter reasonably imply the mechanistic
and functional connections of principal coenzymes. These are oxidation–reduction reactions,
generation of leaving group potential, acyl activation and transfer, carboxylations,
one-carbon transfers, and rearrangements on vicinal carbons. These groupings follow a
conventional pattern of biochemical recognition of what major purposes are served by
vitamin-derived coenzymes.
II. OXIDATION–REDUCTION REACTIONS
A. Nicotinamide Coenzymes
Nicotinamide adenine dinucleotide (NAD) and its phosphate (NADP) are the two natural
pyridine nucleotide coenzymes derived from niacin, as discussed in Chapter 6. Both contain
an N(1)-substituted pyridine-3-carboxamide (nicotinamide) that is essential to function
in redox reactions with a potential near 
0.32 V. The nicotinamide coenzymes function
in numerous oxidoreductase systems, usually of the dehydrogenase/reductase type,
which include such diverse reactions as the conversion of alcohols (often sugars and polyols)
to aldehydes or ketones, hemiacetals to lactones, aldehydes to acids, and certain amino
acids to keto acids (6). The general reaction stoichiometry is written as:
Substrate  NAD(P)
Bproduct  NAD(P)H  H
The common stereochemical mechanism of operation of nicotinamide coenzymes
when associated with enzymes involves the stereospecific abstraction of a hydride ion
(H
) from substrate, with para addition to one or the other side of C-4 in the pyridine
ring of the coenzyme, as shown in Fig. 1. The second hydrogen of the substrate group
oxidized is concomitantly removed, typically from an electronegative atom (e.g., N, O,
or S), as a proton (H) that ultimately exchanges as a hydronium ion. The sidedness of
the pyridine ring is such that the para hydrogen up from the plane, when the carboxyamide
function is near (or to the right) and the pyridine N is on the left (or at the bottom), is
Fig. 1 The stereospecific hydride ion transfer to and from nicotinamide coenzymes generating
prochiral R and S forms.
Mechanisms for Coenzyme Function 201
prochiral R because it is oriented toward the re face (A side). The hydrogen down from
the plane of the ring is then prochiral S and oriented toward the si face (B side). There
are numerous examples of nicotinamide coenzyme–dependent enzymes that are stereospecific
as regards addition/removal of hydride ion from the prochiral R (A) or S (B) position
relative to the pyridine ring. These include, for A sidedness, NAD-dependent alcohol
dehydrogenase and NADP-dependent cytoplasmic isocitrate dehydrogenase; for B sidedness,
NAD-dependent d-glyceraldehyde-3-phosphate dehydrogenase and NADP-dependent
d-glucose-6-phosphate dehydrogenase (7). In general, A-side dehydrogenases bind
a conformation of the coenzyme in which the nicotinamide ring has an anti orientation
with respect to the ribosyl ring, i.e., the carboxamide groups points away; B-side dehydrogenases
usually have syn conformation. This is probably due to stabilization of the dihydronicotinamide
in the boat conformation attributable to orbital overlap between the lone
n pair of electrons on the pyridine nitrogen and the antiboding ? orbital of the ribosyl CO
moiety, as depicted in Fig. 2. The pseudoaxial hydrogens, either pro-R with anti or
pro-S with syn orientation, are then more easily transferred because of orbital overlap in
the transition state.
Nicotinamide coenzymes also participate in other (nonredox) biological reactions
that involve ADP ribosylations; however, these are properly considered as substrate rather
than coenzymic functions. The larger nucleotide-like structure of nicotinamide coenzymes
is important in regard to coenzymic roles primarily to facilitate recognition and specific
binding by the protein apoenzymes with which they interact.
B. Flavocoenzymes
Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and covalently linked
flavocoenzymes (usually 8?-substituted FAD) are the natural coenzymes derived from
riboflavin (vitamin B2), which is discussed in Chapter 7. All contain an isoalloxazine ring
system with a redox potential near
0.2 V when free in solution, but the potential is subject
to considerable variation when bound within functional flavoproteins. This propensity for
being able to shift potential gives flavoenzymes a wider operating range for oxidation–
reduction reactions than is the case for enzymes dependent on nicotinamide coenzymes.
Flavoproteins are variably able to catalyze both one- and two-electron redox reactions.
The ring portion involving nitrogens 1 and 5 and carbon 4a reflects sequential addition
or loss of electrons and hydrogen ions in one-electron processes and addition or
loss of other transitory adducts with two-electron processes. There are nine chemically
discernible redox forms, i.e., three levels of oxidation–reduction and three species for
acid, neutral, and base conditions. Of these only five have biological relevance because
Fig. 2 The syn and anti conformations of bound nicotinamide coenzymes that putatively transfer
pro-(S) and pro-(R) hydrogens, respectively.
202 McCormick
Fig. 3 Biologically important redox states of flavocoenzymes with pKa values for interconversions
of the free species.
of pH considerations (2). These biological forms are summarized in Fig. 3. It should also
be noted that free flavin semiquinones (radicals) are quite unstable, and the free flavin
hydroquinones react very rapidly with molecular oxygen to become reoxidized. This latter
is kinetically dissimilar to reduced nicotinamide coenzymes, which reoxidize more slowly
with O2. Binding to specific enzymes markedly affects the kinetic stability of the halfreduced
and reduced forms of flavocoenzymes and again allows for diverse reactions under
biological situations. An example of a flavoprotein undergoing a one-electron transfer is
with the microsomal NADPH–cytochrome P450 reductase. This enzyme contains both
FAD and FMN, which latter cycles between a neutral semiquinone and fully reduced form
(8). The FAD in the electron-transferring flavoprotein, which mediates electron flow from
fatty acyl coenzyme A (CoA) to the mitochondrial electron transport chain, cycles between
oxidized quinone and anionic semiquinone. In addition, a single-step, two-electron transfer
from substrate can occur in the nucleophilic reactions shown in Fig. 4. Such cases as
hydride ion transfer from reduced nicotinamide coenzymes or the carbanion generated by
Fig. 4 Reaction types encountered with flavoquinone coenzymes and natural nucleophiles to generate
N-5 and C-4a adducts as intermediates.
Mechanisms for Coenzyme Function 203
base abstraction of a substrate proton may lead to attack at the flavin N-5 positions; some
nucleophiles, such as the hydrogen peroxide anion, are added on the C-4a position with
its frontier orbital.
As regards stereochemistry, the transfer of hydrogen on and off N-5 can take place
to or from one or the other face of the isoalloxazine ring system. When visualized with
the benzenoid ring to the left and side chain at top (as in the top of Fig. 3), orientation
is to the si face with re on the opposite side. A fair number of flavoproteins have now
been categorized on this steric basis (7). Examples include the FAD-dependent glutathione
reductase from human erythrocytes, which uses NADPH as substrate and is re side interacting,
and the FMN-dependent spinach glycolate oxidase, which is si side interacting. It
should be noted that reduced flavins free in solution have bent or ‘‘butterfly’’ conformations,
since the dihydroisoalloxazine has a 144° angle between benzenoid and pyrimidinoid
protions. The orientations taken when bound to enzymes, however, may vary and
can influence the redox potential.
There are flavoprotein-catalyzed dehydrogenations that are both nicotinamide coenzyme–
dependent and independent, reactions with sulfur-containing compounds, hydroxylations,
oxidative decarboxylations, dioxygenations, and reduction of O2 to hydrogen peroxide.
The diversity of these systems is covered in periodic symposia on flavins and
flavoproteins (9). The intrinsic ability of flavins to be variably potentiated as redox carriers
upon differential binding to proteins, to participate in both one- and two-electron transfers,
and in reduced (1,5-dihydro) form to react rapidly with oxygen permits wide scope in
their operation.
III. GENERATION OF LEAVING GROUP POTENTIAL
A. Thiamine Pyrophosphate
Thiamine pyrophosphate (TPP) is the coenzyme derived from thiamine (vitamin B1),
which is discussed in Chapter 8. Though the substituted pyrimidyl portion shares a role
in apoenzymic recognition and binding, the thiazole moiety not only is important in that
regard but is involved with substrates to provide a transitory matrix that provides good
leaving group potential (2,10). Specifically, TPP as a Mg2 ternary complex within enzymes
can react as an ylid, resonance-stabilized carbanion that attacks carbonyl functions
as illustrated in Fig. 5.
In all cases, the bond to be labilized (from R? to the C of the original carbonyl carbon)
must be oriented for maximal ?-? orbital overlap with the periplanar system extending to
the electron-deficient quaternary nitrogen of the thiazole ring. There are two general types
of biological reactions in which TPP functions in so-called active aldehyde transfers. First,
in decarboxylation of ?-keto acids, the condensation of the thiazole moiety of TPP with
the ?-carbonyl carbon on the acid leads to loss of CO2 and production of a resonancestabilized
carbanion. Protonation and release of aldehyde occur in fermentative organisms
such as yeast, which have only the TPP-dependent decarboxylase, but reaction of the ?-
hydroxalkyl-TPP with lipoyl residues and ultimate conversion to acyl-CoA occurs in
higher eukaryotes, including humans with multienzymic dehydrogenase complexes (see
Sec. IV.8). The other general reaction involving TPP is the transformation of ?-ketols
(ketose phosphates). Specialized phosphoketolases in certain bacteria and higher plants
can split ketose phosphates, e.g., d-xylulose-5-phosphate, to simpler, released products,
e.g., d-glyceraldehyde-3-phosphate and acetyl phosphate. However, the reaction of impor204
McCormick
Fig. 5 Function of the thiazole moiety of thiamine pyrophosphate. In ?-keto acid decarboxylations,
R? is a carboxylate lost as CO2 and R? is a proton generating an aldehyde. In ketolations
(both trans- and phospho-), R and R? are part of a ketose phosphate generating a bound glycoaldehyde
intermediate and R? is an aldose phosphate or inorganic phosphate generating a different ketose
phosphate or acetyl phosphate, respectively.
tance to humans and most animals is a transketolation. Transketolase is a TPP-dependent
enzyme found in the cytosol of many tissues, especially liver and blood cells, where principal
carbohydrate pathways exist. This enzyme catalyzes the reversible transfer of a glycoaldehyde
moiety (?,?-dihydroxyethyl-TPP) from the first two carbons of a donor ketose
phosphate, i.e., d-xylulose-5-phosphate, to the aldehyde carbon of an aldose phosphate,
i.e., d-ribose-5-phosphate, of the pentose phosphate pathway wherein d-sedoheptulose-7-
phosphate and d-glyceraldehyde-3-phosphate participate as the other substrate–product
pair.
B. Pyridoxal-5?-Phosphate
Two of the three natural forms of vitamin B6, which is discussed in Chapter 10, can be
phosphorylated to yield directly functional coenzymes, i.e., pyridoxal-5?-phosphate (PLP)
and pyridoxamine-5?-phosphate (PMP). PLP is the predominant and more diversely functional
coenzymic form, although PMP interconverts as coenzyme during transaminations
(2–5,11). At physiological pH, the dianionic phosphates of these coenzymes exist as zwitterionic
meta-phenolate pyridinium compounds. Both natural and synthetic carbonyl reagents
(e.g., hydrazines and hydroxylamines) form Schiff bases with the 4-formyl function
of PLP, thereby removing the coenzyme and inhibiting PLP-dependent reactions.
PLP functions in numerous reactions that embrace the metabolism of proteins, carbohydrates,
and lipids. Especially diverse are PLP-dependent enzymes that are involved in
amino acid metabolism. By virtue of the ability of PLP to condense its 4-formyl substituent
Mechanisms for Coenzyme Function 205
with an amine, usually the ?-amino group of an amino acid, to form an azomethine (Schiff
base) linkage, a conjugated double-bond system extending from the ? carbon of the amine
(amino acid) to the pyridinium nitrogen in PLP results in reduced electron density around
the ? carbon. This potentially weakens each of the bonds from the amine (amino acid)
carbon to the adjoined functions (hydrogen, carboxyl, or side chain). A given apoenzyme
then locks in a particular configuration of the coenzyme–substrate compound such that
maximal overlap of the bond to be broken occurs with the resonant, coplanar, electronwithdrawing
system of the coenzyme complex. These events are depicted in Fig. 6.
Selection of the ? bond to be cleaved (R?-C) is achieved by stabilizing a conformation
of the external aldimine in which the leaving group is orthogonal to the plane of the
ring system, thus ensuring maximal orbital overlap with the ? system in the transition
state. The type of stereochemistry involved is characteristic of a given enzyme system.
Both re and si faces of the PLP complex can orient toward a reactive function, such
as a base, contributed by enzyme protein. This is the case, for instance, with aspartate
aminotransferase whereby a base function abstracts a proton from the ? position and
transfers it to the same side of the ? system (syn transfer), adding it to the si face of the
azomethine group in the ketimine. In this case, the hydrogen is incorporated in a pro-S
position at carbon 4? (the methylene of PMP).
Aminotransferases effect rupture of the ?-hydrogen bond of an amino acid with
ultimate formation of an ?-keto acid and PMP; this reversible reaction proceeds by a
double-displacement mechanism and provides an interface between amino acid metabolism
and that for ketogenic and glucogenic reactions. Amino acid decarboxylases catalyze
breakage of the ?-carboxyl bond and lead to irreversible formation of amines, including
several that are functional in nervous tissue (e.g., epinephrine, norepinephrine, serotonin,
and ?-aminobutyrate). The biosynthesis of heme depends on the early formation of ?-
aminolevulinate from PLP-dependent condensation of glycine and succinyl-CoA followed
by decarboxylation. There are many examples of enzymes, such as cysteine desulfhydrase
and serine hydroxymethyltransferase, that affect the loss or transfer of amino acid side
chains. PLP is the essential coenzyme for phosphorylase that catalyzes phosphorolysis of
the ?-1,4 linkages of glycogen. An important role in lipid metabolism is the PLP-dependent
condensation of l-serine with palmitoyl-CoA to form 3-dehydrosphinganine, a precursor
of sphingolipids. The diversity of PLP functions is covered in periodic symposia
Fig. 6 Operation of pyridoxal-5?-phosphate with a biological primary amine. Loss of R? shown
from the aldimine can be generalized for side-chain cleavages and eliminations, loss of CO2 in
decarboxylations, or loss of a proton in aminotransferations and racemizations.
206 McCormick
that now include other carbonyl compounds as cofactors (pyruvyl enzymes, quinoproteins,
etc.) (12).
IV. ACYL ACTIVATION AND TRANSFER
A. Phosphopantetheine Coenzymes
4?-Phosphopantetheine is a phosphorylated amide of ?-mercaptoethylamine and the vitamin
pantothenate, which is discussed in Chapter 9. The phosphopantetheinyl moiety serves
as a functional component within the structure of CoA and as a prosthetic group covalently
attached to a seryl residue of acyl carrier protein (ACP). Because of a thiol (sulfhydryl)
terminus with a pKa near 9, phosphopantetheine and its coenzymic forms are readily oxidized
to the catalytically inactive disulfides.
Esterification of the thiol function to many carboxylic acids is the prelude to numerous
acyl group transfers and enolizations (2–5). In classic terminology, the thiol ester
enhances both ‘‘head’’ and ‘‘tail’’ activations of acyl compounds, as illustrated in Fig.
7. In head activation, the carbonyl function is attacked by a nucleophile and releases the
original thiol. The carbonyl carbon of a thiol ester is more positively polarized than would
be an oxy ester counterpart. Hence, the acyl moiety is relatively activated for transfer.
The phosphopantetheine terminus of CoA and ACP both function in acyl transfers, the
latter only within the fatty acid synthase complex. In tail activation, there is greater tendency
for thio than oxy esters to undergo enolization by permitting removal of an ? hydrogen
as a proton. The enolate is stabilized by delocalization of its negative charge between
the ? carbon and the acyl oxygen, which makes it thermodynamically accessible as an
intermediate. Since the developing charge is also stabilized in the transition state that
precedes the enolate, it is also kinetically accessible. This process leads to facile condensation
whereby nucleophilic addition of the carbanion-like carbon of the enolate to a neutral
activated acyl group (another thiol ester) is a favored process. CoA is a good leaving
group from the tetrahedral intermediate. From the above, it follows that thio esters are
more like a ketone than an oxy ester. The degree of resonance electron delocalization
from the overlap of sulfur p orbitals with the acyl ? bond is less than with oxygen in oxy
esters.
The myriad acyl thio esters of CoA are central to the metabolism of numerous compounds,
especially lipids and the penultimate catabolites of carbohydrates and ketogenic
Fig. 7 Activations of acyl moieties enhanced by formation of thio esters with phosphopantetheine
coenzymes.
Mechanisms for Coenzyme Function 207
amino acids. For example, acetyl-CoA, which is formed during metabolism of carbohydrates,
fats, and some amino acids, can acetylate compounds such as choline and hexosamines
to produce essential biochemicals. It can also condense with other metabolites, such
as oxalacetate, to supply citrate, and it can lead to formation of cholesterol. The reactive
sulfhydryl termini of ACP provide exchange points for acetyl-CoA and malonyl-CoA. The
ACP-S-malonyl thio ester can chain-elongate during fatty acid biosynthesis in a synthase
complex.
B. Lipoic Acid
?-Lipoic (thioctic) acid is not a vitamin for the human and other animals because it can
be biosynthesized from longer chain, essential fatty acids. However, it is indispensable
in its coenzymic role, which interfaces with some of the functions of TPP and CoA (2–
4). The natural d isomer of 6,8-dithiooctanoic (1,2-dithiolane-3-pentanoic) acid occurs in
amide linkage to the -amino group of lysyl residues within transacylases that are core
protein subunits of ?-keto acid dehydrogenase complexes of some prokaryotes and all
eukaryotes. In such transacylases, the functional dithiolane ring is on an extended flexible
arm. The lipoyl group mediates the transfer of the acyl group from an ?-hydroxyalkyl-
TPP to CoA in a cyclic system that transiently generates the dihydrolipoyl residue, as
shown in Fig. 8. Hence, the lipoyl/dihydrolipoyl pair in this cycle serves a dual role of
electron transfer and acyl group vector by coupling the two processes.
The three ?-keto acid dehydrogenase complexes of mammalian mitochondria are
for pyruvate and ?-ketoglutarate of the Krebs citric acid cycle and for the branched-chain
?-keto acids from some amino acids. All involve participation of lipoyl moieties within
core transacylase subunits surrounded by subunits of TPP-dependent ?-keto acid decarboxylase,
which generate ?-hydroxyalkyl-TPP, and are further associated with subunits
of FAD-dependent dihydrolipoyl (lipoamide) dehydrogenase. The number of subunits and
their packing varies among cases. For instance, a single particle of E. coli pyruvate dehy-
Fig. 8 Function of the lipoyl moiety within enzymes involved in transacylations following ?-
keto acid decarboxylations (see Fig. 5). In multienzyme dehydrogenases, there is transfer of an acyl
moiety from an ?-hydroxyalkyl thiamine pyrophosphate to the lipoyl group of a transacylase core
and thence to CoA. This results in formation of the dihydrolipoyl group, which is cyclically reoxidized
by the FAD-dependent dihydrolipoyl dehydrogenase.
208 McCormick
Fig. 9 Function of the biotinyl moiety within enzymes involved in carboxylations with the intermediacy
of the putative carbonyl phosphate.
drogenase consists of at least 24 chains of each decarboxylase and transacetylase plus 12
chains of the flavoprotein.
V. CARBOXYLATIONS
A. Biotin
The characteristics of the vitamin biotin are discussed in Chapter 11. The coenzymic form
of this vitamin occurs only as the vitamin with its valeric acid side chain amide-linked
to the ?-amino group of specific lysyl residues in carboxylase and transcarboxylase (2–
5). The length of this flexible arm (
14 A° ) is similar to that encountered with the lipoyl
attachments in transacylases. Biotin-dependent carboxylases operate by a common mechanism
illustrated in Fig. 9. This involves tautomerization of the ureido 1?-N to enhance
its nucleophilicity. Though the two ureido nitrogens are essentially isoelectronic in the
imidazoline portion of biotin, the steric crowding of the thiolane side chain near the 3?-
N essentially prevents chemical additions to position 3?. Phosphorylation of bicarbonate
by ATP to form carbonyl phosphate provides an electrophilic mixed-acid anhydride. This
latter can then react at the nucleophilically enhanced 1?-N to generate reactive N(1?)-
carboxybiotinyl enzyme. This in turn can exchange the carboxylate function with a reactive
center in a substrate, typically at a carbon with incipient carbanion character (13).
There are nine known biotin-dependent enzymes: six carboxylases, two decarboxylases,
and a transcarboxylase. Of these, only four carboxylases have been found in tissues of
humans and other mammals. Acetyl-CoA carboxylase is a cytosolic enzyme that catalyzes
formation of malonyl-CoA for fatty acid biosynthesis. Pyruvate carboxylase is a mitochondrial
enzyme that forms oxalacetate for citrate formation. Propionyl-CoA carboxylase
forms the d isomer of methylmalonyl-CoA on a pathway toward succinyl-CoA in the
Krebs cycle. Finally, ?-methylcrotonyl-CoA carboxylase forms ?-methylglutaconyl-CoA
in the catabolic pathway from l-leucine.
VI. ONE-CARBON TRANSFER: TETRAHYDROFOLYL COENZYMES
Among natural compounds with a pteridine nucleus, those most commonly encountered
are derivatives of 2-amino-4-hydroxypteridines, which are trivially named pterins. HuMechanisms
for Coenzyme Function 209
mans and other mammals normally require only folic acid as a vitamin of the pterion
type. This is discussed in Chapter 12. Although a number of pterins when reduced to the
5,6,7,8-tetrahydro level function as coenzymes, the most generally utilized are poly-?-
glutamates of tetrahydrofolate (THF) (2,14). Tetrahydropteroylglutamates and forms of
its natural derivatives responsible for vectoring one-carbon units in different enzymic reactions
are shown in Fig. 10. All of these bear the substituent for transfer at nitrogen 5 or
10 or are bridged between these basic centers. The number of glutamate residues varies,
usually from one to seven, but a few to several glutamyls optimize binding of tetrahydrofolyl
coenzymes to most enzymes requiring their function. Less broadly functional, but
essential for some coenzymic roles of pterins, is tetrahydrobiopterin, which cycles with
its quinoid 7,8-dihydro form during O2-dependent hydroxylation of such aromatic amino
acids as in the conversion of phenylalanine to tyrosine. Also, the recently elucidated
molybdopterin functions in some Mo/Fe flavoproteins, such as xanthine dehydrogenase.
Pterion coenzymes, most of them at the tetrahydro level, are sensitive to oxidation.
The interconversions of folate with the initial coenzymic relatives, the tetrahydrofolyl
polyglutamates, involve dihydrofolate reductase, necessary for reducing the vitamin
level compound through 7,8-dihydro to 5,6,7,8-tetrahydro levels. This enzyme is the target
of such inhibitory drugs as aminopterin and amethopterin (methotrexate). A similar dihydropterin
reductase catalyzes reduction of dihydro- to tetrahydrobiopterin. Tetrahydrofolate
is trapped intracellularly and extended to polyglutamate forms that operate with THFdependent
systems. In some cases (e.g., thymidylate synthetase), there is a redox change
in tetrahydro to dihydro coenzyme, which is recycled by the NADPH-dependent reductase.
There are different redox levels for the one-carbon fragment carried by THF systems.
With formate, phosphorylation by ATP leads to formyl phosphate, a reactive mixed-acid
anhydride. This can react with nitrogens at either position 5 or 10 in THF to form the
formyl compounds; upon cyclization, the 5,10-methenyl-THF results. With formaldehyde,
which becomes an electrophilic cation when protonated (CH2OH), the reaction shown
in Fig. 11 ensues. This reaction is initiated at the most basic N-5 (pKa 
 4.8) of THF to
generate an N-hydroxymethyl intermediate. The high electron charge (high basicity) and
large free valence (high polarizability) confer a low activation energy for electrophilic
substitution at N-5. With loss of a hydroxyl and quaternization of this nitrogen, the onecarbon
unit becomes canonically equivalent to a carbocation, which can react either interor
intramolecularly. In the former instance, reaction with a nucleophile can lead to another
Fig. 10 Structures with numbering for tetrahydropteroyl-l-glutamates, including the biological
derivatives for one-carbon transfers.
210 McCormick
Fig. 11 Reaction of formaldehyde with the basic N-5 of tetrahydrofolyl coenzyme to generate
an N-hydroxymethyl intermediate that leads to a mesomeric carbocation. This latter can react intermolecularly
in a Mannich-like reaction or intramolecularly to form 5,10-methylene tetrahydrofolate.
hydroxymethyl compound; in the latter, 5,10-methylene-THF is formed. For the methyl
level, reduction of the methylene-THF occurs.
An overview of some of the major interconnections among the one-carbon-bearing
THF coenzymes and their metabolic origins and roles include a fair range of reaction
types. As mentioned above, there is generation and utilization of formate. The important
de novo biosynthesis of purine includes two steps wherein glycinamide ribonucleotide
and 5-amino-4-imidazole carboxamide ribonucleotide are transformylated by 5,10-methenyl-
THF and 10-formyl-THF, respectively. In pyrimidine nucleotide biosynthesis, deoxyuridylate
and 5,10-methylene THF form thymidylate and dihydrofolyl coenzyme in a
mechanism whereby the tetrahydro coenzyme is oxidized to the dihydro level, as shown
in Fig. 12. There are conversions of some amino acids, namely, N-formimino-l-glutamate
(from histidine catabolism) with THF to l-glutamate and 5,10-methenyl-THF (via 5-
Fig. 12 The 1,3 hydrogen shift and redox nature of 5,10-methylene–tetrahydrofolyl coenzyme
within thymidylate synthase.
Mechanisms for Coenzyme Function 211
formimino-THF), l-serine with THF to glycine and 5,10-methylene-THF, and l-homocysteine
with 5-methyl-THF to l-methionine and regenerated THF.
VII. REARRANGEMENTS ON VICINAL CARBONS: B12 COENZYMES
Though there are several biologically active forms derived from vitamin B12, discussed
in Chapter 13, only a couple warrant attention as coenzymes in higher eukaryotes (2,15).
The coenzyme B12 (COB12) known to function in most organisms, including humans, is
5?-deoxyadenosylcobalamin. A second important coenzyme form is methylcobalamin
(methyl-B12), in which the methyl group replaces the deoxyadenosyl moiety of CoB12.
Some prokaryotes utilize other bases (e.g., adenine) in this position originally occupied
by the cobalt-coordinated cyanide anion in cyanocobalamin, the initially isolated form of
vitamin B12.
The metabolic interconversion of vitamin B12 as the naturally occurring hydroxocobalamin
(B12a) with other vitamin and coenzyme level forms involves sequential reduction
of B12a to the paramagnetic or radical B12r and further to the very reactive B12s. The latter
reacts in enzyme-catalyzed nucleophilic displacements of tripolyphosphate from ATP to
generate CoB12 or of THF from 5-methyl-THF to generate methyl-B12.
Seemingly all CoB12-dependent reactions react through a radical mechanism, and
all but one (Lactobacillus leichmanii ribonucleotide reductase) involve a rearrangement
of a vicinal group (X) and a hydrogen atom. This general mechanism is illustrated in Fig.
13. For the CoB12-dependent mammalian enzyme, l-methylmalonyl-CoA mutase, X is the
CoA-S-CO- group, which moves with retention of configuration from the carboxyl-bearing
carbon of l(R)-methylmalonyl-CoA to the carbon ? to the carboxyl group in succinyl-
CoA. This reaction is essential for funneling propionate to the tricarboxylic acid cycle.
Without CoB12 (from vitamin B12), more methylmalonate is excreted, but also the CoA
Fig. 13 Radical intermediates in vicinal rearrangements catalyzed by enzymes utilizing 5?-deoxyadenosyl-
B12.
212 McCormick
ester competes with malonyl-CoA in normal fatty acid elongation to form instead abnormal,
branched-chain fatty acids. Methyl-B12 is necessary in the transmethylase-catalyzed
formation of l-methionine and regeneration of THF. Without this role, there would not
only be no biosynthesis of the essential amino acid, but increased exogenous supply of
folate would be necessary to replenish THF, which would not otherwise be recovered
from its 5-methyl derivative.
REFERENCES
1. O. H. Hasim and N. Azila Adnan, Coenzyme, cofactor and prosthetic group—ambiguous
biochemical jargon, Biochem. Ed. 22:93 (1994).
2. D. B. McCormick, Coenzymes, biochemistry, in Encyclopedia of Human Biology, Vol. 2 (R.
Dulbecco, ed.-in-chief), Academic Press, San Diego, 1991, pp. 527–545.
3. D. E. Metzler, Coenzymes—nature’s special reagents, in Biochemistry: The Chemical Reactions
of Living Cells, Academic Press, New York, 1977, pp. 428–516.
4. P. A. Frey, Vitamins, coenzymes, and metal cofactors, in Biochemistry (G. Zubay, ed.), Wm.
C. Brown, Dubuque, 1993, pp. 278–304.
5. C. T. Walsh, Enzymatic Reaction Mechanisms, Freeman, San Francisco, 1977.
6. D. Dolphin, R. Poulson, and O. Avamovic (eds.), Pyridine Nucleotide Coenzymes, Parts A
and B, Wiley-Interscience, New York, 1987.
7. D. J. Creighton and N. S. R. K. Murthy, Stereochemistry of enzyme-catalyzed reactions at
carbon, The Enzymes, Vol. 19 (D. S. Sigmon and P. D. Boyer, eds.), Academic Press, San
Diego, 1990, pp. 323–421.
8. A. H. Merrill, Jr., J. D. Lambeth, D. E. Edmondson, and D. B. McCormick, Formation and
mode of action of flavoproteins, Annu. Rev. Nutr. 1:281–317 (1981).
9. B. Curti, S. Ronchi, and G. Zanetti (eds.), Flavins and Flavoproteins, Walter de Gruyter,
Berlin, 1991.
10. H. Z. Sable and C. J. Gubler (eds.), Thiamin. Twenty years of progress, Ann. N.Y. Acad. Sci.,
378, (1982).
11. D. Dolphin, R. Poulson, and O. Avamovic (eds.), Pyridoxal Phosphate, Parts A and B, Wiley-
Interscience, New York, 1987.
12. T. Fukui, H. Kagamiyama, K. Soda, and H. Wada (eds.), Enzymes Dependent on Pyridoxal
Phosphate and Other Carbonyl Compounds as Cofactors, Pergamon Press, Oxford, 1991.
13. J. R. Knowles, The mechanism of biotin-dependent enzymes, Annu. Rev. Biochem. 58:195,
1989.
14. R. L. Blakley and T. J. Benkovic (Vol. 1) and R. L. Blakley and V. M. Whitehead (Vol. 3),
Folate and Pterins, John Wiley and Sons, New York, 1986.
15. D. Dolphin, (ed.), B12, Vols. 1 and 2, Wiley-Interscience, New York, 1982.
6
Niacin
JAMES B. KIRKLAND
University of Guelph, Guelph, Ontario, Canada
JEAN M. RAWLING
University of Calgary, Calgary, Alberta, Canada
I. HISTORICAL PERSPECTIVE
The identification of niacin as a vitamin resulted from an urgent need to cure pellagra,
which ravaged low socioeconomic groups of the southeastern United States in the early
twentieth century and various European populations for the previous two centuries. The
history of pellagra and the progression of early research findings in niacin nutrition are
extensively documented by many writers, including Harris (1) and Carpenter (2). The first
reports of the condition were recorded in the early 1700s in Spain. Corn had been introduced
to Europe from the Americas and quickly became a staple food, as it could produce
more calories per acre than wheat or rye.
In 1735, the Spanish physician Casal became the first to describe the strange new
disease, which he termed mal de la rosa (disease of the rose). He documented the skin
lesions of the disease quite carefully, and the characteristic rash around the neck of pellagrins
is still referred to as ‘‘Casal’s necklace.’’ The disease spread geographically with
the cultivation of corn and became known as pelle agra (rough skin) by the Italian peasantry.
Due to the widespread incidence of pellagra in eighth-century Europe and the dedication
of entire hospitals for victims of the disease, there was a painstaking documentation
of its symptoms (1,2). These include diarrhea and neurological disturbances (dementia)
(3), as well as the sun-sensitive dermatitis, which, along with the eventual death of the
patient, are often referred to as the ‘‘4 D’s’’ of pellagra. The disease was recognized in
populations in Egypt, South Africa, and India in the late 1800s and early 1900s. It reached
epidemic proportions in the United States in the first half of the 1900s, producing at least
213
214 Kirkland and Rawling
250,000 cases and 7,000 deaths per year for several decades in the southern states alone
(4). Serious outbreaks continue to occur in some developing countries (5). An improved
standard of living and supplementation of some breakfast cereals with niacin have limited
the disease in North America and Europe, but cases of pellagra still occur, although they
are likely underreported due to a lack of familiarity of modern physicians with this disease.
It is also likely that subclinical deficiencies of niacin exist in developed countries; 15%
of women surveyed in Malmo, Sweden, had blood nucleotide pools that indicated a suboptimal
niacin intake (6).
Until the first half of the twentieth century, the etiology of pellagra was unknown.
Early theories suggested that it is a type of leprosy, that it results from a toxin in moldy
corn, or that it is an infectious disease communicated through an insect vector. Gradually,
an association between pellagra and corn consumption became apparent, even in the absence
of mold contamination. This was confirmed by Joseph Goldberger (7), who determined
that a pellagra-preventative (‘‘P-P’’) factor, missing from corn, was necessary to
prevent and cure pellagra in humans. In 1937, Elvehjem and colleagues discovered that
nicotinic acid could cure black tongue in dogs, an early animal model for pellagra (8).
Paradoxically, chemical analysis of corn revealed that it is not especially low in nicotinic
acid content. However, Krehl et al. (9) induced niacin deficiency in rats by feeding the
animals a corn-based diet and then alleviated the symptoms with the addition of casein,
a protein source rich in tryptophan. There had been suggestions that tryptophan deficiency
caused pellagra, and in 1921, a pellagrous patient was treated successfully with tryptophan
supplementation (10). It was eventually realized that tryptophan can be used, with low
efficiency, as a substrate for the synthesis of nicotinamide adenine dinucleotide (NAD)
(11). In 1951, Carpenter’s group found that niacin in corn is biologically unavailable and
can be released only following prolonged exposure to extremes in pH (12). These findings
eventually led to a better understanding of the contribution of a corn-based diet to the
development of pellagra; corn-based diets are low in tryptophan, and the preformed nicotinic
acid is tightly bound to a protein that prevents its absorption. The release of niacin
from this protein at extremes of pH explained the good health of native Americans, who
used corn as a dietary staple: in all cases these societies processed their corn with alkali
prior to consumption. Corn was in use throughout North and South America for thousands
of years as a domesticated crop, and methods of preparation ranged from treatment of
corn with ashes from the fireplace, to the well-known use of lime water (water and calcium
hydroxide) in the making of tortillas. Had the explorers brought native American cooking
techniques along with corn to sixteenth-century Europe, pellagra epidemics might never
have occurred.
Pellagra may be difficult to identify, as the symptoms of dermatitis, diarrhea, and
dementia occur in an unpredictable order, and it is uncommon to find all three aspects
until the disease is very advanced. The earliest sign of deficiency is often inflammation
in the oral cavity, which progresses to include the esophagus and eventually the whole
digestive tract, associated with a severe diarrhea (1). Burning sensations often discourage
food consumption, and the patient progresses to a state of marasmus; the cause of death
in most cases is probably a result of the effects of general malnutrition (poor disease
resistance) and diarrhea (1). Because of the sensitivity to sunlight, and possibly due to
dietary variation, the incidence of pellagra is seasonal. The dermatitis can become severe
rapidly, with exposed skin showing hyperpigmentation, bullous lesions, and desquamation
(Fig. 1). The skin may heal during fall and winter, leaving pink scars, only to revert to
open sores the following summer. The other dramatic symptom of pellagra is dementia,
Niacin 215
(a)
(b) (c)
Fig. 1 (a) An Austrian child with pellagra, showing dermatitis on the exposed skin of the face
and hands. Note the unaffected skin on the wrists where the cuffs of the coat are turned up. (b)
Severe pellagrous lesions on the arms of a 32-year-old woman. They did not respond to riboflavin
supplementation but cleared up with the use of Valentine’s whole liver extract, a good source of
niacin. (c) Dermatitis on the legs and feet of a typical pellagra patient, showing the pattern of protection
from the sun afforded by sandals.
[Fig. 1(a) is reproduced with permission from Pellagra: History, Distribution, Diagnosis, Prognosis,
Treatment, Etiology, by Stewart R. Roberts, C.V. Mosby Company, 1914. Fig. 1 (b) and 1 (c) are
reproduced with permission from Clinical Pellagra, by Seale Harris, published by C.V. Mosby, St.
Louis, 1941.]
216 Kirkland and Rawling
which usually goes beyond the type of depression associated with general malnutrition.
Neurological changes in pellagra patients begin peripherally, with signs such as muscle
weakness, twitching and burning feelings in the extremities, and altered gait (13). Early
psychological changes include depression and apprehension, but these progress to more
severe changes, such as vertigo, loss of memory, deep depression, paranoia and delirium,
hallucinations, and violent behavior (14). This type of dementia is very similar to schizophrenia
(15). There are numerous examples of the pellagrous insane committing murder
(16), although it was more common for these patients to turn to suicide. Interestingly,
many insane pellagrins are drawn to water, and their most common form of suicide is
reported to be drowning (16). While there are pathological changes in the spinal cord in
advanced pellagra (17), and some of the motor disturbances are permanent (18), there is
a striking recovery of psychological function when insane pellagra patients are treated
with nicotinic acid, with a disappearance of symptoms in 1–2 days (14). These observations
suggest that a compound derived from niacin is involved in neural signaling pathways.
The first biochemical role established for niacin was its involvement in redox reactions
and energy metabolism. For many years, however, the symptoms of pellagra remained
mysterious when viewed from this perspective. Other nutrients involved in energy
metabolism do cause similar deficiency symptoms. Riboflavin-containing coenzymes are
often coupled with nicotinamide-dependent reactions in the transfer of electrons, but ribo-
flavin deficiency is not very similar to pellagra. Iron is intimately involved in electron
transport and ATP production, and it functions closely with nicotinamide cofactors in
energy metabolism. Iron deficiency and pellagra both cause general weakness, which could
be associated with disrupted energy metabolism, but the pellagra patient displays several
unique and dramatic clinical characteristics. Whereas other nutrient deficiencies cause
dermatitis, only in niacin deficiency is this condition induced by exposure to sunlight.
Although thiamine deficiency also causes changes in energy metabolism and neural function,
it thought to achieve these effects through separate biochemical functions, and this
is likely the case with niacin as well.
Recently, insights into the function of nicotinamide coenzymes in metabolism have
improved our appreciation of the biochemical changes that may underlie the 4 D’s of
pellagra. In 1967, poly(ADP-ribosyl)ation was identified and recognized as a posttranslational
modification of nuclear proteins. Poly(ADP-ribose) synthesis makes use of NAD

as substrate, rather than as an electron-transporting intermediate. Poly(ADP-ribose) formation
has been shown to be important in DNA repair processes, and new knowledge in
this area may soon provide an explanation for the sun-sensitive dermatitis of pellagra.
Mono(ADP-ribosyl)ation, which also uses NAD
 as a substrate, was characterized, beginning
in the mid-1960s, as a mechanism of action for many bacterial toxins. Mono(ADPribosyl)
ation is now thought to be important in the endogenous regulation of many aspects
of signal transduction and membrane trafficking in eukaryotic cells, but these studies are
in their infancy. In 1989, cyclic ADP-ribose was identified as another product of NAD
metabolism and was shown to have the ability to regulate cellular calcium homeostasis,
a central process in neural transmission. Interestingly, the enzymes that make and degrade
cyclic ADP-ribose and the proteins that bind this second messenger are present in the
brain in relatively large quantities compared with other tissues. Even more recently, the
same enzymes that make cyclic ADP-ribose have been found to produce nicotinic acid
adenine dinucleotide phosphate (NAADP), using NADP as a substrate. NAADP appears
to also have distinct functions in the regulation of intracellular calcium stores. The studies
Niacin 217
of mono(ADP-ribosyl)ation reactions and cyclic ADP-ribose and NAADP function are in
their early stages, but they may soon provide explanations for the dementia of the pellagra
patient and the changes in intestinal cell function that lead to diarrhea.
II. CHEMISTRY
A. Nicotinic Acid and Nicotinamide
The term niacin is accepted as a broad descriptor of vitamers having the biological activity
associated with nicotinamide, including nicotinamide, nicotinic acid, and a variety of pyridine
nucleotide structures. In the past, niacin has been used to specifically refer to nicotinic
acid (pyridine-3-carboxylic acid; Fig. 2A), but for the purposes of this discussion, ‘‘niacin’’
is used in reference to all forms with vitamin activity, while ‘‘nicotinic acid’’ refers
to pyridine-3-carboxylic acid. Nicotinic acid is a white crystalline solid, stable in air at
normal room temperature. It is moderately soluble in water and alcohol, but insoluble in
ether. An aqueous solution has a maximum ultraviolet (UV) absorbance at 263 nm.
Like nicotinic acid, nicotinamide (niacinamide; pyridine-3-carboxamide) (see structure,
Fig. 2B) is a white crystalline substance with a maximal UV absorbance at 263 nm.
In contrast to nicotinic acid, nicotinamide is highly soluble in water and is soluble in
ether—characteristics that allow separation of the two vitamers.
Fig. 2 Chemical structures of niacin compounds. (A) Nicotinic acid; (B) nicotinamide; (C) NAD
;
(D) NADP
; (E) site of reduction.
218 Kirkland and Rawling
B. Niacin Coenzymes
The biologically active forms of niacin compounds are the NAD and NADP coenzymes
(Fig. 2C, D). It is the C-4 position on the pyridine ring of the nicotinamide moiety that
participates in oxidation and reduction reactions. Due to the electronegativity of the amide
group and the nitrogen at position 1 on this ring, hydride ions can readily reduce the
oxidized C-4 position. This is the basis for the enzymatic hydrogen transfer reactions that
are ubiquitous among organisms. With respect to the non-redox functions of NAD, the
glycosidic linkage between nicotinamide and ribose is a high-energy bond, and cleavage
of this bond drives all types of ADP-ribose transfer reactions in the forward direction.
The oxidized and reduced forms of the coenzymes are designated NAD
 or NADP

and NADH or NADPH, respectively. The designations NAD and NADP are used to describe
the total pools. This is often necessary if the method of quantification does not
distinguish between oxidized and reduced forms, or if a general statement about the nucleotide
pool is made. The total pool of all four forms may be referred to as NAD(P). Both
NAD and NADP are white powders that are freely soluble in water and poorly soluble
in ether. Both compounds have strong UV absorption at 340 nm in their reduced forms,
with a weaker absorption at 260 nm when oxidized or reduced. The absorbance at 340
nm is often used to monitor the oxidation or reduction of these cofactors in enzyme assays.
III. FOOD CONTENT, DIETARY REQUIREMENTS AND ASSESSMENT
OF STATUS
A. Quantification
The traditional analysis for nicotinic acid entails cleavage of the pyridine ring with cyanogen
bromide (the Koenig reaction), followed by reaction with an aromatic amine to yield
a colored product that can be assayed spectrophotometrically (19). Because hydrolysis of
the nicotinamide pyridine ring with cyanogen bromide is not quantitative, it is advisable
to first deamidate nicotinamide enzymatically to form nicotinic acid before preparing samples
for the Koenig reaction. Microbiological assay of nicotinic acid and nicotinamide is
possible using Tetrahymena pyriformis or Lactobacillus plantarum (20). Nicotinic acid
can also be specifically determined microbiologically with Leuconostoc mesenteroides;
this microorganism is an obligate user of nicotinic acid rather than of nicotinamide (21).
Fluorometric measurement of nicotinamide is very sensitive. In this assay, nicotinamide
is converted to N?-methylnicotinamide, which is then reacted with a carbonyl compound,
commonly acetone or acetophenone, thus producing a fluorescent adduct on ring position
4 (22).
These traditional methods for analysis of nicotinic acid and nicotinamide are gradually
being replaced by a variety of new techniques, including gas chromatography and
mass spectroscopy (23) or high-performance liquid chromatography (HPLC) (24). Because
of the importance of these vitamers as pharmacological agents, more research on
pharmacokinetics following supraphysiological doses is warranted. Specific measurement
of nicotinic acid, nicotinamide, and their metabolites will require the use of these more
advanced analytical techniques.
Measurement of pyridine nucleotides is easier than measurement of the vitamin
precursors. Oxidized forms (NAD
, NADP
) are extracted by acid, usually 1 N perchlorate.
This causes destruction of the reduced forms (NADH, NADPH). The reduced nucleotides
are extracted by base, which causes destruction of the oxidized forms (25). The
Niacin 219
extracted nucleotides are generally quantified by enzyme cycling techniques that recognize
oxidized or reduced nucleotides but are specific to either NAD or NADP (25). The oxidized
forms can also be measured by HPLC techniques that provide additional data on
ATP, ADP, and AMP levels (26).
B. Food Content
The niacin content of human foods is usually expressed as niacin equivalents (NE), which
are equivalent to niacin content (mg) 
 one-sixtieth tryptophan content (mg). This relationship
may not be accurate if the intake of tryptophan is low (see Sec. IV.A). Furthermore,
the conversion of tryptophan appears to be improved when the intake of niacin is
low (2). Niacin in plant products is mainly in the form of nicotinic acid. Animal products
will initially contain mainly NAD and NADP coenzymes, but little is known about breakdown
of these nucleotides during the aging of meats. Under anerobic conditions, NAD

will be converted to NADH, which may protect it from the wide variety of NAD-catabolizing
enzymes in the cell. Analysis of food products for performed niacin is probably best
accomplished by microbiological methods that respond to nicotinic acid, nicotinamide,
and coenzyme forms of the vitamin. Because plant products contain less tryptophan than
animal products and because the nicotinic acid may be largely bound in unavailable forms,
some grain products such as breakfast cereals are supplemented with nicotinic acid. Such
supplementation, together with the widespread occurrence of niacin and tryptophan in a
mixed diet, has greatly diminished the number of clinically obvious cases of pellagra
in developed countries. Subclinical niacin deficiency may still be common in developed
countries (6,23), and clinical pellagra still occurs in association with alcoholism (27).
Outbreaks of pellagra continue in some areas of the world where populations rely on corn
(or maize) as a staple (5). The degree of nutrient deficiency in a population is always
based on the current perception of optimal intake or function. As our understanding of
niacin function evolves, we should be open to revising these end points and reevaluating
niacin status.
C. Dietary Requirements and Assessment of Status
Because of the important roles of NAD and NADP in energy metabolism, the niacin
requirement is related to energy intake. The recommended daily allowance (RDA) is set
at 6.6 NE/1000 kcal/day in the United States. The new Canadian RDAs for niacin range
from 2 mg niacin/day for infants to 18 NE/d during pregnancy, following a similar relationship
with energy requirement. There is also a minimum intake recommended for those
on low-energy diets, and this may reflect the roles of NAD beyond the typical redox
reactions of energy metabolism. Niacin status has traditionally been tested by measuring
the urinary excretion of various niacin metabolites or the urinary ratio of N-methyl-2-
pyridone-5-carboxamide to N-methylnicotinamide (28). The 2-pyridone form decreases
to a greater extent in response to a low dietary intake, and a ratio of less than 1.0 is
indicative of niacin deficiency. More recently, it has been found that the NAD pool in
red blood cells decreases rapidly during niacin deficiency in men, whereas the NADP
pool is quite stable (29). This has led to the suggested use of NAD/NADP, referred to
as the niacin number, as an easily obtained index of niacin deficiency in humans. Studies
using animal models have also shown that blood NAD pools deplete more rapidly and to
a greater extent than those of tissues such as liver, heart, or kidney (30). This suggests
220 Kirkland and Rawling
that a portion of blood NAD may represent a labile storage pool used to support other
tissues in the early stages of deficiency.
IV. PHYSIOLOGY
A. Pathways of Synthesis
Although many microorganisms and plants can synthesize the pyridine ring of NAD de
novo from aspartic acid and dihydroxyacetone phosphate (31), animals do not have this
ability. Nicotinic acid, nicotinamide, pyridine nucleotides and tryptophan represent the
dietary sources from which the pyridine ring structure can be derived by most mammalian
species. Animals may also practice caprophagy to take advantage of colonic synthesis of
niacin by microflora.
In 1958, Preiss and Handler (32) proposed a pathway for the conversion of nicotinic
acid to NAD in yeast and erythrocytes (shown in reactions 9, 5, and 6 of Fig. 3). Initially, it
was believed that nicotinamide was also metabolized through the Preiss-Handler pathway,
following the conversion of nicotinamide to nicotinic acid by nicotinamide deamidase
(reaction 8, Fig. 3). However, it was soon demonstrated by Dietrich that nicotinamide
reacts first with phosphoribosyl pyrophosphate and then ATP to produce NAD directly
(reactions 10 and 11, Fig. 3) (33). Subsequent studies supported this route by showing
that inhibition of NAD synthetase, the final enzyme in the Preiss-Handler scheme (reaction
6, Fig. 3), does not affect the incorporation of nicotinamide into NAD (34). It was also
found that deamidation of nicotinamide to nicotinic acid only occurs at supraphysiological
concentrations of nicotinamide (35), which is not surprising, as the Km of nicotinamide
deamidase for nicotinamide is above physiological nicotinamide levels. It has been concluded,
therefore, that the Dietrich pathway is the major route by which physiological
concentrations of nicotinamide supply NAD in mammalian cells (36). It should be noted,
however, that the predominant pathways of nicotinic acid and nicotinamide utilization
change when the vitamers are consumed in pharmacological quantities, such as those used
in the treatment of cardiovascular disease or insulin-dependent diabetes mellitus (IDDM).
This will be discussed later in this chapter (Sec. VII). Interestingly, there is a common
enzyme in the two routes of NAD synthesis. Nicotinamide mononucleotide (NMN) and
nicotinic acid mononucleotide (NAMN) adenylyltransferase activities reside within a common
protein in the nucleus; this enzyme catalyzes the last step in the conversion of nicotinamide
and the second to last step in the conversion of nicotinic acid to NAD (37).
Quinolinic acid, a tryptophan metabolite, can react with phosphoribosyl pyrophosphate
to produce nicotinic acid mononucleotide. This reaction occurs only in the kidney
and in the liver in mammals, due to the localization of quinolinate phosphoribosyltransferase
in these tissues (38). NAD is then synthesized from nicotinic acid mononucleotide
via the Preiss-Handler pathway. A number of reactions are required to convert tryptophan
to quinolinic acid. Most of the ?-amino-?-carboxymuconic-?-semialdehyde, an intermediate
in this pathway, is catabolized to acetyl CoA and CO2 (39). There is, however, a ratelimiting
step in this degradation sequence, which, when overwhelmed, allows some of
the semialdehyde to transform spontaneously into quinolinic acid (reaction 3, Fig. 3) and
ultimately to produce NAD (40). Therefore, the production of NAD from tryptophan is
favored by a low activity of one enzyme (picolinic carboxylase) and a high activity of
another (quinolinate phosphoribosyltransferase), leading to a wide range in the efficiency
of tryptophan utilization among species.
Niacin 221
Fig. 3 Pathways of NAD
 synthesis in mammals. Reactions 9, 5, and 6 comprise the Preiss-
Handler pathway, while reactions 10 and 11 form the Dietrich pathway. The following enzymes
correspond to the numbered reactions: (1) 5 step conversion; (2) picolinic carboxylase; (3) spontaneous
chemical reaction; (4) quinolinic acid phosphoribosyltransferase; (5) NAMN adenylyltransferase
(enzymes 5 and 11 may be identical proteins); (6) NAD synthetase; (7) NAD glycohydrolases,
various ADP-ribosylation reactions; (8) nicotinamide deamidase; (9) nicotinic acid phosphoribosyltransferase;
(10) nicotinamide phosphoribosyltransferase; (11) NMN adenylyltransferase.
In nutritionally replete humans, there is thought to be a 60 mg/1 mg ratio between
tryptophan supply and niacin formation, although individual variation is significant (41).
Because of this relationship, dietary niacin content is described in niacin equivalents (1
NE  mg niacin 
 1/60 mg tryptophan). More recent work has suggested that humans
may not utilize tryptophan for niacin synthesis when tryptophan levels are limiting in
the diet (29). In these experiments, young men were placed on a diet containing 6 NE/
day, and their blood NAD
 levels decreased by 70% over 5 weeks. Addition of 240 mg/
day of tryptophan to this diet had no effect on blood NAD, although it prevented the
decrease in plasma tryptophan that resulted from consumption of the unsupplemented
diet. This tryptophan represented, in theory, an additional 4 NE/day, but it appears that
222 Kirkland and Rawling
protein turnover takes precedence over niacin synthesis when tryptophan levels are low.
NE calculations for diets with marginal niacin status may be inaccurate because of this
relationship. At the same time, tryptophan supplements have been reported to cure pellagra
and a genetic defect in tryptophan absorption, known as Hartnup’s disease, also
causes pellagra-like signs and symptoms. While the efficiency of conversion of tryptophan
to NAD is thought to be increased by niacin deficiency (2), it is also apparent that
at low tryptophan intakes, the need for protein metabolism dominates over conversion
to NAD (29).
Species vary in their ability to synthesize niacin from tryptophan. Cats, adapted to
a diet high in amino acids, have approximately 50 times greater picolinic carboxylase
activity than humans (40), and they demonstrate extremely poor utilization of tryptophan
as a precursor for NAD (42). Rats, on the other hand, are more efficient than humans in
their use of tryptophan for NAD synthesis (ratio of about 33 mg tryptophan: 1 mg niacin)
(43). Mice are resistant to niacin deficiency, even on diets containing as little as 6% casein,
with 7% gelatin, a protein source lacking tryptophan (unpublished data). Guinea pigs are
very susceptible and will eventually die on a niacin-free diet containing 20% casein, demonstrating
a very poor conversion of tryptophan to niacin (44). The capacity for NAD
generation from tryptophan also depends on adequate status of vitamin B6 and minerals,
including copper, iron, and magnesium, all of which are necessary for reactions in this
pathway. When using experimental animals to model niacin deficiency in humans, it is
important to remember the tremendous variability between species in the utilization of
tryptophan.
B. Absorption
Both nicotinamide and nicotinic acid can be absorbed through the stomach lining, but
absorption in the small intestine is more rapid. For intact nucleotides, pyrophosphatase
activity in the upper small intestine metabolizes NAD to yield nicotinamide mononucleotide,
which is then quickly hydrolyzed to form nicotinamide riboside and eventually free
nicotinamide.
Absorption of both nicotinic acid and nicotinamide at low concentrations appears
to be via sodium-dependent facilitated diffusion (39) or by carrier-mediated transport making
use of proton cotransporters and anion antiporters (45). Higher concentrations of both
forms appear to be absorbed by passive diffusion. Once absorbed from the lumen into the
enterocyte, nicotinamide may be converted to NAD or released into the portal circulation.
Although some nicotinic acid moves into the blood in its native form, the bulk of the
nicotinic acid taken up by the enterocyte is converted to NAD via the Preiss-Handler
pathway (39). As required, NAD glycohydrolases in the enterocytes release nicotinamide
from NAD into the plasma as the principal circulating form of niacin. The kinetics of
nicotinamide and nicotinic acid transport and metabolism are influenced significantly by
pharmacological intakes (see Sec. VII).
C. Distribution and Metabolism
Niacin compounds entering the portal circulation are either internalized by erythrocytes
or transported to the liver. Erythrocytes take up nicotinic acid by facilitated diffusion,
using an anionic transport protein that accounts for 30% of the membrane protein content
(39). In spite of this, erythrocytes appear to favor nicotinamide as a precursor for NAD
synthesis (46).
Niacin 223
The liver is the central processing organ for niacin. Aside from its role in the conversion
of tryptophan to NAD, it receives nicotinamide and some nicotinic acid via the portal
circulation, as well as nicotinamide released from other extrahepatic tissues. In the liver,
nicotinic acid and nicotinamide are metabolized to NAD or to yield compounds for urinary
excretion, depending on the niacin status of the organism.
The liver has some capacity for NAD storage. Because hepatic regulation of nicotinamide
phosphoribosyltransferse by ATP and NAD (positive and negative, respectively)
is less effective than in many other tissues, liver NAD concentrations increase significantly
following dietary nicotinamide administration (39). In rats, nicotinic acid and nicotinamide,
at 1000 mg/kg diet, increase blood and liver NAD
 to a similar and modest extent
(about 50%) (47). NAD glycohydrolases are thought to use the storage pool of hepatic
NAD to produce nicotinamide (48), which is released for replenishment of extrahepatic
tissues. The function of NAD glycohydrolases is controversial, however, and difficult to
distinguish from cyclic ADP-ribose formation and catabolism (see later sections).
The liver plays an important role in the preparation of niacin for urinary excretion,
producing a variety of methylation and hydroxylation products of both nicotinic acid and
nicotinamide. In humans, nicotinamide is primarily methylated to produce N?-methylnicotinamide,
whereas nicotinic acid is conjugated with glycine to form nicotinuric acid. Increasing
levels of the untransformed vitamers can be found in the urine as the level of
niacin ingestion increases (39).
V. BIOCHEMICAL FUNCTIONS
A. NAD Cofactors in Redox Reactions
Oxidation and reduction of the C-4 position on the pyridine ring of NAD coenzymes
is the basis for hydrogen transfer reactions important in oxidative phosphorylation and
biosynthetic reactions. NAD
 is reduced to NADH in glycolytic reactions, oxidative decarboxylation
of pyruvate, oxidation of acetate in the tricarboxylic acid (TCA) cycle, oxidation
of alcohol, ?-oxidation of fatty acids, and a large number of other cellular oxidation
reactions. The electrons derived from these oxidation reactions are transferred to the electron
transport chain through the oxidation of NADH. The energy resulting from these
transfers is used to generate ATP. In contrast to the central role that NAD(H) plays in
energy expenditure, NADP(H) is essential for the biosynthetic reactions involved in energy
storage. NADPH is produced from reduction of NADP
 in reactions of the pentose phosphate
pathway and during the malate/pyruvate shuttle across the mitochondrial membrane.
NADPH then acts as a reducing agent for fatty acid production, cholesterol synthesis,
and manufacture of deoxyribonucleotides. NADPH is also notable for its involvement in
glutathione regeneration.
The extra phosphate group carried on NADP permits the cell to separate the oxidation
and reduction activities of the nicotinamide cofactors by allowing most cellular enzymes
to be specific for one of these coenzyme species. Because of this specificity, the
majority of cellular NADP is maintained in the reduced state by the pentose phosphate
pathway and reduction reactions are favored by mass action. Conversely, NAD is predominantly
oxidized, improving the oxidant capabilities of this cofactor under cellular conditions.
The role of NAD coenzymes in redox reactions has been the function classically
associated with niacin since Warburg and Christian showed that nicotinamide was a component
of NADP in 1935 and, in the following year, demonstrated the presence of nicotin224
Kirkland and Rawling
amide in NAD (49). It is probable that early researchers attributed the pathology associated
with niacin deficiency to disruptions in redox cycling, since this was the only function of
niacin known at that time. However, the distinctive clinical signs of pellagra may be better
explained in relation to the functions of NAD described in the following sections.
B. NAD
 as a Substrate
Although the hydride transfer chemistry of the pyridine nucleotides was initially described
in the 1930s (49), the high-energy glycosidic linkage between nicotinamide and ADPribose
received little attention before the discovery of mono- and poly(ADP-ribosyl)ation
reactions in the 1960s. The energy provided by breaking this bond allows the addition of
ADP-ribose to a variety of nucleophilic acceptors. These include glutamate side chains
and hydroxyl groups of ribose for poly(ADP-ribose) synthesis, a variety of amino acid
side chains in mono(ADP-ribosyl)ation reactions, and an internal ribose linkage for cyclic
ADP-ribose synthesis (Fig. 4). The catabolic activity of NAD glycohydrolase (if this reaction
does exist in isolation) also represents an ADP-ribose transfer, in which water acts
as the nucleophilic acceptor.
C. ADP-Ribose Cyclization and NAADP Synthesis
In 1987, a metabolite of NAD
 was found to cause intracellular calcium mobilization in
sea urchin eggs (50). The molecule was similar to the potent second messenger inositol-
1,4,5-triphosphate (IP3) in its capacity to promote this response. It acted, however, on
different cellular pools of calcium. Eventually, the compound was identified as cyclic
(ADP-ribose) (51). Cells have two types of channels, the IP3 and the ryanodine receptors,
which regulate the release of calcium from internal stores such as the sarcoplasmic or
endoplasmic reticulum and mitochondria. While the IP3 receptors have been characterized,
the ryanodine receptors have been identified by their nonphysiological stimulation by the
plant alkaloid, ryanodine. In the last few years, evidence has accumulated that cyclic ADPribose
is an endogenous second messenger that controls the ryanodine receptors (52) and
that the release of calcium is due to the binding of cyclic ADP-ribose to a small protean,
which then dissociates from the ryanodine receptor, allowing calcium transport (53).
Calcium concentrations are about 10,000-fold higher outside cells than within the
cytoplasm. Transient increases in cytoplasmic calcium are critical to most types of cell
signaling, including nerve transmission and muscle contraction. When a stimulus causes
a small amount of calcium to be released, there is a positive feedback leading to a much
larger flow of calcium into the cytoplasm. Cyclic ADP-ribose and ryanodine receptors
appear to play an important role in this calcium-induced calcium release (CICR) (52).
ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase are the enzyme activities responsible
for synthesis and degradation of cyclic ADP-ribose, respectively. NAD glycohydrolase,
isolated from canine spleen, actually has both cyclase and hydrolase activities,
leading to an overall reaction that appears to be NAD
 glycohydrolysis (54). The leukocyte
cell surface antigen CD38 also has these capacities (55). It is possible that other enzymes
previously identified as NAD glycohydrolases are similar in their abilities to synthesize
and catabolize cyclic ADP-ribose.
At the moment, it is unclear whether cyclic ADP-ribose works as a cofactor to
enhance the sensitivity of ryanodine receptors to calcium (as does caffeine) or if it is a
true second messenger that responds directly to an extracellular stimulus (56). In rat pancreatic
? cells, glucose stimulates the formation of cyclic ADP-ribose, which is thought
Niacin 225
Fig. 4 Structures and origin of cyclic ADP-ribose and NAADP.
to induce insulin secretion via CICR (57). In some systems, there is redundancy between
the function of cyclic ADP-ribose and IP3; in sea urchin eggs, for example, the actions
of both must be inhibited to prevent fertilization (52).
In 1995, a metabolite of NADP was also found to cause release of intracellular
calcium stores; it was identified as nicotinic acid adenine dinucleotide phosphate
(NAADP). Surprisingly, this metabolite is formed by the same enzymes that make cyclic
ADP-ribose, although the reaction mechanism seems quite different. NAADP is synthesized
by exchanging the nicotinamide on NADP with nicotinic acid, forming one of the
most potent calcium-releasing agents known (58). This metabolite is effective at nanomolar
concentrations and appears to be physiologically important in various calcium release
systems, including sea urchin eggs, brain microsomes, and pancreatic cell insulin release
(58). Interestingly, NAADP appears to regulate intracellular calcium pools that are distinct
from those controlled by IP3 or cyclic ADP-ribose.
226 Kirkland and Rawling
There are many questions to be answered concerning the roles of cyclic ADP-ribose
and NAADP in cell signaling and how they might be affected by niacin deficiency. ADPribosyl
cyclase activities are widespread; CD38 has been identified in the plasma membrane
of a wide variety of mammalian tissues, and a soluble cyclase has been characterized
from the testis of Aplysia and of dog (59). Another cyclase enzyme from canine spleen
is localized to the endoplasmic reticulum (54). CD38 presents a unusual picture in that
the catalytic domain is extracellular. This ectoenzyme may obtain NAD
 or NADP
 from
extracellular fluids and release cyclic ADP-ribose or NAADP to interact with extracellular
receptors. Cyclic ADP-ribose or NAADP may also enter the cell to interact with intracellular
binding proteins. Conversely, CD38 could be internalized under appropriate conditions
and function as an intracellular source of cyclic ADP-ribose, drawing on cytosolic NAD

or NADP
 pools. To understand the effect of niacin deficiency on these processes, we
need to determine the subcellular localization of the active forms of these enzymes and
their affinity for nucleotide substrates. Later in this chapter we will try to integrate these
functions with other NAD
-utilizing reactions during niacin deficiency.
The tissue distribution of cyclic ADP-ribose– and NAADP-metabolizing enzymes
is also interesting. Levels of CD38 and cyclic ADP-ribose hydrolase activities are particularly
high in brain tissue (59,60), and preliminary data show that the small proteins that
bind cyclic ADP-ribose are also high in brain tissue (61). Researchers may find over the
next few years that neural functions mediated by cyclic ADP-ribose and NAADP are
impaired during niacin deficiency and lead to the unusual motor disruptions and dementia
of pellagra.
D. NAD
 Glycohydrolysis
NAD
 glycohydrolase enzymes catalyze the donation of the ADP-ribose moiety of NAD

to water, resulting in free ADP-ribose and nicotinamide. There have been many reports
of this activity in mammalian plasma membranes and in the cytosol (62). However, diffi-
culty in isolating the enzymes has prevented clear identification of their form and metabolic
function. The function of these enzymes has never been clearly understood but may
become clearer with the discovery that cyclic ADP-ribose formation and hydrolysis are
often catalyzed by the same enzyme, leading to the misleading appearance of simple NAD
hydrolysis. It is possible that CD38 and other cyclase/hydrolase complexes are identical
to the elusive membrane-bound NAD glycohydrolase (54). To further complicate this
issue, poly(ADP-ribose) polymerase (63), bacterial ADP-ribosylating toxins (64), and endogenous
mono(ADP-ribosyl) transferases (65) all have the ability to use water as an
acceptor of the ADP-ribose moiety, in lieu of a protein, thus leading to glycohydrolaselike
activity. If true NAD glycohydrolase activities exist, their function may be related to
the control of intracellular NAD levels and to the availability of nicotinamide for export
from tissues such as small intestine and liver (62).
E. Mono(ADP-ribosyl)ation
Mono(ADP-ribosyl)ation is the transfer of a single ADP-ribose moiety, derived from
NAD
, to an amino acid residue on an acceptor protein (66,67). Of these enzyme systems,
described in bacteriophages, bacteria, and eukaryotes, bacterial toxins are the best characterized.
Various GTP-binding proteins (G proteins) are the targets for Cholera, Pertussis,
Diphtheria and Pseudomonas toxins. Mono(ADP-ribosyl)ation of G?s by Cholera toxin
results in the stimulation of adenylate cyclase activity, leading to disruption of ion transNiacin
227
port in intestinal epithelial cells and the diarrhea characteristic of this disease. Pertussis
toxin catalyzes the cysteine-specific ADP-ribosylation of several other forms of G proteins,
also leading to an uncoupling of their activities from the associated receptors. Diphtheria
toxin and Pseudomonas exotoxin A ADP-ribosylate a posttranslationally modified histidine
residue, called diphthamide, on polypeptide elongation factor 2 (EF-2), also a G
protein. This single alteration in EF-2 structure renders the enzyme nonfunctional and
halts protein synthesis. Clostridium toxins ADP-ribosylate various actin monomers to prevent
actin polymerization.
The profound metabolic changes elicited by the bacterial toxins indicate that mono
(ADP-ribosyl)ation is a powerful modulator of protein function. Mammalian cells are
believed to contain a variety of endogenous mono-ADP-ribosyltransferases, but most of
these data are based on the labeling of proteins in the presence of radioactive NAD, and the
experiments are usually conducted under nonphysiological conditions. Several transferase
activities have been examined in more detail, including those that modify EF-2, grp78,
rho, and a more general family of arginine-specific transferases. It appears that the list of
endogenous transferases will continue to grow as methodology allows researchers to identify
their protein substrates. Several of the endogenous monotransferase enzymes regulate
the function of G proteins by changing their interaction with GTP, but this is not a universal
mechanism. There is also a broad spectrum of enzyme chemistry, with five or more amino
acid side chains acting as ADP-ribose acceptors. Some of the better characterized cellular
transferases are discussed in separate sections, below.
The best characterized of these activities, with respect to enzyme purification, are
the NAD:arginine ADP-ribosyltransferases and the opposing ADP-ribosylarginine hydrolases.
Several transferases and hydrolases have been purified and characterized from
different species and tissues (67,68). Different transferase proteins are found in nuclear,
cytosolic, and plasma membrane fractions. The enzymes have a fairly broad substrate
specificity in vitro, making it difficult to identify the physiologically important protein
acceptors. Some of the possible acceptor proteins include histones, cytosolic actin, and
Ca2
 ATPase (69), but the metabolic roles of these changes in intact cells or tissues are
uncertain. All of the acceptors are modified on arginine side chains, and most of the
enzymes will ADP-ribosylate free arginine in vitro.
The plasma membrane form of NAD:arginine ADP-ribosyltransferase is generating
the most interest. This enzyme is anchored, via glycosylphosphatidylinositol (GPI), to the
outer surface of the plasma membrane (67). The GPI-anchored form of the enzyme has
been identified in several cell types. A recent report has shown that extracellular NAD

causes a depression in cytotoxic T-cell proliferation and that this response is dependent
on the extracellular GPI-anchored transferase. The ecto activity appears to lead to the
mono(ADP-ribosyl)ation of an intracellular protein (70). This curious chain of events is
similar to the ecto activity of the CD38 family of ADP-ribosyl cyclase enzymes, and it
raises further questions about the role of extracellular NAD pools and how they might
respond to niacin deficiency.
EF-2 is a G protein that is essential to the activity of the peptide elongation cycle. It
has a specific histidine residue that is modified posttranslationally to a unique diphthamide
structure. No other proteins are known to carry this amino acid residue. Furthermore, the
formation of diphthamide on EF-2 occurs in all eukaryotic cells, although it is not necessary
for the elongating activity of EF-2. Diphtheria toxin ADP-ribosylates this diphthamide,
rendering EF-2 inactive and leading to the clinical manifestations of diphtheria infection.
With the idea that humans did not evolve diphthamide synthesis on EF-2 as a site
228 Kirkland and Rawling
for attack by diphtheria toxin, investigators have searched for an endogenous regulatory
system that uses a similar mechanism. Because of an inability to accurately measure the
ADP- ribosylation of EF-2, progress has been slow. It appears that EF-2 is modified by
an endogenous transferase when cultured cells are growth-arrested through serum deprivation
(71). Interestingly, this may be an automodification, as the transferase activity copuri-
fies with EF-2.
Rho is a small molecular weight G protein that is involved in cell division and
differentiation through interactions with the cytoskeletal system. The Clostridium botulinum
C3 toxin ADP-ribosylates rho on a specific asparagine side chain, leading to disruption
of the actin cytoskeleton. An endogenous transferase activity has been identified in
brain cytosol that appears to be specific to rho as an acceptor and modifies the same
asparagine residue (72). No physiological conditions have been identified that cause regulation
of rho ADP-ribosylation.
One of the most interesting proposed mechanisms of cellular regulation using mono-
ADP-ribosylation involves the 78-kDa glucose-regulated-protein (GRP78). GRP78 is a
molecular chaperone that aids in the correct folding of secreted proteins in the lumen of
the endoplasmic reticulum. GRP78 may also bind incorrectly glycosylated proteins and
prevent their secretion. When cultured hepatoma cells are deprived of glucose or an essential
amino acid, GRP78 is mono(ADP-ribosyl)ated by a noncharacterized endogenous
transferase. This modification is freely reversible, suggesting the presence of a hydrolase
enzyme. The ADP-ribosylated form of GRP78 appears to be inactive in its chaperone
functions, and the authors suggest that this is a rapid mechanism to decrease the rate of
protein secretion during times of nutritional stress (73). Of interest, GRP78 expression is
increased in cultured cells grown in niacin-deficient medium (74).
Cysteine-bound ADP-ribose residues have been found in the membrane fraction of
liver tissue (75). An enzyme purified from red blood cell membranes has the ability to
ADP-ribosylate a cysteine residue of Gi?. These observations suggest that an endogenous
transferase similar to Pertussis toxin participates in the regulation of adenylate cyclase
activity.
There are potential artifacts in the study of mono(ADP-ribosyl)ation reactions. For
some time it was believed that nitric oxide induced an endogenous mono(ADP-ribosyl)
ation of glyceraldehyde-3-phosphate dehydrogenase (76). Research eventually showed
that this was actually a nonenzymatic reaction combining an S-nitrosylation with a subsequent
addition of the whole NAD molecule to the active site (77). Free ADP-ribose also
reacts nonenzymatically with a variety of amino acid side chains, creating the illusion of
mono(ADP-ribosyl)ation (78). Careful determination of the enzymatic nature of proposed
mono(ADP-ribosyl)ation reactions will be necessary to avoid artifacts in the future. At
the same time, it may turn out that nonenzymatic reactions of NAD or ADP-ribose with
proteins are physiologically relevant and that these types of reactions could be affected
by dietary niacin status.
F. Poly(ADP-ribosyl)ation
The presence of a novel adenylate-containing compound in liver nuclear extracts was
described by Mandel’s group in 1966 (79). They identified it as a polymer of ADP-ribose
and later confirmed that NAD was the substrate and that nicotinamide was released in the
reaction. The structure, known as poly(ADP-ribose), was found to be covalently attached
Niacin 229
Fig. 5 Synthesis and degradation of poly(ADP-ribose).
to nuclear proteins. [See Figure 5 for an illustration of poly(ADP-ribose) structure and
synthesis.]
1. Poly(ADP-ribose) Synthesis
Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) was the first enzyme identified that
had the ability to synthesize poly(ADP-ribose). PARP contains three functional domains
(80). A 42-kDa DNA-binding domain, containing two zinc fingers, is located at the amino
terminus. Together, these zinc fingers allow the enzyme to bind specifically to strand
breaks in DNA and signal the catalytic portion of the protein to initiate poly(ADP-ribose)
synthesis (81). The 55-kDa carboxy terminus region contains the NAD
 binding and catalytic
sites. Although more than 30 nuclear proteins may act as acceptors, most of the
230 Kirkland and Rawling
poly(ADP-ribose) is synthesized on PARP itself (referred to as automodification). The
middle section of the amino acid sequence of PARP includes a 16-kDa region that has
been identified as an automodification domain. Here there are 15 sites on which PARP
synthesizes poly(ADP-ribose), although more sites for automodification have been found
outside of this area of the protein (82). The synthesis of poly(ADP-ribose) on PARP itself
is critical in the regulation of its interactions with DNA.
The presence of a DNA strand break is detected by the zinc finger close to the amino
terminus, while the second finger is required for catalytic activation (81). Activated PARP
initiates poly(ADP-ribose) synthesis by covalently linking the ADP-ribose portion of an
NAD
 molecule to a glutamate or aspartate residue on an acceptor protein, with the release
of nicotinamide. A linear sequence of ADP-ribose units is synthesized on the initial protein-
bound monomer. At intervals of 40–50 residues, branch points are created on the
parent polymer chain and subsequently serve as sites for elongation (83). These initiation,
elongation, and branching reactions are all carried out by the catalytic domain at the carboxy
terminus of PARP. Automodification of PARP is now thought to occur by two
PARP molecules working together as a dimer (84). As PARP becomes more poly(ADPribosyl)
ated, it takes on an increasingly negative charge because of the accumulation of
phosphate groups. This creates electrostatic repulsion between automodified PARP and
DNA, causing PARP to dissociate from the DNA nick and stop catalytic activity (85).
It is important to note that inhibition of PARP activity by treatment with competitive
inhibitors or removal of NAD from in vitro systems is very different from removing PARP
from the system. Inactive PARP binds to strand breaks, preventing access by repair enzymes
and possibly impeding the signals that the cell uses to regulate DNA replication
and the cell cycle in response to DNA damage. It is not known whether niacin deficiency
in vivo causes a similar situation to occur. This depends upon the degree of depletion of
NAD and the response of other aspects of poly(ADP-ribose) metabolism, including the
rate of degradation of poly(ADP-ribose).
2. Poly(ADP-ribose) Degradation
It has been suggested that two different enzymes are required to degrade poly(ADPribose).
Poly(ADP-ribose) glycohydrolase cleaves the poly(ADP-ribose) in a combined
endo-/exoglycosidic fashion to release free ADP-ribose units and some free oligomers
(86). ADP-ribosyl protein lyase appears to release the final ADP-ribose residue from the
acceptor proteins, but this activity is poorly characterized. Free ADP-ribose is rapidly
degraded to AMP and ribose-phosphate by pyrophosphatase activity; pyrophosphatase
could be considered a third enzyme involved in poly(ADP-ribose) catabolism (87). It is
not certain why free ADP-ribose is catabolized so rapidly, although it may be to encourage
the forward activity of glycohydrolase or to limit the nonenzymatic glycation of proteins,
as discussed previously (78). The activity of the glycohydrolase is modified by the nature
of the poly(ADP-ribose), with a lower Km and a higher Vmax for longer poly(ADP-ribose)
chains (88). On short to medium-length chains, PARP and glycohydrolase will compete
for the free end of the poly(ADP-ribose) chain. As the ADP-ribose polymer chains elongate,
PARP activity decreases (resulting from automodification) and glycohydrolase affinity
increases, favoring catabolism.
3. Metabolic Roles of Poly(ADP-ribosyl)ation
The single-strand breaks required to activate PARP are produced in vivo through the
processes of DNA replication, transcription, and repair (89). The role played by poly(ADPNiacin
231
ribose) metabolism in these processes has been best characterized in DNA repair. Durkacz
et al. (90) demonstrated that poly(ADP-ribosyl)ation was required for cells in culture to
repair DNA damage caused by the carcinogen dimethyl sulfate. Exposure of the cells to
3-aminobenzamide (3-AB), a PARP inhibitor, and depletion of cellular NAD
 (through
the use of nicotinamide-free culture medium) resulted in the same inability to recover from
DNA damage. This landmark study stimulated a wide variety of research to determine
the roles of poly(ADP-ribose) metabolism in the DNA repair process and provided new
perspectives on the possible effects of niacin deficiency in the whole animal.
4. Mechanisms of Action
Poly(ADP-ribose) contributes negative charge to acceptor proteins, and the number of
ADP-ribose units determines the magnitude of this negative charge. The cell controls
poly(ADP-ribose) chain length through the actions of PARP and poly(ADP-ribose) glycohydrolase.
It would appear that the function of poly(ADP-ribose) is dependent on this
anionic nature, which encourages electrostatic repulsion from other polyanions, such as
DNA, and attraction to cations, such as basic DNA-binding proteins.
Quantitatively, much of the poly(ADP-ribose) in whole cells and tissues is associated
with PARP and histones. The extranucleosomal histone, H1, and histone H2B are
modified, leading to localized disruption of the DNA–protein interactions within and
among nucleosomes (89). In a process referred to as ‘‘histone shuttling,’’ the automodifi-
cation of PARP may also contribute to chromatin relaxation by drawing nearby histones
away from the DNA (85). As a result of these modifications, poly(ADP-ribosylation) leads
to a localized relaxation of nucleosomal structure. The exposed DNA is presumably available
for interactions with other DNA-binding proteins such as helicases, topoisomerases,
polymerases, and ligases involved in replication or repair (89).
A number of studies have also demonstrated poly(ADP-ribose) synthesis on enzymes
involved in DNA repair (89). Poly(ADP-ribosyl)ation of most enzymes, including
DNA topoisomerases I and II and DNA polymerases ? and ?, results in inhibition of their
activities (89). In addition, the poly(ADP-ribosyl)ation of Ca2
/Mg2
-dependent endonuclease
has been shown to suppress the rapid, nonspecific DNA-degrading activity of this
enzyme (91), and this may be important in the regulation of apoptosis. In contrast, DNA
ligase, the enzyme required to anneal the strand ends once excision repair is complete,
appears to be stimulated by poly(ADP-ribosyl)ation (92). Noncovalent association of
PARP with DNA polymerase-? has been found to stimulate the activity of DNA polymerase-
? (93).
Many approaches have been used to determine the role of poly(ADP-ribose) metabolism
in DNA repair, replication, and transcription. In some cases, investigators have created
defined in vitro models, in which PARP can be added or removed, and its activity
can be controlled by the removal of NAD or the addition of inhibitors. However, these
systems lack the complexity of chromatin structure and interactions with the nuclear matrix
that exist in the whole cell. Lindhal and co-workers have used a simplified in vitro repair
system to show that the combined addition of PARP and NAD does not increase the rate
of excision repair. However, in the absence of NAD, PARP addition effectively blocks
repair because of its inability to automodify and leave the strand to allow for repair (94).
These authors have suggested that PARP is not involved in excision repair directly but
may play a role in preventing nonhomologous recombination events between two sites of
damage. In support of this, chemical inhibition of PARP in cells has been shown by others
to increase homologous recombination and sister chromatid exchanges (95). At the same
232 Kirkland and Rawling
time, it is apparent that the in vitro models do not contain the complexity of chromatin
structure, including nucleosomal organization, DNA supercoiling, and nuclear matrix interactions,
seen in intact cells. In attempts to extend these findings to more physiological
models, cells expressing PARP antisense mRNA to deplete PARP polypeptide (96) and
cells selected for very low PARP expression (97) have been studied. While both of these
approaches cause an increased susceptibility to DNA-damaging agents, they lead to con-
flicting conclusions regarding whether there is a direct role for PARP in excision repair
vs. a secondary role in the regulation of recombination events. An excellent way to determine
the role of a protein in the whole animal is to delete the gene using recombinant
techniques and, if possible, create a fully homozygous animal lacking any functional expression
of the gene. This is usually done in mice, and the modified strains are referred
to as ‘‘knockouts.’’ In the past 3 years a series of publications characterizing two new
PARP knockout mouse models has appeared (98). Up to 1 year of life, PARP-null mice
appear to be healthy and fertile. In one model, growth of pups is slower and litter size is
smaller, but PARP does not appear to be essential for normal development. However,
x-ray treated thymocytes from the animals are delayed in their recovery, compared with
thymocytes from controls, and there are increases in sister chromatid exchange frequency.
Both PARP knockout models show extreme sensitivity to radiation injury, and the one
that has been tested is very sensitive to the acute toxicity of a chemical carcinogen that
causes DNA alkylation. It will be important to examine the incidence of spontaneous
carcinogenesis in these mice in long-term experiments, as well as determining their response
to low levels of various carcinogens.
PARP-null mice created an unexpected breakthrough in this field through the finding
that they still made small amounts of poly(ADP-ribose), leading to the discovery of four
new enzymes that have PARP activity. These are distinct gene products that have eluded
discovery due to their small quantities and the small amount of poly(ADP-ribose) that
they synthesize. A larger (142 kDa) enzyme, called tankyrase, associates with telomeres
(99) and may help regulate the activity of telomerase or inhibit recombination events at
chromosome tips. Several PARP homologues are approximately 60 kDa in size, lack both
the traditional DNA-binding domain and the automodification zone, but appear to be activated
by DNA damage (100–102). The 193-kDa subunit of mammalian vaults is also
known to have the ability to synthesize poly(ADP-ribose) (103). Vaults are cytoplasmic
ribonucleoprotein complexes of unknown function, but the identification of a cytoplasmic
PARP activity explains some earlier observations regarding the existence of cytoplasmic
poly(ADP-ribose).
The effects of PARP removal are different from those obtained using inhibitors of
PARP activity. It is probably much better for cells to not express PARP at all than to
express inactive forms or to express PARP under conditions in which substrate is limiting.
Niacin deficiency may fall in this later category, as it has the potential to cause inactive
PARP molecules to accumulate on DNA strand breaks, inhibiting their repair. The probability
of mutations occurring would further increase if this binding also impaired the signaling
events that control cell cycling in the presence of DNA damage. There is evidence
that severe niacin deprivation of cells can prevent the DNA damage–induced accumulation
of p53, and the removal of PARP protein has a similar effect in this model (104). Other
experiments have suggested that p53 is a substrate for poly(ADP-ribosyl)ation and that
PARP and p53 may form a complex that binds to certain DNA sequences (98). Increased
p53 levels help to remove the cell from active cycling and either promote efficient DNA
repair or induce apoptosis. Further DNA replication in the presence of DNA damage favors
Niacin 233
the accumulation of mutations that lead to the neoplastic phenotype. Similarly, if apoptosis
of severely damaged cells is prevented, there is a strong tendency for neoplastic transformation.
The responses of the two PARP knockout models differ with respect to p53 induction.
5. DNA Replication and Transcription, Cellular Differentiation
Various roles have been suggested for poly(ADP-ribose) metabolism in other cellular
functions, but the experimental results are not as conclusive as those for DNA repair. The
amounts of PARP and poly(ADP-ribose) vary during the cell cycle, but inhibition of PARP
activity only changes DNA replication or cell division in some experimental models.
PARP could be functioning in the complex of proteins at the replication fork through
protein–protein interactions, without a requirement for poly(ADP-ribose) synthesis. As
mentioned earlier, PARP is known to have protein–protein interactions with DNA polymerase
?. If these roles do exist, there must be redundancy of function, given that the
PARP knockout mouse grows and develops to adulthood normally (98). Tankyrase, for
example, is an alternate PARP that associates with telomerase and could be important in
regulation of telomerase activity following DNA replication (99).
PARP appears to associate with areas of DNA that are undergoing active transcription.
PARP may modify members of the transcription complex, such as RNA polymerase,
or may stabilize cruciform structures in the DNA, but inhibition or removal of PARP from
cells does not appear to have dramatic effects on their overall capacity for transcription
of RNA (98). PARP itself or poly(ADP-ribose) formation may regulate specific genes;
PARP interacts directly with the AP-2 family of transcription factors (89) and also synthesizes
poly(ADP-ribose) on transcription factors that bind to AP-2 (105).
There are some dramatic effects in the area of cellular differentiation, especially in
cells of myeloid origin. PARP protein is absent from circulating neutrophils; this is the
only known example of a nucleated animal cell that does not contain this enzyme (106).
Acute promyelocytic leukemia cells provide a model for in vitro differentiation to either
neutrophils or monocytes. In this model, neutrophilic differentiation is also associated
with a disappearance of PARP protein (106), and when PARP is artificially expressed,
this pathway of differentiation is blocked (107). PARP also appears to play a causative
role in differentiation in other models using transformed cells in culture (82), but the
normal development of the PARP knockout mouse suggests that redundancy exists in the
whole animal. For all of these functions, it is important to remember the difference between
a loss of catalytic activity that could be caused by niacin deficiency and the removal
of PARP protein in the knockout model.
6. Relationship of Poly(ADP-Ribose) Metabolism to Carcinogenesis In
Vivo
Given that poly(ADP-ribose) metabolism plays an important role in DNA repair in cultured
cells, one would expect PARP activity to be important in the in vivo process of
carcinogenesis. There are studies using competitive inhibitors that support this conclusion.
Concurrent treatment with 3-aminobenzamide (3-AB), a competitive inhibitor of PARP,
caused a 10-fold increase in the formation of altered hepatic foci (precancerous lesions)
in rats treated with diethylnitrosamine (108).
It is important to note, for much of the following discussion on PARP inhibition
and carcinogenesis, that there are two very different mechanisms in which PARP may be
234 Kirkland and Rawling
involved in cell death following DNA damage. In the first, low to moderate levels of DNA
damage cause a gradual accumulation of genetic changes that initially prevent the cell
from dividing and may eventually kill a portion of the cells through the loss of function
of essential genes or induction of apoptosis. If one or more mutations occur in genes
involved in cell division, this may also lead to the initiation of carcinogenesis. In a second
mechanism, large quantities of DNA damage activate PARP to such an extent that cellular
NAD is decreased to levels that do not support cellular functions, such as ATP synthesis,
and the cell dies quite rapidly. This type of cell death also tends to follow the pattern of
apoptosis and has been portrayed by some as the central mechanism for induction of
apoptosis via DNA damage. This is not an accurate generalization. Berger proposed the
‘‘suicidal NAD depletion’’ model as a mechanism to encourage cell death when DNA
damage was extreme (109). Under these conditions, the risk of carcinogenesis would be
high if cells survive. Inhibition of PARP increases cell injury in the first model because
of an inhibition of DNA repair processes. In the second model, however, PARP inhibition
can save cells from death by NAD depletion, at least in the short term. This can also lead
to an increase in carcinogenesis, due to the survival of cells with significant levels of
DNA damage.
An in vivo example of this second model is the chemical induction of insulin-dependent
diabetes mellitus (IDDM) in animals. Alloxan and streptozotocin are DNA-damaging
agents, working via hydroxyl radical formation and base alkylation, respectively, and are
fairly selective pancreatic ?-cell toxins. They are commonly used to induce IDDM in
animal models. In these models, alloxan and streptozotocin induce poly(ADP-ribose) synthesis
to the point of NAD depletion and cell death. Treatment with PARP inhibitors,
including nicotinamide and 3-AB, results in conservation of pancreatic ? cells and maintenance
of insulin secretion in rats treated with alloxan and streptozotocin (110). However,
the incidence of pancreatic ?-cell tumors in these animals is essentially 100% later in life
(111). Nicotinamide is a relatively weak PARP inhibitor (Ki  10 mM) (112), and it is
rapidly converted to NAD as it enters the cell, so it is unlikely that it accumulates to a
level at which it acts effectively as a PARP inhibitor in whole-animal models. Nicotinamide
probably functions under these conditions as an NAD precursor (47) and by preventing
extreme NAD depletion in damaged ? cells allows cells to survive and eventually
express a neoplastic genotype.
It is interesting to consider whether suicidal NAD depletion (as a direct response to
PARP activation) is relevant to human carcinogenesis. The extreme level of DNA damage
required to initiate this response suggests that it is not a major factor in chronic disease
states, but there are some interesting exceptions. The concentrated autoimmune attack that
initiates IDDM in humans appears to act by this mechanism (see Sec. VII for further
discussion). In addition, experiments with PARP knockout mice have shown that PARP
activation plays a major role in ischemia-reperfusion injury in the brain (98). The sudden
oxidant stress that occurs when blood flow returns to a hypoxic region of the brain causes
a PARP-dependent depletion of NAD, which leads to neuronal cell death. This model
may provide important clues to the effective treatment of stroke victims. However, most
human cancers result from a gradual accumulation of DNA damage under conditions in
which poly(ADP-ribose) synthesis rates are low and NAD depletion is modest. Even very
aggressive models of chemically induced carcinogenesis cause a high incidence of initiation
without severe depletion of NAD (113). If models of cell injury that cause suicidal
depletion of NAD are used to examine the effect of niacin deficiency, dramatic results
can be obtained (114). This is different from the situation in which a modest amount of
Niacin 235
DNA damage causes initiation of apoptosis and NAD depletion occurs as apoptosis progresses.
Cell culture models have a great contribution to make in understanding the relationship
between niacin status and cellular defense; many parameters can be controlled and/
or measured accurately, including NAD levels, degree of DNA damage, progression of
apoptosis, and transformation to a neoplastic phenotype. However, researchers do need
to relate their in vitro models to the degree of NAD depletion and levels of DNA damage
that are possible in the whole animal.
7. Dietary Niacin Deficiency, Poly(ADP-ribose) Metabolism, and
Carcinogenesis
The native population of the Transkei region in South Africa has a high risk for esophageal
cancer (115). A maize-based, low-protein diet is staple for these people, and pellagra is
common. Esophageal ulcerations and esophagitis, common in pellagrins, have been associated
with development of carcinoma of the esophagus. Van Rensburg et al. found a greater
than fivefold increase in the risk of esophageal cancer in Zulu men who ate maize daily
(116). Frequent consumption of maize by natives of the Henan province of China is also
associated with increased esophageal cancer risk (117). Consumption of maize in northeastern
Italy was associated with increased risk of oral, pharyngeal, and esophageal cancers,
especially with heavy consumption of alcohol (118). The frequency of esophageal
cancer appears to increase when maize replaces sorghum as a basic dietary component
(115,119). Low niacin intake in an American population, in western New York state, was
also associated with increased oral cancer risk, when controlled for smoking and alcohol
consumption (120), which appeared to be the initiating factors in the disease. This is
interesting in that the patients were not deficient in niacin to the degree of showing clinical
symptoms of pellagra, and suggests that subclinical niacin deficiency may increase cancer
risk.
The Linxian province of northern China also has a very high rate of esophageal and
gastric cancers. Recently, a series of nutritional intervention trials was conducted in this
area (121,122). During 5 years of intervention, a combined supplement of ?-carotene,
vitamin E, and selenium decreased total mortality, total cancer mortality, and stomach
cancer mortality. A parallel group receiving riboflavin and niacin did not show any bene-
fits. It is important to note that this population has traditionally been dependent on rice
as a staple rather than maize, and niacin deficiency is less common than thiamine or
selenium deficiency. There is an important need to design an intervention trial in an area
dependent on maize as a staple, with a significant incidence of existing niacin deficiency
in the population.
A limited number of animal experiments have been conducted to test whether niacin
deficiency plays a causal role in the process of carcinogenesis. Miller and Burns studied
the interaction of niacin deficiency, protein-energy malnutrition, and renal carcinogenesis
in rats (123). The diets were low in tryptophan and total protein (25% of recommended
intake), with nicotinamide at zero, requirement, or 10 times requirement levels. The diets
did not affect tumor size or number, although death due to renal tumor burden may have
been accelerated in the deficient animals. Unfortunately, pyridine nucleotides in the liver
and kidney were not decreased by the deficient diet, and it is difficult to determine if niacin
deficiency had any impact in this experiment. This experiment highlights the difficulties in
working with a nutrient that can be synthesized from an amino acid, requiring, in most
species, an imbalanced amino acid diet to create the vitamin deficiency. Parameters must
be controlled carefully to get the desired nutritional status.
236 Kirkland and Rawling
Van Rensburg et al. (124) developed an animal model of malnutrition and esophageal
cancer, induced by N-nitrosomethylbenzylamine (a DNA-alkylating agent) in cornfed
rats. Addition of 20 mg of nicotinic acid per kilogram of basal diet resulted in reduction
of tumor incidence, size, and progression compared with that seen in rats fed the basal
diet alone. NAD and poly(ADP-ribose) levels were not measured in this study. Another
study has shown that lymphocytes from niacin-deficient rats were more susceptible to
oxygen radical–induced DNA damage (125).
We have developed a model of niacin deficiency in rats that maintains a positive
growth rate. The diet is based on components with minimal niacin content and a mixture
of casein (7%) and gelatin (6%) as sources of protein, designed to limit tryptophan content.
Rats fed this diet develop clinical signs of deficiency, including dermatitis, diarrhea, and
ataxia, and also have decreased hepatic NAD
 and poly(ADP-ribose) levels (30). However,
when treated with DEN, hepatic poly(ADP-ribose) accumulation was not affected
by niacin deficiency, and there was no long-term effect of diet on the development of
preneoplastic altered hepatic foci (126). It is apparent that hepatic NAD
 levels, which
decreased from about 900 to about 600 µM in deficient rats, were still adequate to support
the activity of PARP, which has a Km in the range of 20–80 µM (127).
It is likely that, during niacin deficiency, NAD depletion is not uniform in different
organs and tissues. The symptoms of pellagra demonstrate tissue specificity, like most
nutrient deficiencies. One factor that can cause more rapid nutrient depletion is cell turnover.
The bone marrow has the most rapid rate of cell turnover in the body, with a doubling
time of about 12 hours, and these cells leave to other sites in the body, exporting nutrient
resources with them. With this in mind, we recently examined the effect of niacin status
on NAD and poly(ADP-ribose) metabolism in the bone marrow of rats. We found that
NAD depletion in the bone marrow is more dramatic than in any other tissue that we have
measured (20% of control) and that basal poly(ADP-ribose) content is decreased in the
niacin-deficient marrow to almost undetectable levels. The most common problem with
DNA damage to the bone marrow occurs during chemotherapy, so we started to treat
niacin-deficient and control rats with nitrosourea drugs that are known to cause bone marrow
suppression and induce leukemias in the long term. In this model of the side effects
of chemotherapy drugs, niacin deficiency increased the severity of acute bone marrow
suppression (anemia, leukopenia) (128) and increased the rate of development of nitrosourea-
induced leukemias (129). These results may be important, because a large percentage
of cancer patients appear to be niacin deficient (130), and bone marrow suppression
and secondary leukemias are probably the two biggest problems in cancer therapy, beyond
curing the original disease.
Many more experiments have been conducted on the effect of pharmacological supplementation
of nicotinic acid or nicotinamide on various types of carcinogenesis. A wide
range of doses of nicotinamide has been used, in the presence and absence of exogenous
carcinogens, as reviewed by Bryan (131). Nicotinamide alone does not appear to present
risk as a carcinogen, although there may be some risk associated with its use in the prevention
of IDDM (further discussion in Sec. VII). When used in conjunction with carcinogenic
foods or compounds in animal experiments, nicotinamide has a confusing array of effects,
suggesting varied mechanisms of action. When given with diethylnitrosamine, nicotinamide
did not affect liver carcinogenesis, but it did increase kidney neoplasms. Following
streptozotocin treatment, nicotinamide decreased adenoma formation but increased pancreatic
islet cell tumors (111,131). Conversely, nicotinamide has shown significant protecNiacin
237
tive effects against bladder and intestinal cancers when provided in a diet containing
bracken fern.
There is much less information on the effects of large doses of nicotinic acid on
carcinogenesis. Surprisingly, the best information is probably from human studies, derived
from the long-term use of this compound in the treatment of hypercholesterolemia. Nine
years after a 6-year period of nicotinic acid use to treat hypercholesterolemic patients,
there was a significant decrease in mortality in this group, but this did not appear to be
due to a decrease in cancer incidence (132).
We have recently studied animals supplemented with nicotinamide or nicotinic acid
and treated with DEN (47). While both supplements increased liver NAD
 to a modest
extent in the absence of carcinogen treatment, only nicotinamide significantly increased
basal poly(ADP-ribose) levels (before DEN), whereas only nicotinic acid increased poly
(ADP-ribose) levels following DEN. Neither supplement affected the development of
DEN-induced preneoplastic altered hepatic foci. In contrast, pharmacological supplementation
of nicotinamide in mouse diets caused a dramatic increase in skin NAD
 and provided
significant protection against skin cancer induced by UV radiation (see Sec. V.F.9,
below). We have also shown that pharmacological supplementation of both nicotinic acid
and nicotinamide cause large increases in bone marrow NAD
 and poly(ADP-ribose) in
rats, and decrease the long term development of leukemia (unpublished data). As with
deficiency, it is not surprising that niacin supplementation affects cancer susceptibility in
some tissues, like the marrow and skin, and not others, like the liver. We need to continue
to build our knowledge of whole animal models to appreciate the complexities of niacin
metabolism and allow accurate recommendations for human populations.
Although some of the foregoing responses may have been due to changes in the
functions associated with NAD
 utilization, there are also a variety of pharmacological
actions that are not related to NAD
 synthesis. These may be responsible for the inconsistency
of the responses, and some of the potential mechanisms will be discussed in Sec. VII.
8. Niacin Status and Oxidant Lung Injury
Niacin supplementation can decrease the degree of lung injury and fibrosis from a variety
of causes, including exposure to lipopolysaccharide, cyclophosphamide, and bleomycin.
The best defined model uses bleomycin, an antibiotic chemotherapy drug that intercalates
with DNA and induces damage through the local production of oxygen radicals (133).
This is a very severe stress, which causes NAD and ATP depletion, perhaps leading to
suicidal NAD depletion, as hypothesized by Berger (109). Protection by niacin may be
functioning through the maintenance of NAD levels, but nicotinamide is more effective
in increasing NAD, while nicotinic acid is more potent in the reduction of lung pathology
(134). The protection of lung tissue with these supplements may allow much safer use of
bleomycin as a chemotherapy agent, although it is not known whether they will also protect
tumor tissue.
Hyperoxia is another popular model of oxidant stress in the lung and has clinical
relevance to the care of premature infants and patients with adult respiratory distress syndrome.
Hyperoxia has been shown to induce poly(ADP-ribose) synthesis in the lung, and,
although poly(ADP-ribose) synthesis is decreased by niacin deficiency, the deficient state
does not increase the severity of lung damage (135). Consistent with this finding, pharmacological
niacin supplementation is also ineffective in decreasing the severity of hyperoxic
lung damage (136). Why is this response different from that observed following bleomycin
238 Kirkland and Rawling
treatment? Bleomycin induces a more sudden and severe stress to pulmonary cells,
and it targets DNA specifically as an intracellular target, leading to depletion of NAD and
ATP. Hyperoxic damage occurs gradually over a period of 5 or more days (137) and
does not cause NAD depletion (135). The majority of oxygen radicals emanate from the
mitochondria and endoplasmic reticulum, and oxidative damage is distributed among the
intracellular compartments. Perhaps of greatest interest in this model, hyperoxia actually
increases lung NAD content in the niacin-deficient animal to almost that of niacin-replete
controls. This result strongly suggests that enzymes or transport systems involved in NAD
turnover are regulated in response to certain aspects of cellular damage, perhaps via the
oxidant stress response described for the induction of fos and jun (138). The degree of
oxidant stress and the time course of the stress, which differ between hyperoxia and bleomycin
toxicity, are likely to be important in determining the ability of cells or tissues to
adapt.
9. Niacin Status and Skin Injury
Because of the sun sensitivity displayed by pellagrins, there has been a long-standing
interest in the potential of niacin to improve skin health (139). Unfortunately, very little
is known about the effect of niacin deficiency on the susceptibility to UV light or chemical
carcinogens. Rainbow trout are more prone to UV light–induced skin damage when niacin-
deficient (140), but little is known about other nonhuman species, or whether this
finding correlates with a change in ADP-ribose metabolism.
On the other hand, many studies have been conducted using pharmacological doses
of various forms of the vitamin. Nicotinic acid acts as a vasodilator in the skin, leading
to an increase in blood flow through the microvasculature. Interestingly, this occurs with
both oral and topical use and appears, in both cases, to be caused by changes in prostaglandin
production (141,142). Over the years, a wide variety of treatment regimens and different
forms of nicotinic acid have been used to treat various skin disorders (139). The effects
on blood flow occur only at supraphysiological levels of the vitamin and are probably not
caused by modulation of NAD pools. However, nicotinic acid supplementation at 1–10
g/kg of diet was shown to decrease UV-induced skin cancers in mice (143). The decrease
was linear through the supplementation range, as was the increase in skin NAD content.
This work shows that very large dietary doses of niacin may continue to influence cancer
susceptibility through modulation of NAD pools, although the authors also showed that
niacin supplementation improved immune surveillance of tumor cells.
Nicotinamide supplementation has also been shown to protect the skin from DNAdamaging
agents in some animal models. Large doses of nicotinamide, given intraperitoneally,
decreased the skin damage caused by sulfur mustard (144). Used as a chemical warfare
agent in the First World War, this is a DNA-alkylating agent: that causes edema,
necrosis, and microvesical formation in the exposed skin, associated with NAD depletion.
Interestingly, nicotinamide prevents the depletion of NAD and subsequent pathological
changes, without protecting against the earlier pathological changes that precede NAD
depletion.
VI. COMPETITION FOR NAD
 DURING NIACIN DEFICIENCY
It seems obvious that the most critical cellular functions of the niacin-containing nucleotides
are those of electron transport and energy metabolism. A loss in the capacity to
deliver reducing equivalents to the electron transport chain would be similar to poisoning
Niacin 239
the cell with cyanide or suffocating from a lack of oxygen. It makes sense, then, that
these functions will be strongly protected when NAD levels start to deplete during niacin
deprivation. Cultured cells, in the absence of DNA damage, can grow and divide with
less than 5% of control NAD levels (90,104,145), leaving us with a variety of questions
to be answered. How do the other pathways of NAD utilization, including poly-, mono-,
and cyclic ADP-ribose formation, compete for these limiting substrate pools? What is the
nature of this competition at the cellular level with respect to compartmentalization between
the nucleus, cytoplasm, and mitochondria? What is the role of extracellular NAD

in the function of mono(ADP-ribosyl)transferase and ADP-ribosyl cyclase enzymes on
the outer surface of the cell? How are nicotinic acid and nicotinamide distributed among
tissues during deficiency, and does this contribute to the distinctive signs and symptoms
of pellagra? How do these interactions lead to the specific metabolic lesions that cause
the sun-sensitive dermatitis, diarrhea, and dementia?
Possible mechanisms for unequal utilization of NAD at the subcellular level include(
a) variation in the affinity of enzymes for NAD
 (Km) and (b) compartmentalization.
Km values are used to describe the affinity of an enzyme for its substrate and are defined
as the concentration of substrate required to support 50% of the maximal activity. A lower
Km indicates a higher affinity and suggests that an enzyme will compete effectively with
enzymes having a higher Km as NAD
 concentrations fall during deficiency. Some caution
in interpretation is required; enzyme kinetics may change during purification, especially
for membrane-bound proteins.
The Km of PARP for NAD
 is thought to be between 20 and 80 µM (127). In certain
cultured cells, the ability to synthesize poly(ADP-ribose) decreases when cellular NAD
content drops to less than half of control levels (145), showing that the synthesis of poly
(ADP-ribose) is one of the most sensitive pathways of NAD utilization. This is similar
to the proportionate decrease in NAD
 during niacin deficiency in many tissues in vivo,
but tissues vary in their absolute concentrations of NAD
, and direct extrapolation to
intact tissues could be inaccurate. For example, in rats, liver NAD
 decreases by close
to 50% during deficiency but is still at about 500 µM absolute concentration (30).
It has been stated that poly(ADP-ribosyl)ation is the aspect of NAD
 utilization that
is most sensitive to niacin deficiency because of a much higher Km of PARP for NAD
.
Do the Km values of other NAD
-utilizing enzymes suggest that the sensitivity of poly
(ADP-ribose) metabolism may be unique? There are scores of dehydrogenase enzymes
that use NAD
 as an electron acceptor, producing NADH for utilization in the electron
transport chain. Glyceraldehyde phosphate dehydrogenase is a cytosolic enzyme that is
critical to the flow of substrates through glycolysis. It uses NAD
 as an oxidant and has
a Km for this cofactor of 13 µM (146), smaller than that of PARP, indicating a higher
affinity. Other cytosolic enzymes may have slightly higher affinities for NAD
 than PARP,
including alcohol and aldehyde dehydrogenases (17–110 µM and 16 µM, respectively)
(146). In the mitochondria, isocitrate is oxidatively decarboxylated in the TCA cycle by
a dehydrogenase with a Km for NAD
 of 78 µM(146), which is similar to PARP. However,
the mitochondrial form of malate dehydrogenase is also critical to the flow of substrate
through the TCA cycle, and its Km for NAD
 has been reported as 540 µM (146). If these
data are correct, the TCA cycle appears to require compartmentalization of NAD
 during
niacin deficiency, and the role of the mitochondria in this regard will be discussed below.
With respect to cyclic ADP-ribose synthesis, the purified microsomal cyclase from
canine spleen has a Km of 10 µM for NAD
 (54). With access to cytosolic NAD pools,
this enzyme should maintain its catalytic activity during niacin deficiency. The CD38
240 Kirkland and Rawling
cyclase is reported to have a Km of 15 µM for NAD
 (147). While this is a relatively high
affinity for substrate, the curious aspect of this enzyme is that it faces the exterior of the
cell. Does it have a requirement for extracellular NAD or access to intracellular pools?
Cultured kidney epithelial cells synthesize cyclic ADP-ribose, but they require permeabilization
to use NAD
 in the medium and require over 500 µM for a half-maximal response
(148). There are many questions to be answered in this area of research, but the potential
for cyclic ADP-ribose metabolism to be affected by niacin deficiency is worth considering.
Mono(ADP-ribosyl)transferases are a very diverse group. The only published data
on affinity for NAD
 refers to the arginine specific transferases. In a family of transferases
from turkey erythrocytes, two cytosolic enzymes have Km values of 7 and 36 µM, while
a transferase from the membrane fraction has a Km of 15 µM (67). These enzymes would
appear to compete with PARP under conditions of limiting NAD
 pools, but a transferase
from chicken liver nuclei has a Km of between 200 and 500 µM, and a transferase from
mammalian skeletal and heart muscle displays a Km of 560 µM (67). It appears that some
mono(ADP-ribosyl)transferases may be quite sensitive to niacin deficiency. The resulting
changes in cell signaling might not appear as problems in cell culture models but could
nevertheless present significant problems in the whole organism. As discussed earlier,
there is a mono(ADP-ribosyl)transferase anchored to the outer surface of the plasma membrane,
which, like CD38, appears to require extracellular NAD
. This enzyme appears to
cause the ADP-ribosylation of an intracellular protein, leading to a depression in T-cell
proliferation. Although no attempt has been made to determine the Km of this enzyme for
NAD
, levels of NAD
 as low 1 µM in the culture medium are effective in decreasing
cell proliferation (70).
It becomes apparent that the physical partitioning of NAD within the cell is a key
factor in the availability of the molecule for various metabolic functions. The cytoplasmic
pool provides substrate for soluble enzymes, as well as for those on the endoplasmic
reticulum and on the inside of the plasma membrane. These would support a host of redox
reactions and the activity of a variety of poorly defined mono(ADP-ribosyl) transferases
and ADP-ribosylcyclases. The mitochondria isolate a pool of NAD
 that is predominantly
involved in electron transport, although mono(ADP-ribosyl)ation reactions have been reported
in this organelle. Nuclear NAD
 is probably used mainly for poly(ADP-ribosyl)
ation reactions, but mono(ADP-ribosyl)transferases also are located here. The least studied
pool is extracellular NAD
, which appears to play a role in some ADP-ribosylcyclase and
mono(ADP-ribosyl)transferase activities. How distinct are these pools, and how do they
respond to the progression of niacin deficiency?
Because of the presence of nuclear pores, it is unlikely that nuclear and cytosolic
NAD
 concentrations would differ to any great extent. However, the final step in the
synthesis of NAD
 from nicotinamide is catalyzed by a nuclear enzyme (Fig. 3) (36). The
same enzyme catalyzes the second to last step in the conversion of nicotinic acid to NAD
,
but the last enzyme in this pathway is in the cytosol. This means that all of the NAD

synthesized in the cell from newly arrived nicotinamide, or from nicotinamide released
by any of the ADP-ribosylation reactions in the cell, will be available first to nuclear
reactions, of which poly(ADP-ribosyl)ation is likely to predominate. Nicotinic acid will
lead to the production of cytosolic NAD
, which may favor different patterns of utilization.
The mitochondria are well equipped to regulate NAD levels. The inner mitochondrial
membrane is essentially impermeable to all forms of NAD(P). Reducing equivalents
in the form of NADH must be transformed via shuttle mechanisms to enter the mitochondria
for ATP production. How does the mitochondrion produce or obtain NAD, and what
Niacin 241
levels does it maintain? Some researchers believe that mitochondria synthesize NAD (149)
whereas others suggest that slow, high-affinity carriers bring the necessary NAD from the
cytosol (150,151). The net requirement is probably modest, as most of the reactions identi-
fied in this organelle do not degrade the cofactor. The important question concerns the
ability of mitochondria to concentrate NAD
, and it appears that they have potent mechanisms
to accomplish this. NAD
 levels in hepatocyte mitochondria appear to be about
10-fold higher than in the cytoplasm, with absolute concentrations in the neighborhood
of 5 mM (152). These NAD
 concentrations would support enzymes with relatively low
affinities for NAD
, such as malate dehydrogenase (146). With this ability to concentrate
NAD
, the mitochondrial pool could be very well protected during niacin deficiency.
The plasma pool of NAD
 is poorly characterized. Levels of noncellular NAD
 in
blood samples are extremely low (23), and there is no information on the response of this
pool to dietary niacin intake. Further research will be required to determine if this source
of NAD
 has any physiological role.
In addition to the competition for NAD
 at the cellular level, organs and tissues vary
in their ability to conserve NAD pools or compete for precursors during the progression of
niacin deficiency (30). Various tissues also start with different levels of NAD
, which
may act as reserves during deficiency. Blood NADP
 is more stable than NAD
 during
niacin deficiency (29), but it may change in other tissues, and the impact of niacin defi-
ciency on NAADP metabolism is not known. These are some of the concepts that must
be appreciated as we progress toward a better understanding of the biochemical basis of
the pathologies of pellagra, a disease whose clinical symptoms remain unexplained at the
molecular level.
VII. PHARMACOLOGY AND TOXICOLOGY
Levels of niacin in excess of the RNI have been used in attempts to treat Hartnup’s disease,
carcinoid syndrome, poor glucose tolerance, atherosclerosis, schizophrenia, hyperlipidemia,
IDDM, and a variety of skin disorders. In some countries, during the shortages of
proper medical supplies caused by World War II, nicotinic acid became a popular drug
because the dramatic flushing reaction that it caused in the skin was interpreted as a sign
of the potency of the treatment (153). Currently, nicotinic acid and nicotinamide are used
mainly in the prevention of cardiovascular disease and IDDM, respectively. Very few
nutrients are prescribed medicinally in North America for pharmacological purposes that
are mechanistically distinct from their known nutrient functions. Both nicotinic acid and
nicotinamide fall into this category, and the pharmacological effects of these two vitamers
appear to be surprisingly unrelated.
Large oral doses of the two vitamers may be absorbed and distributed quite differently
than when provided at levels found in a normal diet. However, there is very little
information, especially from humans, on the pharmacokinetics of these compounds. In
normal diets, nicotinic acid is obtained mainly from plant products, while nicotinamide
and preformed nucleotides are derived mainly from animal-based food products. The intestine
and liver are active in the conversion of nicotinic acid to NAD and subsequently to
nicotinamide for release into the bloodstream, causing normal plasma levels of nicotinamide
to be greater than those of nicotinic acid (23). Large oral doses of nicotinic acid will
overcome this regulation, but the concentrations that reach peripheral tissues in patients are
poorly defined (153). There are two reasons for this: first, quantification of specific vitamin
forms is laborious, and, second, nicotinic acid was approved as a drug under a ‘‘grand242
Kirkland and Rawling
father’’ clause, which excused it from the rigorous testing required by potential new drugs
on the market.
In the case of nicotinamide, which is the main circulating form of the vitamin under
normal conditions, large doses can have a depressing effect on relative availability. This
is caused by the induction of deamidase enzymes in gut microflora and also by attaining
concentrations in the Km range of similar enzymes in the liver, leading to formation of
nicotinic acid (154). However, pharmacokinetic studies of nicotinamide in humans show
that plasma levels are significantly elevated by large oral doses (155). More work is needed
in defining the pharmacokinetics of both forms of niacin, including an assessment of their
effect on NAD pools and their turnover in various tissues.
A. Nicotinic Acid
1. Nicotinic Acid and Hyperlipidemia
Historically, nicotinic acid has been administered to patients with a variety of disorders,
often more for the dramatic skin reaction than proven curative powers. Its most successful
use is for the treatment of hyperlipidemia (156). At high doses, nicotinic acid causes
several changes in lipid and lipoprotein metabolism, including inhibition of lipolysis in
adipose tissue (157), inhibition of the synthesis and secretion of very low-density lipoprotein
(VLDL) by the liver (158), lowering of serum lipoprotein(a) levels (156), and increasing
serum levels of high-density lipoprotein (HDL) (159).
These mechanisms of action of nicotinic acid appear to be unrelated to the formation
of pyridine nucleotides or to the actions of nicotinamide. Decreased lipolysis in adipose
tissue is due to an inhibition of adenylate cyclase activity (157). The resulting drop in
cAMP levels leads to the decreased mobilization of fatty acids. It is not known whether
nicotinic acid binds to a cellular receptor or interacts directly with adenylate cyclase. The
decrease in fatty acid release from adipose tissue is at least partially responsible for the
drop in VLDL formation by the liver as well as for the subsequent drop in LDL levels,
although there may also be direct effects on liver lipid metabolism. Studies with radioactive
acetate suggest that nicotinic acid inhibits cholesterol synthesis at the level of
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (156), and this may also
play a role in the lowering of VLDL and LDL levels. Unlike many treatments for hyperlipidemias,
nicotinic acid also increases circulating levels of HDL (159), the beneficial lipoprotein
that removes cholesterol from vascular tissue, but the mechanisms involved are
uncertain. The skin flush of the face and upper trunk is a very rapid response to nicotinic
acid, and it appears to be caused by the local formation of prostaglandins (142), although
the underlying mechanism is unclear.
2. Nicotinic Acid Toxicity
There are a few drawbacks to using high levels of oral nicotinic acid. As mentioned above,
the short-term side effects may include vasodilation, burning or stinging sensations in the
face and hands, nausea, vomiting, and diarrhea. In the longer term, there may be varying
degrees of hyperpigmentation of the skin, abnormal glucose tolerance, hyperuricemia,
peptic ulcers, hepatomegaly, and jaundice (156). The chronic doses of time-release nicotinic
acid have been reported to cause more hepatotoxicity (160), but this is controversial.
It should be noted that all drugs used in the treatment of hyperlipidemia have some side
effects, many of which can be managed through changes in dose. Interestingly, nicotinic
acid use for 6 years by patients with cardiovascular disease led to a decrease in all-cause
Niacin 243
mortality measured 8 years after the drug use was discontinued (132). Frequently, the
choice of treatment is financially based, with the cost of nicotinic acid treatment being a
fraction of that of the newer medications.
B. Nicotinamide
In the past, nicotinamide has been used in the treatment of schizophrenia (161), but more
effective drugs have replaced it in this field. It is now being tested as a chemotherapy
agent; in this application, nicotinamide potentiates the cytotoxic effects of chemotherapy
and radiation treatment against tumor cells (162), an action that appears to be due to
increased blood flow and oxygenation of tumor tissue (163). However, most of the current
interest in nicotinamide involves its potential use in the prevention or delay of onset of
IDDM (156,164).
1. Nicotinamide and IDDM
Interest in this area started with the finding that nicotinamide could prevent diabetes induced
by the ?-cell toxins alloxan and streptozotocin (110). It was soon shown that the
? cells were killed by an interesting mechanism; severe DNA damage led to excessive
activation of PARP, which depleted cellular NAD levels to the point that ATP synthesis
could not be maintained. The researchers concluded that nicotinamide was preventing
diabetes by inhibiting PARP activity and preventing the depletion of NAD pools. Since
nicotinamide is not a very high-affinity inhibitor of PARP (112), and cellular levels tend
to stay low due to active conversion to NAD, it seems likely that protection in this model
was due to the use of nicotinamide as a precursor for NAD synthesis. We have shown
that large oral doses of nicotinamide increase NAD
 and poly(ADP-ribose) levels in the
liver (47) and more recently, in the bone marrow (unpublished results). It is unlikely that
oral nicotinamide could act as a PARP inhibitor in the pancreas, under similar conditions.
Interestingly, all of the animals protected from chemically induced diabetes by nicotinamide
developed insulin producing ?-cell tumors (111), a form of cancer that is particularly
lethal in humans (165). It is not surprising that cells rescued from NAD depletion
due to extreme DNA damage, either through inhibition of PARP or through pharmacological
support of NAD pools, would be at risk for neoplastic growth due to the survival of
cells with significant levels of DNA damage.
One concern in promoting the clinical use of nicotinamide in the prevention of
IDDM in humans is that there will also be a long-term risk of pancreatic cancer if survival
of ? cells can be maintained in the face of autoimmune attack. In spite of the theoretical
difficulties and the potential dangers of inhibiting PARP in the face of genotoxic damage
to the pancreas, many investigators in the diabetes field are comfortable with the theory
of PARP inhibition (166). Fortunately, there are other aspects of the response to nicotinamide
that paint a more optimistic picture, although not a very clear one.
In humans, the onset of IDDM occurs spontaneously by immune recognition of ?-
cell antigens. This is associated with leukocyte infiltration and the presence of anti–islet
cell antibodies in the serum. The spontaneous occurrence of IDDM is similar in the nonobese
diabetic (NOD) mouse. When nicotinamide is given to weanling NOD mice, the
onset of diabetic symptoms is prevented or delayed (167). The important distinction is
whether nicotinamide prevents ?-cell death by protecting against autoimmune attack or
by maintaining the ? cell following immune attack. There appear to be several ways in
which nicotinamide can prevent the initial damage to the cells of the pancreas. In some
244 Kirkland and Rawling
experimental models, nicotinamide treatment diminishes some aspects of the immune response
itself, including the ability of monocytes to attack cells labeled with antibodies
(168), the rate of movement of lymphocytes into tissues (169), and the production of
nitric oxide in response to inflammatory cytokines (170). Other studies have shown that
nicotinamide acts as a free-radical scavenger to protect the ?-cell targets even after the
immune response is directed against them (171,172). These mechanisms may provide an
explanation for the prevention of IDDM by nicotinamide and lead to more effective protocols
in the future.
In a very different experimental model, nicotinamide was found to decrease the
severity of diabetes in response to partial pancreatectomy. This appeared to be due to a
stimulation of ?-cell proliferation, leading to an increase in the size of islets (173). The
? cells normally have a low capacity for cell regeneration, and it has been suggested that
this predisposes to the development of human diabetes (110). When fetal pancreas is
pretreated with nicotinamide, there is an acceleration in the reversal of diabetes after transplantation
of the islet cells into diabetic nude mice (174). This is an important model for
future directions in the treatment of fully developed IDDM, and these experiments show
that nicotinamide has a positive influence on ?-cell regeneration and differentiation.
Nicotinamide may have an effect on mono(ADP-ribosyl)ation reactions, either by
acting as an inhibitor or by enhancing substrate (NAD
) levels. In addition, there is a new
feeling that cyclic ADP-ribose could have an important role in immune regulation. Cyclase
enzymes like CD38 are important proteins in leukocytes, and drugs that interfere with
cyclic ADP-ribose regulation of calcium release are immune suppresors (166).
2. Clinical Trials
Encouraged by the type of data summarized above, a number of experiments have been
done with human subjects. In the majority of these studies, patients were recruited in an
early stage of clinically apparent diabetes. Since these subjects retain a varying degree of
?-cell function, it is not surprising that the results have been inconsistent. However, a
number of treatment protocols have been successful in inducing remission in some patients
(175) and increasing the residual level of plasma insulin for up to 2 years after diagnosis
(176).
The animal models show that nicotinamide treatment should start before the disease
process is in an advanced stage. To do this in human populations, researchers must identify
the susceptible population. Blood levels of islet cell antibodies, human leukocyte antigen,
and family history are used as predictors of IDDM for subject recruitment. Several intervention
trials starting in the early stages of the disease have produced interesting results.
In a 2-year study, 13 of 14 high-risk children treated with oral nicotinamide remained
disease-free, while all of the children in the control group became diabetic (177). In another
study, nicotinamide treatment for 8 months protected plasma insulin in high-risk
children, in spite of the fact that anti–islet cell antibody levels were not decreased (178).
This suggests that the autoimmune response is affected downstream of autoantibody production,
or, alternatively, that ?-cell defenses are improved by nicotinamide treatment.
These results have encouraged the organization of several larger studies, including
the European-Canadian Nicotinamide Diabetes Intervention Trial (ENDIT), which will
involve the screening of about 30,000 people for subjects that are anti–islet cell antibody–
positive with normal glucose tolerance (164). These subjects will be treated with nicotinamide
or placebo for 5 years, and the results should provide significant information on
Niacin 245
the prevention of diabetes by nicotinamide, its mechanism of action, and even the longterm
risk of carcinogenesis.
3. Nicotinamide Toxicity
The levels of nicotinamide that are used in the treatment of IDDM (about 3 g/day) have
not been reported to cause any adverse side effects on an acute basis. Larger doses (about
10 g/day) have been known to cause liver injury (parenchymal cell injury, portal fibrosis,
cholestasis) (179). Chronic intake of nicotinamide can also induce a methyl-group defi-
ciency state due to the methylation reactions involved in excretion (180,181), and physicians
recommending nicotinamide therapy should ensure that the subjects have an adequate
intake of methyl donors such as choline and methionine. Methyl donor deficiency
also appears to increase the risk of carcinogenesis, and more work from this perspective
is needed to define the safe use of nicotinamide. The most serious potential side effect
of nicotinamide use would be the induction of pancreatic tumors in patients at risk for
IDDM, and it will take many years to assess this. There is comfort from the fact that studies
of the NOD mouse have not reported pancreatic tumors during long-term nicotinamide
treatment.
VIII. SUMMARY
Niacin deficiency has the potential to alter redox reactions, poly and mono(ADP-ribose)
synthesis, and the formation of cyclic ADP-ribose and NAADP. During niacin deficiency,
the metabolic changes that lead to the dramatic signs and symptoms of pellagra will likely
be tissue-specific and reflect subcellular competition for NAD pools. The effect of chronic
niacin undernutrition on human health, especially the process of carcinogenesis, appears
to be an exciting area that deserves more attention. With a rapidly broadening perspective
on the biochemical roles for niacin in metabolism, identification of optimal niacin nutriture
should be possible in the coming decade.
Supplementation of nicotinic acid and nicotinamide above the dietary requirement
may affect some of the same processes, but these compounds have distinctive pharmacological
properties, some of which may be unrelated to their currently defined nutrient
functions. Future research in models of niacin deficiency and supplementation may lead
us to reevaluate the accepted metabolic roles of niacin and create new guidelines for niacin
intake.
ACKNOWLEDGMENTS
The authors thank Dr. William Bettger, Dr. William Woodward, Dr. Elaine Jacobson, and
Dr. Myron Jacobson for critical reading of this manuscript. Dr. Doug Lanska helped us to
find historical references and early photographs. The Cancer Research Society (Montreal,
Canada) has supported research on niacin status and ADP-ribose metabolism, which made
the writing of this chapter possible.
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7
Riboflavin (Vitamin B2)
RICHARD S. RIVLIN and JOHN THOMAS PINTO
Weill Medical College of Cornell University and Memorial Sloan-Kettering Cancer
Center, New York, New York
I. INTRODUCTION AND HISTORY OF DISCOVERY
In the pioneering studies by McCollum and Kennedy (1) in the early part of the twentieth
century, water-soluble tissue extracts were found to be effective in the prevention of the
deficiency state of pellagra in experimental animals. As studies progressed, it became
evident that there were at least two distinct fractions of these extracts, one of which was
heat-labile and the other heat-stable.
Further studies of this heat-stable fraction showed that it was a complex containing
a yellow growth factor. This factor had fluorescent properties and was later purified and
named riboflavin (B2) (2). Other components of this fraction were later identified as niacin,
which was the true antipellagra compound, and vitamin B6, which was particularly effective
in preventing dermatitis in animals.
The physiological role of the yellow growth factor remained obscure until the landmark
discovery by Warburg and Christian (3) in 1932 of ‘‘yellow enzyme’’ or ‘‘old yellow
enzyme.’’ This protein was found to be composed of an apoenzyme and a yellow cofactor
serving as a coenzyme. This coenzyme was shown subsequently to contain an isoalloxazine
ring (4) and a phosphate group (5).
Synthesis of riboflavin was accomplished by Kuhn et al. (6) and Karrer et al. (7).
The flavin coenzyme riboflavin-5?-phosphate (flavin mononucleotide, FMN), was identi-
fied in 1937 by Theorell (8). In 1938, Warburg and Christian (9) clarified the structure
of flavin adenine dinucleotide (FAD), formed from FMN. While many enzymes utilize
FMN and FAD as cofactors, flavins bound covalently to specific tissue flavoproteins
also have been found to have major biological significance, as reviewed by McCormick
(10).
255
256 Rivlin and Pinto
II. CHEMISTRY
Riboflavin is defined chemically as 7,8-dimethyl-10-(1?-d-ribityl)isoalloxazine. The planar
isoalloxazine ring provides the basic structure not only for riboflavin (vitamin B2) but for
the naturally occurring phosphorylated coenzymes that are derived from riboflavin (Fig.
1). These coenzymes include FMN, FAD, and flavin coenzymes linked covalently to specific
tissue proteins, generally at the 8-? methyl position of the isoalloxazine ring (10).
Among the key mammalian enzymes with covalently bound FAD are included sarcosine
and succinic dehydrogenases, which are located in the matrix and inner mitochondrial
membrane, respectively; monoamine oxidase in the outer mitochondrial membrane;
and l-gulonolactone oxidase, found particularly in liver and kidney microsomes of those
mammalian species capable of synthesizing ascorbic acid from its precursors (11).
The sequence of events in the synthesis of the flavin coenzymes from riboflavin and
its control by thyroid hormones are shown in Fig. 2. Thyroid hormones regulate the activities
of the flavin biosynthetic enzymes (12), the synthesis of the flavoprotein apoenzymes,
Fig. 1 Structural formulas of riboflavin, flavin mononucleotide (riboflavin-5?-phosphate, FMN)
and flavin adenine dinucleotide (FAD).
Riboflavin (Vitamin B2) 257
Fig. 2 Metabolic pathway of conversion of riboflavin into FMN, FAD, and covalently bound
flavin, together with its control by thyroid hormones (12).
and the formation of covalently bound flavins (13). The first biosynthetic enzyme, flavokinase,
catalyzes the initial phosphorylation from ATP of riboflavin to FMN. A fraction
of FMN is directly utilized in this form as a coenzyme. The largest fraction of FMN,
however, combines with a second molecule of ATP to form FAD, the predominant tissue
flavin, in a reaction catalyzed by FAD synthetase, also called FAD pyrophosphorylase.
The covalent attachment of flavins to specific tissue proteins occurs after FAD has been
synthesized. A sequence of phosphatases returns FAD to FMN and FMN, in turn, to
riboflavin (12). Most flavoproteins utilize FAD rather than FMN as coenzyme for a wide
variety of metabolic reactions. Microsomal NADPH–cytochrome P450 reductase is highly
unusual in containing FMN and FAD in equimolar ratios.
Riboflavin is yellow and has a high degree of natural fluorescence when excited by
UV light, a property that can be utilized conveniently in its assay. There are a number of
variations in structure in the naturally occurring flavins. Riboflavin and its coenzymes are
sensitive to alkali and to acid, particularly in the presence of UV light. Under alkaline
conditions, riboflavin is photodegraded to yield lumiflavin (7,8,10-trimethylisoalloxazine),
which is inactive biologically. Riboflavin is photodegraded to lumichrome (7,8-dimethylalloxazine)
under acidic conditions, a product that is also biologically inactive (10). Thus,
an important physical property of riboflavin and its derivatives is their sensitivity to UV
light, resulting in rapid inactivation. Therefore, phototherapy of neonatal jaundice and of
certain skin disorders may promote systemic riboflavin deficiency. The structure–function
relationships of the various biologically active flavins have been comprehensively reviewed
(14).
III. RIBOFLAVIN DEFICIENCY AND FOOD-RELATED ISSUES
A. Riboflavin Deficiency
Clinical riboflavin deficiency is not recognizable at the bedside by any unique or characteristic
physical features. The typical glossitis, angular stomatitis, and dermatitis observed
is not specific for riboflavin deficiency and may be due to other vitamin deficiencies as
258 Rivlin and Pinto
well. In fact, when dietary deficiency of riboflavin occurs, it is almost invariably in association
with multiple nutrient deficits (15).
With the onset of riboflavin deficiency, one of the adaptations that occurs is a fall
in the hepatic free riboflavin pool to nearly undetectable levels, with a relative sparing of
the pools of FMN and FAD that are needed to fulfill critical metabolic functions (16).
Another adaptation to riboflavin deficiency in its early stages is an increase in the de
novo synthesis of reduced glutathione from its amino acid precursors, in response to the
diminished conversion of oxidized glutathione back to reduced glutathione. This may represent
a compensatory reaction resulting from depressed activity of glutathione reductase,
a key FAD-requiring enzyme (Fig. 3).
An emerging concept is that dietary inadequacy is not the only cause of riboflavin
deficiency and that certain endocrine abnormalities, such as adrenal and thyroid hormone
insufficiency, drugs, and diseases, may interfere significantly with vitamin utilization
(17,18). Psychotropic drugs, such as chlorpromazine, antidepressants [including imipramine
and amitriptyline (19)], cancer chemotherapeutic drugs (e.g., adriamycin), and some
antimalarial agents [e.g., quinacrine (20)], impair riboflavin utilization by inhibiting the
conversion of this vitamin into its active coenzyme derivatives. Figure 4 shows the structural
similarities among riboflavin, imipramine, chlorpromazine, and amitriptyline. Alco-
Fig. 3 Diagrammatic representation of metabolic adaptations to riboflavin deficiency. Diminished
conversion of oxidized glutathione to reduced glutathione occurs as a result of decreased activity
of glutathione reductase, an enzyme that utilizes FAD as a coenzyme. Levels of reduced glutathione
are maintained by enhanced de novo synthesis from precursors (31,32).
Riboflavin (Vitamin B2) 259
Fig. 4 Structural similarities among riboflavin, imipramine, chlorpromazine, and amitriptyline.
hol appears to cause riboflavin deficiency by inhibiting both its digestion from dietary
sources and its intestinal absorption (21).
In approaching riboflavin deficiency, as well as other nutrient deficiencies, it may
be useful to think in terms of risk factors. Thus, the consequences of a poor diet may be
intensified if the patient is also abusing alcohol, is using certain drugs for prolonged periods,
is extremely elderly, or has malabsorption or an underlying illness affecting vitamin
metabolism.
In experimental animals, hepatic architecture is markedly disrupted in riboflavin
deficiency. Mitochondria in riboflavin-deficient mice increase greatly in size, and cristae
increase in both number and size (22). These structural abnormalities may disturb energy
metabolism by interfering with the electron transport chain and metabolism of fatty acids.
Villi decrease in number in the rat small intestine; villus length increases, as does the rate
of transit of developing enterocytes along the villus (23). These findings of structural
abnormalities together with accelerated rate of intestine cell turnover (24) may help to
explain why dietary riboflavin deficiency leads to both decreased iron absorption and increased
iron loss from the intestine.
There are many other effects of riboflavin deficiency on intermediary metabolism,
particularly on lipid, protein, and vitamin metabolism. Of particular relevance to vitamin
metabolism is the fact that the conversion of vitamin B6 to its coenzyme derivative, pyridoxal
5?-phosphate, may be impaired (25). Riboflavin deficiency has been studied in many
animal species and has several vital effects, foremost of which is failure to grow. Additional
effects include loss of hair, skin disturbances, degenerative changes in the nervous
system, and impaired reproduction. Congenital malformations occur in the offspring of
female rats that are riboflavin-deficient. The conjunctiva becomes inflamed, the cornea is
vascularized and eventually opaque, and cataract may result (26).
Changes in the skin consist of scaliness and incrustation of red-brown material consistent
with changes in lipid metabolism. Alopecia may develop, lips become red and
260 Rivlin and Pinto
Table
1
Top
Sources
of
Riboflavin
and
Their
Caloric
Content
Riboflavin
Energy
Riboflavin
Energy
Top
sources
(mg/
100
g)
(kcal
/100
g)
Top
food
sources
(mg
/100
g)
(kcal/100
g)
Yeast
baker’s
dry
(active)
5.41
282
Cheese,
pasteurized,
process
American
3.53
375
Liver,
lamb,
broiled
5.11
261
Liver,
chicken,
simmered
1.75
165
Yeast,
torula
5.06
277
Corn
flakes,
w/added
nutrients
1.40
380
Kidneys,
beef,
braised
4.58
252
Almonds,
shelled
0.93
598
Liver,
hog,
fried
in
margarine
4.36
241
Cheese,
natural,
Roquefort
0.59
369
Yeast,
brewer’s,
debittered
4.28
283
Eggs,
chicken,
fried
0.54
210
Liver,
beef
or
calf,
fried
4.18
242
Beef,
tenderloin
steak,
broiled
0.46
224
Brewer’s
yeast,
tablet
form
4.04
—
Mushrooms,
raw
0.46
28
Cheese,
pasteurized,
process
American
3.53
375
Cheese,
natural
Swiss
(American)
0.40
372
Turkey,
giblets,
cooked
(some
gizzard
fat),
simmered
2.72
233
Wheat
flour,
all-purpose,
enriched
0.40
365
Kidneys,
lamb,
raw
2.42
105
Turnip
greens,
raw
0.39
28
Kidneys,
calf,
raw
2.40
113
Cheese,
natural
Cheddar
0.38
402
Eggs,
chicken,
dried,
white
powder
2.32
372
Wheat
bran
0.35
353
Whey,
sweet,
dry
2.21
354
Soybean
flour
0.35
333
Eggs,
chicken,
dried,
white
flakes
2.16
351
Bacon,
cured,
cooked,
drained,
sliced
medium
0.34
575
Liver,
turkey,
simmered
2.09
174
Pork,
loin,
lean,
broiled
0.33
391
Whey,
acid
dry
2.06
339
Lamb,
leg,
good
or
choice,
separable
lean
roasted
0.30
186
Heart,
hog,
braised
1.89
195
Corn
meal,
degermed,
enriched
0.26
362
Milk,
cow’s
dry,
skim,
solids,
instant
1.78
353
Chicken,
dark
meat
w
/o
skin,
fried
0.25
220
Liver,
chicken,
simmered
1.75
165
Bread,
white,
enriched
0.24
270
Liver,
beef
or
calf,
fried
4.18
242
Milk,
cow’s,
whole,
3.7%
0.17
66
Figures
are
given
in
terms
of
the
riboflavin
and
calorie
amounts
in
100
g
(approximately
3.5
oz)
of
the
items
as
usually
consumed.
Portion
size
and
moisture
content
will
differ
among
food
items.
Further
details
can
be
found
in
Table
F-36
of
Ref.
29.
Source:
Ref.
29.
Riboflavin (Vitamin B2) 261
swollen, and filiform papillae on the tongue deteriorate. During late deficiency anemia
develops. Fatty degeneration of the liver occurs. Important metabolic changes occur, so
that deficient rats require 15–20% more energy than control animals to maintain the same
body weight. Thus, in all species studied, riboflavin deficiency causes profound structural
and functional changes in an ordered sequence. Early changes are very readily reversible.
Later anatomical changes, such as formation of cataract, are largely irreversible despite
treatment with riboflavin.
In humans, as noted above, the clinical features of human riboflavin deficiency do
not have absolute specificity. Early symptoms may include weakness, fatigue, mouth pain
and tenderness, burning and itching of the eyes, and possibly personality changes. More
advanced deficiency may give rise to cheilosis, angular stomatitis, dermatitis, corneal vascularization,
anemia, and brain dysfunction. Thus, the syndrome of dietary riboflavin defi-
ciency in humans has many similarities to that in animals, with one notable exception.
The spectrum of congenital malformations observed in rodents with maternal riboflavin
deficiency has not been clearly identified in humans (26).
B. Food-Related Issues
In the United States today, the most significant dietary sources of riboflavin are meat and
meat products, including poultry and fish, and milk and dairy products, such as eggs and
cheese. In developing countries, plant sources contribute most of the dietary riboflavin
intake. Green vegetables, such as broccoli, collard greens, and turnip greens, are reasonably
good sources of riboflavin. Natural grain products tend to be relatively low in riboflavin,
but fortification and enrichment of grains and cereals has led to a great increase in
riboflavin intake from these food items.
The food sources of riboflavin are similar to those of other B vitamins. Therefore,
it is not surprising that if a given individual’s diet has inadequate amounts of riboflavin,
it will very likely be inadequate in other vitamins as well. A primary deficiency of dietary
riboflavin has wide implications for other vitamins, as flavin coenzymes are involved in
the metabolism of folic acid, pyridoxine, vitamin K, niacin, and vitamin D (27).
Several factors in food preparation and processing may influence the amount of
riboflavin that is actually bioavailable from dietary sources. Appreciable amounts of ribo-
flavin may be lost with exposure to UV light, particularly during cooking and processing.
Prolonged storage of milk in clear bottles or containers may result in flavin degradation
(28). Fortunately, most milk is no longer sold in clear bottles. There has been some controversy
as to whether opaque plastic containers provide greater protection than do cartons,
particularly when milk is stored on a grocery shelf exposed to continuous fluorescent
lighting.
It is highly likely that large amounts of riboflavin are lost during the sun-drying of
fruits and vegetables. The precise magnitude of the loss is not known but varies with the
duration of exposure. The practice of adding sodium bicarbonate as baking soda to green
vegetables to make them appear fresh can result in accelerated photodegradation of ribo-
flavin. The riboflavin content of common food items is shown in Table 1 (29).
IV. PHYSIOLOGY
A. Absorption, Transport, Storage, Turnover, and Excretion
Since dietary sources of riboflavin are largely in the form of coenzyme derivatives, these
molecules must be hydrolyzed prior to absorption. Very little dietary riboflavin is found
262 Rivlin and Pinto
as free riboflavin from sources in nature. Under ordinary circumstances the main sources
of free riboflavin are commercial multivitamin preparations.
The absorptive process for flavins occurs in the upper gastrointestinal tract by specialized
transport involving a dephosphorylation-rephosphorylation mechanism, rather
than by passive diffusion. This process is sodium-dependent and involves an ATPase
active transport system that can be saturated (30). It has been estimated that under normal
conditions the upper limit of intestinal absorption of riboflavin at any one time is approximately
25 mg (31). This amount represents approximately 15 times the RDA. Therefore,
the common practice of some megavitamin enthusiasts to consume massive doses of multivitamins
has little benefit with respect to riboflavin, as the additional amounts would be
passed in the stool. Dietary covalent-bound flavins are largely inaccessible as nutritional
sources. In experimental animals, the uptake of riboflavin from the intestine is increased
in dietary riboflavin deficiency (32).
A number of physiological factors influence the rate of intestinal absorption of ribo-
flavin (17). Diets high in psyllium gum appear to decrease the rate of riboflavin absorption,
whereas wheat bran has no detectable effect. The time from oral administration to peak
urinary excretion of riboflavin is prolonged by the antacids aluminum hydroxide and magnesium
hydroxide. Total urinary excretion is unchanged by these drugs, however, and
their major effect appears to be delaying the rate of intestinal absorption rather than inhibiting
net absorption. Alcohol intake interferes with both the digestion of food flavins into
riboflavin and the direct intestinal absorption of the vitamin (21). This observation suggests
that the initial rehabilitation of malnourished alcoholic patients may be accomplished more
efficiently with vitamin supplements containing riboflavin rather than with food sources
comprising predominantly phosphorylated flavin derivatives.
There is evidence that the magnitude of intestinal absorption of riboflavin is increased
by the presence of food. This effect of food may be due to decreasing the rates
of gastric emptying and intestinal transit, thereby permitting more prolonged contact of
dietary riboflavin with the absorptive surface of the intestinal mucosal cells. In general,
delaying the rate of gastric emptying tends to increase the intestinal absorption of riboflavin.
Bile salts also increase the absorption of riboflavin (17).
A number of metals and drugs form chelates or complexes with riboflavin and ribo-
flavin-5?-phosphate that may affect their bioavailability (30). Among the agents in this
category are the metals copper, zinc and iron; the drugs caffeine, theophylline, and saccharin;
and the vitamins nicotinamide and ascorbic acid; as well as tryptophan and urea. The
clinical significance of this binding is not known with certainty in most instances and
deserves further study.
In human blood, the transport of flavins involves loose binding to albumin and tight
binding to a number of globulins. The major binding of riboflavin and its phosphorylated
derivatives in serum is to several classes of immunoglobulins, i.e., IgA, IgG, and IgM
(30).
Pregnancy induces the formation of flavin-specific binding proteins. It has been
known for many years that there is an avian riboflavin carrier protein, genetically controlled,
that determines the amount of riboflavin in chicken eggs (33). Absence of the
protein in autosomal recessive hens results in massive riboflavinuria because there is no
mechanism for retaining and binding the vitamin in serum. Eggs become riboflavin-defi-
cient, and embryonic death occurs between the tenth and fourteenth day of incubation.
Administration of antiserum to the chicken riboflavin-carrier protein leads to termination
of the pregnancy.
Riboflavin (Vitamin B2) 263
A new dimension to concepts of plasma protein binding of riboflavin in mammals
was provided by the demonstration that riboflavin-binding proteins can also be found
in serum from pregnant cows, monkeys, and humans. A comprehensive review of ribo-
flavin-binding proteins covers the nature of the binding proteins in various species and
provides evidence that, as in birds, these proteins are crucial for successful mammalian
reproduction (10). The pregnancy-specific binding proteins may help transport riboflavin
to the fetus.
Serum riboflavin-binding proteins also appear to influence placental transfer and
fetal/maternal distribution of riboflavin. There are differential rates of uptake of riboflavin
at the maternal and fetal surfaces of the human placenta (34). Riboflavin-binding proteins
regulate the activity of flavokinase, the first biosynthetic enzyme in the riboflavin-to-FAD
pathway (16).
The urinary excretion of flavins occurs predominantly in the form of riboflavin;
FMN and FAD are not found in urine. McCormick et al. have identified a large number
of flavins and their derivatives in human urine. Besides the 60–70% of urinary flavins
contributed by riboflavin itself, other derivatives include 7-hydroxymethylriboflavin (10–
15%), 8?-sulfonylriboflavin (5–10%), 8-hydroxymethylriboflavin (4–7%), riboflavinyl
peptide ester (5%), and 10-hydroxyethylflavin (1–3%), representing largely metabolites
from covalently bound flavoproteins and intestinal riboflavin degradation by microorganisms.
Traces of lumiflavin and other derivatives have also been found. These findings
were described fully by McCormick (10).
Ingestion of boric acid greatly increases the urinary excretion of riboflavin (17).
This agent when consumed forms a complex with the side chain of riboflavin and other
molecules having polyhydroxyl groups, such as glucose and ascorbic acid. In rodents,
riboflavin treatment greatly ameliorates the toxicity of administered boric acid. This treatment
should also be effective in humans with accidental exposure of boric acid, although
in practice it may be difficult to provide adequate amounts of riboflavin due to its low
solubility and limited absorptive capacity from the intestinal tract.
Urinary excretion of riboflavin in rats is also greatly increased by chlorpromazine
(19). Levels are twice those of age- and sex-matched pair-fed control rats. In addition,
chlorpromazine accelerates the urinary excretion of riboflavin during dietary deficiency.
Urinary concentrations of riboflavin are increased within 6 h of treatment with this
drug.
V. SPECIFIC FUNCTIONS
The major function of riboflavin, as noted above, is to serve as the precursor of the flavin
coenzymes, FMN and FAD, and of covalently bound flavins. These coenzymes are widely
distributed in intermediary metabolism and catalyze numerous oxidation–reduction reactions.
Because FAD is part of the respiratory chain, riboflavin is central to energy production.
Other major functions of riboflavin include drug and steroid metabolism, in
conjunction with the cytochrome P450 enzymes, and lipid metabolism. The redox functions
of flavin coenzymes include both one-electron transfers and two-electron transfers
from substrate to the flavin coenzyme (10).
Flavoproteins catalyze dehydrogenation reactions as well as hydroxylations, oxidative
decarboxylations, dioxygenations, and reductions of oxygen to hydrogen peroxide.
Thus, many different kinds of oxidative and reductive reactions are catalyzed by flavoproteins.
264 Rivlin and Pinto
A. Antioxidant Activity
In the wake of contemporary interest in the dietary antioxidants, one vitamin that is often
neglected as a member of this category is riboflavin. Riboflavin does not have significant
inherent antioxidant action, but powerful antioxidant activity is derived from its role as
a precursor to FMN and FAD. A major protective role against lipid peroxides is provided
by the glutathione redox cycle (35). Glutathione peroxidase breaks down reactive lipid
peroxides. This enzyme requires reduced glutathione, which in turn is regenerated from
its oxidized form (GSSG) by the FAD-containing enzyme glutathione reductase. Thus,
riboflavin nutrition should be critical for regulating the rate of inactivation of lipid peroxides.
Diminished glutathione reductase activity should be expected to lead to diminished
concentrations of reduced glutathione that serve as substrate for glutathione peroxidase
and glutathione S-transferase, and therefore would limit the rate of degradation of lipid
peroxides and xenobiotic substances (36).
Furthermore, the reducing equivalents provided by NADPH, the other substrate required
by glutathione reductase, are primarily generated by an enzyme of the pentose
monophosphate shunt, glucose-6-phosphate dehydrogenase. Taniguchi and Hara (37), as
well as our laboratory (38), have found that the activity of glucose-6-phosphate dehydrogenase
is significantly diminished during riboflavin deficiency. This observation provides
an additional mechanism to explain the diminished glutathione reductase activity in vivo
during riboflavin deficiency and the eventual decrease in antioxidant activity.
There have been a number of reports (39,40) in the literature indicating that riboflavin
deficiency is associated with compromised oxidant defense and furthermore that supplementation
of riboflavin and its active analogs improves oxidant status. Investigators
have shown that riboflavin deficiency is associated with increased hepatic lipid peroxidation
and that riboflavin supplementation limits this process (37–40). In our laboratory,
we have shown that feeding a riboflavin-deficient diet to rats increases basal as well as
stimulated lipid peroxidation (36).
B. Riboflavin and Malaria
Recent reports provide increasing evidence that riboflavin deficiency is protective against
malaria both in experimental animals and in humans (41,42). With dietary riboflavin defi-
ciency, parasitemia is decreased dramatically, and symptomatology of infection may be
diminished. In a study with human infants suffering from malaria, normal riboflavin nutritional
status was associated with high levels of parasitemia (43,44). In a similar fashion,
supplementation of iron and vitamins, which included riboflavin, to children resulted in
increased malarial parasitemia (45).
Further evidence for a beneficial role of riboflavin deficiency in malaria is provided
by studies utilizing specific antagonists of riboflavin, e.g., galactoflavin and 10-(4?-chlorophenyl)-
3-methylflavin (46). These flavin analogs, as well as newer isoalloxazines derivatives
(47), are glutathione reductase inhibitors and possess clear antimalarial efficacy (46).
The exact mechanism by means of which riboflavin deficiency appears to inhibit malarial
parasitemia is not yet established. One possibility relates to effects on the redox status of
erythrocytes, which is an important determinant of growth of malaria parasites. Protection
from malaria is afforded by several oxidant drugs, vitamin E deficiency, and certain genetic
abnormalities compromising oxidative defense (35).
It is well known that malaria parasites (Plasmodium berghei) are highly susceptible
Riboflavin (Vitamin B2) 265
to activated oxygen species. Parasites are relatively more susceptible than erythrocytes to
the damaging effects of lipid peroxidation (35). We have hypothesized that the requirement
of the parasites for riboflavin should be higher than that of the host cells and therefore
that marginal riboflavin deficiency should be selectively detrimental to parasites. Support
for this hypothesis is that the uptake of riboflavin and its conversion to FMN and FAD
are significantly higher in parasitized than in unparasitized erythrocytes and furthermore
that the rate of uptake of riboflavin is proportional (48) to the degree of parasitemia. These
results strongly suggest that parasites have a higher requirement for riboflavin than do
host erythrocytes.
C. Riboflavin and Homocysteine
A subject of great contemporary interest is the possible role of homocysteine in the pathogenesis
of vascular disease, including cardiovascular, cerebrovascular, and peripheral vascular
disorders (49). Blood levels of folic acid sensitively determine serum homocysteine
concentrations (50). As shown in Fig. 5, N-5-methyltetrahydrofolate is a cosubstrate with
homocysteine in its inactivation by conversion to methionine. Methylcobalamin is also a
coenzyme in this enzymatic reaction. Vitamin B6 is widely recognized for its importance
in the inactivation of homocysteine by serving as coenzyme of two degradative enzymes,
cystathionine ?-synthase and cystathioninase.
However, it is not commonly appreciated that riboflavin also has a role in homocysteine
metabolism, as the flavin coenzyme, FAD, is required by methyltetrahydrofolate
reductase, the enzyme responsible for converting N-5,10-methylenetetrahydrofolate into
N-5-methyltetrahydrofolate. Thus, the efficient utilization of dietary folic acid requires
adequate riboflavin nutrition. Furthermore, a mutation leading to a heat-sensitive form of
methylenetetrahydrofolate reductase has recently been identified (51). Its role in vascular
disease has not been defined. Further research is required to determine whether the serum
levels of homocysteine and the prevalence of vascular disease can be correlated directly
Fig. 5 Diagrammatic representation of homocysteine metabolism, showing the roles of folic acid,
B12, B6, and riboflavin. FAD is the coenzyme for methylenetetrahydrofolate reductase.
266 Rivlin and Pinto
with indices of riboflavin nutrition, and whether effects of marginal as well as overt defi-
ciency of riboflavin are clinically significant with respect to vascular disease.
D. Fat Metabolism
The important role of riboflavin in fat metabolism has been highlighted by recent demonstrations
that in certain rare inborn errors administration of riboflavin may be therapeutic.
In acyl-CoA dehydrogenase deficiency, infants present with recurrent hypoglycemia and
lipid storage myopathy and increased urinary excretion of organic acids. Clinical improvement
has occurred rapidly after riboflavin supplementation (52,53). Three varieties of the
disorder occur, all of which involve flavoproteins of various types. Five patients with a
mitochondrial disorder associated with NADH dehydrogenase deficiency were improved
by riboflavin treatment (54).
VI. PHARMACOLOGY/TOXICOLOGY/CARCINOGENESIS
INTERRELATIONSHIPS
There is general agreement that dietary riboflavin intake at many times the RDA is without
demonstrable toxicity (10,55–57). Because riboflavin absorption is limited to a maximum
of about 25 mg at any one time (10), the consumption of megadoses of this vitamin
would not be expected to increase the total amount absorbed. Furthermore, classical animal
investigations showed an apparent upper limit to tissue storage of flavins that cannot be
exceeded under ordinary circumstances (58). The tissue storage capacity for flavins is
probably limited by the availability of proteins capable of providing binding sites. Thus,
protective mechanisms prevent tissue accumulation of excessive amounts of the vitamin.
Because riboflavin has very low solubility, even intravenous administration of the vitamin
would not introduce large amounts into the body. FMN is more water-soluble than ribo-
flavin but is not ordinarily available for clinical use.
Nevertheless, the photosensitizing properties of riboflavin raise the possibility of
some potential risks. Phototherapy in vitro leads to degradation of DNA and increases in
lipid peroxidation, which may have implications for carcinogenesis and other disorders.
Irradiation of rat erythrocytes in the presence of FMN increases potassium loss (59). Topical
administration of riboflavin to the skin may increase melanin synthesis by stimulation
of free-radical formation. Riboflavin forms an adduct with tryptophan and accelerates the
photo-oxidation of this amino acid (60). Further research is needed to explore the full
implications of the photosensitizing capabilities of riboflavin and its phosphorylated derivatives.
The photosensitization of vinca alkaloids by riboflavin may distort the results of
testing of the efficacy of cytotoxic drugs if the studies are carried out in the presence of
visible light, as is usually done (61). This property of riboflavin needs to be considered
in drug evaluations, inasmuch as cell death will occur even without the addition of the
drug being analyzed.
Riboflavin is capable of reacting with chromate (VI), forming a complex and then
increasing the DNA breaks due to a chromium-induced free-radical mechanism (62).
Treatment of mouse FM3A cells greatly increases mutation frequency and the extent of
cellular DNA damage in the presence of light (63). There appears to be increasing evidence
that in the presence of visible light, riboflavin and its degradative product, lumiflavin,
may enhance mutagenicity (64).
Riboflavin (Vitamin B2) 267
On the other hand, recent studies (65) confirm earlier reports (66) that riboflavin
deficiency may enhance carcinogenesis by increasing activation of carcinogens, particularly
nitrosamines. Riboflavin may possibly provide protection against damage to DNA
caused by certain carcinogens through its action as a coenzyme with a variety of cytochrome
P450 enzymes.
It is important to establish the role of riboflavin as a dietary factor capable of preventing
carcinogenesis while at the same time determining the full implication of the
photosensitizing actions of riboflavin on mutagenesis and carcinogenesis. There are reports
raising the possibility that deficient riboflavin nutritional status, together with shortages
of other vitamins, may possibly enhance development of precancerous lesions of the
esophagus in China (67,68) and in Russia (69).
VII. REQUIREMENTS AND ASSESSMENT
There are a variety of methods available for analysis of riboflavin and its derivatives.
Bioassays measure the growth effect of vitamins but lack the precision of more sensitive
analytical procedures (70). Fluorometric procedures take advantage of the inherent fluorescent
properties of flavins (71). Some degree of purification of the urine or tissues may
be required before analysis as there is often significant interference by other natural substances
that leads to quenching of fluorescence and methodological artefacts. A procedure
has been developed for measuring riboflavin by competitive protein binding that is applicable
to studies in human urine (72). Riboflavin binds specifically to the avian egg white
riboflavin-binding protein (33) and thereby provides the basis for quantitative analysis
(73). Other procedures are also in use based on binding to specific apoenzymes, such as
d-amino acid oxidase. Currently, procedures using high-pressure liquid chromatography
(HPLC) have been widely applied as they have precision and can be utilized for analysis
of riboflavin in pure form as well as in biological fluids and tissues (74). HPLC is the
method most widely employed for determination of flavins in the blood and other tissues.
In clinical studies that involve individual patients as well as population groups, the
status of riboflavin nutrition is generally evaluated by determining the urinary excretion
of riboflavin (75) and the erythrocyte glutathione reductase activity coefficient (EGRAC)
(75). Urinary riboflavin determinations are made in the basal state, in random samples,
in 24-h collections, or after a riboflavin load test. Normal urinary excretion of riboflavin
is approximately 120 µg/g creatinine per 24 h or higher. It is useful to express urinary
excretion in terms of creatinine to verify the completeness of the collection and to relate
excretion to this biological parameter. Expressed in terms of the total amount, riboflavin
excretion in the normal adult is about 1.5–2.5 mg/day, which is very close to the
recommended dietary allowance of the National Academy of Sciences.
In deficient adult individuals, urinary riboflavin excretion is reduced to about 40
µg/g creatinine per 24 hr. Thus, deficient individuals have reduced urinary excretion,
reflecting diminished dietary intake and depleted body stores. Normal urinary excretion
is reduced with age, may be reduced by physical activity (as discussed below), and is
stimulated by elevated body temperature, treatment with certain drugs, and various stressful
conditions associated with negative nitrogen balance (76). Interpretation of urinary
riboflavin excretion must be made with these factors in mind.
Another potential drawback to utilizing urinary riboflavin excretion as an assessment
of nutritional status of this vitamin is that the amount excreted reflects recent intake very
sensitively. Thus, if an individual has been depleted for a long time but consumes a food
268 Rivlin and Pinto
item high in riboflavin, the urinary excretion determined a few hours later may not be in
the deficient range, but is likely to be normal or even elevated.
It is for this reason that attention has been directed to the development of assessment
techniques that more accurately reflect long-term riboflavin status. The method most
widely employed that largely meets these needs is assay of EGRAC as noted above. The
principle of the method is that the degree of saturation of the apoenzyme with its coenzyme,
FAD, should reflect the body stores of FAD. In deficient individuals, relative unsaturation
of the apoenzyme with FAD leads to decreased basal activity of the enzyme. Therefore,
the addition of FAD to the enzyme contained in a fresh erythrocyte hemolysate from
deficient individuals will increase activity in vitro to a greater extent than that observed
in a preparation from well-nourished individuals in whom the apoenzyme is more saturated
with FAD.
The EGRAC is the ratio of enzyme activity with to that without addition of FAD
in vitro. In general, most studies indicate that an activity coefficient of 1.20 or less indicates
adequate riboflavin status, 1.2–1.4 borderline-to-low status, and greater than 1.4 a clear
riboflavin deficiency (75,76).
It must be kept in mind that a number of physiological variables influence the results
of this determination. In the inherited disorder of glucose-6-phosphate dehydrogenase de-
ficiency, associated with hemolytic anemia, the apoenzyme has a higher affinity for FAD
than that of the normal erythrocyte that will affect the measured EGRAC. Thyroid function
affects glutathion reductase activity, the coefficient being elevated in hypothyroidism and
decreased in hyperthyroidism (77), reflecting the fact that hypothyroidism has many biochemical
features in common with those of riboflavin deficiency (17).
The latest RDAs issued by the Food and Nutrition Board (55) call for adult males
aged 19–50 years to consume 1.7 mg/day and 51 years of age or older, 1.4 mg. Adult
females from 19 to 50 years of age should consume 1.3 mg/day and from age 51 years
or older 1.2 mg. It is recommended that intake be increased to 1.6 mg/day during pregnancy,
to 1.8 mg/day early in lactation, and to 1.7 mg/day later in lactation. Infants should
consume 0.4–0.5 mg/day and children 0.8–1.2 mg/day, depending on age.
There has been some concern as to whether these figures are applicable to other
population groups around the world. Chinese tend to excrete very little riboflavin, and
their requirement may be lower than that of Americans (78). Adults in Guatemala appear
to have similar requirements in individuals older than 60 compared to those 51 years or
younger (79). This finding may not necessarily be relevant to populations of other countries.
The requirements of various national groups require further study. Environmental
factors, protein-calorie intake, physical activity, and other factors may have an impact on
riboflavin status. More research is needed on the requirements of the extremely old, who
form an increasingly large proportion of the population. They are also the population
group that consumes the largest number of prescribed and over-the-counter medications.
A point of interest is whether riboflavin requirements are increased in individuals
who exercise compared to those who are sedentary. In women aged 50–67 who exercised
vigorously for 20–25 min/day, 6 days a week, both a decrease in riboflavin excretion and
a rise in the EGRAC was noted, findings consistent with a marginal riboflavin-deficient
state, as shown in Table 2 (80). Supplementation with riboflavin did not, however, improve
exercise performance. These investigators observed compromised riboflavin status as well
in young women exercising vigorously (81). Similar observations of reduced urinary ribo-
flavin excretion and elevated EGRAC were made in young Indian males who exercised
actively (82).
Riboflavin (Vitamin B2) 269
Table 2 Group Means (n 
 7) for EGRAC and Urinary Riboflavin
Excretion During Both Exercise and Nonexercise Periodsa
Group Nonexercise Exercise
Low-riboflavin:
EGRAC 1.224  0.079b 1.283  0.067c
Urinary riboflavin (mg/day) 0.17  0.11 0.14  0.10d
High-riboflavin:
EGRAC 1.070  0.031 1.109  0.045c
Urinary riboflavin (mg/day) 0.66  0.49 0.46  0.21d
a x  SD. EGRAC, erythrocyte glutathione reductase activity coefficient.
b Significantly greater than high-riboflavin group, p  0.0005.
c Significantly different from nonexercise: p  0.0001.
d Significantly different from nonexercise: p  0.01.
Source: Ref. 62.
To determine whether the status of riboflavin nutrition influences metabolic responses
to exercise, blood lactate levels were determined in a group of physically active
college students from Finland before and after the exercise period. A number of the students
were initially in a state of marginal riboflavin deficiency. Following supplementation
with vitamins, including riboflavin, that produced improvement in the elevated EGRAC,
the blood lactate levels were unaffected and were related only to the degree of exercise
(83).
Thus, to date, while exercise clearly produces biochemical abnormalities in riboflavin
metabolism, it has not been shown that these abnormalities lead to impaired performance,
nor has it been shown that riboflavin supplementation leads to improved exercise
performance.
ACKNOWLEDGMENTS
Research was supported in part by the Clinical Nutrition Research Unit Grant 1-PO1-CA-
29502 from the National Institutes of Health, and by grants from the Stella and Charles
Guttman Foundation, the Sunny and Abe Rosenberg Foundation, the Rosenstiel Foundation,
the Isadore Rosenfeld Heart Foundation, and the Frank J. Scallon Foundation and
an industrial agreement with Wakunaga of America Co., Ltd.
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Riboflavin (Vitamin B2) 273
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8
Thiamine
VICHAI TANPHAICHITR
Mahidol University, Bangkok, Thailand
I. HISTORY
Although Neiching, the Chinese medical book, mentioned beriberi in 2697 bc, it was not
known for centuries that this illness was due to thiamine deficiency. In 1884, Takaki, a
surgeon general of the Japanese navy, concluded from his observations that beriberi was
caused by a lack of nitrogenous food components in association with excessive intake of
nonnitrogenous food. Though he did not reach the correct conclusion, the dietary changes
introduced into the Japanese navy resulted in a drastic reduction in the incidence of beriberi.
In 1890, the Dutch physician Eijkman, working in Java, discovered that fowls fed
boiled polished rice developed a polyneuritis that resembled beriberi in humans, and this
polyneuritis could be prevented or cured by rice bran or polishings. He suggested that the
toxic principle was present in polished rice but could be neutralized by some protective
factor in rice polishings. His associate, Grijns, in 1901 extracted a water-soluble protective
factor for polyneuritis from rice bran. In 1911, Funk, a chemist at the Lister Institute in
London, was convinced that he had isolated the antiberiberi principle possessing an amine
function from rice bran extracts and named it ‘‘vitamine.’’ However, his crystalline substance
was shown later to have little antineuritic activity (1–3).
Studies by Frazier and Stanton in 1909–1915 showed that human beriberi was a
deficiency disease that responded to extracts from rice polishings. This led Vedder, a U.S.
army physician, to recommend to Roger Williams, a chemist working in Manila, that the
protective substance be isolated. In 1926, Jansen and Donath, Dutch chemists working in
Java, succeeded in isolating and crystallizing antiberiberi factor from rice bran extracts.
The trivial name ‘‘aneurine’’ was suggested by Jansen. Later, Roger Williams isolated
sufficient quantities of thiamine to elucidate its structure. The chemical synthesis of thiamine
was accomplished in 1936 (1–3).
275
276 Tanphaichitr
Fig. 1 Structural formulas of thiamine and thiamine pyrophosphate.
The biochemical functions of thiamine were identified in 1935 by Thompson and
Johnson who showed that blood pyruvate levels were elevated in persons with thiamine
deficiency. During 1936–1938, Peters and his colleagues at Oxford University demonstrated
that thiamine was essential for pyruvate metabolism; the term biochemical lesion
was coined to describe the failure of thiamine-deficient pigeons to metabolize pyruvate.
In 1937, Lohman and Schuster discovered the active coenzyme form of thiamine to be
thiamine pyrophosphate (TPP; cocarboxylase) (Fig. 1). In 1953, Horecker and his associates
established the coenzymic role of TPP for transkotase (2–5).
II. CHEMISTRY
A. Structure and Nomenclature
The chemical name of thiamine, formerly known as vitamin B1, vitamin F, aneurine, or
thiamine, is 3-(4-amino-2-methylpyrimidin-5-ylmethyl)-5-(2-hydroxyethyl)-4-methylthiazolium
(Fig. 1) (6). The free vitamin is a base. It is isolated or synthesized and handled
as a solid thiazolium salt, e.g., thiamine chloride hydrochloride, thiamine mononitrate.
B. Synthesis
1. Chemical Synthesis
The original synthesis of thiamine was accomplished almost simultaneously in 1936/37
by Williams et al., Todd and Bergel, and Andersag and Westphal by slightly different
procedures that are used today with only slight modifications for commercial production.
Either the pyrimidine and thiazole rings can be prepared separately and condensed via
the bromide, or the pyrimidine ring can be synthesized and the thiazole ring added to it.
Steps of chemical synthesis of thiamine have been reviewed (2).
2. Biosynthesis
Thiamine cannot be synthesized by animals to any significant extent. Many microbial
species can synthesize thiamine if pyrimidine and thiazole are available, and still others
have a dependence on thiamine. Higher plants can synthesize thiamine de novo. In these
Thiamine 277
organisms, the pyrimidine precursor of thiamine is synthesized by a different pathway
from the pyrimidine precursor of the pyrimidine nucleotides (2). Thiamine biosynthesis
requires phosphorylation of pyrimidine and thiazole before synthesis of the thiamine monophosphate
(TMP) molecule by thiamine phosphate pyrophosphorylase. TMP is then
hydrolyzed by phosphatase to yield free thiamine. Thiamine pyrophosphokinase, which
is responsible for the synthesis of TPP from the free thiamine, has been found in yeast
and mammalian tissue. E. coli can synthesize TPP from TMP. Thiamine triphosphate
(TTP) is synthesized in brain by TPP-ATP phosphoryltransferase (Fig. 2). However, studies
of 14C-thiamine turnover show very little incorporation of 14C into TTP. TPP must be
bound to an endogenous protein to act as a substrate, whereas free TPP does not affect
the reaction (3).
C. Chemical Properties
Thiamine hydrochloride is a white crystalline substance. It is readily soluble in water,
only partly soluble in alcohol and acetone, and insoluble in other fat solvents. In the dry
form, it is stable at 100°C. The thiamine stability in aqueous solutions depends upon pH.
Below pH 5 it is quite stable to heat and oxidation; above pH 5 it is rapidly destroyed
Fig. 2 Biosynthetic pathway of thiamine and its phosphorylated derivatives. Pyrimidine, 2-
methyl-4-amino-5-hydroxymethylpyrimidine; thiazole, 4-methyl-5-(2-hydroxymethyl)thiazole; MP,
monophosphate; TTP, thiamine triphosphate.
278 Tanphaichitr
Fig. 3 Structural formula of thiochrome.
by autoclaving, and at pH 7.0 or higher by boiling. Thiamine is readily cleaved at the
methylene bridge into 2-methyl-4-amino-5-methylpyrimidylsulfonate and 4-methyl-5-(2-
hydroxyethyl)thiazole by sulfite treatment at pH 6.0 or above. At pH 8.0 or above, thiamine
turns yellow and is destroyed by a complex series of irreversible reactions. In strong
alkaline solution with the presence of oxidizing agents, e.g., potassium ferricyanide, thiamine
is converted to thiochrome (Fig. 3), which is fluorescent and is used to determine
thiamine in foods, feeds, pharmaceutical preparations, and biological fluids. Thiamine is
precipitated by iron and ammonium citrate, tannin, and various alkaloids (2,4,5).
Thiamine forms esters by combining with various acids at the hydroxyethyl side
chain. The most important esters are TMP, TPP, and TTP. The chemical synthesis of
TPP by phosphorylation of thiamine and the separation from TMP and TTP provide its
commercial quantities (2). The two most common commercial preparations of thiamine are
thiamine hydrochloride and thiamine mononitrate, which are widely available in various
countries. Other commercial forms that are produced by Japanese pharmaceutical industries
include thiamine allyl disulfide (TAD), thiamine propyl disulfide (TPD), thiamine
tetrahydrofurfuryl disulfide (TTFD), and O-benzoyl thiamine disulfide (Fig. 4). They are
more lipid-soluble and less water-soluble, better retained in the body than thiamine hydrochloride,
and readily converted to thiamine in vivo (2,7).
D. Molecular Structure and Biological Activity
Studies in animals using various thiamine analogs have shown that both pyrimidine and
thiazole moieties are needed for its vitaminic activity, which is maximal when only one
methylene group bridges the two moieties. In the thiazole portion, the quaternary nitrogen
and a hydroxyethyl group at C-5 are needed, as is the amino group at C-4 in the pyrimidine
portion (Fig. 1) (2,4,5).
E. Thiamine Analogs with Antagonistic Action
Several synthesized thiamine analogs are shown to be thiamine antagonists, which are
employed to produce thiamine deficiency in animals. They are categorized into compounds
having substitutions on the pyrimidine or thiazole rings, compounds having modifications
of the pyrimidine or thiazole rings, and compounds having anticoccidial action. The representatives
of these groups of thiamine antagonists are oxythiamine, pyrithiamine, and amprolium
(Fig. 5) (2,4).
1. Compounds with Substitutions on the Pyrimidine or Thiazole Rings
Extensive studies of thiamine-catalyzed reactions in both nonenzymatic and enzymatic
systems by Breslow and Krampitz have shown that the catalytic function of thiamine
involves C-2 of the thiazole with its ability to form a carbanion. Thus, substitution at C-
2 of thiazole, i.e., 2-methylthiamines completely eliminates both its catalytic and biologic
activities (2).
Thiamine 279
Fig. 4 Structural formulas of thiamine derivatives.
An amino group at the 4-position on the pyrimidine ring plays an essential role in
the release of the aldehyde adduct from the thiazole C-2 after splitting of the ECECE
bond. Oxythiamine is a potent thiamine antagonist because it can form oxythiamine pyrophosphate,
which can bind with thiamine-dependent apoenzymes but cannot complete the
reaction (2).
2. Compounds with Modifications of the Pyrimidine or Thiazole Rings
Among various compounds having modifications of the pyrimidine or thiazole rings, only
pyrithiamine, in which a ECHCCHE has been substituted for S, is a potent thiamine
antagonist.
Among the aforementioned two groups of thiamine antagonists, only oxythiamine
and pyrithiamine have been studied extensively in animals. Oxythiamine produces most
280 Tanphaichitr
Fig. 5 Structural formulas of thiamine antagonists.
of the usual symptoms and biochemical changes associated with thiamine deficiency. The
anorexia and inanition after oxythiamine treatment appear much earlier and more severe
than those after pyrithiamine treatment or thiamine deprivation. Since oxythiamine is incapable
of crossing the blood–brain barrier, it never produces the neurological manifestations.
On the contrary, pyrithiamine produces chiefly neurological manifestations (2).
3. Compounds Having Anticoccidial Action
Chick anticoccidial compounds contain pyrimidine, which is combined through the methylene
bridge to a quaternary nitrogen of a pyridine ring. Since they do not contain a hydroxyethyl
group they cannot be phosphorylated. Thus, they are not good inhibitors of the TPPdependent
enzymes. A large number of anticoccidial compounds have been produced by
various substitutions on the pyrimidine and pyridine rings. In small doses they inhibit
chiefly thiamine transport across bacterial cell walls and hence exert anticoccidial action,
whereas at larger doses they also inhibit thiamine transport across the chick intestinal wall
and hence produce thiamine deficiency in the chicks. Amprolium, 1-(4-amino-2-n-propyl-
5-pyrimidinylmethyl)-2-picolinium bromide hydrobromide, is most extensively used commercially
(2).
III. FOOD-RELATED ISSUES
A. Food Sources
Table 1 shows thiamine content in milligrams per 100 g edible portion of selected foods
compiled from the food composition table for use in East Asia published by the U.S.
Department of Health, Education and Welfare and the Food and Agricultural Organization
Thiamine 281
Table 1 Thiamine Content of Some Foods
Thiamine Thiamine
Food and description (mg/100 ga) Food and description (mg/100 g)
Yeast Mung bean:
Baker’s, dried 2.33 Whole seeds, dried 0.53
Brewer’s, debittered 15.61 Strip, dried 0.05
Pork Peanut:
Carcass, fresh, lean 0.69 Raw 0.97
Ham, smoked, lean 0.70 Boiled 0.56
Beef Soybean:
Carcass, fresh, very lean 0.07 Whole mature seeds, dried 0.66
Chicken, raw Milk, unsweetened 0.05
Very young birds 0.08 Curd, raw, plain 0.04
Mature birds 0.08 Maize, whole-kernel:
Liver, raw Yellow 0.29
Beef 1.68 Popcorn, popped, oil added 0.12
Chicken 1.92 Starch 0
Duck 1.32 Rice:
Hog 3.57 Bran 1.26
Kidney, raw Home-pounded 0.20
Beef 1.58 Milled 0.10
Hog 1.31 Cooked, milled 0.02
Abalone, raw 0.24 Noodles, freshly made 0.04
Bass, sea, raw 0.15 Noodles, cooked Trace
Catfish, freshwater, raw 0.10 Wheat:
Crab, sea, blue 0.05 Whole grain, hard, red winter 0.37
Lobster, raw 0.01 Germ 2.10
Duck eggs, raw: Bran 0.54
Whole 0.16 Asparagus: green, raw 0.15
White Trace Bamboo shoots, raw 0.09
Yolk 0.54 Cabbage, Chinese, raw 0.07
Hen eggs, raw: Carrot, raw 0.06
Whole 0.10 Apple, raw 0.02
White 0.01 Banana, ripe 0.03
Yolk 0.24 Guava, raw 0.05
Cheese, blue or cheddar 0.04 Orange, raw 0.04
Ice cream, regular 0.05 Butter, salted 0.01
Milk, cow: fluid, 3.5% fat 0.04 Lard 0
Milk, human, fluid, whole 0.02 Soybean oil 0
Tallow 0.07
a Edible portion.
Source: Compiled from Food Composition Table for Use in East Asia (8).
of the United Nations (8). Though thiamine is found in a large variety of animal and
vegetable products, it is abundant in only a few foods. Excellent sources of thiamine are
yeast, especially dried brewer’s yeast; liver and kidney; lean pork; and legumes, including
mung bean, soybean, and peanut. In cereal grains, thiamine is low in the endosperm but
high in the germ. Thus, the thiamine contents in rice bran and home-pounded rice is higher
282 Tanphaichitr
than that in milled rice. In eggs, thiamine is also unevenly distributed, being negligible
in egg white but present in egg yolk. Thiamine is absent in fats, oils, and refined sugars.
Milk and milk products, sea foods, fruits, and vegetables are not good sources of thiamine.
Foods lacking in thiamine are manufactured, i.e., milled rice, cereal flours, refined
sugars, separated animal fats, and separated vegetable oils. It should be emphasized that
Table 1 shows food sources rich in thiamine without consideration to the amount being
regularly consumed. For example, yeast and wheat germ are rich sources of thiamine, but
they are normally eaten in small amounts.
In most animal products, 95–98% of thiamine is present as TMP, TPP, and TTP,
with about 80–85% as TPP, whereas in plant products thiamine occurs in the nonphosphorylated
form (2).
B. Bioavailability
Thiamine status depends not only on the thiamine content of foods but on its bioavailability.
Factors influencing bioavailability are thiamine losses after handling and processing,
ethanol consumption, presence of antithiamine factors (ATFs), and folate and protein status
(2,5,9,10).
1. Thiamine Losses After Handling and Processing of Foods
As noted above, several factors, including pH, temperature, oxidation, solubility, radiation,
and the use of sulfites in handling and processing of foods, affect thiamine losses.
a. pH
Thiamine is stable at acid pH and becomes unstable at pH 7.0 or higher. In cooking
practice, the addition of sodium bicarbonate to green beans and peas to retain green color
or to dried beans to facilitate softening can lead to large losses of thiamine (2,5).
b. Temperature
Thiamine in tissues that is bound to protein is more stable to thermal destruction than is
free thiamine. Thiamine losses on cooking and canning of meats, baking of breads, and
cooking of vegetables are 25–85%, 5–35%, and 0–60%, respectively. The variation depends
on temperature, time, and types of food products. In pasteurization, sterilization,
spray-drying, roller-drying, and condensing of milk, thiamine losses are 9–20%, 30–50%,
10%, 15%, and 40%, respectively. Though freezing has no effect on the thiamine content
of foods, thawing causes thiamine losses into the drip fluid. Thiamine losses on storage
of canned fruits and vegetables at usual room temperatures are 0–20% for a year. Processing
foods at higher temperature and pH in the presence of oxygen or other oxidants
lead to the formation of thiamine sulfides and disulfides, thiochrome, and other oxidation
products. Only thiamine sulfides and disulfides still retain the biological activity of thiamin
(2,5).
Thiamine is cleaved by residual chlorine (0.2–2.0 ppm of chlorine) in proportion
to the rise in temperature, pH, and concentration of residual chlorine. When rice is boiled
in an electric rice cooker, the thiamine in the rice is cleaved by residual chlorine. When
rice is cooked in tap water containing 200 ng/mL of chlorine or distilled water without
chlorine, thiamine losses from rice are 56.5% and 32.2%, respectively. The degradation
of thiamine depends on the amount of ionization of residual chlorine governed by the pH
of water. At pH below 5.0, the residual chlorine is present as HClO and the ratio of ClO

increases in proportion to an increase in pH:
Thiamine 283
HClO s H  ClO

pH 5 < pH 7.5 > pH 10
Thus, ClO
 plays a key role in the cleavage of thiamine to hydroxymethylpyrimidine
and 4-methyl-5-?-hydroxymethythiazide. Though hydroxymethylpyrimidine can induce
convulsions in mice, the amount converted from thiamine in rice by chlorine in tap water
is so small that its toxic effect should be negligible. However, thiamine loss in polished
rice due to residual chlorine during the cooking process needs attention because rice is
the major source of dietary thiamine in Asian population (11).
c. Solubility
Since thiamine is highly water-soluble, significant thiamine losses occur during cooking
of foods when there is the excessive use of water that is then discarded. About 85% of
thiamine is lost by discarding the water after soaking the rice (5,9,10). Though parboiling
rice causes the thiamine to move from the outer layers to the inner layers of the rice
kernel, thus leading to greater thermal stability and smaller losses due to washing of the
thiamine, parboiled rice is not widely available or widely consumed by the Asian population.
d. Oxidation
Processing foods in the presence of oxygen or other oxidants can lead to the formation
of thiamine sulfides and disulfides, thiochrome, and other oxidation products. This can
lead to irreversible loss of thiamine activity (2).
e. Radiation
Thiamine is also destroyed by x-rays, ?-rays, and UV irradiation. UV irradiation leads to
the production of 2-methyl-4-amino-5-aminoethylpyrimidine (2).
f. Sulfites
Sulfites destroy thiamine. Sulfites that form in the treatment of fruits during dehydration
with SO2 destroy most of the thiamine present (5).
2. Ethanol
Excessive alcohol ingestion is associated with thiamine deficiency. Ethanol given orally
or intravenously inhibits intestinal thiamine uptake (2,5,12,13). Ethanol reduces the rate
of intestinal absorption and the net transmural flux of thiamine. Ethanol inhibits only the
active but not the passive component of thiamine transport by impeding the cellular exit
of thiamine across the basolateral or serosal membrane. The impairment of thiamine exit
from the enterocyte correlates with the Na,K-ATPase activity. This relationship may be
at least partly related to the effect of ethanol in increasing the fluidity of enterocyte brushborder
and basolateral membranes (13). The maximum absorption of 35S-thiamine in alcoholics
may be as low as 1.5 mg, similar to that seen in patients with intestinal resection
in whom the reduction is attributable to a decrease in receptor sites (12).
3. ATFs (Antithiamine Factors)
ATFs occurring in foods can alter thiamine structure and reduce the biological activity
of thiamine. ATFs are divided into two groups, according to their stability to heat, i.e.,
thermolabile and thermostable.
284 Tanphaichitr
a. Thermolabile ATFs
The thermolabile ATFs include thiaminase I (EC 2.5.1.2) and thiaminase II (EC 3.5.99.2).
Thiaminase I is found in viscera of freshwater fish (mainly in viscera), shellfish, fern, and
a limited number of sea fish and plants, and produced in microorganisms, e.g., Bacillus
thiaminolyticus and Clostridium thiaminolyticus. It catalyzes the cleavage of thiamine by
an exchange reaction with an organic base or a sulfhydryl compound involving a nucleophilic
displacement on the methylene group of the pyrimidine moiety of thiamine. Thiaminase
II is found in several microorganisms, e.g., Bacillus aneurinolyticus, Candida
aneurinolytica, Trichosporon, and Oospora. It directly catalyzes the hydrolysis of thiamine
(Fig. 6) (14,15).
Thiaminases are usually inactive in living cells. They are activated when they are
homogenized in a water solution around pH 4–8 or excreted from cells or microorganisms
into the medium. The thiamine cleavage in the diets containing thiaminase can occur
during storage or preparation prior to ingestion or during passing through the gastrointestinal
tract. Continual intake of raw freshwater fish with or without fermentation, raw shell-
fish, and ferns are risk factors for the development of thiamine deficiency (5,9,10,14–16).
b. Thermostable ATFs
The thermostable ATFs have been demonstrated in fern, tea, betel nut, and a large number
of other plants and vegetables and some animal tissues. In animal tissues, it is thought
that myoglobin, hemoglobin, and hemin bind thiamine. The ATFs found in plants and
vegetables are related to ortho- and para-polyphenolic compounds, e.g., caffeic acid (3,4-
dihydroxycinnamic acid), chlorogenic acid [3-(3,4-dihydroxycinnamoyl) quinic acid], and
tannic acid (tannin) (5,9). In vitro studies reveal that the antithiamine activity of polyphenols
requires pH 6.5 or higher and oxygen. The high pH is necessary for the ionization
of the polyphenols and the opening of the thiazole moiety of thiamine at C-2 to yield the
SH form of thiamine. Oxygen helps the oxidation and polymerization of polyphenols to
yield active quinones and relatively less active polymerized products. Oxidation of the
SH form of thiamine by the active quinones leads to the formation of thiamine disulfide.
Further hydrolysis and oxidation yield products that do not exhibit biological activity of
thiamine. Ascorbic acid and other reducing agents prevent the formation of quinone, thiamine
disulfide, or both. However, chemical modification of thiamine by the oxidation of
Fig. 6 Clevage of thiamine by thiaminase I and thiaminase II. Thiaminase I activity requires
organic bases or thiol compounds.
Thiamine 285
quinone may not be the only mechanism to reduce thiamine bioavailability. High concentrations
of divalent cations, including calcium and magnesium ions present in water, augment
the precipitation of thiamine by tannin, which makes thiamine less available for
intestinal absorption. Ascorbic, tartaric, and citric acids present in many fruits and vegetables
can lower such precipitation presumably by sequestering divalent cations.
Human studies have shown that tea drinking, tea leaf chewing, coffee or decaffeinated
coffee drinking, and betel nut chewing lead to biochemical thiamine depletion.
Ascorbic acid intake from either pharmaceutical preparations or from foods improves
thiamine status of the subjects (16,17).
Studies in rats have demonstrated that prolonged tea consumption, in contrast to
consumption of water, lowered (60–80%) blood transketolase, brain total thiamine, brain
transketolase activity, as well as ?-ketoglutarate and pyruvate dehydrogenase activities.
Thiamine deficiency caused by prolonged consumption of tea in rats was reversed by
discontinuing tea consumption; brain thiamine and brain and blood transketolase activities
were restored to normal levels within a week, whereas the time required for complete
restoration of pyruvate dehydrogenase and ?-ketoglutarate activities is about 2 weeks.
Tea-treated rats receiving intraperitoneal injection of 4 mg of thiamine 3 h prior to decapitation
had TPP-dependent enzymic activities exceeding the normal levels, which suggests
that the decrease in thiamine-dependent enzymic activities may be due not only to the lack
of TPP but also to the decreased synthesis of apoenzymes (18). Impairment of synthesis of
whole-brain acetylcholine was also observed in rats consuming tea (19).
4. Folate and Protein Status
Folate-deficient rats absorb thiamine less efficiently than pair-fed controls (20). These data
imply that folate may have a role in maintaining the integrity of the active transport process
of thiamine (13). Subjects with folate or protein deficiency show a significant reduction
in the maximum absorption of 35S-thiamine. The interaction of protein-energy malnutrition
and thiamine absorption is demonstrated by an increase in the maximum absorption of
35S-thiamine in malnourished alcoholics, with a decreased maximum absorption of thiamine
after correction of protein-energy malnutrition (21).
IV. PHYSIOLOGICAL RELATIONSHIPS
Ingested thiamine is fairly well absorbed, rapidly converted to phosphorylated forms,
stored poorly, and excreted in the urine in a variety of hydrolyzed and oxidized products
(5).
A. Absorption
The small intestine absorbs thiamine by two mechanisms. At concentrations exceeding 2
µM, thiamine is absorbed by passive diffusion; at concentrations below 2 µM, thiamine
is absorbed by an active process. The findings that the lack of Na or inhibition of ATPase
with ouabain blocks the uptake of thiamine at low concentrations by the intestinal cells
suggest the presence of a specific carrier. This is supported by the isolation of a thiaminebinding
protein (TBP) associated with thiamine transport into and out of the cell of Escherichia
coli. The 4-amino and the imidazole quaternary nitrogen are necessary for thiamine
uptake by the rat small intestine, whereas the 2-methyl and 5-hydroxyethyl appear to be
286 Tanphaichitr
necessary for thiamine binding to the carrier protein. Active thiamine absorption is greatest
in the jejunum and ileum (2,5,9,13,22,23).
A high percentage of the thiamine in the epithelial cells is phosphorylated, whereas
the thiamine arriving on the serosal side of the mucosa is largely free thiamine. Thus, the
entry of thiamine into the mucosal cells is linked with a carrier-mediated system that is
dependent either on thiamine phosphorylation-dephosphorylation coupling or on some
metabolic energetic mechanisms, possibly activated by Na. Thiamine exit from the mucosal
cell on the serosal side is dependent on Na and on the normal function of ATPase
at the serosal pole of the cell (2,5,9,13,23).
Investigations of the absorption of 35S-thiamine in humans indicate that its intestinal
transport is rate-limited and behaves according to Michaelis-Menten kinetics, which
yielded a Vmax of 8.3  2.4 mg and a Km of 12.0  2.4 mg (12). Earlier studies by Japanese
investigators have also shown that a single oral dose of thiamine greater than 6 mg does
not result in increased urinary thiamine excretion. The results imply that the excessive
amount of oral thiamine over 6 mg cannot be absorbed (22) and are consistent with the
subsequent studies in six healthy Australian volunteers receiving an oral dose of 10 mg
of thiamine. In the Australian study, the mean serum thiamine rose only marginally at 30
min from 5.1 to 5.9 µg/L and peaked at 7.2 µg/L—an increase of 42%. Six hours after
the test dose, the serum thiamine concentration had fallen back to its basal level (24). On
the contrary, healthy subjects taking each of the following physiological doses of thiamine
for a consecutive period of 5 days in the following order: 150, 450, 750, 1050, 1350,
1650, 1950, and 2250 µg had a highly significant correlation between the oral dose and
urinary excretion of thiamine (y  41.0239  0.0465, df  68, r  0.8618, P  0.001)
(25). Thus, all of the results indicate that passive absorption of thiamine is not significant
in humans.
B. Transport
Thiamine is carried by the portal blood to the liver. In normal adults, 20–30% of plasma
thiamine is protein-bound, all of which appears to be TPP (24). The transport of thiamine
into erythrocytes seems to be a facilitated diffusion process, whereas it enters other cells
by an active process (2,26). Erythrocytes contain mainly TPP (24).
Like the intestinal absorption of thiamine, thiamine transport across the blood–brain
barrier also involves two different mechanisms. However, the saturable mechanism at the
blood–brain barrier may be dependent on membrane-bound phosphatases. In this regard,
it differs from the energy-dependent processes described for the gut and cerebral cortex
cells. Studies in the rat reveal that a saturable mechanism with a mean Km of 2.2 nmol/
mL and a mean Vmax of 7.3 nmol/g per hour accounts for 95% of cerebellar and 91% of
cerebral cortex uptake at physiological plasma thiamine concentrations. TMP transport
rates are 5–10 times lower than those of thiamine. Thiamine uptake rates are 10 times
the maximal rate of thiamine loss from the brain (3).
A TBP has been identified in chicken egg (white and yolk) and liver (27) as well
as in the serum of pregnant rats (28). The molecular weight of highly purified TBP isolated
from egg white was 38,000. The TBP binds to 14C-thiamine with a molar ratio of 1 and
an association constant of 0.3 µM (27). Estrogen induces hepatic synthesis of TBP and
modulates its plasma level. The vital role of TBP in the transfer of thiamine across the
placenta is suggested by the observation that the passive immunization of pregnant rats
Thiamine 287
(4–16 days) with antibodies to chicken TBP but not ovalbumin resulted in fetal resorption
(28).
C. Tissue Distribution and Storage
The total amount of thiamine in a normal adult is approximately 30 mg. High concentrations
are found in skeletal muscles, heart, liver, kidneys, and brain. About 50% of the
total thiamine is distributed in the muscles. In spinal cord and brain, the thiamine level
is about double that of the peripheral nerves. Leukocytes have a 10-fold higher thiamine
concentration than erythrocytes. Thiamine has a relatively high turnover rate in the body.
The biological half-life of 14C-thiamine is 9–18 days. Besides, thiamine is not stored in
large amounts or for any period of time in any tissue (2,5,9,22).
D. Metabolic Modification
Of the total thiamine in the body, about 80% is TPP, 10% is TTP, and the remainder is
TMP and thiamine. The three tissue enzymes known to participate in formation of the
phosphate esters are thiamine pyrophosphokinase, which catalyzes the formation of TPP
from thiamine and ATP; TPP-ATP phosphoryltransferase, which catalyzes the formation
of TTP from TPP and ATP (Fig. 2); and thiamine pyrophosphatase, which hydrolyzes
TPP to form TMP (2,3,5,29).
At least 25–30 metabolites have been noted to occur in the urine of rats and men
given thiamine labeled in either the pyrimidine or thiazole moiety. Those metabolites that
have been identified are 2-methyl-4-amino-5-pyrimidine carboxylic acid (pyrimidine carboxylic
acid), 2-methyl-4-amino-5-hydroxymethylpyrimidine, 4-methylthiazole-5-acetic
acid (thiazole acetic acid), 3-(2?-methyl-4?-amino-5?-pyrimidylmethyl)-4-methylthiazole-
5-acetic acid (thiamine acetic acid), 2-methyl-4-amino-5-formylaminomethylpyrimidine,
and 5-(2-hydroxyethyl)-4-methylthiazole (thiazole). Of these, pyrimidine carboxylic acid,
thiazole acetic acid, and thiamine acetic acid are the major metabolites excreted in the urine
of rats and men. Rat liver alcohol dehydrogenase is involved in the in vivo metabolism of
thiamine and its thiazole moiety to their corresponding acids. In vitro experiments indicate
that although thiamine itself is a poor substrate for the enzyme, its thiazole moiety is
oxidized at a faster rate than is ethanol (30).
E. Excretion
Thiamine and its metabolites are mainly excreted in the urine. Very little thiamine is
excreted in the bile. Early milk has low thiamine levels. Thiamine administered by oral
or parenteral route is rapidly converted to TPP and TTP in the tissues. Thiamine in excess
of tissue needs, as well as binding and storage capacity, is rapidly excreted in the urine
in the free form (2,4,5,24,25).
V. FUNCTIONS
Functions of thiamine can be categorized into established and plausible functions. The
established functions of thiamine are biochemical functions in which TPP serves as the
coenzyme of biochemical reactions, whereas the plausible functions are neurophysiological
functions.
288 Tanphaichitr
A. Biochemical Functions
In mammalian systems, TPP functions as the Mg2-coordinated coenzyme for the active
aldehyde transfers, which include the oxidative decarboxylation of ?-keto acids and transketolase
reaction (2,4,5,31). The key feature of TPP is that the carbon atom between the
nitrogen and sulfur atoms in the thiazole ring is much more acidic than most CCHE
groups. It ionizes to form a carbanion, which readily adds to the carbonyl group of ?-
keto acids or ketose. The positively charged ring nitrogen of TPP then acts as an electron
sink to stabilize the formation of a negative charge, which is necessary for decarboxylation.
Protonation then gives hydroxyethyl TPP (5).
1. Oxidative Decarboxylation of ?-Keto Acids
Pyruvic, ?-ketoglutaric, and branched-chain ?-keto acids undergo oxidative decarboxylation.
a. Oxidative Decarboxylation of Pyruvic Acid
The net reaction of oxidative decarboxylation of pyruvate catalyzed by the pyruvate dehydrogenase
complex (PDHC) is
Pyruvate  CoA  NAD > acetyl CoA  CO2  NADH  H
In addition to the stoichiometric coenzymes consisting of CoA and NAD, TPP, lipoic
acid, and flavin adenine dinucleotide (FAD) also serve as the coenzymes. The PDHC is
localized in the mitochondrial inner membrane and is an organized assembly of three
enzymes. The conversion of pyruvate to acetyl CoA consists of four steps (2,4,5,9,31).
First, pyruvate is decarboxylated after it combines with TPP is catalyzed by pyruvate
dehydrogenase. Second, the hydroxyethyl group attached to TPP is oxidized by the disul-
fide groups of lipoamide to form an acetyl group and concomitantly transferred to lipoamide
to yield acetyl lipoamide. This step is catalyzed by the lipoic acid-bound dihydrolipoyltransacetylase.
Third, the acetyl group is transferred from acetyl lipoamide to CoA
to form acetyl CoA catalyzed by dihydrolipoyl transacetylase. Fourth, the regeneration of
the oxidized form of lipoamide is catalyzed by the FAD-dependent dihydrolipoyl dehydrogenase;
a hydride ion is transferred to an FAD prosthetic group of the enzyme and then
to NAD (Fig. 7) (31,32).
b. Oxidation Decarboxylation of ?-Ketoglutaric Acid
The net reaction of oxidative decarboxylation of ?-ketoglutarate, taking place in the tricarboxylic
acid (TCA) cycle and catalyzed by the ?-ketoglutarate dehydrogenase complex
(?-KGDHC), is as follows:
?-Ketoglutarate  CoA  NAD > succinyl CoA  CO2  NADH  H
The coenzyme requirements and the steps in formation of succinyl CoA are analogous to
the oxidative decarboxylation of pyruvate (2,4,5,9).
c. Oxidative Decarboxylation of Branched-Chain ?-Keto Acids
The oxidative decarboxylation of the three branched-chain ?-keto acids, i.e., ?-ketoisocaproate,
?-keto-?-methylvalerate, and ?-ketoisovalerate, to yield isovaleryl CoA, ?-
methylbutyryl CoA, and isobutyryl CoA, respectively, is catalyzed by the branched-chain
?-keto acid dehydrogenase complex (BC ?-KADHC), which is analogous to complexes
of pyruvate and ?-ketoglutarate (2,4,5,9).
Thiamine 289
Fig. 7 The pyruvate dehydrogenase complex reactions. TPP, thiamine pyrophosphate; Lip, lipoic
acid.
The aforementioned three ?-keto acid multienzyme complexes play the significant
role in energy generating pathways (Fig. 8). The PDHC irreversibly commits pyruvic acid,
a three-carbon intermediate derived from glucose catabolism, to convert to acetyl CoA,
which participates in three important metabolic fates: complete oxidation of the acetyl
group in the TCA cycle for energy generation; conversion of an excess acetyl CoA into
the ketone bodies; and transfer of the acetyl units to the cytosol with subsequent biosynthesis
of sterols and long-chain fatty acids. Because PDHC occurs at a significant branch
point in the metabolic pathways, its activity is strictly regulated by two separate types of
mechanisms. The first mechanism is competitive end-product inhibition of catalytic PDHC
by acetyl CoA and NADH. The second mechanism involves covalent modification of the
PDHC by phosphorylation/dephosphorylation (inactive PDHC/active PDHC) mechanism,
mediated by a specific protein kinase that is tightly bound to the PDHC and by a specific
phosphoprotein phosphatase that is much less tightly associated with the PDHC (31,32).
The pyruvate dehydrogenase kinase/phosphatase system is regulated by a number
of factors. Inactivation of PDHC is accomplished by an Mg2-ATP-dependent pyruvate
dehydrogenase kinase. Pyruvate dehydrogenase kinase is inhibited by ADP, pyruvate,
CoA, NAD, TPP, and calcium, whereas it is stimulated by NADH and acetyl CoA; TPP
inhibits phosphorylation by binding at the catalytic site of pyruvate dehydrogenase to
promote a conformational change, which in turn causes one of the serine hydroxy groups
on the subunit of pyruvate dehydrogenase to become less accessible to pyruvate dehydrogenase
kinase. The pyruvate dehydrogenase phosphatase reaction requires Mg2. It is stimulated
by Ca2 and inhibited by NADH, and the inhibition is reversed by NAD (31).
The primary metabolic fate of acetyl CoA in most cells is its complete oxidation
in the TCA cycle with the generation of 2 CO2, 2 GTP, and 4 reducing equivalents consisting
of 3 NADH and 1 FADH2, which generate 11 ATP by the electron transport system.
Thus, the integrity of the TCA cycle is critical for the provision of cellular energy. ?-
KGDHC is the only enzyme in the TCA cycle that requires TPP as coenzyme. The equilibrium
of the ?-KGDHC reaction lies strongly toward succinyl CoA formation. In this reaction,
the second molecule of CO2, the second reducing equivalent (NADH  H) of the
290 Tanphaichitr
Fig. 8 Pathways dependent on thiamine pyrophosphate. P, phosphate; TPP, thiamine pyrophosphate;
PDHC, pyruvate dehydrogenase complex; ?-KGDHC, ?-ketoglutarate dehydrogenase complex;
BC ?-KADHC, branched-chain ?-keto acid dehydrogenase complex; TK, transketolase.
TCA cycle, and succinyl CoA, an energy-rich thioester compound, are produced. Succinyl
CoA is conserved in a substrate level phosphorylation reaction in the next step of the
TCA cycle. It is at the level of this reaction where ?-ketoglutarate may leave the TCA
cycle to be converted to glutamate by glutamate dehydrogenase, a mitochondrial enzyme,
in the presence of NADH or NADP and ammonia. Unlike PDHC, the ?-KGDHC is not
regulated by a protein kinase. ATP, GTP, NADH, and succinyl CoA inhibits the ?-
KGDHC activity, whereas Ca2 stimulates its activity (32).
Where the BCAAs valine, leucine, and isoleucine are present in excess over what
is needed for protein synthesis, they are degraded through several steps to provide energy.
The initial step in catabolism is reversible transamination with ?-ketoglutarate to form
the corresponding ?-keto acids, i.e., ?-ketoisovalerate, ?-ketoisocaproate, and ?-keto-?-
Thiamine 291
methylvalerate, respectively. The second step is irreversible oxidative decarboxylation of
the three branched-chain ?-keto acids by BC ?-KADHC to yield CO2, NADH that feeds
electrons to the electron transport system, and isobutyryl CoA, isovaleryl CoA, or ?-
methylbutyryl CoA. These three analogs of fatty acyl CoA are oxidized by specific dehydrogenases
to form the corresponding ?,?-unsaturated compounds. The remainder of the
isoleucine degradation pathway is identical to that of fatty acid oxidation, with the provision
of acetyl CoA and propionyl CoA, whereas that of the valine degradation provides
propionyl CoA. The remainder of the leucine degradation is a combination of reactions
used in ? oxidation and ketone body synthesis with the provision of acetyl CoA and
acetoacetate, which may be converted to two acetyl CoA. The acetyl CoA formed from
the degradation of the BCAAs enters the acetyl CoA pool, which can be used for any of the
aforementioned metabolic functions. Propionyl CoA formed from isoleucine and valine for
the most part is not further oxidized as a fatty acid but is utilized for the formation of
succinyl CoA, which is further metabolized via the TCA cycle. Thus, the three carbon
atoms derived from valine and isoleucine may be completely oxidized to CO2, but they
can be incorporated into carbohydrate by oxidation of succinate to oxaloactate and the
reactions of gluconeogenesis. On the contrary, both acetyl CoA and acetoacetate of leucine
degradation are characteristic of fatty acid oxidation (33).
2. Transketolase Reaction
A TPP-dependent transketolase found in the cytosol catalyzes the reversible transfer of a
glycolaldehyde moiety from the first two carbons of a donor ketose phosphate to the
aldehyde carbon of an aldose phosphate in the pentose phosphate pathway. These reactions
are as follows:
Xylulose-5-phosphate  ribose-5-phosphate s glyceraldehyde-3-phosphate
 sedoheptulose-7-phosphate
Xylulose-5-phosphate  erythrose-4-phosphate s glyceraldehyde-3-phosphate
 fructose-6-phosphate
The metabolic significance of the pentose phosphate pathway is not to obtain energy
from the oxidation of glucose in animal tissues. Its primary purpose is to generate NADPH,
which serves as a hydrogen and electron donor in reductive biosynthetic reactions, including
the biosynthesis of fatty acids; to convert to pentoses, particularly ribose-5-phosphate,
which are components of RNA, DNA, ATP, CoA, NAD, and FAD; and to catalyze the
interconversion of C3, C4, C6, and C7 sugars, some of which can enter the glycolytic
sequence (2).
B. Neurophysiological Functions
There is increasing evidence of the roles of thiamine in neurotransmitter function and
nerve conduction (3,34).
1. Neurotransmitter Function
Though the findings are inconsistent, abnormal metabolism of four types of neurotransmitters
(acetylcholine, catecholamine, serotonin, and amino acids) has been reported in thiamine
deficiency. Acetylcholine, ?-aminobutyric acid (GABA), glutamate, and aspartate
are produced primarily through the oxidative metabolism of glucose (3,34).
292 Tanphaichitr
a. Acetylcholine
Studies in thiamine-deficient rats have revealed that there is no change in regional brain
acetylcholine levels, but there are reductions in acetylcholine turnover and acetylcholine
utilization in the cortex, midbrain, diencephalon, and brain stem. These results suggest
the depression of central cholinergic mechanisms in thiamine-deficient rats. Since choline
acetyltransferase is unaltered in thiamine deficiency, decreased PDHC activity limiting
acetyl CoA production may underlie these changes (3).
b. Catecholamines
Decreased synthesis of catecholamines was demonstrated in severely thiamine-deficient
rat brain; treatment with thiamine normalized catecholamine synthesis within 2 h, although
some neurological signs persist (34). There is also evidence indicating that behavioral
deficits in rats persisting after reversal of thiamine deficiency are linked to significant
reductions in the norepinephrine content of cortex, hippocampus, and olfactory bulbs (3).
c. Serotonin
Decreased uptake of serotonin by cerebellar synaptosomes and significant increase in 5-
hydroxyindoleacetic acid, a serotonin catabolite, occurs in thiamine-deficient rats. These
findings are supported by autoradiographic evidence of decreased serotonin uptake in
indoleaminergic afferents of cerebellum in thiamine-deficient rats (3,34).
d. Amino Acids
The levels of four amino acids with putative neurotransmitter functions, i.e., glutamate,
aspartate, GABA, and glutamine, are decreased in thiamine-deficient rat brain, with
marked changes in the cerebellum (3).
2. Nerve Conduction
That thiamine plays a special role in neurophysiology that is independent of its coenzyme
function was originally postulated by von Muralt (35). The effect of thiamine antagonists
on conduction in peripheral nerves, the localization of thiamine and thiamine pyrophosphatase
in peripheral nerve membranes as opposed to axoplasm, the release of thiamine from
nerve preparations by electrical stimulation, the association of released thiamine with
membrane fragments of brain, spinal cord, and sciatic nerves all support the aforementioned
view (2–5,9,35). This thiamine release appears to be due to hydrolysis of TPP and
TTP. Tetrodotoxin, local anesthetics, acetylcholine, and serotonin have been shown to
cause release of thiamine from nerve and nerve membrane segments (36–38). The finding
that thiamine-phosphorylated derivatives are associated with the sodium channel protein
has led to the hypothesis that TTP plays a fundamental role in the control of sodium
conductance at axonal membranes (39).
VI. FUNCTIONAL CONSEQUENCES OF THIAMINE DEFICIENCY
Cardiac failure, muscle weakness, peripheral and central neuropathy, and gastrointestinal
malfunction have been observed in both animals and humans on diets restricted in thiamine
(9).
Although the precise biochemical defect responsible for the pathophysiological manifestations
of thiamine deficiency is not established, experimental evidence indicates that
thiamine has three major roles at the cellular level. The first relates to energy metabolism
Thiamine 293
and concerns the oxidative decarboxylation of ?-keto acids, the inhibition of which leads
to a failure of ATP synthesis. The second concerns synthetic mechanisms as reflected by
the transketolase reaction yielding NADPH and pentose. The third deals with the functions
of neurotransmitters and nerve conduction. In Wernicke’s disease, failure of energy metabolism
predominantly affects neurons and their functions in selected areas of the central
nervous system. Glial changes may be caused by biochemical lesions that affect transketolase
and nucleic acid metabolism. Membranous structures are altered in a visible manner
and secondary demyelination ensues (9).
VII. PATHOGENESIS OF THIAMINE DEFICIENCY AND HIGH-RISK
CONDITIONS
Thiamine deficiency in free-living populations and hospitalized patients can be caused by
inadequate intake, decreased absorption, and defective transport of thiamine, impaired
biosynthesis of TPP, increased requirement, and increased loss of thiamine. These mechanisms
may reinforce each other.
A. Inadequate Intake of Thiamine
1. Inadequate Intake of Thiamine with Concomitant High Intake
of Carbohydrate
All of the published reports indicate that beriberi, the clinical condition of thiamine defi-
ciency, in Asian populations is always associated with high intake of carbohydrate and
insufficient intake of thiamine (5,9,10,30,40–42).
Recent studies in Thailand (10,43–46) and Japan (47–49) have confirmed that inadequate
thiamine intake accompanied by high intake of carbohydrate derived from milled
rice and refined carbohydrate is still the major cause of thiamine deficiency in Asian
populations. For instance, dietary assessment in a village in northeastern Thailand has
repeatedly shown that the villagers’ protein and energy intakes are mainly supplied from
a rice-based diet; 78% of their energy intake is carbohydrate, mainly derived from milled
rice. Milled rice is a poor source of thiamine (Table 1), and about 85% of its thiamine
content is lost when the water in which it has been soaked is discarded (10,44,50,51).
Thus, their thiamine intake is low, e.g., 0.23 mg/1000 kcal (44). These dietary data are
consistent with the high prevalence of biochemical thiamine depletion in that population
(10,43,44). The adverse effects of the ATFs on thiamine status caused by consumption
of raw fermented fish and chewing betel nuts have also been demonstrated in northeastern
Thais, whereas inadequate thiamine status is also detected in northern Thais who regularly
chew fermented tea leaves (16).
The reappearance of beriberi in Japan in the 1970s is related to high carbohydrate
intake derived from milled rice, instant noodles, and sweet carbonated drinks, coupled
with low thiamine intake (47–49).
2. Infants Breast-Fed by Thiamine-Deficient Mothers
The mean ( SEM) concentrations of thiamine in breast milk from mothers residing in
rural areas of central, northern, and northeastern Thailand were 93  9.9, 102  6.5, and
119  6.3 µg/L. These figures are lower than the concentration of thiamine in breast milk
of 142  1.4 µg/L from the North American mothers. The thiamine concentration in
mothers with complaints suggestive of thiamine deficiency has been reported to be 87 
294 Tanphaichitr
4.8 µg/L (52,53). These findings partly explain the prevalences of infantile beriberi in
Thailand (10,54–58).
3. Chronic Alcoholism
The major cause of thiamine deficiency in Western populations is excessive intake of
alcohol (ethanol). Chronic alcoholics may eat nothing for days, and when they eat their
diet is often high in carbohydrate and low in thiamine (59). Excessive ethanol ingestion
creates a metabolic burden in the human body. The liver is primarily responsible for the
first two steps of ethanol metabolism:
CH3CH3OH  NAD ?> CH3CHO  NADH  H
Ethanol Acetaldehyde
CH3CHO  NAD ?> CH3COO
  NADH  H
Acetaldehyde Acetate
The first step, catalyzed by alcohol dehydrogenase, generates acetaldehyde and NADH,
and occurs in the cytosol. The second step, catalyzed by aldehyde dehydrogenase, generates
acetate and NADH but occurs in the mitochondrial matrix space. The NADH generated
by the second step can be used directly by the mitochondrial electron transfer chain,
whereas the NADH generated by the first step cannot be used directly and must be oxidized
back to NAD by the glycerol phosphate and/or malate-aspartate shuttles. Since the intake
of ethanol demands the hepatic disposal of the NADH, intake of even moderate amounts
of ethanol results in inhibition of important processes requiring NAD, including gluconeogenesis
and fatty acid oxidation (60).
Hepatic mitochondria have a limited capacity to oxidize acetate to CO2 because the
activation of acetate to acetyl CoA requires GTP, which is the product of the succinyl
CoA synthetase reaction. The TCA cycle, and therefore GTP synthesis, is inhibited by
NADH levels during ethanol oxidation. Much of the hepatic acetate generated from ethanol
escapes the blood to other cells containing mitochondria, which oxidize it to CO2 and
H2O through the TCA cycle (Fig. 8). The heart is the most active organ for the disposal
of acetate (60). This process cannot be achieved with an inadequate supply of thiamine.
Acetaldehyde can also escape from the liver. Though its concentration in the blood
is lower than that of acetate, acetaldehyde is a reactive compound that readily forms covalent
bonds with functional groups of biologically important compounds (60).
Not only insufficient thiamine intake but also other factors are involved in the development
of thiamine deficiency in chronic alcoholics. These include ethanol inhibition of
thiamine absorption, decrease in thiamine absorption secondary to protein or folate defi-
ciency, impaired conversion of thiamine to TPP in the presence of alcoholic liver injury,
and reduced hepatic storage of thiamine (2,12,13,21,26,30,61).
4. Patients on Parenteral Nutrition
Inadequate intake of nutrients, including thiamine, is common in hospitalized patients.
The cause of their inadequate thiamine intake can be either primary or secondary. Primary
inadequate intake of thiamine is caused by insufficient supply of food quantitatively and
qualitatively. In secondary inadequate intake of thiamine, illness or treatment affects the
thiamine status by altering a person’s eating behavior or impairing his or her ingestion.
Primary and secondary mechanisms frequently reinforce each other (62). In a dietary assessment
in 656 hospitalized patients over a period of 2 years, 57 of the patients had a
Thiamine 295
low daily intake of thiamine (63), and in 19% the intake was less than half the recommended
daily allowances (RDAs) for thiamine (64). This result warrants the supplementation
of thiamine in hospitalized patients to ensure their adequate thiamine status. Special
attention should be paid to patients on parenteral nutrition in which glucose is the major
source of energy supply because inadequate or absent thiamine creates beriberi (65–73).
B. Decreased Absorption of Thiamine
The ethanol inhibition of thiamine absorption and the defective absorption of thiamine in
folate deficiency states are well documented (2,12,13,21,26,30). Patients with intestinal
resection are also prone to develop thiamine deficiency because jejunum and ileum are
the sites of thiamine absorption (12,74).
C. Defective Transport of Thiamine
Thus far, there is no acquired condition that is known to cause defective transport of
thiamine. However, there is a concern about the adequacy of the transport of thiamine
from mothers across the placenta to the fetus. Measurement of erythrocyte or whole-blood
transketolase activity (ETKA or WBTKA) and the enhancement from added TPP to basal
assays (referred to as the TPP effect, TPPE) is used for thiamine assessment. Using this
approach to assessment, thiamine status in cord blood and in mothers within 24 h of
delivery (75) or 6–12 h before delivery (76) indicates that newborns have better thiamine
status than their mothers. These results are consistent with the findings of previous studies
that (a) cord blood has a higher thiamine content than maternal blood (77) and (b) fetal
thiamine status may be normal even when the mother is thiamine-deficient (78). These
findings imply that there is an efficient transport of thiamine across the placental barrier
and that the fetus can sequester thiamine.
D. Impaired Biosynthesis of TPP
The conversion of thiamine to TPP may be impaired in the presence of alcoholic liver
injury, possibly because of decreased availability or use of ATP, and may be associated
with decreased activities of pyruvate dehydrogenase and transketolase due to apoenzyme
or magnesium deficiency (12,13). Besides, the decreases in hepatic and cerebral transketolase
activities are accompanied by a parallel fall in thiamine concentrations in liver and
brain (79). Since the transketolase activity correlates with TPP concentrations (80), these
findings suggest a lowering in the TPP concentrations. However, thiamine deficiency in
patients with fulminant hepatic failure can be corrected by intravenous administration of
100 mg of thiamine hydrochloride twice daily. This indicates that the bioconversion of
thiamine to TPP is still possible in the presence of acute hepatocellular necrosis when the
patient is treated with pharmacological doses of thiamine (81).
E. Increased Requirement of Thiamine
Increased anabolism occurs in various physiological conditions, including pregnancy, lactation,
infancy and childhood, adolescence, and increased physical activity, whereas a
number of pathological conditions create increased catabolism. Increased anabolism and
catabolism raise the requirements of several nutrients, including thiamine.
296 Tanphaichitr
1. Pregnancy
Studies of urinary excretion of thiamine, blood thiamine levels, and ETKA all indicate
that the requirement for thiamine in women increases during pregnancy. This increase
appears to occur early in pregnancy and to remain constant throughout (64,75,76,82).
Thus women with marginal thiamine status who are pregnant and pregnant women with
inadequate intake of thiamine are vulnerable to develop beriberi. This is well illustrated
in the report on women with hyperemesis gravidarum presenting with Wernicke’s encephalopathy
having the symptom of vomiting for a month, and Wernicke’s encephalopathy
usually occurred following thiamine-free intravenous glucose infusion (83,84).
2. Lactation
Thiamine requirement also increases during lactation due to the loss of thiamine in milk
and increased energy consumption during lactation. The healthy lactating woman secretes
approximately 0.2 mg of thiamine in her milk each day (85).
3. Infancy and Childhood
Because the metabolic rate of infants and children is greater and the turnover of nutrients
more rapid than in adults, the unique nutritional needs for growth and development are
superimposed on higher maintenance requirements than those of adults. Whole-blood total
thiamine concentrations are higher in infants aged 0–3 months than those aged 3–12
months, i.e., 258  63 (mean  SD) and 214  44 nmol/L, respectively. After a large
decline in the first year of life, the concentrations become stable and equal to those in
adults: 187 39 nmol/L. The overall decrease is due mainly to a decrease in the phosphorylated
thiamine. Since these changes occur relatively independently of the changes in
hematocrit characteristically seen in infancy, it may be related to changes in metabolic
activity, growth rate, or other variables of biological activity (86). The findings that
changes in whole-blood thiamine concentrations during infancy and childhood are physiological
are supported by nonsignificant differences in WBTKA and TPPE between healthy
infants and children aged 1–6 years (55). However, inadequate thiamine status is commonly
found in breast-fed infants by thiamine-deficient mothers and children on the ricebased
diets (10,54–58).
4. Adolescence
The nutritional requirements of adolescents are influenced primarily by the normal events
of puberty and the simultaneous spurt of growth. Adolescence is the only time in extrauterine
life when growth velocity increases and exerts a major influence on nutritional requirements.
Since thiamine is involved in energy metabolism, adolescents on milled rice as
their staple diets are vulnerable to develop thiamine deficiency (10,45,46,48,87,88).
5. Increased Physical Activity
For most people, the second largest component of total energy expenditure is the energy
expended in physical activity. Thus, high physical activity can precipitate the development
of beriberi in those with marginal thiamine status (10,88).
6. Increased Catabolism
Increased catabolism raises the requirements for energy and thiamine. Thus, catabolic
conditions, such as fever, infection, trauma, or surgery, can precipitate the development
of beriberi in those with marginal thiamine status (10,89–91).
Thiamine 297
F. Increased Loss of Thiamine
Renal patients on chronic dialysis (92,93) and patients with congestive heart failure treated
with furosemide, a diuretic (94,95), may develop beriberi, which in part is due to an
increased renal excretion of thiamine.
VIII. CLINICAL MANIFESTATIONS OF BERIBERI
The clinical manifestations of beriberi vary with age. Infantile beriberi is commonly found
in infants aged 2–3 months who are being breast-fed by thiamine-deficient mothers. The
clinical manifestations of beriberi in older children and adolescents are similar to those
in adults, and as such are classified as adult beriberi (5,9,10,30,41,96–99).
A. Infantile Beriberi
The predominant symptoms of infantile beriberi include edema, dyspnea, oliguria, aphonia,
cardiovascular disturbances, and gastrointestinal disorders, including loss of appetite,
vomiting, or milk diarrhea. Infantile beriberi may be categorized into four subtypes based
on the dominant features, i.e., cardiac, aphonic, pseudomeningitic, or a combination
(10,41,96–99).
1. Cardiac or Acute Fulminating Form
Infants with cardiac beriberi usually present with acute onset of manifestations, including
a loud piercing cry, cyanosis, dyspnea, vomiting, tachycardia, and cardiomegaly; death
may occur within a few hours after onset unless thiamine is administered.
2. Aphonic Form
Aphonic forms of infantile beriberi are generally less acute. The striking feature is the
tone of the child’s cry, which varies from hoarseness to complete aphonia due to paralysis
of the recurrent laryngeal nerve. A decrease in deep tendon reflexes and blepharoptosis
are detected in some infants.
3. Pseudomeningitic Form
Infants exhibit vomiting, nystagmus, purposeless movement of the extremities, and convulsion.
However, no abnormality of cerebrospinal fluid (CSF) is demonstrated.
4. Mixed Form
Infants with beriberi may present with the combined manifestations of cardiac disturbance,
aphonia, and pseudomeningitis.
In a study of 16 infants with beriberi, the prevalence of cardiovascular system disorders
was 87% with dyspnea, 81% with tachycardia (heart rate more than 130 beats/min),
73% with cardiomegaly (confirmed by chest x-ray), 63% with vomiting, 62% with hepatomegaly,
56% with edema, 19% with cyanosis, and 6% with shock, whereas those of nervous
system disorders included 81% with decreased deep tendon reflexes, 50% with aphonia,
12% with convulsion, and 6% with blepharoptosis (10,56).
B. Adult Beriberi
Older children, adolescents, and adults may present with dry (paralytic or nervous), wet
(cardiac), or cerebral beriberi (5,9,10,30,41,96–99).
298 Tanphaichitr
1. Dry Beriberi
The predominant feature in dry beriberi is peripheral neuropathy, characterized by a symmetrical
impairment of sensory, motor, and reflex functions that affects the distal segments
of limbs more severely than the proximal ones, calf muscle tenderness, and difficulty in
rising from a squatting position (5,10,46,99).
Pathological changes in the peripheral nerve in beriberi neuropathy are unusual axonal
degeneration and accumulation of flattened sacs or tubules in the axoplasm of large
myelinated fibers in untreated patients. In beriberi patients treated with thiamine, there
are numerous Bungner’s bands and clusters of thin myelinated fibers, indicating active
regeneration, which correspond well with clinical improvement (47). Examination of CSF
in patients with dry beriberi reveals normal findings (47,99).
2. Wet Beriberi
In addition to peripheral neuropathy, common signs found in wet beriberi include edema,
tachycardia, wide pulse pressure, cardiomegaly, and congestive heart failure (5,10,46,99).
In some patients, there is a sudden onset of a cardiac manifestation known as acute fulminant
or ‘‘shoshin’’ beriberi, with the predominant features of tachycardia, dyspnea, cyanosis,
cardiac enlargement, and circulatory collapse (41).
a. Electrocardiographic Findings
Considerable attention has been given to the electrocardiographic changes that occur in
wet beriberi (99,100). The serial electrocardiographic study in 11 patients with wet beriberi
whose thiamine deficiency was confirmed by ETKA and TPPE revealed the following
findings: 8 patients had a prolonged QT interval, which became normal in 5 patients within
2 h to 28 days after receiving the thiamine treatment. Seven patients showed T-wave
abnormalities in the precordial leads, consisting of flat or inverted T waves that became
normal within 7–40 days following administration of thiamine. Three patients showed
relatively low QRS complex voltage, which increased after the appearance of diuresis
following the thiamine treatment. The increase of QRS complex voltage and the rapid
reduction of heart size were observed concurrently with clinical improvement. Since the
pattern of the electrocardiographic voltage in wet beriberi resembles that in pericardial
effusion, the transient low QRS voltage in patients with wet beriberi is most likely caused
by pericardial effusion (99,101,102).
b. Hemodynamic Findings
Studies in occidental patients with wet beriberi have shown hemodynamic findings of high
cardiac output with low peripheral vascular resistance (103,104); an acute response to
thiamine administration, i.e., decrease in cardiac output with concomitant increase in total
peripheral resistance, was observed within 37 min (104) and 2 h (103).
A hemodynamic study of six Thai nonalcoholic patients with wet beriberi whose
diagnosis was confirmed by ETKA and TPPE revealed two types of hemodynamic findings.
The first type is consistent with occidental reports (103–105); it consisted of four
patients showing high cardiac output and low peripheral and pulmonary vascular resistance.
Within 15 min of intravenous administration of thiamine HCl, their cardiac output
was reduced and vascular resistance was increased, as was blood pressure (99,106,107).
It appears that the major cause of the high cardiac output is vasomotor depression, the
precise mechanism of which is not understood, but which leads to a reduced systemic
vascular resistance (108).
Thiamine 299
The second type of hemodynamic finding involved one patient with normal cardiac
output and another with low cardiac output. The low cardiac output may be due to myocardial
damage sustained by repeated episodes of thiamine deficiency; the increase in cardiac
output after thiamine treatment suggests the improvement of myocardial metabolism by
the pharmacological dose of thiamine (99,106,107). Thus, cardiac output in wet beriberi
may be variable. Normal or low cardiac output does not exclude the diagnosis.
3. Wernicke–Korsakoff Syndrome
The specific factor responsible for most, if not all, of the clinical manifestations of the
Wernicke–Korsakoff syndrome is a deficiency of thiamine. Wernicke–Korsakoff syndrome
constitutes the most common alcoholic-nutritional affliction of the central nervous
system (3,24,108–110). Although alcoholism is the major cause of thiamine deficiency
in Wernicke–Korsakoff syndrome, iatrogenic causes, including parenteral glucose administration
and chronic dialysis, can aggravate the syndrome in patients with marginal thiamine
status (65–68,83,84).
a. Wernicke’s Disease or Encephalopathy
Wernicke originally described an illness of acute onset characterized by mental disturbance,
paralysis of eye movements, and ataxia of gait. Since Wernicke’s time, views regarding
Wernicke’s disease have undergone considerable modification. The diagnosis is
based on the triad of ocular motor signs, ataxia, and derangement of motor functions
(3,109).
The ocular motor signs are the most readily recognized abnormalities. They include
a paresis or paralysis of abduction accompanied by horizontal diplopia, strabismus, and
nystagmus. In advanced cases, there may be complete loss of ocular movement, and the
pupils may become miotic and nonreacting. Blepharoptosis is rare. The ocular motor signs
are attributable to lesions in the brain stem affecting the abducens nuclei and eye movement
centers in the pons and rostral midbrain.
The ataxia affects stance and gait. Persistent ataxia is related to the loss of neurons
in the superior vermis of the cerebellum; extension of the lesion into the anterior parts of
the anterior lobes accounts for the ataxia of individual movements of the legs.
A derangement of mental function is found in about 90% of patients and takes one
of the following three forms. First, a global confusional-apathetic state is most common,
characterized by profound listlessness, inattentiveness, indifference to the surroundings,
and disorientation. Second, a disproportional disorder of retentive memory, i.e., Korsakoff’s
amnesic state, occurs in some patients. Third, the symptoms of alcohol withdrawal
may be found in a relatively small number of patients. The amnesic defect is related to
lesions in the diencephalon, specifically those in the medial dorsal nuclei of the thalamus.
Most patients with Wernicke’s disease display peripheral neuropathy and some cardiovascular
abnormalities, including tachycardia, exertional dyspnea, and postural hypotension
(3,109).
b. Korsakoff’s Psychosis
Victor (109) has stated that Wernicke’s disease and Korsakoff’s psychosis are not separate
diseases; Korsakoff’s psychosis is the psychic component of Wernicke’s disease. Thus,
the clinical manifestations should be called Wernicke’s disease when the amnesic state
is not evident and the Wernicke–Korsakoff syndrome when both the ocular-ataxic and
amnesic manifestations are present.
300 Tanphaichitr
Though the specific role of thiamine in Wernicke–Korsakoff syndrome is established
and most patients with Wernicke–Korsakoff syndrome are alcoholics, only a few
alcoholics are affected (3,109–111). Genetic abnormalities in ETK may underlie a predisposition
to Wernicke–Korsakoff syndrome (112). However, the investigation on the hysteretic
properties of human transketolase with emphasis on its dependency on TPP concentration
has revealed the substantial lag in formation of active holotransketolase and
interindividual differences and cell type variation from the same individual in the lag
period; these findings suggest the existence of mechanisms for the loss of transketolase
activity during thiamine deficiency and may explain, at least in part, the differential sensitivity
to deficiency demonstrated by tissues and individuals (113).
IX. TREATMENT AND CLINICAL RESPONSE
A. Treatment
Thiamine should be promptly administered to beriberi patients. The dosage usually ranges
from 50 to 100 mg of thiamine HCl given intravenously or intramuscularly for 7–14 days,
after which 10 mg/day can be administered orally until the patient recovers fully. To
prevent the recurrence of beriberi, patients should be advised to change their dietary habits
and to stop their alcohol drinking (5,10,30,46,56).
The rationale to administer a large dose of thiamine is to replenish the thiamine
store, which is consistent with the positive correlation between thiamine concentration in
serum and CSF (114), to stimulate the TPP-dependent reactions optimally, which is consistent
with the aforementioned thiamine-dependent hysteretic behavior of human transketolase
(113), and to improve cardiovascular disorders evidenced by the hemodynamic
studies (99,103–107). The parenteral route is used initially to ensure the bioavailability
of thiamine.
Several thiamine derivatives, especially thiamine propyl disulfide (TPD) and tetrahydrofurfuryldisulfide
(TTFD) (Fig. 4), are useful for oral administration to beriberi patients
because even by the oral route they produce a significantly higher thiamine level in the
blood, erythrocytes, and CSF than thiamine HCl and TPP at the same dose of 50 mg, and
their effectiveness is equivalent to that produced by parenteral administration of thiamine
HCl or TPP in healthy subjects (115). The effective intestinal absorption of TTFD is also
demonstrated at 100- and 180-mg dose levels in healthy subjects (116). The rapid absorption
and transfer to the tissue of TPD is also evident by the increased levels of thiamine
in portal vein, hepatic vein, and femoral artery (117). Oral administration of 50 mg of
TPD to alcoholics with thiamine deficiency also restored their ETKA to normal and eliminated
lateral rectus palsy in patients with Wernicke’s disease (117).
The bioavailability of TTFD in multivitamin tablets has also been shown in healthy
subjects taking the vitamin preparation containing 10 mg of TTFD daily for 7 days and
10 or 20 mg of TTFD daily for 44 weeks. Their baseline blood thiamine concentrations
increased gradually during the long-term administration, with a dose-dependent response
(118). The dramatic response of patients with cardiac beriberi to the daily intravenous
administration of 50 mg of TTFD has also been reported (119).
B. Response to Thiamine Administration
In wet beriberi, within 6–24 h of thiamine administration improvement can be observed
in terms of less restlessness; disappearance of cyanosis; reductions in heart rate, respiratory
Thiamine 301
rate, and cardiac size; and clearing of pulmonary congestion. Dramatic improvement after
thiamine treatment is also observed in infants with cardiac beriberi (10,46,55,56,99).
It is difficult to use the response to thiamine administration as a criterion for immediate
diagnosis in dry beriberi or infants with aphonia because more time elapses before
improvement is observed. In a study of 21 patients with beriberi, impaired sensation disappeared
after 7–120 days of thiamine treatment; motor weakness found in 12 patients was
recovered within 60 days; but absent or hypoactive ankle and knee jerks remained for
several months after treatment (10,46,99).
The response of patients with Wernicke’s disease to thiamine treatment shows a
characteristic pattern. Occular palsies may begin to improve within hours to several days
after the administration of thiamine. Sixth-nerve palsies, ptosis, and vertical gaze palsies
recover completely within 1–2 weeks in most cases, but vertical gaze–evoked nystagmus
may persist for months. Ataxia improves slowly and approximately half of patients recover
incompletely. Apathy, drowsiness, and confusion also recede gradually (109).
The outcome of the Korsakoff amnesic state varies. It ranges from complete or
almost complete recovery in less than 20% of patients to slow and incomplete recovery
in the remainder. The residual state is characterized by large gaps in memory, usually
without confabulation, and an inability of the patient to sort out events in the proper
temporal sequence (109).
X. ASSESSMENT OF THIAMINE STATUS
The sequential development of impaired thiamine status can be divided into four stages.
It begins with an inadequate thiamine intake. The resulting depletion of thiamine in tissues
leads to metabolic derangement of various organs, and clinical manifestations of beriberi
appear (Table 2). Thus, the subject’s history, laboratory tests, and physical examination
are the bases for evaluation of thiamine status in humans (62).
A. Subject’s History
In addition to dietary assessment, demographic data, medical history, family history, and
psychosocial history in each subject should be recorded because all of these factors can
affect the thiamine intake of the subject. Several methods including dietary scan, dietary
record, and/or 24-h dietary recall have been employed to assess the thiamine intake in
various populations, and comparisons are made between the mean thiamine intakes and
RDAs for thiamine. For instance, dietary assessment by dietary record in northeastern
Thai villagers revealed a mean daily energy intake of 2441 kcal, with a mean daily thiamine
intake of 0.56 mg or 0.23 mg per 1000 kcal (44), which were only 51% or 46% of
Table 2 Sequential Development of Inadequate
Thiamine Status
Stages of inadequate thiamine status Methods of assessment
I Inadequate thiamine intake Subject’s history
II Tissue desaturation of thiamine Laboratory assessment
III Metabolic derangement Laboratory assessment
IV Clinical manifestations Physical examination
302 Tanphaichitr
the RDAs for thiamine for the North American population (64). Dietary assessment based
on 24-h recall in the North American male population aged 65–75 years showed a mean
daily energy intake of 1805 kcal and a mean daily thiamine intake of 1.40 mg or 0.68
mg/1000 kcal (120), which are 116% or 136% of the 1989 RDAs for thiamine (64). It
is evident that the northeastern Thai villagers have lower intake of thiamine than the
elderly North American population. These dietary data are consistent with the biochemical
findings on their thiamine status (44,120).
B. Laboratory Tests
Two crucial properties of laboratory tests must be considered when they are employed
for the assessment of thiamine status. The tests must not be overly invasive; they should be
sensitive enough to identify inadequate thiamine status prior to the appearance of clinical
manifestations (Table 1) and to assess the efficacy of subsequent thiamine treatment.
Various biochemical tests have been developed for detecting thiamine deficiency
or assessing the adequacy of thiamine status in humans. These include the measurement
of urinary thiamine excretion; blood thiamine level; thiamine concentration in CFS, blood
pyruvate, lactate, and ?-ketoglutarate levels; and ETKA and TPPE (2,3,5,9,10,26,46,121,
122).
1. Urinary Thiamine Excretion
Prior to the development of the measurement of ETKA and TPPE, the most commonly
used approach to thiamine status assessment was the measurement of urinary levels of
thiamine by the thiochrome method after a one-step purification by ion exchange chromatography.
Microbiological assays were also common and utilized Lactobacillus viridescens
(121). Currently, in addition to ETKA and TPPE measurement, thiamine levels in
urine are estimated by HPLC (122).
There is a reasonably close correlation between the development of a thiamine defi-
ciency and decreased urinary thiamine excretion. Approximately 40–90 and 100 µg or
more of thiamine is excreted in the urine daily in adults on intakes of 0.30–0.36 and 0.50
mg of thiamine per 1000 kcal, respectively, whereas daily urinary thiamine excretion falls
to 5–25 µg with the daily intake of 0.2 mg of thiamine per 1000 kcal. Thus, measurement
of the 24-h urinary thiamine excretion is useful in evaluating human thiamine status. Daily
urinary thiamine excretions of 100, 40–99, and 40 µg are considered to designate
acceptable, low, and deficient thiamine status, respectively. However, it is usually not
feasible to collect 24-h urine samples with large groups under survey conditions. Consequently,
random urine samples are obtained, preferably during fasting state, with the simultaneous
determination of thiamine and creatinine concentrations. A correlation between
the urinary thiamine excretion per gram of creatinine and daily thiamine intake in milligrams
per 1000 kcal has been observed. Urinary thiamine excretions of 66, 27–65, and
27 µg/g of creatinine in adults are considered to designate acceptable, low, or deficient
thiamine status. The corresponding figures in pregnant women during the second trimester
are 55, 23–54, and 23 µg/g creatinine, whereas those during the third trimester are
50, 21–49, and 21 µg/g creatinine (121).
Healthy children have markedly higher levels of urinary thiamine excretion per gram
creatinine than adults. Thus, children aged 1–3, 4–6, 7–12, and 13–15 years are considered
to have acceptable thiamine status when their urinary thiamine excretions are 176,
121, 181, and 151 µg/g creatinine, respectively (121).
Thiamine 303
Though urinary thiamine excretion per gram creatinine may be a useful criterion
for surveying thiamine status in a large population, it is not always a good index for
diagnosing beriberi, e.g., of 15 patients with beriberi prior to thiamine treatment, 8(53%),
3(20%), and 4(27%) had urinary thiamine excretions of 27, 27–65, and 66 µg/g creatinine,
respectively (46).
Although a number of thiamine load tests of a known dose of thiamine administered
orally or intramuscularly have been of value in evaluating the extent of thiamine depletion
in tissue stores, the most commonly used procedure is to administer 5 mg of thiamine
parenterally and to measure the urinary thiamine excretion in the following 4-h period.
Thiamine-deficient subjects usually excrete less than 20 µg of thiamine following the test
dose. However, this procedure is inconvenient in nutrition surveys (121).
2. Blood Thiamine Level
Levels of thiamine in whole blood, plasma or serum, and erythrocytes have been employed
to assess human thiamine status. The thiamine concentrations in these biological samples
can be determined by chemical methods, including the thiochrome procedure and colorimetric
techniques utilizing diazotized p-aminoacetophenone or ethyl-p-aminobenzoate;
microbiological assays using Ochromonas danica or a streptomycin-resistant mutant strain
of Lactobacillus fermenti; and HPLC (2,24,26,86,114,121,122). The specificity, sensitivity,
and other limitations of each method must be carefully reviewed whenever employing
it for assessing blood thiamine levels because blood contains only about 0.8% of the total
body thiamine. Since most of the blood thiamine is in the blood cells, the serum level is
still much lower. The thiamine concentration in leukocytes is about 10 times that in erythrocytes.
In the interpretation of blood thiamine tests, the hematocrit must be taken into
consideration because erythrocyte mass usually contributes 75% or more of the total thiamine
(2,86). Although leukocytes contribute 15% or less of the blood thiamine concentration,
the interpretation of a low-normal blood thiamine level in a subject with leukocytosis
must be done with caution (86).
a. Chemical Methods
The determination of total thiamine concentration in whole blood or serum by the thiochrome
method is based on an oxidative conversion of thiamine (by ferricyanide in an
alkaline solution) to thiochrome (Fig. 3), which is highly fluorescent and is extracted from
the aqueous phase with isobutanol for subsequent fluorometric measurement. Oxidation
of phosphorylated thiamine with ferricyanide yields a thiochrome derivative not extractable
with isobutanol. Pretreatment of whole blood or serum with phosphatase preparations,
e.g., potato acid phosphatase, is necessary for obtaining the total thiamine concentration.
Thus, the total and nonphosphorylated thiamine are measured directly, whereas the phosphorylated
thiamine is calculated from total thiamine minus nonphosphorylated thiamine
(86,123).
The colorimetric method based on the coupling of diazotized p-aminoacetophenone
with thiamine to produce an insoluble purple-red compound that can be measured photometrically.
Uric acid and ascorbic acid interfere with the development of the color. Besides,
the method is not sufficiently sensitive to detect the low concentration of blood
thiamine (2,123).
b. Microbiological Assays
Baker et al. (124) developed a protozoan method using the phytoflagellate Ochomonas
danica to measure thiamine concentrations in whole blood, erythrocytes, leukocytes, CSF,
304 Tanphaichitr
and other tissues. Ochromonas danica has a sensitive and specific intact thiamine requirement;
it can assay 375 pmol/L (100 pg/mL) of thiamine in biological fluids and tissues.
It has been shown that reduced blood thiamine levels determined by this protozoan method
occurred within 2 days in rats on thiamine-deficient diet. At 15 days there was almost no
circulating thiamine, and the thiamine contents in liver and muscle fell to 25% of normal,
whereas the ETKA fell after 10 days, and after only 20 days was reduced to nearly 50%
of normal (125).
Thiamine concentrations in serum and erythrocytes can also be accurately measured
by an automated method using a streptomycin-resistant strain of Lactobacillus fermenti
as the test organism. This method is sensitive to 1.88 nmol/L (0.5 µg/L) of thiamine. The
organism responds to both free and phosphorylated thiamine; its growth response to TPP
is approximately 30% greater than that obtained with thiamine HCl (24,26).
c. High-Performance Liquid Chromatography
HPLC determinations of free and phosphorylated thiamine are now frequently carried out
to assess thiamine status (114,123,126–128); a sensitivity for the detection of 0.05 pmol
of thiamine has been reported (128). Besides, erythrocyte TPP levels in healthy adults
determined by HPLC are in general agreement with those determined by the enzymatic
method utilizing yeast pyruvic carboxylase apoenzyme (123,126). The validity of assessing
thiamine status by erythrocyte TPP levels is supported by the following findings: i.e.,
erythrocyte TPP levels in rats fed a thiamine deficient diet fell more rapidly than ETKA
(123); over an intake range of up to 3.4 mg of thiamine per day in eight hospitalized
patients, their erythrocyte TPP levels increased proportionately with the thiamine intake
(129); and a significantly positive correlation between ETAK and whole-blood TPP levels
in 127 adults (r  0.26, p  0.003) as well as a significantly negative correlation between
?-ETKA (ETKA with added TPP/ETKA without added TPP) (r  
0.44, p  0.0001)
(127).
Unlike urinary thiamine excretion, there is no accepted guideline for the interpretation
of blood thiamine levels. Tables 3 and 4 show normal thiamine levels in biological
fluids determined by the protozoan (125) and HPLC methods (114), respectively.
3. Thiamine Concentration in CSF
In adults, total thiamine in CSF exists in two forms, i.e., 62% as TMP and 38% as thiamine.
The total thiamine, thiamine, and TMP in CSF are four, two, and eight times higher than
the corresponding forms in serum. Besides, there is a correlation between serum and CSF
concentrations of thiamine. TMP is formed from the dephosphorylation of TPP and is
further dephosphorylated into thiamine (Fig. 2). Since TPP is not found in CSF, the high
concentration gradient of TMP from serum to CSF suggests the existence of an active
Table 3 Normal Thiamine Levels in Biological Fluids Determined by the
Protozoan Method Using Ochromonas danica (125)
Thiamine (nmol/L) Thiamine (µg/L)
Specimen Range Mean Range Mean
Whole blood 94–282 120 25–75 32
Serum 563–957 788 150–255 210
CSF 38–113 60 10–30 16
Thiamine 305
Table 4 Concentrations of Various Forms of Thiamine in Blood and Cerebrospinal Fluid in
Adults Determined by HPLC
Subjecta Thiamine formsd (nmol/L)
Age
Author (y) N Specimen Free TMP TTP Total
Tallaksen et al. 23–58 40 Serum 5  23 2–12 — NA
(114) 21–83 31b Serum 8.6  3.9 4.0  1.5 — 12.7  4.2
21–83 45b,c CSF 16.9  8.3 28.1  7.9 — 44.9  14.0
Fidanza et al. 20–82 127 Whole blood 126  35
(127) (64–202)
a All of the subjects were healthy except those with superscripts b and c.
b Blood and CSF were collected simultaneously from 31 patients with severe back pain but were otherwise
healthy during performance of myelogram.
c Only CSF samples were collected from another 14 similar patients.
d Figures are mean  SD or range. NA, not available from the reports.
transport of TMP, which is supported by a saturation mechanism (114). Though simultaneous
measurements of thiamine in serum and CSF reflect the integrity of blood–brain
barrier and thiamine supply to the brain, it is impractical and unjustifiable to assess thiamine
status by the measurement of CSF thiamine in a free-living population. Determination
of CSF thiamine in hospitalized patients with dry beriberi or Wernicke–Korsakoff
syndrome should be based on the risk and benefit of the patients.
4. Blood Pyruvate, ?-Ketoglutarate, and Lactate Levels
In beriberi patients, pyruvic and ?-ketoglutaric acids accumulate in the tissue (Fig. 8) and
their concentration in the blood rises. Part of accumulated pyruvic acid may also be converted
to lactic acid. However, these changes lack specificity and consistency. The fasting
levels of blood pyruvate have frequently been found to be normal in thiamine deficiency
and only rise above normal following a glucose load (99,121). Thus, a glucose load test,
termed the carbohydrate metabolic index (CMI), has been developed; blood glucose (G),
pyruvate (P), and lactate (L) levels are measured at 1 h after the administration of 1.8 g
of glucose per kilogram of body weight and 5 min after completion of a standardized
exercise. CMI is then calculated as follows;
CMI 
(L 
 G/10)  (15P 
 G/10)
2
The upper normal limit of CMI is 15, based on the studies of experimental thiamine
deficiency in humans (130). However, this procedure requires too much cooperation and
technical ability to be useful routinely (121).
5. Blood Transketolase Activity
Blood is a feasible specimen to be obtained for the determination of transketolase activity,
which can be measured in erythrocytes, leukocytes, or whole blood.
a. ETKA and TPPE
Brin and his co-workers (131) have demonstrated that ETKA and its percent stimulation
in vitro by added TPP, called TPPE, are sensitive methods to detect thiamine deficiency
306 Tanphaichitr
both in rats and in humans. Healthy adults fed diets containing 190 µg of thiamine daily
for 8 weeks showed a decline in ETKA as early as 8 days, and their ETKA at 17–20
days was markedly lower than that in the control subjects; their TPPE was 15% and 34%
at days 8 and 31, respectively, whereas TPPE in the control subjects was less than 10%
throughout the study; and following thiamine treatment, their ETKA and TPPE returned
to normal values. These findings indicate the specificity of TPP on the depressed ETKA
in thiamine deficiency in humans. In this study, the urinary thiamine excretion in these
thiamine-deficient subjects was sharply reduced to less than 50 µg/day by the second
week and to less than 20 µg/day by the third week, whereas their CMI was below 15
throughout the study. These findings indicate the sensitivity of ETKA and TPPE in detecting
human thiamine deficiency. Subsequent works have established the validity of
ETKA and TPPE for the diagnosis of various forms of beriberi in infants and adults, and
have proved that they are useful indicators of biochemical thiamine depletion prior to the
appearance of beriberi (2,3,5,9,10,30,43,46,56,121,130). The measurement of ETKA and
TPPE represents a functional test of thiamine adequacy and hence may be a more reliable
indicator of thiamine insufficiency than urinary thiamine excretion (121).
ETKA can be measured by a colorimetric procedure involving incubating hemolyzed
erythrocyte samples for 30 or 60 min at 37–43°C in a buffered medium with an excess
of ribose-5-phosphate with and without added TPP and then determining the amount of
ribose-5-phosphate used, sedoheptulose-7-phosphate formed, or the hexoses produced
(26,121). TPPE is then calculated as follows:
TPPE in % 
ETKA with added TPP 
 ETKA without added TPP
ETKA without added TPP
 100
In some reports, the stimulating effect of TPP on ETKA is expressed as the activity coeffi-
cient of ETKA (?-ETKA), i.e., ETKA with added TPP/ETKA without added TPP (127).
ETKA obtained without the addition of TPP represents the basal level of enzyme
activity and is dependent on the availability of endogenous erythrocyte TPP. The addition
of TPP permits estimation of the amount of apoenzyme uncomplexed as well as the maximum
potential activity. A thiamine deficiency results in a reduction in TPP, which results
in a reduction of the apotransketolase activity (121). This is supported by a correlation
between transketolase activity and TPP concentrations (80).
A modification of the Dreyfus method (132) has been employed for the determination
of ETKA in our laboratory and ETKA is expressed in international units (IU), which
is equivalent to the number of micromoles of sedoheptulose-7-phosphate formed per minute
per liter of erythrocytes.
A colorimetric procedure has been developed that distinguishes transketolase activity
from overall ribose-5-phosphate utilization by measuring ribose disappearance and
sedoheptulose formation in the same color reaction (133). More direct spectrometric methods
have evolved based on the change in absorption at 340 nm due to the decrease in
NADH. For example, glyceraldehyde-3-phosphate oxidation has been used (134). A semiautomated,
continuous-flow procedure for determining unstimulated ETKA and TPP-stimulated
ETKA has also been described (135). The sensitivity, reliability, and precision of
this approach is improved by eliminating hemoglobin interference.
Table 5 shows Brin’s guideline (136) and the guideline of the Interdepartmental
Committee on Nutrition for National Defense (ICNND) (121) for interpreting TPPE levels
in evaluating thiamine status. Both guidelines are agreeable for the TPPE levels of normal
Thiamine 307
Table 5 Guidelines for Interpreting TPPE levels in
Evaluating Thiamine Status in All Ages
Brin’s guideline (136) ICNND’s guideline (121)
Category TPPE (%) Category TPPE (%)
Normal 0–14 Acceptable 0–15
Marginally deficient 15–24 Low 16–20
Severely deficient 25 Deficient 20
or acceptable thiamine status, whereas the TPPE levels for grading severity of thiamine
deficiency are different. The validity of the TPPE levels of 0–15% for acceptable thiamine
status is substantiated by the finding that 94% of beriberi patients prior to thiamine treatment
showed TPPE of 16% or higher (46,99). However, both ETKA and TPPE must be
considered in assessing the adequacy of thiamine status because the patterns of these two
biochemical parameters in acute and chronic thiamine deficiency are different.
Subjects with acute thiamine deficiency have low ETKA with concomitantly high
TPPE levels. Their unstimulated ETKA 1 h after the parenteral administration of thiamine
is equivalent to or higher than the TPP-stimulated ETKA prior to thiamine treatment and
their TPPE is in the acceptable range of 0–15% (10,46,99).
Experimental studies in rats (137) and humans (138) have shown that after a certain
period of thiamine deprivation the TPP added in vitro cannot restore ETKA fully. Thus,
despite low ETKA, high TPPE is not seen. These data are supported by findings that the
TPP added in vitro does not increase ETKA in patients with beriberi prior to thiamine
treatment (46,99) and in free-living populations with chronic inadequate thiamine intake
(43) to the unstimulated ETKA in normal adults. Also, there is no significant difference
in TPPE between subjects with adequate thiamine intake and those with chronically low
intake of thiamine, despite significantly lower ETKA in the latter group (43).
The low unstimulated ETKA in cirrotic patients can be due either to the impaired
conversion of thiamine to TPP or to apotransketolase deficiency. In those with an impairment
of thiamine conversion to TPP, there is no increase in the low ETKA after following
thiamine treatment; only the added TPP in vitro raises their ETKA. In those with apotransketolase
deficiency, there are no increases in ETKA after thiamine treatment and the
addition of TPP in vitro; in some instances, the transketolase level returns to normal when
positive nitrogen balance is restored (125).
Apotransketolase deficiency, evidenced by decreases in unstimulated ETKA and
TPP-stimulated ETKA with no change in TPPE, has also been shown in patients with
insulin-dependent diabetes mellitus, patients with non–insulin-dependent diabetes mellitus
(139), and uremic patients (140).
Elevated ETKA with normal TPPE is documented in patients with pernicious anemia
(139,141,142), whereas patients with megaloblastic anemia caused by folate deficiency
or fish tapeworm do not exhibit elevated ETKA (141). Since TPPE is only rarely increased
in pernicious anemia with or without neuropsychiatric manifestations, thiamine deficiency
should not be a feature of this disease and does not take part in the genesis of hyperpyruvicacidemia
(142). Because transketolase activity is normally greater in young erythrocytes
than in older cells, it has been suggested that patients with pernicious anemia have
a larger population of young erythrocytes than healthy individuals (139,142). This suggestion
is supported by findings of increased ETKA in patients with iron deficiency anemia
308 Tanphaichitr
responding to the iron treatment, in most patients with congenital hemolytic anemia, and
in some patients with acute leukemia; and the normalization of unstimulated ETKA after
3–5 weeks of vitamin B12 treatment as well as the significantly negative correlation between
hemoglobin levels and TPP-stimulated ETKA (142).
b. Leukocyte Transketolase Activity
Erythrocytes have a limited ability to respond to stimuli because they have a long halflife
and lack a nucleus, whereas leukocytes would be expected to respond more rapidly
to changes in nutritional status and perhaps more nearly reflect the status of other cells
in the body. Study in the rats has shown leukocyte transketolase activity to be a reliable
indicator of changes in thiamine status. It is not affected by dietary levels of carbohydrate,
protein, and fat; it responds well to changes in dietary thiamine levels; it reaches a maximum,
which does not increase further when excess thiamine is fed; and there is a parallel
change of transketolase activity in leukocytes and liver (143). However, the use of leukocytes
in assessing thiamine status in humans imposes greater technical difficulties than
the use of erythrocytes.
c. WBTKA and TPPE
Dreyfus (132) has demonstrated that WBTK and TPPE can be employed to assess thiamine
status in humans and to confirm the diagnosis of Wernicke’s disease. The usefulness of
WBTK and TPPE is substantiated by subsequent studies in assessing thiamine status in
pregnant women and their cord blood (76), in infants and children (55), in patients with
thyrotoxicosis (144), and patients with wet beriberi (145). Thus, in certain circumstances,
whenever the separation of erythrocytes is not feasible, a whole-blood specimen can be
used to determine transketolase activity and TPPE.
C. Physical Examination
Though physical signs of beriberi appear in the last stage of thiamine deficiency (Table
2), physicians should be able to detect such abnormalities in patients for immediate and
long-term nutritional management (62). The delay in diagnosing beriberi affects the morbidity
and mortality rates of the patients. Beriberi should be suspected in infants being
breast-fed by thiamine-deficient mothers; such infants would have a loud piercing cry,
dyspnea, cyanosis, cardiac failure, and aphonia (10,97).
Common suggestive signs in dry beriberi include glove-and-stocking hypoesthesia
of pain and touch sensations, loss of ankle and/or knee reflexes, tenderness of calf muscle,
difficulty in rising from squatting position, and aphonia. However, other possible known
causes of peripheral neuropathy must be carefully ruled out (10,46,97,99).
Patients with wet beriberi exhibit both peripheral neuropathy and edema. Severe
cases show tachycardia, wide pulse pressure, cardiac enlargement, and pulmonary congestion
(10,46,97,99).
Wernicke’s disease must be suspected in chronic alcoholics presenting with the
triad of ocular motor signs, ataxia, and derangement of motor functions. The diagnosis
of Wernicke–Korsakoff syndrome should be made in those having both the ocular-ataxic
and amnestic manifestations (109).
As already mentioned in Sec. IX.B, drastic responses to thiamine treatment are observed
in patients with cardiac beriberi or Wernicke’s disease, whereas more time elapses
before improvement is observed in patients with dry beriberi or Korsakoff’s psychosis.
Laboratory tests are required in the latter conditions to confirm the diagnosis.
Thiamine 309
XI. SUBCLINICAL THIAMINE DEFICIENCY
There is concern about the impact of persisting subclinical or borderline thiamine defi-
ciency on the health status of the population. The major hazard for subjects with subclinical
thiamine deficiency is the increased risk of beriberi when faced with extreme physiological
or pathological conditions, e.g., pregnancy, lactation, high physical activity, infection, or
surgery. This is well illustrated in a study in Japanese university students (49). Routine
physical examination in 766 students revealed irritability with mild cardiovascular signs
in 42 students. Of these 42 students, 16 had whole-blood thiamine levels below 50 ng/
mL. A review of 2754 chest x-ray films indicated that 93 students had cardiothoracic
ratios greater than 50%. Of these 93 students, 44 had whole-blood thiamine levels below
50 ng/mL. The analysis of lifestyle in 59 students with low whole-blood thiamine levels
revealed that 50–60% ate their meals in restaurants and 39–47% did not eat breakfast;
86% of students with cardiac enlargement and low blood thiamine levels undertook strenuous
daily exercise; only 20% were aware of their own abnormalities. The improvement
of cardiothoracic ratio was obtained in 63% of 83 subjects by eliminating strenuous exercise
and administering sound nutritional advice.
More studies are needed to verify subclinical thiamine deficiency in persons having
psychological, cardiovascular, and neurological symptoms. The application of dietary assessment
and laboratory tests should be conducted in populations with chronic low intake
of thiamine and appropriate dietary guidelines be implemented to improve their thiamine
status.
XII. REQUIREMENTS AND RECOMMENDED INTAKES
Thiamine is essential in all phases of metabolism. The recommended thiamine intake is
expressed in terms of total caloric intake. The current U.S. recommended thiamine allowances
are 0.5 mg/1000 kcal for children, adolescents, and adults, and 0.4 mg/1000 kcal
for infants. These recommendations are based on the assessment of the effects of varying
levels of dietary thiamine on the occurrence of deficiency signs, on the excretion of thiamine
or its metabolites, and on ETKA. A minimum thiamine intake of 1.0 mg/day is
recommended for adults who consume less than 2000 kcal daily. An additional thiamine
intake of 0.4 mg/day is recommended throughout pregnancy to accommodate maternal
and fetal growth and increased maternal caloric intake. To account for both the thiamine
loss in milk and increased energy consumption during lactation, an increment of 0.5 mg/
day is recommended throughout lactation (64).
XIII. THIAMINE-RESPONSIVE DISEASES
Vitamin-responsive or vitamin-dependent diseases are a group of genetically determined
metabolic disorders in which either a vitamin-dependent enzymatic step or a reaction
involving the conversion of a vitamin to its active cofactor form is defective, causing the
abnormal accumulation of metabolites or substrates in the blood. In these diseases, blood
vitamin levels are normal. The basic metabolic defect involves the structure of the apoenzyme,
its coenzyme binding sites, or some aspect of coenzyme synthesis (146).
Thiamine-responsive diseases include maple syrup urine disease, pyruvate decarboxylase
deficiency, subacute necrotizing encephalopathy (Leigh’s disease), and thiamineresponsive
megaloblastic anemia. Such patients require pharmacological doses of thiamine
to alleviate their clinical manifestations (3,26,146,147).
310 Tanphaichitr
XIV. TOXICITY
Excess thiamine is easily cleared by the kidneys. Although there is some evidence of
toxicity from large doses given parenterally, there is no evidence of thiamine toxicity by
oral administration; oral doses of 500 mg taken daily for a month were found to be nontoxic
(64).
XV. CONCLUSION
Although much has been accomplished in terms of the nutritional, biochemical, physiological,
pharmacological, and molecular aspects of thiamine, humans, especially those on
rice-based diets and chronic alcoholics, are still facing the problem of inadequate thiamine
status, ranging from subclinical thiamine deficiency to beriberi. To combat the problem
of thiamine deficiency, existing nutritional knowledge should be disseminated to motivate
the public to consume appropriate foods in their daily life. To achieve this dietary goal,
both governmental and private agencies must work closely together and recognize the
role of community participation.
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9
Pantothenic Acid
NORA S. PLESOFSKY
University of Minnesota, St. Paul, Minnesota
I. INTRODUCTION
In 1933, R. J. Williams gave the name pantothenic acid to a substance that he proved
necessary for the growth of the yeast Saccharomyces cerevisiae (1). Independently, a
factor was isolated from liver that effectively cured dermatitis in chickens caused by a
deficiency in B vitamins (2). Pantothenic acid was biochemically separated from pyridoxine
(vitamin B6), a copurifying component of the vitamin B2 complex, by its lack of adsorption
to Fuller’s earth (3). Once separated, these two vitamins were shown to reverse different
animal B2 complex deficiency disorders, with pantothenic acid curing chicken
dermatitis and vitamin B6 curing dermatitis in rats.
In 1940, pantothenic acid was successfully synthesized by R. J. Williams and R. T.
Major (4). However, its biologically functional form was not discovered until 1947, when
F. Lipmann and his colleagues (5) demonstrated that pantothenic acid was contained in
coenzyme A (CoA), an essential cofactor for such acetylation reactions as that of sulfonamide
in the liver and choline in the brain. The biochemical structure of CoA was published
in 1953 (6); and the central roles of CoA in the mitochondrial tricarboxylic acid
cycle, fatty acid synthesis and degradation and other metabolic processes were elucidated.
Within the past 15 years, there has been increasing evidence for a novel role of CoA in
donating acetyl and acyl groups to many cellular proteins. This chapter will discuss the
newly discovered role of CoA in the modification of proteins, a role that may ultimately
be linked to specific effects of pantothenate deficiency.
II. CHEMISTRY
Microorganisms synthesize pantothenic acid by joining pantoic acid and ?-alanine in
amide linkage (7). The main route for human biosynthesis of CoA (7) is through phos-
317
318 Plesofsky
phorylation of pantothenic acid to pantothenic acid 4?-phosphate, whose condensation
with cysteine in an ATP-dependent reaction forms 4?-phosphopantothenoylcysteine.
Decarboxylation yields 4?-phosphopantetheine, whose metabolite pantetheine is an essential
growth factor for the yogurt-producing bacterium Lactobacillus bulgaricus. The anhydride
addition of adenosine 5?-monophosphate to 4?-phosphopantetheine, followed by
phosphorylation of the ribose 3?-hydroxyl, produces CoA (Fig. 1). The CoA sulfhydryl
group, which is derived from cysteine, is the active site of esterification to acetate or acyl
groups. As an alternative to being attached to diphosphoadenosine in CoA, 4?-phosphopantetheine
may be covalently linked to a protein (8). Proteins that participate in fatty acid
metabolism, such as the acyl carrier protein of bacteria and mitochondria and the eukary-
Fig. 1 Coenzyme A and intermediates.
Pantothenic Acid 319
otic fatty acid synthetase, are linked to 4?-phosphopantetheine by a phosphodiester bond.
Citrate lyase of anaerobic bacteria and enzymes that are involved in the nonribosomal
synthesis of peptide antibiotics, such as tyrocidin and gramicidin S, are also linked to
phosphopantetheine.
The two types of chemical reactions in which CoA and other pantothenate-containing
molecules participate are acyl group transfer and condensation (9). In acyl group
transfer, there is nucleophilic addition to the carbonyl group thioesterified to CoA, followed
by new ester bond formation and displacement of CoA. Condensation reactions
involve acidification of the ? carbon of the acyl group thioesterified to CoA and attachment
of the ? carbon to an electrophilic center, leading to carbon–carbon bond formation or
cleavage. The first step in the tricarboxylic acid cycle of respiratory metabolism is the
condensation of acetyl-CoA with oxaloacetic acid to yield citric acid. Fatty acid synthesis
involves both types of reactions (10). Malonate is transferred from CoA to the enzymelinked
phosphopantetheine, and acetate is transferred to an enzyme sulfhydryl group. The
introduced acetate or the growing fatty acid chain condenses with the pantetheine-linked
malonate.
III. FOOD SOURCES AND REQUIREMENT
Since pantothenic acid is essential to all forms of life, it is widely distributed in nature
and readily available in food sources. Foods that are particularly rich in pantothenic acid
(11) are liver, kidney, yeast, egg yolk, and broccoli, which contain at least 50 µg of the
vitamin per gram dry weight. Extremely high levels of pantothenate are found in royal
bee jelly (511 µg/g) and ovaries of tuna and cod (2.32 mg/g). The pantothenate content
of human milk increases fivefold within the first 4 days after parturition, from 2.2 to 11.2
µmol/L (48 to 245 µg/dL), a level similar to that found in cow’s milk. Although pantothenic
acid is relatively stable at neutral pH, cooking is reported to destroy 15–50% of
the vitamin in meat, and vegetable processing is associated with pantothenate losses of
37–78% (12). Multivitamin preparations commonly contain the alcohol derivative of pantothenic
acid, panthenol, because it is more stable than pantothenate to which it is converted
by humans (13). Calcium and sodium salts of d-pantothenate are also available as
vitamin supplements.
Formal recommended daily allowances (RDAs) have not been established for pantothenic
acid, but the recommended daily intake is 18–32 µmol (4–7 mg) for adults. Among
younger age groups, 9 µmol (2 mg) pantothenate daily is recommended for infants and
18–23 µmol (4–5 mg) for children 7–10 years of age (14). The pantothenate content of
the average American diet, estimated at 5.8 mg/day (15), is consistent with these recommendations
(12). Even when the intake of pantothenate was less than 4 mg/day, as indicated
by a study of adolescents (16), blood concentrations of the vitamin were in the
normal range (0.91–2.74 µmol/L). The excess appeared to be excreted, since pantothenate
excretion was highly correlated with dietary intake.
IV. ABSORPTION AND METABOLISM
CoA that is ingested from dietary sources is hydrolyzed in the intestinal lumen to pantothenic
acid, which is absorbed into the bloodstream by a sodium-dependent transport
mechanism (17). After being circulated in the plasma, pantothenate is taken up into most
cells by cotransport with sodium ions; sodium cotransport is also responsible for placental
320 Plesofsky
absorption of pantothenate from maternal circulation (18). However, the level of CoA
synthesized in tissues does not reflect the amount of pantothenate available (12). Phosphorylation,
the first step in the conversion of pantothenic acid to CoA, which is catalyzed by
pantothenate kinase, is the primary regulatory site of CoA synthesis in bacteria and rat
hearts (19). Feedback inhibition of pantothenate kinase by the products CoA and acyl-
CoA was found to be relieved by carnitine, the carrier of fatty acids into mitochondria (20).
All of the enzymes required for CoA synthesis are located in the cytoplasm. Nevertheless,
mitochondria must also be a final site for CoA synthesis, since 95% of CoA is found in
mitochondria and CoA itself does not cross the mitochondrial membranes (19). CoA is
ultimately hydrolyzed to pantothenate in multiple steps, with the final, unique step being
the hydrolysis of pantetheine to cysteamine and pantothenate, which is excreted in the
urine (21).
V. BIOCHEMICAL FUNCTIONS IN METABOLISM
AND PROTEIN MODIFICATION
A. Metabolism and Synthesis of Biological Molecules
CoA plays a central role (9) in the energy-yielding oxidation of glycolytic products and
other metabolites through the tricarboxylic acid cycle. As acetyl-CoA condenses with
oxaloacetate, in the first step of the cycle, to yield citrate, and as succinyl-CoA it provides
the energy for substrate level phosphorylation of guanosine diphosphate (GDP). Furthermore,
the synthesis of many essential molecules depends on CoA, as does their degradation.
Fatty acids and membrane phospholipids, including the regulatory sphingolipids,
require CoA for synthesis, and the synthesis of amino acids such as leucine, arginine, and
methionine includes a pantothenate-dependent step. CoA participates, as well, in the ?-
oxidation of fatty acids and the oxidative degradation of amino acids, making the catabolic
products available to the respiratory tricarboxylic acid cycle. Pantothenic acid, incorporated
in CoA, is also required for synthesis of isoprenoid-derived compounds, such as
cholesterol, steroid hormones, dolichol, vitamin A, vitamin D, and heme A. Through succinyl-
CoA, pantothenate is essential to the synthesis of ?-aminolevulinic acid, which is a
precursor of the corrin ring in vitamin B12 and the porphyrin rings in hemoglobin and the
cytochromes. CoA donates an essential acetyl group to the neurotransmitter acetylcholine,
as well as to the sugars N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic
acid, components of glycoproteins and glycolipids.
B. N-Terminal Acetylation of Proteins
The exposed N-terminal amino acid of 50–90% of soluble eukaryotic proteins is modified
with acetate donated by CoA (22). Usually the terminal methionine is cleaved and the
second amino acid, typically alanine or serine, is cotranslationally acetylated on its ?-
amino group. In experiments suggesting that this acetylation may protect proteins from
proteolytic degradation, certain acetylated proteins were resistant to ubiquitin-dependent
proteolysis in vitro, whereas their unacetylated counterparts were degraded (23). More
sophisticated experiments, however, resulted in the proteolysis of acetylated proteins as
well. An additional factor found to be required for degradation of N-terminally acetylated
proteins was the protein synthesis elongation factor EF-1? (24). Despite its uncertain
function, the importance of N-terminal acetylation was demonstrated genetically in Saccharomyces
cerevisiae, where cell cycle progression and sexual development were disPantothenic
Acid 321
rupted in a strain defective in N-terminal acetyltransferase activity (25). The unacetylated
proteins in this mutant strain did not evince destabilization due to lack of acetylation.
Acetylation alters the structure of certain proteins. For example, it increases the N-terminal
?-helical content of calpactin I, a calcium-binding protein, which requires acetylation to
assemble with its regulatory subunit (26).
A unique type of N-terminal acetylation occurs during processing of certain mammalian
peptide hormones from their polyprotein precursors, and this acetylation strongly
affects hormone activity. Both adrenocorticotropic hormone (ACTH), a steroidogenic hormone,
and ?-lipotropin, a lipolytic hormone, are processed from the common precursor
pro-opiomelanocortin. ACTH is processed, in turn, to ?-melanocyte-stimulating hormone
(MSH), and ?-lipotropin is processed to the opioid ?-endorphin (27). Both ?-MSH and
?-endorphin become acetylated, but they differ in terms of the tissues in which they are
acetylated and the effect of acetylation on activity. Both become N-terminally acetylated
in the intermediate pituitary, but only MSH is acetylated in brain, and neither hormone
is acetylated in the anterior pituitary (8). Acetylation stimulates the activity of ?-MSH
(28) but inactivates ?-endorphin by inhibiting its binding to opioid receptors (29). A single
N-acetyltransferase activity, identified in the intermediate but not the anterior lobe of rat
and bovine pituitary, may be responsible for modifying both hormones (30), providing a
mechanism for differentially activating two distinct products of a single precursor.
C. Internal Acetylation of Proteins
1. Histones
The histones and ?-tubulin are two major groups of proteins that undergo selective, reversible
acetylation on the 
-amino group of internal lysine residues. Other DNA-binding
proteins, including the high-mobility group proteins HMG1 and HMG2 and protamines,
are also subject to acetylation. Within the nucleosome are a tetramer of histones H3 and
H4 and two dimers of histones H2A and H2B, around which is wrapped 146 bp of DNA.
These core histones of the octamer become acetylated within their basic amino terminal
regions, with H3 and H4 each having four possible acetylation sites. A study of preferred
acetylation sites in mammalian histones (31) showed that in H3, lysine 14 was acetylated
first, followed by lysine 23 and lysine 18. Monoacetylated H4 was modified exclusively
at lysine 16, with further additions progressing from lysine 12 to lysine 8 and lysine 5.
In histone H2B, lysine 12 and lysine 15 were the preferred acetylation sites over lysine
5 and lysine 20. Histone H2A has only one acetylation site.
Histones that become highly acetylated are associated chiefly with DNA that is
either newly replicated or contains genes that are actively transcribed (8,32). Different
acetylation sites may be utilized in these two processes. In the ciliated protozoan Tetrahymena
thermophila, where these activities are easily distinguished, histone H4 becomes
diacetylated on lysines 4 and 7 in transcriptionally active chromatin, whereas newly assembled
chromatin contains H4 that is diacetylated on lysines 4 and 11 (33). In Physarum
polycephalum, replicating DNA has acetylation in all four core histones, whereas only
histones H3 and H4 have high levels of acetylation in actively transcribed genes (34).
The temporal patterns of acetylation also differ. Newly synthesized histones are only transiently
acetylated as they assemble with replicating chromatin, whereas histones in transcriptionally
active chromatin are continually and dynamically acetylated.
The presence of highly acetylated H3 and H4 noticeably affects nucleosomal structure,
in part through charge neutralization of the modified lysines. The more open, unfolded
322 Plesofsky
configuration of acetylated chromatin is indicated by its increased sensitivity to nucleases
and its increased salt solubility (35). Agarose gel electrophoresis also showed that histone
acetylation leads to decompaction of the chromatin fiber (36). Hyperacetylation of H3 and
H4 has been demonstrated to lead to a reduction in the linking number change per nucleosome,
indicating a decrease in the negative supercoiling within nucleosomes (37). For the
H2A/H2B dimer, acetylation makes its nucleosomal association particularly labile, which
may facilitate its exchange with newly synthesized histones (38).
Acetylated histones are enriched in genes that are being actively transcribed. The
acetylated chromatin fraction from chick embryo erythrocytes, selected by an antibody
against the acetylated lysine of histones, was strongly enriched in actively transcribed ?-
d-globin gene sequences (39). However, histone acetylation does not correspond exactly
to the transcribed sequences, either spatially or temporally. Acetylation extends into the
interspersed, nontranscribed DNA of the ?-globin locus, as well as being present in the
transcribed region (40). Furthermore, acetylation does not respond to the switch in globin
gene transcription that occurs between 5 and 15 days of embryo development, since acetylated
histones were associated with both globin genes at both stages of development
(39). Similarly, acetylated histones were associated with the gene for the platelet-derived
growth factor B chain before its induction by phorbol esters (41). Histone acetylation,
therefore, may be a precondition for transcription, rather than specifying expression of
particular genes. In vitro experiments indicate that histone acetylation is required for specific
proteins to bind to chromatin. For example, acetylation was required for transcription
factor IIIA (TFIIIA) binding to the 5S RNA gene (42). Acetylation of core histones also
indirectly facilitated the binding of transcription factors USF and GAL4-AH to nucleosomes,
by inhibiting the binding of the internucleosomal histone H1, which represses
transcription factor binding (43).
The chromosomal distribution of acetylated histone H4 was observed in the polytene
chromosomes of Drosophila larvae by immunofluorescence, using antibodies developed
against specific, acetylated lysines (44). The single X chromosome in male larvae,
which is very actively transcribed, was the chief location of H4 acetylated at lysine 16,
an acetylated form that was not observed in male autosomes or in any chromosomes
of female larvae. Immunolabeling of mammalian chromosomes showed that acetylated
H4 was concentrated in chromosomal regions enriched in coding DNA, but acetylated
H4 was largely absent from the inactive X chromosome of female mammalian cells; the
few immunolabeled bands corresponded mainly to regions containing known expressed
genes (45).
That acetylation of histone H4 affects the transcription of certain genes was demonstrated
in S. cerevisiae, where mutations were made in H4 that either delete the N-terminal
tail or substitute its lysine residues. Deletion of the basic tail or an uncharged substitution
of lysine 16 led to derepression of the silent mating locus, suggesting that the chargeneutralizing
acetylation of lysine 16 may be responsible for activating transcription (46).
In contrast, transcription of the GAL1 and PHO5 genes was inhibited by similar mutations
of H4, in which multiple lysines were substituted (47). However, a neutralizing mutation
was still much less inhibitory to transcription than an arginine substitution. Mutations of
the N-terminal sequences of the other three core histones had no or little effect on transcription
of these genes or the mating type locus.
Unlike gene transcription, the replication of DNA does not require histone acetylation
for polymerase enzymes to gain access to the nucleosomal DNA. However, newly
synthesized histones that assemble with the replicated DNA become transiently acetylated
Pantothenic Acid 323
on their N-terminal tails, a modification that is detected in the presence of the deacetylase
inhibitor butyrate. Histones are deposited onto DNA in a two-step process that begins
with the deposition of H3/H4 tetramers, followed by H2A/H2B dimers. Diacetylation of
H4 precedes its deposition onto DNA and its assembly with newly synthesized H3, H2A,
and H2B (48). Histone H2B is also highly acetylated in newly assembled nucleosomes
(49). In the absence of DNA synthesis, newly synthesized H2A and H2B exchange preferentially
into acetylated chromatin regions, assembling with acetylated H3 and H4, but
newly synthesized H3 and H4 remain cytosolic (48). Deposition-related histone acetylation
inhibits the binding of histone H1 to newly assembled chromatin, thereby reducing
higher order nucleosome interactions (50).
Other chromatin-associated molecules that undergo acetylation include HMG1 and
HMG2, which are acetylated by the same N-acetyltransferase as the histones (51). It is
apparently the acetylated form of HMG1 that serves as a histone assembly factor and
stimulates DNA polymerase activity (52). The same acetyltransferase also acetylates polyamines,
which interact with chromatin during DNA synthesis (51).
2. ?-Tubulin
A subunit of microtubules is the other class of proteins that becomes acetylated on the
?-amino group of lysine residues. Microtubules are essential structural components of
eukaryotic cells that are central to chromosome segregation in nuclei and to the cytoplasmic
cytoskeleton, where they affect cell shape, motility, and organelle movement.
They also constitute the major component of flagella and cilia. Microtubles are assembled
from ?-tubulin/?-tubulin dimers that polymerize and depolymerize dynamically. A subset
of microtubules has been found to contain ?-tubulin that is acetylated on lysine 40 (53).
Acetylation occurs on the ?-tubulin after it is incorporated into the microtubule, which
is a better substrate than the tubulin dimer for the isolated acetylase activity (54), and
deacetylation appears to be coupled to microtubule depolymerization. The acetylated microtubules
are more stable to depolymerizing agents, such as colchicine, than the unacetylated
microtubules; in turn, drugs that stabilize microtubules, such as taxol, induce ?-
tubulin acetylation (55). Tubulin turnover in neurons was found to be much slower in
areas where acetylated ?-tubulin was concentrated, such as the neurite shafts, than in the
cell body and growth cones (56). These observations suggest that acetylation may help
to stabilize microtubules.
The distribution of acetylated ?-tubulin in various types of cells has been detected
by an antibody specific for the modified tubulin and has been found to be nonrandom. In
cerebellar neurons, which have abundant microtubules, acetylated ?-tubulin was found to
be concentrated in axons, relative to dendrites, during early neuronal development (57),
although in mature neurons it was present also in thick dendritic trunks. It was excluded
from growth cones at neurite tips in dorsal root ganglion neurons, and acetylated ?-tubulin
was also absent from leading edge microtubules of migrating 3T3 cells. Acetylated microtubules
underlie motor endplates in chick muscle fibers and they contribute to vesicle and
organelle transport, whereas growing or motile regions of the cells appear to be depleted
of acetylated microtubules (58). Individual axonal microtubules actually contain separate
regions of acetylated and unacetylated ?-tubulin along their length, with the older regions
corresponding to the acetylated domains (59). It was suggested that the acetylation of ?-
tubulin may be a step in cell commitment. When fibroblast cells were induced to become
neuron-like, they formed an acetylated microtubule bundle that subsequently extended
into a neurite (60).
324 Plesofsky
Cells undergoing mitosis and meiosis also show nonrandom, varying distribution
of acetylated microtubules. During arrest in meiotic metaphase, acetylated ?-tubulin was
found predominantly at the poles of unfertilized mouse oocytes (61). At meiotic anaphase,
the spindle became labeled, but by telophase only the meiotic midbody microtubules were
acetylated. Changes in the distribution of acetylated microtubules were also described for
the preimplantation development of mouse embryo cells (62). Acetylated microtubules
were generally associated with the cell cortex before differentiation. As cellular asymmetry
developed, the acetylated microtubule subpopulation relocated to the basal part of the cell
cortex and, after asymmetrical cell division, became concentrated in the inside cells rather
than the outside cells.
D. Acylation of Proteins
During the past 15 years it has been discovered that a wide variety of cellular proteins
are modified with long-chain fatty acids, donated by CoA (8). This type of covalent modi-
fication affects the location and activity of many proteins (Table 1), including those that
Table 1 Proteinsa That Are Modified with Fatty Acids
Palmitate 
Palmitate Myristate myristate
Rasv, Rasc Transducin Go, Gi protein,
Gs protein ? subunit ARF proteins ? subunit
Receptors: Srcv, Srcc p56lck, p59fyn
Rhodopsin Protein kinase A Insulin receptor
Dopamine (D1, D2) Calcineurin ecNOS
?2A-, ?2-Adrenergic Recoverin
Thyrotropin-releasing hormone MARCKS
Choriogonadotropin Band 4.2
Iron-transferrin IgM heavy chain
Nicotinic acetylcholine Interleukin-1?, 1?
CD4, CD44, CD36, P-selectin TNF-?
HLA-B heavy chain NADH-cytochrome b5 reductase
Band 3 NADH dehydrogenase, B18
Ankyrin cytochrome c oxidase, su 1
Spectrin
Vinculin
Fibronectin
Actin
Gap junction proteins
Myelin proteolipid subunit
Cysteine string proteins
Acetylcholinesterase
Glutamic acid decarboxylase65
GAP-43, SNAP-25
Methylmalonate semialdehyde
dehydrogenase
Aldehyde and glutamate dehydrogenases
a Discussed in text.
Pantothenic Acid 325
have a central role in signal transduction. The two fatty acids that are usually added to
proteins are myristic acid, a rare 14-carbon saturated fatty acid, and the more common
16-carbon palmitic acid. These two fatty acids are attached to proteins by distinct mechanisms,
and they affect proteins differently. The myristoylation that has been best characterized
occurs cotranslationally, by amide linkage to the ?-amino group of an N-terminal
glycine, and it is irreversible. Palmitate, in contrast, is added posttranslationally and forms
a reversible ester bond with a cysteine or serine. The greater hydrophobicity of palmitate
results in strong membrane association of the acylated protein, whereas myristoylation
leads either to weak membrane association or interactions with proteins. The reversibility
of palmitoylation allows this modification to be regulatory.
There are stringent sequence requirements for a protein to become N-terminally
myristoylated (63). An N-terminal glycine, originally in the second position, is absolutely
required, and there is preference for a small neutral subterminal residue and a serine in
the sixth position. The myristoyltransferase that has been isolated shows a specific preference
for myristoyl-CoA over other acyl-CoA donors (63). The N-terminal myristoylation
was shown to be essential for viability in S. cerevisiae, where gene disruption of N-myristoyltransferase
proved to be recessively lethal (64). In contrast, enzymes that transfer palmitate
show little substrate specificity, either for the fatty acyl group transferred from
CoA or the peptide sequence required for protein acylation.
1. GTP-Binding Proteins
The small GTP-binding proteins comprise an extensive group of influential cellular
proteins that become acylated with myristic acid and/or palmitic acid. This group of
nucleotide-binding proteins includes the monomeric Ras superfamily of proteins, heterotrimeric
G proteins, and ADP-ribosylating factors (ARFs). Several of these proteins are also
modified by isoprenoid groups whose synthesis requires CoA, although it is not the lipid
donor.
Covalent modification of the Ras proteins, both cellular and viral, has been extensively
studied. Most Ras proteins undergo two types of modifications at their carboxyl
end, isoprenylation and methylation on the processed terminal cysteine and palmitoylation
of a nearby cysteine; in some proteins a basic region functionally replaces palmitoylation
(65). The viral Ras proteins, such as those of Harvey murine and Kirsten sarcoma viruses,
are deficient in GTP hydrolysis and are oncogenic. Both isoprenylation and palmitoylation
are required for viral Ras proteins to bind to the plasma membrane and transform cells,
with palmitoylation strengthening the weak activity conferred by isoprenylation (65). A
15-carbon farnesyl group modifies Ras proteins, and farnesyltransferase inhibitors are effective
antitumor agents in Ras-transformed cells. However, the Ras-related proteins and
most cellular proteins are isoprenylated with the 20-carbon geranylgeranyl (66). The cellular
Ras protein is a critical component of the mitogen-activated protein (MAP) signaling
kinase cascade, serving to direct the protein kinase Raf to the plasma membrane. When
Raf itself was experimentally engineered to contain a farnesylation signal, Ras proved to
be unnecessary for signal transduction (67).
The mammalian Rab proteins are Ras-related proteins that are proposed to regulate
specific stages of vesicular transport to the cell surface (68). These proteins appear to target
vesicles to specific acceptor membranes by binding to the vesicles until their delivery to
membranes. The cycling of Rab proteins between membranes and cytosol is related to
the membrane budding and fusion that drives vesicular movement. Rab proteins are modi-
fied at their C termini with geranylgeranyl, which is required for their association with
326 Plesofsky
organellar membranes, as well as for interaction of a Rab protein with its GDP dissociation
inhibitor (GDI), which releases the Rab protein from membranes (69). A defect in a protein
with sequence homology to GDIs has been implicated in retinal degradation or choroideremia
in humans (66).
The ? subunits of several heterotrimeric G proteins, which mediate transmembrane
signaling, are esterified to palmitate near their N termini on a third-position cysteine. The
Gs? subunit, which mediates hormonal stimulation of cAMP synthesis, requires palmitoylation
both for binding to membranes and for stimulation of adenylyl cyclase (70). Hormonal
activation leads to GTP binding by ?s and to its rapid depalmitoylation, which is
accompanied by relocalization of ?s to the cytosol. In addition to palmitate, myristic acid
is added to the N terminus of those G-protein ? subunits that are members of the Gi and
Go subfamilies. Prior myristoylation increases these proteins’ membrane affinity, which
is required for the addition of palmitate (71). Both types of acylation were found to contribute
to the membrane affinity of the ?o subunit and its binding to the ?? subunits. Mutation
of the palmitoylated cysteine of Go1? disrupted its exclusive localization to membranes,
even in the presence of N-myristoylation, whereas mutation of the myristoylated glycine,
preventing both fatty acylations, caused ?o to become chiefly cytosolic (72). It has been
suggested that dual acylation of these G-protein ? subunits and certain Src-related proteins
(discussed below) directs their localization to plasma membrane caveolae, possibly via
association with glycosylphosphatidylinositol (GPI)-anchored membrane proteins (72). In
addition to myristate, heterogeneous fatty acids, such as laurate and unsaturated 14-carbon
fatty acids, modify the N terminus of the ? subunit of transducin, the photoreceptor G
protein that is stimulated by photolyzed rhodopsin. The nature of the modifying fatty acid
appears to affect the strength of the interaction of transducin ? with ?? and may influence
the speed of visual excitation (73). Since the ? subunits of G proteins are isoprenylated
at their C termini (66), increasing their membrane affinity, these heterotrimeric G proteins,
which play a central role in cell signaling, undergo all three types of modifications with
hydrophobic moieties.
The small GTP-binding proteins that are involved in vesicular transport, known as
ARFs, are N-terminally myristoylated, and this modification is essential for their functions
in transport. In mammalian cells the ARF protein is the principal coat component of non–
clathrin-coated vesicles, and its binding to the Golgi membrane appears to mark the site
of budding of a future vesicle. Whereas recombinant myristoylated ARF5 bound to Golgi
membranes in a GTP-dependent and temperature-dependent manner, the nonmyristoylated
ARF5 did not bind to membranes at all (74). The N-terminal region of ARF1 may be
stabilized by myristoylation in an amphipathic ? helix, conducive to membrane binding.
Myristoylation also apparently stabilizes the interaction of ARF1 with GTP in the presence
of membranes and enables ARF1 to release GDP at high physiological concentrations of
Mg2 (75).
In addition to the myristoylation of ARFs, reconstitution of in vitro transport through
the Golgi stacks requires palmitoyl-CoA (76). The addition of acyl-CoA was necessary
for the budding of vesicles from donor Golgi and for the fusion of these vesicles with
acceptor Golgi cisternae. A nonhydrolyzable analog of palmitoyl-CoA or an inhibitor of
the acyl-CoA synthetase inhibited vesicle budding (76). The source of this acyl group
requirement is not known.
2. Protein Kinases and Phosphatases
Like palmitoylation of Ras proteins, myristoylation of the Src protein provided the first
model of this type of acylation. Src, first identified in Rous sarcoma virus, is a tyrosine
Pantothenic Acid 327
kinase that has both cellular and viral homologues. The viral Src protein is responsible
for cell transformation by the oncogenic viruses, and its N-terminal myristoylation is necessary
both for the protein’s association with the plasma membrane and for its transforming
capability. Expression of a mutant, nonmyristoylated pp60src protein resulted in a
small group of membrane-associated proteins showing decreased tyrosine phosphorylation
(77). pp60src was also prevented from associating with membranes by addition of a less
hydrophobic analog of myristic acid, which is a possible candidate for an antitumor agent
(78). Mutation of the cellular Src protein at its N-terminal glycine to prevent myristoylation
proved inhibitory to its activity in mitosis, presumably because c-Src is activated by a
membrane-bound phosphatase (79).
Although pp60src itself is not additionally acylated with palmitate, several Src-related
tyrosine kinases, such as p56lck and p59fyn, are dually acylated at their N termini (80).
These kinases associate with proteins that are anchored in the outer plasma membrane by
glycosylphosphatidylinositol modification, and dual acylation of the kinases is necessary
for this association. When the palmitoylated cysteines at positions 3 and 6 in p59fyn and
at positions 3 and 5 in p56lck were mutated to serines, these two kinases no longer associated
with the GPI-anchored protein DAF (decay-accelerating factor). On the other hand,
when the reverse mutations were made in pp60src, from serines at positions 3 and 6 to
cysteines, the Src protein uncharacteristically associated with DAF (81). GPI-anchored
proteins, present in the membranes of T cells, B cells, monocytes, and granulocytes, are
thought to transduce external activation signals through their interaction with tyrosine
kinases in the inner membrane layer.
The catalytic subunit of cAMP-dependent protein kinase is also myristoylated at its
N terminus. Myristoylation does not appear to affect its association with membranes, since
the catalytic subunit is anchored in the membrane by the regulatory subunit until dissociation
by cAMP. A comparison of the properties of the myristoylated vs. nonmyristoylated
catalytic subunit indicated that acylation increased the thermal structural stability of the
kinase, possibly by stabilizing an intramolecular interaction between the N-terminal domain
and an internal hydrophobic surface (82). N-terminal myristoylation also occurs in
the B subunit of calcineurin (83), a calmodulin-dependent phosphatase that is the target
of the immunosuppressant cyclosporin A.
3. Membrane Receptors
Many membrane-anchored receptors are acylated with palmitic acid. Heterotrimeric Gprotein–
coupled receptors all have a similar domain structure, consisting of an extracellular
N terminus, seven transmembrane ? helices, and an intracellular C terminus, leading
to three intracellular and three extracellular loops. Near the seventh transmembrane region,
the C-terminal tail has a conserved cysteine whose palmitoylation has been characterized
in rhodopsin, the dopamine (D1 and D2) receptors, the ?2-adrenergic receptor, the ?2Aadrenergic
receptor, thyrotropin-releasing hormone receptor, and the choriogonadotropin
receptor. This acylation appears to lead to the formation of a fourth intracellular loop.
Fluorescence quenching studies with rhodopsin, the retinol-binding photoreceptor of retinal
rod cells, showed that two cysteines at positions 322 and 323, modified by palmitic
acid, are integrated into the cell membrane (84). Furthermore, the light excitation of rhodopsin
is regulated by recoverin, an N-myristoylated protein that responds to changing
levels of calcium. Calcium binding alters the conformation of recoverin to expose the Nmyristoyl
group, which can then bind to membranes (85).
The regulatory functions of receptor palmitoylation have been studied in the dopamine
and adrenergic receptors. The human receptor for dopamine (D1) incorporates palmi328
Plesofsky
tate shortly after cells are exposed to hormone, concurrent with cellular desensitization
to dopamine (86). The receptor also becomes phosphorylated and dissociates from the
cell surface. By mutating the palmitoylated cysteine of the ?2-adrenergic receptor, it was
determined that acylation is important both for receptor coupling with Gs (the stimulatory
G protein) and for agonist-promoted desensitization (87). However, the corresponding
mutation in the ?2A-adrenergic receptor had a different effect. Mutation of the cysteine
abolished the downregulation of receptor number that occurs after prolonged exposure to
hormone (88). In contrast, mutational analysis showed that palmitoylation of thyrotropinreleasing
hormone and choriogonadotropin receptors slowed their internalization after prolonged
agonist exposure (89).
Other transmembrane receptors that are palmitoylated include the iron-transferrin
receptor, the insulin receptor, and the nicotinic acetylcholine receptor. In addition to palmitoylation,
the insulin receptor is also reported to be linked to myristic acid (90). Rat adipocytes
contain a membrane glycoprotein, related to CD36, that becomes palmitoylated
on an extracellular domain in response to insulin and energy depletion (91). Several cells
of blood and the immune system, such as T lymphocytes, macrophages, and platelets,
contain receptors that become palmitoylated. CD4, a surface glycoprotein of T lymphocytes
and macrophages that functions in cell adhesion and signaling during antigen recognition,
also serves as receptor for the human immunodeficiency virus (HIV). CD4 becomes
palmitoylated on two cysteines at the junction of its transmembrane and cytoplasmic domains,
but this modification does not apparently affect either membrane localization of
the receptor or its interactions with proteins such as p56lck (92). However, palmitoylation
of a lymphoma glycoprotein, closely related to CD44 of T lymphocytes, was found to be
required for its in vitro binding to ankyrin, the protein that mediates attachment of the
cell membrane to the cytoskeleton (93). The heavy chain of the human histocompatibility
antigen HLA-B is thioesterified to palmitate. An adhesion receptor in human platelets, Pselectin
(CD62), is palmitoylated on a cysteine in its cytoplasmic tail, a domain that is
responsible for the sorting of P-selectin into secretory granules and its endocytosis into
cells (94).
4. Proteins Associated with the Cytoskeleton
An N-myristoylated protein, rich in alanine, that is a major substrate of protein kinase C
(MARCKS) has been identified as a protein that cross-links actin filaments. MARCKS
is thought to be involved in the cytoskeletal rearrangements that occur during neuronal
transmitter release, leukocyte activation, and growth factor–induced mitosis. The initial
binding of MARCKS to the plasma membrane requires myristoylation, which provides
one of its two membrane binding sites. Phosphorylation within the second membrane
binding site occurs upon cell activation and results in dissociation of MARCKS from the
membrane (95).
Membrane proteins in erythrocytes are linked to the cytoskeleton by a series of
acylated proteins. The anion transport protein Band 3, which is palmitoylated (96), is
linked to Band 4.2, which is a major myristoylated protein of the membrane (97). Band
4.2, in turn, is linked to ankyrin, the cytoskeleton attachment protein, which is itself palmitoylated
(8). In addition, the cytoskeletal protein spectrin becomes palmitoylated in its ?
subunit, and this acylated subpopulation is more tightly membrane-associated than the
unmodified spectrin (98). Other cytoskeleton-associated proteins whose palmitoylation has
been reported are vinculin, fibronectin, and a subpopulation of actin. Gap junction proteins
of heart and eye lens also become palmitoylated (99).
Pantothenic Acid 329
5. Neuronal Proteins
One of the first proteins found to be covalently modified by palmitate was the proteolipid
subunit of brain myelin, in which palmitate is esterified to a threonine residue (8). Cysteine
string proteins (csp) are located in synaptic terminals and are required for neurotransmitter
release. Fatty acylation of multiple cysteines, 10 in Torpedo, appears to be a mechanism
for attachment of Tcsp to vesicles, where they interact with presynaptic calcium channels
in the plasma membrane (100). Palmitoylation of acetylcholinesterase, which degrades
the neurotransmitter acetylcholine, results in its anchorage in the cell membrane. Palmitate
attachment to an esterase subpopulation correlated with its attachment to the outer cell
surface, whereas most of the expressed esterase was unmodified and secreted from cultured
cells (101). A major isoform of glutamic acid decarboxylase (GAD65) is located in synaptic
vesicles of GABA-secreting neurons and in microvesicles of pancreatic ? cells. Palmitoylation
of GAD65 in its N-terminal domain is reported to increase its membrane affinity,
possibly regulating its membrane anchorage (102).
Protein acylation appears to play an important role during neuronal development.
The reversible palmitoylation of both GAP-43 and SNAP-25, in developing brains, was
proposed to influence growth cone motility and process outgrowth (103). GAP-43 is a
major component of the growth cone membranes of elongating axons, and SNAP-25 is
a synaptic protein involved in later stages of axon growth. Whereas the nonacylated form
of GAP-43 stimulates the G protein, Go, palmitoylation at two cysteines near its N terminus
inhibits this activity and keeps GAP-43 membrane-bound (104). Nitric oxide inhibits the
acylation of GAP-43 and SNAP-25, along with that of other neuronal proteins (103), and
it inhibits the growth of cultured neurites by causing growth cone collapse. It was suggested
that these effects of nitric oxide reflects its role in regulating process outgrowth
and remodeling in vivo (103). The endothelial form of nitric oxide synthase (ecNOS),
which is involved in smooth-muscle relaxation, is itself regulated by dual acylation (105).
N-terminal myristoylation is required for the membrane localization of ecNOS and for its
reversible modification with palmitate. Depalmitoylation follows addition of the agonist
bradykinin and results in ecNOS relocalizing to the cytosol and becoming phosphorylated.
6. Myristoylated Proteins of the Immune System
Several proteins of the immune system are modified with myristate on internal lysine
residues. These include the heavy chain of µ immunoglobulins, which becomes acylated
during transport to the surface of developing B cells (106). Precursors of the cytokines
interleukin (IL)–1? and 1? are myristoylated on lysines (107), as is the tumor necrosis
factor (TNF) ? precursor (108). After processing, the mature forms of IL-1? and IL-1? are
secreted and bind to receptors. The precursor of IL-1?, however, which is the predominant
myristoylated species, also exists as a plasma membrane–associated protein (107). The
acylated lysines in the propiece of the TNF-? precursor are near a membrane-spanning
segment and are thought to facilitate membrane insertion of the precursor, which, like the
mature TNF-?, is active in mediating inflammation (108).
7. Mitochondrial Proteins
Mitochondrial proteins involved in oxidation–reduction reactions have been reported to
become fatty acylated. The NADH–cytochrome b5 reductase, located at the outer mitochondrial
membranes of animal cells, as well as at microsomes, is N-terminally myristoylated
(109). In the inner membrane, there is N-myristoylation of subunit B18 of complex
330 Plesofsky
I of the electron transport chain, the NADH:ubiquinone oxidoreductase, which was detected
by cDNA sequencing and electrospray mass spectrometry (110). Complex I also
contains the pantothenate-containing mitochondrial acyl carrier protein, identified as an
enzyme subunit by its stoichiometry (111). The core catalytic subunit 1 of cytochrome c
oxidase, the terminal electron carrier of the respiratory chain, is modified by myristic acid
on an internal lysine residue (112). The oxidase subunit 1 is one of only a few proteins
that are encoded by mitochondrial genes and synthesized in mitochondria; its modification
suggests that there is a mitochondrial N-myristoylating activity.
Dehydrogenases involved in oxidative catabolism also become acylated, a modification
that inhibits their enzymatic activity and may be a mechanism for feedback regulation
(113). A decrease in mitochondrial energy level was found to correlate with increased
acylation of an active-site cysteine in methylmalonate semialdehyde dehydrogenase
(MMSDH). It was proposed that long-chain fatty acids accumulate when MMSDH and
other dehydrogenases utilize the NAD required to oxidize fatty acids. The accumulation
of these fatty acids, in turn, promotes acylation of the dehydrogenases, whose inhibition
allows ?-oxidation of fatty acids to proceed. Aldehyde dehydrogenase and glutamate dehydrogenase
are two other mitochondrial NAD-dependent dehydrogenases that become
acylated (113).
VI. PHARMACOLOGY
The human effects of pantothenate deficiency have been detected chiefly by the administration,
either intentional or unintentional, of pantothenic acid antagonists. Hopantenate is
an analog of pantothenic acid, which contains GABA in place of ?-alanine. Also an agonist
of GABA, hopantenate was used in Japan as a cerebral stimulant for retarded individuals
and to alleviate symptoms of tardive dyskinesia induced by tranquilizers. It was found,
however, that treatment with hopantenate produced severe side effects in patients, including
lactic acidosis, hypoglycemia, and hyperammonemia (114). It ultimately led to acute
encephalopathy with hepatic steatosis resembling Reye’s syndrome. A study in which
dogs were administered hopantenate over several weeks showed that they suffered similar
effects (115). This study also characterized the abnormalities that developed in hepatic
mitochondria, which became enlarged, had increased numbers of cristae, and contained
crystalloid inclusions. These mitochondrial alterations differed from those associated with
Reye’s syndrome. The side effects of hopantenate treatment are apparently caused by
induction of pantothenic acid deficiency. Dogs that were given an equivalent amount of
pantothenic acid at the same time as calcium hopantenate did not develop these disorders
(115).
When individuals who suffer seizures are treated with the anticonvulsant drug
valproic acid, liver damage may follow in association with decreased levels of CoA
and acetyl-CoA. These effects appear to be due to sequestration of CoA in esters of valproic
acid and its metabolites (20). Coadministration of pantothenic acid, carnitine, and
acetylcysteine, along with valproate or its derivative, relieved these metabolic defects in
developing mice (20). Skeletal muscles of the murine model of Duchenne’s muscular
dystrophy show reduced energy metabolism, as indicated by reduced heat production,
relative to control muscles. The addition of pantothenate resulted in increased cytoplasmic
synthesis of CoA and increased the thermogenic response to glucose in the diseased muscles
(116). Pantothenic acid also protected rats against liver damage and peroxidation
produced by carbon tetrachloride (117). Furthermore, surgical wound healing was greatly
Pantothenic Acid 331
improved by the administration of pantothenic acid, apparently due to its anti-inflammatory
properties. In vitro studies show that calcium pantothenate dampens the responses
of neutrophils (polymorphonuclear leukocytes) to activation by stimulatory peptides and
cytokines (118).
Analogs of pantothenic acid have been used experimentally to induce pantothenate
deficiency. The antagonist ?-methylpantothenate, in combination with a pantothenatedeficient
diet, produced deficiency symptoms in humans that included headache, fatigue,
insomnia, intestinal disturbances, and paresthesia of hands and feet. A decrease in the
eosinopenic response to ACTH, loss of antibody production, and increased sensitivity to
insulin were also reported (119). Naturally occurring pantothenate deficiency has been
detected only under conditions of severe malnutrition. World War II prisoners in the Philippines,
Japan, and Burma suffered from a disease in which they experienced numbness
in their toes and painful burning sensations in their feet. Pantothenic acid was specifically
required to relieve these symptoms of nutritional melalgia (120).
Many studies have documented the physical effects on animals of a pantothenaterestricted
diet (11). Rats develop hypertrophy of the adrenal cortex, which is followed by
hemorrhage and necrosis; their resistance to certain viral infections is increased. Pantothenate
deficiency in dogs produces hypoglycemia, gastrointestinal symptoms, rapid respiration
and heart beat, and convulsions. Monkeys show depressed heme synthesis and become
anemic. During exercise, deficient mice display lower stamina, and their liver and muscle
glycogen levels are reduced (12). Chickens develop dermatitis and poor feathering, as
well as axon and myelin degeneration within the spinal cord. Dermatitis and graying in
mice, induced by pantothenate deficiency, were reversed by administration of pantothenic
acid, but this treatment did not prove successful at restoring hair color in humans. Calcium
pantothenate is not toxic to rats, dogs, rabbits, or humans at high doses, but the lethal
dose for mice, leading to respiratory failure, was determined to be 42 mmol (10 g)/kg
(11).
VII. CONCLUSIONS
Pantothenic acid, through incorporation into phosphopantetheine and CoA, is essential to
many metabolic conversions and to all forms of life. It is widely available in dietary
sources, and human deficiency in pantothenic acid is rare. Nevertheless, the administration
of pantothenate is helpful in counteracting the inhibitory effects certain drugs and diseases
have on respiratory metabolism and other activities. It has long been known that pantothenate
plays a central role in energy generation and molecular syntheses. It is now clear,
in addition, that pantothenic acid participates in regulating numerous proteins by donating
acetyl- and fatty acyl-modifying groups, which alter the location and/or activity of the
acylated protein. Proteins that are acetylated constitute major structural components of
chromatin and microtubules. Myristoylated and palmitoylated proteins are central components
of signal transduction systems, vesicular transport, and the cytoskeleton, and they
are seemingly common to all eukaryotic cells.
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10
Vitamin B6
JAMES E. LEKLEM
Oregon State University, Corvallis, Oregon
I. INTRODUCTION AND HISTORY
Vitamin B6 is unique among the water-soluble vitamins with respect to the numerous
functions it serves and its metabolism and chemistry. Within the past few years the attention
this vitamin has received has increased dramatically (1–8). Lay publications (9) attest
to the interest in vitamin B6.
This chapter will provide an overview of vitamin B6 as it relates to human nutrition.
Both qualitative and quantitative information will be provided in an attempt to indicate
the importance of this vitamin within the context of health and disease in humans. As a
nutritionist, my perspective no doubt is biased by these nutritional elements of this vitamin.
The exhaustive literature on the intriguing chemistry of the vitamin will not be dealt with
in any detail, except as related to the function of vitamin B6 as a coenzyme. To the extent
that literature is available, reference will be made to research in humans, with animal or
other experimental work included as necessary.
As we leave the twentieth century behind, there may be a tendency to lose the sense
of excitement of discovery that Gyorgy and colleagues experienced when they began to
unravel the mystery of vitamin B complex. Some of the major highlights of the early
years of vitamin B6 research are presented in Table 1. Paul Gyorgy was first to use the
term vitamin B6 (10). The term was used to distinguish this factor from other hypothetical
growth factors B3, B4, B5 (and Y). Some 4 years later (1938), in what is a fine example
of cooperation and friendship, Gyorgy (11) and Lepkovsky (12) reported the isolation of
pure crystalline vitamin B6. Three other groups also reported the isolation of vitamin B6
that same year (13–15). Shortly after this, Harris and Folkers (16) as well as Kuhn et al.
(17) determined that vitamin B6 was a pyridine derivative and structurally identified it as
3-hydroxy-4,5-hydroxymethyl-2-methylpyridine. The term pyridoxine was first intro-
339
340 Leklem
Table 1 Historical Highlights of Vitamin B6 Research
1932 A compound with the formula of C3H11O3N was isolated from rice polishings.
1934 Gyorgy shows there was a difference between the rat pellagra preventive factor and
vitamin B2. He called this vitamin B6.
1938 Lepkovsy reports isolation of pure crystalline vitamin B6. Keresztesky and Stevens,
Gyorgy, Kuhn and Wendt, and Ichibad and Michi also report isolation of vitamin B6.
1939 Chemical structure determined and vitamin B6 synthesized by Kuhn and associates and
by Harris and Folkers.
1942 Snell and co-workers recognize existence of other forms of pyridoxine.
1953 Snyderman and associates observe convulsions in an infant and anemia in an older child
fed a vitamin B6 deficient diet.
duced by Gyorgy in 1939 (18). An important aspect of this early research was the use of
animal models in identification of vitamin B6 (as pyridoxine in various extracts from rice
bran and yeast). This early research into vitamin B6 then provided the ground work for
research into the requirement for vitamin B6 for humans and the functions of this vitamin.
Identification of the other major forms of the vitamin B6 group, pyridoxamine and
pyridoxal, occurred primarily through the use of microorganisms (19,20). In the process
of developing an assay for pyridoxine, Snell and co-workers observed that natural materials
were more active in supporting the growth of certain microorganisms than predicted
by their pyridoxine content as assayed with yeast (20). Subsequently, this group observed
enhanced growth-promoting activity in the urine of vitamin B6 deficient animals fed pyridoxine
(20). Treatment of pyridoxine with ammonia also produced a substance with
growth activity (21). These findings subsequently led to the synthesis of pyridoxal and
pyridoxamine (22,23). The availability of these three forms of vitamin B6 reduced further
research into this intriguing vitamin possible.
II. CHEMISTRY
Since Gyorgy first coined the term vitamin B6 (10), there has been confusion in the terminology
of the multiple forms of the vitamin. ‘‘Vitamin B’’ is the recommended term for
the generic descriptor for all 3-hydroxy-2-methylpyridine derivatives (24). Figure 1 depicts
the various forms of vitamin B6, including the phosphorylated forms. Pyridoxine
(once referred to as pyridoxal) is the alcohol form and should not be used as a generic
Fig. 1 Structure of B6 vitamers.
Vitamin B6 341
Table
2
Physical
Properties
of
B
6
Vitamers
a
Percent
stability
compared
to
solution
in
dark
(24).
8
h,
15
h


length
of
time
exposed
to
light.
bFrom
Storvick
et
al.
(25);
pH
7.0.
c
pH
3.4,
0.01
N
acetic
acid.
dpH
10.5,
0.1
N
NH4
OH,
lactone
of
4-PA.
e
Data
are
for
PN-HCL,
PL-HCL,
PM-2HCl,
PLP
monohydrate,
PMP
dihydrate
(26).
342 Leklem
name for vitamin B6. The trivial names and abbreviations commonly used for the three
principal forms of vitamin B6, their phosphoric esters, and analogs are as follows: pyridoxine,
PN; pyridoxal, PL; pyridoxamine, PM; pyridoxine-5?-phosphate, PNP; pyridoxal-5?-
phosphate, PLP; pyridoxamine-5?-phosphate, PMP; 4-pyridoxic acid, 4-PA. As will be
discussed later, other forms of vitamin B6 exist, particularly bound forms.
The various physical and chemical properties of the phosphorylated and nonphosphorylated
forms of vitamin B6 are given in Table 2. Detailed data on fluorescence (28)
and ultraviolet (27) absorption characteristics of B6 vitamers are available. Of importance
to researchers as well as to food producers and consumers is the relative stability of the
forms of vitamin B6. Generally, as a group B6 vitamers are labile, but the degree to which
each is degraded varies. In solution the forms are light-sensitive (25,29), but this sensitivity
is influenced by pH. Pyridoxine, pyridoxal, and pyridoxamine are relatively heat-stable
in an acid medium, but they are heat-labile in an alkaline medium. The hydrochloride and
base forms are readily soluble in water, but they are minimally soluble in organic solvents.
The coenzyme form of vitamin B6, PLP, is found covalently bound to enzymes via
a Schiff base with an ?-amino group of lysine in the enzyme. While nonenzymatic reactions
with PLP or PL and metal ions can occur (30), in enzymatic reactions the amino
group of the substrate for the given enzyme forms a Schiff base via a transimination
reaction. Figure 2 depicts the formation of a Schiff base with PLP and an amino acid.
Because of the strong electron-attracting character of the pyridine ring, electrons are withdrawn
from one of the three substituents (R group, hydrogen, or carboxyl group) attached
to the ? carbon of the substrate attached to PLP. This results in the formation of a quinonoid
structure. There are several structural features of PLP that make it well suited to
form a Schiff base and thus act as a catalyst in a variety of enzyme reactions. These
features have been detailed by Leussing (31) and include the 2-methyl group, which brings
the pKa of the proton of the ring pyridine closer to the biological range; the phenoxide
oxygen (position 3), which aids in expulsion of a nucleophile at the 4-position; the 5-
phosphate group, which functions as an anchor for the coenzyme and prevents hemiacetal
formation and the drain of electrons from the ring; and the protonated pyridine nitrogen
that is para to the aldehyde group aids in delocalizing the negative charge and helps regulate
the pKa of the 3-hydroxyl group. A recent publication has extensively reviewed the
chemistry of pyridoxal-5? phosphate (4).
PLP has been reported to be a coenzyme for over 100 enzymatic reactions (32). Of
these, nearly half involve transamination-type reactions. Transamination reactions are but
Fig. 2 Schiff base formation between pyridoxal-5?-phosphate and an amino acid.
Vitamin B6 343
Table 3 Enzyme Reactions Catalyzed by Pyridoxal-5?-Phosphate
Type of reaction Typical reaction or enzyme
Reactions involving ? carbon
Transamination Alamine > pyruvate  PMP
Racemization d-Amino acid - l-amino acid
Decarboxylation 5-OH tryptophan > T-OH tryptamine  CO2
Oxidative deamination Histamine > imidazole-4-acetaldehyde  NH
Loss of the side chain THF  serine > glycine  N5,10-methylene THF
Reactions involving ? carbon
Replacement (exchange) Cystein synthetase
Elimination Serine and threonine dehydratase
Reaction involving ? carbon
Replacement (exchange) Cystathionine > cysteine  homoserine
Elimination Homocysteine desulfhydrase
Cleavage Kynurenine > anthranilic acid
one type of reaction that occur as a result of Schiff base formation. The three types of
enzyme reactions catalyzed by PLP are listed in Table 3 and are classified according to
reactions occurring at the ?, ?, or ? carbon.
III. METHODS
The measurement of B6 vitamers and metabolites is important in evaluating vitamin B6
metabolism and status. Methods used in measuring B6 vitamers in foods are complicated
not only by the numerous forms but by the various matrices. Reviews of the methods
currently used are available (33–35). HPLC techniques are more common today than
other methods, such as microbiological (26,36) and enzymatic techniques. B6 vitamers in
biological fluids can be determined by a variety of HPLC techniques (35,37). These methods
involve nonexchange or paired-ion reversed-phase procedures. Determination of the
active coenzyme form, PLP, in plasma and tissue extracts is conveniently done by a radioenzymatic
technique (38). The advantage of this type of procedure is that it allows for
analyses of a large number of samples in one assay.
Determination of vitamin B6 in foods and biological samples can be done microbiologically
(36). Yeast growth assays using Saccharomyces uvarum (ATCC 9080) are most
commonly used. While it has been reported that the three forms respond differently to
yeast (35), in my laboratory we do not observe this if the yeast grows rapidly. In all of
the methods mentioned above, adequate extraction of the forms of vitamin B6 is critical.
TCA and perchloric acid are effective extractants.
Methods for the determination of the glycosylated form of vitamin B6 PN-glucoside
(PNG), in foods are available (35,39). Both microbiological-based (40) and HPLC (35)
procedures have been utilized. All procedures for B6 vitamers should be conducted under
yellow lights to minimize photodegredation.
IV. OCCURRENCE IN FOODS
To appreciate the role of vitamin B6 in human nutrition, one must first have knowledge
of the various forms and quantities found in foods. A microbiological method for determining
the vitamin B6 content of foodstuffs was developed by Atkin in 1943 (32). While this
344 Leklem
Table 4 Vitamin B6 Content of Selected Foods and Percentages of the Three Forms
Vitamin B6
a Pyridoxineb Pyridoxalb Pyridoxamineb
Food (mg/100 g) (%) (%) (%)
Vegetables
Beans lima, frozen 0.150 45 30 25
Cabbage, raw 0.160 61 31 8
Carrots, raw 0.150 75 19 6
Peas, green, raw 0.160 47 47 6
Potatoes, raw 0.250 68 18 14
Tomatoes, raw 0.100 38 29 33
Spinach, raw 0.280 36 49 15
Broccoli, raw 0.195 29 65 6
Cauliflower, raw 0.210 16 79 5
Corn, sweet 0.161 6 68 26
Fruits
Apples, Red Delicious 0.030 61 31 8
Apricots, raw 0.070 58 20 22
Apricots, dried 0.169 81 11 8
Avocados, raw 0.420 56 29 15
Bananas, raw 0.510 61 10 29
Oranges, raw 0.060 59 26 15
Peaches, canned 0.019 61 30 9
Raisins, seedless 0.240 83 11 6
Grapefruit, raw 0.034 — — —
Legumes
Beans, white, raw 0.560 62 20 18
Beans, lima, canned 0.090 75 15 10
Lentils 0.600 69 13 18
Peanut butter 0.330 74 9 17
Peas, green, raw 0.160 69 17 14
Soybeans, dry, raw 0.810 44 44 12
Nuts
Almonds, without skins, shelled 0.100 52 28 20
Pecans 0.183 71 12 17
Filberts 0.545 29 68 3
Walnuts 0.730 31 65 4
Cereals/grains
Barley, pearled 0.224 52 42 6
Rice, brown 0.550 78 12 10
Rice, white, regular 0.170 64 19 17
Rye flour, light 0.090 64 14
Wheat, cereal, flakes 0.292 79 11 10
Wheat flour, whole 0.340 71 16 13
Wheat flour, all-purpose white 0.060 55 24 21
Oatmeal, dry 0.140 12 49 39
Cornmeal, white and yellow 0.250 11 51 38
Bread, white 0.040 — — —
Bread, whole wheat 0.180 — — —
Vitamin B6 345
Table 4 Continued
Vitamin B6 Pyridoxine Pyridoxal Pyridoxamine
Food (mg/100 g) (%) (%) (%)
Meat/poultry/fish
Beef, raw 0.330 16 53 31
Chicken breast 0.683 7 74 19
Pork, ham, canned 0.320 8 8 84
Flounder fillet 0.170 7 71 22
Salmon, canned 0.300 2 9 89
Sardine, Pacific canned, oil 0.280 13 58 29
Tuna, canned 0.425 19 69 12
Halibut 0.430 — — —
Milk/eggs/cheese
Milk, cow, homogenized 0.040 3 76 21
Milk, human 0.010 0 50 50
Cheddar 0.080 4 8 88
Egg, whole 0.110 0 85 15
aValues from Ref. 43, Table 1.
bValues from Ref. 43, Table 2.
method has been refined (26,42,35), it still stands as the primary method for determining
the total vitamin B6 content of foods and has been the basis for most of the data available
on the vitamin B6 content of foods. There are various forms of vitamin B6 in foods. In
general, these forms are a derivative of the three forms: pyridoxal, pyridoxine, and pyridoxamine.
Pyridoxine and pyridoxamine (or their respective phosphorylated forms) are
the predominant forms in plant foods. Although there are exceptions, pyridoxal, as the
phosphorylated form, is the predominant form in foods. Table 4 contains data for the
vitamin B6 content of a representative sample of food commonly consumed in the United
States. Data on the amount of each of the three forms are also listed (43). While the
phosphorylated forms are usually the predominant forms in most foods, the microbiological
methods used to determine the level of each form measure the sum of the phosphorylated
and free (nonconjugated) forms.
In addition to the phosphorylated forms, other conjugated forms have been detected
in certain foods. A glycosylated form of pyridoxine has been identified in rice bran (44)
and subsequently quantitated in several foods (40). The glycosylated form isolated from
rice bran has been identified as 5?-O-?-d-pyridoxine (44) (Fig. 3). Suzuki et al. have shown
that the 5?-glucoside can be formed in germinating seeds of wheat, barley, and rice cultured
on a pyridoxine solution (45). In addition, a small amount of 4?-glucoside was also detected
Fig. 3 Structure of 5?-O-(?-d-glucopyranosyl)pyridoxine.
346 Leklem
Table 5 Vitamin B6 and Glycosylated Vitamin B6 Content of
Selected Foods
Glycosylated
Vitamin B6 vitamin B6
Food (mg/100 g) (mg/100g)
Vegetables
Carrots, canned 0.064 0.055
Carrots, raw 0.170 0.087
Cauliflower, frozen 0.084 0.069
Broccoli, frozen 0.119 0.078
Spinach, frozen 0.208 0.104
Cabbage, raw 0.140 0.065
Sprouts, alfalfa 0.250 0.105
Potatoes, cooked 0.394 0.165
Potatoes, dried 0.884 0.286
Beets, canned 0.018 0.005
Yams, canned 0.067 0.007
Beans/legumes
Soybeans, cooked 0.627 0.357
Beans, navy, cooked 0.381 0.159
Beans, lima, frozen 0.106 0.039
Peas, frozen 0.122 0.018
Peanut butter 0.302 0.054
Beans, garbanzo 0.653 0.111
Lentils 0.289 0.134
Animal products
Beef, ground, cooked 0.263 n.d.
Tuna, canned 0.316 n.d.
Chicken breast, raw 0.700 n.d.
Milk skim 0.005 n.d.
Nuts/seeds
Walnuts 0.535 0.038
Filberts 0.587 0.026
Cashews, raw 0.351 0.046
Sunflower seeds 0.997 0.355
Almonds 0.086 -0-
Fruits
Orange juice, frozen concentrate 0.165 0.078
Orange juice, fresh 0.043 0.016
Tomato juice, canned 0.097 0.045
Blueberries, frozen 0.046 0.019
Banana 0.313 0.010
Banana, dried chips 0.271 0.024
Pineapple, canned 0.079 0.017
Peaches, canned 0.009 0.002
Apricots, dried 0.206 0.036
Avocado 0.443 0.015
Raisins, seedless 0.230 0.154
Vitamin B6 347
Table 5 Continued
Glycosylated
Vitamin B6 vitamin B6
Food (mg/100 g) (mg/100g)
Cereals/grains
Wheat bran 0.903 0.326
Shredded wheat cereal 0.313 0.087
Rice, brown 0.237 0.055
Rice, bran 3.515 0.153
Rice, white 0.076 0.015
Rice cereal, puffed 0.098 0.007
Rice cereal, fortified 3.635 0.382
n.d., none detected.
Sources: Data taken from Ref. 40 and Leklem and Hardin, unpublished.
in wheat and rice germinated seeds, but not in soybean seeds. Also of interest is an asyet-
unidentified conjugate of vitamin B6 reported by Tadera and co-workers (46). This
conjugate released free vitamin B6 (measured as pyridoxine) only when the food was
treated with alkali and then ?-glucosidase. Tadera et al. have also identified another derivative
of the 5?-glucoside of pyridoxine in seedlings of podded peas (47). This derivative
was identified as 5?-O(6-O-malonyl-?-d-glucopyranosyl)pyridoxine. The role of these
conjugates in plants is unknown. Table 5 lists the total vitamin B6 content of pyridoxine
5?-glucoside content of various foods. There is no generalization that can be made at this
time as to a given class of foods having high or low amounts of pyridoxine-5?-glucoside.
The effect of the 5?-glucoside of vitamin B6 nutrition will be addressed in the section on
bioavailability and absorption.
Food processing and storage may influence the vitamin B6 content of food (48–57)
and result in production of compounds normally not present. Losses of 10–50% have
been reported for a wide variety of foods. Heat sterilization of commercial milk was found
to result in conversion of pyridoxal to pyridoxamine (49). Storage of heat-treated milk
decreases the vitamin B6 content presumably due to formation of bis-4-pyridoxyldisulfide.
The effect of various processes on the vitamin B6 content of milk and milk products has
been reviewed (57). Losses range from 0 to 70%. Vanderslice et al. have reported an
HPLC method for assessing the various forms of vitamin B6 in milk (58), which aids in
understanding the effects of processing on the vitamin B6 content of milk and milk products.
DeRitter (59) has reviewed the stability of several vitamins in processed foods, including
vitamin B6, and found that the vitamin B6 added to flour and baked into bread is
stable. This has been confirmed by Perera et al. (60).
Gregory and Kirk have found that during thermal processing (61) and low-moisture
conditions of food storage (54), there is reductive binding of pyridoxal and pyridoxal 5?-
phosphate to the ?-amino groups of protein or peptide lysyl residues. These compounds
are resistant to hydrolysis and also possess low vitamin B6 activity. Interestingly, Gregory
(62) has shown that ?-pyridoxyllysine bound to dietary protein has anti–vitamin B6 activity
(50% molar vitamin B6 activity for rats).
348 Leklem
V. ABSORPTION AND BIOAVAILABILITY
The questions of how much vitamin B6 is biologically available (i.e., absorbed and utilizable)
and what factors influence this are important in terms of estimating a dietary requirement.
Before considering the factors that influence bioavailability, a brief description of
absorption of the forms of vitamin B is appropriate.
Absorption of the various forms of vitamin B6 has been studied most extensively
in animals, particularly rats. However, gastrointestinal absorption of pyridoxine has been
examined with guinea pig jejunum preparations (63), intestine, cecum, and crop of the
chicken (64), and intestine of the hamster (65).
In the rat, Middleton (65–67) and Henderson and co-workers (68) have conducted
extensive research on intestinal absorption of B6 vitamers. The evidence to date indicates
that pyridoxine and the other two major forms of vitamin B6 are absorbed by a nonsaturable,
passive process (68). Absorption of the phosphorylated forms can occur (69,70), but
to a very limited extent. The phosphorylated forms disappear from the intestine via hydrolysis
by alkaline phosphatase (67,70), and a significant part of this takes place intraluminally.
Prior intake of vitamin B6 in rats over a wide range (0.75–100 mg PN-HCl per kg
diet) was found to have no affect on in vitro absorption of varying levels of PN-HCl
(71). This study provides further support for passive absorption of B6 vitamers. However,
Middelton has questioned the concept of a nonsaturable process (72). Using an in vivo
perfused intestinal segment model, he found there was a gradient of decreasing rates of
uptake from the proximal to the distal end of the intestine and that there was a saturable
component of uptake, especially in the duodenum.
The various forms of vitamin B6 that are absorbed into the rat intestinal cell (intracellular)
can be converted to other forms (i.e., PL to PLP, PN to PLP, and PM to PLP), but
that which is ultimately transported to other organs via the circulation system primarily
reflects the nonphosphorylated form originally absorbed (69,70). A similar pattern of uptake
and metabolism has been observed in mice (73); however, in mice given PN, pyridoxal
was the major form detected in the circulation. Portal blood was not examined. The
liver was likely the primary organ that further metabolized the PN absorbed and released
PL to the circulation.
Bioavailability of a nutrient from a given food is important to an organism in that
it is the amount of a nutrient that is both absorbed and available to cells. The word available
is key here in that the vitamin may not be needed by the cell and simply excreted
or metabolized to a nonutilizable form, such as 4-pyridoxic acid in the case of vitamin B6.
Methods used to evaluate the bioavailability of nutrients such as vitamin B6 include
balance studies in which input and output are determined. Included in these studies is the
use of stable isotopic techniques (74). A second approach is to measure an in vivo response,
such as growth, after a state of deficiency has been created. The third type of
study is the examination of blood levels of the nutrient or a metabolite of the nutrient
over a specified period of time after a food is fed. The concentration of a metabolite, such
as PLP, is then compared with concentrations after ingestion of graded amounts of the
crystalline form of vitamin B6. Gregory and Ink (74) and Leklem (75) have reviewed
vitamin B6 bioavailability.
One of the early studies that suggested a reduced availability of vitamin B6 involved
feeding canned combat rations that had been stored at elevated temperatures (75). Feeding
diets containing 1.9 mg of total vitamin B6 resulted in a marginal deficiency based on
urinary excretion of tryptophan metabolites. Some 18 years later, Nelson et al. observed
that the vitamin B6 in orange juice was incompletely absorbed by humans (77). These
Vitamin B6 349
authors suggested that a low molecular weight form of vitamin B6 was present in orange
juice and responsible for the reduced availability. Kabir and co-workers (40) subsequently
found that approximately 50% of the vitamin B6 present in orange juice is the pyridoxine-
5?-O-glucoside.
Leklem et al. conducted one of the first human studies that directly determined
bioavailability of vitamin B6 (78). In their study, nine men were fed either whole wheat
bread, white bread enriched with pyridoxine (0.8 mg), or white bread plus a solution
containing 0.8 mg of pyridoxine. After feeding each bread for a week, urinary vitamin
B6 and 4-pyridoxic and fecal vitamin B6 excretion were measured to assess vitamin B6
bioavailability. Urinary 4-pyridoxic acid excretion was reduced when whole wheat bread
was fed compared to the other two test situations, and the vitamin B6 from this bread was
estimated to be 5–10% less available than the vitamin B6 from the other two breads. While
this relatively small difference in bioavailability may not be nutritionally significant by
itself, in combination with other foods of low vitamin B6 bioavailability, vitamin B6 status
may be compromised.
In other studies in humans, feeding 15 g of cooked wheat bran slightly reduced
vitamin B6 bioavailability (79). Using urinary vitamin B6 as the sole criterion, Kies and
co-workers estimated that 20 g of wheat, rice, or corn bran reduced vitamin B6 availability
35–40% (80). Since various brans are good sources of vitamin B6, it is not possible to
determine if the vitamin B6 in the bran itself was unavailable or if the bran may have
been binding vitamin B6 present in the remainder of the diet.
The bioavailability of vitamin B6 from specific foods or groups of foods has been
examined utilizing balance and blood levels (dose response) studies. Tarr et al. estimated
a 71–79% bioavailability of vitamin B6 from foods representing the ‘‘average’’ American
diet (81). Using a triple-lumen tube perfusion technique, Nelson et al. found that the
vitamin B6 from orange juice was only 50% as well absorbed as crystalline pyridoxine
(82). In our laboratory, Kabir et al. compared the vitamin B6 bioavailability from tuna,
whole wheat bread, and peanut butter (83). Compared to the vitamin B6 in tuna, the vitamin
B6 in whole wheat bread and peanut butter was 75% and 63% as available, respectively.
The level of glycosylated vitamin B6 in these foods was inversely correlated with vitamin
B, bioavailability as based on urinary vitamin B, and 4-pyridoxic acid(84). We have observed
an inverse relationship between vitamin B6 bioavailability as based on urinary 4-
pyridoxic acid excretion and the glycosylated vitamin B6 content of six foods (85). These
foods and their respective availabilities were as follows: walnuts (78%), bananas (79%),
tomato juice (25%), spinach (22%), orange juice (9%), and carrots (0%). While the glycosylated
vitamin B6 content of foods appears to be a significant contributor to bioavailability,
the presence of other forms of vitamin B6 and/or binding of specific forms of vitamin
B6 to other components in a food may also contribute to availability. The question of the
extent to which vitamin B6 bioavailability affects vitamin B6 status (and thus requirement)
has been studied in women (86). When diets containing 9% of the vitamin B6 as PNG
were compared with diets containing 27% PNG it was observed that vitamin B6 status
was decreased. The decreased bioavailability was consistent with that observed in humans
by Gregory et al. (87) who estimated that the bioavailability of PNG may be as low as
58% of the bioavailability of free pyridoxine.
VI. INTERORGAN METABOLISM
Extensive work by Lumeng and Li and co-workers in rats (88) and dogs (89) has shown
that the liver is the primary organ responsible for metabolism of vitamin B6 and supplies
350 Leklem
the active form of vitamin B6, PLP, to the circulation and other tissues. The primary
interconversion of the B6 vitamers is depicted in Fig. 4. The three nonphosphorylated
forms are converted to their respective phosphorylated forms by a kinase enzyme (pyridoxine
kinase EC 2.7.1.35). Both ATP and zinc are involved in this conversion, with ATP
serving as a source of the phosphate group. The two phosphorylated forms, pyridoxamine-
5?-phosphate and pyridoxine-5?-phosphate, are converted to PLP via a flavin mononucleoticle
(FMN)–requiring oxidase (90). A review of the interrelation between riboflavin and
vitamin B6 is available (91).
Dephosphorylation of the 5?-phosphate compounds occurs by action of a phosphatase.
This phosphatase is considered to be alkaline phosphatase (92) and is thought to be
enzyme-bound in the liver (93). PL arising from dephosphorylation or that taken up from
the circulation can be converted to 4-pyridoxic acid by either an NAD-dependent dehydrogenase
or an FAD-dependent aldehyde oxidase. As discussed below, in humans only aldehyde
oxidase (pyridoxal oxidase) activity has been detected in the liver (94). The conversion
of pyridoxal to 4-pyridoxic acid is an irreversible reaction. Thus, 4-pyridoxic acid
is an end product of vitamin B6 metabolism. A majority of ingested vitamin B6 is converted
to 4-pyridoxic acid (95–97).
The interconversion of vitamin B6 vitamers in human liver has been extensively
studied by Merrill et al. (94,98,99). Although only five subjects were examined, this study
(94) provides the first detailed work in humans on the activities of enzymes involved in
Fig. 4 Metabolic interconversions of the B6 vitamers.
Vitamin B6 351
vitamin B6 metabolism. The activities of pyridoxal kinase, pyridoxine (pyridoxamine)-5?-
phosphate oxidase, PLP phosphatase, and pyridoxal oxidase are summarized in Table 6.
These activities are optimal ones and, as Merrill et al. have pointed out, at the physiological
pH of 7.0 pyridoxal phosphatase activity was less than 1% of the optimal activity at pH
9.0. Considering this, the kinase reaction would be favored and, hence, formation of PLP.
The kinase enzyme is a zinc-requiring enzyme. The limiting enzyme in the vitamin B6
pathway appears to be pyridoxine-5?-phosphate oxidase. Since this enzyme requires FMN
(108), a reduced riboflavin status may affect the conversion of PN and PM to PLP. Lakshmi
and Bamji have reported that whole-blood PLP in persons with oral lesions (presumably
riboflavin deficiency) were normal, and supplemental riboflavin had no significant
effect on these levels (100). Madigan et al. found that riboflavin supplementation (25 mg/
day) of elderly people improved plasma PLP concentration (101). In studies of red cell
metabolism of vitamin B6, Anderson and co-workers have shown that riboflavin increases
the conversion rate of pyridoxine to PLP (12). In addition, PLP feeds back and inhibits
the oxidase. This may be a mechanism by which cells limit the concentration of the highly
reactive PLP.
Another riboflavin-dependent (FAD) enzyme, aldehyde oxidase (pyridoxal oxidase),
was suggested by Merill et al. (94) to the enzyme that converts pyridoxal to 4-pyridoxic
acid. The activity of the aldehyde (pyridoxal) oxidase in humans appears to be sufficient,
so that PL which arises from hydrolysis of PLP or that which is taken up into liver would
be readily converted to 4-pyridoxic acid. Such a mechanism may prevent large amounts
of the highly reactive PLP from accumulating.
The PLP that is formed in liver (and other tissues) can bind via a Schiff base reaction
with proteins. The binding of PLP to proteins may be the predominant factor influencing
tissue levels of PLP (93). This binding of PLP to proteins is thought to result in metabolic
trapping of PLP (vitamin B6 in cells (88,93). PLP synthesized in liver cells is released
and found bound to albumin. Whether the PLP is bound to albumin prior to release from
the liver or released unbound and subsequently binding to albumin has not been determined.
The binding of PLP to albumin in the circulation serves to protect it from hydrolysis
and allows for the delivery of PLP to other tissues (104). This delivery process of PLP
to other tissues is thought to involve hydrolysis of PLP and subsequent uptake of PL into
the cell (92). Hydrolysis occurs by action of phosphatases bound to cellular membranes.
Other forms of vitamin B6 are present in the circulation (plasma). Under fasting conditions,
the two aldehyde forms compose 70–90% of the total B6 vitamers in plasma, with PLP
making up 50–75% of the total (Table 7). The next most abundant forms are PN, PMP,
and PM. Interestingly, pyridoxine-5?-phosphate is essentially absent in plasma.
Table 6 Activity of Human Liver Enzymes
Involved in Vitamin B6 Metabolism
Enzyme (activity) Per gram liver
Pyridoxal kinase (nmol/min) 11.2
Pyridoxine-5?-P-oxidase (nmol/min) 2.4
Pyridoxal-5?-P-phosphatase (nmol/min) 0.1–2.1
Pyridoxal oxidase (nmol/min) 16.5
Source: Data taken from Refs. 94 and 99.
352 Leklem
Table 7 B6 Vitamers in Plasma (nmol/L)
PLP PL PNP PN PMP PM
Coburn (n 
 38)a 57  26 23  10 0 19  33 8 8 2 2
Lumeng (n 
 6)b 62  11 13  4 n.d. 32 7 33 6 1
Hollins (n 
 10)c 61  34 5  9 n.d. n.d. n.d. n.d.
aFrom Ref. 103.
bFrom Ref. 104.
cFrom Ref. 105.
n.d., none detected.
All data obtained by HPLC methods.
Within the circulating fluid (primarily blood), the erythrocyte also appears to play
an important role in the metabolism and transport of vitamin B6. However, the extent of
these roles remains controversial (5,106). Both PN and PL are rapidly taken up by a simple
diffusion process (107). In the erythrocytes of humans, PL and PN are converted to PLP
because both kinase and oxidase activity are present (107). The PLP formed can then be
converted to PL by the action of phosphatase; however, this may not be quantitatively
important because the phosphatase is considered to be membrane-bound. Any role that
the erythrocyte might play in transport of vitamin B6 is complicated by the tight binding
of both PLP and PL to hemoglobin (108,109). PL does not bind as tightly as PLP, and
each is bound at distinct sites (110). In comparison to the binding to albumin, PL is bound
more tightly to hemoglobin (5). As a result, the PL concentration in the erythrocyte is up
to four to five times greater than that in plasma (111).
The PLP and PL in plasma, as well as perhaps the PL in erythrocytes, represent the
major B6 vitamers available to tissues. To a limited extent, PN would be available following
a meal if the uptake was high enough and if the PN escaped metabolism in the liver.
Another situation in which PN would be available is following ingestion of vitamin B6
supplements (primarily as PN-HCl). While PN can be converted to PNP in most tissues,
conversion to PLP does not take place in many tissues because the oxidase enzyme is
absent (112). Human muscle contain PMP oxidase activity (113), but the activity is lower
than that of the liver (99). A majority of the vitamin B6 in muscle is present as PLP bound
to glycogen phosphorylase (114). Coburn et al. calculated that approximately 66% and
69% of the total vitamin B6 in muscle is present as PLP in males and females, respectively
(115). Furthermore, they estimated that the total vitamin B6 pool in muscle was 850 and
900 µmol in males and females, respectively. This pool plus the pool of vitamin in other
tissues and circulation would total about 1 nmol (1000 µmol). Previous estimates of totalbody
pools have been made based on metabolism of a radioactive dose of pyridoxine
(116,117). These pools ranged from 100 to 700 µmol.
The precise turnover time of these pools in humans in not known. However, Shane
has estimated that there are two pools: one with a rapid turnover of about 0.5 day and a
second with a slower turnover of 25–33 days (117). Johansson et al. have also shown
that the change in blood levels following administration of tritium-labeled pyridoxine was
consistent with a two-compartment model (116). Johansson et al. further suggested that
the slow turnover compartment was a storage compartment, but they did not determine
the nature of this storage compartment. Figure 5 shows a semilog plot of the decrease in
plasma PLP concentration with time in 10 control females and 11 oral contraceptive users
fed a diet low in vitamin B6 (0.19 mg, 1.1 µmol) for 4 weeks (95). There was an initial
Vitamin B6 353
Fig. 5 Semilog plot of plasma pyridoxal-5?-phosphate concentration over 4 weeks of feeding a
vitamin B6-deficient diet and 4 weeks of repletion with pyridoxine in control subjects and oral
contraceptive users. (From Ref. 86.).
rapid decline in plasma PLP concentration followed by a slower decrease. Extrapolation
of the slope for the slowly decreasing portion of the curve for each of the two groups and
determination of the plasma t1/2 PLP revealed a value of 28 days for the control females
and 46 days for the oral contraceptive users. The value for controls is consistent with the
data of Shane (117). The longer t1/2 for oral contraceptive users may reflect higher levels
of enzymes with PLP bound to them (118). Coburn (119) has discussed the turnover and
location of vitamin B6 pools, based in part on modeling calculations.
Muscle has been suggested as a possible storage site for vitamin B6. This is based
in part on the B6 content of muscle and the total muscle mass of animals. As previously
mentioned, in muscle a majority of vitamin B6 is present as PLP bound to glycogen phosphorylase
(114,115). In contrast, glycogen phosphorylase accounts for only about 10%
of the vitamin B6 content of liver (120). Black and co-workers examined the storage of
vitamin B6 in muscle by studying the activity of muscle glycogen phosphorylase (121).
In their studies, Black et al. found that feeding rats a diet high in vitamin B6 (70 g of
vitamin B6 per kilogram of diet) resulted in a high vitamin B6 content and a high glycogen
phosphorylase content in muscle. This increase in content and enzyme level occurred
in concert for 6 weeks, whereas the level of alanine and aspartic aminotransferase increased
for the first 2 weeks and then plateaued. In subsequent work (122), these same
researchers found that muscle phosphorylase content (and thus vitamin B6 content) decreased
only when there was a caloric deficit and not necessarily with a deficiency of
vitamin B6. This observation of muscle not acting as a mobile reservoir during a vitamin
B6 deficiency was also observed in adult swine (123). Coburn et al. (113) observed that
human muscle vitamin B6 pools are resistant to depletion. In their study there was a nonsignificant
increase in muscle vitamin B6 content when subjects were supplemented with
354 Leklem
0.98 µmol pyridoxine HCl per day as compared to the muscle vitamin B6 content during
depletion.
In humans, indirect evidence for muscle serving as a vitamin reservoir has come
from my laboratory (124). We have observed an increase in plasma PLP concentration
during and immediately after exercise (125,126). Strenuous exercise results in a metabolic
state of acute caloric deficit and increased need for gluconeogenesis. Thus, the increased
circulating levels of PLP following exercise may reflect PLP released from muscle glycogen
phosphorylase. Such a mechanism for this release would mean either that (a) PLP
must cross the muscle cell membrane or (b) PLP is hydrolyzed, PL released, and the PL
rapidly converted to PLP in liver. Because phosphorylated compounds are thought not to
cross membranes easily, the direct release of PLP is considered unlikely by some. Others
(127) suggest that this increase in plasma PLP is a release of PLP from liver or interstitial
fluid. However, PLP formed in liver is released, and studies of uptake of phosphorylated
B6 vitamers have examined only uptake and not possible transport out of the cell. Further
work in my laboratory has shown that in rats starved for 1–3 days there is an increased
plasma PLP concentration and an increased PLP concentration in liver, spleen, and heart
tissue (unpublished observations). Thus, both direct studies in animals and indirect evidence
in humans suggest that vitamin B6 is stored in muscle and released in times of
decreased caloric intake and/or increased need for gluconeogenesis.
VII. ASSESSMENT OF STATUS
The assessment of vitamin B6 status is central to an understanding of vitamin B6 nutrition
in humans. A variety of methods have been utilized to assess vitamin B6 status. These
methods are given in Table 8 and are divided into direct, indirect, and dietary methods
(128–130). Direct indices of vitamin B6 status are those in which one or more of the B6
vitamers or the metabolite 4-pyridoxic acid are measured. These are usually measured in
plasma, erythrocytes, or urine samples because tissue samples are not normally available.
Indirect measures are those in which metabolites of metabolic pathways in which PLP is
required for specific enzymes are measured, or in which activities of PLP-dependent enzymes
are determined. In this latter case, an activity coefficient is often determined by
measuring the enzyme activity in the presence and absence of excess PLP.
Dietary intake of vitamin B6 itself is not sufficient to assess vitamin B6 status, especially
if only a few days of dietary intake are obtained. In addition to the inherent problems
in obtaining accurate dietary intakes, the nutrient databases used in determining vitamin
B6 content of diets are often incomplete with respect to values for vitamin B6. Thus, reports
of vitamin B6 status based only on nutrient intake must be viewed with caution. Some of
the suggested values for the evaluation of status given in Table 8 are based on the relationship
of vitamin B6 and tryptophan metabolism (95). Plasma pyridoxal-5?-phosphate concentration
is considered one of the better indicators of vitamin B6 status (131). Lumeng
et al. (104) have shown that plasma PLP concentration is a good indicator of tissue PLP
levels in rats. In humans, plasma PLP concentration is significantly correlated with dietary
vitamin B6 intake (97). Table 9 contains mean plasma PLP values reported by several
laboratories for males and females. These are selected references drawn from reports in
which the sex of the subjects was clearly identified. The means reported range from 27
to 75 mnol/L for males and 26 to 93 µmol/L for females. These ranges should not necessarily
be considered as normal since the values given in Table 9 reflect studies in which
dietary intake was controlled and other studies in which dietary intake was not assessed.
Vitamin B6 355
Table 8 Methods for Assessing Vitamin B6 Status and
Suggested Values for Adequate Status
Suggested value for
Index adequate status
Direct
Blood
Plasma pyridoxal-5?-phosphatea 30 nmol/La
Plasma pyridoxal NV
Plasma total vitamin B6 40 nmol/L
Erythrocyte pyridoxal-5?-phosphate NV
Urine
4-Pyridoxic acid 3.0 µmol/day
Total vitamin B6 0.5 µmol/day
Indirect
Blood
Erythrocyte alanine aminotransferase 1.25b
Erythrocyte aspartate aminotransferase 1.80b
Urine
2 g Tryptophan load test; xanthurenic acid 65 µmol/day
3 g Methionine load test; cystathionine 351 µmol/day
Oxalate excretion NV
Dietary intake
Vitamin B6 intake, weekly average 1.2–1.5 mg/day
Vitamin B6: protein ratio 0.02
Pyridoxine-?-glucoside NV
Other
EEG pattern NV
aReference values in this table are dependent on sex, age, and protein intake
and represent lower limits (130).
bFor each aminotransferase measure, the activity coefficient represents the ratio
of the activity with added PLP to the activity without PLP added.
NV, no value established; limited data available, each laboratory should establish
its own reference with an appropriate healthy control population.
As discussed by Shultz and Leklem (97), dietary intake of both vitamin B6 and protein
influences the fasting plasma PLP concentration. Miller et al. (136) have shown that
plasma PLP and total vitamin B6 concentrations in males were inversely related to protein
intake (see Table 9) in males whose protein intake ranged from 0.5 to 2 g/kg per day.
Similar results from metabolic studies in women support these findings in men (151).
Other factors that may influence plasma PLP and should be considered when using
this index as a measure of vitamin B6 status include the physiological variables of age
(133,147,153), exercise (124), and pregnancy (143). Rose et al. determined the plasma
PLP concentration in men ranging in age from 18 to 90 years (133). They observed a
decrease in plasma PLP with age, especially after 40 years of age. However, one must
keep in mind that the PLP concentration was determined 1–2 hours after a meal. The
intake of vitamin B6 may have influenced the data. Also, the carbohydrate intake could
have resulted in a depressed plasma PLP concentration (124). Hamfelt has reviewed the
effect of age on plasma PLP and observed that investigators in several countries (153)
have seen decreased vitamin B6 status with increasing age. The mechanism of this decrease
356 Leklem
Table 9 Selected Mean Plasma Pyridoxal-5?-Phosphate Concentrations Reported for Healthy
Males and Females
No. Age
Ref. subjects (years) Dieta PLP (nmol/L) Methodb
Males
Wachstein (132) 27 —c — 35.2  9.3 TDC
Chabner (38) 17 20–34 SS, F 74.8  22.2 TDC
7 35–49 SS, F 63.9  13.3
Rose, 1976 (133) 26 18–29 SS, NF 59.1  28.9 TDC
43 30–39 SS, NF 59.9  29.2
82 40–49 SS, NF 53.4  22.0
152 50–59 SS, NF 46.9  21.7
59 60–69 SS, NF 49.4  24.9
65 70–79 SS, NF 47.7  26.1
24 80–89 SS, NF 31.1  19.8
Contractor (134) 5 — — 54.6  11.7 FL
Wozenski (96) 5 27  3 SS, F 35  14 TDC
Leklem (78) 8 27  4 SS, F 51.5  14.1 TDC
Met, (1.55), F 33.8  11.2
Shultz (97) 35 38  14 SS, (2.0  8), F 51.9  19.3 TDC
Shultz (135) 4 22–35 Met, (1.60), F 59.9  41.7 TDC
Leklem (125) 7 16  1 SS, F 47.6  18.7 TDC
Lindberg (79) 5 27  6 Met, (1.60), F 43.3  6.5 TDC
Kabir (83) 9 25  4 SS, F 81.5  36.0 TDC
Met, F 65.0  23.3
Miller (136) 8 27  4 Met 1, (1.6)LP, F 43.5  19.4 TDC
Met, (1.6)MP, F 33.7  9.0
Met, (1.6)HP, F 27.9  11.5
Leklem (137) 8 20–30 Met, (1.6)F 38.8  10.9 TDC
Swift (138) 9 57 SS, (1.9), F 45.5  15.0 TDC
5 60 SS, (1.5), F 39.2  22.4
Tarr (81) 6 21–35 Met, (1.1), F 27.5  2.7 TDC
(2.3), F 55.0  5.7
(2.7), F 114.5  7.0
Ribaya-Mercado (139) 4 63.6  0.8 SS, (1.34), F 25.7  5.1 TDC
SS, (1.96), F 40.3  7.2
SS, (2.88), F 48.0  9.4
Females
Wachstein (132) 20 — — 34.0  10.1 TDC
Chabner (38) 12 20–34 SS, F 67.9  14.6
7 35–49 SS, F 46.1  13.8
Reinken (40) 29 — SS, — 36.8  8.9 TDC
Miller (141) 11 20–29 SS, F 25.9  15.4 TDC
Lumeng (142) 77 29  8 SS, NF 38.0  17.0 TDC
Brown (95) 6 22  2 Met, (0.8), F 22.9  13.9 TDC
3 22 2 Met, (1.8), F 60.7  20.2 TDC
Brophy (143) 4 20–34 SS, — 68.4 TDC
Cleary (144) 58 20–34 SS, — 43.4 TDC
Prasad (145) ? — SS, (1.19), — 51.8  30.7 TDC
? — SS, (1.02), — 46.5  24.3 TDC
Shultz (135) 4 24–32 SS, F 38.4  15.8 TDC
Vitamin B6 357
Table 9 Continued
No. Age
Ref. subjects (years) Dieta PLP (nmol/L) Methodb
Shultz (97) 41 50  14 SS, (1.6  0.5), F 37.7  14.7 TDC
Guilland (146) 23 27 SS, (1.1), F 92.8  7.3 LC/FL
29 84 Met, (1.0), F 52.0  4.1
Lee (147) 5 24  3 SS, F 35.5  14.8 TDC
5 55 4 SS, F 31.3  13.3
5 24 3 Met, (2.3), F 61.7  25.6
5 55 4 Met, (2.3), F 40.5  12.2
5 24 3 Met, (10.3), F 202  45
5 55 4 Met, (10.3), F 168  38
Driskell (148) 41 (C)d 12 SS, (1.23), F 48.1  17.9 TDC
32 (C) 14 SS, (1.23), F 44.5  15.8
23 (C) 16 SS, (1.25), F 43.7  15.3
32 (B) 12 SS, (1.30), F 46.1  15.8
39 (B) 14 SS, (1.24), F 42.1  15.8
19 (B) 16 SS, (1.17), F 46.5  13.9
Ubbink (149) 9 — SS, F 31.7  19.4 HPLC
Huang (150) 8 28–34 Met, (1.60), F 58.19  16.28 HPLC
Met, (0.45), F 32.40  10.50
Met, (1.26), F 38.31  9.68
Met, (1.66), F 45.43  16.24
Met, (2.06), F 53.65  10.94
Hansen (151) 10 27.5  6.8 Met, (1.03), F 27.9  11.4 TDC
Met, (1.33), F 32.4  11.6
Met, (1.73), F 41.0  14.8
Met, (2.39), F 58.9  25.3
6 28.2  2.6 Met, (0.84), F 26.5  12.4
Met, (1.14), F 29.4  12.5
Met, (2.34), F 52.6  22.3
Ribaya-Mercado (139) 4 63.6  0.8 SS, (0.89), F 21.6  4.4 TDC
SS, (1.29), F 27.0  5.2
SS, (1.90), F 36.6  2.1
Kretsch (152) 8 21–30 animal  plant protein TDC
CF, (0.5), F 8.68  6.13
CF, (1.0),(0.5), F 18.66  8.14
CF, (1.5)(1.0), F 30.44  14.76
CF, (2.0)(1.5), F 42.33  22.11
aThe notations for diet indicate if the blood samples were obtained from subjects who self-selected (SS) their
diets or were receiving a controlled intake (Met) and the amount of vitamin B6 consumed (value given as mg/
day in parentheses), if known. F indicates that the blood sample was collected after a fast of at least 8 h; NF
indicates nonfasting. LP, MP, HP refer to grams of protein as 0.5, 1.0, and 2.0 g/kg body weight.
bTDC, tyrosine apodecarboxylase; HPLC, high-performance liquid chromatography; FL, fluorimetry.
cA dash indicates data was not given in the respective reference.
dC, Caucasian; B, black.
358 Leklem
Table
10
Urinary
4-Pyridoxic
Acid
and
Vitamin
B
6
Excretion
in
Males
and
Females
No.
Age
4-Pa
UB6
Ref.
subjects
(years)
Diet
(nmol
/
day)
(µ
mol/day)
Males
Kelsay
(158)
5
20–25
Met
(1.66
mg,
P


150)
5.51

1.75
—
6
18–35
(1.66
mg,
P


54)
4.80

0.60
(50)
a
Mikai-Devic
(159)
10
16–51
—
5.7

1.4
—
Leklem
(78)
8
27

4
Met
(1.55)
4.04

0.85
(44)
0.76

0/17
Wozenski
(96)
5
27

3
SS
5.4

0.5
0.8

0.1
Shultz
(135)
4
22–35
Met
(1.6)
5.71

1.08
0.76

0.10
Shultz
(97)
35
35

14
SS
(2.0

0.8)
7.46

4.34
(63)
0.92

0.49
Lindberg
(79)
10
26

4
SS
4.78

1.40
0.81

0.18
10
26

4
Met
(1.60)
3.62

0.59
0.76

0.10
Kabir
(83)
9
25

4
Met
(1.55)
4.89

1.10
(53)
1.05

0.20
Dreon
(160)
6
28

6
Met
(4.2

0.4)
11.15

1.86
(45)
Miller
(136)
8
27

4
Met
(1.60)
4.37

0.89
(LP)
0.77

0.14
3.58

0.54
(MP)
0.71

0.09
2.74

0.71
(HP)
0.68

0.15
Females
Mikai-Devic
(159)
15
18–47
SS
4.5

0.9
Contractor
(134)
26
—
SS
6.62

4.6
Reinken
(140)
29
25
SS
8.32

1.30
Brown
(95)
6
22

2
Met
(0.82)
1.98

0.81
(41)
322

2
Met
(1.81)
6.03

2.04
(56)
Donald
(161)
8
18–23
Met
(1.54)
2.4

0.1
(26)
0.33

0.3
(2.06)
3.78

0.41
(31)
Vitamin B6 359
Shultz
(97)
41
50

14
SS
(1.6

0.5)
5.57

3.09
(59)
0.76

0.24
Lee
(147)
5
24

3
Met
(2.3)
6.89

0.55
(50)
1.12

0.29
Ubbink
(149)
9
—
SS
5.48

2.93
Huang
(150)
8
28–34
Met,
(1.60),
F
3.48

0.93
(37)
—
Met,
(0.45),
F
0.93

0.30
(35)
Met,
(1.26),
F
2.52

0.32
(33

2)
Met,
(1.66),
F
3.01

0.61
(33

2)
Met,
(2.06),
F
4.24

0.74
(33

2)
Hansen
(151)
10
27.5

6.8
Met,
(1.03),
F
3.23

0.73
(60)
0.54

0.12
Met,
(1.33),
F
4.00

0.91
(52)
0.61

0.10
Met,
(1.75),
F
5.89

0.77
(54)
0.75

0.09
Met,
(2.39),
F
9.51

1.08
(69)
0.95

0.14
6
28.2

2.6
Met,
(0.84),
F
2.87

0.47
(58)
0.48

0.08
Met,
(1.14),
F
3.35

0.65
(50)
0.54

0.09
Met,
(2.34),
F
7.88

0.81
(57)
0.79

0.11
Hansen
(86)
5
29

6
High
PNG:
Met,
(1.52),
F
3.60

0.84
(40)
0.587

0.082
4
Low
PNG:
Met,
(1.45),
F
4.02

0.58
(47)
0.660

0.116
Kretsch,
(152)
4
21–30
animal
protein:
CF,
(0.5),
F
0.62

0.28
—
CF,
(1.0)(0.5),
F
1.76

0.41
(32)
CF,
(1.5)(1.0),
F
3.55

0.43
(43)
CF,
(2.0)(1.5),
F
5.47

0.71
(50)
4
plant
protein:
CF,
(0.5),
F
1.35

0.47
—
CF,
(1.0)(0.5),
F
2.29

0.27
(39)
CF,
(1.5)(1.0),
F
4.01

0.25
(49)
CF,
(2.0)(1.5),
F
5.98

0.36
(55)
aThe
number
in
parentheses
refers
to
the
percent
of
intake
excreted
as
4-PA.
Abbreviations
are
as
used
in
Table
9,
except
for
4-pyridoxic,
which
is
4-PA
and
urinary
vitamin
B
6,
which
is
UB6.
360 Leklem
remains to be determined. There is one controlled metabolic study that has evaluated
vitamin B6 status in different age groups. Lee and Leklem (147) studied five women age
20–27 years and eight women age 51–59 years under conditions in which the women
received a constant daily vitamin B6 intake of 2.3 mg for 4 weeks followed 10.3 mg per
day for 3 weeks. Compared with the younger women, the older women had a lower mean
plasma PLP, plasma and urinary total vitamin B6, and a slightly higher urinary 4-pyridoxic
acid excretion with the 2.3-mg intake. Interestingly, there was no difference in urinary
excretion of xanthurenic or kynurenic acid following a 2-g l-tryptophan load. Thus, while
there may be age-related differences in vitamin B6 metabolism, there is no significant age
effect on functional activity of vitamin B6 when intake is adequate. The metabolism of
vitamin B6 has been studied in elderly men and women older than 60 years. While younger
individuals were not examined in the same study, the researchers concluded that the elderly
had an increased vitamin B6 requirement, indicative of increased metabolism. Kant et al.
(154) observed no age-related impairment in the absorption or phosphorylation of vitamin
B6. However, there was an increase in plasma alkaline phosphatase activity with age that
would increase hydrolysis of PLP.
The use of plasma PLP as a status indicator has been questioned (155) and the
determination of plasma PL recommended. Others have also suggested that plasma PL
may be an important indicator of status. When Barnard et al. (156) studied the vitamin
B6 status in pregnant females and nonpregnant controls, they found that plasma PLP concentration
was 50% lower in pregnant females but that the concentration of the total of
PLP and PL was only slightly lower. When concentrations of PLP and PL were expressed
on a per-gram-albumin basis, there was no difference between groups. In contrast, in
pregnant rats both plasma PLP and PL decreased, as did liver PLP, in comparison with
nonpregnant control rats (157). These studies are in direct opposition to each other but
do provide support for the need to determine several indices of vitamin B6 status
(130,131,155).
Urinary 4-pyridoxic acid excretion is considered a short-term indicator of vitamin
B6 status. In deficiency studies in males (158) and females (159), the decrease in urinary
4-pyridoxic acid paralleled the decrease in plasma PLP concentration. Table 10 lists values
for urinary 4-pyridoxic acid and vitamin B6 in males and females. As reflected in the
studies in which dietary intake was assessed or known, 4-pyridoxic acid excretion accounts
for about 40–60% of the intake. Because of the design of most studies and the limited
number of studies done with females compared with males, it is not possible to determine
if there is a significant difference between males and females. The limited data in Table
11 suggest that there is little difference. However, males consistently had higher plasma
PLP and total vitamin B6 concentrations as well as higher excretion of 4-pyridoxic acid
and total vitamin B6. Urinary total vitamin B6 (all forms, including phosphorylated and
Table 11 Plasma Pyridoxal-5?-Phosphate and Total Vitamin B6 Concentration, and Urinary 4-
Pyridoxic Acid and Vitamin B6 Excretion in Males and Females Consuming 2.2 mg Vitamin B
PLP TB6 4-PA UB6
Subject (nmol/L) (nmol/L) (µmol/day) (µmol/day)
Males (n 
 4) 78.4  27.0a 86.2  37.1 7.86  0.74 0.92  0.20
Females (n 
 4) 58.5  12.6 71.5  15.8 7.02  0.78 0.82  0.19
aMean  SD.
Vitamin B6 361
glycosylated) excretion is not a sensitive indicator of vitamin B6, except in situations
where intake is very low (158).
Erythrocyte transaminase activity (alanine and aspartate) has been used to assess
vitamin B6 status in a variety of populations (133,142,146,162–168), including oral contraceptive
users (95,166). Transaminase activity is considered a long-term indicator of vitamin
B6 status. Most often the transaminase activity has been measured in the presence
and absence of excess PLP (163). Table 8 indicates suggested norms for activity coeffi-
cients for alanine and aspartate aminotransferase. While transaminase activity is used to
assess status, there is not unanimous agreement, and some consider this measure to be
less reliable than other indices of vitamin B6 status (95,168). The long life of the erythrocyte
and tight binding of PLP to hemoglobin may explain the lack of a consistent signifi-
cant correlation between plasma PLP and transaminase activity or activity coefficient. An
additional consideration that complicates the use of aminotransferases is the finding of
genetic polymorphism of erythrocyte alanine aminotransferase (169).
Urinary excretion of tryptophan metabolites following a tryptophan load, especially
excretion of xanthurenic acid, has been one of the most widely used tests for assessing
vitamin B6 status (170,171). Table 8 gives a suggested normal value for xanthurenic acid
excretion following a 2-g l-tryptophan load test. The use of the tryptophan load test for
assessing vitamin B6 status has been questioned (172,173), especially in disease states or
in situations in which hormones may alter tryptophan metabolism independent of a direct
effect on vitamin B6 metabolism (174).
Other tests for status include the methionine load (175), oxalate excretion, and electroencephalographic
tracings (176). These tests are used less often but under appropriate
circumstances provide useful information. The review by Reynolds (155) provides an
excellent critique of methods currently in use for assessment of vitamin B6 status.
VIII. FUNCTIONS
A. Immune System
The involvement of PLP in a multiplicity of enzymatic reactions (177) suggests that it
would serve many functions in the body. Table 12 lists several of the known functions
of PLP and the cellular systems (137) affected. PLP serves as a coenzyme for serine
transhydroxymethylase (178), one of the key enzymes involved in one-carbon metabolism.
Alteration in one-carbon metabolism can then lead to changes in nucleic acid synthesis.
Such changes may be one of the keys to the effect of vitamin B6 on immune function
Table 12 Cellular Processes Affected by Pyridoxal-5?-Phosphate
Cellular process or enzyme Function/system influenced
One-carbon metabolism, hormone modulation Immune function
Glycogen phosphorylase, transamination Gluconeogenesis
Tryptophan metabolism Niacin formation
Heme synthesis, transamination, O2 affinity Red cell metabolism and formation
Neurotransmitter synthesis, lipid metabolism Nervous system
Hormone modulation, binding of PLP to lysine on Hormone modulation
hormone receptor
362 Leklem
(179,180). Studies in animals have shown that a vitamin B6 deficiency adversely affects
lymphocyte production (179) and antibody response to antigens (180). Additional studies
in animals support an effect of vitamin B6 on cell-mediated immunity (181). Talbot et al.
found in 11 elderly women whose immune response has impaired that treatment with 50
mg pyridoxine per day for 2 months improved their immune system, as judged by lymphocyte
response (182). However, in humans a diet-induced marginal vitamin B6 status for
11 weeks was not found to significantly influence cellular or humoral immunity (183).
These two studies differed in their experimental design. The study by van den Berg et al.
(183) employed a diet marginally deficient in vitamin B6 in young adults; that of Talbot
et al. (182) utilized a treatment of elderly individuals with an excess of vitamin B6. This
excess intake may be necessary for increased activity of certain cell types of the immune
system in the elderly. Meydani et al. examined immune response in healthy elderly adults
fed graded levels of vitamin B6 and found that a deficiency impairs in vitro indices of
cell-modulated immunity, especially interleukin-2 production (184). A review of vitamin
B6 and immune competence is available (185).
B. Gluconeogenesis
Gluconeogenesis is key to maintaining an adequate supply of glucose during caloric defi-
cit. Pyridoxal-5?-phosphate is involved in gluconeogenesis via its role as a coenzyme for
transamination reactions (177) and for glycogen phosphorylase (114). In animals a defi-
ciency of vitamin B6 results in decreased activities of liver alanine and aspartate aminotransferase
(186). However, in humans (females) a low intake of vitamin B6 (0.2 mg/
day), as compared with an adequate intake (1.8 mg/day), did not significantly influence
fasting plasma glucose concentrations (187). Interestingly, the low vitamin B6 intake was
associated with impaired glucose tolerance in this study.
Glycogen phosphorylase is also involved in maintaining adequate glucose supplies
within liver and muscle and, in the case of liver, a source of glucose for adequate blood
glucose levels. In rats a deficiency of vitamin B6 has been shown to result in decreased
activities of both liver (188) and muscle glycogen phosphorylase (114,122,188). Muscle
appears to serve as a reservoir for vitamin B6 (114,122,123), but a deficiency of the vitamin
does not result in mobilization of these stores. However, Black et al. (122) have shown
that a caloric deficit does lead to decreased muscle phosphorylase content. These results
suggest that the reservoir of vitamin B6 (as PLP) is only utilized when there is a need for
enhanced gluconeogenesis. In male mice the half-life of muscle glycogen phosphorylase
has been shown to be approximately 12 days (189). In contrast to low intake of vitamin
B6, rats given an in injection of a high dose of PN, PL, or PM (300 mg/kg) showed a
decrease in liver glycogen and an increase in serum glucose (190). This effect is mediated
via increased secretion of adrenal catecholamines. The extent to which lower intake of
B6 vitamers has this effect or if this occurs in humans remains to be determined.
C. Erythrocyte Function
Vitamin B6 has an additional role in erythrocyte function and metabolism. The function
of PLP as a coenzyme for transaminases in erythrocytes has been mentioned. In addition,
both PL and PLP bind to hemoglobin (107,108). The binding of PL to the ? chain of
hemoglobin (191) increased the O2 binding affinity (192), while the binding of PLP to
the ? chain of hemoglobin S or A lowers the O2 binding affinity (193). The effect of PLP
and PL on O2 binding may be important in sickle cell anemia (194).
Vitamin B6 363
Pyridoxal-5?-phosphate serves as a cofactor for ?-aminolevulinic acid synthetase
(195), the enzyme that catalyzes the condensation between glycine and succinyl-CoA to
form ?-aminolevulinic acid. This latter compound is the initial precursor in heme synthesis
(196). Therefore, vitamin B6 plays a central role in erythropoiesis. A deficiency of vitamin
B6 in animals can lead to hypochromic microcytic anemia. Furthermore, in humans there
are several reports of patients with pyridoxine-responsive anemia (197,198). However,
not all patients with sideroblastic anemia (in which there is a defect in ?-aminolevulinic
acid synthetase) respond to pyridoxine therapy (199).
D. Niacin Formation
One of the more extensive functions of vitamin B6 that has been researched is its involvement
in the conversion of tryptophan to niacin (171). This research is in part related to
the use of the tryptophan load in evaluating vitamin B6 status. While PLP functions in at
least four enzymatic reactions in the complex tryptophan–niacin pathway (Fig. 6), there
is only one PLP-requiring reaction in the direct conversion of tryptophan to niacin. This
step is the conversion of 3-hydroxykynurenine to 3-hydroxyanthranilic acid and is catalyzed
by kynureninase. Leklem et al. have examined the effect of vitamin B6 deficiency
on the conversion of tryptophan to niacin (200). In this study, the urinary excretion of
N?-methylnicotinamide and N?-methyl-2-pyridone-5-carboxamide, two metabolites of niacin,
was evaluated in women. After 4 weeks of a low-vitamin B6 diet, the total excretion
of these two metabolites following a 2-g l-tryptophan load was approximately half that
when subjects received 0.8–1.8 mg vitamin B6 per day. This suggests that low vitamin
B6 has a moderate negative effect on niacin formation from tryptophan.
E. Nervous System
In addition to the effect of vitamin B6 on tryptophan-to-niacin conversion, there is another
tryptophan pathway that is vitamin B6–dependent. The conversion of 5-hydroxytryptophan
Fig. 6 Tryptophan–niacin pathway, B6 indicates the steps in the pathway in which pyridoxal-5?-
phosphate functions as a coenzyme.
364 Leklem
to 5-hydroxytryptamine is catalyzed by the PLP-dependent enzyme 5-hydroxytryptophan
decarboxylase (201). Other neurotransmitters, such as taurine, dopamine, norepinephrine,
histamine, and ?-aminobutyric acid, are also synthesized by PLP-dependent enzymes
(201). The involvement of PLP in neurotransmitter formation and the observation that
there are neurological abnormalities in human infants (202,203) and animals (204,205)
deficient in vitamin B6 provide support for a role of vitamin B6 in nervous system function.
Reviews on the relationship between nervous system function and vitamin B6 are available
(201,206,207).
In infants fed a formula in which the vitamin B6 was lost during processing, convulsions
and abnormal electroencephalograms (EEGs) were observed (202). Treatment of
the infants with 100 mg of pyridoxine produced a rapid involvement in the EEGs. In these
studies reported by Coursin, the protein content of the diet appeared to be correlated with
the vitamin B6 deficiency and the extent of symptoms. Other evidence for a role of vitamin
B6 comes from studies of pyridoxine-dependent seizures, disorders, which is an autosomal
recessive disorder. Vitamin B6 dependency, though a rare cause of convulsions, has been
reported by several investigators (208–211). The convulsions occur during the neonatal
period, and administration of 30–100 mg of pyridoxine is usually sufficient to prevent
convulsions and correct an abnormal EEG (211,212). However, there are atypical patients
who present a slightly different clinical picture and course but are responsive to pyridoxine
(213).
Vitamin B6 deficiency in adults has also been reported to result in abnormal EEGs
(176,214), especially in individuals on a high-protein (100 g/day) intake. In one study
(215), subjects were receiving a diet essentially devoid of vitamin B6 (0.06 mg). In a
separate study, Grabow and Linkswiler fed to 11 men a high-protein diet (150 g) and 0.16
mg of vitamin B6 for 21 days (215). No abnormalities in EEGs were observed, nor were
there changes in motor nerve conduction times in five subjects who had this measurement.
Kretsch et al. (176) observed abnormal EEG patterns in two of eight women after 12 days
of a low (0.05 mg/day) vitamin B6 diet. Feeding 0.5 mg/day corrected the abnormal pattern.
While there were differences in the length of deficiency in these studies, which may
explain the differences observed, it appears that long-term very low vitamin B6 intakes
are necessary before abnormal EEGs are observed in humans.
Another aspect of the relationship of vitamin B6 (as PLP) to the nervous system is
the development of the brain under conditions of varying intakes of vitamin B6. Kirksey
and co-workers have conducted numerous well-designed studies in this area. These studies
have utilized the rat model to examine the development of the brain, especially during
the critical period when cells are undergoing rapid mitosis. Early experiments showed
that dietary restriction of vitamin B6 in the dams was associated with a decrease in alanine
aminotransferase and glutamic acid decarboxylase activity and low brain weights of progeny
(216). Alterations in fatty acid levels, especially those involved in myelination, were
observed in the cerebellum and cerebrum of progeny of dams fed a low (1.2 mg/kg daily)
vitamin B6 diet (217). Kurtz et al. found a 30–50% decrease in cerebral sphingolipids of
progeny of dams fed a vitamin B6 deficient diet (218). In the progeny of dams fed graded
levels of vitamin B6, a decrease in the area of the neocortex and cerebellum as well as
reduced molecular and granular layers of the cerebellum were noted (219). Myelination
was reduced in the progeny of severely deficient dams (220). Noting in previous studies
that Purkinje cells were dispersed from the usual noncellular layer in progeny of severely
vitamin B6-deficient dams, Chang et al. (221) carried out other studies and found that a
maternal vitamin B6 deficiency interferes with normal development of Purkinje cells, seen
Vitamin B6 365
as reduced total length of Purkinje cell dendrites. At the biochemical level, a vitamin B6
deficiency led to reduced GABA levels, which were associated with impaired function of
the extrapyramidal motor system (222). Both Wasynczuk et al. (223) and Kurtz et al.
(218) found that with vitamin B6 deficiency amino acid levels were altered in specific
regions of the brain. Glycine, leucine, isoleucine, valine, and cystathionine levels were
elevated, whereas alanine and serine levels were reduced. Other studies have shown that
PLP levels in certain areas of the central nervous system are more dramatically affected
by vitamin B6 deficiency than others. Levels in the spinal cord and hypothalamus were less
affected than these in the corpus striatum and cerebellum (233). These findings suggested
metabolic trapping in a caudal-to-rostral direction.
F. Lipid Metabolism
One of the more intriguing and controversial aspects of vitamin B6 is its role in lipid
metabolism (225). Studies conducted more than 60 years ago suggested a link between fat
metabolism and vitamin B6 (226). These studies showed a similarity (visual) in symptoms
between a lack of essential fatty acids and a deficiency of vitamin B6. Other early studies
found that a vitamin B6 deficiency in rats resulted in a decrease in body fats (227). Subsequent
research showed that liver lipid levels were significantly lower in vitamin B6-defi-
cient vs. pair-fed rats (228). The changes were due mainly to lower trigylceride levels,
whereas cholesterol levels were not different. In contrast, Abe and Kishino showed that
rats fed a high-protein (70%), vitamin B6-deficient diet developed fatty livers and suggested
that this was due to impaired lysosomal degradation of lipid (229). The synthesis
of fat in vitamin B6-deficient rats has been reported to be greater (230), normal (231,232),
or depressed (233). The observed differences may be related to the meal pattern of the
animals (234).
The effect of vitamin B6 deprivation on fatty acid metabolism has also received
attention. A pyridoxine deficiency may impair the conversion of linoleic acid to arachidonic
acid (202,235). Cunnane and co-workers (202) found that phospholipid levels of
both linoleic and ?-linolenic acid were increased in vitamin B6-deficient rats, but the level
of arachidonic acid was decreased as compared with that of control levels in plasma, liver,
and skin. They suggested that both linoleic desaturation and ?-linoleic acid elongation
may be impaired by a vitamin B6 deficiency. She et al. (236) have observed decreased
activity of terminal ?6-desaturase in the linoleic acid desaturation system in rats fed a
vitamin B6-deficient diet. They also found a positive correlation between phosphatidylcholine
(PC) content and ?6-desaturase activity in liver microsomes, suggesting that altered
PC may affect linoleic acid desaturation and thus decreased arachidonic acid synthesis.
Subsequent work by She et al. suggests that altered S-adenosylmethionine (SAM) to Sadenosylhomocysteine
is involved in these changes (237). In one of the few studies of
vitamin B6 and fatty acid metabolism in humans, desoxypyridoxine was utilized to induce
a vitamin B6 deficiency (238). Xanthurenic acid excretion following a 10-g d,l-tryptophan
load indicated a moderate vitamin B6-deficient state. Only minor changes in fatty acid
levels in plasma and erthyrocytes were observed as a result of the deficiency produced.
The pattern of fatty acids observed was interpreted by the authors to support the findings
by Witten and Holman (234). Delmore and Lupien also observed a decreased proportion
of arachidonic acid in liver phospholipids and an increased level of linoleic acid in vitamin
B6-deficient rats (239). They suggested that changes were based on the decrease in PC
via methylation of phosphoethanolamine. Support for this comes from the studies of Loo
366 Leklem
and Smith (240), in which it was found that a deficiency of vitamin B6 resulted in decreased
phospholipid methylation in the liver of rats. The level of SAM in livers of vitamin B6-
deficient rats was nearly five times higher than that in livers of pair-fed animals. This
change in SAM is secondary to the inhibition of the catabolism of homocysteine, a PLPdependent
process. Negative feedback of SAM on the conversion of phosphatidylethanolamine
to PC may thus explain the changes seen in fatty acid metabolism. The work
of She et al. (237) supports this. This provides a plausible mechanism because the primary
metabolic steps in fatty acid metabolism do not involve nitrogen-containing substrates, a
feature common to most PLP-dependent enzymatic reactions.
The change observed in arachidonic acid levels and the role it plays in cholesterol
metabolism (241) may have clinical implications (242). The effect, if any, of vitamin B6
on cholesterol metabolism remains controversial. The increase in plasma cholesterol in
monkeys made vitamin B6-deficient (243) provided much of the impetus for research
relating vitamin B6, cholesterol, and atherosclerosis. Studies by Lupien and co-workers
have shown that the rate in incorporation of [14C] acetate into cholesterol was increased
in vitamin B6-deficient rats as compared to controls (244). However, the amount of cholesterol
in plasma and liver of rats and other species has been reported to be increased, not
changed, or even decreased (244–247). In humans, a deficiency of vitamin B6 did not
result in a significant change in serum cholesterol (248). Significant positive correlation
between plasma PLP and high-density lipoprotein (HDL) cholesterol and negative correlations
with total cholesterol and low-density lipoprotein (LDL) cholesterol have been reported
in monkeys fed atherogenic Western diets and a ‘‘prudent’’ Western diet (249).
However, the diets fed to the monkeys contained distinctly different amounts of vitamin
B6. The use of supplemental vitamin B6 in reduction of blood cholesterol has not been
definitively tested. Serfontein and Ubbink reported decreased serum cholesterol (0.8
mmol/L) in 34 subjects given a multivitamin containing 10 mg of pyridoxine (250). The
reduction was mainly as LDL cholesterol. In another study, pyridoxine (50 mg/day) administration
prevented the increase in serum cholesterol seen when disulfiram was administered
(251). Controlled trials of pyridoxine are needed to resolve the role of vitamin B6
in modifying serum cholesterol levels.
The role of vitamin B6 in lipid metabolism remains unclear. Evidence to data suggests
a role of vitamin B6 in modifying methionine metabolism and thus an indirect effect
on phospholipid and fatty acid metabolism. This effect (240) and an effect of vitamin B6
on carnitine synthesis (252) appear to be the primary effects of vitamin B6 on lipid/fatty
acid metabolism.
G. Hormone Modulation/Gene Expression
One of the more intriguing functions of PLP is as a modulator of steroid action (253,254).
Reviews of this interaction are available (255,256). PLP can be used as an effective tool
in extracting steroid receptors from the nuclei of tissues on which the steroid acts (257).
Under conditions of physiological concentrations of PLP, reversible reactions occur with
receptors for estrogen (258), androgen (259), progesterone (260), and glucocorticoids
(261,262). PLP reacts with a lysine residue on the steroid receptor. As a result of the
formation of a Schiff base, there is inhibition of the binding of the steroid–receptor complex
to DNA (253). Holley et al. found that when female rats were made vitamin B6-
deficient and injected with [3H]-estradiol, a greater amount of the isotope accumulated in
the uterine tissues of the deficient animal than in the tissues of control rats (263). Bunce
Vitamin B6 367
and co-workers studied the dual effect of zinc and vitamin B6 deficiency on estrogen
uptake by the uterus (264). They found that there was an increased uptake of estrogen in
both the vitamin B6- and the zinc-deficient animals. A combined deficiency to the two
nutrients resulted in even greater retention of estrogen. The number of estrogen receptors
was not altered by the deficiency of vitamin B6. This study suggests that there would be
increased sensitivity of the uterus (or other end-target tissues) to steroids when vitamin
B6 status was abnormal.
Sturman and Kremzner found enhanced activity of ornithine decarboxylase in testosterone-
treated vitamin B6-deficient animals as compared to control animals (265). DiSorbo
and Litwack observed increased tyrosine aminotransferase activity in hepatoma cells
raised on a pyridoxine-deficient medium and treated with triamcinolone acetonide as compared
to pyridoxine-sufficient cells treated with the same steroid (266). Allgood and Cidlowski
(267) have used a variety of cell lines and a range of intracellular PLP concentrations
to show that vitamin B6 modulates transcriptional activation by several (androgen,
progesterone, and estrogen) steroid hormone receptors. This supports the role of vitamin
B6 as a physiological modulator of steroid hormone action.
Oka et al. (268) have found that in vitamin B6-deficient rats the level of albumin
mRNA was increased sevenfold once that of control rats. They suggest that PLP modulates
albumin gene expression by inactivation of tissue-specific transcription factors. Oka and
co-workers have also observed a sevenfold increase in the level of mRNA for cystosolic
aminotransferase in the level of vitamin B6-deficient rats as compared to that of vitamin
B6-sufficient rats (269). Subsequent work by Oka et al. (270) shows an inverse relationship
between intracellular PLP concentration and albumin in RNA in rats given amino loads.
Thus, PLP may be a modulator of gene expression in animals, especially under conditions
of altered amino acid supply. Given the intimate relationship of vitamin B6 and an amino
acid metabolism, these studies open a new area of metabolic regulation via altered intracellular
nutrient (PLP) concentration.
IX. REQUIREMENTS
Considering the numerous functions in which vitamin B6 is involved, assessment of the
requirement for this vitamin becomes important. Reviews of vitamin B6 requirements are
available (271–273). Each of the last three recommended dietary allowances (RDA) publications
(274–276) has evaluated the requirements for vitamin B6. While vitamin B6 has
been known for over 70 years, only in the past 40 years has a requirement been established
in the United States. There are numerous factors that may contribute to the requirement
for vitamin B6, several of which are listed in Table 13. Many of these have not been
experimentally tested, whereas others have received greater attention and been examined
in more detail.
Given the central role of PLP in amino acid metabolism, it is not surprising that
there is an intimate relationship between vitamin B6 requirements and protein intake. Historically,
the establishment of the requirement for vitamin B6 has been based primarily
on this protein–vitamin B6 relationship. Early studies by Canham et al. (214), Baker et
al. (277), and Miller and Linkswiler (278) in men utilized l-tryptophan load tests and the
excretion of tryptophan metabolites as an indicator of vitamin B6 adequacy. In a study
by Park and Linkswiler (279), a methionine load was used to assess vitamin B6 adequacy.
In all of these studies the intake of vitamin B6 used ranged from 0.1 to 2.2 mg. There are
several important observations relative to use of these studies in subsequent estimation
368 Leklem
Table 13 Factors Affecting Vitamin B6 Requirement
1. Dietary
a. Physical structure of a food
b. Forms of vitamin B6 natural; those due to processing
c. Binding of forms of vitamin B6
2. Defect in delivery to tissues
a. Impaired gastrointestinal absorption
b. Impaired transport—albumin synthesis and binding, impaired phosphatase activity
3. Physiological/biochemical
a. Physical activity—increased loss, gluconeogenesis
b. Protein—enzyme induction
c. Increased catabolism turnover—phosphatase activity, illness
d. Impaired phosphorylation and/or interconversion, competing pathways, nutrient deficiencies,
drugs
e. Pregnancy—demand of fetus
f. Growth—increased cell mass, repair
g. Lactation—adequate levels in milk
h. Excretion rate—urinary, sweat, menstrual loss
i. Sex-differences in metabolism
j. Age differences in metabolism
4. Genetic
a. Apoenzyme defects—altered binding to apoenzyme
b. Altered enzyme levels—biochemical individuality
5. Disease prevention/treatment
a. Which? heart disease, cancer, diabetes, PMS, kidney disease, alcoholism
of a vitamin B6 requirement: (a) they were done only in men; (b) the men were fed two
levels of protein: 30–40 g/day at the low-protein intake levels and 80–100 g/day for the
high-protein; intake level; (c) subjects were usually fed a low-vitamin B6 diet (0.06–0.16
mg/day) for several weeks (4–6 weeks) before vitamin B6 supplements were given; (d)
often the diets were not representative of a typical American diet and could be considered
ones in which the vitamin B6 was highly bioavailable (given that the intake of vitamin
B6 from food was low); (e) the tryptophan load tests were not uniform between studies
(2 g of the l form or 10 g of dl form); and (f ) most of the studies did not include
measurement of plasma PLP concentration.
Based on data from these studies of the vitamin B6 protein interrelationship and
urinary metabolite excretion, the amount of vitamin B6 considered to be adequate (normalization
of amino acid post-load loose metabolite excretion) ranged from 1.0 to 2.0 mg of
vitamin B6. For diets containing 100–150 g of protein per day the requirement was judged
to be 1.5–2.0 mg/day. At protein intakes less than 100 g of protein per day the requirement
was judged to be 1.0–1.5 mg/day (281).
In a subsequent study by Miller et al. (136), the effect of three levels of protein on
vitamin B6 status indices was evaluated in men. An important feature of this study was
that it was not a depletion/repletion study. There was a significant effect of protein (inverse
relationship) on plasma PLP and total vitamin B6 and on urinary 4-pyridoxic acid excretion.
The authors pointed out that protein intake should be considered when evaluating
requirement.
Vitamin B6 369
It should be noted that in some of these early studies one or more of the following
indices of B6 adequacy were utilized: urinary B6 excretion, erythrocyte transaminase activity,
urinary cystathionine excretion, urinary 4-pyridoxic excretion, and electroencephalograms.
Each of these indices varies with respect to its sensitivity to changes in vitamin
B6 status and its usefulness in establishing vitamin B6 requirements (130).
Subsequent studies in women focused on evaluating the effect of oral contraceptives
on vitamin B6 status (95,200,281). These studies utilized depletion/repletion-type designs.
The length of the depletion periods varied (14–43 days) as did the amount of vitamin B6
fed (0.16–0.34 mg/day). In addition, the protein intake ranged from 57–109 g/day. The
vitamin B6 repletion levels ranged from 0.6 to 1.85 mg over periods of 3–28 days. Vitamin
B6 status (adequacy of vitamin B6 intake) was assessed with a variety of indices, including
plasma PLP, plasma vitamin B6, urinary 4-pyridoxic acid excretion, tryptophan metabolite
excretion, and erythrocyte transaminase activity and stimulation.
The differences in vitamin B6 intake and protein intake in these studies are critical
in evaluating the subsequent vitamin B6 requirements derived from them. Also, these were
depletion/repletion experiments. The effect of this experimental design on the length of
time for the various vitamin B6 status indices to stabilize during the repletion period could
have an effect on the estimation on vitamin B6 requirement. As an example, 4-pyridoxic
acid excretion is very sensitive to vitamin B6 intake and establishes a plateau relatively
quickly (3–4 days) after B6 intake has been changed. However, plasma PLP takes longer
(up to 10 days) before a plateau is achieved. Transaminase activity (and stimulation) would
be expected to take longer to achieve a plateau at a given vitamin B6 intake due to the
long half-life of the erythrocyte (200).
Several studies have been done since the publication of the 1989 RDAs that are
relevant to establishing the next RDA for vitamin B6. These studies have been carried
out in both young and elderly adults and in males and females (139,150–152). While
some of these studies are similar to previous ones in that they employed depletion/repletion
design (139,150,152) and diets with high B6 bioavailability, others have used diets
more representative of the usual U.S. diet (151).
What is also different about some of these studies is that they have included additional
measurements that may be indicative of intercellular function of PLP. Meydani et
al. (184) examined the effect of different levels of vitamin B6 (pyridoxine added to a low-
B6 food diet) on immune function. They observed that adequate immune function in elderly
women was not achieved until 1.9 mg/day of vitamin B6 was fed. Men required
2.88 mg/day to return function to baseline levels. In addition, several indices of vitamin
B6 status were measured. Based on when these values for these indices returned to predepletion
levels, the requirement for vitamin B6 was estimated to be 1.96 and 1.90 for
men and women, respectively (also see discussion of this study below).
Kretsch et al. (152) fed four graded doses of vitamin B6 to eight young women
following a depletion diet (for 11–28 days). A variety of clinical, functional, and biochemical
measures were conducted. No abnormal clinical evidence of vitamin B6 deficiency
was observed at the 0.5 mg/day intake. Based on this and other studies, less than 0.5 mg/
day is needed to observe clinical signs of vitamin B6 deficiency. Functional signs, such
as abnormal EEGs, were only seen with an intake lower than 0.5 mg/day. Various biochemical
measures, including the functional tests of tryptophan metabolite excretion
(xanthurenic acid) and erythrocyte transaminase (EAST) stimulation, were normalized at
the 1.5 and 2.0 mg/day level, respectively. The authors stated that if all currently used
370 Leklem
biochemical measures are to be normalized, then more than 0.020 mg of vitamin B6 per
gram of protein is required.
Hansen et al. (151) used a different approach in evaluating the effect of graded doses
of vitamin B6 on status. First, rather than feeding a diet deficient in vitamin B6 a diet
containing a level that is low but within the realm of what individuals might normally
consume was fed. Various levels of pyridoxine (as an oral solution) were then added in
addition to the basal diet (range 0.8–2.35 mg B6/day). Based on both direct and indirect
measures (including tryptophan metabolite excretion) it was concluded that a B6/protein
ratio greater than 0.20 was required to normalize all vitamin B6 status indices. Ribaya-
Mercado et al. (139) evaluated the vitamin B6 requirements of elderly men and women
in a depletion/repletion study. Individuals were fed 0.8 or 1.2 g/kg protein and increasing
amounts of vitamin B6. Several indices of vitamin B6 status were measured and predepletion
values used to evaluate when these indices were normalized. Men who ingested about
120 g of protein per day required 1.96 mg (for xanthurenic acid and PLP) to 2.88 mg (for
EAST-AC and 4-PA excretion) of vitamin B6 to normalize these indices. For women
ingesting 78 g of protein per day, 1.90 mg/day of vitamin B6 was needed to normalize
the same indices. The authors concluded that the vitamin B6 requirements of elderly men
and women are about 1.96 and 1.90, respectively. The vitamin B6 (pyridoxine) fed to
these subjects was in a highly bioavailable form.
Another metabolic study in young women evaluated the requirement for vitamin B6
(150). Again, a depletion/repletion design was used and several indices of vitamin B6
status were measured. These indices included urinary 4-PA excretion, plasma PLP, erythrocyte
PLP, and erythrocyte alanine aminotransferase (EALT) and erythrocyte aspartic
aminotransferase (EAST) activity coefficients. Using predepletion baseline levels (after 9
days of feeding 1.60 mg/day) of these indices as a basis for comparison in determining
adequacy, the amount of vitamin B6 required to normalize these indices was 1.94 mg/
day (B6 to protein ratio of 0.019 mg/day).
An important consideration relative to many of these metabolic studies that have
been used in establishing the adult vitamin B6 RDA is the composition of the diets used.
Most diets were ones in which the amount of vitamin B6 from food was low and of
relatively high bioavailability. Vitamin B6 was added back to the diets in the form of
pyridoxine hydrochloride and thus is considered 100% bioavailable. Therefore, the total
vitamin B6 in the diets is probably 95–100% bioavailable. Taken together, these four
recent metabolic studies support a higher vitamin B6 requirement for women and men
than is currently employed. A value of 1.9 mg/day for women and 2.2 mg/day for men
is recommended. Since the vitamin B6 in these studies was highly available, the inclusion
of a factor for bioavailability would further increase the RDA (86). The studies that have
been used in determining vitamin B6 are summarized in Table 14 along with suggested
values for requirement.
The above discussion has focused on the vitamin B6 requirement for adults aged
18–70. There has been little research to support a statement of recommendations for children
(aged 1–10) or adolescents (aged 11–18). Extrapolating from adults and taking into
account muscle accretion (since this is the major body pool of vitamin B6, a requirement
for these populations could be estimated (119). Of the factors listed in Table 13, pregnancy
and lactation are the ones in which additional needs could be estimated from the total
vitamin B6 content of the fetus and vitamin B6 content of human milk, respectively. While
the former is not entirely feasible except in nonhuman primates, the latter is possible
because the vitamin B6 content of human milk has been determined. Table 15 lists reported
Vitamin B6 371
Table
14
Studies
in
Which
Vitamin
B6
Requirements
Have
Been
Investigated
Durationa
No.
of
Age
Diet.
Energy
Suggested
Ref.
subjects
Sex
(years)
Wt.
(kg)
B6
(mg)
protein
(kcal)
Food
types
c
Adj.
Defic.
Repletion
Tests
b
requirements
Canham
(205)
6
M
—
—
0
80
—
SS
7(2)
28
14(10)
10g
dl-Try,
XA;
NS
0
40
—
SS
SGOT;
NME;
Urinary
B6
Baker
(291)
5
M
0.06
30
Liq.
formula
7(4)
49
(1.5)
10g
dl-Try;
XA;
1.0
6
M
0.06
100
21
Urinary
B6
EEG
1.5
Harding
(76)
9
M
20
–28
65–83
4.28
149
3800
SS
24
10
g
dl-try,
XA
1.97
–2.76
2.76
164
AR
(34)
24
1.93
165
AR(100)
24
Canham
(277)
—
M
—
—
0.34
100
3000
SS
—
8
5
g
l-Try,
XA;
0.35;
0.35
EEG
/EKG
GPT
submarginal
(WB)
Swan
(292)
6
M
24
–35
61–82
0.16
100
2500–
3000
SS
5-SSd
28–
41
9–
19(0.8
to
1.1)
5
g
l-Cysteine;
NS
Yess
(293)
2gl-try;
TM,
NS
Brown
(294)
NMe,
QA;
PLP
Baysal
(248)
Miller
(278)
5
M
21
–25
63–73
0.16
54
2800–
3600
SS
6(1.66)
16
7(0.76)
2
g
l-Try,
TM,
4-
NS(0.8)
PA:
Urinary
B6
Kelsay
(158)
6
M
19
–31
63–71
0.16
150
2800–
3600
18(1.66)
16
16(0.76)
2
g
l-Try,
TM;
EEg;
4-PA;
Urinary
B6;
QA;
NMe
Cheslock
(295)
7
F
18
–20
—
0.41
26
2045
Natural
—
52
—
5
g
l-Try,
XA,
0.5
1
M
—
—
0.50
36
2724
Natural
Blood
B
6
Aly
(296)
5
F
21
–31
—
1.3
81
—
Liquid/solid
6
—
—
2
g
l-Try,
XA;
Uri-
NS
nary
B6;
Amino
Acids
Donald
(297)
8
F
21
–31
56
0.34
57
1300
Natural
—
44
7(0.94)
Urinary
4-PA,
B6;
1.5
3(1.54)
E-B6
EGOT;
Amino
Acids
Shin
(298)
5
F
23
49
0.16
109
SS
7(2.16)
14
14(2.16)
2
g
l-Try,
TM;
Uri-
NS
nary
B6;3gl-
Meth
372 Leklem
Table
14
Continued
Durationa
No.
of
Age
Diet.
Energy
Suggested
Ref.
subjects
Sex
(years)
Wt.
(kg)
B6
(mg)
protein
(kcal)
Food
types
c
Adj.
Defic.
Repletion
Tests
b
requirements
Leklem
(95,
200)
10
F
22
60
0.19
78
1992
SS
4
28
28(0.83)
2
g
l-Try,
TM;
4-
0.83
PA;
PLP;
EGOT,
EGPT;
3
g
Meth;
NMe
Hansen
(86)
5
F
29

6
55.4

10.1
0.96
72
—
Met
8
Urinary
4-PA;
UB6,
Needs
to
take
into
4
1.52
90
18
UPNG,
PLP,
E-B6
account
the
PNG
0.96
72
High
PNG
8
Fecal
B
6
,
EALT,
content
of
the
1.45
84
Low
PNG
18
EAST
diet;
PNG
v15–
18%
vitamin
B
6
bioav.
Ribaya-Mercado
6
M
61
–70
94.8

64
—
—
2755

453
Natural
5
5
g
l-Tryp
1.90
–1.96
(139)
6
F
61
–71
66.7

4.1
0.003
0.8
g
/
kgbw
1918

30
Liq.
formula
20
Urinary
4-PA;
0.015
SS
21
PLP;
East-AC;
0.0225
SS
21
XA
.03375
SS
21
6
M
—
—
3051

232
Natural
5
6
F
0.003
1.2
g
/
kgbw
1980

120
Liq.
formula
20
0.015
SS
21
0.0225
SS
21
.03375
SS
21
Meydani
(184)
4
M
63.6

0.8
94.8

6.4
—
1.2
g
/
kgbw
—
Natural
5
5
g
l-Try
1.6
0.17

0.01
726.4

55.3
Liq.
formula
20
WBC;
PLP
XA;
M:
2.88

0.17
1.34

0.08
SS
21
PBMCs;
IL-2
F:
1.90

0.18
SS
21
SS
21
4
F
66.7

4.1
1.96

0.11
—
Natural
5
2.88

0.17
471.4

28.5
Liq.
formula
20
—SS
21
0.10

0.01
SS
21
0.89

0.08
SS
21
1.29

0.12
1.90

0.18
Vitamin B6 373
Huang
(150)
8
F
28
–34
61.8

5.4
1.60
1.55
g
/
kgbw
1576

110
Met
9
Urinary
4-PA;
PLP,
1.94
0.45
27
PL;
E-PLP,
PL,
1.26
21
PMP;
EALT-AC,
1.66
21
EAST-AC
2.06
14
Hansen
(151)
10
F
27.5

6.8
73.6

23.2
1.03
85
2000
Met
15
No
defic.
2
g
l-Try;
Urinary
1.33
–1.73
(70.016
1.33
12
4-PA,
UBy;
Fecal
mg/g)
1.73
12
B6
PLP,
TB6-E-
28.2

2.6
2.39
12
PLP
EALT,
6
F
69.3

12.8
0.84
12
EAST;
KA,
XA,
1.14
10
VA
2.34
10
Kretsch
(152)
4
F
21
–30
—
Variable
—
38
–42
kcal/
CF
4
4
g
l-Try
PLP,
0.015–0.020
mg/g
2
animal
kgbw
Formula
3
PAST,
PALT;
protein
0.05
protein
Formula
EAST,
EALT;
Uri-
(2)
1.55
g
/
kgbw
CF
14
nary
4-PA,
UTB6
0.5
CF
14
UFB6,
XA
1.0
(0.5)
CF
21
1.5
(1.0)
CF
14
2.0
(1.5)
4
F
Variable
—
CF
4
2
plant
protein
Formula
3
0.05
(2)
1.55
g/
kg
bw
Formula
11–
28
0.5
CF
14
1.0
(0.5)
CF
14
1.5
(1.0)
CF
21
2.0
(1.5)
CF
14
aFood
types:
The
types
of
foods
used
in
the
metabolic
study.
SS,
semisynthetic;
AR,
army
ration
stored
at
34°
C
(100°
F).
bDuration
refers
to
length
of
any
adjustment
(Adj)
period,
deficiency
(Defic)
period,
or
repletion
period
given
as
days.
Values
in
parentheses
are
milligrams
of
vitamin
B6
fed.
cTests:
Biochemical
tests
used
to
evaluate
vitamin
B6
status.
Abbreviations:
XA,
xantherneic
acid;
SGOT,
EGOT
serum
or
erythrocyte
glutamic
oxalacetate
transaminase;
NMe,
n-methylnicotinamide;
EEG,
electroencephalogram;
EKG,
electrocardiogram;
GPT
(WB),
glutamic
pyruvate
transaminase
in
whole
blood;
TM,
tryptophan
metabolites;
QA,
quinolinic
acid;
PLP,
pyridoxal-5?-phosphate;
4-PA,
4-pyridoxic
acid;
Try,
tryptophan;
Meth,
methionine;
PNG,
pyridoxine
glucoside,
IL-2,
interleukin-2.
NS,
none
suggested
by
authors.
374 Leklem
Table 15 Vitamin B6 Content of Human Milk
Vitamin B6 Vitamin B6
No. of Stage of intakec contentd
Ref. subjectsa lactationb (mg) (µmol/L)
Thomas et al. (282) 6 5–7 d 1.45 0.76
7 5–7 d 5.69 1.3
6 43–45 d 0.84 1.21
7 43–45 d 5.11 1.40
Roepke and Kirksey (283) 9 3 d 2.19 0.05
42 3 d 7.65 0.10
9 14 d 2.19 0.24
38 14 d 7.65 0.33
Sneed et al. (285) 7 5–7 d 1.52 0.72
9 5–7 d 5.33 1.46
7 43–45 d 1.41 0.70
9 43–45 d 5.21 1.42
Styslinger and Kirksey (286) 6 77 d 2.0 0.55
6 77 d 4.4 1.13
6 77 d 11.3 1.46
6 77 d 21.1 2.44
Barnji et al. (287) 27 6–30 d ND 0.12
26 7–12 mo ND 0.42
18 18 mo ND 0.37
Borschel et al. (296) 8 1–6 mo 3.6 0.87–1.25
9 1–6 mo 14.0 2.21–3.16
aNo. of subjects in which milk samples assayed.
bDays after birth.
cIntake of mother.
d1 µmol/L 
 169 µg/L.
ND, not determined.
values for the vitamin B6 content of human milk and shows the relationship between
vitamin B6 intake and the change in content over time of lactation. While a gradual increase
in vitamin B6 content of milk in the first several months of lactation has been observed
(288), a gradual decrease in content from 7 to 25 months of lactation has been reported
(289). Intake of vitamin B6 is reflected in milk vitamin B6 concentration, especially at
intakes above 5 mg/day. When considering the vitamin B6 requirement for infants and
lactating mothers, the recent publication by Borschel (290) provides a comprehensive
review. Borschel recommends a vitamin B6 intake greater than 10 mg/day (as PN-HCl)
for lactating mothers. An infant’s need in the first 112 days of life when growth is most
rapid is estimated to be 0.12–0.16 mg of vitamin B6 per day. Current requirements during
the first year of life may be set too high according to Borschel. The estimate for vitamin
B6 during growth of the infant may be estimated based on the recent recommendation of
Coburn, which is 0.02 nmol/g (119).
X. DISEASE AND TOXICITY
Several books (2,3,7,8) and reviews (6) have examined the relationship between specific
diseases and vitamin B6 nutrition in detail. There are numerous diseases or pathological
Vitamin B6 375
conditions in which vitamin B6 metabolism is altered. The primary indicator of an alteration
in vitamin B6 metabolism has been in evaluation of tryptophan metabolism or the
plasma PLP concentration. As previously discussed, the first of these is an indirect measure
of status and the second is a direct measure. Furthermore, using only PLP as a measure
really begs the question of whether vitamin B6 metabolism is actually altered. Conditions
in which tryptophan metabolism has been shown to be altered and in which vitamin B6
(pyridoxine) administration was used include asthma (299), diabetes (300), certain cancers
(173), pellagra (301), and rheumatoid arthritis (302). Diseases and pathological conditions
in which plasma PLP levels have been shown to be depressed include asthma (303), diabetes
(304), renal disorders (305), alcoholism (306), heart disease (307), pregnancy
(132,144,156,308), breast cancer (309), Hodgkin’s disease (310), and sickle cell anemia
(194). Hypophosphatasia is an example of a condition in which plasma PLP levels are
markedly elevated in some individuals (92). Relatively few of these studies have exhaustively
evaluated vitamin B6 metabolism.
Vitamin B6 in the form of pyridoxine hydrochloride has been used as a therapeutic
agent to treat a variety of disorders. Examples of disorders that have been treated with
pyridoxine include Down’s syndrome (311), autism (312), hyperoxaluria (313), gestational
diabetes (314), premenstrual syndrome (315,316), carpal tunnel syndrome (317,318), depression
(319), and diabetic neuropathy (329). It should be emphasized that the extent to
which pyridoxine was effective in treating these diseases or reducing symptoms has been
variable. In addition, in the treatment of these diseases, the amount of pyridoxine given
often varied, as did the length of time over which the pyridoxine was given. These two
variables, as well as the important consideration of whether a double-blind placebo-controlled
design was used, are necessary considerations in evaluating the effectiveness of
vitamin B6 therapy (129). Reynolds (321) has reviewed the use of vitamin supplements,
including vitamin B6, for the treatment of various diseases.
A. Coronary Heart Disease
A relationship between vitamin B6 and coronary heart disease can be viewed from both
an etiological perspective and that of the effect of the disease state on vitamin B6 metabolism.
With respect to an etiological role, an altered sulfur amino acid metabolism has been
suggested to result in vascular damage. A poor vitamin B6 status can result in an increased
circulating concentration of homocysteine (322). In the transsulfuration pathway, serine
and homocysteine condense to produce cystathionine. This reaction is catalyzed by the
PLP-dependent enzyme cystathionine ?-synthase. In genetic disorders of this enzyme,
homocysteine accumulates in the plasma (323). An increased incidence of arteriosclerosis
has been associated with this enzyme defect (324). In addition, elevated levels of homocysteine
in the plasma have been observed in people with ischemic heart disease (325–327).
There has been an explosion in the number of papers, suggesting that elevated plasma
homocysteine is a risk factor for heart disease and stroke. While folic acid is most effective
in reducing the plasma concentration of homocysteine (327,328), vitamin B6 has been
shown to be most effective in reducing plasma homocysteine when a methionine load is
given. A recent European study of 750 patients with vascular disease and 800 control
subjects found that increased fasting homocysteine (more than 12.1 µmol/L) was associated
with elevated risk of vascular disease (329). In this study, a plasma concentration of
PLP below the 20th percentile (less than 23 nmol/L) for controls was associated with
increased risk. This relationship between plasma PLP and atherosclerosis was independent
376 Leklem
of homocysteine levels. Other studies (326,328) have also found an increase in coronary
artery disease risk and low PLP levels in plasma.
While some animal experiments have shown that rhesus monkeys made vitamin
B6-deficient develop atherosclerotic lesions (243,330), other studies did not reveal any
pathological lesions (331). In humans at risk for coronary heart disease, a negative correlation
between dietary vitamin B6 and bound homocysteine has been observed (138). For
some people with homocystinuria, treatment with high doses of vitamin B6 reduces the
plasma concentration of homocysteine in certain patients but does not totally correct methionine
metabolism (332), especially when there is an increased methionine intake. Thus,
if vitamin B6 therapy is to be successful in reducing vascular lesions, diet modification
with a lowered methionine intake may be necessary. The extent to which supplemental
vitamin B6 intake (beyond normal dietary intakes) may reduce the risk for coronary heart
disease is not known.
A second aspect of coronary heart disease is the relationship between the presence
of the disease and vitamin B6 status. Several recent studies have found that the plasma
PLP concentrations in people with coronary heart disease are significantly lower (21–41
mnol/L) than in healthy controls (32–46 mnol/L) (250,307,333,334). However, Vermaak
et al. have found that the decrease in plasma PLP concentration is only seen in the acute
phase of myocardial infarction (335). Unfortunately, other measures of vitamin B6 status
have not been evaluated in this disease. In one study (334), cardiac patients given vitamin
B6 supplements (amounts not given) resulted in plasma PLP levels well above normal.
The effect of long-term vitamin B6 therapy on recurrence of coronary artery disease has
not been evaluated.
Elevated plasma cholesterol concentration has been strongly associated with an increased
risk for coronary heart disease. As previously reviewed, vitamin B6 may influence
cholesterol metabolism. Serfontein and Ubbink (250) have found that use of a multivitamin
supplement containing about 10 mg of pyridoxine for 22 weeks by hypercholesterolemic
adult men resulted in a significant decrease in cholesterol levels, with most of the reduction
due to a decreased level of LDL cholesterol. Smoking is an additional risk factor of coronary
heart disease. Interestingly, smokers have decreased plasma levels of PLP (250,336).
Evidence to date suggests a link between several risk factors for coronary heart disease
and altered vitamin B6 status and a potential beneficial effect of increased vitamin B6
intake on cholesterol levels. Furthermore, well-controlled studies are needed before the
therapeutic effect of vitamin B6 can be evaluated for this disease.
B. HIV/AIDS
Vitamin B6 status (337–340) and, to a limited extent, metabolism (341) has been examined
in persons with human immunodeficiency virus (HIV). Because of the link between immune
function and vitamin B6 (185), one would expect that maintaining an adequate vitamin
B6 status is critical for HIV patients. Several studies have evaluated vitamin B6 intake
(339,342,343) and the progression of the disease as related to intake of nutrients, including
vitamin B6 (342). These studies generally found low intakes of vitamin B6, and one study
(344) reported an inverse relationship between vitamin B6 intake and progression.
Biochemical assessment of vitamin B6 status has been made in several studies (337–
339,343) and been found to be low. In most of these studies (337–339), ?-EAST activity
and stimulation was used as an index of status. In the studies samples were frozen, which
may have compromised the data and subsequent evaluation. Although other researchers
Vitamin B6 377
have measured and reported low levels of ‘‘serum vitamin B6,’’ they failed to specify
what form was being measured (343,344). Therefore, given the complexities of nutritional
well-being in HIV/AIDS patients and methodological problems in these studies, it is diffi-
cult to assess the role of vitamin B6 in HIV/AIDS.
In vitro studies suggest that PLP may play a role in HIV/AIDS. Salhany and
Schopfer (345) found that PLP binds to the CD4 receptors at a site that is competitive
with a known antiviral agent, (4,4?-diisothiocyanato-2; 2?-stilbenedisulfonate). Other investigators
have found that PLP is a noncompetitive inhibitor of HIV-1 reverse transcriptase
(346,347). Based on these in vitro studies, clinical trials with vitamin B6 appear
warranted.
C. Premenstrual Syndrome
Premenstrual syndrome (PMS) is another clinical situation for which vitamin B6 supplementation
has been suggested (348). Estimates of 40% of women being affected by this
syndrome have been made (349). Using a wide variety of parameters, no difference in
vitamin B6 status was observed in women with PMS compared to those not reporting
symptoms (172,350,351). Nevertheless, beneficial effects of B6 administration on at least
some aspects of PMS have been reported.
Treatment of PMS with vitamin B6 has been based in part on the studies of Adams
et al. (352) in which PN was used to manage the depression observed in some women
taking oral contraceptives. Of the several studies in which PN was used to treat PMS,
there have been open-type studies that were double-blind placebo-controlled. Open studies
are prone to a placebo effect error, often as high as 40%. Of the well-controlled type,
one study showed no effect of pyridoxine therapy (353), whereas three studies reported
significant improvement of at least some of the symptoms associated with PMS. In one
study, 21 of 25 patients improved (354). The other study found that about 60% of 48
women showed improvement with pyridoxine (200 mg/day) and 20% showed improvement
with placebo (355). The fourth study (356) reported involvement in some symptoms
in 55 women treated daily with 150 mg of pyridoxine. Brush (348) reported results of
studies he has conducted using vitamin B6 alone and vitamin B6 plus magnesium. His
data suggest that doses of 150–200 mg of vitamin B6 are necessary before a significant
positive effect is observed. In addition, the combination of vitamin B6 plus magnesium
appears to be beneficial. The complexity of PMS and the subjective nature of symptom
reporting continue to result in contradictions and controversy in the lay and scientific
literature. Kleijnen et al. (357) have reviewed 12 controlled trials in which vitamin B6
was used to treat PMS. They concluded that there is only weak evidence of a positive
effect of vitamin B6. There may be a decrease in the availability of vitamin B6 during
PMS, possibly due to cell transport competition from fluctuating hormone concentrations.
An increase in vitamin B6 concentration could overcome competition and may explain the
relief of symptoms seen in some women following high-dose vitamin B6 supplementation.
D. Sickle Cell Anemia
Low levels (18 µmol/L) of plasma PLP have been reported in 16 persons with sickle cell
anemia (194). Treatment of 5 of these patients with 100 mg of pyridoxine hydrochloride
per day for 2 months resulted in a reduction of severity, frequency, and duration of painful
crises in these persons. The mechanism by which vitamin B6 acts is not known, but it
may be related to pyridoxal and PLP binding to hemoglobin.
378 Leklem
E. Asthma
Depressed levels of plasma and erythrocyte PLP have also been reported in persons with
asthma (303). Of significance was the fact that all persons were receiving bronchodilators.
Treatment of seven asthmatics with 100 mg of pyridoxine hydrochloride per day resulted
in a reduction in the duration, occurrence, and severity of their asthmatic attacks. Subsequent
work by one of these authors has not fully supported the earlier findings (358).
Treatment of 15 asthmatics with vitamin B6 did not result in a significant difference in
symptom scores, medication usage, or pulmonary function tests as compared to placebo
treatment. Ubbink et al. (359) have shown that theophylline lowers plasma and erythrocyte
PLP. Pyridoxal kinase is inhibited by theophylline and was responsible for the decreased
PLP level in the plasma and presumably intracellularly.
F. Carpal Tunnel Syndrome
At least five placebo-controlled trials from four different laboratories have shown that
administration of PN relieved the symptoms of carpal tunnel syndrome (pain and/or numbness
in hands) (317,360,361). In one study no significant improvement was observed (362).
Since supplementation with vitamin B6 well in excess of the RDA was required for improvement
(generally 50–150 mg), it would seem that individuals with this disorder have
a high metabolic demand or that the vitamin is active in some non-coenzyme role. Two
recent studies examined the relationship between plasma PLP and carpal tunnel syndrome.
One study (363) found no relationship between symptoms of carpal tunnel syndrome and
plasma PLP, but a study by Keniston et al. (364) found a significant inverse univariate
relationship between plasma PLP concentration and the prevalence of pain, the frequency
of tingling, and nocturnal awakening.
G. Drug–Vitamin B6 Interaction
Treatment of persons with various drugs may also compromise vitamin B6 status and
hence result in an increased need for vitamin B6. Table 16 lists several drugs and their
effect on vitamin B6 status. Bhagavan has reviewed these interactions in detail (365). A
common feature of these drug interactions is their adverse effect on central nervous system
function. In addition, many of these drugs react with PLP via a Schiff base formation.
Table 16 Drug–Vitamin B6 Interactions
Drug or drug Examples Mechanism of interaction
Hydrazines Iproniazid, isoniazid, hydralazine React with pyridoxal and PLP to form
a hydrazone
Antibiotic Cycloserine Reacts with PLP to form an oxime
l-DOPA l-3,4-dihydroxyphenylalanine Reacts with PLP to form tetrahydroquinoline
derivatives
Chelator Penicillamine Reacts with PLP to form thiazolidine
Oral contraceptives Ethinyl estradiol, mestranol, increased
enzyme levels in liver and other tissues;
retention of PLP
Alcohol Ethanol Increased catabolism of PLP, low
plasma levels
Vitamin B6 379
Table 17 Toxicity Symptoms Reported to Be Associated with Chronic Use of High-Dose
Pyridoxine
Ref. Symptoms
Coleman et al. (311) Motor and sensory neuropathy; vesicular dermatosis on regions of the
skin exposed to sunshine
Schaumburg (376) Peripheral neuropathy; loss of limb reflexes; impaired touch sensation
in limbs; unsteady gait; impaired or absent tendon reflexes; sensation
of tingling that proceeds down neck and legs
Brush (348) Dizziness; nausea; breast discomfort or tenderness
Bernstein (320) Photosensitivity on exposure to sun
This reaction can result in decreased levels of PLP in tissues, such as the brain, leading
to a functional deficiency. In most cases, supplemental vitamin B6 reverses the adverse
consequences of the drug. Oral contraceptives do not react directly with PLP but do induce
enzyme synthesis. Some of these enzymes are PLP-dependent and as a result PLP is metabolically
trapped in tissues. This may then lead to a depressed plasma PLP concentration
(366). In addition, the synthetic estrogens specifically affect enzymes of the tryptophan–
niacin pathway, resulting in abnormal tryptophan metabolism (200). There may be a need
for extra vitamin B6 above the current RDA in a small proportion of women using oral
contraceptives and consuming low levels of vitamin B6. Any drug that interacts with the
reactive molecule PLP in a Schiff base reaction should be considered an instigator of
resultant adverse effects on vitamin B6 status and a subsequent negative influence on
central nervous system function.
H. Hazards of High Doses
With the therapeutic use of pyridoxine for various disorders and self-medication has come
the potential problem of toxicity. Shaumburg et al. have identified several individuals who
developed a peripheral neuropathy associated with chronic high-dose use of pyridoxine
(367). Subsequent to this, other reports of toxicity related to pyridoxine ingestion have
been made (368,369). The minimal dose at which toxicity develops remains to be determined.
Other toxicity symptoms have been identified. These symptoms and those reported
by Schaumburg et al. are listed in Table 17. These symptoms are relatively rare, and the
use of pyridoxine doses of 2–250 mg/day for extended periods of time appears to be
safe (370).
In rats given high doses of pyridoxine hydrochloride for 6 weeks there was a decrease
in testis epididymis and prostate gland at the 500 and 1000 mg/kg dose (371). There
was also a decrease in mature spermatid counts. This high intake would be equivalent to
1.5–2.0 g of vitamin B6 for a human (372). Thus, the application of these data to human
nutrition is not clear.
XI. SUMMARY
In the more than 60 years since vitamin B6 was elucidated, a great deal of information
about its functional and metabolic characteristics has been gathered. The involvement of
the active form of vitamin B6, PLP, in such a wide spectrum of enzymatic reactions is
an indication of the importance of this vitamin. In addition to the involvement of PLP in
380 Leklem
amino acid metabolism and carbohydrate metabolism, its reactivity with proteins points
to the diversity of this vitamin. Further research is needed about the factors controlling
the metabolism of vitamin B6 and determination of vitamin B6 needs of specific population.
With knowledge of the functional properties of vitamin B6 and quantitation of the metabolism
of vitamin B6 under various physiological and nutritional conditions, the health and
well-being of individuals, can be improved.
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11
Biotin
DONALD M. MOCK
University of Arkansas for Medical Sciences, Little Rock, Arkansas
I. HISTORY OF DISCOVERY
Although a growth requirement for the ‘‘bios’’ fraction had been demonstrated in yeast,
Boas was the first to demonstrate the requirement for biotin in a mammal (1). In rats fed
protein derived from egg white, Boas observed a syndrome of severe dermatitis, hair loss,
and neuromuscular dysfunction known as ‘‘egg-white injury.’’ A factor present in liver
cured the egg-white injury and was named protective factor X. It is now recognized that
the critical event in this egg-white injury of both the human and the rat is the highly
specific and very tight binding (Kd 
 1015 M) of biotin by avidin, a glycoprotein found
in egg white. Native avidin is resistant to intestinal proteolysis in both the free and biotincombined
form. Thus, dietary avidin (e.g., in diets containing uncooked egg white) is
thought to bind and prevent the absorption of both dietary biotin and any biotin synthesized
by intestinal bacteria.
II. CHEMISTRY OF BIOTIN
A. Structure
As reviewed by Bhatia et al. (2) and Bonjour (1), the structure of biotin (Fig. 1) was
established by Ko?yl and his group in Europe and by du Vignraud and his collaborators
in the United States between 1940 and 1943. Because biotin has three asymmetrical carbons
in its structure, eight stereoisomers exist; of these, only one, designated d-()-biotin,
is found in nature and is enzymatically active. This compound is generally referred to
simply as biotin or d-biotin. Biocytin (?-N-biotinyl-l-lysine) is about as active as biotin
on a mole basis in mammalian growth studies.
Biotin is a bicyclic compound. One of the rings contains a ureido group (ENE
COENE), and the other contains sulfur and is termed a tetrahydrothiophene ring. The
397
398 Mock
Fig. 1 Biotin metabolism. The specific systems leading to the sulfoxides have not been defined.
ATP, adenosine triphosphate; AMP, adenosine monophosphate; HS-CoA, coenzyme A; PPi pyrophosphate;
*, site of attachment of carboxyl moiety. (Courtesy of International Life Sciences Institute,
Washington, D.C., M. Brown, ed.)
Biotin 399
two rings have a boat configuration with respect to each other. The tetrahydrothiophene
ring has a valeric acid side chain. Based on binding of biotin analogs by avidin and on
x-ray crystallography of the biotin–avidin complex (3), the ureido ring of the molecule
is the most important region in terms of the extraordinarily tight binding of biotin to avidin
and to streptavidin, a protein similar to avidin that is excreted by Streptomyces avidinii.
Other studies (3) suggest that the length of the side chain or the apolar nature of the
ECH2Emoieties in the side chain also play a role in the binding of biotin to the hydrophobic
site on avidin.
B. Chemical Synthesis of Biotin
The structure of biotin was confirmed by de novo chemical synthesis by Harrison and coworkers
in the 1940s (1). As reviewed recently (1,4), all of the early synthetic methods
suffered from the disadvantage that either the yield of the proper stereospecific isomer
was low or that special intermediates were required to obtain stereospecificity. These
shortcomings are avoided by the stereospecific synthesis developed by Goldberg and
Sternbach in 1949 in the laboratories of Hoffman-LaRoche (5). The Goldberg–Sternbach
synthesis, or modifications thereof, is the method by which biotin is synthesized commercially
(1). Additional stereospecific methods of synthesis have been published recently
(6,7).
III. PHYSIOLOGY OF BIOTIN
A. Digestion of Protein-Bound Biotin
Neither the mechanism of intestinal hydrolysis of protein-bound biotin nor the relationship
of the digestion of protein-bound biotin to its bioavailability has been clearly defined. The
content of free biotin and protein-bound biotin in foods is variable, but the majority of
biotin in meats and cereals appears to be protein-bound. Biotinidase is the enzyme that
catalyzes the cleavage of biotin from its covalent attachment to protein (e.g., the biotindependent
enzymes) during cellular turnover of these proteins. Wolf et al. have postulated
that biotinidase plays a critical role in the release of biotin from dietary protein (8). Biotinidase
in pancreatic juice might be responsible for release of biotin during the luminal phase
of proteolysis. Mucosal biotinidase might release biotin from biotinyl oligopeptides, the
presumed products of intestinal proteolysis. The role of mucosal biotinidase is not certain
because the activity is not enriched in intestinal brush-border membranes (8).
In view of the observations suggesting that a free carboxyl group is necessary for
binding to the intestinal biotin transporter (see discussion below), significant uptake of
biotinyl oligopeptides by the biotin transporter seems unlikely. This conclusion is supported
by recent studies on the uptake of biocytin (9) but is not universally accepted (10).
Alternatively, biotinyl oligopeptides might be absorbed directly by a nonspecific pathway
for peptide absorption.
In patients with biotinidase deficiency, doses of free biotin that do not greatly exceed
the estimated dietary intake (e.g., 50–150 µg/day) appear adequate to prevent the symptoms
of biotinidase deficiency, presumably by preventing biotin deficiency (11). These
observations are consistent with a mechanism in which biotinidase deficiency contributes
to biotin deficiency through impaired intestinal digestion of protein-bound biotin or impaired
renal salvage of ultrafiltered biotin or both.
400 Mock
B. Intestinal Absorption of Biotin
Early studies using low-specific-activity radiolabeled biotin and intact tissues, such as
everted gut sacs, concluded that intestinal and renal transport of biotin occurred by simple
diffusion (3). However, given the small amounts of biotin in foodstuffs and animal tissues,
and the efficient conservation of biotin by the body under most circumstances, simple
diffusion is teleologically unattractive (12). Within the last decade, studies using higher
specific activity radiolabeled biotin, brush-border membrane vesicles, and cell culture systems
have greatly expanded current knowledge of intestinal and renal transport of biotin
(3–17). A biotin transporter is present in the intestinal brush-border membrane. According
to most studies, the carrier is structurally specific, requiring both a free carboxyl group
on the valeric acid side chain and an intact ureido ring (12,17); however, not all structure–
activity studies (18) have confirmed the need for a free carboxyl group. Based on studies
with normal human tissue, human intestinal cell lines, and intestinal tissue from rabbit
and rat, the transport of biotin is temperature-dependent and occurs against a concentration
gradient. Biotin transport is electroneutral because of the 1:1 coupling of biotin
(RECOO) with Na. Biotin transport also occurs by simple diffusion; diffusion predominates
at higher (pharmacologic) concentrations. These recent studies of the biotin transporter
cast into doubt the conclusion drawn from earlier studies that biotinidase is the
principal biotin-binding protein in the intestinal brush-border membrane (3).
In rats, biotin transport is upregulated with maturation from the suckling to adult
rat, and the site of maximal transport by the biotin transporter increases aborally with
age, shifting from the ileum to the jejunum. Although carrier-mediated transport of biotin
was most active in the proximal small bowel of the rat, Bowman and Rosenberg (19)
concluded that the absorption of biotin from the proximal colon was still significant, supporting
the potential nutritional significance of biotin synthesized by enteric flora. Clinical
studies have also provided some evidence that biotin is absorbed from the human colon
(20,21).
In intact rats and Caco-2 cells, upregulation of biotin transport occurs in response
to biotin deficiency; the mechanism for most of the change appears to be an increased
Vmax (presumably mediated by an increased number of carriers) rather than a change
in carrier affinity. Based on studies of rabbit intestinal brush-border membrane transport,
histidine residues and sulfhydryl groups are important in the normal function of the
transporter. The histidine residues are probably located at or near the biotin binding site
(22).
In contrast to most other investigators, Leon-Del-Rio and co-workers observed only
passive diffusion of biotin in the rat, but saturable transport for biocytin. Moreover, they
reported saturable transport of biotin but passive diffusion of biocytin in the hamster (10).
They have proposed that protein-bound biotin is absorbed mainly in its free form in the
hamster but at least partially as biocytin in the rat.
The exit of biotin from the enterocyte (i.e., transport across the basolateral membrane)
is also carrier-mediated, but basolateral transport is independent of Na, is electrogenic,
and does not accumulate biotin against a concentration gradient (23). To investigate
the mechanism leading to reduced plasma biotin concentration in a substantial portion of
alcoholics, Said and co-workers (24) studied the effect of chronic ethanol feeding and
acute ethanol exposure on intestinal transport of biotin using everted intestinal sacs from
the rat. Acute ethanol exposure inhibited intestinal transport in biotin; chronic ethanol
feeding reduced intestinal transport of biotin and decreased plasma concentrations of bioBiotin
401
tin. These authors speculated that the effects of ethanol on intestinal transport of biotin
may contribute to the impaired biotin status associated with chronic alcoholism.
C. Transport of Biotin from the Intestine to Peripheral Tissues
Little has been definitively established concerning the transport of biotin to the liver and
peripheral tissues from the site of absorption in the intestine. Investigation of the binding
of biotin to proteins in plasma and serum has proceeded along two distinct lines: (a)
investigation of the biotin binding properties of biotinidase and (b) empirical assessment
of the covalent and reversible binding of biotin to whole plasma and fractionated plasma
proteins.
Wolf et al. (25) originally hypothesized that biotinidase might serve as a biotinbinding
protein in plasma or perhaps even as a carrier protein for the transport of biotin
into the cell. Chuahan and Dakshinamurti (26) provided evidence to support that hypothesis.
Biotin binding to purified biotinidase, albumin, ?- and ?-globulins, and fractionated
human serum was assessed using [3H]biotin, ammonium sulfate precipitation, and equilibrium
dialysis. These investigators concluded that biotinidase is the only protein in human
serum that specifically binds biotin. Others reached a different conclusion. Using [3H]biotin,
centrifugal ultrafiltration, and dialysis to assess reversible binding in plasma from the
rabbit, pig, and human (27,28), Mock and co-workers found that less than 10% of the total
pool of free plus reversibly bound biotin is reversibly bound to plasma macromolecules
(presumably proteins). A similar biotin-binding system was detected by Mock et al. (28)
in experiments with physiological concentrations of human serum albumin. Additional
studies determined the proportion of biotin covalently bound to plasma protein (27). Using
acid hydrolysis and [3H]biotinylalbumin to assess the completeness of biotin release, these
investigators found that an additional 12% of biotin was released by hydrolysis after extensive
dialysis. Those investigators concluded that the percentages of free, reversibly
bound, and covalently bound biotin in human serum are approximately 81% to 7% to
12% (27).
Results of the two approaches discussed above apparently conflict; the conflict may
arise from differences in the experimental approach, the definition of binding, or both.
The importance of either type of biotin binding to the transport of biotin from the intestine
to the peripheral tissues is not yet clear. The binding detected by Mock and co-workers
may represent a structurally nonspecific interaction between the hydrophobic portions of
the biotin molecule and one or more of the hydrophobic binding sites on a serum protein,
such as albumin. This binding may be analogous to the binding of apolar optical and
fluorescent dyes to the hydrophobic binding sites on albumin and avidin (29–34).
D. Transport of Biotin to the Liver
The uptake of biotin by liver and peripheral tissues from mammals has been the subject
of several investigations. Studies of 3T3-L1 fibroblasts (35), rat hepatocytes isolated by
collagenase profusion (17,36), basolateral membrane vesicles from human liver (37,38),
and Hep G2 human hepatoma cells (39) indicate that uptake of free biotin is mediated
both by diffusion and by a specialized carrier system that is dependent on an Na gradient,
temperature, and energy. Transport is electroneutral (Na/biotin 
 1:1) and specific for
a free carboxyl group, but transport is not strongly specific for the structure in the region
of the thiophene ring (39). Different results were found in studies of isolated cultured
hepatocytes (40); these cells did not exhibit a carrier-mediated transport system.
402 Mock
Additional studies in cultured rat hepatocytes demonstrated trapping of biotin, presumably
covalently bound in holocarboxylase enzymes (41). These studies confirm earlier
studies by McCormick and recapitulate the importance of metabolic trapping of watersoluble
vitamins as a mechanism for an intracellular accumulation (17). After entering
the hepatocyte, biotin diffuses into the mitochondria via a pH-dependent process (42).
Said and co-workers have postulated that biotin enters the mitochondria in the neutral
protonated form and dissociates into the anionic form in the alkaline mitochondrial environment,
thus becoming trapped by the charge (42).
E. Transport of Biotin into the Central Nervous System
Using an in situ rat brain perfusion technique and tracer [3H]biotin, Spector and Mock
(43) demonstrated that biotin is transported across the blood–brain barrier by a saturable
system; the apparent Km was about 100 µmol/L (a value several orders of magnitude
greater than the concentration of free biotin in plasma). Inhibition of transport by structural
analogs suggested that the free carboxylate group in biotin was important to transport,
presumably due to structural specificity of a biotin transport protein. Transfer of biotin
directly into the cerebral spinal fluid (CSF) via the choroid plexus did not appear to be
an important mechanism of biotin entry into the central nervous system. In additional
studies using either intravenous or intraventricular injection of [3H]biotin into rabbits (44),
Spector and Mock found that [3H]biotin was cleared from the CSF more rapidly than
mannitol, suggesting specific transport systems for biotin uptake into the neurons after
biotin crosses the blood–brain barrier. Whether infused intravenously or injected intraventricularly,
unlabeled biotin competed with [3H]biotin for transport. Two hours after intraventricular
injection, little metabolism of biotin or covalent binding to brain proteins was
observed; however, after 18 h, approximately one-third of the biotin had been incorporated
into brain protein. These findings suggest that biotin enters the brain by a saturable transport
system that does not depend on the subsequent metabolism of biotin or immediate
‘‘trapping’’ by incorporation into brain proteins.
Mock and co-workers have measured the concentrations of free ‘‘biotin’’ (i.e., total
avidin-binding substances) in human CSF and ultrafiltrates of plasma; the ratio was
0.85  0.5 for 11 subjects (3). This result is similar to the CSF plasma ratios determined
for biotin by Spector and Mock in the rabbit, an animal that has a specific system for
biotin transport across the blood–brain barrier (44).
F. Renal Handling of Biotin
Specific systems for the reabsorption of water-soluble vitamins from the glomerular filtrate
may make an important contribution to conservation of the water-soluble vitamins (12).
A biotin transport system has been identified by Podevin and Barbarat in both brush-border
and basolateral membrane vesicles from rabbit kidney cortex (45). Uptake by brush-border
membrane vesicles was saturable, occurred against a biotin concentration gradient, and
was dependent on an inwardly directed Na gradient. The Km was 28 µmol/L, and transport
exhibited structural specificity. In contrast, the uptake of biotin by basolateral membrane
vesicles was not sensitive to an Na gradient. In rat kidney, Spencer and Roth demonstrated
a similar system with a Km of 0.2 µmol/L that was inhibited by equimolar concentrations
of biocytin (46).
In vitro studies of biotin transport by renal tissue preparations in humans have not
been published. Baumgartner and co-workers have measured the renal clearance of biotin
in vivo and have calculated biotin/creatinine clearance ratios (47–49). In normal adults
Biotin 403
and children who are not receiving biotin supplementation, the clearance ratio is approximately
0.4. In patients with biotinidase deficiency, renal wasting of biotin and biocytin
occurs; biotin/creatinine clearance ratios typically exceed 1, and half-lives for biotin clearance
are about half of the normal value. The mechanism for the increased renal excretion
of biotin in biotinidase deficiency has not been defined, but this observation suggests that
there may be a role for biotinidase in the renal handling of biotin. For example, abnormal
plasma biotinidase might (a) bind biotin less tightly, increasing the glomerular sieving
coefficient; (b) serve less effectively as a reclamation transporter in the renal tubule; or
(c) alter cellular salvage of biotin during turnover of renal holocarboxylases.
G. Placental Transport of Biotin
Specific systems for transport of biotin from the mother to the fetus have recently been
reported (50–52). Studies using microvillus membrane vesicles and cultured trophoblasts
(50,51) detected a saturable transport system for biotin that was dependent on Na and
actively accumulated biotin within the placenta with slower release into the fetal compartment.
However, in the isolated perfused single cotyledon (50,51), transport of biotin across
the placenta was slow relative to placental accumulation. Little evidence of accumulation
on the fetal side was detected, suggesting that the overall placental transfer of biotin is
most consistent with a passive process. Membrane vesicle transport was sensitive to shortterm
exposure to ethanol, but overall transfer was not (51). Further studies using fetal
facing (basolateral) membrane vesicles detected a saturable, Na-dependent, electroneutral,
carrier-mediated uptake process that was not as active as the biotin uptake system in
the maternal facing (apical) membrane vesicles (52).
H. Transport of Biotin to Human Milk
Using an avidin-binding assay, Mock and co-workers have concluded that greater than
95% of the biotin (i.e., total avidin-binding substance) is present in the skim fraction of
human milk rather than in the cell pellet or fat fraction (53). Less than 3% of the biotin
was reversibly bound to macromolecules, and less than 5% was covalently bound to macromolecules.
Thus, almost all of the biotin in human milk is free in the aqueous compartment
of the skim fraction. The concentration of biotin in human milk remains fairly constant
between the fore, middle, and hind milk in the same feeding but varies substantially
over 24 h in some women (54). Though no single postpartum pattern is followed by all
women, a steady increase in the biotin concentration was observed during the first 18 days
post partum in half of the women studied. However, rather than reaching a stable plateau
in mature milk, biotin concentrations vary substantially in most women after 18 days post
partum. The cause of this variation is not known.
When separated from other biotin catabolites by high-performance liquid chromatography
(HPLC) and measured by an avidin-binding assay, the concentration of biotin
in the aqueous phase of human milk exceeds the concentration in serum by one to two
orders of magnitude (55). It seems likely that a transport system exists that conveys biotin
from the plasma to human milk against a concentration gradient.
Measurements of the biotin and metabolite in human milk using an HPLC separation
and avidin-binding assay indicate that bisnorbiotin accounts for approximately 50% and
biotin sulfoxide about 10% of the total biotin plus metabolites in early and transitional
human milk.With maturation post partum the biotin concentration increases, but the bisnorbiotin
and biotin sulfoxide concentrations still account for 25% and 8% at 5 weeks post
partum (55). Studies to date provide no evidence for trapping by a biotin-binding protein.
404 Mock
IV. SPECIFIC FUNCTIONS
In mammals, biotin serves as an essential cofactor for four carboxylases, each of which
catalyzes a critical step in intermediary metabolism (see Chapter 5). All four of the mammalian
carboxylases catalyze the incorporation of bicarbonate into a substrate as a carboxyl
group. Four similar carboxylases, two other carboxylases, two decarboxylases, and a transcarboxylase
are found in nonmammalian organisms. All of these biotin-dependent enzymes
appear to work by a similar mechanism.
A. Incorporation into Carboxylases
Attachment of the biotin to the apocarboxylase (Fig. 1) is a condensation reaction catalyzed
by holocarboxylase synthetase. The holocarboxylase synthetase reaction is driven
thermodynamically by hydrolysis of ATP to inorganic phosphate. An amide bond is
formed between the carboxyl group of the valeric acid side chain of biotin and the ?-
amino group of a specific lysyl residue in the apocarboxylase; these regions of the protein
backbone contain sequences of amino acids that tend to be highly conserved within and
between species for the individual carboxylases.
Holocarboxylase synthetase (EC 6.3.4.10) is present in both the cytosol and the
mitochondria. Studies of human mutant holocarboxylase synthetase indicate that both the
mitochondrial and cytoplasmic forms are encoded by one gene (57–60). However, other
investigators have concluded that two different holocarboxylase synthetases catalyze the
biotinylation of the mitochondrial and cytosolic carboxylases in rat and chicken liver (61–
63). This conclusion is based on differences in pH optima, nucleotide specificity, and
tetrapeptide sequence in the areas containing the lysine residues, as well as on site-specific
mutagenesis studies.
B. Regulation of Intracellular Carboxylase Activity by Biotin
Currently, the role of biotin in regulating activity of the four mammalian carboxylases at
the gene level remains to be elucidated. However, the interaction of biotin synthesis and
production of holoacetyl-CoA carboxylase in Escherichia coli has been extensively studied
and reviewed (64,65). The biotin-protein ligase (specifically a holoacetyl-CoA carboxylase
synthetase in E. coli) catalyzes formation of the covalent bond between biotin and
a specific lysine residue in the biotin carboxylase carrier protein (BCCP) of acetyl-CoA
carboxylase. The biotin binding domain of BCCP of E. coli acetyl-CoA carboxylase has
been sequenced (66). As with the four mammalian carboxylases (Fig. 1), the biotinylation
of the apocarboxylase proceeds in two steps. First, holocarboxylase synthetase reacts with
biotin and ATP to form a complex between the synthetase and biotinyl-AMP, releasing
pyrophosphate. The stoichiometric amounts of biotinyl-AMP are synthesized quickly and
diffuse off the enzyme slowly in the absence of apocarboxylase to complete the two-step
reaction (67). If a suitable amount of the BCCP portion of acetyl-CoA carboxylase is
present, the holocarboxylase is formed and AMP is released. If insufficient apocarboxylase
is present, the holocarboxylase synthetase:biotinyl-AMP complex acts to repress further
synthesis of biotin by binding to the promoter regions of the biotin operon (‘‘bio’’). These
promoters control a cluster of genes that encode enzymes that catalyze biotin synthesis;
these enzymes include biotin synthetase, the enzyme complex that converts dethiobiotin
to biotin. In its role as a repressor of the bio operon, the holocarboxylase synthetase has
been named BirA; this name arose from initial observations on biotin intracellular retenBiotin
405
tion properties and was found to be allelic to repression of biotin synthesis by biotin
(‘‘bioR’’). Biotinyl-AMP acts as a corepressor through its role in the BirA:biotinyl-AMP
complex. Thus, the rate of biotin synthesis is responsive to both the supply of apo-BCCP
and the supply of biotin as reflected in the biotinyl-AMP concentration.
Additional research has focused on conversion of dethiobiotin (or an earlier precursor
pimelic acid) to biotin (68). This is an unusual enzymatic reaction that closes the
tetrahydrothiophene ring by inserting a sulfur. These studies used E. coli or Bacillus
sphaericus bioB transformants that overproduce biotin. Ifuku and co-workers have sequenced
several point mutations leading to bioB transformants in E. coli (69). Such studies
have provided evidence that biotin synthetase is a two-iron/two-sulfur enzyme that requires
NADPH, S-adenosylmethionine, and Fe3 or Fe2 (70–72). Flavodoxin is also required,
probably as an electron donor (73). The source of the sulfur in the thiophene ring
is probably cysteine or a derivative (74–76). Additional studies used cell-free systems to
establish the cofactor and metal ion requirements (74,75,77). Alanine and acetate serve
as carbon sources; carbon dioxide liberated via the tricarboxylic acid cycle may serve as
well (78). The first eukaryotic biotin synthetase has now been cloned and sequenced from
the yeast Saccharomyces cerevisiae (79).
C. The Four Mammalian Carboxylases
In the carboxylase reaction, the carboxyl moiety is first attached to biotin at the ureido
nitrogen opposite the side chain; next, the carboxyl group is transferred to the substrate.
The reaction is driven by the hydrolysis of ATP to ADP and inorganic phosphate. Subsequent
reactions in the pathways of the four mammalian carboxylases release CO2 from the
products of the various carboxylase reactions. Thus, these reaction sequences rearrange the
substrates into more useful intermediates but do not violate the classic observation that
mammalian metabolism does not result in the net fixation of carbon dioxide. The mechanism
for the carboxylase reaction is discussed in detail in Chapter 5, ‘‘Bioorganic Mechanisms
Important to Coenzyme Functions,’’ by Donald McCormick.
Three of the four biotin-dependent carboxylases are mitochondrial; the fourth (acetyl-
CoA carboxylase, ACC) is found in both the mitochondria and the cytosol. Based on
a series of observations by Allred and co-workers (63,80–83), ACC (EC 6.4.1.2) exists
in an active cytosolic form and two largely inactive mitochondrial forms with molecular
weights of approximately 264 and 234 kDa, respectively. The mitochondrial forms are
postulated to serve as storage forms that leave the mitochondria and are transformed into
the active cytosolic ACC during periods of restricted biotin availability. The timing of
decrease of the mitochondrial form during maintenance of the activity of cytosolic ACC
is consistent with the hypothesis of Allred and co-workers (63). However, the reported
maintenance of normal amounts of the three mitochondrial enzymes after 4 weeks of eggwhite
feeding is not consistent with the reports of abnormal organic aciduria after as little
as 2 weeks of egg-white feeding in rats (84) and humans (85).
ACC catalyzes the incorporation of bicarbonate into acetyl-CoA to form malonyl-
CoA (Fig. 2). This three-carbon compound then serves as a substrate for the fatty acid
synthetase complex. The net result is the elongation of the fatty acid substrate by two
carbons and the loss of the third carbon as CO2.
Pyruvate carboxylase (PC; EC 6.4.1.1) catalyzes the incorporation of bicarbonate
into pyruvate to form oxaloacetate, an intermediate in the Kreb’s tricarboxylic acid cycle
(Fig. 2). Thus, PC catalyzes an anapleurotic reaction. In gluconeogenic tissues (i.e., liver
406 Mock
Fig. 2 Lactate and pyruvate metabolism. Deficiencies of PC and ACC can redirect intermediates
into unusual pathways . Abbreviations as in Fig. 1. ADP, adenosine diphosphate; Pi,
inorganic phosphate.
and kidney), the oxaloacetate can be converted to glucose. Deficiency of PC has been
proposed as the cause of the lactic acidemia, central nervous system lactic acidosis, and
abnormalities in glucose regulation observed in biotin deficiency and biotinidase defi-
ciency as discussed below.
Methylcrotonyl-CoA carboxylase (MCC; EC 6.4.1.4) catalyzes an essential step in
the degradation of the branch-chained amino acid leucine (Fig. 3). Deficient activity of this
enzyme (whether due to the isolated genetic deficiency, multiple carboxylase deficiency, or
biotin deficiency per se) leads to metabolism of its substrate 3-methylcrotonyl-CoA by
an alternate pathway to 3-hydroxyisolvaleric acid, 3-methylcrotonylglycine, or both
(3,86). Thus, increased urinary excretion of these abnormal metabolites in the urine reflects
deficient activity of MCC and can reflect biotin depletion at the tissue level in genetically
normal individuals.
Propionyl-CoA carboxylase (PCC; EC 6.4.1.3) catalyzes the incorporation of bicarbonate
into propionyl-CoA to form methylmalonyl-CoA, which undergoes isomerization
to succinyl-CoA and enters the tricarboxylic acid cycle (Fig. 4). The three-carbon propionic
acid moiety originates from several sources:
1. Catabolism of the branch-chained amino acids isoleucine, valine, methionine,
and threonine
2. The side chain of cholesterol
3. Oxidation of odd chain length saturated fatty acids, and
4. Metabolism of dietary carbohydrate by intestinal flora
In a fashion analogous to MCC deficiency, deficiency of PCC leads to increased
urinary excretion of 3-hydroxypropionic acid and 2-methylcitric acid (3,86).
PCC consists of 12 subunits: 6 ? subunits and 6 ? subunits (87,88). Each subunit
is transported into the mitochondria, cleaved to a smaller ‘‘mature’’ subunit, and assemBiotin
407
Fig. 3 Leucine degradation. A deficiency of MCC causes increased urinary excretion of 3-
methylcrotonylglycine and 3-hydroxyisovaleric acid. Abbreviations as in Fig. 1 and 2.
Fig. 4 Propionate metabolism. Propionate is derived primarily from amino acid degradation and
to a lesser extent from cholesterol and odd-chain fatty acids, a deficiency of PCC causes increased
urinary excretion of methylcitric and 3-hydroxypropionic acids. Abbreviations as in Fig.
1–3.
408 Mock
bled into the active enzyme. Inborn errors affecting the two subunits lead to the two major
complementation groups (pcc A and pcc BC) of propionic acidemia (89). Differential
rates of degradation of the ? and ? subunits explain observations concerning the presence
or absence of the corresponding mRNA, synthesis rates of the subunits, and steady-state
levels of the subunits (90). The ? subunit can be transported to the mitochondria before
or after biotinylation in the cytosol, providing evidence that biotinylation is not required
for transport of carboxylase subunits into mitochondria and that holocarboxylase synthetase
is active in both cytosol and mitochondria (91). Glycine and valine residues near the
biotinylated lysine residue are highly conserved among all known biotinylated peptides
and among many lipoylated proteins as well. One interpretation concerning conservation
of this amino acid sequence is that these residues allow the biotinylated (or lipoylated)
peptide to swing the carboxyl (or acetyl) group from the site of activation to the receiving
substrate (91). Indeed, there is a close functional analogy between these two enzyme cofactors.
Each has a valeric acid side chain joined to a lysine residue on one end; the other
end joins to a complex sulfur containing ring(s) that accept and transfer the activated
moiety (i.e., carboxyl for the carboxylases and acetyl for the pyruvate dehydrogenase
complex). Avidin binds to the lipoyl domains in the pyruvate dehydrogenase complex
(92). However, binding of lipoic acid analogs to avidin appears to be weak compared to
biotin binding to avidin because the interaction is not detectable in our sequential, solidphase
avidin-binding assay (Zempleni, Mock, and McCormick, unpublished observations).
Genetic deficiencies of holocarboxylase synthetase and biotinidase cause the two
distinct types of multiple carboxylase deficiency that were previously designated as the
neonatal and juvenile forms. Biotinidase deficiency is discussed in several sections of
this chapter because studies of these patients have provided important insights into biotin
nutrition and the pathogenesis of the clinical findings of biotin deficiency. In the normal
turnover of cellular proteins, holocarboxylases are degraded to biotin linked to lysine
(biocytin) or biotin linked to an oligopeptide containing at most a few amino acid residues
(Fig. 1). Because the amide bond between biotin and lysine is not hydrolyzed by cellular
proteases, a specific hydrolase is required to release biotin for recycling. This enzyme is
biotinidase (biotin–amide hydrolase, EC 3.5.1.12). Current opinion is that serum biotinidase
and lipoamidase (the enzyme that cleaves lipoic acid from the dihydrolipoyl dehydrogenase
component of the various multienzyme ?-keto acid dehydrogenase complexes) are
the same enzyme. This conclusion is based on the following observations: (a) greater than
95% inactivation of lipoamidase by a monospecific polyclonal antibody against biotinidase
(93) and (b) less than 2% of normal lipoamidase activity in the serum of patients with
biotinidase deficiency (93,94).
The gene for human biotinidase has been cloned, sequenced,and characterized (95).
The biotinidase gene is a single-copy gene of 1629 bases encoding a 543-amino-acid
protein; the mRNA is present in multiple tissues, including heart, brain, placenta, liver,
lung, skeletal muscle, kidney, and pancreas. Highest biotinidase activities are found in
serum, liver, kidney, and adrenal gland. On the basis of decreased serum concentrations
of biotinidase in patients with impaired liver function, Grier et al. have concluded that
the liver is the source of serum biotinidase (96).
D. Biotin Catabolism
Instead of being incorporated into carboxylases after entering the pools of biotin and its
intermediary metabolites, dietary biotin or biotin released by carboxylase turnover may
Biotin 409
be catabolized. For example, biotinyl-AMP can be converted to biotinyl-CoA by biotinyl-
CoA synthetase (97,98). Biotinyl-CoA synthetase also catalyzes the formation of biotinyl-
AMP from biotin and ATP; thus, biotinyl-CoA synthetase catalyzes a two-step process
converting biotin to biotinyl-CoA. The relation between biotinyl-CoA synthetase and holocarboxylase
synthetase, as well as the existence and location of intracellular pools of
biotinyl-AMP and biotinyl-CoA (if any), remains unclear. Biotinyl-CoA is oxidized to
bisnorbiotin and tetranorbiotin (metabolites with two and four fewer carbons in the valeric
acid side chain, respectively; Fig. 1).
Contrary to the tacit assumption of many early biotin balance studies (3), it now
appears that about half of biotin undergoes metabolism before excretion, and thus a significant
proportion of the total avidin-binding substances in human urine and plasma and
rat urine is attributable to biotin metabolites rather than to biotin per se. The findings of
a pioneering study of biotin metabolites in human urine (99) using paper chromatography
and bioassays have been confirmed by recent studies that take advantage of the greater
sensitivity and reproducibility of HPLC separation and avidin-binding assays (3,100,101).
Such studies have indicated that biotin, bisnorbiotin, and biotin sulfoxide are present in
mole ratios of approximately 3:2:1 in human urine and plasma. Recent observations provide
evidence that biotin catabolism is induced in some individuals during pregnancy and
by anticonvulsants, thereby increasing the ratio of biotin catabolites to biotin (102,103).
As discussed below, these observations emphasize the importance of distinguishing biotin
from its catabolites when assaying physiological fluids.
E. Other Roles for Biotin
Effects of biotin on cell growth, glucose homeostasis (104,105), DNA synthesis, and expression
of the asialoglycoprotein receptor (106) have been reported. For these effects, a
direct relationship to biotin’s role as a cofactor for the four carboxylases has not been
defined. Whether one or more of the effects will ultimately prove to be the indirect result
of carboxylase deficiency remains unclear.
V. REQUIREMENT AND ASSESSMENT
A. Circumstances Leading to Deficiency
That the normal human has a requirement for biotin has been clearly documented in two
situations: (a) prolonged consumption of raw egg white and (b) parenteral nutrition without
biotin supplementation in patients with short-gut syndrome and other causes of malabsorption
(3). Biotin deficiency also has been clearly demonstrated in biotinidase deficiency
(107). The mechanism by which biotinidase deficiency leads to biotin deficiency probably
involves several processes:
1. Gastrointestinal absorption of biotin may be decreased because deficiency of
biotinidase in pancreatic secretions leads to inadequate release of protein-bound
biotin.
2. Salvage of biotin at the cellular level may be impaired during normal turnover
of proteins to which biotin is linked covalently.
3. Renal loss of biocytin and biotin is abnormally increased.
The clinical findings and biochemical abnormalities caused by biotinidase deficiency
are similar to those of biotin deficiency; the common findings include periorificial derma410
Mock
titis, conjunctivitis, alopecia, ataxia, and developmental delay (107,108). These clinical
similarities support the hypothesis that the pathogenesis of biotinidase deficiency involves
a secondary biotin deficiency. However, the reported signs and symptoms of biotin defi-
ciency and biotinidase deficiency are not identical. Seizures, irreversible neurosensory
hearing loss, and optic atrophy have been observed in biotinidase deficiency (109–111)
but have not been reported in biotin deficiency. However, cerebral atrophy and apparent
stretching of the optic nerve have been reported in one patient with biotin deficiency (112).
Moreover, Heard et al. have reported that biotin deficiency causes impaired auditory brain
stem function in young rats (113).
On the basis of lymphocyte carboxylase activity and plasma biotin levels, Velazquez
and co-workers have reported that biotin deficiency occurs in children with severe protein
energy malnutrition (114,115). These investigators have speculated that the effects of biotin
deficiency may be responsible for part of the clinical syndrome of protein energy
malnutrition.
Accumulating data provide evidence that long-term anticonvulsant therapy in adults
can lead to biotin depletion and that the depletion can be severe enough to interfere with
amino acid metabolism. In the initial reports, Krause and co-workers (116,117) reported
decreased plasma concentrations of biotin (as determined using the Lactobacillus plantarum
bioassay). The later demonstration of increased urinary excretion of 3-hydroxyisovaleric
acid in some adults receiving long-term anticonvulsant therapy (118) has been
confirmed by Mock and Dyken (103) and provides evidence that biotin is depleted at the
tissue level.
The mechanism of biotin depletion during anticonvulsant therapy is not known. The
anticonvulsants implicated include phenobarbital, phenytoin, carbamazepine, and primidone.
These drugs each have a carbamide (ENHECOE) moiety in their structures, as
does biotin; in some cases, they incorporate a full ureido group (ENHECOENHE).
Said and co-workers (16,119) have demonstrated that therapeutic concentrations of primidone
and carbamazepine specifically inhibit biotin uptake by brush-border membrane vesicles
from human intestines; these investigators have suggested that these anticonvulsants
compete with biotin for binding to the intestinal transporter leading to biotin malabsorption
and biotin deficiency. Mock and co-workers have recently reported substantial increases
in the urinary excretion of biotin catabolites, especially bisnorbiotin, in these patients
(103,120). These urinary losses are sufficient to waste a substantial proportion of dietary
intake and thus eventually deplete total-body pools of biotin; hence, these investigators
have suggested that accelerated catabolism of biotin may also contribute to reduced biotin
status in these individuals (103). Chuahan and Dakshinamurti (26) have also demonstrated
that phenobarbital, phenytoin, and carbamazepine displace biotin from biotinidase. In this
manner, these anticonvulsants could conceivably affect plasma transport of biotin, renal
handling of biotin, or cellular uptake of biotin.
Biotin deficiency has also been reported or inferred in several other circumstances:
1. Leiner’s disease: A severe form of seborrheic dermatitis that occurs in infancy.
Although a number of studies have reported prompt resolution of the rash with biotin
therapy (3), biotin was ineffective in the only double-blind therapeutic trial (121).
2. Sudden infant death syndrome: Biotin deficiency in the chick produces a fatal
hypoglycemic disease dubbed ‘‘fatty liver–kidney syndrome’’; impaired gluconeogenesis
due to deficient activity of PC is the cause of the hypoglycemia. Hood et al. have proposed
that biotin deficiency may cause sudden infant death syndrome (SIDS) by an analogous
pathogenic mechanism (122,123). They have supported their hypothesis by demonstrating
Biotin 411
that hepatic biotin is significantly lower at autopsy in SIDS infants than in infants dying
from other causes. Additional studies (e.g., levels of hepatic PC, urinary organic acids,
and blood glucose) are needed to confirm or refute this hypothesis.
3. Pregnancy: Concerns about the teratogenic effects of biotin deficiency (see below)
have led to studies of biotin status during human gestation. Some of these studies
have detected low plasma concentrations of biotin; others have not (3). Recent studies by
Mock et al. have detected increased urinary excretion of 3-hydroxyisovaleric acid in more
than half of normal women by the third trimester of pregnancy; urinary excretion of biotin
was abnormally decreased in about half of the pregnant women studied (102).
4. Dialysis: Patients undergoing chronic hemodialysis have been reported to have
low plasma concentrations of biotin (124). Yatzidis et al. (125) have reported nine patients
on chronic hemodialysis who developed encephalopathy (four patients) or peripheral neuropathy
(five patients); all responded to biotin therapy. Blood concentrations of biotin and
lactic acid and urinary excretion rates of the characteristic organic acids were not reported.
Braguer and co-workers have reported that microtubule formation, at least in vitro, is
inhibited by toxins partially purified from the plasma of uremic patients (126). This inhibition
is completely reversed by the addition of biotin. These investigators speculated that
impaired tubulin formation might be involved in uremic neuropathy and that reversal of
impaired tubulin formation could be a partial explanation for the clinical improvement
seen with biotin in uremic neuropathy. However, other investigators have reported that
plasma and red cell concentrations of biotin are significantly increased in patients receiving
chronic hemodialysis (127). The etiological role of biotin in uremic neurological disorders
and the general applicability of these results remain to be determined.
5. Gastrointestinal diseases or alcoholism: Reduced blood or liver concentrations
of biotin or urinary excretion of biotin have been reported in alcoholism (1,128), gastric
disease (1), and inflammatory bowel disease (129).
6. Brittle nails: Because pathological hoof changes in horses and swine have responded
to biotin supplementation, Colombo and co-workers treated women with brittle
fingernails with 2.5 mg biotin per day orally (130). They reported a 25% increase in nail
thickness and improved morphology by electron microscopy. The biotin status of the subjects
was not assessed.
B. Clinical Findings of Frank Deficiency
Whether caused by egg-white feeding or omission of biotin from total parenteral nutrition,
the clinical findings of frank biotin deficiency in adults and older children have been
similar to those reported by Sydenstricker in his pioneering study of egg-white feeding
(3,131). Typically, the findings began to appear gradually after weeks to several years for
egg-white feeding Six months to 3 years typically elapsed between the initiation of total
intravenous feeding without biotin and the onset of the findings of biotin deficiency
(3,132). Thinning of hair, often with loss of hair color, was reported in most patients. A
skin rash described as scaly (seborrheic) and red (eczematous) was present in the majority;
in several, the rash was distributed around the eyes, nose, and mouth. Depression, lethargy,
hallucinations, and paresthesias of the extremities were prominent neurological symptoms
in the majority of adults.
In infants who developed biotin deficiency, the signs and symptoms of biotin defi-
ciency began to appear 3–6 months after initiation of total parenteral nutrition. This earlier
onset may reflect an increased biotin requirement because of growth. The rash typically
412 Mock
appeared first around the eyes, nose, and mouth; ultimately, the ears and perineal orifices
were involved (‘‘periorificial’’). The appearance of the rash was similar to that of cutaneous
candidiasis (i.e., an erythematous base and crusting exudates); typically, Candida
could be cultured from the lesions. The rash of biotin deficiency is similar in appearance
and distribution to the rash of zinc deficiency. In infants, hair loss was noted after 6–9
months of parenteral nutrition; within 3–6 months of the onset of hair loss, two infants
had lost all hair, including eyebrows and lashes. These cutaneous manifestations, in conjunction
with an unusual distribution of facial fat, have been dubbed ‘‘biotin deficiency
facies.’’ The most striking neurological findings in biotin-deficient infants were hypotonia,
lethargy, and developmental delay. A peculiar withdrawn behavior was noted and may
reflect the same central nervous system dysfunction diagnosed as depression in adult patients.
C. Laboratory Findings of Biotin Deficiency
1. Methodology for Measuring Biotin
Methods for measuring biotin at pharmacological and physiological concentrations have
been reviewed (3). In this chapter, assays for biotin and metabolites will be discussed
briefly with two objectives in mind: (a) to provide the reader with sufficient knowledge
of methods to understand the implications of recent studies of biotin nutritional status and
(b) to review recent analytical advances.
For measuring biotin at physiological concentrations (i.e., 100 pmol/L to 100
nmol/L), a variety of assays have been proposed, and a limited number have been used
to study biotin nutriture. All the published studies of biotin nutriture have used one of
three basic types of biotin assays: bioassays (most studies), avidin-binding assays (several
recent studies), or fluorescent derivative assays (two published studies).
Bioassays generally have adequate sensitivity to measure biotin in blood and urine.
Precision is limited for the turbidity methods, but a recent modification of the L. plantarum
assay uses agar plates previously injected with L. plantarum to obtain better precision
(133). Radiometric bioassays offer both sensitivity and precision. However, the bacterial
bioassays (and perhaps the eukaryotic bioassays as well) suffer interference from unrelated
substances and variable growth response to biotin analogs; these bioassays can give con-
flicting results if biotin is bound to protein (3). For some bioassay organisms, prior acid
or enzymatic hydrolysis (or both) is required to release the biotin from protein and thus
make the biotin available to the assay organism. For other organisms (e.g., Klockera
brevis), the detectable biotin decreases with enzymatic hydrolysis (134), suggesting that
some destruction of biotin may occur during acid hydrolysis (53).
Avidin-binding assays generally measure the ability of biotin to do one of the following:
(a) to compete with [3H]biotin, [125I]biotin, or [14C]biotin for binding to avidin (isotope
dilution assays), (b) to bind to [125I]avidin and thus prevent [125I]avidin or enzyme-coupled
avidin from binding to a biotinylated protein adsorbed to plastic (sequential, solid phase
assay), or (c) to prevent the binding of a biotinylated enzyme to avidin and thereby prevent
the consequent inhibition of an enzyme activity. Other methods detect the postcolumn
enhancement of fluorescence activity caused either by the mixing of the column eluate
with fluorescent-labeled avidin or derivatization of biotin and metabolites by a fluorescent
agent before separation by HPLC (135,136). Avidin-binding assays using novel detection
systems, such as electrochemical detection (137), bioluminescence linked through glucose-
6-phosphate dehydrogenase (138), or a double-antibody technique (139), have been reBiotin
413
ported recently and may offer some advantages in terms of sensitivity. Avidin-binding
assays have been criticized for being cumbersome, requiring highly specialized equipment
or reagents, or performing poorly when applied to biological fluids. Avidin-binding assays
detect all avidin-binding substances, although the relative detectability of biotin and analogs
varies between analogs and between assays, depending on how the assay is conducted
(e.g., competitive vs. sequential). In a manner analogous to the pioneering work of Wright
et al. (99), assays that couple chromatographic separation of biotin analogs with subsequent
avidin-binding assays of the chromatographic fractions are both sensitive and chemically
specific. These assays have been used in several recent studies that provide new
insights into biotin nutrition.
A problem in the area of biotin analytical technology that remains unaddressed is
the disagreement among the various bioassays and avidin-binding assays concerning the
true concentration of biotin in human plasma. Reported mean values range from approximately
500 pmol/L to more than 10,000 pmol/L.
Although commonly used to assess biotin status in a variety of clinical populations,
the putative indices of biotin status had not been previously studied during progressive
biotin deficiency. To address this issue, Mock and co-workers (85) induced progressive
biotin deficiency by feeding egg white; then the urinary excretion of biotin, bisnorbiotin,
and 3-hydroxyisovaleric acid was measured. The urinary excretion of biotin declined dramatically
with time on the egg-white diet, reaching frankly abnormal values in eight of
nine subjects by day 20 of egg-white feeding. Bisnorbiotin excretion declined in parallel,
providing evidence for regulation of catabolism of the biotin metabolic pools. By day 14
of egg-white feeding, 3-hydroxyisovaleric acid excretion was abnormally increased in all
nine subjects, providing evidence that biotin depletion decreases the activity of the biotindependent
enzyme MCC and alters intermediary metabolism of leucine earlier in the
course of experimental biotin deficiency than previously appreciated. The time course for
development of metabolic abnormalities was similar to that observed in the egg-white–
fed rat (84,140). Serum concentrations of free biotin as measured by HPLC separation
and avidin-binding assay decreased to abnormal values in less than half of the subjects.
Thus, these studies provide objective confirmation of the impression of many investigators
in this field (141) that blood biotin concentration is not an early or sensitive indicator of
impaired biotin status.
Plasma concentrations of biotin (i.e., total avidin-binding substances) are higher in
term infants than older children and, for reasons that are not simply related to dietary
intake, decline after 3 weeks of breast-feeding or feeding a formula containing 11 µg/L
of biotin. Infant formulas supplemented with 300 µg/L produce plasma concentrations
approximately 20-fold greater than normal (142); consequences of these higher levels, if
any, are unknown.
Odd-chain fatty acid accumulation is a marker of biotin deficiency, as shown by
the work of Kramer et al. (143), Suchy et al. (144), and Mock et al. (145). These groups
independently demonstrated increases in the percentage composition of odd-chain fatty
acids (e.g., 15:0, 17:0, etc.) in hepatic, cardiac, or serum phospholipids in the biotindeficient
rat; similar accumulation has been reported in the liver of the biotin-deficient
chick (146). Further, Mock and co-workers reported the accumulation of these odd-chain
fatty acids in the plasma of patients who developed biotin deficiency during parenteral
nutrition (147). The accumulation of odd-chain fatty acid is thought to result from propionyl-
CoA carboxylase (PCC) deficiency, based on the observation that the isolated genetic
deficiency of PCC and related disorders cause an accumulation of odd-chain fatty acids
414 Mock
in plasma, red blood cells, and liver (148,149). Apparently, the accumulation of propionyl-
CoA leads to the substitution of propionyl-CoA moiety for acetyl-CoA in the ACC reaction
and hence to the ultimate incorporation of a three-carbon, rather than a two-carbon, moiety
during fatty acid elongation (150).
2. Biochemical Pathogenesis
The mechanisms by which biotin deficiency produces specific signs and symptoms remain
to be completely delineated. However, several studies have given new insights into the
biochemical pathogenesis of biotin deficiency. The tacit assumption of most of these studies
is that the clinical findings of biotin deficiency result directly or indirectly from defi-
cient activities of the four biotin-dependent carboxylases.
Sander and co-workers initially suggested that the central nervous system (CNS)
effects of biotinidase deficiency (and, by implication, biotin deficiency) might be mediated
through deficiency of PC and the attendant CNS lactic acidosis (151). Because brain PC
activity declined more slowly than hepatic PC activity during progressive biotin deficiency
in the rat, these investigators discounted this mechanism. However, subsequent studies
suggest their original hypothesis is correct. Diamantopoulos et al. (11) expanded the hypothesis
by proposing that deficiency of brain biotinidase (which is already low in the
normal brain) combined with biotin deficiency leads to a deficiency of brain pyruvate
carboxylase and, in turn, to CNS accumulation of lactic acid. This CNS lactic acidosis
is postulated to be the primary mediator of the hypotonia, seizures, ataxia, and delayed
development seen in biotinidase deficiency. Additional support for the CNS lactic acidosis
hypothesis has come from direct measurements of CSF lactic acid in children with either
biotinidase deficiency or isolated pyruvate carboxylase deficiency and from the rapid resolution
of lactic acidemia and CNS abnormalities in patients who have developed biotin
deficiency during parental nutrition (3). The work of Suchy, Wolf, and Rizzo has provided
evidence against an etiologic role for disturbances in brain fatty acid composition in the
CNS dysfunction (144,152).
Several studies have demonstrated abnormalities in the metabolism of fatty acids
in biotin deficiency and have suggested that these abnormalities are important in the pathogenesis
of the skin rash and hair loss. Initially, the similarity between cutaneous manifestations
of biotin deficiency and essential fatty acid deficiency as well as the established role
of biotin in lipid synthesis suggested a relationship that has led to dietary intervention
with polyunsaturated fatty acid. For example, Munnich et al. (153) have described a 12-
year-old boy with multiple carboxylase deficiency; in retrospect, the enzymatic defect
was almost certainly biotinidase deficiency (154). The child presented with alopecia and
periorificial scaly dermatitis. Oral administration of ‘‘unsaturated fatty acids composed
of 11% C18:1, 71% C18:2, 8% C18:3, and 0.3% C20:4’’ at a rate of 200–400 mg/day
plus twice-daily topical administration of the same mixture of fatty acids ‘‘resulted in
dramatic improvement of the dermatologic condition’’ and hair growth. Lactic acidosis
and organic aciduria remained the same. These investigators speculated that the ACC
deficiency led to impaired synthesis or metabolism of long-chain polyunsaturated fatty
acids (PUFAs), which was treated by the topical and oral administration of PUFAs.
Three studies in the rat support the possibility of abnormal PUFA metabolism as a
result of biotin deficiency and as a cause of the cutaneous manifestations. Kramer et al.
(143) and Mock et al. (145) reported significant abnormalities in the n-6 phospholipids
of blood, liver, and heart. Watkins and Kratzer also found abnormalities of n-6 phospholipBiotin
415
ids in liver and heart of biotin-deficient chicks (146). It has been speculated (145–156)
that these abnormalities in PUFA composition might result in abnormal composition or
metabolism of the prostaglandins and related substances derived from these PUFAs. However,
these studies do not directly address the question of an etiological role. To address
that question, Mock (157) examined the effect of supplementation of the n-6 PUFAs (as
Intralipid) on the cutaneous manifestations of biotin deficiency in a nutrient interaction
experiment. Supplementation of n-6 PUFAs prevented the development of the cutaneous
manifestations of biotin deficiency in a group of rats that were as biotin-deficient (based
on biochemical measurements) as the biotin-deficient control group. The rats not receiving
the supplemental n-6 fatty acids did develop the classic rash and hair loss. These investigators
concluded that an abnormality in n-6 PUFA metabolism plays a pathogenic role in
the cutaneous manifestations of biotin deficiency and that the effect of the n-6 PUFAs
cannot be attributed to biotin sparing.
D. Other Effects of Deficiency
Subclinical biotin deficiency has been shown to be teratogenic in several species, including
chicken, turkey, mouse, rat, and hamster (3). Fetuses of mouse dams with biotin deficiency
too mild to produce the characteristic cutaneous or CNS findings developed micrognathia,
cleft palate, and micromelia (158–160). The incidence of malformation increased with the
degree of biotin deficiency to a maximum incidence of approximately 90%. Differences
in teratogenic susceptibility among rodent species have been reported; a corresponding
difference in biotin transport from the mother to the fetus has been proposed as the cause
(161). Bain et al. have hypothesized that biotin deficiency affects bone growth by affecting
synthesis of prostaglandins from n-6 fatty acid (162). This effect on bone growth might
be the mechanism for the skeletal malformations caused by biotin deficiency.
On the basis of studies of cultured lymphocytes in vitro and of rats and mice in
vivo, biotin is required for normal function of a variety of immunological cells. These
functions include production of antibodies, immunological reactivity, protection against
sepsis, macrophage function, differentiation of T and B lymphocytes, afferent immune
response, and cytotoxic T-cell response (3). In humans, Okabe et al. (129) have reported
that patients with Crohn’s disease have depressed natural killer all activity caused by
biotin deficiency and are responsive to biotin supplementation. In patients with biotinidase
deficiency, Cowan et al. (163) have demonstrated defects in both T-cell and B-cell immunity.
E. Diagnosis of Biotin Deficiency
The diagnosis of biotin deficiency has been established by demonstrating reduced urinary
excretion of biotin, increased urinary excretion of the characteristic organic acids discussed
earlier, and resolution of the signs and symptoms of deficiency in response to biotin supplementation.
Plasma and serum levels of biotin, whether measured by bioassay or by avidinbinding
assay, have not uniformly reflected biotin deficiency (141). The clinical response
to administration of biotin has been dramatic in all well-documented cases of biotin defi-
ciency. Within a few weeks, healing of the rash was striking, and by 1–2 months, growth
of healthy hair was generally present. Within 1–2 weeks in infants, hypotonia, lethargy,
and depression generally resolved; accelerated mental and motor development followed.
416 Mock
F. Requirements and Allowances
Data providing an accurate estimate of the biotin requirement for infants, children, and
adults are lacking (164); as a result, recommendations often conflict (3). Data providing
an accurate estimate of the requirement for biotin administered parenterally are also lacking.
For parenteral administration, uncertainty about the true metabolic requirement for
biotin is compounded by a lack of information concerning the effects of infusing biotin
systemically and continuously (rather than the usual postprandial absorption into intestinal
portal blood). Despite these limitations, recommendations for biotin supplementation have
been formulated for oral and parenteral intake from preterm infants through adults (164–
166). These recommendations are given in Table 1. One published study of parenterally
supplemented infants (167) found normal plasma levels of biotin in term infants supplemented
at 20 µg/day and increased plasma levels of biotin in preterm infants supplemented
at 13 µg/day. [Note that the unit for plasma biotin should be picograms per milliliter in
this publication (167).]
An important factor in the current uncertainty concerning the biotin requirement is
the possibility that biotin synthesized by intestinal bacteria (referred to hereafter as ‘‘bacterial
biotin’’) may contribute significantly to absorbed biotin. If so, the required intake
would be reduced and might be dependent on factors that influence the density and species
distribution of intestinal flora. For example, it is conceivable that interruption of absorption
of bacterial biotin is a critical event in both biotin deficiency caused by egg-white ingestion
and biotin deficiency that develops during biotin-free parenteral feeding. Binding of bacterial
biotin by avidin may be occurring in the former, and reduced intestinal surface for
absorption, rapid transit time, and antibiotic suppression of gut bacteria may lead to reduced
absorption of bacterial biotin in the latter. Unfortunately, few data are available for
assessing the actual magnitude of the absorbed bacterial biotin.
G. Dietary Sources of Biotin
There is no published evidence that biotin can be synthesized by mammals; thus, the
higher animals must derive biotin from other sources. The ultimate source of biotin appears
Table 1 Recommended Daily Intake of Biotin (µg/kg)
Safe and adequate Parenteral
Age oral intakesa intakesb
Preterm infantsc 5 5–8 µg ? kg1
Infants up to 6 mo 5 20
Infants 0.5–1 y 6 20
Children 1–3 y 8 20
Children 4–8 y 12 20
Older children, 9–13 y 20 20
Older children 14–18 y 25 60
Adults 30 —
Pregnancy 30 —
Lactation 35 —
a Ref. 164
b Ref. 165.
c Ref. 166.
Biotin 417
to be de novo synthesis by bacteria; primitive eukaryotic organisms, such as yeast, molds,
and algae; and some plant species.
Most measurements of the biotin content of various foods have used bioassays.
Despite the limitations due to interfering substances, protein binding, and lack of chemical
specificity as discussed above, there is reasonably good agreement among the published
reports (168–172), and some worthwhile generalizations can be made. Biotin is widely
distributed in natural foodstuffs, but the absolute content of even the richest sources is
low when compared with the content of most other water-soluble vitamins. Foods relatively
rich in biotin include egg yolk, liver, and some vegetables. Based on the data of
Hardinge (168), the average dietary biotin intake has been estimated to be approximately
70 µg/day for the Swiss population. This result is in reasonable agreement with the estimated
dietary intake of biotin in a composite Canadian diet (62 µg/day) and the actual
analysis of the diet (60 µg/day) (173). Calculated intake of biotin for the British population
was 35 µg/day (174,175).
VI. PHARMACOLOGY AND TOXICITY
A. Treatment of Biotin Deficiency
Pharmacological doses of biotin (e.g., 1–10 mg) have been used to treat most patients.
For two patients, parenteral administration of 100 µg of biotin per day was adequate to
cause resolution of the signs and symptoms of biotin deficiency and to prevent their recurrence
(3). However, abnormal organic aciduria persisted for at least 10 weeks in one
patient receiving 100mg/day, suggesting that this dosemaynot have been adequate to restore
tissue biotin levels to normal over that time. Could organic aciduria be an indication that
biotin status at the tissue level has not been restored to normal?Could this degree of deficiency
be less severe but sufficient to cause significant, subtle morbidity? If so, should a loading
dose of 1 or 10 mg for 1 or 2 weeks be given as initial therapy for acquired biotin deficiency?
There are currently no data on which to base answers to these questions.
B. Toxicity
Daily doses up to 200 mg orally and up to 20 mg intravenously have been given to treat
biotin-responsive inborn errors of metabolism and acquired biotin deficiency; toxicity has
not been reported.
C. Pharmacology
Mounting reports of biotin deficiency in commercial animals and humans have led to
several studies of plasma levels, pharmacokinetics, and bioavailability after acute or
chronic oral, intramuscular, or intravenous administration of biotin in cattle (176), swine
(177,178), and human subjects (179–181).
Studies using bioassays to measure biotin in blood and urine suggest that doses of
biotin less than 150 mg are suitable as loading doses to assess biotin status (180). Doses
greater than 300 mg result in high biotin concentrations in blood and urinary excretion
of a large proportion of the administered biotin as the unchanged vitamin (179–181).
When metabolites are measured separately from biotin per se, increased blood concentrations
(181) and urine excretion rates (unpublished observations) of bisnorbiotin and biotin
418 Mock
sulfoxide as well as biotin are observed. These observations are consistent with the metabolites
originating from human tissues rather than from enteric bacteria.
ACKNOWLEDGMENTS
Many thanks to Nell Mock and Celia Bernheimer for preparing the art work and to Gwyn
Hobby for typing the manuscript.
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12
Folic Acid
TOM BRODY and BARRY SHANE
University of California, Berkeley, California
I. HISTORY
A dietary factor that prevented megaloblastic anemia of pregnancy and animal growth
factors and lactic acid bacteria growth factors was studied during the 1930s. Liver, yeast,
and spinach proved to be good sources of these factors. Because of the relative ease in
performing bacterial growth assays, the bacterial growth factors were extensively purified
from liver and yeast and crystallized. Eventually, it was determined that the various antianemia
and growth factors all had a common structure, and they were named folate. Folic
acid (Fig. 1) was identified and synthesized in 1946 using techniques previously used to
study other pteridines, such as butterfly pigments. The isolation of folate allowed its use
for the treatment of anemia. Because folate and vitamin B12 deficiency lead to an identical
and indistinguishable megaloblastic anemia in which blood cells are enlarged due to a
derangement of DNA synthesis, folate was originally thought to be the only factor required
for the treatment of megaloblastic anemia, and was used to ‘‘cure’’ pernicious anemia.
With the isolation of vitamin B12 several years later, it became clear that vitamin B12 was
a second anti-anemia factor and it also became clear that while folate could ameliorate
the anemia of vitamin B12 deficiency, it could not prevent the neurological manifestations
of vitamin B12 deficiency.
The biochemical functions of folate were first determined with bacteria. Purines or
thymine could partly replace the nutritional requirements for folate or for p-aminobenzoic
acid (PABA). Similarly, the inhibition of bacterial growth by folate analogues could be
overcome by adding purines or thymine to the growth medium. Studies with [14C]formate
and [14C]formaldehyde disclosed the role of folate in transferring 1-carbon units. For example,
[14C]formate was incorporated into specific positions of the purine ring in studies of
427
428 Brody and Shane
Fig. 1 Structure of (A) folic acid (PteGlu) and (B) 5-methyl-tetrahydrofolate pentaglutamate (5-
Methyl-H4PteGlu5). Although folic acid is not naturally occurring, it is readily transported and reduced
to the natural forms of the vitamin. One-carbon substituents can be at the N-5 and/or N-10
positions of the reduced folate molecule.
animals. In studies with liver extracts, it was shown that tetrahydrofolate stimulated the
incorporation of [14C]formaldehyde into the amino acid serine.
Folates were originally isolated as polyglutamates (Fig. 1), and these forms were
found to be the major intracellular form of the vitamin. The polyglutamate had the ‘‘property’’
of supporting animal growth but not bacterial growth. The monoglutamate supported
growth of both. The value of the poly-?-l-glutamyl chain was not recognized until the
1960s and 1970s, when it was determined that the folate polyglutamates were the preferred
substrates for folate-dependent enzymes and appeared to be more active metabolically in
cells and also were better retained in cells than folate monoglutamates.
Soon after the isolation of folate, various chemically forms were shown to be folate
antagonists, and one of these, methotrexate, proved to be very effective in the treatment
and cure of childhood leukemia. Because of the role of folate coenzymes in the synthesis
of DNA precursors, a multitude of folate antagonists have found clinical use as anticancer
and antimicrobial agents. More recently, the demonstration that periconceptional folic acid
supplementation reduces the incidence of birth defects in humans has led to fortification
of the U.S. food supply with folic acid.
An excellent comprehensive multivolume series on folate and pterins was published
by Blakley et al. in the 1980s (1,2), and is a valuable source of background information
and references. A more recent book by Brody (3) details the metabolic pathways that
require folate.
II. CHEMISTRY
A. Isolation
Folates in natural sources should be extracted and isolated under conditions that preserve
the oligo-?-glutamyl chain and the reductive state of the folate. For maximal recovery of
intact folates, the biological sample may be minced, then heated for 5–10 min in a boiling
Folic Acid 429
water bath in the presence of antioxidants to denature folate-metabolizing enzymes, such
as ?-glutamyl hydrolase, as well as folate-binding proteins. The boiled sample should then
be cooled and homogenized (4). Suitable antioxidants for preserving reduced folates are
0.2 M 2-mercaptoethanol or 1.0% sodium ascorbate. One might be cautioned that heating
folates in ascorbate alone can alter the folates themselves, though this should not be a
problem where sulfhydryl agents are also present (5).
Although folates covalently bound to macromolecules have not yet been discovered,
heat treatment may be needed to release folates bound to such components as folatebinding
proteins (6,7), membranes (8), and viruses (9). The selective absorption to charcoal
and solvent extractions were originally used to purify folates. These have given way to
ionic exchange column chromatography, molecular sieve column chromatography, and
high-pressure liquid chromatography.
B. Structure and Nomenclature
Folic acid (pteroylmonoglutamate, PteGlu) consists of a 2-amino-4-hydroxy-pteridine
(pterin) moiety linked via a methylene group at the C-6 position to a p-aminobenzoylglutamate
moiety (Fig. 1). Natural folates occur in the reduced, 7,8-dihydro- and 5,6,7,8-
tetrahydro-forms. Folates bearing one 1-carbon units are 5-methyl-, 10-formyl-, 5-formyl-,
5,10-methenyl-, 5,10-methylene-, and 5-formiminotetrahydrofolate. Folates in nature
occur as pteroyloligo-?-l-glutamates (PteGlun) of from one to nine or more glutamates
long. The subscript ‘‘n’’ indicates the total number of glutamate residues. ‘‘Folate’’ is a
generic term for the above compounds.
The C-6 position of the pterin ring of tetrahydrofolates is an isometric center. This
carbon in the naturally occurring forms of H4PteGlu, 5-formyl-H4PteGlu, and 5-methyl-
H4PteGlu has the S configuration, while 10-substituted folates such as 10-formyl-H4Pte-
Glun, 5,10-methylene-H4PteGlu and 5,10-methenyl-H4PteGlu have the R configuration
(10,11).
C. Synthesis
Folic acid was first synthesized at Lederle Laboratories by the condensation of triamino-
6-hydroxypyrimidine, dibromopropionaldehyde, and p-aminobenzoylglutamic acid (12).
Folic acid and pterins in general have been synthesized by the method of Taylor et al.
(13). This method uses 2-amino-3-cyano-5-chloromethylpyrazine as a key intermediate
and avoids a contaminating isomer formed in earlier procedures. The l-glutamyl group
of folic acid may racemize in the above method, and a strategy for avoiding this problem
has been suggested (14). Pteroyloligo-l-?-glutamates (folate polyglutamates) were first
isolated by the Lederle group (15) and were synthesized by Meienhofer et al. (16). Baugh
et al. (17), introduced a solid phase synthesis method.
Folic acid can be reduced to the dihydro- form (18) with dithionite or to the tetrahydro
form with hydrogen using a platinum catalyst. Folic acid can be formylated (19)
followed by reduction to 10-formyl-H4PteGlu (20) and reduced further to 5-methyl-
H4PteGlu (21). The natural isomers of reduced folates can be made enzymatically. These
derivatives, including the polyglutamate forms, are now commercially available.
D. Chemical Properties
Folic acid is yellow, has a molecular weight of 441.4, and is slightly soluble in water in
the acid form but quite soluble in the salt form. Tetrahydrofolates in solutions are sensitive
430 Brody and Shane
to oxygen, light, and pH extremes, and can break down to p-aminobenzoylglutamic acid
and dihydroxanthopterin, pterin-6-carboxaldehyde, or pterin-6-carboxylic acid (22). One
oxidation product of 5-methyl-H4PteGlu is 5-methyl-H2PteGlu (23). When acidified, 10-
formyl- and 5-isomerize to the relatively oxygen stable folate, 5,10-methenyl-H4PteGlu
(24). When neutralized, the 5,10-methenyl- H4PteGlu isomerizes to 10-formyl-H4PteGlu,
one of the less stable folate compounds. The formyl group can be removed under anaerobic
conditions (19). 5-Formimino- H4PteGlu, although stable to oxygen, is readily hydrolyzed
with the production of ammonia (25). Formaldehyde condenses reversibly with H4PteGlu
to form 5,10-methylene-H4PteGlu (26). The concentration of this folate can be maintained
only when excess formaldehyde is in the solution. 5,10-Methylene-H4PteGlu is stable at
pH 9.5 but is in somewhat rapid equilibrium with formaldehyde at neutral and lower pH
(27). This equilibrium can be disturbed by the reaction of thiols with formaldehyde (26).
E. Chemical Degradation of Folates
Tetrahydrofolates and N10-substituted tetrahydrofolates are unstable to oxygen. In contrast,
folic acid and tetrahydrofolates substituted at N5 (or N5 and N10) are relatively
stable to oxygen. All folates are degraded by light. H4PteGlu in oxygenated solutions
breaks down to form 6-formyl-pterin (pterin-6-carboxyaldehyde), H2pterin, pterin, and
small amounts of xanthopterin. Cleavage at the C9-N10 bond is rapid, forming p-aminobenzoylglutamic
acid (pABAGlu) as a by-product. In mild acid, H2pterin is the initial
major product while pterin is the eventual major product. At neutral and alkaline pH, 6-
formyl-pterin is the eventual major product. H2PteGlu accumulates momentarily as a major
product only at alkaline pH (28), while 6-formyl-H2pterin may be the major breakdown
product of H2PteGlu (29). PteGlu is only a minor breakdown product of H4PteGlu (1),
but under some conditions it may be a major breakdown product of H2PteGlu (5). 10-
Formyl-H4PteGlun breaks down under oxygen to produce 10-formyl-H2PteGlu and 10-
formyl-PteGlu. Folates are usually extracted from biological sources by boiling (5–10
min) in 0.2 M 2-mercaptoethanol, with or without ascorbate. Most forms of folate are
fairly stable under these conditions, where recoveries are 70–95 percent (5).
III. BIOSYNTHESIS
Folates are synthesized from GTP by microorganisms and plants as the 7,8-dihydrofolate
form, and all naturally occurring folates are reduced derivatives. The biosynthesis pathway
was elucidated by G. M. Brown, T. Shiota, and others (reviewed in Refs. 30,31).
GTP cyclohydrolase I catalyzes the conversion of GTP to 7,8-dihydro-neopterin triphosphate
and formic acid. The reaction is complex, apparently occurring in four steps, one
of which is an Amadori rearrangement. The E. coli cyclohydrolase is a 210 kDal protein
and contains four apparently identical subunits. GTP cyclohydrolase I also occurs in mammals
where it is used for the first step in biopterin synthesis. However, mammals do not
contain the subsequent enzymes of the folate biosynthetic pathway. The second step of
folate synthesis involves an unidentified phosphatase, which converts dihydroneopterin-
PPP to H2neopterin. Dihydroneopterin aldolase then converts dihydroneopterin to 6-hydroxymethyl-
dihydropterin and glycoaldehyde, a 2-carbon by-product. Hydroxymethyldihydropterin
pyrophosphokinase (32) catalyzes the fourth step in the pathway, the
ATP-dependent conversion of 6-hydroxymethyl-7,8-dihydropterin to 6-hydroxymethyl-
7,8-dihydropterin pyrophosphate, with AMP as the by-product. Dihydropteroate synthase
Folic Acid 431
(34 kDal) catalyzes the condensation of 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate
and pABA to form 7,8-dihydropteroic acid, with the release of PP1. Dihydrofolate
synthetase (46 kDal) catalyzes the ATP-dependent condensation of 7,8-dihydropteroic
acid and glutamate to form H2PteGlu. In E. coli, the latter enzyme is bifunctional and
occurs as dihydrofolate synthase/folylpoly-?-glutamyl synthase (33), where the latter activity
catalyzes the addition of additional glutamate residues to folate derivatives. Some
microorganisms contain separate dihydrofolate synthetase and folylpolyglutamates synthetase
proteins. Mammals contain a folylpolyglutamate synthetase activity but lack the
dihydrofolate synthetase enzyme. Some bacteria also contain a second folylpolyglutamate
synthetase enzyme that adds glutamates residues in ?-peptide linkages to H4folyltriglutamate
to produce longer chain length folates (34), although the role of these ?-linked forms
is not known. In bacteria, hydroxymethyl-dihydroneopterin pyrophosphokinase, dihydropteroate
synthase, and dihydrofolate synthase occur as separate proteins. However, in
Pneumocystis carinii, the enzymes responsible for carrying out these sequential reactions
occur as a single trifunctional polypeptide of 84 kDal (35).
Archaea may utilize unusual folate-like coenzymes for mediating one-carbon metabolism.
H4Methanopterin, rather than H4PteGlu, mediates one-carbon metabolism in methanogens.
Methanopterin is a cofactor used in the reduction of CO2 through formyl, methenyl,
methylene, and methyl stages to methane (36). Certain thermophilic archaea contain
unique versions of folate bearing an oligosaccharide chain, rather than an oligoglutamyl
chain. Sarcinapterin is a monoglutamated version of methanopterin and occurs in unique
species of methanogens.
IV. ANALYTICAL PROCEDURES
The method used to identify folates depends on whether one needs to know the identities
of the one-carbon unit and reductive state of the cofactor, the length of the oligo-glutamyl
chain of the folate, or both. For identification of polyglutamate distributions, folates can
be cleaved at the C9-N10 bond to yield p-aminobenzoylpolyglutamate derivatives, which
can be separated and identified by HPLC analysis (37–39). If the one-carbon distribution
is required, folates can be converted to monoglutamates by treatment with a neutral ?-
glutamyl hydrolase and the monoglutamates separated by reverse phase HPLC (5). Care
must be taken to ensure that the experimental conditions used do not cause interconversion
of folate one carbon forms (40). Identification of one-carbon distribution and polyglutamate
chain length of all folates is complicated by the large number of potential derivatives.
Selhub (41) devised a method for the affinity purification of extracted folates using immobilized
milk folate binding protein prior to chromatographic analysis by reverse phase
HPLC. Although complete resolution of all folates is not obtained, the high purity of the
folate sample applied to the HPLC column allows quantitation of individual folates in
overlapping peaks by their spectral properties. This method works well for tissues, such
as liver, that contain relatively high levels of folate. Alternately, eluted folates can be
detected by microbiological assay or by fluorescence.
The folate content of natural sources, as well as that in fractions recovered after
chromatographic procedures, can be measured by microbiological assay (42). The hydrolysis
of the oligo-?-glutamyl chain by treatment with ?-glutamyl hydrolase is required to
support a maximal growth response of the test microorganism (43), usually Lactobacillus
casei, as this organism responds to all folate one carbon forms. The method involving
treatment with ?-glutamyl hydrolase followed by assay with L. casei has been called an
432 Brody and Shane
assay for ‘‘total’’ folate. The amount of 5-methyl-tetrahydrofolates can be assessed by
subtracting the value obtained with Streptococcus fecium from that obtained with L. casei,
where the sample was treated with ?-glutamyl hydrolase. 5-Methyl-H4PteGlu does not
support growth of S. fecium. Another lactic acid bacterium, Pediococcus cerevisiae, does
not respond to nonreduced folate, i.e., PteGlu, or to 5-methyl-H4PteGlu. The recent development
of a microtiter plate assay procedure has greatly simplified the microbiological
assay of folate (42).
V. CONTENT IN FOOD AND BIOAVAILABILITY
The major source of folate in the U.S. diet is fortified cereals, followed by vegetables,
bread and bread products, citrus fruits and juices, and meat, poultry and fish. Fully oxidized
folic acid is only found in the diet when foodstuffs are fortified with folic acid or when
dietary folates are oxidized. Reduced folates are less stable than folic acid and large losses
in food folate can occur during food preparation such as heating, particularly under oxidative
conditions. Additional losses can also occur by leaching out folate during food preparation.
When using food tables, attention should be given to the manner in which the test
food was prepared, i.e., cooked or raw.
Absorption of orally administered folate mono- and polyglutamates and of dietary
folate has been followed by measuring the appearance of vitamin appearing in the mesenteric
vein (44) or in the urine (45). The availability of food folate can range from 30–
80% that of PteGlu. This was found to be the case for folates extracted from yeast (46)
and folates in the presence of certain foods, such as yeast or cabbage (47). Synthetic
PteGlu in cooked food may be absorbed at a lesser rate than PteGlu administered in water
(48). These rates were determined from increases in serum folates levels in subjects presaturated
with folic acid prior to test doses (48). Stronger data on the biological availability
of folic acid are those (49,50) showing that synthetic folic acid in cooked food was 55%
as available as folic acid in tablet form, as determined by changes in red blood cell folate
content. The test foods used by Colman et al. (48–50) were maize, rice, and bread. Certain
foods such as cabbage and legumes contain glutamyl hydrolase inhibitors, which can decrease
the availability of folylpolyglutamates. The bioavailability of folic acid when given
as a supplement or in fortified food is high (51). However, the bioavailability of food
folate is less than 50% and may be significantly lower than this because recent studies
have suggested that methods commonly used for the analysis of folate in foodstuffs may
have underestimated the folate content (52). The general consensus of these studies is that
food folate bioavailability averages about 50%, while that of folic acid, either given as a
supplement or added to fortified foods, is in excess of 90%. Pharmacological doses of
folate are well absorbed but most of the vitamin is not retained in the body due to a limited
capacity of tissues to retain large amounts of folate.
Many of the values shown in food tables for folate content of foods were obtained
after treating food extracts with glutamyl hydrolase. Some recent studies suggest many
of these values may be underestimates as treatment of food extracts with three enzymes,
?-glutamyl hydrolase, ?-amylase and protease, resulted in significant increases in folate
values, up to twofold, increases that were dependent on the food stuff being analyzed
(52). There is some disagreement whether differences in extraction procedures may have
confounded these results and validation of the tri-enzyme procedure is currently underway.
If these new data hold up, this would suggest that folate bioavailability from unfortified
food may be significantly less than 50%.
Folic Acid 433
VI. METABOLISM
A. Absorption
Dietary folates are predominantly pteroylpolyglutamate derivatives and are hydrolyzed to
monoglutamates by a ?-glutamylhydrolase activity (sometimes called conjugase) prior to
their absorption across the intestinal mucosa. In some species, such as in humans and
pigs, the hydrolase activity is a membrane-bound exopeptidase (53) while in other species,
such as the rat, the enzyme activity is secreted in the bile. This latter activity is similar
to a hydrolase activity found in the lysosomes of all tissues. The human jujenal membranebound
enzyme has recently been cloned (54). In common with many proteases, ?-glutamylhydrolase
can be shown to catalyze the reverse of the hydrolytic reaction (ligase), as well
as a transpeptidase reaction (55).
The mechanism by which folate crosses the mucosal cell and is released across
the basolateral membrane into the portal circulation is not well understood. Intestinal
folate absorption occurs mainly in the jejunum. Absorption of folate monoglutamate
is via a saturable carrier-mediated process although at high folate concentrations a diffusion-
like process occurs. Transport is maximal at pH 5–6 with a rather sharp pH optimum.
The intestinal transporter, which is encoded by the reduced folate carrier gene
(RFC1), is a transmembrane protein that is expressed in most, if not all, tissues (56,57)
but the specificity of this transporter for various folates differs between tissues and between
the apical and basolateral membranes of tissues. Affinities for reduced folates are
in the low micromolar range while affinities for folic acid are similar for some tissues
such as the intestine but can be 100-fold lower in other tissues. These differences may
reflect tissue specific differences in posttranslational modification of the reduced folate
carrier protein. A folate binding protein (sometimes called the folate receptor) has also
been detected in the intestine. This protein, which has been shown to mediate endocytosis
of folate in the kidney and other tissues, has high affinity for folates, in the nanomolar
range (57). It has been shown that the binding protein and the membrane transporter are
located on opposing membranes in some polarized cells (58). It is attractive to speculate
that the high affinity folate binding protein is involved in the movement of folate into
the intestinal cell across the apical membrane while transport across the basolateral
membrane to the portal circulation is mediated by the lower affinity RFC transmembrane
protein.
Most dietary folate is metabolized to 5-methyl-H4PteGlu during its passage through
the intestinal mucosa although this metabolism is not required for transport (59–61). The
degree of metabolism in the intestinal mucosa is dependent on the folate dose given. When
pharmacological doses of folic acid, or other folates, are given, most of the transported
vitamin appears unchanged in the portal circulation. During passage through the liver,
immediate conversion of the PteGlu to 5-methyl-H4PteGlu occurs, with release of much
of the converted vitamin back to the bloodstream (60,61). With large oral doses of PteGlu,
substantial amounts of the vitamin are recovered, unchanged, in the urine.
B. Transport and Tissue Accumulation
Pteroylmonoglutamates, primarily 5-methyl-H4PteGlu, are the circulating forms of folate
in plasma, and mammalian tissues cannot transport polyglutamates of chain length three
and above (62). After folate absorption into the portal circulation, much of the folate can
be taken up by the liver via the reduced folate carrier. In the liver, it is metabolized to
434 Brody and Shane
polyglutamate derivatives and retained, or it may be released into blood. Plasma folate
levels in humans are usually in the 10–30 nM range, while hepatic levels, practically all
polyglutamates, are about 20 µM. Some folate is secreted in bile, but this can be reabsorbed
in the intestine via an enterohepatic circulation (63). The predominance of 5-methyl-
H4PteGlu in plasma probably reflects that this is the major cytosolic folate in mammalian
tissues. The extent of release of short chain folylpolyglutamates from tissues is unknown.
Plasma contains a soluble ?-glutamylhydrolase activity, and any polyglutamate released
into plasma would be hydrolyzed to the monoglutamate.
Folic acid, naturally occurring reduced folates and folate analogs such as methotrexate
may share a common transport system in mammalian cells (64), which is thought to
be the low affinity reduced folate carrier. However, the specificity and kinetics of transporters
in different tissues varies considerably, although thus far only one RFC gene has been
identified (65–69). The hepatic transporter has similar affinity for folic acid and reduced
folate monoglutamates, while many other tissues have a greatly decreased affinity for folic
acid.
Some plasma folate is bound to low-affinity protein binders, primarily albumin.
Plasma also contains low levels of a high-affinity folate binder, the levels of which are
increased in pregnancy and in some leukemia patients. The high-affinity binder is a soluble
form of a second membrane-associated folate transporter known as the folate-binding
protein or the folate receptor (70–72). The folate receptor is encoded by at least three
distinct genes in humans and two genes in mice with most tissues expressing the ? form.
The encoded protein is usually attached to the plasma membrane of cells via a glycosylphosphatidylinositol
anchor. Levels are highest in the choroid plexus, kidney proximal
tubes, placenta, and in a number of human tumors, while lower levels have been found
in many other tissues. As described above, in polarized cells the binding protein is often
found on the opposing membrane to the reduced folate carrier (58). Although the physiological
function of this binding protein has not been established for all tissues, its role in
the receptor-mediated reabsorption of folate in the kidney has been well documented (73).
The function of the soluble form of folate binding protein, which is expressed at high
levels in milk, is not understood, but may also play a role in folate transport.
Red blood cells contain higher levels of folate than plasma (normally 0.5–1 µM).
Mature red cells do not transport folate and their folate stores are formed during erythropoiesis
and are retained, probably due to binding to hemoglobin, through the life span of the
human red cell. Red cell folate levels are often used as a measure of long-term folate
status.
Most of the folate in tissues is found in the mitochondrion and cytosol. Mitochondria
contain a transporter that is specific for reduced folates and which differs from the plasma
membrane transporter in that it will not transport folic acid or methotrexate (74,75). This
transporter has only been characterized kinetically. The major hepatic mitochondrial folates
are 10-formyl-H4PteGlunand H4PteGlun, and much of these are found bound to two
folate enzymes, dimethylglycine dehydrogenase and sarcosine dehydrogenase. 5-Methyl-
H4PteGlun, is the major cytosolic folate, and much of this is bound to glycine N-methyltransferase
in liver, while much of the cytosolic H4PteGlun is bound to 10-formyltetrahydofolate
dehydrogenase.
C. Intracellular Metabolism and Storage
Folate metabolism involves the reduction of the pyrazine ring of the pterin moiety to the
coenzymatically active tetrahydro form, the elongation of the glutamate chain by the addiFolic
Acid 435
tion of l-glutamate residues in an unusual ?-peptide linkage, and the acquisition and oxidation
or reduction of one-carbon units at the N-5 and/or N-10 positions. Folates are substrates
for multiple enzymes and the interconversion of folate one-carbon forms is
intertwined with the metabolic roles of folate as described below. Folates in cells and
tissues occur almost exclusively as reduced folate oligo-?-glutamates. The polyglutamates
are more effective substrates than pteroylmonoglutamates of most folate-dependent enzymes
and usually exhibit greatly increased affinities for these enzymes. For most folatedependent
enzymes the major kinetic advantages are achieved by elongation of the glutamate
chain to the triglutamate (reviewed in Ref. 76). Longer polyglutamate forms are
required for the enzymes involved in the methionine re-synthesis cycle. Folates are metabolized
to polyglutamates of chain lengths considerably longer than the triglutamate form
required for folate retention. The glutamate chain length varies with the species with the
pentaglutamate predominating in the rat, hexaglutamate in the mouse, and hexa- and heptaglutamates
as well as longer chain length derivatives, up to the decaglutamate, in human
cells (77).
The accumulation of folate in the mitochondrion and cytosol of tissues requires
their conversion to polyglutamates, which is catalyzed by the enzyme folylpolyglutamate
synthetase (76, reaction 17 Fig. 2).
MgATP 
 folate(glun) 
 glutamate > MgADP 
 folate(glun
1) 
 phosphate
Glutamate residues are added one at a time and the products of the reaction have to be
released from the enzyme following each catalytic cycle before rebinding and further chain
extension. Tetrahydrofolate and its polyglutamate forms are the preferred substrates for
human folylpolyglutamate synthetase, while 5-substituted folates such as 5-methyl-
H4PteGlu are poor substrates. The affinity of folates for the human enzyme drops off as
the chain length is extended beyond the diglutamate. Because 5-methyl-H4PteGlu, the
major folate transported into most tissues, is a very poor substrate and its diglutamate
derivative is almost inactive, the extent of folate accumulation is dependent on the tissue’s
ability to metabolize 5-methyl-H4PteGlu to H4PteGlu via the methionine synthase reaction
(Fig. 4). The cell can rapidly release any folate that is not converted to the triglutamate
(78). Folylpolyglutamate synthetase is a low abundance protein and its activity is ratelimiting
for folate retention and accumulation by some tissues, and also limits the ability
of tissues to accumulate large stores of folate. With pharmacological doses of folate, competition
by the entering monoglutamate limits the glutamate chain extension of cellular
folates and much of the folate is converted to the diglutamate and released from the tissue,
while the predominant chain length of folate that is retained by the tissue is shortened.
Folylpolyglutamate synthetase is encoded by a single human gene, and cytosolic
and mitochondrial isozymes are generated by alternate transcription start sites for the gene
(79,80) and by alternate translational start sites for its mRNA (81). Cells that lack folylpolyglutamate
synthetase activity are unable to accumulate folate and are auxotrophic for
products of one carbon metabolism (82,83). Cells that lack mitochondrial folylpolyglutamate
synthetase activity are defective in mitochondrial one carbon metabolism and are
unable to accumulate folate in the mitochondria despite possessing normal cytosolic folate
pools.
Polyglutamylation of antifolates such as methotrexate is required for their accumulation
and cytotoxic efficacy and clinical resistance to methotrexate can result from decreased
folylpolyglutamate synthetase activity (84,85). The level of folylpolyglutamate
synthetase activity in human leukemia blasts varies over a very wide range and this may
explain some of the differences in sensitivity of tumors to antifolate agents. Tumor cells
436 Brody and Shane
Fig. 2 Metabolic cycles of one-carbon metabolism in the cytosol of mammalian tissues. The
numbers refer to reactions catalyzed by the following enzymes: (1) dihydrofolate reductase, (2)
dihydrofolate reductase, (3) serine hydroxymethyltransferase, (4) 5,10-methylene-tetrahydrofolate
dehydrogenase, (5) 5,10-methenyltetrahydrofolate cyclohydrolase, (6) 10-formyl-tetrahydrofolate
synthetase, (7) glycinamide ribonucleotide transformylase, (8) aminoimidazole carboxamide ribonucleotide
transformylase, (9) thymidylate synthase, (10) 5,10-methylene-tetrahydrofolate reductase,
(11) methionine synthetase, (12) tetrahydrofolate formiminotransferase, (13) formiminotetrahydrofolate
cyclodeaminase, (14) glutamate transformylase, (15) 5-formyl-tetrahydrofolate isomerase,
(16) 10-formyl-tetrahydrofolate dehydrogenase, (17) folyl-polyglutamate synthetase, and (18) ?-glutamyl
hydrolase. In mammals, reactions 12 and 13 are catalyzed by a single bifunctional enzyme,
and reactions 4, 5, and 6 are catalyzed by a trifunctional enzyme. dUMP, deoxyuridine monophosphate;
dTMP, deoxythymidine monophosphate (thymidine monophosphate); PLP, pyridoxal phosphate;
FIGLU, formiminoglutamic acid; fAICAR, formyl-aminoimidazole carboxyamide ribonucleotide;
AICAR, aminomidazole carboxamide ribonucleotide; GAR, glycinamide ribonucleotide;
FGAR, formyl-glycinamide ribonucleotide; and B12, vitamin B12 (cobalamin).
that are especially sensitive to methotrexate appear to have an enhanced ability to form
methotrexate polyglutamates (86).
Tissues contain a soluble lysosomal ?-glutamylhydrolase activity that converts folylpolyglutamates
to the mono- or diglutamate form (reaction 18, Fig. 2). The enzyme also
hydrolyzes p-aminobenzoylpolyglutamates, and can hydrolyze poly(?-glutamate) although
this is much poorer substrate. Lysosomes contain a transporter that can transport folate
and methotrexate polyglutamates (87). This transporter may be involved in the subsequent
hydrolysis of folylpolyglutamates with their subsequent release from the tissue. However,
the physiological role of this lysosomal system may be related to the catabolism of folate
(see Section D, below).
D. Catabolism and Excretion
The daily urinary excretion of intact folates is only between 1 and 12 µg, which accounts
for only a small fraction of the total folate intake. Fecal folate levels are quite high, someFolic
Acid 437
times much higher than the estimated intake, which presumably reflects folate production
by the gut microflora. The major urinary excretory product of folate is N-acetyl-p-aminobenzoylglutamate
(88) with smaller amounts of p-aminobenzoylglutamate. These compounds
can arise from cleavage of the labile C9-C10 bond of reduced folates, which would
yield p-aminobenzoylpolyglutamates and pterin derivatives, followed by their subsequent
hydrolysis by ?-glutamylhydrolase, and N-acetylation of the resulting p-aminobenzoylglutamate
(89).
Although this catabolic pathway was initially thought to be initiated by a nonenzymatic
cleavage of labile folate derivatives, recent studies have suggested that several enzyme-
mediated systems may be involved in this process and that formyl derivatives of
folate may be the immediate substrates for the cleavage reactions (P. Stover, personal
communication). Metabolic conditions or manipulations that cause an accumulation of 5-
or 10-formyl-H4PteGlun result in increase folate catabolism and heavy chain ferritin has
been isolated as a activity that cleaves folates to pterin derivatives (P. Stover, personal
communication).
VII. BIOCHEMICAL FUNCTIONS
Folates are used as cofactors and serve as acceptors and donors of one-carbon units in a
variety of reactions involved in amino acid and nucleotide metabolism. The one-carbon
units can be at the oxidation levels of methanol, formaldehyde, and formate but not carbon
dioxide. These reactions, known as one-carbon metabolism, are shown in Fig. 2, which
emphasizes the cyclical nature of folate metabolism. Although the major pathways of
methionine, thymidylate, and purine synthesis occur in the cytosol, folate metabolism also
occurs in the mitochondria and mitochondrial folate metabolism plays an important role
in glycine metabolism and in providing one-carbon units for cytosolic one-carbon metabolism
(90–93, Fig. 3). Folate coenzymes are cosubstrates in these reactions. Consequently,
folate metabolism and its regulation is interwoven with the regulation of the synthesis of
products of one-carbon metabolism and factors that regulate any one cycle of one-carbon
metabolism would be expected to influence folate availability for the other cycles of onecarbon
metabolism. The C-3 of serine is the major source of one-carbon units for folate
metabolism. Other sources include formate, much of which is derived from serine metabolism
in the mitochondria, and the C-2 of histidine. Many of the enzymes involved in
folate metabolism are multifunctional or are part of multiprotein complexes which allows
channeling of polyglutamate intermediates between active sites without release of intermediate
products from the complex (94).
Some folate-requiring enzymes are used for biosynthetic purposes, whereas others
are used only for interconversion of the various forms of the vitamin. The folate molecule
does not remain enzyme bound, but acts rather as a cosubstrate. The available studies
have shown that, within the cytosol, the folylpentaglutamates and folyhexaglutamates have
an equal tendency to participate in one-cabon metabolism (209,210).
A. Amino Acid Metabolism
1. Serine-Glycine Interconversion and Metabolism
Serine hydroxymethyltransferase (E.C. 2.1.2.1; reaction 3, Fig. 2) catalyzes the transfer
of formaldehyde from serine to H4PteGlun as follows:
serine 
 H4PteGlun - glycine 
 5,10-methylene-H4PteGlun
438 Brody and Shane
Fig. 3 Compartmentalization of folate-dependent one-carbon metabolism between mitochondria
and cytoplasm. (From Ref. 93, adapted from Ref. 145.)
The enzyme contains bound pyridoxal 5?-phosphate and is present as two distinct isozymes
in the cytosol and mitochondria of all tissues (95,96, reaction 14, Fig. 3). The cytosolic
enzyme is the predominant species in liver although the mitochondrial form predominates
in cultured cells. The highest levels are in the liver and kidney. In mammalian tissues,
the ?-carbon of serine is the major source of one-carbons for folate metabolism. The 5,10-
methylene-H4PteGlun formed in this reaction plays a central role in one-carbon metabolism
because it can be directed into the three cytoplasmic one-carbon cycles of methionine, de
novo purine, and thymidylate synthesis (Fig. 2). The hydroxymethyltransferase reaction,
which is freely reversible, is a major pathway for serine catabolism. In kidney and in liver
under gluconeogenic conditions net synthesis of serine from glycine may occur via serine
hydroxymethyltransferase catalyzed reactions.
The role of the two serine hydroxymethyltransferase isozymes is not completely
understood. Serine, a nonessential amino acid, is derived from glucose. Some tissues are
net producers of glycine while others, such as kidney, are net producers of serine from
glycine. Glycine is a gluconeogenic amino acid and, in liver and kidney at least, the net
flux through one of the hydroxymethyltransferase isozymes should be in the direction of
serine synthesis under normal conditions of net gluconeogenesis. Mammalian cell mutants
that lack mitochondrial serine hydroxymethyltransferase enzyme activity but have normal
levels of cytosolic activity require exogenous glycine for growth (97–100) and overexpression
of the cytosolic isozyme increases serine synthesis (unpublished data). Most of the
one carbons used in cytosolic one-carbon metabolism in cultured cells are derived from
serine metabolism in the mitochondria (unpublished data) indicating that the mitochondrial
isozyme is required for net glycine synthesis, while the cytosolic enzyme catalyzes a net
Folic Acid 439
flux from glycine to serine, in mammalian cells at least. Whether this is true of all normal
tissues remains to be ascertained.
Different genes encode the two serine hydroxymethyltransferase isozymes. The
cDNA for human cytosolic serine hydroxymethyltransferase codes for a 483 amino acid
protein of 53 kDal. The cytosolic and mitochondrial enzymes share about 63% amino
acid identity. The genes coding for the cytosolic and mitochondrial enzymes lie on human
chromosomal regions 17p11.2 and 12q13, respectively (101–103).
Serine hydroxymethyltransferase also catalyzes the irreversible hydrolysis of 5,10-
methenyl-H4PteGlun to 5-formyl-H4PteGlun (104). 5-Formyl-H4PteGlun was originally
thought to be an artifact of folate extraction procedures. However, it is not found in cells
that lack serine hydroxymethyltransferase activity, and overexpression of the hydroxymethyltransferase
increases the level of this folate derivative, and this reaction probably
accounts for the low level of 5-formyl-H4PteGlun found in many biological sources. 5-
Formyl-H4PteGlun is a potent inhibitor of some folate-dependent enzymes, including serine
hydroxymethyltransferase (95), and is the substrate for the folate catabolism enzyme
(P. Stover, personal communication). However, it is not used directly as a substrate in
one-carbon transfer reactions.
5-Formyl-H4PteGlun can be reconverted to 5,10-methenyl-H4PteGlun by methenyltetrahydrofolate
synthetase (EC 6.3.3.2; reaction 15, Fig. 2):
5-formyl-H4PteGlun 
 MgATP > [5,10-methenyl-H4PteGlun]
 
 MgADP 
 Pi
A human cDNA for by methenyl-tetrahydrofolate synthetase has been isolated and
encodes a 27 kDal cytosolic enzyme (104). 5-Formyl-H4PteGlu is used clinically and
experimentally as a source of reduced folate because it is more stable than other reduced
folates.
The major pathway of glycine catabolism is via the glycine cleavage system (reaction
13, Fig. 3) which is located in the mitochondria and catalyzes the following reaction:
glycine 
 H4PteGlun 
 NAD
 > 5,10-methylene-H4PteGlun

 NADH 
 CO2 
 NH4


This complex, which is present in high concentrations in liver and kidney, can provide
an additional one carbon derived from C-2 of glycine to the folate pool (105,106). The
glycine cleavage system is composed of four associated proteins, P,T,L, and H. P protein,
which contains pyridoxal phosphate, catalyzes glycine decarboxylation and transfer of
methylamine to lipoic acid on H protein. The lipoic acid is reduced and the carbon moiety
from glycine is oxidized to the level of formaldehyde. T protein catalyzes the transfer of
formaldehyde to H4PteGlun, and the reduced lipoate on H protein is reoxidized by NAD

in a reaction catalyzed by L protein. Although potentially reversible, the glycine cleavage
system does not appear to play a role in the synthesis of glycine. Coupling of the serine
hydroxymethyltransferase and glycine cleavage systems provides a mechanism for the net
synthesis of serine from two molecules of glycine, with the C-1 and C-2 of serine derived
directly from one molecule of glycine and the C-3 derived from 5,10-methylene-H4PteGlun
arising glycine cleavage.
The cDNA encoding the proteins of the complex have been isolated and contain
mitochondrial leader sequences (107,108). A human genetic disease involving a mutation
in T-protein results in a disease called ‘‘nonketotic hyperglycinemia’’ and results in the
accumulation of glycine in body fluids and early death.
440 Brody and Shane
2. Homocysteine Methylation and Methionine Synthesis
A major cytosolic cycle of one-carbon utilization involves the reduction of 5,10-methylene-
H4PteGlun to 5-methyl-H4PteGlun and the transfer of the methyl group to homocysteine
to form methionine and to regenerate H4PteGlun (Fig. 4). This cycle is catalyzed by two
enzymes, methylenetetrahydrofolate reductase and methionine synthase, although serine
hydroxymethyltransferase can also be considered part of the cycle.
5,10-Methylenetetrahydrofolate reductase (E.C. 1.1.1.68; reaction 10, Figure 2; reaction
9, Figure 4) catalyzes the conversion of one-carbon units at the oxidation level of
formaldehyde to that of methanol as follows:
5,10-methylene-H4PteGlun 
 NADPH 
 H
 > 5-methyl-H4PteGlun 
 NADP

The enzyme is a flavoprotein and catalyzes the committed step in methionine synthesis
and folate-dependent homocysteine remethylation in mammalian tissues (109,110). The
reaction is physiologically irreversible under in vivo conditions due to the redox state of
the NADPH/NADP couple (111).
The human gene for methylenetetrahydrofolate reductase has been cloned (112). A
number of common polymorphisms have been described, one of which (C677T) is associated
with lower tissue levels of the enzyme (113). This polymorphism results in an A to
V change in the coding region of the protein. Although the kinetic properties of the protein
are unchanged, the affinity for the flavin cofactor is reduced and the apoenzyme is unstable
and degraded (114). The mammalian protein is a dimer and consists of two domains, an
N terminal catalytic domain that shares extensive homology with the analogous bacterial
proteins, and a C terminal regulatory domain that binds adenosylmethionine, an allosteric
inhibitor (109).
Fig. 4 The folate-dependent methionine resynthesis cycle and its relationship to transmethylation
and transsulfuration cycles in tissues. The numbers refer to reactions catalyzed by the homocysteine
remethylation cycle (8, serine hydroxymethyltransferase; 9, methylenetetrahydrofolate reductase;4,
B12-dependent methionine synthase; 5, betaine methyltransferase), the transmethylation cycle (1,
adenosylmethionine synthetase; 2, R-methyltransferase; 3, adenosylhomocysteine hydrolase), and
the transsulfuration pathway (6, cystathionine ?-synthase). Genetic defects in key enzymes of homocysteine
metabolism that lead to hyperhomocysteinemia and homocystinuria are indicated by the
blocks. (From Ref. 125.)
Folic Acid 441
The next enzyme in this cycle, methionine synthase (E.C.2.1.1.13; reaction 11, Fig.
2; reaction 4, Fig. 4), is one of only two B12-dependent mammalian enzymes and catalyzes
the transfer of the methyl group from 5-methyl-H4PteGlun to homocysteine (115):
5-methyl-H4PteGlun 
 homocysteine > H4PteGlun 
 methionine
The methionine synthase reaction is the only reaction in which the methyl group of 5-
methyl-H4PteGlun can be metabolized in mammalian tissues. Although methionine is an
essential amino acid and is required in the diet, the methionine synthase reaction plays a
major role in methyl group metabolism as it allows the reutilization of the homocysteine
backbone as a carrier of methyl groups derived primarily from the C-3 of serine. The
enzyme contains tightly bound cob(I)alamin, and the reaction proceeds via methylation
of the cofactor to the methylcob(III)alamin intermediate by 5-methyl-H4PteGlun, and then
by transfer of the methyl group from methylcob(III)alamin to homocysteine in a heterolytic
cleavage to generate methionine and H4PteGlun and regenerate the enzyme cob(I)alamin
form. The enzyme is a Zn metalloprotein.
Human cDNAs and the gene for human methionine synthase have been cloned (116–
118). The cDNA encodes a 140 kDal protein. The enzyme is a monomer and has a three
domain structure. The N terminal domain encodes the catalytic site with homocysteine,
folate, and Zn binding sites, the central domain binds the B12 cofactor, and the C terminal
domain interacts with adenosylmethionine and accessory proteins (see below).
Methionine synthase in mammalian tissues is normally present as the holoenzyme
form, containing a tightly bound B12-cofactor. The cob(I)alamin cofactor is highly reactive
and the cofactor is occasionally oxidized to the nonfunctional cob(II)alamin form during
catalysis. The enzyme is reactivated by one or several poorly characterized accessory
proteins that catalyze the AdoMet and NADPH-dependent reductive methylation of enzyme
bound cob(II)alamin to methylcob(III)alamin. Bacteria possess two methionine synthase
accessory proteins, flavodoxin and flavodoxin reductase, that use NADH, FAD, and
FMN as cofactors. Flavodoxins are not present in mammalian tissues. Mammalian methionine
synthase, when isolated, contains tightly bound soluble cytochrome b5 and, in an
in vitro anaerobic assay system, soluble cytochrome b5 and cytochrome P450 reductase
catalyzed the AdoMet and NADPH dependent reactivation of the enzyme (119). The gene
for a single putative human methionine synthase reductase protein that contains binding
sites for NADPH, FAD, and FMN has recently been cloned (120) by searching the databases
for a novel gene that contained binding sites for all three cofactors. The gene encodes
a novel member of the P450 reductase and NO synthase family. While it has not yet been
demonstrated that the gene product reactivates methionine synthase in a cell free assay
system, genetic evidence strongly points to this protein as the physiological reactivator
of methionine synthase. DNA from patients with severe genetic disease due to a failure
to reactivate methionine synthase contains mutations in conserved regions of the putative
methionine synthase reductase (121). It is difficult to reconcile two systems for the reactivation
of methionine synthase with a single genetic defect that causes loss of activity. It
remains to be established which of these systems is the one that functions under physiological
conditions.
Although cytosolic methionine synthase and mitochondrial methylmalonyl CoA mutase
are the only enzymes that use B12 as a cofactor in mammalian tissues, a large number
of other proteins are involved in the transport and metabolism of vitamin B12. Genetic
defects in many of these as well as in the methionine synthase structural gene can result
in defective methionine synthase activity.
442 Brody and Shane
Methionine synthase can also catalyze the reduction of the anesthetic gas nitrous
oxide to nitrogen. During this process, a hydroxyl radical is formed, which can lead to
destruction of the polypeptide backbone of the protein and inactivation of the enzyme.
Nitrous oxide is sometimes used to inactivate methionine synthase in experimental animals
to generate a model for the metabolic effects of vitamin B12 deficiency (209).
Homocysteine arises from hydrolysis of adenosylhomocysteine (AdoHcy;reaction 3,
Figure 4), the product of adenosylmethionine (AdoMet)-dependent methylation reactions
(reaction 2, Fig. 4), and is not normally found in the diet. Homocysteine can be metabolized
to cysteine via reactions catalyzed by PLP-dependent cystathionine ?-synthase (reaction
6, Fig. 4) and cystathioninase in a transsulfuration pathway. Alternatively, homocysteine
can be remethylated back to methionine via the methionine synthase reaction or by
betaine-homocysteine methyltransferase, which catalyzes the transfer of one of the methyl
groups of betaine to homocysteine to generate methionine and dimethylglycine (122; reaction
5, Fig. 4). Betaine is a product of choline oxidation in liver mitochondria. Although
cytosolic betaine methyltransferase is a high abundance protein in liver, it has a very
limited tissue distribution in other tissues and is present in the kidney of humans but not
in the rat, while methionine synthase is present in all tissues. The high abundance probably
reflects the low catalytic activity of this enzyme, which does not use a cobalamin cofactor.
Many microorganisms, including yeast, express a sluggish B12-independent methionine
synthase that is absolutely specific for 5-H4PteGlu polyglutamates and normally found at
very high concentrations in the cell.
The dimethylglycine product of the betaine methyltransferase reaction is converted
to glycine in the mitochondria via folate-dependent reactions. The flavoproteins dimethylglycine
dehydrogenase and sarcosine dehydrogenase catalyze the oxidative demethylation
of dimethylglycine to sarcosine and sarcosine to glycine, respectively, with the generation
of 5,10-methylene-H4PteGlun (reaction 12, Fig. 3). Most of the folate in mitochondria is
associated with these proteins (123). Thus, although the betaine methyltransferase reaction
is folate-independent, the product of the reaction can generate two one-carbon moieties
that can potentially be used for folate-dependent homocysteine remethylation. Human betaine
methyltransferase has been cloned and the deduced protein sequence shares some
limited homology with the N terminal domain of methionine synthase (124), reflecting
that both enzymes bind homocysteine and Zn.
Tissue levels of homocysteine are normally in the low micromolar range and increased
homocysteine causes elevated AdoHcy, an inhibitor of many methylation reactions.
The extent of homocysteine remethylation or transsulfuration is tissue dependent,
and many tissues export homocysteine and cystathionine into the circulation. The major
sites of homocysteine remethylation and for transsulfuration are believed to be the liver
and kidneys, but this also appears to be species specific, and has not been completely
clarified (125). Remethylation, in liver at least, is also dependent on the methyl status of
the animal. The major regulator of the folate-dependent methionine cycle is AdoMet,
which is a potent allosteric inhibitor of methylenetetrahydrofolate reductase (Fig. 3). Liver
contains a high Km adenosylmethionine synthetase (reaction 1, Fig. 4), the product of the
MATI gene, and hepatic levels of AdoMet vary with hepatic methionine status. Elevated
levels of AdoMet inhibit the reductase, reducing 5-methyl-H4PteGlun formation and remethylation
of homocysteine, and activate cystathionine ?-synthase, stimulating transsulfuration
of homocysteine to cysteine. When methionine levels are low, AdoMet levels are
reduced relieving the inhibition of methylenetetrahydrofolate reductase, and remethylation
of homocysteine is favored and transsulfuration is inhibited. The adenosylmethionine synFolic
Acid 443
thetase in nonhepatic tissues is the product of the MATII gene and has a lower Km for
methionine. Consequently, AdoMet levels and the methionine synthesis cycle in nonhepatic
tissues are less sensitive to changes in methionine levels.
Liver, kidneys, and pancreas also contain a very high abundance cytosolic protein,
glycine N-methyltransferase, that acts as a sink for excess methyl groups. This enzyme
catalyzes the AdoMet-dependent methylation of glycine to sarcosine. Although folate is
not a substrate, 5-methyl-H4PteGlun is a potent inhibitor and the protein is a major cytosolic
folate binding protein (126,127). When AdoMet levels are high, methylenetetrahydrofolate
reductase is inhibited, which reduces 5-methyl-H4PteGlun formation and relieves
inhibition of glycine methyltransferase, allowing removal of excess methyl groups. At
low methionine and AdoMet concentrations, methylenetetrahydrofolate reductase is more
active, 5-methyl-H4PteGlun accumulates, and glycine methyltransferase is inhibited. Glycine
N-methyltransferase binds polycyclic aromatic hydrocarbons and may also function
as a receptor protein for these compounds as small amounts of the protein are found in
the nucleus (128).
Methionine synthesis requires higher levels of folate than other metabolic cycles of
one-carbon metabolism and longer polyglutamate forms than the other metabolic cycles
(129). Although methylenetetrahydrofolate reductase is considered the major regulatory
enzyme in the folate-dependent methionine cycle, 5-methyl-H4PteGlun is the major cytosolic
folate in tissues. This suggests that the methionine synthase reaction is also partially
rate limiting this cycle. Both methylenetetrahydrofolate reductase and methionine synthase
are present at considerably lower concentrations than most of the other enzymes involved
in the metabolism of folate coenzymes. 5-Methyl-H4PteGlu is the major form of folate
taken up by tissues. Removal of the methyl group, via the methionine synthase reaction,
is required before the entering folate can be utilized in other reactions of one-carbon
metabolism, or can be metabolized to the polyglutamates that are retained by cells. As
the entering monoglutamate has to compete for methionine synthase with the preferred
5-methyl-H4PteGlu polyglutamate substrate present in tissues, accumulation of exogenous
folate by tissues is repressed by high intracellular folate or by expansion of the 5-methyl-
H4PteGlun pool.
The ubiquitous nature of the folate-dependent methionine cycle in all tissues probably
reflects its importance in maintaining methyl group status despite normal intakes of
methionine, and possibly because most tissues lack any other mechanism for disposing of
homocysteine, although the homocysteine can be exported to the circulation. As discussed
below, the elimination of this cycle in the methylenetetrahydrofolate knockout mouse
results in viable but sick offspring with many defects including birth defects (R. Rozen,
personal communication). The methionine synthase knockout is embryonically lethal, with
embryos dying very early in gestation (unpublished data). This would suggest that although
this cycle is clearly of physiological importance in homocysteine remethylation
and/or methionine synthesis, the role of methionine synthetase in the conversion of 5-
methyl-H4PteGlun to H4PteGlun, is of more importance to the tissue.
3. Histidine Catabolism
C-2 of the imidazole ring of histidine provides one-carbon units at the oxidation level of
formate for one-carbon metabolism. Cytosolic formiminotransferase (E.C.2.1.2.5; reaction
12, Fig. 2) catalyzes one of the final steps in histidine catabolism. The formimino group
of formiminoglutamate is transferred to H4PteGlun as follows:
formiminoglutamate 
 H4PteGlun > 5-formimino-H4PteGlun 
 glutamate
444 Brody and Shane
The formimino moiety is converted to 5,10-methenyl-H4PteGlun in a formimino-tetrahydrofolate
cyclodeaminase (E.C.4.3.1.4; reaction 13, Fig. 2) catalyzed reaction:
5-formimino-H4PteGlun 
 H
 > [5,10-methenyl-H4PteGlun]
 
 NH3
The transferase and cyclodeaminase activities are found on a single bifunctional protein
in mammalian tissues (94). The protein contains a single binding site for the polyglutamate
tail of folate and channeling between active sites has been demonstrated for polyglutamate
substrates, with the most effective channeling occurring with the longer polyglutamates
normally found in mammalian tissues (94,130). Channeling is abolished with
the pteroylmonoglutamate substrate. Under folate or vitamin B12 deficiency conditions,
formiminoglutamate is excreted in urine.
B. Nucleotide Metabolism
1. Thymidylate Synthesis
Folate in not involved in the de novo synthesis of pyrimidines but is required for the
synthesis of thymidylate. Thymidylate synthase (E.C.2.1.1.45) catalyzes the conversion
of dUMP to dTMP, where the donated one-carbon unit is at the oxidation level of formaldehyde
(reaction 9, Fig. 2):
5,10-methylene-H4PteGlun 
 dUMP > H2PteGlun 
 dTMP
The reaction is unique among folate-dependent reactions in that it not only involves
the transfer of the hydroxymethyl group from folate to the 5 position of dUMP, it also
involves the transfer of the hydride from 6 position of H4PteGlun to the 5 position of the
nucleotide. During catalysis, a binary covalent complex is formed between enzyme and
nucleotide through an active site cysteine, and then a ternary complex is formed with
folate (131,132). The use of the tetrahydrofolate molecule as a reductant results in its
conversion to H2PteGlun. This is the only reaction in which the oxidation state of folate
changes from the tetrahydro to the dihydro form. H2PteGlun is inactive as a coenzyme
and has to be reduced back to H4PteGlun in a reaction catalyzed by dihydrofolate reductase
(E.C.1.5.1.3; reaction 2, Fig. 2) before it can play a further role in one-carbon metabolism:
H2PteGlun 
 NADPH 
 H
 > H4PteGlun 
 NADP

The sole physiological role of dihydrofolate reductase in mammalian tissues is the reduction
of H2PteGlun formed as a result of thymidylate synthesis. In microorganisms, the
enzyme is also required for the reduction of H2PteGlu formed as the end product of folate
biosynthesis. Fortuitously, the enzyme will also reduce unnatural folic acid to H2PteGlu
(reaction 1, Fig. 2), thus making it available for folate metabolism, although folic acid is
a poorer substrate than H2PteGlu.
Thymidylate synthase activity is only expressed in replicating tissues and expression
of the synthase and dihydrofolate reductase mRNA is highest during the S phase of the
cell cycle. Many folate antagonists that inhibit these enzymes have been developed and
used widely as anticancer agents. The drug 5-fluoro-deoxyuridylate acts initially as a substrate
for the thymidylate synthase reaction and forms a covalent complex with enzyme
and folate. Because the fluorine atom cannot be abstracted to complete the reaction, this
drug acts as a covalent inhibitor of the enzyme. Folate binding enhances the stability of
the complex and drug efficacy is improved if pharmacological doses of folate, usually
as 5-formyl-H4PteGlu, are also given. Drug resistance often develops due to increased
Folic Acid 445
thymidylate synthase enzyme. The mechanism for this is somewhat unusual as it involves
translational activation (133). Thymidylate synthase protein binds to its cognitive mRNA
and regulates its own translation. 5-Fluoro-deoxyuridylate binding to thymidylate synthase
inhibits mRNA binding, which leads to derepression of translation. Methotrexate, a 4-
aminofolic acid analog, is a potent inhibitor of dihydrofolate reductase. Treatment of rapidly
growing cells with this drug causes trapping of folate in the nonfunctional dihydrofolate
form. Slowly growing tissues, which have negligible or low levels of thymidylate
synthase activity, do not convert reduced folate to the dihydrofolate form as rapidly, so
are less affected by a dihydrofolate reductase inhibitor. Clinical resistance to methotrexate
often develop by a number of mechanisms. These include mutations in the reduced folate
carrier that result in decreased methotrexate uptake, amplification of the dihydrofolate
reductase gene, which results in a corresponding amplification of reductase mRNA and
protein levels, and decreased folylpolyglutamate synthetase activity, which reduces accumulation
of the drug by the tissue.
2. Purine Synthesis
The C-8 and C-2 positions of the purine ring are derived from 10-formyl-H4PteGlun in
reactions catalyzed by glycinamide ribonucleotide transformylase (GART, E.C.2.1.2.2;
reaction 7, Fig. 2) and aminoimidazolecarboxamide ribonucleotide transformylase (AICART,
E.C.2.1.2.3; reaction 8, Fig. 2), as follows:
10-formyl-H4PteGlun 
 GAR > H4PteGlun 
 formyl-GAR
10-formyl-H4PteGlun 
 AICAR > H4PteGlun 
 formyl-AICAR
GART catalyzes the third step in purine biosynthesis. GART is part of a 110 kDal mammalian
trifunctional protein that also catalyzes two other steps in the purine biosynthetic
pathway (GAR synthase and AIR synthase, 134), while AICART is part of a bifunctional
protein that also possess inosinicase, which catalyzes the final ring closure step of purine
synthesis. The gene for human GAR transformylase is on the q22 band of chromosome
21 (135). A second novel GAR transformylase occurs in E. coli that utilizes formate and
ATP (and not folate) for the synthesis of formyl-GAR (134).
Aminoimidazolecarboxamide (AIC) is excreted in elevated amounts by animals and
humans with a deficiency in folate or vitamin B12 (136). In fact, AIC was discovered
because it accumulated when bacteria were treated with folate antagonists (137).
The 10-formyl-H4PteGlun required for purine synthesis can be formed by the oxidation
of 5,10-methylene-H4PteGlun, which is reversibly, catalyzed by methylene-tetrahydrofolate
dehydrogenase (E.C. 1.5.1.5; reaction 4, Fig. 2; reaction 3, Fig. 3) and methenyltetrahydrofolate
cyclohydrolase (reaction 5, Fig. 2; reaction 4, Fig. 3):
5,10-methylene-H4PteGlun 
 NADP
 - [5,10-methenyl-H4PteGlun]
 
 NADPH
[5,10-methenyl-H4PteGlun]
 
 H2O - 10-formyl-H4PteGlun 
 H

Alternatively, 10-formyl-H4PteGlun can be obtained by the direct formylation of H4PteGlun
(reaction 6, Fig. 2; reaction 8, Fig. 3), catalyzed by formyltetrahydrofolate synthetase:
formate 
 MgATP 
 H4PteGlun > 10-formyl-H4PteGlun 
 MgADP 
 Pi
The dehydrogenase, cyclohydrolase and synthetase activities are associated on a
single trifunctional protein in mammalian tissues that is called C1 synthase (138,139). The
446 Brody and Shane
synthase consists of two separate domains: One contains the dehydrogenase and cyclohydrolase
activities and the other the synthetase activity.
This activity plays a central role in one-carbon metabolism as it controls the oxidation
levels of one-carbon units that can be channeled into purine biosynthesis or into the
synthesis of thymidylate and methionine. It is widely distributed, with very high levels
in liver and kidney. In common with many folate enzymes, it is absolutely specific for
NADP(H). The cDNA for the human protein encodes a 935 amino acid protein (101.5
kDal, 140). The gene for the human enzyme lies on chromosome 14q24. The Km for
formic acid for the synthetase is more favorable when the folylpolyglutamate substrate is
used, demonstrating that the length of the polyglutamyl tail can affect the kinetic properties
of substrates other than the folate itself (141). Yeast also possess a monofunctional methylenetetrahydrofolate
dehydrogenase activity that uses NAD(H) as a substrate, but this activity
has not been observed in mammalian tissues (142).
Mitochondria can also interconvert 5,10-methylene-H4PteGlun and 10-formyl-
H4PteGlun, and this may be catalyzed by a mitochondrial C1 synthase (Fig. 3). This activity
has been well established in yeast, and very low levels of this activity have been reported
in rat liver mitochondria (91), although some investigators have not been able to observe
this activity. No mitochondrial C1 synthase gene has thus far been identified. A separate
bifunctional 5,10-methylenetetrahydrofolate dehydrogenase-cyclohydrolase that uses
NAD
 rather than NADP
 as the acceptor has also been described and its gene cloned
(143). This mitochondrial enzyme activity is found in embryonic, undifferentiated, or
transformed tissues and cells. One suggested role for this enzyme is to increase the onecarbon
flux into purine biosynthesis and away from other one-carbon cycles such as methionine
synthesis.
Isolated mammalian liver mitochondria can oxidize the ?-carbon of serine to formate
and CO2, the proportion of which is dependent on the respiratory state of the organelle
(144). Formate formed can leave the mitochondria and be reincorporated into the folate
one carbon pool by 10-formyltetrahydrofolate synthetase. NMR studies in yeast suggest
that mitochondrial formate generated from mitochondrial serine oxidation is the major
provider of one-carbon units for purine synthesis in the cytosol and that the cytosolic C1
synthase operates in the reductive direction (145,146). Mammalian cells with defects in
mitochondrial folate metabolism are glycine auxotrophs, have depleted cytosolic one-carbon
pools, require higher levels of folate to support purine synthesis, and are defective
in homocysteine remethylation (unpublished data). Recent mass spectrometry studies with
mammalian cells have suggested that over 95% of the one-carbon moieties that are incorporated
into methionine via the cytoplasmic folate-dependent methionine cycle are derived
from mitochondrial serine metabolism to formate. This mimics the directionality for onecarbon
pathways suggested for yeast. Whether this is also true for normal mammalian
tissues remains to be explored.
C. Disposal of One-Carbon Units
One-carbon moieties are oxidized to CO2 in a reaction catalyzed by 10-formyl-tetrahydrofolate
dehydrogenase (E.C.1.5.1.6; reaction 16, Fig. 2; reaction 7, Fig. 3):
10-formyl-H4PteGlun 
 NADP
 
 H2O > H4PteGlun 
 CO2 
 NADPH 
 H

The purified enzyme also catalyzes the hydrolysis of 10-formyl-H4PteGlun to H4PteGlun
and formate (147,148). H4PteGlun is a potent product inhibitor of the enzyme. The metaFolic
Acid 447
bolic flux through this reaction in liver may be regulated by the 10-formyl-H4PteGlun/
H4PteGlun ratio, rather than by the tissue concentration of 10-formyl-H4PteGlun, as the
concentrations of these folates are in excess of their Km and Ki values for the enzyme
under physiological conditions. The physiological role of this protein would appear to be
to regulate the proportion of folate present in the H4PteGlun form, presumably to make
it available for other reactions of one-carbon metabolism, and to dispose of excess onecarbon
moieties. The enzyme is a major H4PteGlun cytosolic folate binding protein in
liver (149,150). It appears to bind 10-formyl-H4PteGlun, which turns over to H4PteGlun
when the protein is isolated.
The rat cDNA encodes a 902 amino acid protein of 100 kDal (151). The N-terminal
200 amino acids share some homology with GAR transformylase while the final C-terminal
400 amino acids share homology with aldehyde dehydrogenases and the protein possesses
a low NADP-dependent aldehyde dehydrogenase activity.
Mice lacking this activity have been generated by random neutron bombardment
(152). These animals have elevated 10-formyl-H4PteGlun levels and reduced tissue folate,
the latter being consistent with recent studies suggesting that formyl folate derivatives are
substrates for the enzymes that catabolize folates to inactive derivatives. These animals
should serve as a useful model for studying the metabolic role of this protein, although
the possibility of other lesions in these animals can not be eliminated.
D. Mitochondrial Protein Synthesis
Protein biosynthesis in mitochondria, chloroplasts, and bacteria is initiated by a special
type of transfer RNA, N-formyl-methionyl—tRNAfmet. The formylation of the methionine
residue bound to the tRNA is catalyzed by methionyl-tRNA transformylase (reaction
9, Fig. 3):
10-formyl-H4PteGlun 
 methionyl—tRNAfmet
> H4PteGlun 
 N-formyl-met-tRNAfmet
This enzyme is essential in bacteria and interruption of the gene in yeast causes defects
in mitochondrial protein synthesis. Although it is assumed that an analogous protein is
required for normal mitochondrial function in mammalian cells, it is not clear whether
this is the case. Mammalian cells will grow at normal rates in the complete absence of
folate if glycine, purines, thymidine, and methionine, the major products of one-carbon
metabolism, are provided, which would suggest that formylation of methionyl-tRNAfmet
is not required in mammalian cells.
VIII. DEFICIENCY EFFECTS
The classical symptom of folate deficiency in humans is a megaloblastic anemia that is
indistinguishable from that caused by vitamin B12deficiency. Folate deficiency symptoms
are usually due to a dietary insufficiency, although they can arise from other causes such
as malabsorption syndromes or drug treatment. Cases of increased requirement due to
genetic variation have been identified, including some individuals who require increased
folate in early pregnancy to reduce the risk of birth defects. Many of the clinical effects
of folate deficiency can be explained by the metabolic role of folate coenzymes in pathways
leading to DNA precursor synthesis and methyl group homeostasis. Because of these
roles, symptoms of deficiency are often expressed first in rapidly growing tissues. De448
Brody and Shane
pressed folate status has also been associated with increased cancer and vascular disease
risk (153,154). Neurological manifestations of deficiency have been suggested, although
the evidence for this is not convincing. However, in rare cases of genetic disease resulting
in severe defects in enzymes of one-carbon metabolism, neurological symptoms have been
clearly documented with many cases of mental retardation.
Serum folate falls below normal after three weeks of experimental folate deprivation
in humans. After seven weeks there is an increase in the average number of lobes of the
nuclei of the neutrophils (hypersegmentation). Red blood cell folates gradually fall and
reach subnormal levels after four months of folate deprivation, reflecting the life span of
the red cell. At about 4.5 months the bone marrow becomes megaloblastic and anemia
occurs (155). Lesions throughout the intestinal tract may occur in subjects receiving antifolate
chemotherapy (156).
Folate deficiency can be induced in experimental animals by feeding diets lacking
in or deficient in the vitamin. Deficiency symptoms appear earlier in the young growing
animal than in the adult. In some animals, such as the rat, folate deficiency is difficult to
achieve unless the diet contains an antibacterial drug to prevent synthesis of folate by the
gut microflora (157). Although coprophagy may explain the need for antibacterials, it
appears that rats are able to absorb folate biosynthesized toward the distal end of the
intestine. Addition of labeled p-aminobenzoic acid to the diet of the rat resulted in labeled
folates in the liver (158). The extent to which bacterially synthesized folates can contribute
to the animal’s vitamin stores has not been evaluated. The signs of folate deficiency in
animals includes anorexia, diarrhea, cessation of growth, weakness, decreased leukocyte
count, and low red blood cell counts (anemia), and eventually death. Although leukopenia
develops universally in rats fed folate-free sulfa drug-containing diets, anemia occurs less
frequently.
A. Megaloblastic Anemia
Megaloblastic anemia is a reflection of deranged DNA synthesis in blood cells. It is characterized
by enlarged red cells and hypersegmentation of the nuclei of circulating polymorphonuclear
leukocytes with reduced cell number. Megaloblastic changes also occur in
other tissues such as the small intestine but the condition is usually detected clinically by
the anemia. Cellular DNA content is increased, but the DNA contains strand breaks suggesting
a defect in DNA synthesis or repair. Cells growth is arrested in the G2 phase of
the cell cycle just prior to mitosis, preventing cell division. If cell division occurs, many
of the cells undergo apoptosis (159,160).
These defects are thought to be a result of uracil misincorporation into DNA in
place of thymidylate. dUTP is not normally incorporated into DNA but can arise by deamination
of cytosine. This is a potentially mutagenic event as uracil is recognized as thymine
and can form a base pair with adenine. Replication of the DNA will change the C-G base
pair to a U(T)-A base pair. This change is repaired by uracil-DNA glycosylase, which
removes the uracil base (211). A few additional bases are removed on either side of the
damage and the DNA is repaired by complementary base pairing with a C being reinserted
opposite the G. Direct dUTP incorporation into DNA is also minimized by a dUTPase
activity, which hydrolyzes dUTP to dUMP and keeps cellular dUTP pools very low.
In severe experimental folate deficiency induced by antifolate drugs, thymidylate
pools are depressed and dUTP pools are increased leading to increased dUTP incorporation
instead of dTTP (160). Increased repair by the glycosylase would lead to more transient
Folic Acid 449
single-stranded breaks. In addition, repair of the damage by reinsertion of thymidine is
defective due to the lower thymidylate pools and the probability of uracil being reinserted
by mistake is higher, which would lead to prolongation of the single-stand breaks. A
double-strand break can occur when uracil is misincorporated on both DNA strands in
close proximity. Although this mechanism has been established in cell culture with antifolate
drugs and is believed to be responsible for inducing the megaloblastosis of human
folate deficiency, technical problems in the measurement of uracil in DNA have prevented,
until recently, confirmation that this occurs in folate-depleted humans. Damaged blood
cells, which might be expected to demonstrate an increased uracil content, are normally
removed by the spleen. In a recent study using a more sensitive assay for uracil in DNA
and involving splenectomized subjects, individuals with low red cell folate levels had an
increased uracil content and double-strand breaks in their DNA, and folate supplementation
reversed these abnormal findings (161).
B. Vitamin B12 Interactions
The pernicious anemia that results from vitamin B12 deficiency is identical to that observed
in folate deficiency, and vitamin B12 deficiency induces many metabolic changes in onecarbon
metabolism that are identical to those observed in folate deficiency. The interrelationship
between these two vitamins is best explained by the methyl trap hypothesis (162).
The two vitamins are substrates or cofactors for the methionine synthase reaction. A block
in this enzyme would cause accumulation of folate in the 5-methyl-H4PteGlun form. As
5-methyl-H4PteGlun cannot be metabolized by any other mechanism, this would result in
the trapping of folate in a nonfunctional form with a concomitant reduction in the level
of other folate coenzymes required for other reactions of one-carbon metabolism. Because
5-methyl-H4PteGlu is a poor substrate for folypolyglutamate synthetase, the ability of
tissues to polyglutamate and retain entering folate would be greatly diminished (163,164),
and a true folate deficiency would be superimposed on the functional deficiency caused
by the methyl trap.
Methionine synthase activity in bone marrow of pernicious anemia patients is reduced
over 85%, and most of the protein is present in the apoenzyme form (165). Patients
with severe genetic defects in methionine synthase or methionine synthase reductase that
cause gross impairment of methionine synthase activity develop early onset megaloblastic
anemia (166,167). A defect in methionine synthesis or methyl group status cannot explain
this anemia as patients with severe genetic disease involving methylenetetrahydrofolate
reductase, a defect that would block the methionine cycle but not result in a methyl trap,
do not develop megaloblastosis (168). As discussed previously, deletion of both copies
of the mouse methionine synthase gene causes embryonic lethality while the methylenetetrahydrofolate
reductase knockout is viable. This illustrates that the methyl trap can cause
a very severe derangement of one-carbon metabolism and that no mechanism exists that
can compensate for impaired methionine synthase activity.
Vitamin B12 deficiency can be induced in the rat by feeding a vitamin-free diet and
the metabolic effects of a severe deficiency can be mimicked by nitrous oxide exposure,
which causes essentially total loss of methionine synthase activity. Nitrous oxide treatment
causes a gross impairment in tissue folate levels due to an inability to retain folate, an
increase in plasma folate levels, and an increase in the proportion of hepatic folate in the
5-methyl-H4PteGlun form, all of which are consistent with the methyl trap mechanism
(210). Although not all hepatic folate is trapped as 5-methyl-H4PteGlun, essentially all
450 Brody and Shane
cytosolic folate is converted to this form, and the nonmethyl folate remaining in the tissue
is located in the mitochondria (unpublished data). Addition of high levels of methionine
or ethionine to the diet, or their injection, ameliorates the effects of nitrous oxide (169)
on hepatic folate levels and one-carbon distributions, consistent with adenosyl-methionine
or adenosyl-ethionine inhibition of methylenetetrahydrofolate reductase. Megaloblastic
changes also occur in the blood profiles of subjects treated with the anesthetic nitrous
oxide as a result of destruction of methionine synthase. After treatment is terminated,
enzyme levels gradually return to normal due to synthesis of new protein.
C. Cancer
Epidemiological studies have suggested that folate deficiency is associated with increased
risk for certain types of cancer, including colon cancer (154). While the mechanism for
this has not been established, uracil misincorporation arising from defective thymidylate
synthesis has been hypothesized as one possibility. Transcription of many genes is turned
off during development by methylation of their promoter regions’ and changes in gene
methylation, both hyper and hypomethylation, have been observed in tumors. As folate
deficiency impairs the remethylation of homocysteine to methionine and alters AdoMet/
AdoHcy ratios, it has also been proposed that the increased cancer risk in folate deficiency
may be due to hypomethylation of DNA. It has been shown that methionine deficiency
causes hypomethylation of DNA (170). A clear demonstration that folate deficiency results
in DNA hypomethylation remains to be carried out.
The increased cancer risk in subjects with poorer folate status is reduced in subjects
homozygous for a common polymorphism in methylenetetrahydrofolate reductase (154)
and, somewhat surprisingly, this polymorphism had an even greater beneficial effect on
risk in subjects with good folate status. This polymorphism causes decreased enzyme
activity and presumably impaired conversion of 5,10-methylene-H4PteGlun to 5-methyl-
H4PteGlun. It is speculated that this may allow a redirection of more of the folate onecarbons
into the cycles of nucleotide biosynthesis in these subjects, but this remains to
be established.
D. Folate Antagonists—Cancer Treatment and Other Diseases
Folate antagonists that are inhibitors of thymidylate synthase, dihydrofolate reductase, and
de novo purine biosynthetic enzymes have been used extensively for the treatment of a
variety of cancers. These metabolic inhibitors cause a functional folate deficiency and
generally show a selective toxicity for rapidly growing tumors because of the increased
rates of DNA synthesis in these tumors. The most widely used drug is methotrexate, a 4-
amino-folate, which is a potent inhibitor of dihydrofolate reductase. This drug was not a
‘rationally designed drug’ as it was first synthesized and used for chemotherapy almost
60 years ago, long before dihydrofolate reductase was identified. ‘‘Rescue’’ therapy is
sometimes employed to increase the efficacy of methotrexate. In this treatment, a large
toxic dose of the drug is given and this is followed by giving a large dose of folinic acid
(5-formyl-H4PteGlu) as an antidote to rescue normal cells. Another commonly used drug
is 5-flurouracil, an inhibitor of thymidylate synthase described earlier.
In experimental animals, toxicity and drug effectiveness is reduced by the provision
of purines and thymidine. Uracil misincorporation and apoptosis have been demonstrated
as the mechanism for cell death. Uracil misincorporation is also potentially mutagenic,
Folic Acid 451
and successful treatment with antifolates, or with many other drugs used for cancer chemotherapy,
carries the risk of further cancers after ten to twenty years.
Antagonists of folate biosynthesis, such as sulfa drugs, have been used extensively
as antimicrobiol agents with limited side effects, as most of these drugs do not interfere
with mammalian folate metabolism.
Recently there has been a renewed interest in the treatment of rheumatoid arthritis
using low dose methotrexate treatment (171). Here, as with the treatment of cancer and
other diseases such as psoriasis, the patient should be monitored for anemia and other
forms of methotrexate toxicity.
E. Hyperhomocysteinemia and Vascular Disease
Patients with severe genetic disease involving enzymes in the homocysteine remethylation
and transsulfuration pathways are homocystinuric, display very marked hyperhomocysteinemia,
and suffer from a variety of clinical symptoms including early onset occlusive
cardiovascular and cerebrovascular disease (172). These genetic diseases include methylenetetrahydrofolate
reductase deficiency and cystathionine ?- synthase deficiency (Fig.
4). The beneficial effects of folate and betaine on disease progression in patients with
severe B6-nonresponsive cystathionine-?-synthase deficiency is the strongest evidence that
elevated homocysteine or a homocysteine metabolite is the primary cause of the vascular
complications in these patients (173). These treatments can not rescue the metabolic defect
but they can divert homocysteine into methionine. Elevated homocysteine increases proliferation
of smooth muscle cells and inhibits proliferation of endothelial cells by a mechanism
that is not understood (174). Many potential reasons for the adverse effects of homocysteine
have been described. These include effects on transcription factors involved in
regulation of cell growth, covalent modification via disulfide bond formation of proteins
such as apolipoprotein B100, modulation of NO synthase activity, and increased cellular
adenosyl homocysteine levels. It has not been established which, if any, of these potential
adverse changes is responsible for the vascular disease.
Recently, it has been recognized that chronic mild hyperhomocysteinemia may also
be a major risk factor for occlusive vascular disease (175). Most of the evidence for this
has come from case control studies and prospective studies have been less convincing.
Plasma homocysteine concentrations in patients with vascular disease were about 30%
higher than in controls, and carotid artery stenosis was positively correlated with plasma
homocysteine concentrations over the entire range of normal and abnormal homocysteine
values (176). Prospective assessment of vascular disease risk in men with higher homocysteine
concentrations indicated that plasma homocysteine levels only 12% above the upper
limit of normal levels were associated with a threefold increase in acute myocardial infarction
(177).
Fasting homocysteine levels have been inversely correlated with both plasma folate
levels and food folate intake (178). Increased folate intake lowers the mean homocysteine
of groups, and the lowering effect is greatest in subjects with the highest plasma homocysteine
levels. Folate is less effective in reducing elevated homocysteine in renal disease
patients, suggesting that the kidney is a major site of homocysteine metabolism. A common
polymorphism (Ala to Val) in methylenetetrahydrofolate reductase that results in a
heat-labile enzyme, and decreased enzyme activity in tissues has been implicated as one
reason for the folate-responsiveness of a subset of hyperhomocysteinemic subjects (113).
The incidence of the val/val homozygote (around 10 to 15% in most populations) is sig452
Brody and Shane
nificantly increased in subjects with the highest deciles of homocysteine levels. Although
the contribution of this polymorphism to elevated homocysteine levels has varied from
study to study, and the incidence of the polymorphism varies between different population
groups, the val/val polymorphism may at most contribute to or be associated with 30%
of hyperhomocysteinemia. While case control studies indicate an association between this
polymorphism and vascular disease risk, this relationship was not observed in a large
prospective study (179). It is also interesting to note that this potential genetic risk factor
for vascular disease is the same polymorphism that epidemiological studies have suggested
lowers the risk of cancer (described above). About 50% of the general population are
heterozygous or homozygous for this polymorphism, and it is particularly interesting that
a simple nutritional intervention may ameliorate at least some of the adverse effects of a
potentially deleterious genetic trait.
Impaired vitamin B12 status has also been associated with the highest decile of fasting
plasma homocysteine in the general population, but B12 is quantitatively a less important
risk factor than folate for hyperhomocysteinemia (178). Again, this is consistent with the
role of cobalamin as a cofactor for methionine synthase. Dietary vitamin B12 content had
little effect on homocysteine levels, reflecting that defects in B12 absorption rather than
dietary vitamin B12 content play a greater role in the development of impaired B12 status.
PLP is a cofactor for cystathionine ?-synthase. However, vitamin B6 status has little effect
on fasting homocysteine levels (178), but improved vitamin B6 status reduces the increase
in plasma homocysteine following a methionine load or a meal, and also nonfasting homocysteine
levels.
Very high levels of homocysteine are clearly associated with severe vascular disease,
but it remains to be established whether the increased risk associated with mildly elevated
homocysteine is due directly to homocysteine. The increased risk may be due to some
other metabolic disturbance or change in vitamin status for which homocysteine acts as
an indicator. Consequently, although homocysteine is associated with risk for vascular
disease, and increased intake of folate reduces circulating homocysteine levels, it remains
to be determined whether increased folate intake reduces vascular disease risk. A number
of clinical intervention trials are currently in progress to ascertain whether vitamin supplementation
influences the incidence of stroke and of cardiovascular disease, which should
help in answering some of these questions.
F. Pregnancy
Megaloblastic anemia due to folate insufficiency has long been recognized as a complication
of pregnancy (180) with an incidence of 3–5% in developed countries and much
higher incidence in Africa, Southeast Asia, and South America. The development of this
syndrome is due to the increased nutritional need due to the growth of the fetus. The
anemia presents as hematocrit values below the accepted norm for pregnancy, and folate
deficiency as indicated by low red blood cell folates.
G. Birth Defects
Neural tube defects (NTDs), including most forms of spina bifida, are the most common
birth defects in humans, affecting about 0.1% of births, although in some regions the
numbers are much higher. The reoccurrence rate for this condition is about 2%. Neural
tube defects are a family of birth defects involving the brain or spinal cord. They arise from
incomplete closure of the neural tube during the fourth week of pregnancy. Neurulation is
Folic Acid 453
the first organogenetic process to be initiated and completed in humans, and occurs during
the fourth week of pregnancy. In this process, a flat structure called the neural plate forms
two parallel ridges. These ridges fold over and move towards each other and seal and
form the neural tube. Closure of the tube begins separately at three sites, the cervicalhind
brain boundary, the forebrain-midbrain boundary, and the rostral extremity of the
forebrain. Closure spreads to the intervening areas and a failure of closure at any of the
regions can lead to a NTD. Any drug, nutrient imbalance, or genetic defect that interferes
with this stage of development may prevent normal closure of the tube, resulting in defects
in the newborn. At its least severe, an NTD may be undetectable except by examination
of the spine by the fingers of a physician. At its most severe, an NTD involves the absence
of the brain. The most common NTDs are spina bifida (spinal cord at the lumbar vertebra
not covered with bone) and anencephaly (no brain).
The observation that peri-conceptual folate supplementation with folic acid reduced
the incidence of NTDs by about two-thirds (181), which has now been confirmed in a
number of studies (182,183), has led to fortification of the U.S. food supply with folic
acid (184). Folate supplementation is only useful if given very early in pregnancy at a
time when many women do not realize they are pregnant. Although folate status affects
the risk for neural tube defects, this condition is not thought to be a result of folate defi-
ciency per se. It appears to be a genetic disease, probably multigenetic, with a phenotype
that can be modified by increased folate in the subset of individuals that are folate responsive.
As the mechanism behind the disease in not known, there is currently no screening
technique to identify individuals at risk.
A defect in homocysteine metabolism has been proposed as a mechanism, although
the evidence supporting this proposal is far from conclusive. Plasma homocysteine levels
are slightly higher in affected mothers (185). Homocysteine can cause teratogenic effects
in embryo culture, but very high levels were used in these studies. An increased incidence
of the homozygous Ala to Val polymorphism in methylenetetrahydrofolate reductase has
been reported in a number of studies but this could account for, at most, 15% of neural
tube defect risk (186). One epidemiological study suggested that vitamin B12 status is an
independent risk factor for neural tube defects (187), which would implicate methionine
synthase, or a gene encoding a product that influences methionine synthase activity, as
another possible locus for the defect. A common polymorphism in the methionine synthase
gene has a modest influence on homocysteine levels (188). However, no polymorphisms
or mutations in the methionine synthase gene or in the methionine synthase reductase
gene have been identified thus far that track with neural tube defects.
A number of folate responsive mouse models of neural tube defects have been developed.
Disruption of the FBP1 gene, which encodes one of the two mouse folate binding
proteins, causes NTDs, the incidence and severity of which are reduced by folate supplementation
(189). However, no polymorphisms in the equivalent human FBP? gene were
observed in human NTD cases (190). The methylenetetrahydrofolate reductase knockout
also exhibits neural tube defects that are alleviated to some extent by high folate. The
methionine synthase knockout embryo does not survive to day 8.5 of gestation when the
neural tube closes in the mouse, even when the mother is supplemented with very high
levels of folate and products of one-carbon metabolism. Because of the role of folate in
pathways involved in cell growth and DNA metabolism, it is likely that disruption of
many of the genes encoding folate enzymes in mice would cause birth defects. Which,
if any, of the genes is responsible for human birth defects remains to be discovered. None
of the polymorphisms that are known for human cystathionine ?-synthase track with NTD
454 Brody and Shane
cases and total disruption of this gene in mice does not have any apparent effect on fetal
development (191). The animals become quite sick soon after birth when homocysteine
levels become greatly elevated. However, they die of hepatic failure rather than vascular
disease. The animals can be maintained for longer periods if the diet is fortified with
choline. Although this might suggest that homocysteine is not involved in the etiology of
NTDs, the synthase enzyme is not expressed during early fetal development and the fetus
appears to be dependent on remethylation for metabolism of homocysteine.
The folate intervention trials that established the protective effect of folic acid, coupled
with other dietary surveys, have indicated that 400 µg of supplemental folic acid in
addition to customary dietary folate intake is sufficient to provide the maximum benefit
in reducing the incidence of folate-responsive birth defects (reviewed in Ref. 192). It is
not known whether lower levels would be as effective, or whether disease risk could be
reduced by dietary folate alone, as food folate is less bioavailable than folic acid. The
fortification of the U.S. food supply with folic acid is designed to provide an average
intake of 100 µg of supplemental folic acid in addition to normal dietary folate intake.
This represents a compromise between the needs of the relatively small population at risk
for birth defects and the relatively larger population at risk for masking of symptoms of
vitamin B12 deficiency by high folate (see below). Preliminary studies have suggested that
folate supplementation may also have a beneficial effect on other pregnancy outcomes.
Although the fortification of the food supply with folic acid has just started, it is already
clear that this had a major impact on indicators of folate status in the U.S. population.
The average additional intake of folic acid is considerably higher than 100µg because of
overage by food producers; in part to allow for losses in storage. As folic acid is almost
twice as bioavailable as food folate, the average increased intake is equivalent to about
250 to 300 µg food folate, or about a doubling of the folate intake. This has led to large
increases in plasma folate levels and a significant drop in plasma homocysteine levels,
particularly in individuals who were at the higher end of the homocysteine distribution
previously (193). Whether this fortification has a positive effect of birth defect incidence
or on vascular disease incidence remains to be seen.
While the beneficial effect of increased folate is probably due to its correction of
a metabolic defect(s), other possibilities cannot be excluded. One of the intervention trials
that demonstrated a protective effect of folate for neural tube defects also noted an increased
spontaneous abortion rate in supplemented subjects. A large proportion of embryos
abort very early in pregnancy, and it has been suggested the folate effect may be due to
an increased abortion rate of impaired embryos in early pregnancy rather than a correction
of a metabolic imbalance.
H. Anticonvulsants
A small percentage of epileptics treated with diphenylhydantoin develop a folate defi-
ciency and anemia (194,195). Studies with mice (196) and humans (197) suggest that
increased folate excretion may be a contributing factor. Large doses of folic acid (5–30
mg/day) have been used to reverse the hematological signs of folate deficiency (198),
though the folic acid may exacerbate the seizures in certain patients.
I. Ethanol
Low serum folates are commonly encountered in alcoholics, largely because of poor nutritional
habits. Chronic alcoholism is probably the major cause of folate deficiency in the
Folic Acid 455
United States. An antifolate effect of ethanol was shown in studies of humans (199).
Megaloblastic anemia could be induced with a low-folate diet after 6–10 weeks, whereas
the inclusion of alcohol with this diet provoked anemia at an earlier time, 2–3 weeks
(199).
J. Malabsorption Syndromes
Diseases of the intestinal tract such as tropical sprue and nontropical sprue (gluten enteropathy)
lead to general malabsorption syndromes including folate deficiency. Folate is normally
absorped in the jejunum. In some sprue cases, the malabsorption in the jejunum is
partially alleviated by increased absorption lower down the intestine. Folate deficiency
can also occur in Crohn’s disease and ulcerative colitis because of the malabsorption (200).
Congenital disorders associated with folate malabsorption have been reported, implying
a specific transport system for the vitamin (201). The resulting deficiency can be relieved
by large oral doses of the vitamin (40–100 mg).
IX. METHODS OF NUTRITIONAL ASSESSMENT
A. Assay of Folate Derivatives in Plasma and Serum
Folate status is most commonly assessed by plasma or serum folate levels. This can be
measured by microbiological assay using L. casei as the test organism, but this test can
be confounded if the subject is on antibiotic treatment. Other bacteria such as S. fecium
or P. cerevisiae are not suitable for the assay of serum folates as they do not respond to
5-methyl-H4PteGlu. Serum folate levels of less than 3 ng/ml indicate a folate deficiency,
levels of 3–6 ng/ml a marginal deficiency, and levels above 6 ng/ml adequate folate status.
One problem in interpreting serum folate values is that they reflect recent dietary intake,
and a vitamin deficiency can be ascribed only where serum folate remains low over a
period of time.
Radioassay procedures for the measurement of serum folate have been developed
and radioassay kits are used extensively in clinical laboratories. These competitive proteinbinding
assays are easier to perform than microbiological assays and are not affected by
antibiotics, which give false low values in microbiological assays. However, the affinities
of different folate monoglutamates for the binding proteins vary considerably, making this
assay method useful only for those tissues in which one form of folate predominates,
i.e.,serum or plasma (202). In addition, serum folate values obtained by using the different
commercially available kits fluctuate considerably. Because of this, no absolute values
can be given to indicate folate deficiency. Each laboratory has to define its own lower
limits based on a large number of sample assays from a representative population of normal
subjects. In the measurement of serum folate levels by both microbiological and radioassay
methods, great care must be taken to prevent hemolysis of the samples, as red
cell levels of folate are considerably higher than serum levels.
B. Assay of Red Cell Folates
Red cell folate levels reflect the body folate stores at the time of red cell formation and
hence reflect a more accurate and less variable index of folate status than plasma folate
levels. Folates in the red cell are polyglutamate derivatives and must be hydrolyzed to
monoglutamates prior to their assay by microbiological or radioassay techniques. As prac456
Brody and Shane
tically all the whole blood folate is located in the red cell, this is usually accomplished
by lysing whole blood and allowing plasma glutamyl hydrolase to hydrolyze the polyglutamate
derivatives. As in the assay of serum folates, it is essential to protect the vitamin
with reducing agents, such as ascorbate. Red blood cell folate levels of less than 140 ng/
ml packed cells, measured by microbiological assay, indicate folate deficiency, levels of
140–160 ng/ml suggest marginal status, and levels above 160 ng/ml indicate normal folate
status.
Subjects homozygous for the C677T polymorphism in methylenetetrahydrofolate
reductase exhibit a changed folate one-carbon distribution in their red cells (203). As
the radioassay procedure responds differently to different folate forms (202), it may be
confounded under conditions such as this that alter folate one-carbon distributions.
C. Histidine and Methionine Load Tests
The levels of metabolites such as urinary formiminoglutamate and plasma homocysteine
following a loading dose of histidine or methionine can be used as a measure of folate
status (193). However, the levels of these metabolites are also abnormal under conditions
of vitamin B12, and in some cases, vitamin B6 deficiency.
D. Deoxyuridine Suppression Test
Labeled thymidine incorporation into the DNA of bone marrow cells is reduced in the
presence of deoxyuridine due to the conversion of dUMP to dTMP, and the consequent
competition between labeled and unlabeled dTTP (204). The extent of the competition is
a measure of thymidylate synthase activity, which depends on the level of functional folate
in the cell. Under conditions of low cellular folate, the conversion of dUMP to dTMP is
reduced and the suppression of labeled thymidine incorporation into DNA is reduced.
This test, known as the dU suppression test, can distinguish between folate and vitamin
B12 deficiency as, in the former case, addition of any folate to the medium increases the
suppression rate. In the case of a vitamin B12 deficiency, addition of methylcobalamin or
any folate (with the exception of 5-methyl-H4PteGlu) increases the suppression. Although
this test has been used experimentally, it has not found widespread usage, partly because
of the difficulty in obtaining marrow cells from patients on a routine basis. Attempts to
extend this test to other cells, such as lymphocytes, have met with mixed success.
X. NUTRITIONAL REQUIREMENTS
The current RDA is 150–300 µg for children, 400 µg for the adolescent, adult, and the
elderly, 600 µg for pregnant women, and 500 µg for lactating women (192). Higher levels
may be required to minimize the risk of birth defects. The recommendation for women
of child bearing age, who are capable of becoming pregnant, is to take 400 µg folic acid
per day, derived from supplements plus fortified food, in additional to their normal food
folate intake. Requirements for the infant are an Adequate Intake (AI) of 65–80 µg, based
on the folate content of milk of well-nourished mothers. Increases in dietary intake of
folate do not affect maternal milk levels.
XI. HAZARDS OF HIGH DOSES
No toxicity of high doses of folate has been reported. In adults no adverse effects were
noted after 400 mg/day for 5 months or after 10 mg/day for 5 years (205). The acute
Folic Acid 457
toxicity (LD50) is about 500 mg/kg body weight for rats and rabbits (206). Chronic doses
of 10–75 mg PteGlu/kg body weight (IP) can injure the kidneys, probably because of
precipitation of the PteGlu at acidic pH (207,208).
Large doses of folic acid can produce a hematological response in subjects with
megaloblastic anemia due to vitamin B12 deficiency, although it does not correct the severe
neurological symptoms of vitamin B12 deficiency. As large doses of folate may prevent
or delay the development and diagnosis of anemia in vitamin B12-deficient subjects, there
is an increased risk that these subjects are only recognized when they develop frank neurological
symptoms. Megadose levels of folate should be avoided and an upper limit of 1
mg has been suggested (192). The intakes of many individuals now exceed this level due to
fortification of the food supply and vitamin pill intake. Whether this leads to an increased
incidence of neurological disease in the elderly will be closely watched, particularly as
there continues to be on ongoing debate about whether the current levels of fortification
is sufficient to eliminate all folate-responsive birth defects.
LIST OF ABBREVIATIONS
PteGlu, pteroylglutamic acid, folic acid
H4PteGlu, tetrahydrofolate
H4PteGlun, tetrahydropteroylpolyglutamate, n indicating the number of glutamate
residues
AdoMet, S-adenosylmethionine
AdoHcy, S-adenosylhomocysteine
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13
Cobalamin (Vitamin B12)
WILLIAM S. BECK
Massachusetts General Hospital and Harvard University, Cambridge,
Massachusetts
I. HISTORY
Cobalamin, the preferred name for the family of derivatives familiarly known as vitamin
B12, is a distinctive molecular species that shares many structural features with other compounds.
Nonetheless, it possesses numerous biochemically unique attributes. The chronicle
of discoveries leading to current knowledge of cobalamin and its deficiency states
embraces a distinguished chapter in the history of biomedical science, one that still needs
an ending. It is worth noting that the influence of these discoveries has reached far into
many and diverse fields, ranging through evolutionary biology, taxonomy, microbial metabolism,
organic chemistry, nutrition, animal husbandry, and clinical medicine.
The familiar story of the discovery of cobalamin (1,2) began with Addison’s description
in 1835 of the disorder known in the English-speaking world as pernicious anemia
(3,4). Until Minot’s demonstration in 1926 of the successful treatment of pernicious anemia
by liver feeding (5,6), this disease was often fatal. In 1929, Castle discovered intrinsic
factor in a study still viewed as a model clinical investigation (7).
There ensued the unsuccessful efforts of two decades to isolate and identify the liver
principle by crude methods—mainly in the laboratory of E. J. Cohn at Harvard University
[though a stimulating new account vividly recounts the contribution of Y. A. Subbarow
at Lederle Laboratories (8)]. As a result of these efforts, parenterally administered liver
extracts early replaced ingested liver in the treatment of pernicious anemia. But great
difficulties plagued investigators attempting to isolate and identify the antipernicious anemia
principle of liver. For example, their failure to discover a naturally occurring animal
disease resembling pernicious anemia meant that purification studies could be guided only
by tiresome assays performed on pernicious anemia patients in relapse.
Soon bacterial and animal nutritionists independently found three factors that led
to the discovery of cobalamin (2): (a) LLD factor, a factor in yeast and liver extracts
463
464 Beck
essential in the nutrition of Lactobacillus lactis Dorner and other microorganisms; (b) an
animal protein factor obtained from tissue extracts and animal feces that promotes growth
of pigs and poultry receiving only vegetable rations; and (c) a ruminant factor, whose
lack causes a wasting disease of ruminants grazing in cobalt-poor pastures and whose
replacement is effected by oral feeding of cobalt salts (or by dusting cobalt on pastures),
parenteral cobalt being ineffective.
The discovery and crystallization of cobalamin in 1948 by Rickes and associates of
Merck Laboratories (9) followed the astute observation by Mary Shorb of proportionality
between the nutrient activity of liver extracts in cultures of L. lactis Dorner and their
therapeutic activity in pernicious anemia (10,11). A swiftly devised, simple microbiology
assay hastened the final purification and identification of the vitamin. Shortly thereafter,
cobalamin was isolated from liver by H. G. Wijmenga and associates, following the unwitting
use of cyanide-activated papain as a proteolytic ferment, with cyanide converting
various forms of the vitamin to the stable and readily crystallizable cyano derivative (12).
Isolation was also achieved in 1948 by E. L. Smith in England, without benefit of microbiological
assay and cyanide (13). Animal protein factor and cobalt-dependent ruminant
factor were promptly identified with cobalamin. West (14) was the first to show that injections
of cobalamin, supplied to him by Rickes et al., induced a dramatic beneficial response
in patients with pernicious anemia. Folkers has presented a stimulating review of these
events (15).
The elucidation of the structure of cobalamin (16) by Dorothy Hodgkin at Oxford
University was a pioneering contribution of the x-ray crystallographer. The discovery by
Barker, Weissbach, and Smyth of the first cobalamin coenzyme (17) was also a tour de
force of biochemical ingenuity. However compelling these works, we can here consider
them only briefly. Historical reviews will be found elsewhere (1,2,6,15).
Early workers isolated two crystalline ‘‘vitamin B12’’ preparations from cultures of
Streptomyces aureofaciens (18). One had an absorption spectrum similar to that earlier
reported for vitamin B12 (19,20); the other had a different spectrum and was termed vitamin
B12. Because vitamin B12, in contrast to vitamin B12 (21), lacked cyanide, it was named
hydroxocobalamin (22). There were no differences in the antipernicious anemia effects
and microbiological nutritional activity of hydroxo- and cyanocobalamin (23). As noted
below, adventitious cyanide arising in the preparatory process probably converted hydroxocobalamin
to cyanocobalamin.
II. CHEMICAL ASPECTS
A number of reviews dealing with aspects of cobalamin chemistry (24–29) may be consulted
for additional references on topics in the following sections.
A. Structure
Despite intensive investigation, elucidation of cobalamin’s unusual chemical structure required
7 years (16). The structure has several unique features (I, Fig. 1). The cyanocobalamin
molecule (C83H88O14N14PCo; mol wt 1355) has two major portions; a planar group,
which bears a close but imperfect resemblance to the porphyrin macro ring, and a nucleotide,
which lies nearly perpendicular to the planar group (II). The porphyrin-like moiety
contains four reduced pyrrole rings (designated A–D) that link to a central cobalt atom,
the two remaining coordination positions of which are occupied by a cyano group (above)
Cobalamin 465
Fig. 1 Cobalamin. I, chemical structures of cyanocobalamin. II, semidiagrammatic representation
of three-dimensional structure showing relations of planar and nucleotide moieties. Hydrogen atoms
and a number of oxygen atoms are omitted.
and a 5,6-dimethylbenzimidazolyl moiety (below the planar group). With one exception,
the pyrrole rings are connected to one another by methylene carbon bridges similar to those
found in porphyrin precursors. The exception is the direct linkage between the carbon of
rings A and D. Another dissimilarity from the porphin structure is the relatively saturated
character of the pyrrole rings, which are extensively substituted with methyl groups or
longer acetamide and propionamide residues.
The macro ring of cobalamin and related compounds is termed corrin; the major
corrin derivatives are known as corrinoid compounds. The term corrin was proposed originally
to refer to the ‘‘core’’ of the vitamin B12 structure. Its first two letters do not denote
the presence of cobalt. That fact is implied by the ‘‘cob’’ of cobalamin.
A biological relation between corrin and porphin macro rings is seen in the fact that
both are synthesized from ?-aminolevulinic acid (30,31). Porphobilinogen, a precursor of
uroporphyrin, coprophyrin, and protoporphyrin, is incorporated into the corrin system by
certain microorganisms.
The several brightly colored forms taken by cyclic tetrapyrroles in nature (Fig. 2),
including heme, chlorophyll, bacteriochlorophyll, siroheme, corrinoids, and F430, the recently
discovered coenzyme of bacterial methanogenesis (32), give striking notice of the
many diverse assignments given to this molecular form in the course of evolution. Indeed,
the cobalt in corrinoids is but one of five metals now known to occur in cyclic tetrapyrroles
with biological functions. The others are iron in hemes and siroheme; magnesium in chlorophyll;
copper in turacin, a pigment of turaco bird feathers; and nickel in F430. Functionally,
as described below, the corrinoids most closely resemble F430 in that both can accept
466 Beck
Fig. 2 Cyclic tetrapyrroles with biological functions. Stippled areas show double bonds and resonating
conjugated bond systems. (From Ref. 32.)
a methyl group when the metal is in the 
1 oxidation state. However, the stability of
these organometallic bonds differs greatly.
Among the biochemically unusual features of cobalamin is a nucleotide, whose
base—5,6-dimethylbenzimidazole—is not found elsewhere in nature. Others include its
base–ribose linkage, which sterically is an ?-glycoside unlike the ? linkages typical of
nucleic acid and coenzyme nucleotides. The ribose is phosphorylated at C-3, one of the
rare natural occurrences of a ribose 3-phosphate structure. There are two connections
between the planar and nucleotide moieties; (a) an ester linkage between the nucleotide
phosphate and a 1-amino-2-propanol moiety that is joined in turn in amide linkage with
a propionic side chain in ring D; and (b) the coordinate linkage between cobalt and the
glyoxalinium nitrogen atom of benzimidazole.
B. Nomenclature
Of many known corrinoid compounds, a certain number occur naturally, whereas others
are prepared by chemical transformation or by manipulation of microbial biosynthetic
systems. A few received trivial names before their structures were known. To abate confusion,
systematic nomenclature was introduced in 1959 (33). Additional rules for naming
corrinoids were proposed in 1966 (34) and 1973 (35). Numbering and ring designations
of the corrin system are summarized in Fig. 3. Nucleotide derivatives of the corrinoids
are named by adding ‘‘-yl’’ to the name of the nucleotide base; e.g., ?-(5,6-dimethylbenzimidazolyl)
cyanocobamide. Since in ordinary parlance complex systematic names cannot
displace trivial or semisystematic names, the semisystematic term cobalamin, introduced
before its chemical structure was known, came to be used to refer to the combining form
of cobalamin that lacks a ligand in the cobalt-? position (i.e., above the plane). Once
lacking in precise meaning, cobalamin is now defined as a cobamide containing a 5,6-
dimethylbenzimidazolyl moiety. In recent years, editors have preferred cobalamin to the
inexact term vitamin B12.
In chemical relatives of cobalamin (e.g., cyanocobalamin), various ligands are covalently
bound to cobalt above the plane. Other such compounds are hydroxocobalamin (or
the basic product of its combination with H
, aquacobalamin) and nitritocobalamin. The
ligand below the plane can also be replaced. In strong acid, a second H2O displaces
the 5,6-dimethylbenzimidazolyl moiety to form diaquocobalamin. Pseudovitamin B12, a
cyanocobalamin analog containing adenine in place of 5,6-dimethylbenzimidazole, is active
in some microorganisms but inert in animals.
C. Cobalamins in Animal Cells
Four cobalamins are of major importance in animal cell metabolism (36). Two are equivalents
of the natural vitamin—cyanocobalamin and its analog hydroxocobalamin—abbreviCobalamin
467
Fig. 3 Systematic nomenclature of cobalamin and related compounds. Inset shows porphyrin
structure for comparison. Note that pyrrole rings of the corrin macro rings are designated A–D.
Substituent acetamide and propionamide groups are designated a–g.
ated CNCbl and OHCbl (35). The other two are alkyl derivatives that are synthesized
from the vitamin and serve as coenzymes. In one, a 5?-deoxy-5?-adenosyl (in short form,
adenosyl) moiety replaces CN as the ligand of cobalt above the plane (Fig. 4). This compound
is usually called adenosylcobalamin (AdoCbl). The other coenzymatic derivative
is methylcobalamin (MeCbl).
Fig. 4 The coenzyme synthetase reaction. ATP adenosylates cobalamin to form adenosylcobalamin.
468 Beck
Knowledge of AdoCbl derived from classic studies of Barker and associates on the
conversion of glutamate to ?-methylasparate by extracts of Clostridium tetanomorphum
(17,24). Purified preparations of the glutamate mutase contained a coenzyme shown to
be a novel derivative of pseudovitamin B12, ?-(7-adenyl)adenosylcobamide. When C. tetanomorphum
was cultivated in the presence of free exogenous 5,6-dimethylbenzimidazole,
the biosynthetic product was AdoCbl. Proplonibacterium shermanii was found to produce
AdoCbl naturally and the same compound was soon found in mammalian liver. In AdoCbl
the 5?-methylene carbon atom of the 5?-deoxy-5?-adenosyl moiety is linked directly to the
cobalt atom (37).
In the second cobalamin with coenzyme activity, the ligand of cobalt is a methyl
group (38). MeCbl is the major form of cobalamin in human blood plasma (39,40). As
noted below, the CCo bond in both coenzymes is labile to light, cyanide, and acid. It
is likely that the cyanocorrinoids encountered in nature, including CNCbl, arise mainly
from the cyanolysis of corrinoid coenzymes.
CNCbl and OHCbl are converted to AdoCbl in tissues by a ‘‘coenzyme synthetase’’
system (Fig. 4) (41–43). The reaction requires a thiol or dithiol, a reduced flavin, and
ATP—the biological alkylating agent. The 5?-deoxy-5?-adenosyl moiety of ATP is transferred
to the vitamin, and the three phosphates of ATP are released as inorganic triphosphate.
Reducing agents are required in a preliminary step that converts the trivalent cobalt
of cobalamin [cyanocob(III)alamin] through the bivalent state [vitamin B12r or cob(II)
alamin] to the univalent state, vitamin B12 (or cob(I)alamin, which has nucleophilic properties).
D. Biosynthesis
A detailed summary of the biosynthetic pathway of cobalamin in microorganisms is beyond
the scope of this chapter. We note here only that the basic precursor, uroporphyrinogen
III (also the precursor of other natural pigments), is channeled toward cobalamin by
successive methylations yielding precorrins 1, 2, 3A, 4, 5, and 6A. A reduction step produces
precorrin 6B. The last two of the required eight methylations plus a decarboxylation
step yields precorrin 8x, an isomer of hydrogenobyrinic acid. A 1,5-methyl shift from
C-11 to C-12 then creates the conjugated system typical of the corrin macrocycle. Several
amidation steps followed by cobalt insertion, cobalt adenosylation, and creation of the
nucleotide loop generate the coenzyme AdoCbl. A review, vividly describing both the
step-by-step pathway and the remarkable developments that led to its elucidation, has
appeared (29).
E. Chemical Properties
The extensively studied chemical properties of cobalamin have been well reviewed
(14,33,44,45).
The absorption spectrum of cyanocobalamin shows three characteristic maxima that
are relatively independent of pH. Extinction coefficients are E278  16.3  102; E361 
28.1  103; and E550  8.7  103.
From the biological and clinical viewpoint it is significant that the cyano moiety of
cyanocobalamin is readily replaced by other groups to form hydroxocobalamin, chlorocobalamin,
nitrocobalamin, sulfitocobalamin, adenosylcobalamin, methylcobalamin, and
others. Each of these derivatives is freely reconverted to cyanocobalamin on exposure to
CN. A purple compound formed on addition of excess cyanide to alkaline solutions of
Cobalamin 469
cyanocobalamin is called dicyanocobalamin. In this labile compound, two cyano groups
are coordinated to the cobalt atom.
The two coenzymes AdoCbl and MeCbl are strikingly unstable to light, which rapidly
causes spectral changes and loss of coenzyme activity, owing to the homolytic cleavage
of the CCo bond. In contrast to the photolysis of AdoCbl, the photolysis of MeCbl
and other alkylcorrinoids is accelerated by ambient oxygen. The reaction proceeds slowly
under anaerobic conditions. CNCbl is slowly decomposed by strong visible light. The
cyano group is split off, yielding OHCbl. Prolonged exposure to light causes irreversible
decomposition and inactivation.
Mild acid hydrolysis of CNCbl induces removal of the nucleotide, whereas vigorous
acid hydrolysis liberates ammonia, 5,6-dimethylbenzimidazole, d-1-amino-2-propanol,
and cobyrinic acid. Dilute acids split amides from the side chains to yield mono- and
polycarboxylic acids.
CNCbl, a relatively durable cobalamin derivative, is stable in air and, in dry form,
is relatively stable at 100°C for a few hours. Aqueous solutions at pH 4–7 can be autoclaved
at 120°C. Crystalline CNCbl can be safely mixed with a wide variety of therapeutic
and nutritional agents. In solution, thiamine and nicotinamide, or nicotinic acid, slowly destroy
CNCbl; the addition of small amounts of iron or thiocyanate appears to protect it.
III. ASSAY METHODS
Assays of serum cobalamin came into use in the 1950s, but for a number of years they
were performed only in specialized laboratories. For a time, cobalamin levels could be
measured only by microbiological methods, using Lactobacillus leichmannii ATCC 7830
(46–49), Eugelena gracilis strain Z (50–52), or, less frequently, Ochromonas malhamensis
(53), or the cobalamin/methionine auxotrophic mutant Escherichia coli 113-3 (54–
56). Of the several microbiological procedures, the one employing O. malhamensis is
most specific, since this organism is supported almost exclusively by cobalamins in the
growth medium, whereas cobalamins and certain other corrins (or nucleosides) support the
growth of the other three organisms—L. leichmannii, E. gracilis, and the E. coli mutant.
Nonetheless, comparative studies show that the several assay organisms yield nearly identical
results in serum assays in a clinical setting (57–60). In the writer’s view, the most
reliable of the microbiological procedures employs L. leichmannii. Details of this procedure
are presented elsewhere (61).
The radioisotope dilution (RID) principle was first applied to the assay for serum
cobalamin in 1961 (62). Though performed in many variations, all of these methods rest
on the following principles: (a) extraction of serum cobalamins from binding proteins;
(b) addition to the extract of a standard amount of 57Co-CNCbl; (c) exposure of the mixture
of native and labeled cobalamin to a cobalamin-binding agent; and (d) removal by centrifugation
of the saturated binder and radioassay of the pellet. The level of serum cobalamin
is then read from an appropriate calibration curve (63).
In the early years, these assays regularly yielded higher values than microbiological
assays. It was shown (64,65) that serum contains corrinoid compounds, termed cobalamin
analogs, that are not utilized by assay organisms but are bound by R protein, then the
commonly used binder in RID assays. When intrinsic factor (a more specific cobalamin
binder) replaced R proteins in assay protocols, the two procedures—microbiological and
RID methods—gave comparable results.
Interpretation of serum cobalamin levels is discussed below.
470 Beck
IV. NUTRITIONAL ASPECTS
A. Sources
Cobalamin is synthesized only by certain microorganisms; hence, it is a unique vitamin.
Wherever it occurs in nature, it can be traced to bacteria or other microorganisms growing
in soil, sewage, water, intestine, or rumen. Animals depend ultimately on microbial synthesis
for their cobalamin supply; foods in the human diet that contain cobalamin are essentially
those of animal origin—liver, seafood, meat, eggs, and milk. Although the nitrogen-
fixing bacteria associated with leguminous plants are cobalamin-dependent, cobalamin is
not found in plant tissues.
The most intensive natural synthesis occurs in rumen bacteria (66). Of the microorganisms
that synthesize cobalamin, many do so in quantities just sufficient for their own
needs. However, species such as the rumen organism P. shermanii and the antibioticproducing
molds Streptomyces griseus and Streptomyces aureofaciens synthesize amounts
sufficient to make them feasible commercial sources. Some microorganisms that cannot
synthesize cobalamin (e.g., L. lactis, L. leichmannii) require an exogenous supply and
hence are useful for the microbiological assay of cobalamin. Others cannot synthesize
cobalamin and appear not to require it (e.g., E. coli).
B. Daily Requirements
The daily diet in Western countries contains 5–30 µg of cobalamin—with an average
of 7–8 µg/day ingested by adult men, 4–5 µg/day by adult women, and 3–4 µg/day
by children 1–5 years of age. An additional nondietary source of small amounts of absorbable
cobalamin may be synthesis by intestinal microorganisms. Only 1–5 µg is absorbed
(67). Less than 250 ng of cobalamin and cobalamin analogs appears in the urine; the
unabsorbed remainder appears in the faces. Long-term studies, employing radioactive
cobalamin, showed a total daily loss of 0.66–2.1 µg, with a mean of 1.3 µg (68). Of
an administered oral dose of 0.5–2 µg of pure cobalamin, 60–80% is absorbed. As the
oral dose increases, the percent absorbed decreases; at a dose of 5 µg, 30% or less is
absorbed.
Total-body content is 2–5 mg in an adult man (69,70). Of this approximately 1 mg
is in the liver. Thus, the concentration in adult liver is about 0.7 µg per gram wet weight.
Kidney is also rich in cobalamin (approximately 0.4 µg per gram wet weight) (71).
It has been suggested that cobalamin has a daily rate of obligatory loss approximating
0.1% (range 0.05–0.2) of the total body pool, irrespective of its size (67). This conclusion
implies (a) that the daily dietary requirement is 2–5 µg and (b) that a deficiency
state will not develop for several years after cessation of cobalamin intake. The officially
recommended daily allowance for adults is 2 µg (72); for infants during the first year, the
recommended allowance is 0.3 µg/day. It had earlier been held that the minimal daily
requirement was 0.6–1.2 µg. However, this amount is adequate to maintain nutritional
balance only in subjects with low body stores (in whom daily obligatory losses are proportionately
low).
Growth, hypermetabolic states, and pregnancy increase daily requirements. Because
of the buffering effects of body stores, it has been difficult to obtain precise nutritional
data in these conditions. A diet containing 15 µg/day will gradually replenish depleted
body stores (72). Average U.S. diets appear to meet this requirement.
Cobalamin 471
C. Absorption
Intrinsic factor (IF) is the name given long ago by Castle (7) to a normal constituent of
gastric juice needed to facilitate the hematopoietic effects of the essential dietary ingredient
(i.e., ‘‘extrinsic factor’’) now recognized as cobalamin. Later work showed that IF facilitates
absorption in the ileum of cobalamins ingested at physiological dose levels. Knowledge
of the molecular and functional properties of IF has expanded dramatically in recent
years (73–75).
For decades after its discovery, efforts to purify IF encountered difficulty. Substantial
purifications, based on affinity chromatography, were not achieved until 1973 (76–
78). This elegant work opened a significant new era in the study of IF and other cobalaminbinding
proteins. Human IF was shown to be an alkali-stable glycoprotein [or class of
proteins (79)] that avidly binds cobalamin (cyano, hydroxo, or adenosyl derivative). Its
properties are summarized in Table 1. Bound vitamin alters the conformation of IF, producing
a more compact form that resists proteolysis.
In fact, gastric juice contains several cobalamin-binding proteins. Only one possesses
IF activity (such activity being defined as the capacity to promote intestinal absorption
of cobalamin). Others lack it. In zone electrophoresis of gastric juice at least two
immunologically nonidentical binders or classes of binders are observed, one with slow
and one with rapid mobility (74). These were designated, respectively, S-type and R-type
protein(s). IF activity resides in the S-type class. R-type proteins are a large class found
in serum, leukocytes, saliva, and virtually all body cells. The name cobalophilin has been
proposed for all R-type proteins (80). Currently under active study, they are discussed
below in the context of plasma cobalamin transport proteins.
IF is secreted by the parietal cells of the fundic mucosa in man, guinea pig, cat,rabbit,
and monkey; by the chief cells in the rat; and by glandular cells of the pylorus and duodenum
in the hog. Secretion of IF usually parallels that of HCl. It is enhanced by histamine,
methacholine, and gastrin.
Food cobalamins are liberated by peptic digestion in the stomach and bound there
to IF. Proteins or peptides bound to naturally occurring cobalamins are competitively
displaced by IF at the low pH of gastric juice. The stable IF-Cbl complex subsequently
encounters specific mucosal receptors of the microvilli of the ileum. A specific site on
Table 1 Properties of Human Intrinsic Factor
Property Value
Mr (approximate) 44,000
E1%
1 cm at 279 nm 9.5
So
20.w 5.75
Cyanocobalamin-binding capacity, µg/mg 30.1
18.6
Association constant for cyanocobalamin, M1 1.5  1010
Composition:
Carbohydrate content, % 15.0
Hexoses, including fucose, % 6.9
Hexosamine, residues/mol 4.1
Sialic acid, residues/mol 1.7
472 Beck
the IF molecule (other than the cobalamin binding site) attaches to a receptor; binding
requires neutral pH, Ca2
, or other divalent cations, but no energy (81). Mucosal receptors
accept IF-Cb1 in preference to free IF (82). The specific mucosal receptor has been isolated
in soluble form (83). It appears to be a polypeptide containing critical disulfide bonds.
Pancreatic secretions may contain a factor that promotes cobalamin absorption (84,85).
The factor appears to be a proteolytic enzyme that acts to protect IF-Cb1 (86).
Following binding of IF-Cbl to receptor, the vitamin either passes into the cell
leaving IF behind, in analogy with the passage of transferrin-bound iron into a normoblast,
or the complex enters a mucosal cell by pinocytosis and then dissociates. Unlike
the rapid attachment of IF-Cbl to surface receptors, passage of vitamin into the mucosal
cell is a slow, energy-requiring process (87). Cobalamin entering the ileal cell is bound
there to a macromolecule that is immunologically similar to IF (88). It is of interest
that in the guinea pig cobalamin in the ileal cell accumulates in the mitochondrial fraction
(89).
Cobalamin (without IF) is eventually transferred to portal blood. After a small oral
dose (e.g., 10–20 µg), cobalamin first appears in the blood in 3–4 h, reaching a peak level
at 8–12 h. Larger oral doses are absorbed by simple diffusion that is not mediated by IF.
In these instances, vitamin appears in blood within minutes.
At least two types of anti-IF antibodies are recognized: (a) blocking antibodies,
which prevent binding of cobalamin by IF, and (b) binding antibodies (AB II), which
combine with IF-Cb1 complex or with free IF without impairing its ability to bind cobalamin.
D. Transport of Cobalamin in Plasma
Normal plasma contains 150–450 pg of cobalamin per mL (the range of normal varying
with the assay method and laboratory, as noted below). All plasma cobalamin is bound
to transport proteins of unusual physiological complexity.
1. Properties of Plasma Cobalamin–Binding Proteins
Plasma and extracellular fluids contain two major cobalamin-binding proteins that have
been known by different names over the years. The original terms transcobalamin I
(TC I) and transcobalamin II (TC II) (90–93) were criticized by workers who urged that
‘‘transcobalamin’’ be reserved for the protein previously termed TC II and that TC I be
renamed ‘‘haptocorrin’’ (94). Other synonyms for this protein in the literature include
cobalophilin (80), R binder, granulocyte vitamin B12-binding protein, and salivary binder.
None of the newer terms has found general acceptance. We here use the terms haptocorrin
and transcobalamin (or TC).
Plasma also contains a quantitatively minor protein (or class of proteins) that has
also been variously named. A component of what was once termed TC III (95–97) was
given the noncommittal name ‘‘void volume binder’’ (91). Properties of these proteins,
summarized in Table 2, have been extensively reviewed (73,74,94,98–102).
Cobalamin-binding proteins in plasma are either complexed with cobalamin (holo
binder) or free of cobalamin (apo binder). Holo binder is assayed by the mass of endogenous
cobalamin bound to it (101). Apo binder is assayed by the amount of additional
cobalamin which it binds in vitro.
The R-type binders compose a class of immunologically similar glycoproteins that
are found in various tissues and body fluids (including plasma, tears, gastric juice, saliva,
Cobalamin 473
Table 2 Properties of the Cobalamin-Binding Proteins of Plasma
Property Haptocorrin Transcobalamin
Electrophoretic mobility (pH 8.6) ?1 ?2?
Mr (approximate) 120,000a 38,000a
Cyanocobalamin binding capacity, µg/mg 12.2 28.6
Protein type (R or S) R S
Composition:
Carbohydrate content, % 33–40 0
Sialic acid, residues/mol 18 0
Fucose, residues/mol 9 0
Portion of plasma vitamin B12 bound, % 75 25
(approximate)
Portion of binder unsaturated, % (approxi- 50 98
mate)
T1/2 of TC-B12 complex 9–12 days 60–90 min
Reacts with:
Anti-TC II No Yes
Anti-TC I Yes No
Anti-saliva R protein Yes No
a Mr is 150,000 dalton by gel filtration and 95,000–100,000 by sodium dodecyl sulfate electrophoresis.
and milk). All have the same amino acid sequence in their polypeptide portions, with
differences within the class being attributable to qualitative and quantitative differences
in their carbohydrate content (103). Notably, IF and haptocorrin contain the same carbohydrates
(94).
Haptocorrin was long thought to derive from granulocytes, investigators having observed
increased levels of plasma R-type binders in association with myeloproliferative
and other granulocytic diseases (95,99,103–107). Some held that R-binder accumulating
in these diseases is different from haptocorrin and transcobalamin; hence, the names
‘‘granulocyte binder’’ and TC III (109). So-called ‘‘granulocytic binder’’ is now considered
indistinguishable from haptocorrin (97,102). Some plasma R binder appears to arise
in vitro from granulocytes after blood has been collected, i.e., during clotting (96,104).
Thus the pattern of R binders may differ in plasma and in serum. It now seems likely that
haptocorrin has many sources, including erythrocyte precursors, hepatoma cells (105,106),
salivary gland cells, and granulocytes (107). Unsaturated haptocorrin is found both intracellularly
and in all extracellular fluids.
The source of human TC is not certain. Some data suggest hepatic synthesis
(110,111); others suggest that synthesis occurs in small intestinal mucosa cells (112,113).
There is also evidence of secretion by endothelial cells (114).
Although the nature of the cobalamin binding sites in cobalamin-binding proteins
has attracted interest (115,116), most past studies on the types of corrinoids bound were
performed with inadequately purified binding proteins. A recent study of the binding of
corrinoids to human intrinsic factor, transcobalamin, and haptocorrin escapes this criticism
(117). p-Cresolyl cobamide and 2-aminovitamin B12 (complete corrinoids, whose nucleotide
at the lower face of the corrin ring is not coordinated to Co) are 103 times less
efficiently recognized by intrinsic factor or transcobalamin than vitamin B12 itself, which
contains a Co-coordinated nucleotide.
474 Beck
In summary, evidence suggests that cobamide binding to IF and TC is affected by
the CoN coordination bonds of their lower cobalt nucleotide ligands, probably because
this bond positions the nucleotide at a critical distance to the corrin ring, which is recognized
by the binding proteins. However, haptocorrin reveals greater selectivity among
differing corrinoid structures. This protein binds all corrinoids with comparable avidity,
independent of the strength of their CoN coordinations or the structures of their lower
Co? ligands. Hence, the corrin ring, rather than a structural feature induced by CoN
coordination, appears responsible for corrinoid binding to haptocorrin.
Workers have recently cloned and characterized the human gene encoding transcobalamin
(118). The gene spans a minimum of 18 kbp and contains nine exons and eight
introns, with a polyadenylation signal sequence located 509 bp downstream from the termination
codon and a transcription initiation site beginning 158 bp upstream from the
ATG translation start site. The 5? flanking DNA does not have a TATA or CCAAT regulatory
element, but a 34-nucleotide stretch beginning just upstream of the CAP site contains
four tandemly organized 5?-CCCC-3? tetramers. This sequence is a motif for a trans-active
transcription factor that regulates expression of the epidermal growth factor receptor gene,
which also lacks TATA and CCAAT regulatory elements. A number of the exon/intron
splice junctions of human transcobalamin, haptocorrin, and IF genes are located in homologous
regions of these proteins, suggesting that these genes have evolved by duplication
of an ancestral gene. Characterization of the TC gene should facilitate identification of
the mutation(s) responsible for the genetic abnormalities of TC expression (see below).
2. Functions of Plasma Cobalamin-Binding Proteins
Haptocorrin and TC are present in plasma in trace quantities. The concentration of haptocorrin
is approximately 60 µg/L (400  1012 mol/L). TC concentration is about 20 µg/
L (600  1012 mol/L). In fasting plasma, three-quarters of the circulating cobalamin
(mainly MeCb1) is bound to haptocorrin. Nonetheless, haptocorrin has substantial unsaturated
binding capacity, ranging range from 79 to 939 pg/mL of plasma (mean  330)
(119). Thus, in normal plasma more than half of the binding capacity of haptocorrin is
saturated with cobalamin; the remaining 8.5–47.1% is free. Apo-R binder in plasma has
a more basic isoelectric point than holo-binder owing to differing sialic acid levels.
TC, the only true transport protein of the several plasma cobalamin-binding proteins,
binds only 10–25% of the total plasma cobalamin (97,119,120). The unsaturated binding
capacity of normal plasma is 611–1505 pg/mL (mean  986) (91). A small fraction of
plasma TC is saturated at a given moment; TC accounts for at least two-thirds of the
unsaturated cobalamin-binding power of plasma. Hence, it is the major apo-binder of
plasma.
Known functions of the cobalamin binders include (a) transport of cobalamins
through cell membranes; (b) protection of bound MeCbl from photolysis (121); and (c)
prevention of loss of cobalamin in urine, sweat, and other body secretions. When a small
dose of cobalamin (injected or orally administered) enters the blood, it is initially bound
by transcobalamin. Indeed, more than 90% of recently absorbed cobalamin is carried by
TC (120). TC-Cbl complex is then cleared from plasma in minutes (98,122), with ligand
and protein moiety disappearing at comparable rates (123). In contrast, haptocorrin, which
carries most of the cobalamin in plasma, clears slowly from plasma (98,124), with a halflife
of 9–10 days (125). Despite the rapid disappearance of most TC-bound freshly absorbed
cobalamin, a small portion of the circulating cobalamin continues to be carried by
Cobalamin 475
TC long after its intestinal absorption (119). Cobalamin evidently recirculates to an extent,
and recycling vitamin is bound to TC.
Interest in the mechanism of membrane transport facilitation has stimulated studies
of the TC receptor (126). In hog kidney membranes binding of TC-Cbl complex to solubilized
TC receptor requires bivalent cations and is influenced by salt concentration. The
highly specific binding of TC-Cbl to TC receptor has an association constant of 4.6  109
L/mol. Studies on subcellular fractions supported the view that the receptor was located on
the brush-border membrane. Similar cation requirements and kinetic constants were found
in earlier studies of the binding of transcobalamins to subcellular fractions of rat liver
(127).
A persisting conundrum is the failure of haptocorrin to behave as an effective transport
protein. Indeed, congenital absence of haptocorrin is seemingly innocuous (128); in
contrast, a severe, even lethal, megaloblastic anemia occurs in infants lacking TC
(129,130). Only TC promotes cellular uptake of cobalamin (131,132). Cobalamin is assimilated
in many cells; liver cells have a notably high affinity for TC-bound cobalamin.
In summary, current evidence suggests that (a) TC turns over rapidly and is the
major transport protein of recently absorbed cobalamin; (b) haptocorrin turns over slowly
and transports cobalamin only after it has been circulating for some time; (c) there are
no other functional transport proteins; (d) TC is a membrane transferase that binds to a
specific membrane receptor and functions uniquely in promoting entry of cobalamin into
cells; and (e) R-type proteins include many immunologically indistinguishable proteins
that arise in many tissues and differ widely in their carbohydrate content and hence in
their molecular weight and isoelectric focusing properties.
The following abnormalities of plasma cobalamin-binding proteins have been described
in various disease states: (a) plasma TC and haptocorrin levels rise in myeloproliferative
disorders, e.g., chronic granulocytic leukemia (73,95,119,133,134) and polycythemia
vera (108,135), and occasionally in hepatocellular carcinoma (105,136) and other
solid tumors (137); (b) haptocorrin is undersaturated in pernicious anemia and other cobalamin
deficiency states, occurring early in patients with impaired cobalamin absorption
(138); (c) the sum of unsaturated haptocorrin and transcobalamin—sometimes termed
serum unsaturated B12 binding capacity, or UBBC—may be decreased in cirrhosis and
infectious hepatitis (139); (d) TC is an acute-phase reactant that rises nonspecifically in
infectious or inflammatory states (140,141); and (e) UBBC tends to rise in transient neutropenia
(142); however, the situation is unclear in neutropenia. No strict correlation has been
found between serum cobalamin level, unsaturated or total cobalamin binding capacity, or
any serum binder, on the one hand, and neutrophil count, bone marrow findings, total
blood granulocyte pool, or granulocyte turnover rate, on the other (142).
The fact that TC contains less sialic acid than haptocorrin (Table 2) may explain
its low level in plasma. Asialoglycoproteins have been shown to be cleared more rapidly
from plasma by the liver than sialoglycoproteins (143), which may explain why it is
cleared more rapidly than haptocorrin.
Cobalamin undergoes enterohepatic circulation. In humans, 0.5–9 µg of cobalamin
daily enters the gut via the bile stream (144,145). Of this, 65–75% is reabsorbed. Reabsorption
is IF-dependent (146).
In rabbits, cobalamin bound to R-type proteins is processed by hepatocytes before
reentering the plasma or being excreted in the bile (143). If human hepatocytes also preferentially
process cobalamin bound to R proteins, such as haptocorrin (147,148), the liver
476 Beck
may play a major role in clearing the circulation of inactive cobalamin analogs produced
by bacteria in infectious foci and then transported on R proteins derived from granulocytes
in those foci. Conceivably, the liver disposes of such analogs by secreting them preferentially
into the bile. Since IF binds a narrow range of cobalamin analogs (115,116), most
are probably not reabsorbed from the intestine.
V. METABOLIC FUNCTIONS
The two coenzymatic cobalamin derivatives described above—adenosylcobalamin
(AdoCbl) and methylcobalamin (MeCbl)—participate in the functioning of a dozen or
more separate enzyme systems. It is a conspicuous fact that only two—AdoCbl-dependent
methylmalonyl CoA mutase and the MeCbl-dependent methyltetrahydrofolate-homocysteine
methyltransferase (now termed methionine synthase)—occur in animal cells. All
other known systems are found in monerans and protista. Advances in understanding of
the metabolic functions of cobalamin coenzymes have been surveyed extensively (149–
158).
A. Adenosylcobalamin-Dependent Reactions
As noted above, the first enzyme shown unequivocally to require cobalamin was the
glutamate mutase of the unusual bacterium Clostridium tetanomorphum (Fig. 5)—a
notable achievement in 1958 of H. A. Barker and co-workers at Berkeley (17). At that
time, parallel studies of propionic acid metabolism in animal tissues (159–162) revealed
the existence of methylmalonyl CoA isomerase (later termed methylmalonyl CoA mutase).
Earlier work had demonstrated an ATP-dependent conversion of propionate to succinate
by mitochondrial enzymes of rat liver (163). The observation that extracts of
pig heart converted propionate to methylmalonate (a compound then virtually unknown
Fig. 5 Glutamate mutase, the first enzyme shown to require AdoCbl as coenzyme. It is here
compared with methylmalonyl CoA mutase.
Cobalamin 477
to biochemists), whereas rat liver or sheep kidney converted it to methylmalonate and
succinate, led to the resolution of a remarkable two-step pathway: a carboxylation of
propionate to methylmalonate followed by a novel isomerization of methylmalonate to
succinate. Then Barker and co-workers showed not only that glutamate isomerase is cobalamin-
dependent; they discovered three forms of the first known cobalamin coenzyme—
5?-deoxyadenosyl cobalamin (or adenosylcobalamin)—then abbreviated DBCC and later
AdoCbl (164).
The resemblance of the reactions catalyzed by that enzyme and by methylmalonyl
CoA mutase was striking and obvious (Fig. 5). Workers soon demonstrated the AdoCbl
dependence of methylmalonyl CoA mutases from vitamin B12-deficient rats (165,166) and
propionibacteria (166,168). It was now possible to write a pathway of propionic acid
catabolism (Fig. 6) that in a few steps embodies important novelties. Among them is
the fact revealed by later work (169,170) that carboxylation of propionyl CoA (a biotindependent
reaction) yields an epimer of methylmalonyl CoA that must then be enzymatically
racemized to another epimer, which is the true substrate of the mutase. In propionibacteria,
traffic along this pathway is heavily in the opposite direction. Indeed, these
organisms excrete propionic acid—hence, their genus name. Cox and White soon observed
striking methylmalonic aciduria in cobalamin-deficient patients (171). Were it not
for difficulties noted below in assaying methylmalonate, this would have been a more
widely used clinical test for cobalamin deficiency.
Fig. 6 Pathway of propionate metabolism.
478 Beck
Table
3
Adenosylcobalamin-Dependent
Enzymes
R1E
C
| R
2
| H
E
'
:
C
| H
| H
ER
3
Mode
of
hydrogen
Enzyme
transfer
R1
R2
R3
Biological
occurrence
Glutamate
mutase
Intramolecular
H
CH(NH2)COOH
COOH
Clostridium
Methylmalonyl
CoA
mutase
Intramolecular
H
COCoA
COOH
Animal
cells,
propionibacteria
Leucine
2,3-amino
mutase
Intramolecular
(CH3)3CH
NH2
COOH
Animal
cells,
bacteria
Dioldehydrase
Intramolecular
CH3
OH
OH
Aerobacter
HNH2
OH
Glycerol
dehydrase
Intramolecular
CH2OH
OH
OH
Aerobacter
Ethanolamine
deaminase
Intramolecular
NH2
OH
Clostridium
?-Lysine
mutase
Intramolecular
—
NH2
CH2CH(NH2)CH2COOH
Clostridium
Ribonucleoside
triphosphate
Intermolecular
Ribonucleotide


R(SH)2
>
deoxyribonucleotide


R(S)2
reductase
Lactobacilli,
protists
Cobalamin 479
The discovery of adenosylcobalamin, the first known cobalamin coenzyme, inspired
important observations, summarized above, on the mechanism of cobalamin coenzyme
synthesis by adenosylating enzyme (Fig. 4). In lactobacilli, this enzyme is on the ribosomal
surface (43), a deft method of gathering the small number of cobalamin molecules present
in these cells (172,173) to a restricted activation site. In liver cells, cobalamin is more
plentiful, but the adenosylating enzyme is still localized. Here it is mainly in mitochondria
(174).
A dozen or so AdoCbl-dependent enzymes have been identified (Table 3). In most,
hydrogen transfer occurs intramolecularly; in one, hydrogen transfer occurs intermolecularly
(Fig. 7). Reactions in which there is a cobalamin-mediated intramolecular transfer
of hydrogen forms a new carbon–hydrogen bond. The apparent diversity of these reactions
was rationalized by recognition that in all the coenzyme is an acceptor–donor of hydrogen,
with the locus of the transfer being the 5?-deoxyadenosyl carbon atom next to the cobalt
atom (175,176).
The one known AdoCbl-dependent reaction in which hydrogen is transferred intermolecularly—
hence it is a reduction—is ribonucleoside triphosphate reductase, which
converts ribonucleotides to deoxyribonucleotides at the triphosphate level (177–180). In
this reaction, so far demonstrated only in lactobacilli, Euglena gracilis, and other cobalamin-
requiring protists, hydrogen for the reduction of the ribonucleotide substrate comes
from a dithiol (181,182). Later work showed that this reductase catalyzes a reversible
cleavage of the unique C-Co bond (183,184).
The strategic significance of the reductase reaction for the synthesis of DNA precursors
in a cobalamin-requiring lactobacillus is evident in the scheme in Fig. 8. In lactobacilli,
the cobalamin-dependent reaction is the link between ribosyl and deoxyribosyl nucleotides.
This explains the occurrence of unbalanced growth, i.e., impaired DNA synthesis
Fig. 7 Parallel mechanisms in reactions catalyzed by dioldehydrase and ribonucleotide reductase.
In both, an H displaces an OH. The H derives from an outside reductant only in the reductase
reaction. Note: DBCC is AdoCbl.
480 Beck
Fig. 8 Pathways of nucleotide and nucleic acid in Lactobacillus leichmannii.
and inability to divide, and unimpaired RNA and protein synthesis (185,186). The resulting
forms are elongated filaments with elevated RNA/DNA ratios that are analogous to those
of the megaloblasts of human cobalamin delivery.
Great biological interest attaches to the fact that evolution has produced at least
three major classes of ribonucleotide reductases, of which only one is cobalamin-dependent
(187). Animal cells and aerobically grown E. coli have a cobalamin-independent
ribonucleoside diphosphate reductase. This enzyme, unlike the triphosphate reductase of
L. leichmannii (a single polypeptide chain), consists of two separate homodimers named
R1 and R2 (188). R1 contains the catalytic sites and two types of allosteric sites that bind
nucleoside triphosphate effectors. R2 contains two dinuclear iron centers with associated
stable tyrosyl free radicals, generation of which is oxygen-dependent. Surprisingly, anaerobically
grown E. coli was recently shown to contain a ribonucleotide triphosphate reductase
requiring strict anaerobiosis. A homodimer, the active form of this enzyme contains
an oxygen-sensitive glycyl free radical within its protein structure (Gly-681) and an iron–
sulfur center (189–191). Radical generation requires the presence of S-adenosylmethionine,
NADPH, and a reducing enzyme system (flavodoxin and its reductase) (192).
Aside from their important evolutionary implications, these enzymes have in common
their dependence on free-radical generators. It is now clear that the cobalamin coenzymes
are effective and facile generators of free radicals. The reason, it appears, is the
unique and notably fragile CCo bond. Its bond energy is only 26  2 kcal/mol (193),
and many agents, including light, can rupture it. In an enzyme-catalyzed homolytic cleavage
reaction, one electron of the CCo bond remains with C and the other with Co. As
a result, an unusual free radical (CH2 ?) forms around a temporarily free carbon atom
and efficiently abstracts H from a substrate molecule, thus triggering a chain of events
that ends in the homolytic cleavage of the 3? CH bond on ribonucleotides followed by
cleavage of the 2? COH bond, i.e., a stereospecific exchange of H for OH. Similar
mechanisms explain the two other types of cobalamin-dependent reactions: intramolecular
Cobalamin 481
rearrangements and methylations. Significantly, a similar mechanism appears to underlie
to role of S-adenosylmethionine in the anaerobic reductase reaction (192). The structural
homology of this compound and adenosylcobalamin has not escaped notice. For that reason,
one reviewer termed S-adenosylmethionine ‘‘a poor man’s adenosylcobalamin’’
(194).
B. Methylcobalamin-Dependent Reactions
Participation of methylcobalamin in the reactions listed in Table 4 have been well reviewed
(28,155,195). Unlike the carbon skeleton rearrangements catalyzed by AdoCbl-dependent
enzymes, the salient feature of the MeCbl-dependent enzymes is removal of a methyl
group from a tertiary amine. Nonetheless, the mechanism rests on the ability of a cobalamin
prosthetic group to form a C-Co bond. In cases at hand, this bond links cobalt to a
methyl group in MeCbl; in those discussed above, the bond links to a cyano group in
CNCbl or to a 5?-deoxyadenosyl group in AdoCbl. AdoCbl-linked enzymes catalyze group
migrations that are ordinarily initiated by homolytic cleavage of the C-Co bond to form
an adenosyl radical and cob(II)alamin. In contrast, MeCbl-linked enzymes catalyze methyl
group transfers involving heterolytic cleavage of the C-Co bond to form cob(I)alamin,
with transfer of the methyl to an acceptor substrate.
Methionine synthase, the prototypic and best studied MeCbl-dependent enzyme
(Fig. 9), in essence catalyzes two methyl transfers in which enzyme-bound MeCbl is successively
demethylated and remethylated:
CH3-cob(III)alamin 
 homocysteine > cob(I)alamin 
 methionine (1)
Cob(I)alamin 
 N5-methyltetrahydrofolate > CH3-cob(III)alamin (2)

 tetrahydrofolate
A remaining issue concerns the means by which the reactivity of cobalamin is made
to favor heterolytic cleavage of the C-Co in one class of enzymes and homolytic cleavage
in the other. Some light may have been shed on this question by a remarkable recent
achievement (196)—the first resolution by x-ray diffraction of how MeCbl binds to one
domain (27 kDa) of this 236-kDa protein, earlier crystallized from E. coli (197). As noted
by Stubbe (198), the ability of methionine synthase to use cob(II)alamin might provide
a link with the class of AdoCbl enzymes, which use cob(II)alamin as the catalytically
active form of the cofactor. Studies of methionine synthase and two AdoCbl-dependent
mutases suggest a common binding motif for the cobalamin cofactor.
Having shown that cobalamin-dependent methionine synthase is a large enzyme
composed of structurally and functionally distinct regions, workers have recently begun
to define the roles of several regions (199). X-ray crystallographic determination of the
Table 4 Reactions Requiring Methylcobalamin
Enzyme Reaction catalyzed
1. N5-Methyltetrahydrofolate homocysteine CH3-THFA 
 HSCH2CH2CH(NH2)
methyltransferase (methionine synthetase) COOH > CH3SCH2CH2CH(NH2)COOH 
 THFA
2. Methane synthetase 2CH3OH 
 H2 > CH4 
 2H2O
3. Acetate synthetase 2CO2 
 4H2 > CH3COOH 
 2H2O
THFA, tetrahydrofolic acid.
482 Beck
Fig. 9 Methylcobalamin-dependent pathway of methionine synthesis, showing essential role of
cobalamin-dependent methyltransferase in conversion of N5-methyl FH4 to FH4. If folate is
‘‘trapped’’ as N5-methyl FH4, it cannot be converted to N5,10-methylene FH4, the cofactor of thymidylate
synthetase.
structure of a 27-kDa cobalamin-binding fragment of the enzyme from E. coli has revealed
the motifs and interactions responsible for recognition of the cofactor. The amino acid
sequences of several AdoCbl-dependent enzymes, the methylmalonyl coenzyme A mutases
and glutamate mutases, show homology with the cobalamin binding region of methionine
synthase and retain conserved residues that are determinants for the binding of the
prosthetic group, suggesting that these mutases and methionine synthase share common
three-dimensional structures.
MeCbl-dependent methionine synthesis occurs in bacteria (38,200) and animal cells
(201,202). In humans, this pathway, one of several means by which the body acquires
methionine, serves also as a mechanism for converting N5-methyltetrahydrofolate to tetrahydrofolate.
The level of this pathway in a cell appears related to its proliferative capacity
(203,204).
Methylcobalamin functions in many exotic bacterial species in the curious reactions
on which their survival depends. For example, it is an intermediary in the biosynthesis
of methane by the several methane bacteria. In Clostridium aceticum and other anaerobic
clostridia, it participates in acetate synthesis.
C. Pending Claims
Another proposed role for cobalamin in animal cells merits brief mention. An interesting,
if fragmented, literature has suggested its possible participation in the metabolism of cyanide
(205) for at least three reasons: (a) cyanide readily converts the various cobalamins
with coenzymatic functions to metabolically inert cyanocobalamin; (b) chronic cyanide
exposure (like cobalamin deficiency) has certain neuropathological effects (to be discussed
Cobalamin 483
below); and (c) there is evidence of the participation of cobalamin in the conversion of
cyanide to thiocyanate (206). These considerations gain importance when it is realized
that the body normally must deal with a continuing burden of cyanide from sources such as
tobacco smoke and foods containing cyanogenic glycosides (e.g., fruit, beans, and nuts).
VI. ASPECTS OF COBALAMIN DEFICIENCY
Cobalamin deficiency, whatever its cause, induces two clinical complexes: the megaloblastic
state (with anemia and related phenomena) and a characteristic neuropathy
(156,207,208). Each complex evidently reflects distinctive and incompletely understood
pathogenetic mechanisms, the study of which has engendered much controversy. One
source of contention has concerned laboratory tests used in the diagnosis of cobalamin
deficiency.
A. Causes of Cobalamin Deficiency
The many disorders leading to megaloblastic anemia occur in three broad etiological categories:
(a) those due to cobalamin deficiency that respond to cobalamin therapy, (b) those
due to folate deficiency that respond to folic acid therapy, and (c) refractory marrow disorders
not due to cobalamin or folate deficiency and not reversed by their administration.
It is rarely possible to infer the underlying cause from clinical features of the anemia
alone.
Cobalamin deficiency, the subject at hand, has many underlying causes. As in all
vitamin deficiencies, it may result from (a) inadequate dietary intake; (b) increased metabolic
requirements; or (c) impaired vitamin activation or utilization in tissues. Poor diet,
a rare cause of cobalamin deficiency, occurs mainly in vegetarians who abstain from dairy
products and eggs as well as from meat.
Most cobalamin deficiencies result from diminished intestinal absorption of various
etiologies. In pernicious anemia, a gastric mucosal defect diminishes intrinsic factor synthesis.
Other causes include total (occasionally subtotal) gastrectomy; pancreatic disease;
overgrowth of intestinal bacteria in the ‘‘blind-loop’’ syndrome, anastomoses, diverticula,
and other conditions producing intestinal stasis; HIV infection; advanced age (209–211);
nitrous oxide anesthesia (212,213); infestation with the cobalamin-utilizing fish tapeworm
Diphyllobothrium latum; and organic disease of the ileum (or other digestive organs) that
interferes with cobalamin absorption despite the adequate presence of intrinsic factor.
Cobalamin deficiency resulting from increased requirements occurs mainly in pregnancy
(214), especially when fetal demands supervene in a setting of poor nutrition. Impaired
utilization of cobalamin occurs in various genetic defects, involving deletions or
defects of methylmalonyl CoA mutase, transcobalamin, and enzymes in the pathway of
cobalamin adenosylation (215–217).
All of these disorders cause tissue deficiencies of cobalamin coenzymes that are
usually correctable by cobalamin repletion. Hematopoiesis then reverts from megaloblastic
from normoblastic. Diagnostic study of megaloblastic anemia is imperative because it
guides the choice of therapy and often discloses a significant underlying disorder.
B. Diagnostic Approach
In a clinical setting, after recognition of the presence of megaloblastic changes (e.g., bone
marrow failure with characteristic morphological changes), diagnosis requires; first, serum
484 Beck
vitamin assays and other tests to be described to elucidate the broad etiological category;
second, appropriate studies aimed at elucidating the underlying cause; and third, specific
treatment and observation of response (218). Too often, patients with megaloblastic anemia
are given cobalamin and folic acid and dismissed without further investigation.
1. Is There a Deficiency of Cobalamin?
Serum Cobalamin Levels. Leaving aside the well-known difficulties of establishing
meaningful ranges of normal, which are discussed elsewhere (61), and the problem of
precision and comparability of various assay kits (219,220), it is important to recognize
that in differing settings the term ‘‘vitamin deficiency’’ may imply (a) total-body vitamin
deficiency; (b) low serum vitamin level; or (c) decreased levels of the intracellular coenzymes
derived from the vitamin in question.
There are no practical methods for measuring total-body cobalamin. Clearly, serum
cobalamin levels are not measures of total-body levels. A corollary of defining ‘‘defi-
ciency’’ by serum levels is the practical need of physicians to designate a given level as
a cutoff, below which they will initiate further diagnostic studies. A problem with this
definition is that low serum levels (assuming the validity of the ‘‘range of normal’’) may
occur without clinical morbidity (221,222)—and vice versa.
When deficiency is defined as a decrease in coenzyme levels within body cells suffi-
cient to inhibit relevant coenzyme-dependent enzymes, one is dealing with the deficiency
that leads to clinical signs and symptoms. Newer diagnostic tests are now available to
assess cobalamin and folate coenzyme levels in a clinical setting.
These considerations are relevant in considering available diagnostic tests, both old
and new. Confronted with clinical signs suggestive of cobalamin deficiency (e.g., megaloblastosis,
myelopathy, etc.), one asks first whether or not cobalamin deficiency exists. If
it does, then one seeks its cause. The first question initially requires assay of the serum
cobalamin level. This may be considered a screening test. Basic principles of the assay
procedures were summarized above. However, aspects of this familiar clinical test merit
additional comment.
Assay of serum cobalamin has long been a diagnostic mainstay (223), despite the
discovery in 1978 that normal human serum contains cobalamin analogs, which were
assayed as cobalamin by commercial radioisotope dilution assay (RIDA) kits containing
R protein as binder (64,65). These analogs do not support the growth of L. leichmannii or
other cobalamin-dependent microorganisms. Hence, laboratories, which performed routine
assays by microbiological methods, were obtaining lower but more accurate results. After
1978, kit makers replaced R-protein binders with IF. Thereafter, the two methods yielded
comparable results (61). The nature and possible pathophysiological significance of serum
cobalamin analogs remain unclear.
As noted above, cited ranges of normal serum cobalamin levels vary with the assay
method and the laboratory. The following points merit emphasis: (a) serum cobalamin
levels generally fall before the appearance of megaloblastosis or neuropathy; (b) these
clinical manifestations are commonly (but not invariably) present when the serum level
has fallen below 100 pg/mL*; (c) since a level of less than 150 pg/mL probably indicates
a deficiency state, many physicians regard 200 pg/mL as the level below which further
* Cobalamin levels are expressed here in picograms per milliliter, though some prefer picomoles per liter in
accordance with the SI system (224). In this system, a level of 200 pg/mL would be equivalent to 147 pmol/L.
Cobalamin 485
studies are initiated; (d) several conditions (e.g., pregnancy, transcobalamin deficiency,
etc.) can lower serum cobalamin without inducing intracellular cobalamin deficiency
(225); and (e) several conditions (e.g., coexisting myelocytic leukemia, other myeloproliferative,
disease, etc.) can elevate serum cobalamin levels enough to invalidate them as
indicators of intracellular cobalamin levels.
Studies of the relation between serum and tissue cobalamin levels in normal subjects
(226), postgastrectomy patients (227), and patients with pernicious anemia (228–230)
indicate that although the correlation is fairly good in normal subjects, serum levels are
often poor indicators of tissue levels. One reason is the poorly understood influence of
serum cobalamin–binding proteins. Also, kinetic studies confirm that as deficiency develops
serum levels are maintained at the expense of the tissue level. Because cobalamin
reserves are normally large, a low serum cobalamin level implies a long-term abnormality.
It is, therefore, a compelling reason for further investigation.
Although serum cobalamin levels may be normal in the presence of intracellular
cobalamin deficiency (and the clinical signs it produces), the opposite pattern—low serum
cobalamin levels in the absence of megaloblastic anemia—occurs. Some of these patients
display neurological disturbances (231).
To better assess the state of cobalamin nutrition, workers have studied the relation
of plasma holotranscobalamin (holoTC) (232) with levels of red blood cell holoTC and
cobalamin (RBC-Cb1), accepted measures of tissue cobalamin content, and studied the
relationship between RBC-B12 and plasma holoTC II levels. Plasma holoTC and RBCCbl
concentrations were concomitantly assayed in 20 hematologically normal controls
and cancer patients. In normal controls, the mean value of RBC-Cbl was 241  51 pg/
mL of packed erythrocytes (range 180–355). In cancer patients, values of holoTC and
RBC-Cbl were subnormal. RBC-Cbl was greater than 180 pg/mL packed erythrocytes
only when the holoTC level was above 70 pg/mL.
Other Tests for Cobalamin Deficiency. Methylmalonic aciduria indicates cobalamin de-
ficiency (171,233) except in rare cases in which it is due to an inborn metabolic error
(215–217). Urinary methylmalonate has been assayed colorimetrically (234), by paper
(235), thin-layer (236), or gas (237) chromatography, or by mass spectrometry (238,239).
Unfortunately, none of these methods is ideally suited for many diagnostic laboratories.
Normal subjects excrete only trace amounts of methylmalonate: 0–3.5 mg (0–38
µmol) per 24 h. Excretion increases in cobalamin deficiency, sometimes to 300 mg (3260
µmol) or more per 24 h. In many studies, urinary methylmalonic acid (measured with and
without an oral loading dose of dl-valine (240)) has proved to be normal in folate defi-
ciency and elevated in cobalamin deficiency, often rising before cobalamin levels has
fallen below 200 pg/mL. Hence, elevated urinary methylmalonic acid may be considered
an earlier indication of cobalamin deficiency than depressed serum cobalamin (241) and
thus a better indicator of intracellular cobalamin deficiency.
For years, the closest approach to a test for cobalamin coenzyme levels was the
urinary methylmalonic acid. Lacking that datum, which was hard to obtain, it was necessary
to guess whether intracellular cobalamin deficiency exists on the basis of serum cobalamin
levels and clinical signs. A pitfall of this reasoning is that the clinical signs of intracellular
cobalamin deficiency (megaloblastosis, neuropathy) have other possible causes.
For example, megaloblastic anemia can be caused by folate deficiency, various drugs, and
so forth, and the neuropathy is mimicked by other disorders.
Two new tests—serum methylmalonic acid and serum homocysteine—appear to
486 Beck
go far toward permitting a direct assessment of intracellular cobalamin levels, though
these tests are still not readily available.
Proceeding on the premise that intracellular cobalamin deficiency should be reflected
most sensitively by elevated concentrations of the unmetabolized substrates of the two
cobalamin-dependent enzymes, and on the basis of earlier work showing elevations of
urinary methylmalonic acid and intracellular homocysteine (242) in cobalamin deficiency,
stabler and co-workers in the laboratories of Allen and Lindenbaum demonstrated the
diagnostic value of serum methylmalonic acid and total homocysteine (free plus bound)
assays performed by innovative methods employing capillary gas chromatography and
mass spectrometry (243–247). Although these techniques are still beyond the scope of
routine laboratories, the assays are increasingly available in commercial and reference
laboratories. Significantly, their precision and careful exploration has redefined the inquiry
and in consequence opened a new chapter in the study of an old problem.
These studies revealed the following normal ranges (248):
Mean  2 SD Mean  3 SD
Serum methylmalonic acid (nmol/L) 73–271 53–376
Serum total homocysteine (µmol/L) 5.4–16.2 4.1–21.3
Data suggest that these tests are more sensitive than the serum cobalamin level in detecting
intracellular cobalamin deficiency. Early studies (246,247) of normal and deficient subjects
showed that about 95% of cobalamin-deficient patients had elevated serum methylmalonic
acid and total homocysteine levels. Of 86 consecutive cobalamin-deficient patients with
serum cobalamin levels below 200 pg/mL, 77% had marked elevations (more than 3 SD
above the normal mean) of both metabolites, whereas 9% had elevated methylmalonic
acid alone and 8% had a marked elevation of homocysteine alone. Only 6% had normal
levels of both metabolites.
In a study of 40 nonanemic cobalamin-deficient patients (231), only 22 had serum
cobalamin levels under 100 pg/mL, yet all but one had elevated serum methylmalonic
acid levels and all but two had elevated homocysteine levels. Later work (247) indicated
that 95% of patients who relapse because of suboptimal therapy display early elevations
of serum methylmalonic acid, total homocysteine, or both metabolites, compared to 69%
with low serum cobalamin levels. Of 419 consecutive patients with recognized significant
cobalamin deficiency, 12 had serum cobalamin levels greater than 200 pg/mL, mild or
absent anemia, and (in 5) prominent neurological signs that responded to cobalamin. In
all 12 cases, both serum methylmalonic acid and total homocysteine were increased. Observations
by others (248,249) confirm the value of total homocysteine assays in cobalamin
deficiency. Such data, it would appear, establish that serum cobalamin is normal in a
significant minority of cobalamin-deficient patients and that assay of these serum metabolites
is diagnostically essential.
The so-called deoxyuridine suppression test is an isotopic procedure that assays the
ability of nonradioactive deoxyuridine to suppress the incorporation of labeled thymidine
into DNA via a pathway that is impaired in folate and cobalamin deficiency. The test has
proved useful in investigative settings, particularly in detecting the effect of various added
metabolites or temporal changes. Though it is said to become abnormal prior to the emerCobalamin
487
gence of clinical signs (250), the complexity of its metabolic basis has been cause for
concern (251,252).
The diagnostic approach to suspected cobalamin deficiency has been usefully summarized
by reviewers (218,225,253).
2. Determining the Cause of Cobalamin Deficiency
Malabsorption, by far the most common cause of cobalamin deficiency, was opened to
investigation by classic studies of Schilling (254). His demonstration that a large parenteral
dose (1 mg) of nonradioactive cobalamin promotes renal excretion of an ingested tracer
dose of radioactive cobalamin, presumably by blocking cobalamin binding sites in plasma
and liver, led to a standard procedure for assessing cobalamin absorption. It is emphasized
that the Schilling test estimates cobalamin absorption, not cobalamin repletion. Thus, it
may explicate the basis of cobalamin deficiency by revealing the typical absorptive patterns
of such disorders as pernicious anemia and ileal disease. Though a bedrock of diagnosis,
the Schilling test was long more popular in the United States than in Europe, where
assays of IF in gastric juice were generally preferred. Today, it is universally used despite
several potential sources of error.
The Schilling test procedure is as follows: After voiding, a fasting patient ingests
0.5 µCi (0.5–2.0 µg) 57Co-cyanocobalamin in water at time zero when a 24-h urine collection
is begun. At 2 h, 1 mg of nonradioactive cyanocobalamin—the so-called flushing
dose—is administered intramuscularly. The subject may then take food. An adequate sample
of pooled urine is assayed for radioactivity and the percentage of administered radioactivity
excreted in the first 24 h is calculated. With a 1-µg dose of labeled cobalamin,
normal subjects excrete 7% or more of the administered radioactivity in the first 24 h. A
greater percentage is excreted when a smaller dose is given. It is important that laboratories
performing this test establish a normal range in suitable control subjects.
If excretion of radioactivity is slow, the second part of the Schilling test is performed
in no less than 5 days. The procedure is the same except that 60 mg of demonstrably
active hog IF (equivalent to 1 NF unit) is given orally with the radioactive cobalamin. If
poor excretion in the first part was due to IF deficiency, the result in the second part should
be normal. If excretion in the second part is still abnormal, other explanations must be
found for cobalamin malabsorption.
The major source of error is incomplete urine collection. Completeness of urine
collection may be assessed by a determination of total creatinine content in a 24-h urine
specimen. The lower limit of normal is 15 mg/kg of body weight per day.
The kidneys excrete cyanocobalamin and inulin in a similar manner. Indeed, radioactive
cobalamin is useful in measurements of the glomerular filtration rate (255). Renal
disease associated with impaired glomerular filtration may delay excretion of radioactivity
in the Schilling test (256). To circumvent that difficulty, various modifications have been
proposed, e.g., a 72-h urine collection with flushing doses of cobalamin every 24 h. Wholebody
counting may be the only satisfactory technique in severe renal insufficiency
(257,258). In this procedure, the flushing dose is omitted. Normal subjects retain 45–80%
of administered radioactivity following an oral dose of radioactive cobalamin. In one such
procedure (259), the plasma of normal subjects 8 h after an oral dose of 57Co-cobalamin
contained 1.4–4.1% of administered radioactivity per liter of plasma, with a mean of 2.3%.
The pernicious anemia group had a range of 0.0–0.6%, with a mean of 0.2%.
Over the years, laboratories performing both Schilling tests and serum cobalamin
assays accumulated data on two seemingly anomalous patterns unlike the majority pattern
488 Beck
that displayed a generally linear relation in the lower range of values between Schilling
test results and serum cobalamin levels (61). One anomalous pattern—a low Schilling
test with a normal serum cobalamin—is seen in subjects with early pernicious anemia.
The other—a normal Schilling test and a low serum cobalamin—proved more interesting.
Some patients in this group had parasitic competitors (fetus or tumor); others were revealed
by later work to malabsorb food cobalamin, while absorbing normally the pure cobalamin
used in the standard Schilling test (260–267).
This pattern, first described by Doscherholmen (266), is common in atrophic gastritis,
post vagotomy (268), and in subtotal gastrectomy (268). It is also seen in subjects
taking cimetidine or other inhibitor of gastric secretion (270) and in the elderly. In many,
there is no overt evidence of gastric dysfunction.
In later years, modified Schilling tests were developed in which the labeled cobalamin
is bound to egg yolk (264). Variously known as the food Schilling test or egg-yolk
cobalamin absorption test, the procedure at first involved ingestion of labeled cobalamin
derives from eggs produced by hens injected with labeled cobalamin (266). Later workers
substituted labeled cobalamin bound in vitro to dried egg proteins. Now generally available
in most major medical centers, this procedure has accounted for otherwise unexplained
low serum cobalamin levels and frank cobalamin deficiency in many cases. Indeed, this
patient group is at least as large as the group with pernicious anemia or other classic
causes of cobalamin deficiency. One study (264) reported 47 patients with low serum
cobalamin levels and normal Schilling test results. Egg test results were significantly lower
than normal, whereas routine Schilling test results were normal. Twenty subjects had egg
test excretions below 1.5%. No other clinical features distinguished them from the 27
who excreted more than 1.5% other than the presence of lower ratios of pepsinogen I:II.
Interestingly, 60% of tested patients had neurologic, cerebral, or psychiatric abnormalities.
If food cobalamin malabsorption is often associated with otherwise unexplained low cobalamin
levels, low cobalamin levels in the presence of normal Schilling test results should
not be dismissed without testing for food cobalamin malabsorption, whether or not there
is known gastric dysfunction.
Assays of IF in gastric juice can also be based on IF-mediated (a) enhancement of
cobalamin uptake by intestinal cell preparations in vitro (271,272), (b) inhibition of an AdoCb1-
dependent enzyme (273), and (c) various immunological attributes (274,275). The
use of such techniques led to discovery of a physiologically inert IF molecule in a child with
cobalamin malabsorption (276). Studies of the abnormal IF molecule isolated by affinity
chromatography (277) demonstrated nonidentity of the cobalamin- and ileum receptor–binding
sites on the IF molecule. The genetic abnormality was confined to the latter site.
C. Clinical and Metabolic Features
1. Megaloblastic Anemia and Related Phenomena
The metabolic derangement that leads to megaloblastic anemia is a defect of DNA synthesis—
without impairment of RNA synthesis (150). The ribonucleotide reductase of bone
marrow and other animal cells was shown to be cobalamin-independent (278–280); hence,
it became necessary to seek other explanations for the observable fact that cobalamin
deficiency leads to impaired DNA synthesis.
‘‘Methylfolate Trap’’ Hypothesis. Of the hypotheses offered to account for the role of
cobalamin in DNA synthesis, one (with the vernacular name ‘‘methylfolate trap hypotheCobalamin
489
sis’’) was proposed in 1961 to account for abnormalities of folate metabolism in cobalamin-
deficient rats (281). Because methionine supplementation decreases the proportion
of N5-methyltetrahydrofolate in rat liver from 80–90% of total folate to about 50%, it was
suggested that a similar shift might follow suppression of MeCb1-dependent methionine
synthesis in cobalamin deficiency (Fig. 9). It was then suggested that in human cobalamin
deficiency (282) accumulation (‘‘trapping’’) of folate as N5-methyltetrahydrofolate sequesters
folate in this form, thus blocking its conversion to tetrahydrofolate, a precursor
of N5,N10-methyltetrahydrofolate, the cofactor of thymidylate synthetase. Hence, conversion
of dUMP to dTMP—and thus DNA synthesis—is impaired.
The following evidence seemed to favor this view: (a) changes in folate metabolism
often, but not always, occurring in cobalamin deficiency, e.g., the altered partitioning of
tissue folate compounds referred to above (281); elevation of the N5-methyltetrahydrofolate
in serum (282); increased urinary excretion of formininoglutamic acid after an oral
histidine loading dose (283); and elevated urinary excretion of aminoimidazole carboxamide
(284); (b) megaloblastosis in a patient with congenital methyltransferase deficiency
(285); (c) diminished methyltransferase activity in cobalamin-deficient rats (286); (d) correction
of defective DNA synthesis in cobalamin deficiency with tetrahydrofolate (287);
and (e) apparent impairment of the conversion of dUMP to dTMP in bone marrow cells
in cobalamin-deficient subjects, the so-called dU suppression test (250–252).
Despite this evidence, opposition to this hypothesis stemmed from certain contrary
findings, the following among them: (a) lack of urinary excretion of formiminoglutamic
acid and aminoimidazole carboxamide in many cobalamin-deficient subjects (288); (b)
clearance studies by some investigators (289,290)—but not others (291)—showing normal
rates of N5-methyltetrahydrofolate utilization in cobalamin deficiency, suggesting that
elevation of serum N5-methyltetrahydrofolate is due to translocation rather than ‘‘trapping’’;
(c) normal 14CO2 production from (14C-methyl)-N5-methyltetrahydrofolate in cobalamin-
deficient rats (292); (d) evidence that a cobalamin-independent pathway exist for
the release of methyl groups from N5-methyltetrahydrofolate in neural tissue (293,294)
and blood cells (295); (e) depression in cobalamin deficiency of total folate in red cells
(296) and liver cells (294), contrary to predictions of the ‘‘methylfolate trap hypothesis’’;
(f) questions about the validity of the so-called dU suppression test (250–253), which has
been widely employed (297–302) without critical scrutiny; and (g) evidence in children
with severe homocystinuria, cystathioninuria, hypomethionimemia, and methylmalonic
aciduria (303–305) of defective formation of both AdoCbl and MeCbl, possibly resulting
from a defect in membrane transport of cobalamin-binding protein. Significantly, these
children did not have megaloblastic anemia. Cultured skin fibroblasts showed depressed
methyltransferase activity and correspondingly impaired conversion of homocysteine to
methionine—precisely the defect envisioned in the ‘‘methylfolate trap hypothesis.’’
Evidence that intracellular folates are in the form of polyglutamates (a device preventing
leakage from cells) and that N5-methyltetratetrahydrofolate is, among folylmonoglutamates,
a uniquely poor substrate for folylpolyglutamate synthetase added another facet
to this story. If cobalamin deficiency traps folate as N5-methyltetrahydrofolate, this may
account for unpredicted decreases of tissue folates in this setting. In sum, the trap hypothesis
is now widely accepted, though reservations persist.
Clinical Features. Although anemia is a prominent feature of the megaloblastic state,
there is often pancytopenia associated with a familiar morphological pattern in blood and
bone marrow cells—and indeed in all proliferating cells—that includes gigantism of these
490 Beck
cells and various signs of impaired cell division (207,208). The anemia is typically macrocytic,
though not all macrocytic anemias are megaloblastic and not all megaloblastic anemias
are macrocytic. Megaloblastic blood cell precursors (megaloblasts) contain a normal
or increased amount of DNA and an increased amount of RNA per cell, which accounts
for cytoplasmic basophilia in Wright’s-stained smears.
Misincorporation of Uracil into DNA. Defective DNA replication generally reflects impaired
conversion of dUMP to dTMP, as a result of which there is decreased intracellular
dTMP and dTTP and increased dUMP and dUTP. Thus, the dUTP/dTTP ratio rises (306–
308) and dUTP is misincorporated into DNA (309,310). DNA uracil is removed by uracil-
DNA-glycosylase (311), but dTTP is unavailable for repair and DNA becomes increasingly
fragmented. This irreversibly impairs cell division and causes eventual cell death.
2. Neurological Abnormalities
Aspects of the neuropathy of cobalamin deficiency have been reviewed (312–317).
Classic Syndrome. In its classic form, the neurologic syndrome of cobalamin deficiency
(which does not occur in folate deficiency) consists of symmetrical paresthesias in feet and
fingers, with associated disturbances of vibratory sense and proprioception, progressing to
spastic ataxia with ‘‘subacute combined system’’ disease of the spinal cord, i.e., degenerative
changes of the dorsal and lateral columns (318). In fact, the picture is more often
chronic than subacute—and, as noted below, the clinical perimeter of this syndrome may
be wider than previously thought.
The earliest recognizable pathological changes in the cord consist of small vacuolated
areas in the myelin with focal swellings of individual myelinated fibers. Later, the
lesions coalesce into larger foci involving many fiber systems, but not in a systematic
manner (319).
Usually, so we once believed, the spinal cord is initially affected and predominantly
involved throughout the clinical course. Typically, the process begins at the cervicothoracic
junction of the spinal cord, attacking the posterior columns first. From there, it spreads
up and down the cord as it takes in the more anterior portions.
In a common onset, patients experience distressing and persistent paresthesias of the
hands and feet. Often, when something is touched with hands or feet, a burst of paresthetic
sensations make their way up the limb. Characteristically, paresthesias begin first in the
hands and then the feet. Without treatment, all limbs and body parts are eventually affected
until the full-blown classic picture emerges: posterior column, corticospinal tract, spinothalamic
tract, and ultimately peripheral nerve disease, which occurs late and in a relatively
few cobalamin-deficient patients.
A patient is often brought to a physician by persistence of the paresthesias throughout
the day and night. This is a manifestation of posterior column disease. The patient
soon notices ataxia, as he brusquely places widely spread feet upon the floor and totters
from side to side, sometimes falling. This sensory ataxia, resulting from lost proprioception,
is to be distinguished from cerebellar ataxia, which is vertiginous. By the time the
gait is affected, impairment of vibratory disturbance is advanced.
Corticospinal tract disease is characterized in advanced cases by weakness or paralysis
of voluntary movement, with impaired motor function and spasticity, increased tendon
reflexes, even clonus and a positive Babinski’s sign.
Occasionally, the disease begins by affecting the optic nerve with impaired vision,
scotomata, and sometimes blindness. That is a rare but well-established syndrome, typiCobalamin
491
cally occurring in heavy smokers; hence, the name tobacco amblyopia. It was traditionally
taught that in advanced disease there can be deranged mental function and in some cases
structural changes of the white matter of the brain resembling those in the white matter
of the spinal cord. More will be said of this syndrome later.
Differential diagnosis of the clinical neurological syndrome includes cerebellar disease,
which typically displays dysmetria on heel-to-shin testing, nystagmus, and impaired
tandem walking; multiple sclerosis and other demyelinating diseases; subacute myelooptic
neuropathy; tropical ataxic neuropathy; Leber’s hereditary optic atrophy; and a variety
of other disorders. An essential diagnostic datum, obviously, is proof of cobalamin
deficiency.
Typically, patients with untreated pernicious anemia display megaloblastic anemia
first and then a complex of neurological signs that, if untreated, progress to spastic ataxia
and beyond. If treated early, these changes are generally reversible, but every experienced
hematologist will recall cases in which therapy came late and neuropathies became tragically
irreversible. Significant neuropathy was once observed in virtually every new patient
with pernicious anemia. Today, perhaps because of earlier diagnosis and treatment, the
classic neurological syndrome is seen less frequently.
The appearance of neurological signs before the appearance of anemia (318) has
been attributed to administration of folate (without cobalamin). This stimulates marrow
function enough to maintain adequate erythropoiesis while inciting neuropathy or failing
to prevent its progression. Those who develop neurological signs as a first manifestation
will invariably show hematologic signs within weeks or months of the onset if not treated
with cobalamin.
Newer Perspectives. Recently, workers have claimed that newer, more sensitive diagnostic
tests justify the attribution of such common complaints as memory loss, fatigue and
weakness, and personality and mood changes to cobalamin deficiency, even in the absence
of depressed serum cobalamin levels and megaloblastic anemia.
It is an old idea that cobalamin deficiency can cause a variety of neuropsychiatric
symptoms beyond the classic ones described above. A huge literature of several decades,
consisting mainly of clinical reports (320–328), described cerebral dysfunction; EEG
changes; irritability; somnolence; perversion of taste and smell; and all manner of psychological
derangements, even ‘‘megaloblastic madness’’ (329), in cobalamin-deficient patients.
However, much of this work lacked adequate controls, i.e., double-blinded therapeutic
studies on groups of deficient and nondeficient subjects with similar symptoms,
and adequate diagnostic testing.
In 1988, Lindenbaum, Allen, and co-workers published an interesting study (231)
of 141 consecutive patients in whom various neuropsychiatric abnormalities had been
attributed to cobalamin deficiency. Of these, 40 patients (28%) had no anemia or macrocytosis.
Although these patients had serum cobalamin levels that were often equivocal,
serum methylmalonic acid and homocysteine levels were markedly elevated in many—
and patients benefited from cobalamin therapy, displaying improvement of neuropsychiatric
abnormalities, of abnormal blood counts, and of elevated levels of serum methylmalonic
acid and/or total homocysteine.
The new element in this work was the sensitivity of diagnostic testing and the evidence
of therapeutic response. However, double-blinded controls were lacking, a possible
significant omission because such symptoms wax and wane, and are thus notoriously dif-
ficult to deal with quantitatively. Despite this reservation, it seems clear that determination
492 Beck
of serum methylmalonic acid and total homocysteine provides a new diagnostic standard
against which other procedures are henceforth to be compared and evaluated. The use of
these tests has provided compelling evidence that intracellular cobalamin deficiency can and
often does exist in the absence of megaloblastic anemia or low serum cobalamin levels.
Pathogenesis. The neuropathy has proved a difficult target for investigators. The main
impediment to its study has been the unavailability of affected tissues or suitable animal
models, although recent efforts appear to have produced neurological lesions in cobalamindeficient
fruit bats (330) and primates (331,332). Unfortunately, such materials are as hard
to come by as incisive hypotheses.
It is of interest that cobalamin deficiency in most laboratory animals produces a
picture quite different from that in deficient humans. It does not include megaloblastic
anemia, even when body cobalamin stores have been totally consumed. Perhaps hematopoietic
precursors in these species are richer in cobalamin-binding proteins than human
marrow cells. Neurological abnormalities in deficient laboratory animals are also peculiar
in character or difficult to recognize.
Although it is generally accepted that neurologic involvement is associated with a
defect in myelin synthesis, its mechanism remains unknown. Nor is it clear whether this
is the primary neuropathic event or whether that putative event is a consequence of impaired
myelin synthesis, destruction of existing myelin, or another process. Three major
theories have been advanced to explain the neuropathy of cobalamin deficiency: (a) depressed
myelin synthesis and incorporation into myelin of abnormal fatty acids; (b) impaired
DNA synthesis; and (c) chronic cyanide intoxication.
Abnormal Fatty Acid Synthesis. In view of evidence that cobalamin deficiency impairs
one of the body’s two cobalamin-dependent enzymes—methyltransferase—in causing the
defect in DNA synthesis underlying megaloblastosis, it seemed logical that impairment
of the other cobalamin-dependent enzyme—methylmalonyl CoA mutase—might account
for abnormalities of myelin synthesis associated with neural lesions, especially since affected
neurons are not dividing cells engaged in DNA synthesis.
Some experimental work supports that view. The lipids of myelin turn over rapidly
(333,334), and myelin replacement is heavily dependent on fatty acid synthesis. Normally,
the de novo synthesis of fatty acids consists of the repetitive sequential addition of twocarbon
units (deriving from malonyl CoA, a three-carbon compound) to a growing chain
that begins with acetyl CoA, a two-carbon compound. The substantial accumulation of
methylmalonyl CoA in cobalamin-deficient tissues suggested that methylmalonyl CoA (a
branched four-carbon molecule) might inhibit fatty acid synthesis or compete with malonyl
CoA or acetyl CoA in the initiation of fatty acid synthesis and thus generate abnormal
branched fatty acids or fatty acids with an odd number of carbon atoms. This hypothesis
gained credibility from the observed toxicity of valine, a precursor of methylmalonyl CoA,
in cobalamin-deficient pigs. Doses of valine that have no effect on normal pigs can be
fatal to cobalamin-deficient pigs (335).
Further support is found in the observation that rat glial cells cultured in cobalamindeficient
medium produce increasing amounts of two unusual fatty acids (336)—unbranched
acids with 15 and 17 carbon atoms—as cells progressively lose the ability to
metabolize propionic acid. Levels of 15- and 17-carbon acids return to normal when cultures
are supplemented with cobalamin. Studies of the in vitro effect of methylmalonyl
CoA on fatty acid synthesis in rat liver fractions showed that methylmalonyl CoA inhibits
Cobalamin 493
fatty acid synthesis and can be incorporated into branched fatty acids (335). Similar results
were obtained in unpublished studies of lipid extracts of plasma and red cells from patients
with untreated pernicious anemia (337).
Further confirmation came from experiments (338,339) demonstrating synthesis of
abnormal 15- and 17-carbon fatty acids from [14C]propionate in the nerves excised from
patients with pernicious anemia. Finally, branched and odd-numbered fatty acids were
found in the neural tissues of a patient with methylmalonic aciduria and deranged cobalamin
metabolism (340). Additional evidence of a role for accumulated methylmalonyl CoA
comes from recent studies, discussed below, of methylmalonic acid levels in cerebrospinal
fluid (341).
These results, which curiously have stimulated little further work, are provocative,
yet they fail to prove that such fatty acids in myelin directly account for the neuropathy
of cobalamin deficiency. Moreover, several considerations may argue against the pathogenetic
importance if this pathway: (a) the relatively low level of propionate metabolism
in humans, especially since valine and other amino acids are not major precursors of
methylmalonyl CoA (342); (b) failure to demonstrate such changes in animal models in
which neuropathy is induced by exposure to nitrous oxide (315); and (c) the unimpressive
correlation between methylmalonic aciduria and the presence or severity of neurological
disease. In the various genetically induced methylmalonic acidurias in infants and children,
the only neurological signs are lethargy and mental retardation (216).
Impaired DNA Synthesis. It is generally believed that blocked DNA synthesis, which
causes megaloblastosis in cobalamin deficiency, does not account for damaged neurons,
which are nondividing. However, the cells that synthesize myelin divide, and recent preliminary
evidence has implicated impairment of cobalamin-dependent methyltransferase
in a neuropathy resulting from N2O exposure (213,343,344).
A suggestive case study (345) reported presumed genetic depression of methyltransferase
that was associated with homocystinuria, mild megaloblastic anemia, and neurological
deficits appearing at age 21. Methylmalonyl CoA mutase activity was normal. In this
case, decreased methyltransferase activity may have induced the neuropathy, but proof of
causality and mechanistic details remain to be demonstrated.
Impaired Methylation. According to Scott, Weir, and co-workers (346,347), the neuropathy
of cobalamin deficiency may be determined by abnormalities in the ratio of the methyl
donor, S-adenosylmethionine, to the coproduct, S-adenosylhomocysteine. This so-called
methylation ratio is thought to control the activity of tissues methyltransferases. They
found that inactivation of cobalamin-dependent methionine synthase reduces the methylation
ratio in rats and pigs in vivo. Methylation ratios found in the neural tissues of cobalamin-
inactivated pigs (i.e., after prolonged exposure to nitrous oxide) significantly inhibits
protein methyltransferases of pigs and humans, whereas the altered methylation ratio in
deficient rats only marginally inhibits the equivalent rat methyltransferases. This may be
consistent with the induction of a myelopathy by such treatment in pigs and humans, but
not in rats. Dietary supplements of methionine are given to cobalamin-inactivated pigs
to prevent the myelopathy in vivo by elevating neural S-adenosylmethionine levels and
normalizing the methylation ratio. It is proposed that reduction of the methylation ratio
in the brain of pigs as a consequence of methionine synthase inhibition leads to brain
hypomethylation, which could affect critical neural components and induce the vacuolar
myelopathic changes and demyelination seen in the spinal cord of these animals, which
494 Beck
mimic those of human subacute combined degeneration. The validity of this thesis remains
to be established (315).
Possible Role of Cyanide. The hypothesis that neuropathy can occur in cobalamin defi-
ciency as a consequence of chronic occult cyanide intoxication rests on fragmentary evidence
that includes (a) the relatively high incidence of tobacco amblyopia, retrobulbar
neuritis, and optic atrophy in cobalamin-deficient tobacco smokers (348–350) and carriers
of the fish tapeworm Diphyllobothrium latum (351); (b) the relatively elevated cyanocobalamin
fraction reported in the plasma cobalamin of smokers (352,353); and (c) the reported
association of neurological disorders with chronic cyanide exposure or abnormalities of
cyanide metabolism (350,354,355).
Tobacco smoke contains 6 ppm of cyanide. Far higher cyanide levels arise from
the heating or cooking of many plant materials, notably including cassava, a root tuber
(Manihot esculenta) widely used as food in the tropics, which is rich in the cyanogenic
glycoside linamarin (356,357). Some circumstantial evidence links these cyanogens to
various tropical neuropathies (350,358). Conceivably, an exogenous cyanide load would
convert cobalamin coenzymes to cyanocobalamin, which is metabolically inactive, less
firmly held by binding proteins, and hence more readily lost to renal excretion that other
cobalamins. Any such theory might require the assumption that neurons are more vulnerable
to such effects than bone marrow cells. It has been suggested, but not proved (359,360),
that certain neural elements, especially those of the eye, are uniquely sensitive to cyanide
exposure.
Much of the literature proposing a neuropathic role for cyanide comes from British
and African writers of the 1960s and 1970s. Though speculative, these ideas are evocative
enough to justify further investigation with current methods.
Role of Folate. A word is in order on the curious role of folic acid in the neuropathy
of cobalamin deficiency. Despite long contention (361,362), a convincing case has yet to
be made for a neuropathy due to folate deficiency alone. However, it is well established
that administration of folate to a cobalamin-deficient patient can induce or worsen neurological
abnormalities, even as a partial (and temporary) hematological remission is taking
place. Folate has no neuropathic effect in cobalamin-repleted individuals (363).
An explanation for this phenomenon still eludes us. In the author’s view, it is plausible
to suppose that pharmacological doses of folic acid stimulate DNA biosynthesis previously
limited by reduced methyltransferase activity. That would account for a partial hematological
remission. Increased cell division in marrow and other cells would likely
increase the cobalamin uptake of these cells from already depleted plasma carriers and
storage depots. This would probably promote transfer of cobalamin from nondividing cells
(such as neurons) to dividing cells in bone marrow. The result would be an abrupt appearance
or worsening of neuropathy. Though a defensible hypothesis, it would be difficult
to test without a suitable animal model.
D. Response to Treatment
The clinical response to cobalamin therapy is another useful diagnostic datum. Following
parenteral administration of cobalamin to deficient subjects, elevated plasma bilirubin,
iron, and lactic dehydrogenase levels fall promptly (Fig. 10). Decreasing plasma iron turnover
and fecal urobilinogen excretion reflects cessation of ineffective erythropoiesis.
Within 8–12 h, the appearance of a bone marrow aspirate begins to convert from megaloCobalamin
495
Fig. 10 Effect of cyanocobalamin on reticulocyte count, serum iron, serum bilirubin, stool urobilinogen,
and plasma iron turnover.
blastic to normoblastic. Transformation is complete in 48–72 h. The population of accumulated
megaloblasts is probably converted to normoblasts ineffectively, i.e., a majority
of the megaloblasts die within the marrow or soon after delivery into the circulation.
Abrupt reticulocytosis begins on the third to fifth day, reaching a climax on the fourth to
tenth day. Cells released at this time apparently come from new normoblasts, not from
converted megaloblasts. The intensity of the reticulocyte crisis is roughly proportional to
the severity of the anemia.
Hemoglobin levels begin to rise, though the increase during the reticulocyte crisis
is smaller than the increase in circulating red cells as judged by the reticulocyte count.
The discrepancy is attributable to the fact that the unusually young reticulocytes delivered
after sudden remission of a megaloblastic process undergo prolonged maturation in the
blood. Hence, the count at a given moment reflects the accumulated reticulocyte output
of several days.
When maturation delay in the marrow is corrected by cobalamin, a new condition
is established in which a still severe anemia elicits an intensive erythropoietin-mediated
stimulation of erythropoiesis. In later stages of hemoglobin restoration, hypochromia and
other signs of iron deficiency may appear. In such instances, the plasma iron level decreases
as the cobalamin level becomes normal. A second reticulocyte response may then
be produced by iron administration.
As documented elsewhere (208), other changes produced by repletion of cobalamin
deficiency include the following: (a) striking and prompt improvement in sense of wellbeing;
(b) rise in serum alkaline phosphatase (which is often depressed in cobalamin defi-
ciency; (c) positive nitrogen balance; (d) sharp rise in serum and urine uric acid (variably
depressed in cobalamin deficiency) within 24 h of the start of therapy, a peak occurring
496 Beck
24 h before the peak of the reticulocyte crisis; (e) decrease in serum folate; (f) decrease
in urine phosphorous after cobalamin administration, increase during reticulocytosis, and
then normal; (g) rise in serum cobalamin; and (h) sharp drop in serum potassium, in some
cases severe enough to warrant replacement therapy. Failure to provide such replacement
has occasionally led to sudden death during treatment for cobalamin deficiency.
A comment should be made on the therapy of the neuropathy of cobalamin defi-
ciency. Basic principles of therapy are well known: early administration of sufficient cobalamin
at a slow rate to replete reserves, followed by administration to meet ongoing
needs. Several writers argued in the past that a monthly cobalamin dose of 100 µg should
be more than adequate. However, this author has seen dramatic evidence that that is bad
advice, e.g., a middle-aged person presenting with classic hematological signs of pernicious
anemia without neuropathy then developed neuropathy for the first time during the
first year of therapy at a monthly dose of 100 µg, a dose that produced hematological
remission. Reinducing therapy and increasing the monthly dose to 1000 µg reversed the
neurological syndrome.
Physicians have been urged to look for cobalamin deficiency in all patients with
‘‘unexplained’’ neuropsychiatric disorders or, if that is not possible, to administer cobalamin
therapy anyway. This, of course, would add new costs to medical care, but if real
benefits await these patients the costs are justified. At least one can hope that physicians
will try to gather meaningful data before giving cobalamin uncritically—and that they
will observe the results of cobalamin therapy. Although recent findings are provocative
and encouraging, they need extensive confirmation.
It is of interest that methylcobalamin (Methylcobal, Eisai) is widely prescribed in
Japan for various peripheral nerve disorders. A large Japanese literature amply demonstrates
its value in many cases but fails to show its superiority over cyano- or hydroxocobalamin.
Some have claimed that hydroxocobalamin is superior to cyanocobalamin in the
therapy of cobalamin deficiency neuropathy. To the extent that this is true it has been
speculatively attributed to the ability of hydroxocobalamin to bind cyanide, which is a
putative neurotoxin. This has periodically led those who are persuaded of the neuropathic
role of cyanide in cobalamin deficiency to demand the withdrawal of cyanocobalamin
from the market. They may be right.
ADDENDUM
My manuscript, as it appears in print, was originally submitted in 1995. For circumstances
that appear not to have been under anyone’s control, I was not informed until November
2000 of the opportunity to update this chapter. Regrettably, such notice was insufficient.
Consequently, this chapter is outdated in some respects wih regard to recent developments
in this field. Although my preference was to withdraw the chapter, with reluctance, I
append this addendum to provide the diligent reader with a road map to major develops
in this field.
Given that space is limited, I will cite only two bodies of work. First, there are the
published proceedings of the fourth international Innsbruck symposium Vitamin B12 and
B12-proteins. B. Kra?utler, D. Arigoni, and B. T. Golding, editors. Weinheim: Wiley-VCH
1998. This useful volume authoritatively updates readers on the current state of B12 research—
with emphasis on what we of medicine call basic science. There is a fine overview
Cobalamin 497
of the field by Kra?utler, and excellent presentations on: (1) biosynthesis; (2) enzymatic
methyl transfer; (3) B12-dependent enzymatic rearrangements (wherein I was relieved to
learn from JoAnne Stubbe that adenosylcobalamin-dependent ribonucleotide reductase is
still amazing, but no longer confusing); (4) B12 structure and reactivity; (5) reflections on
the role of B12 and like molecules in the ancient world; and finally (5) a small concession
to medical aspects.
This leads to my second suggestion regarding important recent biological developments,
not anticipated in my chapter, i.e., the work of Moestrup and colleagues on cubilin.
See, for example, (1) Renata Kozyraki, Mette Kristiansen, Asli Silahtaroglu, Claus Hansen,
Christian Jacobsen, Niels Tommerup, Pierre J. Verroust, and Soren K. Moestrup,
The human intrinsic factor-vitamin B12 receptor, cubilin: molecular characterization and
chromosomal mapping of the gene to 10p within the autosomal recessive megaloblastic
anemia (MGA1) region, Blood 91: 3593 (1998); and (2) Mette Kristiansen, Maria Aminoff,
Christian Jacobsen, Albert de la Chapelle, Ralf Krahe, Pierre J. Verroust, and Soren K.
Moestrup, Cubilin P1297L mutation associated with hereditary megaloblastic anemia 1
causes impaired recognition of intrinsic factor-vitamin B12 by cubilin, Blood 96: 405
(2000). Ebba Nexo’s excellent work on cobalamin-binding proteins also deserves mention.
To readers, I apologize for circumstances that appear not to have been under anyone’s
control.
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14
Choline
STEVEN H. ZEISEL and MINNIE HOLMES-McNARY
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
I. HISTORY
Choline is a dietary component that is important for normal functioning of all cells (1). We
believe that humans require dietary choline for sustaining normal life (2). It is ubiquitous in
foods, is required for synthesis of essential components of membranes, is a precursor for
biosynthesis of the neurotransmitter acetylcholine, and is an important source of labile
methyl groups (3). Choline was discovered in 1862 and was chemically synthesized in
1866 (4). It was known to be a component of phospholipids, but the pathway for its
biosynthesis was first described in 1941 by duVigneaud (5). The route for its incorporation
into phosphatidylcholine (lecithin) was not elucidated until 1956 (6).
The importance of choline as a nutrient was first appreciated during early research
on the functions of insulin (7,8) when it was found to be the nutrient needed to prevent
fatty liver and death in dogs lacking a pancreas. The term ‘‘lipotropic’’ was coined to
describe choline and other substances that prevented deposition of fat in the liver. In 1946,
Copeland (9) first observed that rats fed diets deficient in choline develop liver cancer.
In the subsequent 50 years, this model has been used to develop an understanding of the
mechanisms which initiate and promote carcinogenesis (10–12). In 1975, it was discovered
that administration of choline accelerated the synthesis and release of acetylcholine
by neurons (13–18). This led to an increased interest in dietary choline and brain function.
II. ASSAY
Choline can be measured using gas chromatography–mass spectrometry (19). If choline,
acetylcholine, phosphocholine, glycerophosphocholine, cytidine diphosphocholine, lysophosphatidylcholine,
and phosphatidylcholine are to be determined, extraction and isola-
513
514 Zeisel and Holmes-McNary
tion of these metabolites can be accomplished using high-pressure liquid chromatography
(20). This assay can be used with a nitrogen-phosphorus or a flame ionization detector
instead of with the mass spectrometer (21), but internal standards are not as easy to use.
Other methods for measurement of choline include high-performance liquid chromatography
combined with continuous-flow fast-atom bombardment mass spectrometry (22) or
with a postcolumn reaction converting the choline to betaine and then forming hydrogen
peroxide detected by an electrochemical detector (23–26), a biological assay using the
thermophilic enteric yeast Torulopsis pintolopessi (27), a chemiluminescence method (28),
and a radioenzymatic method (29,30).
III. CONTENT IN FOOD
Many foods eaten by humans contain significant amounts of choline and esters of choline.
No information is available about the phosphocholine or glycerophosphocholine content
of foods, but it is likely that they are present in significant amounts. Some of this choline
is added during food processing [especially during preparation of infant formula (31)].
Average choline dietary intake (as choline and choline esters) in the adult human (as free
choline and the choline in phosphatidylcholine and other choline esters) is more than 7–
10 mmol/day (32,33). A kilogram of beef liver contains 50 mmol of choline moiety; thus,
it is easy to consume a diet of normal foods (eggs, liver, etc.) that delivers much more
choline per day than the calculated average intake or 700 mg/day (34). The human infant
consumes a choline-rich diet. Breast milk contains approximately 1.5 mmol/L choline
and choline esters; only recently did we discover that the major forms of choline in mature
human and rat milk are phosphocholine and glycerophosphocholine (35,36). Thus, an
infant consuming 500 mL breast milk ingests 750 µmol choline. Choline is routinely added
to commercially available infant formulas (approximately 1 mmol free choline per L).
IV. METABOLISM
There are several comprehensive reviews of the metabolism and functions of choline
(33,37,38) (Figs. 1–3; Table 1). A small fraction of dietary choline is acetylated (39,40)
in cholinergic neurons (41) and in such nonnervous tissues as the placenta (42). In brain
it is unlikely that choline acetyltransferase is saturated with either of its substrates, so that
choline (and possibly acetyl-CoA) availability determines the rate of acetylcholine synthesis
(43). Some investigators report that administration of choline or phosphatidylcholine
results in the accumulation of acetylcholine within brain neurons (13–16), whereas others
observe that such acceleration of acetylcholine synthesis by choline administration can
only be detected after pretreatments with agents that cause cholinergic neurons to fire
rapidly (17,44–48). Increased brain acetylcholine synthesis is associated with an augmented
release of this neurotransmitter into the synapse.
The metabolisms of choline, methionine, and methyl folate are closely interrelated,
and the use of choline molecules as methyl donors is probably the major factor that determines
how rapidly a diet deficient in choline will induce pathology (10). The pathways
of choline and one-carbon metabolism intersect at the formation of methionine from homocysteine.
Betaine-homocysteine methyltransferase catalyzes the methylation of homocysteine
using the choline metabolite betaine as methyl donor (49–51). In an alternative
pathway, 5-methyltetrahydrofolate-homocysteine methyltransferase regenerates methionine
using a methyl group derived de novo from the one-carbon pool (50,52). Perturbing
metabolism of one of the methyl donors results in compensatory changes in the other
Choline 515
Fig. 1 Choline metabolic pathway is interrelated with folate, methionine, and B12 metabolism. SAdoMet,
S-adenosylmethionine; CDP-Choline, choline–cytidine diphosphocholine; PtdEtn, phosphatidylethanolamine;
GPC, glycerophospocholine; LysoCho, lysophosphatidylcholine; S-AdoHcy,
S-adenosylhomocysteine.
Fig. 2 Myoinositol metabolic pathway. Mg
2, magnesium; PI kinase, phosphoinositol kinase;
Pase A1, phospholipase A1; Pase A2, phospholipase A2; Co-A, coenzyme A; PIP, phosphoinositol
phosphate; PIP2, phosphoinositol biphosphate; PIP3, phosphoinositol triphosphate.
516 Zeisel and Holmes-McNary
Fig. 3 Carnitine metabolic pathway. CO2, carbon dioxide; O2, oxygen; NAD
 (NADH), nicotinamide
dehydrogenase; Fe
2, ferrous iron; PLP, pyridoxyl phosphate.
Table 1 Pathophysiology Associated with Required Carnitine Supplementation
Altered biosynthesis Increased excretion Altered transport
Malnutrition Immaturity Malnutrition
Immaturity Organic aciduria Immaturity
Genetic carnitine deficiency Genetic carnitine deficiency Genetic carnitine deficiency
Liver disease Liver disease Liver disease
Renal disease Renal disease
Drug treatment (e.g., valproate)
Complied from P. R. Borum (1991) in The Handbook of Vitamins.
Choline 517
methyl donors due to the intermingling of these metabolic pathways (53–61). Redundant
parallel pathways protect the capacity to donate methyl groups. Clearly, if we had established
that choline was essential first, we would believe that methionine is not an essential
amino acid because it can be replaced by homocysteine and choline. The amounts of
choline, methyl folate, or methionine required in the diet are dependent on the availability
of the other two. Does this mean that other methyl donors can completely substitute for
choline? Many investigators assume that this is true, but this question has never been
adequately investigated.
Synthesis of phosphatidylcholine occurs by two pathways. In the first, choline is
phosphorylated, then converted to cytidine diphosphocholine (CDP-choline) in the regulated
step in phosphatidylcholine biosynthesis (62). This high-energy intermediate, in combination
with diacylglycerol, forms phosphatidylcholine and cytidine monophosphate (6).
In the alternative pathway, phosphatidylethanolamine is sequentially methylated to form
phosphatidylcholine, using S-adenosylmethionine as methyl donor (63). This is the only
source of choline other than diet.
V. FUNCTIONS OF CHOLINE AND CHOLINE PHOSPHOLIPIDS
The numerous functions of choline as a methyl donor and of choline phospholipids as
structural elements of cells have been reviewed extensively elsewhere (33). Choline and
its relationships to acetylcholine synthesis also have been thoroughly reviewed elsewhere
(64). We focus on new observations about physiologic roles for these compounds.
A. Hepatic Secretion of Very Low Density Lipoprotein
The triacylglycerol produced by liver is delivered to other tissues mainly in the form of
very low density lipoprotein (VLDL). Phosphatidylcholine is a required component of the
VLDL particle (65,66), and in choline deficiency the diminished capacity of liver cells
to synthesize new phosphatidylcholine molecules results in the intracellular accumulation
of triglycerides. Methionine can substitute for choline in VLDL secretion, but only as
long as phosphatidylethanolamine-N-methyltransferase activity (see earlier discussion) is
active (65). Secretion of high-density lipoprotein (HDL) from hepatocytes does not require
the synthesis of new phosphatidylcholine molecules (67). Choline-deficient humans have
diminished plasma low-density lipoprotein cholesterol (LDL; derived from VLDL) (2).
This observation is consistent with the hypothesis that in humans, as in other species,
choline is required for VLDL secretion.
B. Platelet-Activating Factor: A Choline-Containing Phospholipid
with Hormonal Functions
Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a choline
phospholipid characterized by a fatty alcohol (usually hexadecanol) at the sn-1 position
of the glycerol backbone and an acetate residue at the sn-2 position. It has diverse
biological functions that are exerted through specific receptors located on cells of many
different types. Receptor-mediated synthesis of PAF requires calcium and is mediated
by a G-protein. The effects of PAF include the activation of phosphoinositide-specific
phospholipase C, and the release of fatty acids from phospholipids, possibly by activation
of phospholipase A2 (68). Besides activating platelet aggregation, the actions of PAF include
lowering of blood pressure; increasing of vascular permeability; activation of mono518
Zeisel and Holmes-McNary
cytes, macrophages, and polymorphonuclear neutrophils; and stimulation of hepatic glycogenolysis.
PAF may be one of the mediators of parturition, and it mediates many processes
of inflammation and allergy. Many excellent reviews describe the biosynthesis, turnover,
and actions of PAF (68–71).
C. Signal Transduction
Our understanding of choline phospholipid–mediated signal transduction has been vastly
improved during the past decade. Stimulation of membrane-associated receptors activates
neighboring phospholipases, resulting in the formation of breakdown products that are
signaling molecules either by themselves (i.e., they stimulate or inhibit the activity of
target macromolecules), or after conversion to signaling molecules by specific enzymes.
Much of signaling research focused on minor membrane phospholipid components, particularly
phosphatidylinositol derivatives [signal transduction via receptor-mediated hydrolysis
of phosphatidylinositol biphosphate has been extensively reviewed elsewhere (72,73)].
However, metabolism of choline phospholipids, especially phosphatidylcholine and sphingomyelin,
results in biologically active molecules that can amplify external signals or that
can terminate the signaling process by generating inhibitory second messengers (74).
For example, activation of heptahelical receptors leads to altered conformation of
the receptor so that it can activate a GTP-binding protein (G-protein). The activation of
the G-protein results in the subsequent activation of phospholipase C activity within the
plasma membrane. Phospholipase C hydrolyzes intact phospholipids to generate 1,2-sndiacylglycerol
and an aqueous soluble head group. The action of phospholipase C triggers
the next event in the signal cascade, which is activation of protein kinase C (PKC; serine/
threonine kinase). Products generated by phosphatidylinositol-bis-phosphate-phospholipase
C include inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) and diacylglycerol. Ins-1,4,5-
P3 is a water soluble product, which acts to release calcium from stores in the endoplasmic
reticulum. Some PKC isotypes are Ca2
-dependent (PKC ?, ?2, and ?). PKC ?, ?, ?, ?,
and ? lack a calcium binding domain (75). Calcium, by increasing membrane occupancy
of PKC, places the enzyme in close proximity to phosphatidylserine, a cofactor for PKC
activation. Diacylglycerol, the other product of phospholipase C, remains in the plasma
membrane, and is both a messenger molecule and an intermediate in the metabolism of
lipids. Normally, PKC is folded so that an endogenous ‘‘pseudosubstrate’’ region on the
protein is bound to the catalytic site, thereby inhibiting activity. The combination of diacylglycerol
and Ca2
 causes a conformational change in PKC, causing flexing at a hinge
region that heads to withdrawal of the pseudosubstrate and unblocking of the PKC catalytic
site. The appearance of diacylglycerol in membranes is usually transient, and therefore
PKC is activated for a short time after a receptor has been stimulated. The generation of
diacylglycerol from membrane phosphatidylcholine amplifies the signal. Phospholipase
C and, indirectly, phospholipase D generate this diacylglycerol (76–79). Other products
of phosphatidylcholine hydrolysis, such as phosphatidic acid, lysophosphatidylcholine,
and free fatty acids, also are second messengers (76,80). Phosphatidic acid can act as a
mitogen (81). Lysophosphatidylcholine stimulates PKC activity (75), but it is a membranelytic
detergent with potential toxic effects. Lysophosphatidylcholine generation is important
in chemotaxis, relaxation of smooth muscle, and activation of T lymphocytes (75).
Recent data indicate that phosphatidylcholine may synergize with diacylglycerol and calcium
to activate PKC in lymphocytes (82) and that modulation of PKC isozymes by phosphatidylcholine
may be isoform-specific (83).
Choline 519
The characterization of events that occur downstream from PKC is just beginning.
Serine-threonine kinases and tyrosine kinases catalyze phosphorylation of target proteins
distal to PKC. Phosphorylation alters the biochemical properties of these substrates, resulting
in a range of cellular responses. These phosphorylation cascades serve to enhance
amplification of the original signal. PKC signals impinge on several known intracellular
control circuits (84). The targets for phosphorylation by PKC include receptors for insulin,
epidermal growth factor, and many proteins involved in control of gene expression (85,86).
Although choline sphingolipids are ubiquitous components of mammalian cells, it
has only recently been proven that they are necessary for cellular survival and growth
(87). While phosphatidylcholine hydrolysis generates a series of messengers that sustain
the PKC phosphorylation cascade, hydrolysis of sphingomyelin generates messengers that
terminate the cascade. Sphingomyelin metabolism has been reviewed extensively (88).
The activation of sphingomyelinase may constitute one of the signal transduction pathways
for triggering differentiation (89,90). Activation of sphingomyelinase causes elevations
in cellular ceramide levels, a compound with potent biological activities including the
triggering of programmed cell death (apoptosis) (91). The formation of sphingosine from
ceramide may be critical for regulating PKC signal transduction.
VI. DEFICIENCIES
A. Choline Deficiency in Animals
The liver dysfunction and fatty liver associated with choline deficiency has been discussed
earlier. Choline deficiency compromises renal function, with abnormal concentrating ability,
free water reabsorption, sodium excretion, glomerular filtration rate, renal plasma flow,
and gross renal hemorrhage (92–95). Diets low in choline content also cause infertility,
growth impairment, bone abnormalities, decreased hematopoiesis, and hypertension (96–
99).
B. Choline Deficiency in Humans
Humans require choline for sustaining normal life. Is a dietary source of choline required?
As discussed above, normal diets deliver sufficient choline. When healthy humans were
fed a choline-deficient diet for 3 weeks they developed biochemical changes consistent
with choline deficiency (2). These included diminished plasma choline and phosphatidylcholine
concentrations, as well as diminished erythrocyte membrane phosphatidylcholine
concentrations. Serum alanine transaminase (ALT) activity, a measure of hepatocyte damage,
increased significantly during choline deficiency. This experiment established a requirement
for choline in the diet of normal humans.
This requirement may be more apparent in special human populations. The demand
for choline in normal adults is likely to be smaller than the demand for choline in the
infant, as large amounts of choline must be used to make phospholipids in growing organs
(37). The observed changes that occurred in choline-deficient adult humans might have
been greater had we studied growing children. Malnourished humans, in whom stores of
choline, methionine, and folate have been depleted (100,101), are also likely to need more
dietary choline than did our healthy adult subjects. The liver is the primary site for endogenous
synthesis of choline. Alcoholics with liver cirrhosis have diminished plasma choline
concentration, alone with fatty liver which resolves when patients are supplemented with
520 Zeisel and Holmes-McNary
choline (101). In baboons, treatment with phosphatidylcholine can prevent alcoholinduced
liver fibrosis (102).
C. Carcinogenesis
Choline is the only nutrient for which dietary deficiency causes development of hepatocarcinomas
in the absence of a known carcinogen (10). It is interesting that choline-deficient
rats not only have a higher incidence of spontaneous hepatocarcinoma but are markedly
sensitized to the effects of administered carcinogens (10). Choline deficiency is therefore
considered to have both cancer-initiating and promoting activities.
There are several mechanisms suggested for the cancer-promoting effect of a
choline-devoid diet. In the choline-deficient liver there is a progressive increase in cell
proliferation, related to regeneration after parenchymal cell death (103). Cell proliferation,
with associated increased rate of DNA synthesis, could be the cause of greater sensitivity to
chemical carcinogens (104). Stimuli for increased DNA synthesis increase carcinogenesis;
hepatectomy and necrogenic chemicals are examples. However, the overall rate of liver
cell proliferation could be dissociated from the rate at which preneoplastic lesions formed
during choline deficiency (105), suggesting that cell proliferation is not the sole condition
acting as a promoter of liver cancer. Methylation of DNA is important for the regulation
of expression of genetic information. Undermethylation of DNA, observed during choline
deficiency (despite adequate dietary methionine), may be responsible for carcinogenesis
(106,107). Another proposed mechanism derives from the observation that, when rats
eat a choline-deficient diet, increased lipid peroxidation occurs within liver (108). Lipid
peroxides in the nucleus could be a source of free radicals that could modify DNA and
cause carcinogenesis. We have proposed that choline deficiency perturbs PKC signal transduction,
thereby promoting carcinogenesis. Recently, we reported that a defect in cell
suicide (apoptosis) mechanisms may contribute to the carcinogenesis of choline defi-
ciency.
As discussed earlier, choline deficiency causes massive fatty liver (see Sec. V.A).
We have observed that 1, 2-sn-diacylglycerol accumulates in this fatty liver (11,109). In
plasma membrane from livers of choline-deficient rats, diacylglycerol reaches values
higher than those occurring after stimulation of a receptor linked to phospholipase C activation
(e.g., vasopressin receptor). This results in a stable activation of PKC and/or an
increase in the total PKC pool in the cell (11) with changes in several PKC isotypes (at
6 weeks of choline deficiency, amounts of PKC ? and ? increased 2-fold and 10-fold,
respectively). The accumulation of diacylglycerol and subsequent activation of PKC
within liver during choline deficiency may be the critical abnormality that eventually contributes
to the development of hepatic cancer in these animals (11). Abnormalities in PKCmediated
signal transduction may trigger carcinogenesis (86).
The process of carcinogenesis involves an initiating event that induces genetic damage,
followed by survival and progression of selected clones of the mutant cells to form
tumors. In order to study the underlying mechanisms involved in progression of carcinogenesis
after initiation, we developed a cell culture model using immortalized CWSV-1
rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen. Many cancers
are p53-defective (110), suggesting that their precursor (initiated) cells also share this
defect. Previously, we showed that choline deficiency (CD) medium induced apoptosis
in CWSV-1 cells via a p53-independent pathway (111). In normal tissues, apoptosis proCholine
521
vides a physiological way to eliminate terminally differentiated, damaged, or genetically
altered cells, thus facilitating tissue remodeling following cell injury (112). Apoptosis is
an important defensive barrier that inhibits carcinogenesis by eliminating initiated cells,
usually via p53-dependent mechanisms (113). However, in p53-defective cells, alternative,
p53-independent apoptosis pathways may serve as a mechanism(s) for eliminating initiated
cells. When both p53-dependent and p53-independent apoptosis are inactivated, an environment
is created in which initiated cells may have a high survival rate, significantly
enhancing carcinogenesis. We observed that CWSV-1 cells that are gradually deprived
of choline can adapt and become resistant to CD apoptosis. These adapted cells express
a tumorigenic phenotype, and treatment with an antioxidant during the CD adaptation
process can substantially abrogate the acquisition of a transformed phenotype (114).
D. Brain Development and Function
Given the central role of choline as a precursor of acetylcholine and phosphatidylcholine,
nature has developed a number of mechanisms to ensure that a developing animal gets
adequate amounts of choline. In mammals, the placenta transports choline to the fetus
(115). At birth, all mammals studied, including the human, have plasma choline concentrations
that are much higher than those in adults (116). The capacity of brain to extract
choline from blood is greatest during the neonatal period (117). There is a novel phosphatidylethanolamine-
N-methyltransferase (synthesizes choline de novo) in neonatal rat brain
that is extremely active (118). Human and rat milk contain especially large amounts of
choline (35,36).
There are two sensitive periods in development of rat brain during which treatment
with choline results in long-lasting facilitation of spatial memory. The first occurs during
embryonic days 12–17 and another during postnatal days 16–30 (119–121). Choline supplementation
during these critical periods elicits a constant percentage improvement in
choice performance at all stages of training for a visuospatial task (12-arm radial maze),
suggesting a memory rather than a learning rate effect. This effect is apparent months
after the brief exposure to extra choline. The two sensitive periods correlate with neurogenesis
of cholinergic cells (prenatal) and with synaptogenesis (postnatal) (122–124).
The observation that perinatal supplementation with choline results in improved
memory performance of adult rats suggests that choline availability is critical for brain
development. The choline content of rat chow has been established based on choline requirements
of nonpregnant adult rats. We do not know what the minimal requirement is
for the pregnant or lactating female, or for the fetus. Perhaps changes in eating behaviors
during pregnancy allow rats in the wild to adjust their choline intake. These considerations
are also important when considering the choline requirements for humans.
VII. NUTRITIONAL STATUS
There are no established approaches for determining nutritional status for choline. Plasma
choline and phosphatidylcholine concentrations fall when humans are fed a cholinedeficient
diet (2), but this also occurs in marathon runners (125). Even in severe deficiency,
plasma choline concentrations do not fall below 50% of normal. We have observed that
liver phosphocholine concentrations are the most accurate indicator of acute changes in
the choline content of diet (60).
522 Zeisel and Holmes-McNary
VIII. FACTORS THAT MAY INFLUENCE NUTRITIONAL STATUS
Amino acid–glucose solutions used in total parenteral nutrition of humans contain no
choline (100,126). The lipid emulsions used to deliver extra calories and essential fatty
acids during parenteral nutrition contain choline in the form of phosphatidylcholine (20%
emulsion contains 13.2 mmol/L). Humans treated with parenteral nutrition required 1–
1.7 mmol of choline-containing phospholipid all day during the first week of parenteral
nutrition therapy to maintain plasma choline levels (100). Others (127) reported that
plasma choline concentrations decreased in parenteral nutrition patients at the same time
that they detected liver dysfunction. Conditions that enhance hepatic triglyceride synthesis
(such as carbohydrate loading) increase the requirement for choline needed for export of
triglyceride from liver (128). Thus, treatment of malnourished patients with high-calorie
parenteral nutrition solutions at a time of depleted choline stores might enhance the likelihood
of hepatic dysfunction. A recent clinical trial supports the requirement for supplemental
choline during total parenteral nutrition (129,130).
ACKNOWLEDGMENTS
Some of the work described herein was supported by grants from the National Institutes of
Health (AG09525, DK56350, DK55865) and the American Institute for Cancer Research.
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of recent evidence. Trends NeuroSci. 14, 220–223.
125. Conlay, L. A., Wurtman, R. J., Blusztajn, K., Coviella, I. L., Maher, T. J. and Evoniuk, G.
E. (1986). Decreased plasma choline concentrations in marathon runners [letter]. N. Engl.
J. Med. 315, 892.
126. Chawla, R. K., Berry, C. J., Kutner, M. H. and Rudman, D. (1985). Plasma concentrations
of transsulfuration pathway products during nasoenteral and intravenous hyperalimentation
of malnourished patients. Am. J. Clin. Nutr. 42, 577–584.
127. Burt, M. E., Hanin, I. and Brennan, M. F. (1980). Choline deficiency associated with total
parenteral nutrition. Lancet 2, 638–639.
128. Carroll, C. and Williams, L. (1982). Choline deficiency in rats as influenced by dietary energy.
Nutr. Rep. Int. 25, 773–782.
129. Buchman, A. L., Dubin, M., Jenden, D., Moukarzel, A., Roch, M. H., Rice, K., Gornbein,
J., Ament, M. E. and Eckhert, C. D. (1992). Lecithin increases plasma free choline and
decreases hepatic steatosis in long-term total parenteral nutrition patients. Gastroenterology
102, 1363–1370.
130. Buchman, A. L., Moukarzel, A., Jenden, D. J., Roch, M., Rice, K. and Ament, M. E. (1993).
Low plasma free choline is prevalent in patients receiving long term parenteral nutrition and
is associated with hepatic aminotransferase abnormalities. Clin. Nutr. 12, 33–37.
15
Ascorbic Acid
CAROL S. JOHNSTON
Arizona State University East, Mesa, Arizona
FRANCENE M. STEINBERG and ROBERT B. RUCKER
University of California, Davis, California
I. INTRODUCTION AND HISTORY
Ascorbic acid (vitamin C) functions as a redox cofactor and catalyst in a broad array of
biochemical reactions and processes. In humans, vitamin C cures and prevents scurvy,
hence the designation ascorbic acid (1). Scurvy or ‘‘scurfy’’ (Old English) was probably
derived from the Scandinavian terms, skjoerberg and skorbjugg, meaning rough skin.
Aside from famine, scurvy has caused the most suffering of nutritional origin in human
history.
Those without access to fresh fruits and vegetables are susceptible to scurvy. Onehalf
of the original 60 colonists of Plymouth died of scurvy in 1628. The military of the
seventeenth and eighteenth centuries often adhered to dietary protocols that promoted
scurvy. During the Civil War, poor nutrition resulting in scurvy, as well as pellagra, also
took its toll (2,3). Excellent accounts of the history of scurvy have been presented by
Carpenter (4) and Clemetson (5), and the Treatise on the Scurvy by James Lind published
in 1753 (6) is considered the first recorded account of a controlled clinical trial.
By the end of the 1800s, the connection between scurvy and diet was established.
The observation in 1907 that guinea pigs were susceptible to scurvy was an important
breakthrough in the understanding of scurvy. It was also one of the earliest examples of
the use of an animal model to study a nutritional disease. By 1915, Zilva and his associates
of the Lister Institute in London had isolated antisorbutic activity from a crude fraction
of lemon. Work using animal assays demonstrated that the activity was destroyed by
oxidation and protected by reducing agents (8). Important to the evolving nomenclature
529
530 Johnston et al.
for vitamins, it was suggested that the new antiscorbic factor be designated ‘‘factor or
vitamin C’’ since ‘‘A’’ and ‘‘B’’ had been previously designated as potential health and
growth factors or vitamins (9).
Throughout the 1930s, work progressed rapidly with validation and identification
of vitamin C in a number of foods. Early papers by Szent-Gyorgyi, Haworth, King, and
their colleagues document this effort and provide chemical identification and elucidation
of ascorbic acid structure (10–15).
II. CHEMICAL FEATURES
A. Nomenclature and Structure
Ascorbic acid or l-ascorbic acid is the IUPAC-IUB Commission designation for vitamin
C (2-oxo-l-theo-hexono-4-lactone-2, 3-enediol). The chemical structures of ascorbic acid
and selected derivatives are given in Fig. 1. Ascorbic acid has a near planar five-member
ring; the two chiral centers at positions 4 and 5 determine the four stereoisomers. Dehydroascorbic
acid, the oxidized form of ascorbic acid, retains some vitamin C activity and
can exist as a hydrated hemiketal, or as a dimer (16).
B. Physical and Chemical Properties
Table 1 summarizes physical and chemical features of ascorbic acid. Data are also available
from x-ray crystallographic analysis, [1H] and [13C] NMR spectroscopy, IR- and UVspectroscopy,
and mass spectroscopy (see 16–19 and references cited). The reversible
oxidation to semidehydro-l-ascorbic acid (and further to dehydro-l-ascorbic acid) is the
most important chemical property of ascorbic acid. This property is the basis for its known
physiological activities. The proton on oxygen-3 is acidic (pK1 
 4.17) and contributes
to the acidic nature of the vitamin.
Degradation reactions of l-ascorbic acid in aqueous solutions depend on a number
of factors such as pH, temperature, and the presence of oxygen or metals. Alkali-catalyzed
degradation results in over 50 compounds, mainly mono-, di-, and tricarboxylic acids
(15,18,19). The vitamin can be stabilized in biological samples with trichloracetic acid
or metaphosphoric acid (20). In general, ascorbic acid is not stable in aqueous media, e.g.,
culture media or parenteral/enteral solutions, at typical room temperatures. Ascorbic acid
is reasonably stable in blood, when stored at or below 20 C (20–22).
The ascorbate radical is an important intermediate in redox reactions (15,18,23,24).
The physiologically dominate ascorbic acid mono anion (pK1 
 4.1) and dianions (pK2

 11.79) are shown in Fig. 1.
The ascorbate/ascorbate monoanion reduction potential is low in comparison to
other redox systems (25). Ascorbic acid readily scavenges reactive oxygen species; rate
constants 104–108 ? mol1/L ? s1 (23,24). Moreover, since the ascorbate radical has a high
disproportion rate under physiological conditions and converts rapidly to ascorbic acid
and dehydroascorbic acid without reacting with adjacent molecules, ascorbic acid is well
suited as a physiological antioxidant (25). In biological systems, accessory enzymatic
systems are present to reduce dehydroascorbic acid. This recycling of ascorbic acid helps
to maintain ascorbic acid in tissues. Further, the process diminishes the possibility of an
abnormal excess of ascorbic acid radical and nonspecific oxidations.
In plants, NADH: monodehydroascorbate reductase (EC 1.6.5.4) evolved to maintain
ascorbic acid in its reduced form, and plays a major role in the stress response of
Ascorbic Acid 531
Fig. 1 Ascorbic acid and various oxidation products. The two predominant forms of ascorbic
acid and their associated oxidation products are shown. In solution, ascorbic acid probably exists
as the hydrated hemiketal.
plants. In animal tissues, glutathione dehydroascorbate reductase (EC 1.8.5.1) is the primary
enzyme that serves this purpose (26). Dehydroascorbic acid (DHA) is a more stable
oxidation product of the ascorbyl radical. Dehydroascorbic acid is thought to exist mainly
in vivo as the hydrated hemiketal and has two possible metabolic fates (Fig. 1). It may
either be reduced to ascorbate or irreversibly hydrolyzed to a variety of metabolites including
oxalate. The reduction of dehydroascorbic acid is therefore very important in maintaining
cellular ascorbate levels.
532 Johnston et al.
Table 1 Selected Physical Properties of Ascorbic Acid
Empirical formula C6H8O6
Molar mass 176.13
Crystalline form Monoclinic, mix of platelets and needles
Melting point 190–192°C
Optical rotation [?] 25/D 20.5° to 21.51°
(cm 
 1 in water)
pH, at 5 mg/ml 
3
at 50 mg/ml 
2
pK1 4.17
pK2 11.57
Redox potential (dehydroascorbic acid/ascorbate) 174 mV
(ascorbate •E, H/ascorbate) 282 mV
Solubility, g/ml
water 0.33
ethanol, abs. 0.02
ether, chloroform, benzene Insoluble
Absorption spectra
at pH 2 Emax (1%, 10 mm) 695 at 245 nm
at pH 6.4 Emax (1%, 10 mm) 940 at 265 nm
C. Isolation
Ascorbic acid is stable in many organic and inorganic acids. Metaphosphoric acidcontaining
ethylenediamine tetra-acetic acid (0.5–2%), oxalic acid, dilute trichloracetic
acid or dilute perchloric acid containing reducing agents, such as 2,3-dimercaptopropanol,
are often used as solvent for tissue extraction (15,20). Extraction of ascorbic acid should
be carried out under subdued light and an inert atmosphere to avoid the potential for
degradation (20).
D. Chemical Synthesis
The chemical pathway for the industrial synthesis of ascorbic acid from glucose was first
developed in the 1930s and continues to be used (Fig. 2). Initially, glucose is converted
to l-sorbitol by hydrogenation; subsequent fermentation with Acetobacter suboxidans
yields l-sorbose. Next, a carboxyl group is introduced at the C1 position with derivatization
to diacetone-2-keto-l-gulonic acid. Removal of acetone and heating under acid conditions
yields l-ascorbic acid, which may be crystallized in high purity.
The need for and utilization of commercial sources of ascorbic acid is high. In the
United States, the annual per capita use is 25–30 mg of ascorbic acid, 9–10 mg of sodium
ascorbate, 6 mg of erythorbic acid, the d-isomer of ascorbic acid, and 6–8 mg sodium
erythorbic acid (27). Erythorbic acid is a common food additive. Both forms of ascorbic
acid are inhibitors of enzymatic browning reactions and are used as preservative antioxidants.
Note that erythorbic acid does not possess antiscorbutic activity, nor does it act as
an ascorbic acid antagonist in vivo (28).
Selective derivatization of ascorbic acid can be difficult, because of delocation of
the negative charge of ascorbate in its anionic form. If the C-2 and C-3 hydroxyl groups
are protected, however, base-promoted alkylation or acylation can take place at the more
sterically accessible primary hydroxyl group on C-6, but not at C-5. Reactions at the CAscorbic
Acid 533
Fig. 2 Chemical synthesis of ascorbic acid from d-glucose.
5 position often require derivatizations at the C-2, C-3, and C-6 positions (25,26). The
formation of acetates or ketals of ascorbic acid is useful for protection of the molecule
while reactions at the other carbons are carried out.
E. Analysis
Numerous high-performance liquid chromatographic methods have been developed for
determination of ascorbic acid and related isomers or derivatives. Electrochemical detection
is generally used for measuring ascorbic acid and derivatives. For example, chromatographic
approaches include both the ion exchange, gas, and reversed-phase chromatography
(29–36).
534 Johnston et al.
Colorimetric assays of ascorbic acid in crude mixtures include the 2,2?-dipytidyl
calorimetric method, which is based on the reduction of Fe (III) to Fe (II) by ascorbic
acid (33), and the 2,4-dinitrophenylhydrazine method (37). Methods based on fluorometric
and chemiluminescence detection also provide highly sensitive approaches for the determination
ascorbic acid (33,34). Further, conventional and isotope ratio mass spectrometry
techniques have been used to analyze ascorbic acid when 13C ascorbic acid is available
for use as a reference or standard in the analysis of complex matrices (35).
As a final point, since ascorbic acid is a reductant and can cause nonspecific color
formation, the presence of ascorbic acid may interfere with many chemical tests, including
the analysis of glucose, uric acid, creatinine, bilirubin, glycohemoglobin, hemoglobin A,
cholesterol, triglycerides, leukocytes, and inorganic phosphate (36,38–41).
F. Sources of Ascorbic Acid
Ascorbic acid occurs in significant amounts in vegetables, fruits, and animal organs such
as liver, kidney, and brain. In many European cultures, potatoes and cabbage have been
important sources of vitamin C. Typical values are given in Table 2.
III. BIOCHEMICAL FUNCTIONS
A. Plants
Ascorbic acid is detected in yeast and prokaryotes, with the exception of cyanobacteria
(15). In higher plants, ascorbic acid is synthesized from d-glucose and functions chiefly
as a reductant, protecting and participating in many metabolic processes. For example,
ascorbic acid is crucial for the scavenging of H2O2 in plants (42) and functions to maintain
the ?-tocopherol pool, which scavenges radicals in plant membranes (43). Ascorbic acid
is the cofactor for violaxanthin depoxidase, which forms zeaxanthin, an important factor
in the stress response of plants (44).
B. Animals
The pathway for ascorbic acid synthesis from glucose in animals is shown in Fig. 3. A
key enzyme in the synthesis, l-gulonolactone oxidase (GLO, EC 1.1.3.8), resides in the
kidney of reptiles, but during the course of evolution, the activity was transferred to the
liver of mammals. For reasons that are not clear, the ability to express l-glulonolactone
oxidase is absent in the guinea pig, some fruit-eating bats, and most primates, including
man. In these animals, signs of ascorbic acid deprivation include a range of symptoms
that can be related to the failure of specific enzymatic steps and processes that require
ascorbic acid as cofactor (Table 3). As given in Fig. 3, ascorbic acid can be converted to
ascorbic acid 2-sulfate and methylated ascorbic acid derivatives. Although the exact role
of such derivatives is unknown, they may be important to intracellular partitioning, transport,
or storage or protection from cellular excesses of ascorbic acid. Decarboxylation at
the C-1 position or cleavage to oxalic acid and four carbon fragments occur when ascorbic
acid is in excess (5,15).
1. Ascorbic Acid and Glutathione Interrelationships
Glutathione l-glutamyl-l-cysteine-glycine (GSH) is widely distributed in plant and animal
cells and functions predominately as an antioxidant scavenging reactive oxygen radicals.
Ascorbic Acid 535
Table 2 Vitamin C in Selected Foods
Animal products mg/100 g of edible portion
Cow’s milk 0.5–2
Human milk 3–6
Beef 1–2
Pork 1–2
Veal 1–1.5
Ham 20–25
Liver, chicken 15–20
Beef 10
Kidney, chicken 6–8
Heart, chicken 5
Gizzard, chicken 5–7
Crab muscle 1–4
Lobster 3
Shrimp muscle 2–4
Fruits mg/100 g of edible portion
Apple 3–30
Banana 8–16
Blackberry 8–10
Cherry 15–30
Currant, red 20–50
Currant, black 150–200
Grape 2–5
Grapefruit 30–70
Kiwi fruit 80–90
Lemon 40–50
Melons 9–60
Mango 10–15
Orange 30–50
Pear 2–5
Pineapple 15–25
Plums 2–3
Rose hips 250–800
Strawberry 40–70
Tomato 10–20
Vegetable mg/100 g of edible portion
Asparagus 15–30
Avocado 10
Broccoli 80–90
Beet 6–8
Beans, various 10–15
Brussels sprout 100–120
Cabbage 30–70
Carrot 5–10
Cucumber 6–8
Cauliflower 50–70
Eggplant 15–20
Chive 40–50
Kale 70–100
Lettuce, various 10–30
536 Johnston et al.
Table 2 Continued
Onion 10–15
Pea 8–12
Potato 4–30
Pumpkin 15
Radish 25
Spinach 35–40
Spices and condiments mg/100 g of edible portion
Chicory 33
Coriander (spice) 90
Garlic 16
Horseradish 45
Leek 15
Parsley 200–300
Papaya 39
Pepper, various 150–200
GSH is synthesized by a two-step reaction involving l-glutamyl cysteine synthetase and
GSH synthetase. In addition, GSH-dependent reduction of intracellular dehydroascorbic
acid to ascorbate is believed to play a crucial role in ascorbic acid recycling in vivo,
thereby enhancing the antioxidant reserve as it relates to ascorbic acid (45–48).
When GSH synthesis is blocked, e.g., by use of inhibitors, such as l-buthionine-
(SR)-sulfoximine, newborn animals die within a few days due to oxidative stress (45).
The cellular damage involves mostly abnormal alterations in mitochondria that lead to
proximal renal tubular damage, liver damage, and disruption of lamellar bodies in lung
(45). Furthermore, the buthionine-(SR)-sulfoximine-induced loss in GSH is associated
with a marked increase in dehydroascorbic acid. Interestingly, ascorbic acid administration
ameliorates most signs of chemically induced GSH deficiency in rats (45). In guinea pigs,
treatment with GSH ester significantly delays the onset of scurvy (49). The sparing effect
is probably due to the need for both ascorbic acid and GSH in counteracting the deleterious
effects of reactive oxidant species (Fig. 4).
2. Norepinephrine Synthesis
The dopamine-?-hydroxylase mediated conversion of dopamine to norepinephrine (Fig.
5) is dependent on ascorbic acid (50,51) and this may explain in part the high concentration
of ascorbic acid in adrenal tissue. Dopamine ?-hydroxylase (EC 1.14.17.1) is present
in catecholamine storage granules in nervous tissues and in chromaffin cells of adrenal
medulla, the site of the final and rate-limiting step in the synthesis of norepinephrine.
Dopamine-?-hydroxylase is a tetramer containing two Cu (I) ions per monomer that consumes
ascorbate stoichiometrically with O2 during its cycle. At a steady state the predominant
enzyme form is an enzyme-product complex, and the primary function of ascorbate
is to maintain copper in a reduced state. Only the reduced enzyme seems to be catalytically
competent, with bound cuprous ions as the only reservoir of reducing equivalents.
3. Hormone Activation
Many hormones and hormone-releasing factors are activated by posttranslational steps
involving ?-amidations (52,53). Examples of hormone activation include melanotropins,
Ascorbic Acid 537
Fig. 3 Pathway for ascorbic acid synthesis from glucose in animal cells and examples of common
adducts and degradation products.
calcitonin, releasing factors for growth hormone, corticotrophin and thyrotropin, pro-
ACTH, vasopressin, oxytocin, cholecystokinin, and gastrin. Petidylglycine ?-amidating
mono-oxygenase (EC 1.14.17.3), which catalyzes ?-amidations, is ascorbic acid dependent
(54) and found in secretory granules of neuroendocrine cells in the brain, pituitary,
thyroid, and submaxillary glands. The activation process involves C-terminal amidation
and release of glyoxylate (Fig. 5) and requires copper (53).
4. Ascorbic Acid as an Antioxidant
The Food and Nutrition Board’s panel on Dietary Antioxidant and Related Compounds
of the NAS has defined an antioxidant as ‘‘any substance that, when present at low concentrations
compared to those of an oxidizable substrate (e.g., proteins, lipids, carbohydrates,
and nucleic acids), significantly delays or prevents oxidation of that substrate’’ (55).
Ascorbic acid readily scavenges reactive oxygen and nitrogen species, such as superoxide
and hydroperoxyl radicals, aqueous peroxyl radicals, singlet oxygen, ozone, peroxynitrite,
nitrogen, dioxide, nitroxide radicals and hypochlorous acid. Moreover, ascorbic acid supplementation
has been associated with reduced lipid, DNA, and protein oxidation in experimental
systems (23,24).
538 Johnston et al.
Table 3 Functions of Ascorbic Acid Associated with Specific Enzymes
Associated mechanism
Function Associated enzyme(s) and features
Extracellular matrix maturation Prolyl-3-hydroxylase Dioxygenase; Fe2
(Collagen biosynthesis) Prolyl-4-hydroxylase
Lysyl hydroxylase
Cq1 complement synthesis Prolyl-4-hydroxylase Dioxygenase; Fe2
Carnitine biosynthesis Dioxygenase; Fe2
6-N-Trimethyl-l-lysine hydroxylase
?-Butyrobetaine hydroxylase
Pyridine metabolism Pyrimidine deoxyribonucleoside Dioxygenase; Fe2
Hydroxylase (fungi)
Cephalosporin synthesis Deacetoxycephalosporin C Dioxygenase; Fe2
Synthetase
Tyrosine metabolism Tyrosine-4- Dioxygenase; Fe2
hydroxyphenylpyruvate hydrolase
Norepinephrine biosynthesis Dopamine-?-monooxygenase or Monooxygenase; Cu1
hydrolase
Peptidylglycine-?-amidation in Peptidylglycine-?-amidating Monooxygenase; Cu1
the activation of hormones Monooxygenase
Lipids. Although mostly inferential, numerous in vitro and in vivo studies have examined
on the ability of ascorbic acid to reverse lipid peroxidation (61–69). When lipid peroxyl
radicals are generated in lipoprotein fractions, vitamin C is consumed faster than other
antioxidants, e.g., uric acid, bilirubins, and vitamin E (56–63). Ascorbic acid was 103
times more reactive than polyunsaturated fatty acids in reacting with peroxyl radicals (63),
but ascorbic acid was not as effective in scavenging hydroxyl or alkoxyl radicals.
In assays for lipid peroxidation, low density lipoprotein (LDL) particles are often
used as the lipid source (56–63). LDL oxidation is estimated by the lag time and propagation
rate of lipid peroxidation in LDL exposed to copper ions or other catalyst. Numerous
studies in smokers, nonsmokers, hypercholesterolemic subjects or healthy controls, as well
as in animal models, support the observation that ascorbic acid generally retards peroxyl
radical formation in LDL. The data are particularly convincing when ascorbic acid is
combined with vitamin E. In studies utilizing only vitamin C, however, the effect on LDL
oxidation is more varied (see Ref. 65 for excellent summary of work in this area).
Fig. 4 Interaction between ascorbic acid and glutathione in its reduced (GSH and oxidized) forms
(GSSG).
Ascorbic Acid 539
In vitro, both ascorbic acid and vitamin E may display pro-oxidant properties and
promote lipid peroxidation (66). Ascorbate and tocopherol radicals at the surface of LDL
particles are capable of initiating free-radical reactions (66). However, in the complexity
of biological systems such as serum, vitamin C does not appear to have pro-oxidant effects
(67).
DNA. In somatic cells, oxidative damage to DNA increases the risk of mutations, and
in turn, is a risk factor in cancer or birth defects. Deoxyguanosine is a common target
of oxidative modifications with 8-oxoguanosine and its respective nucleoside, 8-oxodeoxyguanosine
as products. These modified nucleic acid bases are found in all cells and
urine (68–72). Based on measurements of 8-oxo-6-deoxyguanosine derivatives and other
modified bases, tissues appear to have the potential to repair 104 to 105 oxidative lesions
per cell per day.
Fraga et al. (73,74) were among the first to demonstrate a significant decrease in
human sperm 8-oxo-deoxyguanosine levels following vitamin C supplementation. However,
the nucleus of many cells contains relatively low concentrations of ascorbic acid,
and whether ascorbic acid serves to protect DNA from oxidative damage remains unresolved
(75–77).
Protein. Examples of protein oxidation include the life-long oxidation of long-lived proteins,
such as the crystallins in the lens of the eye, the oxidation of ?-proteinase inhibitor,
and the advanced glycation end products associated with diabetes. Tyrosine, N-terminal
amino acids, and cysteine are often targets of such reactions (78–81). Although data are
limited, ascorbic acid supplementation may protect some proteins from oxidation. Protein
carbonyl formation is reduced upon ascorbic acid repletion in guinea pigs (82).
Fig. 5 Reactions catalyzed by mono- and dioxygenases for which ascorbic acid and iron or copper
are catalyst. Reaction sequences for hydroxyprolyl synthesis, norepinephrine synthesis, and Cterminal
amidation formation are indicated.
540 Johnston et al.
Fig. 6 Steps in carnitine biosynthesis.
Vitamin C supplementation has also been shown to reduce to formation of nitrotyrosine
levels in patients with Helicobacter pylori gastritis (81).
5. Carnitine Biosynthesis
It has been suggested that early features of scurvy (fatigue and weakness) may be attributed
to carnitine deficiency (83). Ascorbate is a cofactor of two-enzyme hydroxylation in the
pathway of carnitine biosynthesis, ?-butyrobetaine hydroxylase and ?-N-trimethyllsine hydroxylase
(84, Fig. 6). High doses of ascorbic acid in guinea pigs fed high-fat diets enhanced
carnitine synthesis (85), and ascorbate deficiency was associated with up to a 50%
decrease in heart and skeletal muscle carnitine compared to control guinea pigs (37,83,86).
6. Collagen Assembly
The role of ascorbic acid in extracellular matrix (ECM) regulation is fundamental to understanding
the physiology of scurvy and is addressed in Chapter 16. In brief, ascorbic acid
maintains iron in the reduced state, enhancing prolyl and lysyl hydroxylase activity and
hence collagen assembly (88, Fig. 5).
IV. ASCORBIC ACID METABOLISM AND REGULATION
Many cells accumulate ascorbic acid against a concentration gradient. Intracellular concentrations
of ascorbic acid are up to 40-fold higher than plasma concentrations (89, Table
4). When activated, neutrophils accumulate ascorbate with intracellular ascorbic acid levels
that range from 2 mM to as much as 10 mM. Although simple diffusion accounts for
some of this movement, ascorbic acid transport is primarily carrier-mediated (90–93).
Ascorbic acid enters cells on a sodium- and energy-dependent transporter, and with the
exception of intestine, simple diffusion is of minor importance.
Most of the intracellular ascorbate, however, is derived from rapid conversion of
dehydroascorbic acid (20,94). Dehydroascorbic acid enters cells on GLUT 1, 2, or 4 transporters,
and this transport is inhibited by glucose in vitro. Of clinical significance, it has
Ascorbic Acid 541
Table 4 Tissue Concentrations of Ascorbic Acid
Tissue Rat (mg/100 g) Human (mg/100 g)
Adrenal glands 280–400 30–40
Pituitary gland 100–130 40–50
Liver 25–40 10–16
Spleen 40–50 10–15
Lungs 20–40 7
Kidneys 15–20 5–15
Testes 25–30 3
Thyroid 22 2
Thymus 40
Brain 35–50 13–15
Pancreas 10–16
Eye lens 8–10 25–31
Skeletal muscle 5 3–4
Heart muscle 5–10 5–15
Bone marrow 12
Plasma 1.6 0.4–1.0
Saliva 0.07–0.09
been postulated that diabetics may have compromised ascorbic status due in part to the
inference by glucose of dehydroascorbic acid uptake (93,94).
V. CLINICAL FEATURES INFLUENCED BY CHANGES IN ASCORBIC
ACID STATUS
A. Defining Ascorbic Acid Status
Although leukocyte concentrations are generally considered to be the best indicator of
vitamin C status (95,96), the measurement of leukocyte vitamin C is technically complex
(95,96). The varying amounts of ascorbic acid in differing leukocyte fractions and the
lack of standardized reporting procedures (95–99) also complicate interpretation of data.
Hence, the measurement of plasma vitamin C concentration is currently the most widely
applied test for vitamin C status. Plasma concentrations between 11–28 µmol/L represent
marginal vitamin C status. At this level, there is a moderate risk for developing clinical
signs of vitamin C deficiency due to inadequate tissue stores of the vitamin.
Data from the Second National Health and Nutrition Examination Survey, 1976–
1980, (NHANES II) (99) indicated that the prevalence of vitamin C deficiency (plasma
vitamin C concentrations less than 11 µmol/L) ranged from 0.1% in children (3–5 years
of age) to 3% in females (25–44 years of age) and 7% (in males 45–64 years of age). A
decade later, the more sensitive measures utilized by NHANES III indicated that the prevalency
of vitamin C deficiency was 9% for adult females and 13% for adult males (100).
Marginal vitamin C status (plasma vitamin C concentrations greater than 11 µmol/L and
less than 28 µmol/L) was noted in 17% of adult females and 24% of adult males (100).
Smokers are more likely to have marginal vitamin C status compared to nonsmoking
adults. Several studies suggest that smokers required over 200 mg vitamin C daily to
maintain plasma vitamin C concentrations at a level equivalent to nonsmokers consuming
542 Johnston et al.
60 mg vitamin C daily (cf. ref. 101 and references cited therein). The current vitamin C
recommendation for smokers is about 40% greater than that for nonsmokers, 110 and 125
mg daily for females and males, respectively (102).
In its most extreme form, scurvy is characterized by subcutaneous and intramuscular
hemorrhages, leg edema, neuropathy and cerebral hemorrhage, and, if untreated, the condition
is ultimately fatal. Presently, even in affluent countries, scurvy should be considered
when cutaneous and oral lesions are observed, particularly in alcoholics, the institutionalized
elderly, or persons who live alone and consume restrictive diets containing little or
no fruits and vegetables. Patients may also complain of lassitude, weakness, and vague
myalgias, and seek medical attention following the appearance of a skin rash or lower
extremity edema. Below are descriptions of the functional consequences of ascorbic acid
deficiency, and represent the signs and symptoms of scurvy.
B. Clinical Features Influenced by Changes in Ascorbic Acid Status
1. Immune Function
Since the publication of Vitamin C and the Common Cold (103) by Pauling, the role of
vitamin C in immune function has been the topic of lively debate. The high concentration
of vitamin C in leukocytes and the rapid decline of vitamin C in plasma and leukocytes
during stress and infection has been used as evidence that vitamin C plays a role in immune
function (103). Descriptive data in vitro suggest that vitamin C functions as an antiviral
agent (106,107), presumably by aiding in the degradation of phage/viral nucleic acids
(108). Intracellular HIV replication in chronically infected T-lymphocyte cell cultures is
also inhibited by ascorbic acid. Ascorbic acid suppresses reverse transcriptase activity
(106), as may be the case for a number of strong reducing agents.
Dozens of clinical trials have been conducted attempting to resolve the role, if any,
of vitamin C in common cold infections (see 104,105). Ultramarathon runners often suffer
upper respiratory tract (URT) infections in the immediate post-race period (109). In a
prospective double-blind, placebo-controlled trial, vitamin C supplementation (600 mg/
day), beginning 21 days prior to the start of the race, reduced the incidence of post-race
URT infections by 50%. Sedentary control subjects were not affected by vitamin C supplementation,
although the duration of symptoms was significantly less in control subjects
receiving vitamin C supplements, i.e., 1-2 days. Others have also reported a decrease in
the duration of URT infections in subjects consuming 1 g vitamin C daily (105). In addition,
elderly patients hospitalized with acute respiratory infections (receiving 200 mg vitamin
C daily) have been shown to fare better than patients receiving placebo (110).
The mechanisms by which vitamin C reduces the severity of URT infections are
not well established, but must be related to any number of redox sensitive signals and
sites associated with enzymes and receptors. Further, reducing agents and proton donors
are needed to drive the activation of phagocytes. Vitamin C can also accelerate the destruction
of histamine, a mediator of allergy and cold symptoms in vitro (111–113). Vitamin
C supplementation consistently reduces blood histamine concentrations 30% to 40% in
adult subjects (111–112). An acute dose of vitamin C (e.g., 2 g) can also reduce bronchial
responsiveness to inhaled histamine in patients with allergy (113). Thus, the antihistamine
effect of vitamin C may attenuate the severity of symptoms associated with respiratory
tract infections.
Ascorbic acid can also influence neutrophil chemotaxis (114–118); however, enhancement
occurs only at nonphysiological concentrations of vitamin C. Neutrophil cheAscorbic
Acid 543
motaxis is enhanced following an intravenous injection of 1 g ascorbic acid (118) and
following high doses of ascorbic acid (2 or 3 g/d for one or more weeks) (119,120).
Ascorbic acid supplementation (1 g/d) has also been shown to significantly improve leukocyte
chemotaxis in patients with Chediak-Higashi syndrome, a disease characterized by
impaired neutrophil microtubule assembly (121,122) and patients with chronic granulomatous
disease, a condition characterized by depressed neutrophil activation (123). In guinea
pigs, vitamin C deficiency does not affect neutrophil activity, but it does decrease neutrophil
killing of internalized pathogens (124).
Data regarding the effect of vitamin C on lymphocyte proliferation in vitro are equivocal.
Vitamin C depletion for a nine-week period in human subjects did not appear to
alter T-cell number or T-cell proliferation in vitro (125). In a carefully controlled, metabolic
depletion-repletion study, vitamin C ingested daily in amounts ranging from 5 to
250 mg for 92 days did not affect mitogen-induced lymphocyte proliferation in vitro (126).
However, a delayed hypersensitivity skin response to seven recall antigens was suppressed
in subjects receiving the lower levels of vitamin C. Repletion with vitamin C (60 or 250
mg/d) improved responses in about half of the subjects. In separate studies, mitogeninduced
lymphocyte proliferation in vitro was significantly enhanced in subjects ingesting
high levels of vitamin C (2-3 g daily) (120,127).
Vitamin C may also influence other immune system parameters. Vitamin C status
in guinea pigs is directly related to serum concentrations of the complement component
Clq, a protein that, in association with other complement proteins, mediates nonspecific
humoral immunity (128,129). Finally, several investigators have suggested that the antihistamine
effect of vitamin C may indirectly enhance immunoresponsiveness. Ascorbic acid
enhances mitogen-dependent lymphocyte blastogenesis by inhibiting histamine production
in spleen cell cultures (130).
2. The Progression of Selected Chronic Diseases
Atherosclerosis. Epidemiologic studies have shown that death due to cardiovascular disease
is inversely related to regular use of vitamin C supplements (65,131). Males with
vitamin C deficiency (plasma vitamin C  0.2 mg/dL) were at significantly increased
risk of myocardial infarction after controlling for potentially confounding variables (131).
The oxidation of low-density lipoproteins has been implicated in the etiology of atherosclerosis
and serum lipid peroxides were significantly reduced in patients hospitalized with
acute myocardial infarction after consuming diets rich in vitamin C (132). In smokers,
acute smoking (5–7 cigarettes in 90 min) increased LDL lipid peroxidation twofold; vitamin
C supplementation (1.5 g daily) reversed LDL lipid smoking-induced peroxidation
(133). In rabbits fed atherogenic diets for 11 weeks, the addition of vitamins C and E to
diets significantly reduced the severity of atherosclerotic lesions (134). Since vitamin C
can potentially regenerate vitamin E from its tocopherol radical, the protective effects of
vitamin C supplementation on LDL lipid oxidation may be related to maintenance of
vitamin E.
Cancer. In epidemiologic studies, cancer incidence and deaths appear inversely related
to regular use of vitamin C supplements (65). Diets rich in fruits and vegetables are consistently
related to reduced risk for cancers (65,135–138). Although it is likely that ascorbic
acid may have some limited effect given the complexity of protective mechanism(s), the
relative importance of ascorbic acid in various cancers has yet to be resolved. Ascorbic
acid may simply be a marker for fruit and vegetable consumption (139).
544 Johnston et al.
Cataracts. Epidemiologic studies have also shown that the risk of cataract, particularly
posterior subcapsular cataract, is significantly higher in individuals with moderate to low
blood concentrations of vitamin C (odds ratio, 3.3 to 11.3 after adjustment for age, gender,
race, and diabetes) (140,141). After controlling for potentially confounding variables, including
diabetes, smoking, sunlight exposure and regular aspirin use, taking vitamin C
supplements for 
10 years was associated with reduced risk for early (odds ratio, 0.23;
95% CI, 0.99-0.60) and moderate (odds ratio, 0.17; 95% CI, 0.03-0.87) age-related lens
opacities in women (141). When consumed for less than 10 years little or no association
to cataract formation has been observed.
Bone Density. Vitamin C intake is positively associated with bone mineral density (142–
145). This association is independent of other nutrients correlated with dietary vitamin C,
including vitamin A and ?-carotene. The relationship is particularly strong at high calcium
intakes in postmenopausal women (144,145). Among persons with low vitamin E intake
(6 mg per day) and those with modest vitamin C intake (70 mg per day or less), the odds
of sustaining a fracture were 2–4 times greater for current smokers than for women who
never smoked. Among persons with low intakes of both vitamins, the odds of fracture
were nearly five times greater among current smokers than among women who never
smoked. Compared with those who have never smoked, the odds of fracture were not
increased among smokers who also had high intakes of vitamin E and/or vitamin C, e.g.,
200 mg/day (143–145).
3. Wound Healing and Connective Tissue Metabolism
As discussed, the mechanisms that link ascorbic acid intake to connective regulation and
deposition are related to its role as an enzymatic cofactor (and stabilizing factor) for prolyl
and lysyl hydroxylases.
VI. REQUIREMENTS, ALLOWANCES, AND UPPER LIMITS
Plasma vitamin C concentrations in people who regularly consume vitamin C supplements
are 60–70% higher than those who do not take supplements (75–80 and 45–50 µmol/L,
respectively (146–149). A daily intake of 500–1000 mg is necessary to maintain plasma
vitamin C concentrations at 75–80 µmol/L. The recently revised RDA for vitamin C, 75
mg daily for adult females and 90 mg daily for adult males, represents a 25–50% increase
over the 1989 RDA, 60 mg (102). Exclusively breastfed infants ingest approximately 10
mg vitamin C/kg body weight (149).
Serum vitamin C concentrations in newly captured vervet monkeys range from 100 to
115 µmol/L (150), and rats have serum vitamin C concentrations ranging from 60 to 100
µmol/L (150). These data indicate that high tissue levels of vitamin C are well tolerated in
mammalian systems. Approximately 70% of a 500 mg dose is absorbed. However, much of
the absorbed dose (50%) is excreted unmetabolized in urine.With a dose of 1250 mg, only
50% of the dose absorbed and nearly all (85%) of the absorbed dose is excreted (151 and
references cited).These factors support the contentionthat ascorbic acid is relativelynontoxic.
A. Rebound Scurvy
There is some evidence that accelerated metabolism or disposal of ascorbic acid may
occur after prolonged supplementation of high doses. Presumably, when vitamin C suppleAscorbic
Acid 545
mentation ceases abruptly, the accelerated disposal of vitamin C creates a vitamin Cdeficient
state, i.e., ‘‘rebound scurvy.’’ The concerns regarding rebound scurvy come
largely from work by Cochan (152). Of 42 cases of infantile scurvy at Children’s Hospital
in Halifax, Nova Scotia (from October 1959 to January 1961), two could not be attributed
to inadequate dietary vitamin C. The possibility of rebound scurvy was considered, because
the mothers of both of the infants reported taking vitamin C supplements during
pregnancy, i.e., 400 mg of vitamin C daily. An intake of 400 mg per day is not remarkable
and is currently consumed by 5–10% of the U.S. population. In guinea pigs fed diets
containing 0.1% vitamin C by weight, intraperitoneal administration of vitamin C (1 g/
kg body weight per day for four weeks) was associated with an increased rate of vitamin
C turnover (153). The mean plasma vitamin C concentrations fell significantly below
control values during the second and fifth week following the abrupt withdrawal of vitamin
C treatment; however, plasma vitamin C concentrations measured after vitamin C treatment
remained within normal ranges. Thus, the extent to which ‘‘rebound scurvy’’ actually
occurs may be exaggerated.
B. Oxalic Acid and Uric Acid
About 75% of kidney stones contain calcium oxalate; another 5–10% are composed of
uric acid. High doses of vitamin C have been shown to increase urinary excretion of both
oxalic acid and uric acid; and thus, theoretically promote the formation of kidney stones
(154–161). However, calculi may form in the absence of hyperoxaluria or hyperuricosuria,
and conversely, patients with hyperoxaluria or hyperuricosuria often do not form stones.
Unrelated factors, such as a lack of inhibitors of crystal formation in urine, changes in
urinary pH, decreased urine volume, and the presence of bacteria, all influence the risk
for stones.
Chronic daily ingestion of 1000 mg vitamin C increased urinary uric acid by 
30%
(155). The physiologic relevance of this moderate rise in urinary uric acid is not known.
More than half of patients with urate calculi do not have hyperuricemia or hyperuricosuria;
rather, they have a tendency to excrete acidic urine. Although ascorbic acid may be prescribed
as a urinary acidifying agent, this property is more accurately described as preventing
the alkalization of urine. Vitamin C supplementation (1–6 g/day) had little effect
on urinary pH in subjects with nonalkaline urine (157).
Levine et al. (161) demonstrated that mean urinary oxalate was not significantly
affected by the chronic ingestion of 200 or 400 mg vitamin C in seven healthy young
men. Although chronic ingestion of 1000 mg vitamin C did cause a rise in urinary oxalate,
the mean oxalate concentration remained within the reference range. Early reports that
attempted to connect vitamin C intake to urinary oxalate often used methods that permitted
oxalate generation from the ascorbate present in urine. (Oxalate is an alkaline degradation
product of ascorbic acid (159).) In preserved urine samples, no significant increase in
oxalate excretion is usually noted.
Epidemiologic data do not support an association between vitamin C supplementation
and kidney stones. In the Harvard Prospective Health Professional Follow-Up Study
involving over 45,000 men from 40–75 years of age, 751 incident cases of kidney stones
were documented over a six-year period (160). The age-adjusted relative risk for men
consuming 1500 mg or more vitamin C/day compared with those consuming less than
251 mg/day was 0.78 (95% confidence interval, 0.54–1.11).
546 Johnston et al.
C. Iron-Related Disorders
Red cell hemolysis, related to glucose-6-phosphate dehydrogenase (G6PD) deficiency, has
been reported as a toxic effect of ascorbic acid (162). Although the mechanism is not
clear, it is possible that ascorbic acid in excess can act as a pro-oxidant catalyst in the
presence of available iron and the absence of an important source of reducing equivalents
in red cells, i.e., the NADPH that is generated by G6DP.
Enhanced iron absorption has been associated with ascorbate intakes (163–168),
and a significant relationship between serum ferritin and dietary vitamin C in elderly has
been shown (166). In iron-depleted young adults, vitamin C supplementation (e.g., 500
mg at two–three times per day with meals) raised apparent iron absorption from a single
test meal 30–40% (164,165). Vitamin C in meals in the range of 50–100 mg had a signifi-
cant effect on improving iron absorption. However, higher intakes of vitamin C had little
further effect. If hemochromatosis is recognized, a high ascorbic acid intake may not be
prudent. However, omitting or reducing dietary iron sources, e.g., meat consumption,
would be more effective.
D. Vitamin B-12
Herbert et al. (169) reported that patients who received ascorbic acid in high doses had
low serum vitamin B12. These data have not been replicated by others, and this concern
has never been confirmed. The long-term use of supplemental vitamin C is not likely to
affect serum vitamin B12 concentration (170).
VII. SUMMARY
Ascorbic acid is the cell’s universal reducing agent. It is a dietary essential for humans
and several other species that have mutations in the gene for l-gulonolactone oxidase.
Ascorbic acid maintains specific enzyme activities, notably the hydroxylase enzymes involved
in collagen assembly and carnitine biosynthesis. In the promotion of antioxidant
defense, ascorbate metabolism is linked to the metabolism of glutathione and, probably,
?-tocopherol.
Ascorbic acid deficiency results in reduced mono- and dioxygenase activities. The
consequences of severe deficiency are profound, since growth, extracellular matrix, and
hormonal regulation are all impaired. Low intakes of ascorbic acid may accentuate and
exacerbate chronic disease, e.g., atherosclerosis and those for which defective immune
responsiveness is a component. Recently, the RDA for vitamin C was set at 75 mg daily
for adult females and 90 mg daily for adult males, a 25–50% increase over the previous
recommendation. Gram doses (1–2 g/day) of vitamin C are well tolerated by most individuals.
A number of studies suggest that optimal health benefits are achieved at intakes of
100–200 mg/day.
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16
Ascorbic Acid Regulation of
Extracellular Matrix Expression
JEFFREY C. GEESIN
Johnson and Johnson, Skillman, New Jersey
RICHARD A. BERG
Collagen Corporation, Palo Alto, California
I. INTRODUCTION
Ascorbic acid is an essential cofactor in many biochemical reactions. Several of these
reactions demand that ascorbic acid provide electrons, either directly or indirectly, to enzymes
requiring prosthetic metal ions in a reduced form for activity. These reactions are
accelerated by ascorbic acid but do not involve its direct participation. Included in this
group of reactions are the hydroxylation of proline and lysine residues found characteristically
in collagen, but also in elastin, C1q of complement, and acetylcholinesterase. The
role of ascorbic acid in the synthesis of these proteins is thought to occur in these hydroxylation
reactions.
Recently, the regulation of these proteins has been investigated because of results
indicating that ascorbic acid regulates their expression by regulating transcription of the
relevant genes. The mechanisms responsible are different from ascorbate’s function as
antioxidant in known hydroxylation reactions. In the following discussion, the effect of
ascorbic acid on the regulation of the synthesis of extracellular matrix (ECM) and ECMassociated
proteins will be described and potential mechanisms that regulate their synthesis
discussed.
555
556 Geesin and Berg
II. ASCORBIC ACID EFFECT ON EXTRACELLULAR MATRIX
PRODUCTION IN VITRO
Collagen, which is the predominant structural protein in animals, is found in a large number
of tissues, including bone, cartilage, tendon, and skin. It is also the predominant protein
produced by human dermal fibroblasts, which incorporate the molecule into the ECM
found in the dermis. At least 18 types of collagen have been identified to date, with more
being described (1). These collagen types are often subdivided into subgroups based on
common characteristics (2). Prominent in the subgroups are the fibrillar collagens, which
include types I, II, III, V, and XI (1–7). The fibrillar collagens are characterized by the
triple-helical ropelike structure without interruptions of the unique collagen amino acid
sequence (8). Type I collagen is composed of two identical polypeptide chains, designated

1(I), and one 
2(I) protein. Types II and III collagen are composed of three identical
proteins, whereas types V and XI collagen are composed of three different proteins (2,8).
Formation of the triple helix in these molecules requires that the helical region have the
sequence of glycine-x-y where every third amino acid is glycine and many of the other
residues are prolines. For the three peptides to form a stable helix, some minimum number
of prolines must be hydroxylated (9–12). This hydroxylation is produced by the enzyme
prolyl hydroxylase in the presence of ferrous ion, ascorbic acid, and ?-ketoglutarate (8,13–
15). The exact mechanism of this reaction is thought to include the production of an active
oxygen species, possibly a superoxide radical, at the active site (16–19). Ascorbic acid
functions in this reaction as an antioxidant that keeps the supply of iron molecules in the
ferrous (2) state, which appears to be a requirement for hydroxylation (18,20,21).
Elastin is another ECM protein that, like collagen, contains hydroxyproline; however,
unlike collagen, these hydroxyproline residues represent a much smaller portion of
the elastin molecule and have not been implicated in the cellular control of elastin secretion
(22,23). Elastin is present in the ECM of a large number of tissues, but it is most prominent
in tissues that require elasticity and are repeatedly forced to deform as part of their function.
Tissues containing large amounts of elastin include lung, large blood vessels, and
skin (22). Elastin is the major component of elastic fibers, which are the functional units
providing elasticity to tissues (24). These fibers are complex structures composed of micro-
fibrillar proteins, proteoglycans, and lysyl oxidase in addition to elastin.
Along with its role in proline hydroxylation, ascorbate and related analogs have
been shown to stimulate the rate of synthesis of collagen by a number of different cell
types, including human dermal (25–51) and lung (52) fibroblasts, bovine (53) and rat aortic
smooth-muscle cells (54,55), rat perisinusoidal stellate cells (56), rabbit chondrocytes (57)
and keratocytes (58), mouse (59) and porcine (60) osteoblasts, mouse BALB (61) and L1
(62) 3T3 cells, chick fibroblasts (61), neural crest cells (63), chondrocytes (64), tendon
cells (65–72), and chick tibia organ culture (73,74). This process occurs with no change
in the rate of degradation of the protein (37) but involves increased transcription of the
collagen peptides (45,47,48,55,66). In addition, the effect of ascorbic acid on collagen
synthesis appears to be independent of cell density or serum concentration, and does not
involve any change in the intracellular proline pool or the rate of incorporation of radioactive
proline into collagen (46).
Elastin synthesis and accumulation are also affected by ascorbic acid (54,55,75).
Most of the evidence to date indicates that ascorbic acid inhibits the accumulation of
insoluble, matrix-associated tropoelastin, possibly due to an increased level of hydroxyproline
found in elastin after ascorbate treatment without a change in transcription or mRNA
Ascorbic Acid 557
stability (54,55). Therefore, short-term treatment with ascorbate produced a shift in the
partitioning of elastin from an insoluble, matrix-associated form to a soluble, media component
(55). This effect has been demonstrated in both fibroblasts and smooth-muscle
cells (54,55). However, recent evidence indicates that elastin production can be inhibited
at both the levels of transcription and reduced mRNA stability with chronic ascorbic acid
treatment (75).
A number of other molecules have been described that are regulated at the protein
or mRNA level by ascorbic acid. In bovine aortic smooth-muscle cells, the levels of the
minor collagen types V and VI were found to be reduced as a result of ascorbic acid
treatment (53). Type IV collagen was similarly induced coincident with adipose conversion
of 3T3-L1 cells (62). Cartilage maturation, along with inorganic pyrophosphate and
type X collagen secretion, is induced by ascorbic acid (76–78). The production of glycosaminoglycans
and gangliosides was enhanced by ascorbic acid treatment in cultured human
skin fibroblasts (39,79), calf aortic smooth-muscle cells (80), and chick wing mesenchyme
micromass cultures (81). The protein and mRNA levels for the acetylcholine
receptor are induced in L5 muscle cells by ascorbic acid by a mechanism that is not
dependent on changes in collagen synthesis (82–84).
III. POSSIBLE MECHANISMS FOR THE EFFECT OF ASCORBIC ACID
ON MATRIX GENE EXPRESSION IN VITRO
There are a number of lines of evidence pointing to a role of ascorbate in stimulating
collagen synthesis that is independent of its role in hydroxylation. The amount of ascorbic
acid required to produce maximal activity of the prolyl hydroxylase enzyme (0.1 µM)
is at least 100-fold less than that necessary for stimulation of collagen gene expression
(50 µM) (46). In addition, although d-ascorbic acid and d-isoascorbic acid can support
hydroxylation reactions at equimolar concentrations to l-ascorbic acid (85), it takes at
least a 10-fold greater concentration of these analogs to stimulate collagen synthesis maximally
(86). Also, the effect of ascorbic acid on collagen synthesis is sensitive to the age
of the donor, whereas there is no effect of age on prolyl or lysyl hydroxylase activities
in culture (46). This phenomenon involves a stimulation of transcription of collagen genes
(45,47,48,66), which reside on different chromosomes (1), indicating a more direct effect
on gene expression than would be implied by mechanisms involving the role of ascorbate
in hydroxylation. Interestingly, ascorbate treatment produces cellular remodeling of the
rough endoplasmic reticulum (87,88), which may be involved in permitting increased
secretion rates (61,69). In addition, calvarial bones from scorbutic guinea pigs regain normal
prolyl hydroxylase activity with ascorbate treatment without a change in collagen
synthesis (89). In some cells the role of ascorbate in the hydroxylation of collagen can
be replaced by other cellular reductants, but collagen synthesis is still ascorbate-inducible
(37,43,90).
The mechanism by which ascorbic acid stimulates collagen gene expression is unclear,
although several possible explanations have been proposed, including increased transcription
(32,46,66,68), increased mRNA stabilization (66,75), and release of a posttranslational
block (32,46,69). One effect of high doses of ascorbic acid that has recently been
considered as a means of altering collagen synthesis is the capacity to cause lipid peroxidation
in cells (29,34–36,91–98). The mechanism by which ascorbic acid produces lipid
peroxidation has received extensive study (97,99,100) and is outlined in Fig. 1. Ascorbic
acid is potentially capable of assisting Fenton chemistry in the production of hydroxyl
558 Geesin and Berg
Fig. 1 Mechanism of ascorbate-induced lipid peroxidation (99).
radicals (OH•) from molecular oxygen (O2) and ferrous ions (Fe2). Hydroxyl radical is
thought to be the species that extracts a proton from available lipid molecules (LH). This
step, which results in the formation of the first lipid radical (L•), is called initiation. In
the presence of oxygen, these lipid radicals form peroxide radicals (LOO•), which can
react with neighboring lipid molecules to form hydroperoxides (LOOH) and lipid radicals
(L•). Since ascorbic acid has been shown to enhance the decomposition of lipid hydroperoxides
to form alkoxyl radicals (LO•), ascorbic acid may also participate in this process,
referred to as propagation.
Lipid peroxidation induces a wide range of effects. It has been associated with alterations
in second-messenger pathways and cell proliferation (101–109). Also, ascorbic
acid–induced cell membrane alterations have been shown to affect the ability of extracellular
molecules to bind to cell surface receptors, leading to altered responses to those molecules
(110–122). Aldehyde and hydroperoxide products of membrane peroxidation have
been shown to modify or cross-link proteins, sugars, or DNA (123–129). Any of these
changes could be involved in the regulation of collagen synthesis. With this in mind, it
is noteworthy that ascorbate induction of lipid peroxidation (94) follows a concentration
profile consistent with that of ascorbate-induced collagen synthesis (31,43) (Fig. 2) and
that inhibitors of ascorbate-stimulated lipid peroxidation also inhibit ascorbate-induced
collagen synthesis (Table 1).
Several investigators have demonstrated a link between the ability of ascorbic acid
to stimulate both the production of collagen and lipid peroxidation (29,33–36,95,130–
136), whereas only one report has produced results in conflict with this correlation (137).
In addition, the ability of oxidized lipids (136) or aldehyde products of lipid peroxidation
(29,130,131) to stimulate collagen synthesis in a number of cell types indicates that these
products of lipid oxidation may be responsible for mediating the response. The mechanism
by which these products of lipid peroxidation affect collagen synthesis is not clear; howAscorbic
Acid 559
Fig. 2 Effect of ascorbic acid on collagen synthesis and lipid peroxidation in human dermal fibroblasts
(34).
ever, a number of potential mechanisms have been suggested. The histone H1 has been
shown to bind chromatin and regulate gene expression, particularly collagen gene expression.
This binding can be prevented by modification by aldehydes of sensitive sites on
the histone (138). One investigator has demonstrated the ability of malondialdehyde and
oxidative stress to stimulate specifically c-myb expression in hepatic stellate cells. Antisense
RNA to c-myb blocked the stimulation of collagen synthesis by ascorbic acid, indicating
that this protein may be a mediator of the effect of oxidative stress on gene expression
in these cells (139).
Another potential mediating mechanism involves a collagen-specific stress protein
known as Hsp47 (140–148). Hsp47 has also been shown to function as a collagen-binding
560 Geesin and Berg
Table 1 Correlation Between Various Modes of Inhibition of
Ascorbate-Induced Lipid Peroxidation and Ascorbate-Induced Collagen
Synthesis in Cultured Human Dermal Fibroblasts
Measurement Technique
Collagen Lipid
Treatment synthesis peroxidation
Initiation inhibitors
Iron chelators
Dipyridyl Inhibit Inhibit
o-Phenanthroline Inhibit Inhibit
Iron competitors
Cobalt chloride Inhibit Inhibit
Oxygen radical scavengers
Dimethyl sulfoxide No Effect No Effect
Isopropanol No Effect No Effect
Ethanol No Effect No Effect
SOD and catalase No Effect No Effect
PEG-SOD & PEG-catalase No Effect No Effect
Propagation inhibitors
Antioxidants
Mannitol No Effect No Effect
Tocopherol Inhibit Inhibit
Propyl gallate Inhibit Inhibit
Naphthol Inhibit Inhibit
Retinoic acid Inhibit Inhibit
Retinol Inhibit Inhibit
SOD, Superoxide dismutase; PEG, polyethylene glycol
Compiled from previously published experiments (34–36).
chaperone protein located in the endoplasmic reticulum of cells. This heat shock protein
has been shown to respond not only to heat shock, but is also induced by carbon tetrachloride
treatment of animals (146). There have been other reports of the ability of oxygen
radical–producing agents, such as carbon tetrachloride, to stimulate collagen synthesis or
induce fibrosis (149–158). Recently, Hsp47 has been shown to play an unexpected role
in regulating collagen gene expression because antisense RNA for Hsp47 inhibits collagen
gene expression as well as collagen secretion and trafficking (159). Although no link has
been established between the ability of ascorbic acid to induce oxidative stress and increased
levels of Hsp47,it is an interesting possible mechanism that could regulate collagen
gene expression.
A number of other potential, oxygen radical–mediated mechanisms for regulating
collagen synthesis have been proposed. Poly-ADP ribosylation has recently received attention
as a proposed mediator of the oxidative events involved in the stimulation of collagen
synthesis by ascorbic acid. Ascorbic acid has been reported to stimulate the activity of
poly-ADP-ribose synthetase, which has been reported to either inhibit (159) or stimulate
(160–162) collagen synthesis and to inhibit (161,163) or stimulate (159,160,162) prolyl
hydroxylase activity. ADP ribosylation is a posttranslational modification of specific proteins
that has been shown to produce alterations in protein structure and function (164).
Ascorbic Acid 561
Collagen is known to be susceptible to modification or cleavage by oxygen radicals in
solution (165–173) and in vivo (165,174). Consequently, collagen cleavage by ascorbic
acid–induced free radicals could alter cell matrix interactions which have been shown to
be capable of regulating collagen synthesis (175). Finally, the redox state within cells has
been shown to be crucial for determining the activity of some transcription factors (176–
182) and could be a mechanism by which ascorbic acid alters gene expression. Presumably,
changes in oxidation within the cell resulting from ascorbic acid uptake or metabolism
or effect on free-radical formation could alter the function of oxidation-sensitive
transcription factors involved in regulating collagen synthesis.
IV. ASCORBIC ACID EFFECT ON EXTRACELLULAR MATRIX
PRODUCTION IN VIVO
The role of ascorbic acid in the production of ECM synthesis in vivo is also not clearly
defined and involves a number of proposed mechanisms. One factor contributing to the
confusion probably involves the lack of free iron in the circulation under normal conditions.
Since many of the in vitro effects of ascorbic acid are iron-dependent, it is perhaps
not a surprise that these specific phenomena are not reproduced readily in vivo. Since
ascorbic acid was originally discovered as the causative agent in scurvy (218), a disease
that involves deficiencies in matrix production as part of its pathological process, it is
clear that ascorbic acid is crucial for the production of ECM in animals. The most obvious
animal model for studying the role of ascorbic acid would seem logically to be one with
an ascorbic acid deficiency. However, few animals can be made ascorbic acid–deficient.
Most animals synthesize their own ascorbic acid. Only humans, other primates, guinea
pigs, passiformes birds, and flying mammals must obtain it from their diet. This requirement
derives from their apparent inability to catalyze the conversion of l-gulonolactone
to 2-keto-l-gulonolactone due to a deficiency in gulonolactone oxidase (219).
By far the largest literature in this area has been produced in guinea pigs. In scorbutic
guinea pigs, ascorbic acid supplementation was shown to stimulate proline hydroxylation
without increasing collagen production in healing wounds (183). The wounds contained
cells that possessed an increase in the number of membrane-associated polyribosomes
specific for collagen. The decrease in collagen synthesis was later shown to correlate with
weight loss in the affected animals (184) and could be reproduced in fasted, ascorbic acid–
supplemented guinea pigs (185). In addition to reduced collagen synthesis, proteoglycan
synthesis was also reduced in cartilage and was correlated with altered levels of circulating
hormones (186–188). Of particular interest are the levels of insulin-like growth factors
(IGF) and their binding proteins. IGF-I and II are known to be induced during wound
repair (189); however, the circulating levels of two IGF-binding proteins, IGFBP-1 and
IGFBP-2,were elevated in response to fasting and vitamin C deficiency and capable of
inhibiting the action of IGF-I (190–193). These differences in circulating IGFBP levels
do not seem to explain the differences noted in wound repair (194). Ascorbic acid defi-
ciency has also been associated with altered expression of iron-related proteins, which
has been proposed as a potential factor contributing to altered angiogenesis during wound
repair (194–195).
Another in vivo pathological condition that involves alterations in ascorbic acid
utilization is diabetes mellitus. Competition for membrane transport of ascorbic acid in
vitro and in vivo has been demonstrated in a number of cell types, including lymphocytes,
granulocytes, and fibroblasts (196,213). Chronic reduction in ascorbic acid levels also
562 Geesin and Berg
contributed to reduced chemotaxis by affected polymorphonuclear leukocytes and mononuclear
lymphocytes (196). Diabetes mellitus was further shown to be associated with
reduced plasma levels of ascorbic acid without changes in dehydroascorbic acid (197–
199). This altered ascorbic acid metabolism could only be partially reversed by dietary
supplementation of ascorbic acid (198). In addition, although free-radical mechanisms
have been implicated in the pathology of diabetes, specifically microangiopathy (197,200),
supplementation with various antioxidants, such as vitamins E and C and ?-carotene, produced
no reduction in a marker of protein oxidation, namely, glycosylated hemoglobin
(201). Other markers of diabetes, such as fasting glucose levels and cholesterol and triglyceride
levels, did benefit from ascorbic acid supplementation (202), but not in all studies
(203,204). Finally, ascorbic acid supplementation was also shown to inhibit erythrocyte
sorbitol levels, consistent with an inhibitory effect on aldose reductase, an enzyme involved
in the polyol pathway (203,204).
Experimental diabetes was induced in rats treated with streptozotocin. Animals
treated in this way have shown reduced levels of hydroxyproline in nascent type I collagen
isolated from skin (205,206), tendon (207), and periodontum (208). This underhydroxylated
collagen correlated with an increased susceptibility to intracellular degradation (205–
208) and could be reversed by ascorbic acid supplementation (205). As with clinical diabetes
mellitus, streptozotocin-treated rats have deficiencies in plasma ascorbic acid levels
that can be reversed by supplementation with ascorbic acid (211,212). This loss in plasma
ascorbic acid can also be prevented by inhibitors of aldose reductase, consistent with the
observed role of the polyol pathway in regulating ascorbic acid metabolism (211,212).
These changes in collagen production and degradation lead to reduced levels of
collagen deposition in different skin wound healing models (209,210). Specifically,
changes in collagen deposition have been correlated with reduced granulation tissue formation
in steel-mesh cylinders implanted under the skin of diabetic rats, and the granulation
tissue formed is specifically deficient in collagen content (209). This lack of collagen
deposition during wound repair also correlated with altered breaking strength of incisional
wounds placed in the skin of affected rats (210). The results, which indicated impaired
wound repair, have not been addressed to date in clinical studies in diabetic patients;
however, clinical trials in both patients undergoing tattoo removal surgery and patients
with decubitus ulcers have been performed with no obvious benefit from ascorbic acid
treatment (214,215).
Finally, experiments have been conducted to investigate the administration of high
doses of ascorbic acid to otherwise healthy rats (216) or to mice induced to form pulmonary
fibrosis by bleomycin treatment (217). In normal rats, collagen synthesis was unaffected
whereas elastin accumulation was inhibited, consistent with the in vitro effect of
ascorbic acid on elastin synthesis (216). In bleomycin-treated mice, ascorbic acid treatment
did not prevent the effects of bleomycin on prolyl hydroxylase activity or collagen or
elastin deposition (217). Interestingly, there was instead some indication that high-dose
ascorbic acid treatment increased the tissue response to bleomycin (217).
V. SUMMARY
The effect of ascorbic acid on the synthesis of ECM proteins has been studied for decades
and there are still many questions to be answered. It is clear that the previously accepted
mechanism involving the role of ascorbic acid in the hydroxylation of collagen, which
permits appropriate folding and secretion of the molecule, is certainly one of the mechanisms
involved. This mechanism does not explain all of the effects of either ascorbic acid
Ascorbic Acid 563
deficiency or supplementation by high doses produced both in vitro and in vivo. It is also
clear that direct correlations between in vitro and in vivo observed phenomena concerning
the role of ascorbic acid are not often possible due to the effect that other environmental
factors play in regulating cellular and tissue responses to ascorbic acid. Consequently,
additional ongoing research in the area of ascorbic acid will presumably lead to enhanced
understanding of its role in ECM synthesis, degradation, and secretion. Such research is
likely to provide insight into other related mechanisms for regulating matrix synthesis,
such as the role of growth factors and their modulation by free radicals or other forms of
cellular stress. Although much is known about ascorbic acid, there is much to be discovered.
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17
Nutrients and Oxidation: Actions,
Transport, and Metabolism of
Dietary Antioxidants
J. BRUCE GERMAN
University of California, Davis, California
MARET G. TRABER
Oregon State University, Corvallis, Oregon
I. INTRODUCTION
Free-radical–mediated oxidation reactions are ubiquitous in biological cells and fluids.
Oxidation reactions that follow free-radical pathways are vital to cells in several areas:
(a) Energy metabolism; the production of energy-rich intermediates that drive most of
life’s processes is largely accomplished by the free-radical transfer of electrons from highenergy
organic substrates to oxygen, forming carbon dioxide and water. (b) Biosynthesis;
many necessary macromolecules are partially synthesized by enzymes that utilize freeradical
pathways to accomplish difficult reactions. (c) Detoxification; many toxic compounds
are converted to inert metabolites using free-radical reactions. (d) Signaling pathways;
various inter- and intracellular communication systems utilize the very rapid and
energetic reactions of free-radical chemistry to produce oxidation products as signals,
second messengers, and autocoids. However, oxidation reactions are also potentially catastrophic.
With the ability to produce devastating cellular and extracellular damage, freeradical
reactions can destroy virtually all biologically important molecules, and hence
control of free-radical reactions is critical for the existence of life.
Once thought to be solely the deteriorative reactions of dead tissues and organic
molecules, free-radical oxidation reactions are now recognized as important chemical
pathways for a great many constitutive biological processes as well as acute response and
569
570 German and Traber
elaboration of signaling pathways. This recognition of oxidation in the fields of biochemistry
and cell biology has been followed by an appreciation that various nutrients, whose
chemical structures allow them to interact with free radicals, become important in the
regulation and especially the limitation of these oxidative processes. As a result of the
utility and potential destructiveness of free-radical reactions, biological cells have evolved
a genuinely spectacular array of defensive, preventive, responsive, and repair systems to
minimize the damage associated with carrying out free-radical reactions and producing
free-radical intermediates. Not surprisingly, both the extent of free-radical production and
the efficacy of the defense and repair systems are influenced by an individual’s nutritional
status generally and the abundance of redox active nutrients specifically.
This chapter describes the basic chemistry of oxidation reactions and their prevention
via readily oxidized molecules (typically referred to as antioxidants), and discusses
the role of antioxidants in preventing and delimiting these reactions in human cells and
tissues. This chapter reviews the chemistry of lipids as biological molecules unusually
susceptible to oxidation reactions and highlights the potentially devastating effects of these
reactions within certain biological environments, notably the cellular membranes. The
combination of this chemical susceptibility, the solubility properties of lipids, and the
structural diversity of cellular and tissue lipid components demonstrates not only the importance
of the chemical properties of certain antioxidants but also the importance of
biological transport systems that deliver antioxidants to the sites of oxidative reactions in
cells and tissues. The chapter thus summarizes the nutritional importance of both providing
and transporting these antioxidant molecules from the diet to the tissues in which they
accomplish their actions.
II. CHEMISTRY OF OXIDATION
Oxidation is the chemical process by which an atom or molecule (termed the reductant)
with a surfeit of outermost orbital electrons loses an electron(s) to another atom or molecule
such as oxygen (termed the oxidant) that accepts the electron(s) into its orbital sphere
of influence. There are always two participants in an oxidation reaction: the species that
is oxidized and the species that is reduced. The oxidation–reduction reaction requires
neither that the reductant and corresponding oxidant be on separate molecules nor that they
dissociate into separate species after the electron transfer event. Normally, an oxidation–
reduction reaction takes place when one molecule encounters another molecule and the
electrons are transferred from one to another. In organic reactions, a pair of electrons are
transferred. The reaction is considered a free-radical reaction when just one electron is
transferred. This net transfer of electron(s) proceeds spontaneously in the direction in
which the net energy of the overall system is lower. In essence, a ‘‘poor’’ oxidant requires
a ‘‘good’’ reductant and vice versa. In chemical terms, this strength of molecules to undergo
oxidation or reduction reactions is quantified by the oxidation–reduction potential
of the molecules. Oxygen is arguably the most important biological oxidant due to the
substantial energy given up when electrons are transferred from reduced carbon–based
macromolecules to molecular oxygen. The paradox to modern life of humans and other
animals is that the free-radical oxidative reactions involving oxygen are both life sustaining
by being coupled to energy production in the mitochondria of living cells and life threatening
due to the damage and deterioration of living cells that they can cause.
The evidence is compelling that throughout the course of evolution, biology has
found the power of free-radical reactions to be a seductive strategy, and higher organisms
Nutrients and Oxidation 571
use free-radical chemistry to accomplish many necessary biochemical reactions. As a result,
a complex spectrum of biochemical systems that either utilize or detoxify products
of free-radical chain reactions has gradually developed over the course of the evolution
of organisms living in an oxygen atmosphere.
Science is only now beginning to understand the interactions between systems that
generate and those that utilize oxidants. Information about these interactions will eventually
lead to an understanding of where in the chemical process normal reactions begin to
deviate toward pathological reactions and where antioxidants can provide protection
against cell damage and disease.
A. Oxidative Stress
Oxidant production or exposure and antioxidant depletion are general phenomena that
together are described as ‘‘oxidative stress.’’ This is an important concept that is frequently
poorly defined. One can consider appropriate oxidative status as the gradual oxidation of
energy-rich molecules and expenditure of energy consistent with the normal functioning
of cells and tissues. These various oxidative processes invariably produce a finite number
of free radicals whose release and detoxification are rapidly accomplished without any
apparent deleterious effects on the organism. In all cells, radicals are both made and successfully
eliminated on a continual basis. Oxidative stress occurs when either the production
rate increases or the elimination rate decreases, causing free-radical damage to unprotected
and/or non-repairable molecules. Increases in both acute catastrophic events and
natural processes compose the production side of oxidative stress (1). Oxidative stress
induced by chemical toxicity, acute physical injury, or even iron overload (2–4) causes
the production of excessive and frequently unusual free radicals, which results in gross
tissue damage. Also, normal responses to nonradical stresses can increase oxidative stress.
The most obvious example is the release of activated oxygen species by immune cells,
such as macrophages, designed to kill invading pathogens. Although oxidation chemistry
is an endogenously initiated process, it can result in localized damage to surrounding
tissues. In this case, the cost of a defensive strategy that generates chemically toxic oxygen
radicals is balanced by the benefit of protection against the threat of pathogenic infection.
However, the power of free-radical oxidative chemistry to do harm is seen in the devastating
consequences of chronic inflammation of several autoimmune disorders that create
pathological damage due to the constant production of oxidants by the host’s own immune
cells. This seemingly two-edged sword underscores both the protective power of oxidative
chemistry and the risks associated with these naturally evolved biological reactions.
The risks associated with acute oxidant exposure are being increasingly recognized;
however, another factor that is important to oxidant balance is the overall and local concentrations
of antioxidant protectors. Antioxidants are important because normal energetic
metabolism, primarily through electron transport in the mitochondria, results in the continuous
inadvertent release of a significant number of free-radical oxidants. Even in the absence
of an acute oxidant challenge, antioxidant systems are constantly responsible for
delimiting and repairing the cost of doing business, as it were, with free-radical chemistry.
Given the normal production rate of free-radical oxidants, which has been estimated at
106 free radicals released per cell per day (5), the importance of maintaining a substantial
concentration of antioxidant molecules of various types in all tissues is clear.
As will be described further below, many of the antioxidant protectors must be
concentrated in areas of high oxidation and/or the production of active oxidants. This
572 German and Traber
means that both the intake and delivery of antioxidants can be important. Because many
of the protectors at the front line of defense are redox-active molecules that rapidly reduce
damaging radicals to relatively benign radicals, their abundant presence at the sites of
radical generation is important. Such redox-active molecules are typically complex phenolic
molecules. Many of this class of protector molecules are not made in humans and
thus are present in tissues as a function of their abundance in the diet. Vitamins C and E
(ascorbate and ?-tocopherol) are the most obvious examples of redox-active molecules
whose abundance in the tissue sites of interest is critical to the overall balance of oxidative
stress, and for which a deficiency has literally catastrophic consequences in terms of oxidative
damage to tissue.
The notion of a balance between the benefits and risks of free-radical oxidative
chemistry implies that this balance can be shifted by changing the predominance of prooxidative
processes and antioxygenic protection (6). The implications of this relationship
have been tested by mechanistic research, animal trials, and epidemiological studies of
various populations wherein disease incidence has been related to genetic and environmental
variables. The results are in general quite convincing that the balance of oxidation can
be upset with deleterious consequences. The effects of acute oxidative stress or acute
antioxidant deficiency are now accepted by the scientific community. In some cases, an
imbalance in oxidation is accepted as a compelling rationale for recommending antioxidant
therapy for overt pathologies that produce oxidative stress, such as iron overload, ischemia
reperfusion injury, and certain chemical toxicities.
One additional issue emerges from the scientific perspective of oxidant–antioxidant
balance. If acute oxidative stress is ameliorated by antioxidant therapy, is it possible that
prolonged chronic imbalances in oxidative protection may lead to repetitive damage to
sensitive biological tissues? An imbalance in oxidants–antioxidants with an excess of
oxidants would, for example, lead to a buildup of potentially toxic byproducts, which over
long periods leads to loss of function. Such long-term accumulation of oxidant byproducts
is now believed to be an important aspect of chronic and degenerative diseases, such as
coronary artery disease. This possibility has led to a consensus among the research community
that antioxidant nutrients represent one of the nutritional attributes of a diet rich in
plant material, i.e., fruits and vegetables.
The concept of oxidant–antioxidant balance has implications for the etiology of
many chronic diseases. Atherosclerosis is an example of a disease in which oxidative
damage and the molecules that result promote a metabolic dysfunction (7). LDLs become
atherogenic when their basic surface structure is modified and they become substrates
for uptake by macrophages located in the subendothelium of artery walls. An important
mechanism by which this modification is thought to take place is oxidation. Thus, the
oxidation of LDL is a part of the gradual development of atherosclerosis. Antioxidants
retard oxidative modification of LDL, and it has been proposed that part of the value of
diets rich in fruits and vegetables in reducing coronary disease is the provision of antioxidants
as protectors against LDL oxidation.
Another example of a means by which the oxidative balance is inordinate is environmental
insult to antioxidant protection. The elimination of essential protectors, such as
ascorbate, through chemical insult (tobacco smoking) has been shown to increase the
damage to the DNA of sperm (8). The increase in cancer incidence in the offspring of
smoking male parents that was predicted by molecular results has been observed in human
populations whose vitamin C intakes are low (9).
Nutrients and Oxidation 573
The chemistry of free-radical oxidation is highly complex and, due to its aggressive
nature, tends to be nonspecific in both reaction targets and products. While all macromolecules
are targets of oxidative chemistry, the chemistry of lipid oxidation is described below
in detail as this is the area most understood.
B. Free-Radical Lipid Oxidation
The free-radical oxidation of lipids is a complex series of reactions with three distinct
phases: initiation, propagation, and termination. Lipid oxidation should be recognized as
a highly dynamic and complex result of the interplay between substrates, circumstances,
and time (10).
C. Hydroperoxide Formation
During the initiation phase of oxidation, free-radical species are generated on unsaturated
lipids. During the propagation phase, lipid radicals react with oxygen, forming peroxy
radicals that in turn react with more lipids, generating more radicals. The propagation
phase is thus a classic example of an autocatalytic chain reaction. The termination phase
is the point in which two free radicals react with each other, eliminating the free radical
on each.
Thermodynamic equilibrium strongly favors the net oxidation or transfer of electrons
from reduced, carbon-based biomolecules to molecular oxygen. That is, biological
macromolecules, proteins, polynucleotides, carbohydrates, and lipids are all reduced carbon-
based molecules that contain considerably more energy than their constituent atoms
in their more oxidized states, e.g., carbon dioxide. The tendency of virtually all biological
molecules to oxidize is therefore driven by the strong energetic advantage to the reaction
with oxygen. Life is possible because this direct oxidation reaction is so slow at the ambient
temperature of the planet. This so-called kinetic stability of all biological molecules
in an oxygen-rich atmosphere results from the unique spin state of the unpaired electrons
in ground-state molecular (triplet) oxygen in the atmosphere. Whereas most biologically
important carbon-based molecules contain an even number of electrons in which their
electrons are paired in what is referred to as antiparallel arrangement. Oxygen, on the other
hand, contains an even number of electrons, but in its lowest energy state the outermost two
electrons occupy separate orbitals. Thus, the stable form of oxygen is a biradical in which
the outermost two electrons are unpaired. This property renders atmospheric oxygen relatively
inert to direct oxidation reactions with reduced, carbon-based biomolecules. Hence,
even though thermodynamically favored, reactions between oxygen and protein, lipids,
polynucleotides, carbohydrates, and so forth proceed at an insignificantly slow rate. The
kinetic limitation can be overcome by converting oxygen to a nonradical or by converting
carbon-based molecules to radicals, i.e., by removing a single electron from one of their
pairs. The removal of an electron from a carbon-based molecule can be accomplished by
active, single-electron oxidants.
There are various sources of free-radical oxidants, as shown in a table of free-radical
redox half potentials (11). Free-radical oxidants that are important to lipid oxidation are
those that will abstract a single electron from an unsaturated fatty acid and thus have a
half-cell redox potential approaching 1 V. An oxidant that initiates lipid oxidation chain
reactions in polyunsaturated fatty acids (PUFAs) must have sufficient redox potential to
abstract the hydrogen from the most easily oxidized carbon, a methylene interrupted car574
German and Traber
bon (the carbon between two double bonds). An oxidant that can initiate the oxidation of
monounsaturated fatty acids must have a slightly higher redox potential due to the greater
energy required to abstract a hydrogen from a carbon that is alpha to only a single double
bond.
Oxygen in the ground state is a very poor single-electron oxidant, with a half-cell
redox potential of 
0.33 V. Superoxide is a better oxidant but still of insufficient potential
to oxidize unsaturated lipids. Hydrogen peroxide is a poor oxidant; however, in the presence
of reduced metals, it oxidizes a reduced transition metal, such as cuprous or ferrous
ion. The reduction of hydrogen peroxide causes it to split homolytically, yielding two free
radicals. The electron from the reduced metal reduces one free radical, but the other free
radical remains. This free-radical species is the hydroxyl radical. This species has a redox
potential above 2 V. The hydroxyl radical will readily oxidize virtually any organic molecule,
including fatty acids. This is the main reaction pathway that generates powerful
oxidants in cells. The production of hydroxyl radicals then initiates free-radical chain
reactions within polyunsaturated lipids.
Molecules that can remove a single electron from reduced macromolecules, such
as lipids, are potentially very deleterious because as initiators of oxidation they eliminate
the reactive impediments imposed by the spin restrictions of ground-state oxygen. By
converting stable organic molecules, RH, to free-radical–containing molecules, R?, they
initiate chain reactions that continue to oxidize subsequent lipid molecules. Oxygen reacts
readily with the unpaired free-radical species on fatty acids to form the peroxy radical,
ROO?. The peroxy radical contains unpaired electrons and hence is active as a free radical
oxidant that oxidizes other reduced molecules: ROO?  RH > ROOH  R?. Initiators
of lipid oxidation are relatively ubiquitous, primarily single-electron oxidants, and they
include trace metals, hydroperoxide cleavage products, and ultraviolet light. Polyunsaturated
fatty acids are oxidized by the ROO? species to yield another free radical, R?, and
a lipid hydroperoxide, ROOH. This effectively sets up a self-propagating free-radical chain
reaction, R?  O2 > ROO? > ROOH  R?, that can lead to the complete consumption
of PUFAs (12). The ability of the peroxy radical to act as an initiating, single-electron
oxidant drives the destructive and self-perpetuating reaction of PUFA oxidation.
D. Hydroperoxide Decomposition
The reduced products of the immediate reaction of a lipid radical with oxygen, the hydroperoxides,
are not radicals and hence are more stable. However, their chemical formula,
ROOH, is similar to that of hydrogen peroxide, HOOH, and the reactivity of the peroxides
are similar. Both are readily attacked by reduced transition metals, causing a homolytic
cleavage reaction. The decomposition stage of oxidation occurs when lipid hydroperoxides
encounter a transition metal. From lipid hydroperoxide cleavage, the oxygen-based radical
formed is termed an alkoxyl radical, RO. This free-radical species is analogous to the
hydroxyl radical and has similar reactivity. The RO radical is a very strong, single-electron
oxidant and will react with adjacent molecules to abstract an electron, thus oxidizing the
target molecule to a free radical. In this way, hydroperoxide decomposition reactions are
the most important free-radical initiators in most lipid-containing systems. The alkoxyl
radical can also react internally with the lipid molecule itself, leading to a host of decomposition
products (13).
Decomposition of hydroperoxides yields alcohols, aldehydes, ketones, and hydrocarbons
as fragments of the original lipid hydroperoxides. While it is the reactivity of the
Nutrients and Oxidation 575
alkoxy radical that is considered most damaging to cells and other macromolecules, the
other decomposition products of lipid oxidation can also be deleterious. The release of
these oxidative products provides the most discernible property of oxidized lipids, the off-
flavors of rancidity. However, off-flavors are not the only consequences of lipid decomposition
reactions. Aldehydes are weak electrophiles and will react with a variety of biological
nucleophiles. There are many such nucleophiles in cells, including parts of proteins,
vitamins, and polynucleotides. The adducts of these aldehydes with proteins in most cases
render the proteins inactive.
1. Consequences of Lipid Oxidation
Lipid hydroperoxides formed during lipid oxidation decompose to various products, including
short-chain aldehydes, ketones, and alcohols. These products, as well as radicals,
compromise cell and tissue health in a number of ways. (a) The direct oxidation of susceptible
molecules can result in loss of function. For example, oxidation of membrane lipids
alters membrane integrity, promotes membrane leakage, and affects the conformation and
activity of membrane-bound proteins. The direct oxidation of proteins results in a loss or
alteration of enzyme catalytic activity and/or regulation. (b) Reaction of the products of
lipid oxidation with protein or other macromolecules leads to adduct formation with loss
of native function and, in some cases, accession to novel actions. As a well-described
example, the oxidative modification of the apoB protein molecule on LDL by lipid aldehydes
changes its uptake properties completely. The formation of these adducts on apoB
prevents the uptake of LDL by the LDL receptor and stimulates uptake by the unregulated
scavenger receptor. (c) Oxidation can cleave DNA, causing point, frame-shift, and deletion
mutations, as well as base damage. This oxidative cleavage impairs or destroys normal
functionality. (d) Oxidative reactions can liberate signal molecules or analogs that elicit
inappropriate responses, such as activation of platelet aggregation, promotion of cell proliferation,
and down-regulation of vascular relaxation.
The susceptibility and overall rate of oxidation of a lipid molecule is related to the
number of double bonds on the fatty acids. First and foremost, the rate of oxidation is
determined by the ease of hydrogen abstraction. Thus, oxidation of saturated fatty acids
is uncommon because hydrogen abstraction is extremely rare. The addition of a single
double bond renders the alpha hydrogens substantially more susceptible to oxidation. The
formation of a cis interrupted pair of double bonds renders the hydrogens on this carbon
much more susceptible to oxidation. Additional double bonds do not increase the susceptibility
of individual hydrogens to abstraction, but they do increase the number of potential
sites of abstraction and oxidation. These three considerations result in the relative oxidative
rates of fatty acids. An increase in the number of double bonds increases the oxidation
rate for the fatty acids 18:1, 18:2, 18:3, and 20:4 such that the relative oxidation rates for
purified fatty acids are 1, 50, 100, and 200. Diets high in PUFAs require higher than
normal amounts of antioxidant nutrients to prevent oxidation and rancidity. Consumption
of high-PUFA diets by animals increases the antioxidant requirement. A diet enriched in
highly unsaturated fatty acids increases the tendency to oxidation, and this would be predicted
to increase the incidence of oxidation-associated chronic degenerative diseases.
The metabolic effects of PUFA in altering lipoprotein metabolism and lowering serum
cholesterol, for example, has led to their increased incorporation in human diets. This
metabolic benefit does not abrogate their susceptibility to oxidation, however, and the
importance of increasing dietary antioxidants to protect tissues from high intakes of polyunsaturated
fat diets is implicit from their chemical susceptibility.
576 German and Traber
III. BIOLOGY OF OXIDATION: A BALANCE BETWEEN
PRO- AND ANTIOXIDANTS
Organisms need to cascade and amplify chemical signals to develop appropriate responses
to many stressors, including oxidants. Many of these signaling systems are oxidant-generating
pathways, such as the enzymatic systems that oxidize specific PUFA moieties to form
potent signaling molecules called oxylipins. These molecules, including prostaglandins,
leukotrienes, etc., signal a state of stress to adjacent or responsive cells. Enzymatically
produced oxidized lipids act on higher order brain functions such as pain and even sleep.
Oxidation of some protein transcription factors allows binding to ‘‘oxidant response elements’’
within DNA and directly affects its transcription. This cascading proliferation of
oxidized molecules, which accomplishes the tasks of intracellular and multicellular signaling,
also places a burden on oxidant defense systems. These chemical signals clearly coevolved
with the increasingly sophisticated and necessary oxidant repair systems. Perhaps
the presence of these oxidant defenses allowed the proliferation of nonlethal uses of oxidants
in higher organisms.
A. Oxidant Production In Vivo
Oxidation is initiated in cells, tissues, and fluids by a host of chemical and protein, i.e.,
enzyme, factors. Oxidation events, such as reactions involving Fenton chemistry, mitochondrial
electron transport, respiratory bursts, oxygenating enzymes, reductive cleavage
of peroxides, and xenobiotic metabolism, are initiated by organisms because they are either
essential for or beneficial to the success of the organism. As delineated below, the pathogenic
potential for these reactions can be considerable.
1. Metal-Catalyzed Recycling of Radicals
The univalent reduction of hydroperoxides by transition metals, especially ferrous and
cuprous salts, yields a radical: an unpaired electron, as either the alkoxyl or hydroxyl
radical. A highly destructive free radical–initiating system has been shown by the presence
of free metals and oxidized lipids in atherosclerotic plaque (7).
2. Mitochondrial Electron Transport
Mitochondria transfer billions of electrons per day to oxygen in single-electron steps that
eventually converts metabolizable hydrocarbon into carbon dioxide and water. Even with
transfer equal to 99.999% of total electrons transferred, leakage from this system is a
significant source of reactive oxygen species. Release of free radicals has been estimated
to be in excess of 104 free-radical species per cell per day (5). This imperfection of mitochondrial
oxidative coupling is the basis of the mitochondrial damage theory of aging.
3. Respiratory Burst
The immune system has developed an oxygen-reducing system that releases superoxide,
which is toxic to bacteria, to combat pathogenic organisms. Stimulation of this oxidase
system in phagocytes produces a burst of oxygen consumption that stoichiometrically
progresses to production of superoxide radicals. Superoxide leads to the death of bacterial
cells in the immediate vicinity of the immune cells that produce it. The action of superoxide
is not highly specific and can damage host cells as well as invading pathogens. This
class of reactive biochemistry as an immune response is a good example of the overall
cost–benefit equation that is inherent to stress responses in both animals and plants.
Nutrients and Oxidation 577
4. Oxygenating Enzymes
Many, perhaps all, cells respond to external stimuli by liberating PUFAs from their membranes.
Arachidonic acid is a particularly active fatty acid. This fatty acid initiates a signal
cascade that uses enzymatically catalyzed free-radical reactions to produce oxygenated
derivatives of itself that are broadly termed eicosanoids. These molecules produce local
signals that activate that cell and its immediate neighbors, thus acting as part of the cellular
stress response system in higher organisms. Chronic activation or overactivation of this
system constitutes an oxidative stress that produces inflammation and activates additional
response systems.
5. Reductive Cleavage of Peroxides
A general class of enzymes called peroxidases eliminates peroxides as a general detoxifying
mechanism. Certain peroxidases catalyze oxidant production as a result of this reaction.
Peroxidases produce oxidants as a response to pathogen invasion and so are considered
to be additional elements in the killing mechanisms of immune cells. Chronic activation
of the oxidant-producing systems of immune cells constitutes an independent oxidative
stress to many humans suffering from inflammatory diseases, autoimmune diseases, or
chronic infection.
6. Xenobiotic Metabolism
The primary tissue involved in the conversion of toxic chemicals into excretable compounds
is the liver. This conversion is effected by inducible enzymes that are typically
oxidases. As a result, toxin metabolism can and does produce free-radical species. The
poisonous properties of a host of pesticides and other chemical products are now recognized
to result from the secondary byproducts produced during the metabolism of xenobiotics.
The above described use of oxidation by cells and tissues represents a clear risk–
benefit relationship. Such risks are acceptable, at least in the short term, because oxidation
provides a net benefit. The long-term consequences may only be relevant to aging organisms
and poorly defended tissues, both of which occur in humans.
The current scientific view is that many chronic diseases exhibit secondary effects
of unprotected or aberrant oxidation. Oxidant damage accumulating over a lifetime can
greatly influence the health of the individual. Developments in oxidant biology indicate
that antioxidant requirements need to be reevaluated in relation to cellular dysfunction.
The requirement for oxidant defense will vary with oxidant stress. Antioxidant effects
defined under conditions of zero stress are unlikely to be meaningful for any system either
under brief, acute stress (such as viral infections, inflammation, trauma, and exposure to
environmental pollutants) or during states of chronic and sustained oxidant stress (such as
autoimmunity, chronic infection, chronic inflammation, elevated circulating lipoproteins,
diabetes, or mild antioxidant deficiency). Thus, estimated requirements for antioxidant
defense of a population would be better if based on individuals exposed to an ‘‘average’’
or ‘‘typical’’ amount of oxidant stress than based on individuals assumed to lack stress.
However, the concept of what constitutes ‘‘typical’’ oxidant stress is undefined, and such
definition will be difficult because, as described above, a variety of insults can elicit an
oxidant stress both directly and indirectly.
Free-radical reactions are extremely powerful and absolutely necessary for a large
number of biochemical and cellular processes. Cells generate energy, assemble and disas578
German and Traber
semble necessary molecules, signal within and between cells and tissues, and eliminate
pathogens, all using free-radical reactions and reaction products. Biology has evolved a
very complex system of catalysts whose function is to produce free-radical oxidants and
reductants, and all have the potential to promote deleterious reactions if not controlled
and regulated. This chemistry of free radicals was clearly a major evolutionary pathway
in which successful exploitation provided selective biochemical advantage. However, this
proliferation of free-radical generating systems in biology placed a substantial priority on
balancing systems that could control, delimit, and repair the consequences of free-radical
reactions.
B. Antioxidant Control of Oxidation
The chemistry of free-radical oxidations is multistage and complex. Oxidation is not a
single catastrophic event. There is no single initiating oxidant that generates all free radicals;
there are a great many sources of single-electron oxidants. Similarly, there is no
single reactive product of oxidation; there are entire classes of oxidative products, many
of which are either selectively or even broadly damaging. Free radicals and their products
react with virtually all biological molecules, and there is no single defense against all
targets of oxidative damage. Thus, organisms have evolved a spectrum of mechanisms to
prevent or to respond to oxidative stresses and free radicals and their products at one or
more of the many steps of oxidation. With this diversity of oxidative chemistry acting at
a host of biological points, the use of the single term ‘‘antioxidant’’ is misleadingly simple.
Nevertheless, the basic principle that an antioxidant prevents or slows the production or
lowers the duration or minimizes the damage associated with free-radical oxidative reactions
is still accepted. Now, however, it is possible to categorize the types of protection
into several broad classes.
The potential health effects of antioxidant protectors must be considered in the context
of the overall response of living organisms to oxidation. As illustrated by specific
examples below, many complex biochemical pathways have evolved to prevent oxidation,
to delimit the chain reactions of oxidation, and to respond to oxidation once it has occurred.
1. Scavenging of Activated Oxidants
Many free radicals are highly reactive, single-electron oxidants. Since most organic molecules
contain electrons as pairs, the abstraction of an electron from an organic molecule
produces another radical. This is the insidious nature of free-radical reactions, i.e., they
keep going. The ability of certain organic molecules to reduce single-electron oxidants,
and in so doing form a free radical that can no longer act as a strong single electron
oxidant, is the major action of a classic scavenging antioxidant. Primary chain reaction–
breaking antioxidants include vitamin E, coenzyme Q (reduced form, ubiquinol; oxidized
form, ubiquinone), ascorbate, uric acid, polyphenolics, various flavonoids and their polymers,
amino acids, and protein thiols.
There are three key ingredients to a successful scavenging antioxidant. For a freeradical
reaction in which an oxidant, O•, reacts with a target, RH, to produce the reduced
oxidant, OxH, and a new radical, R•, the success of an antioxidant, AH, depends on (a)
its ability to successfully compete with RH for the oxidant, Ox•, (b) the effective concentration
or location of AH at the site of the reaction of Ox• with RH; and (c) the stability of
the radical formed, A•, and the ease with which it reacts as an oxidant with other RH
targets.
Nutrients and Oxidation 579
?-Tocopherol (vitamin E), because of its high biological activity and high tissue
concentrations compared with other forms, is a uniquely successful antioxidant in the
protection of membrane PUFAs. First, ?-tocopherol reacts with the most prevalent free
radical oxidant in membranes, the peroxy radical (ROO•). Even more important to its role
as an antioxidant in membranes is that it reacts with the peroxy radical quite quickly and
hence very competitively with the most abundant target in membranes, PUFA (RH). In
fact, ?-tocopherol is so competitive with PUFAs that one ?-tocopherol molecule per
10,000 PUFA molecules provides effective protection. Second, ?-tocopherols are insoluble
molecules that partition preferentially into the membrane bilayer of cells, precisely
where the abundance of PUFAs renders them particularly susceptible to free-radical chain
reactions. Third, the redox potential of the ?-tocopherol radical (11) is such that the significant
redox couple that reduces it under biological conditions is possibly ascorbate,
which in effect regenerates the ?-tocopherol molecule and produces an ascorbyl radical
that is even more stable than the ?-tocopherol radical and that can itself be further reduced
enzymatically back to ascorbate.
2. Prevention of Oxidant Formation
Compartmentation of Subcellular Reactions and Targets. Oxidative reactions are not
carried out homogeneously within cells or organisms. Mitochondria and peroxisomes are
specialized cellular organelles that limit the generation and transfer of electrons, and these
organelles contain enzymes and free-radical scavenging activities that can dispose of toxic
intermediates and the products of metabolism. This topological separation of toxic reactions
from sensitive targets is an important advantage of multicellular protection; however,
it carries an additional energetic requirement in that energy is required to assemble structures
and concentrate reaction substrates, enzymes, and intermediates. One of the most
deleterious aspects of tissue disruption and necrotic cell death is the loss of this exquisite
cellular topology and the release of reactive catalysts into surrounding tissues.
Inactivation of Reactive Precursors. Several classes of molecules are capable of generating
or propagating free-radical reactions. For most of these molecules that capability depends
on the structures in which they are bound. Transition metals are examples of highly
reactive species that, when free, readily propagate free-radical reactions. However, when
they are bound within specific proteins, all of the potentially reactive metal ligands are
occupied, thus inactivating the metal as an oxidation catalyst. Transferrin and ceruloplasmin
are plasma proteins that not only bind and transport transition metals but actively
sequester iron and copper ions, which are otherwise capable of initiating oxidation, and
inactivate them. Lactoferrin similarly inactivates iron and prevents its participation in redox
cycling reactions.
Reduction of Reactive Intermediates. Higher animals possess enzyme systems that scavenge
active oxygen, including superoxide dismutase, catalase, and several peroxidases.
Other enzymes that detoxify reactive intermediates include catalase, a scavenger of hydrogen
peroxide; glutathione peroxidase, which catabolizes hydroperoxides; and superoxide
dismutase, which reduces superoxide anion. Natural food constituents with antioxidant
activity can also act as free-radical quenchers, antioxidants, and or protectors/regenerators
of other antioxidants. Synergistic (14) and antagonistic effects among mixtures of antioxidant
compounds are possible based on the nature of redox couples formed by compounds
present in tissues. Phenolic antioxidants stabilize some enzymes, enhancing some activities
and inhibiting others.
580 German and Traber
The complexity and interdependence of the systems described above indicate that
oxidative stress could increase requirements for not only direct antioxidants but also for
those nutrients essential for proper up-regulation of oxidant defense and repair mechanisms.
IV. PLANT PHENOLICS
‘‘Antioxidant’’ is a broad classification for molecules that interrupt or decelerate oxidation
reactions either prior to or during a free-radical chain reaction, at initiation, propagation,
termination, decomposition, or the subsequent reaction of oxidation products with sensitive
targets. Antioxidants by definition are protectors against oxidation reactions. Similarly,
antioxidant molecules are heterogeneous, both in chemical structures and in biochemical
actions. Although various antioxidant compounds have known actions and
functions apart from their ability to protect specific targets of oxidation, these functions
are not the subject of this discussion. However, one should keep in mind that various
molecules have dual actions, and the relative importance of these two actions in vivo
depends on the immediate environment and physiological state of the tissue.
Antioxygenic compounds can participate in several of the protective strategies described
above for higher animals. Differences in the chemical reactivity and the physical
location between different antioxidants are not trivial, and the reactivity and location in-
fluence the efficacy of a given compound to act as a net antioxidant or protector. How
different molecules act can also affect the impact of oxidation and its inhibition on biological
function and damage. Plant phenolics vary in their ability to interrupt a free-radical
chain reaction, with functional differences being detectable among different lipid systems,
oxidation initiators, and other antioxygenic components. These chemical differences,
which do not include aspects of biological structure, make it relatively clear that the most
effective protection from oxidation in vivo would be afforded by a mixture of antioxidant
molecules that differ in both chemical and physical properties, rather than by a single,
however excellent, chemical antioxidant.
Certain plant phytochemicals also provide an interesting benefit to cells in addition
to their interaction with oxidation processes in that they induce various repair pathways.
Emerging from both epidemiological and mechanistic studies is the realization that induction
of repair prior to an oxidative or toxic challenge can substantially decrease the damage
associated with that event. ‘‘Forewarned is forearmed,’’ so to speak. Since many oxidative
insults lead to DNA damage and ultimately can promote proliferative disorders, such as
cancer, the ingestion of phytochemicals that induce DNA protection and repair systems
is thought to explain the action of these compounds in slowing the development of some
cancers.
A. Classes of Plant Phenolics
Ostensibly, phenolic molecules in animals, whether essential or not, are derived from
plants. Important examples include phenolic amino acids as well as vitamins K and E.
Plant secondary metabolism produces a host of phenolic molecules that are further modi-
fied and complexed during plant growth, harvesting, and processing. These molecules can
be divided into three overall classes: simple phenolics, flavonoids, and high molecular
weight tannins.
Nutrients and Oxidation 581
1. Simple Phenolics
Simple phenolics consist of various polyhydroxylated phenolics derived from further metabolism
of primarily cinnamic and benzoic acids. The most abundant of these compounds
in plants are chlorogenic, gallic, and caffeic acids. The ability of these and similar compounds
to inhibit potentially deleterious free-radical chain reactions has been recognized
by the food industry for decades. Thus, largely as extracts of the plants in which they are
abundant, plant phenolics have been used to stabilize food materials for much of recent
food processing history. These simple phenols are also building blocks for more complex
molecules, forming polymers with many other classes of plant metabolites. The formation
of more complex molecules may or may not affect their actions as free-radical scavenging
antioxidants.
2. Flavonoids
The flavonoids are an extremely broad class of plant secondary metabolites that are all
related to a single flavone nucleus and are responsible for much of the distinctive coloring
of vascular plants. Flavonoids occur not only in multiple variants due to simple substitutions
of the A, B, or C rings but in complexes with other molecules to form an astonishingly
rich variety of polyphenolic structures (over 4000 different molecules) in the plant kingdom.
Among the flavonoids are morin, quercetin, fisetin, myricetin, kaempferol, apigenin,
and luteolin. They are an integral but highly variable part of the human diet, and their
effects on human nutrition are thus of intense interest and potential importance.
3. Tannins
Tannins consist of highly complexed polymers. The monomeric units of these polymers
consist either of simple phenolics, such as gallic acid, which as polymers are referred to
as the hydrolyzable tannins, or of the flavonoids, which as complex polymers up to 5000
daltons are the condensed tannins. While these compounds have been pursued with intense
interest for their antitumor activities, two perspectives on this class of molecules argue
for some caution in applying optimistic results in animals to humans. While apparently
benign when consumed, the tannins are toxic when injected. Their ability to inhibit protein
synthesis is blamed for liver failures in World War II patients to whom tannins were
applied as open-wound dressings. Their lack of toxicity when ingested is due to their not
being absorbed or metabolized. In fact, selection appears to have responded to the need
to avoid absorbing tannins by evolving modifications in salivary protein production. Both
animals and humans produce a spectrum of conspicuously proline-rich proteins that actively
bind tannins and prevent their absorption. Humans appear to express these proteins
constitutively; however, animals require an exposure to dietary tannins to induce their
production. The pre-exposure to tannins significantly lowers the toxicity of tannins to
animals, implying that these induced proteins are highly protective.
The myriad structures of the polyphenolic phytochemicals from plants dictate not
only their functions to the plants but their functions, biological activities, and ultimately
nutritional benefits and toxicities to humans.
B. Chemical Actions of Phenolics
Polyphenolics exhibit a variety of chemical interactions with biologically important classes
of molecules, particularly with potentially reactive species. Examples of these are summa582
German and Traber
Table 1 Actions of Polyphenolics with Biological Molecules
Action Effect of action
Redox-active Reduce/oxidize essential compounds, i.e., ascorbate
Free-radical scavengers Halt propagation reactions, i.e., catechin reduces peroxy radicals
Active oxygen quenchers Quench singlet oxygen, i.e., quercetin quenches photosensitized
oxidation
Transition metal chelators Chelation and its effects are structure-specific; redox cycling is
both inhibited and stimulated by polyphenols
Form complexes with proteins, Binding to proteins and subsequent effects are structure-
DNA, polycyclic aromatics specific, both antioxidant/pro-oxidant
Flavonoid-DNA complexes retard and promote strand cleavage
depending on structure
React with diol epoxides; complex and lower absorption of
benzpyrenes
Nitrite reactants Compete with amines for nitrosation
rized in Table 1. The phenolic compounds are primary antioxidants that act as free-radical
scavengers (reductants) and chain breakers. The key to any phenolic acting as a freeradical
chain-propagating inhibitor is that the phenolic radical generated as a result of
scavenging a peroxy radical is kinetically stable as a free-radical oxidant. This is a logical
extension of their antioxidant ability, but the actual fate of the various polyphenolic radicals
that could be generated by the insertion of these compounds in a free-radical chain
reaction remains to be determined. Polyphenols include salicylic, cinnamic, coumaric, and
ferulic derivatives and gallic esters. In grapes alone, the following phenolics have been
identified: phenolic acids (hydroxybenzoic, salicylic, cinnamic, coumaric and ferulic derivatives,
and gallic esterols), flavonols (kaempferol and quercetin glycosides), flavan-3-
ols (catechin, epicatechin, and derivatives), flavanonols (dihydroquercetin, dihydrokaempferol,
and hamnoside), and anthocyanins (cyanidin, peronidin, petunidin, malvidin, coumarin,
and caffeine glucosides). In compounds that are derivatives of benzoic and cinnamic
acids as well as flavonoids, the degree and position of hydroxylation are important
in determinating antioxidant efficiency. Evaluation of antioxidative activity of naturally
occurring substances has been of interest; however, there is a lack of knowledge about
their molecular composition, the amount of active ingredients in the source material, and
relevant toxicity data.
C. Biochemical Actions of Phenolics
Largely as a consequence of the chemical interactions described above, phenolics exert
a multiplicity of effects on catalytic systems and structure of biochemical systems in living
cells. Summarized in Table 2 is a small subset of the multiple biochemical pathways that
are influenced by the presence and/or abundance of either crude mixtures of plant phenolics
or highly purified single molecules.
Based on the redox activity and potential protector or antioxidant actions of the
myriad molecules discussed above, it is compelling to argue that these molecules are
valuable as routine dietary constituents and that they constitute an important asset of a
complex diet rich in fruits and vegetables. However, the value of polyphenolics is not
Nutrients and Oxidation 583
Table 2 Acions of Phenolics on Some Biochemical Pathways
Biochemical pathway Effects of phenolics
Redox enzymes Inhibit activity of lipoxygenases, cyclooxygenases, monooxygenases,
cytochromes, etc.
Inhibit activity of proteases
Slow proteolysis; slow digestion
Inhibit bleaching reactions of lipoxygenases and peroxidases
Cellular reductants Sparing action of catechin decreases severity of tocopherol defi-
ciency in animal models
Transcription of proteins Various proteins exhibit selective induction by flavonoids
just nonspecific. One class of molecules is so critical to the protection of biological membranes
that a protein system has evolved to ensure delivery of these compounds to sites
of oxidative requirement. These are the tocopherols, or vitamin E.
V. WHY IS THE PLANT PHENOLIC, VITAMIN E, REQUIRED?
Vitamin E is the collective name for molecules that exhibit the biological activity of ?-
tocopherol (see Chapter 4 and Ref. 15). Vitamin E occurs naturally in eight different
forms: four tocopherols and four tocotrienols, which have similar chromanol structures;
trimethyl (?-), dimethyl (?- or ?-), and monomethyl (?-). Tocotrienols differ from tocopherols
in that they have an unsaturated side chain. In general, all plant phenolics inhibit
lipid hydroperoxide formation catalyzed by metals, radiation, and heme compounds, and
also scavenge peroxy, alkoxy, and hydroxy radicals and singlet oxygen. ?-Tocopherol in
oxidizing lipid systems is spared by flavonoids. If ?-tocopherol is an essential antioxidant
that acts where no other compound can, the sparing effect of nonessential antioxidants
may be one of their most important actions.
A. Vitamin E Deficiency
The free-radical scavenging properties of vitamin E are likely the basis for its essentiality
and the pathologies associated with its deficiency. That the basis for the essentiality of
tocopherols lies in their ability to prevent oxidative damage raises several important nutritional
questions. For example, is there value in consuming higher than simply adequate
levels? Can oxidation be inhibited too much? Can other molecules with similar actions
provide added benefits? Many phytochemicals have been implicated as being capable of
interfering with and inhibiting free-radical chain reactions of lipids, which again raises
the question, What is the function of ?-tocopherol?
In humans, vitamin E deficiency results in a peripheral neuropathy. That is, the
large-caliber axons in the sensory neurons die as a result of free-radical–mediated damage.
This is a progressive disorder with increasing severity as the damage moves up the spinal
cord and into the cerebellum (16). Initially, the disorder causes deficits in sensory perception
with increasing loss of function and, ultimately, death. Patients are often diagnosed
with spinocerebellar ataxia; the symptoms are nearly identical to those of Friedreich’s
ataxia.
Remarkably, animals fed experimental vitamin E–deficient diets are more likely to
display anemia and muscle degeneration, and only at a late stage to demonstrate nerve
584 German and Traber
degeneration. This suggests that the delivery mechanisms are important in determining
the susceptible tissues. In experimental animals, the lack of dietary vitamin E results in
continuous low plasma vitamin E concentrations. In contrast, humans with a genetic defect
in the ?-tocopherol transfer protein (?-TTP) have normal absorption of vitamin E but are
unable to maintain plasma vitamin E concentrations (17,18). As a result, plasma concentrations
fall within hours upon cessation of vitamin E consumption.
B. Delivery Systems—Intestinal Absorption
Vitamin E is present in the diet, largely in fat-containing foods. Not surprisingly, absorption
of this fat-soluble vitamin is dependent on micellarization with bile acids and lipolytic
products. Once taken up into the intestinal cell, vitamin E is incorporated into chylomicrons
that are secreted into the lymph. This is apparently an unregulated process; all forms
of vitamin E are absorbed in proportion to that in the diet. The absorption of other lipophilic
dietary components is likely similar. Thus, absorption of carotenoids or other fatsoluble
vitamins may be dependent on micellarization for intestinal cell uptake and chylomicron
assembly for secretion into the circulation.
C. Delivery to Tissues in the Postprandial State
The mechanisms for delivery of vitamin E to tissues are largely the same mechanisms
that deliver peroxidizable fats. Thus, chylomicrons that contain dietary fat, vitamin E, and
other lipophilic compounds are catabolized in the circulation by lipoprotein lipase. During
the initial pass through the circulation, all of the absorbed forms of vitamin E could reach
peripheral tissues. This is likely an important process for delivery of vitamin E to muscles,
skin, adipose tissue, and perhaps brain. Similarly, there may be distribution of other fatsoluble
nutrients to these tissues as a result of the lipolytic process.
D. Role of the Liver the Antioxidant Defense:
Delivering Antioxidants
Vitamin E serves as an interesting example of how the liver regulates antioxidant defenses.
The intestine appears to efficiently absorb a variety of fat-soluble compounds, allowing
for the potential absorption of molecules with no apparent value and that produce toxicity
only. These molecules ‘‘hitch-hike’’ aboard the chylomicron catabolic system that serves
to deliver the absorbed fatty acids to peripheral tissues. Certainly, some fat soluble molecules,
including vitamin E and perhaps some carotenoids, are also deposited in peripheral
tissues during the clearance of chylomicrons, but because adipose tissue contains ‘‘white
fat,’’ delivery of the fat-soluble compounds largely does not occur during lipolysis.
Chylomicron remnants deliver to the liver a variety of potentially helpful and potentially
toxic compounds. In the transport of vitamin A, the retinal-binding protein serves
to accomplish the distribution from the hepatocytes. This complex subject is considered
in other chapters of this text.
Unlike vitamin A, vitamin E is transported in the plasma in all lipoproteins. The
ready solubility of vitamin E in membranes and the apparent ease of its exchange between
membranes and lipoproteins made it appear likely that vitamin E distribution was a nonspecific
process. Thus, the description of patients who were vitamin E–deficient without
having fat malabsorption were critical to the recognition of a specific vitamin E transport
process (17). Now it is clear that tocopherols are not easily exchanged between memNutrients
and Oxidation 585
branes, and specific protein-based mechanisms are necessary for efficient quantitative
transport of tocopherol between cellular and tissue membranes.
The hepatic ?-TTP (30–35 kDa) was purified from rat (19,20) and human liver
(21,22), and the amino acid sequence of both has been reported (19,22). All of the following
features of vitamin E are required for recognition and transfer by ?-TTP (19,23,24):
(a) the fully methylated, intact chromane ring with a free 6-OH group; (b) the presence
of the phytyl side chain; and (c) the stereochemical configuration of the phytyl tail in the
2R position.
In vitro, purified ?-TTP transfers ?-tocopherol between liposomes and microsomes
(24,25). This ability to recognize ?-tocopherol and to transfer it suggests that ?-TTP is
likely responsible for the export of ?-tocopherol from the liver, and nascent lipoproteins
isolated from perfused monkey livers were preferentially enriched in RRR-?-tocopherol
(26). However, hepatocytes overexpressing ?-TTP were able to export ?-tocopherol even
when lipoprotein synthesis was inhibited by Brefeldin A. Thus, the mechanism by which
?-TTP exports ?-tocopherol from the liver remains unknown.
Once chylomicron remnants deliver vitamin E to the liver, the ?-tocopherol transfer
protein is necessary for its export to the plasma (17,18). Recent discoveries of humans with
vitamin E deficiency–like diseases demonstrate that defects in ?-TTP result in vitamin E
deficiency (27,28). Thus, delivery of tocopherols to the liver is nonspecific, as it is for
many of the fat-soluble compounds ingested. With respect to vitamin E form, delivery to
the liver is nonspecific for the different isomers, but export from the liver is selective. An
exquisite advantage of such a system acting within the liver is that, because of the continuous
recycling of vitamin E with lipoprotein metabolism, the entire plasma pool of vitamin
E is replaced daily, thereby assuring sustained vitamin E concentrations.
E. Liver-Detoxifying Systems
Because of the relative nonselectivity of intestinal absorption of fat-soluble molecules,
the liver becomes the site of clearance of unneeded, unwanted, and potentially toxic compounds.
That excess vitamin E is metabolized to a chain-shortened metabolite, ?-tocopherol,
2,5,7,8-tetramethyl-2(2?carboxyethyl)-6-hydroxychroman, is also known (29). This
metabolite is sulfated and glucuronidated to form a water-soluble product that is excreted
in urine. Excretion of this metabolite increases linearly with the dose of supplemental
vitamin E. Where or how the metabolite is synthesized is unknown, but the liver seems
a likely site.
F. Non-antioxidant Functions of Vitamin E
In addition to its antioxidant function, reported structure-specific effects of ?-tocopherol
on specific enzyme activities or on membrane properties have been reviewed (30). The
most convincing work in this respect is on the suppression of arachidonic acid metabolism
via phospholipase A2 inhibition (31) and on the regulation of vascular smooth-muscle cell
proliferation and protein kinase C (PKC) activity (32–35). Reportedly, ?-tocopherol, but
not ?-tocopherol, prevents the phosphorylation of PKC-?, and this is the mechanism by
which ?-tocopherol decreases PKC-? activity (32).
Interestingly, PKC? inhibition by ?-tocopherol has been demonstrated not only in
smooth-muscle cells but also in monocytes (36) and platelets (37)—three cell types involved
in atherosclerosis. Thus, these observations begin to provide a mechanism for the
586 German and Traber
beneficial effects reported for vitamin E in heart disease and perhaps a mechanism for
the specific vitamin requirement for ?-tocopherol.
VI. CONCLUSIONS
Free radical–mediated redox reactions are a fact of life, especially in an oxygen-rich atmosphere.
Oxidation reactions are essential not only to the continuous transduction of energy
from carbon-rich molecules to cellular processes, but also to the successful responses of
living organisms to potentially catastrophic environmental, toxic, and pathogenic challenges.
However, oxidation reactions are also fundamentally damaging, with the potential
to destroy the functions of ostensibly all biologically important molecules.
The control of free-radical reactions by biological cells and tissues is effected by
overlapping and redundant biochemical processes that prevent excessive oxidant production,
protect susceptible molecular targets, and repair or replace damaged molecules.
However, the key protective molecules as a portion of this overall control are largely
phenolic molecules that are not made by cells but that must be consumed in the diet.
Thus, in the biological battle against oxidation in humans, the quality and abundance of
these phenolic molecules in the diet is an important variable to ongoing and especially
long-term success.
Although not recognized until relatively recently, oxidation is not only an ongoing
chemical insult but is also a highly localized stress. Thus, biological processes that deliver
protective molecules to the sites of oxidation reactions are as important as the presence
of these molecules in the diet. The identification of populations of humans with perfectly
adequate intakes of vitamin E but who suffer from devastating vitamin E deficiency as
they age, solely because they have a defect in the protein that is responsible for successfully
delivering vitamin E to peripheral tissues, emphasizes the importance of this new
dimension to lipid metabolism to the nutritional value of antioxidants and the overall diet.
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the ?-tocopherol transfer protein. Nature Gene 1995; 9:141–145.
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kinase C activity and vascular smooth muscle cell growth by ?-alpha-tocopherol. Biochim
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36. Devaraj S, Li D., Jialal I. The effects of alpha tocopherol supplementation on monocyte function.
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37. Freedman J. E., Farhat J. H., Loscalzo J., Keaney J. F. J. Jr. Alpha-tocopherol inhibits aggregation
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2434–2440.
589
Index
Abetalipoproteinemia, 174
Acetaldehyde, 294
Acetylation reactions, 320
Acetyl-CoA carboxylase, 404, 406
Acetylcholine, 285, 292, 329
conversion from choline, 513–514
Acyl carrier protein (ACP), 206, 318
Acyl-CoA dehydrogenase, 266
Adenosylcobalamin, 467–468
reaction requiring, 476–481
s-Adenosylmethionine, 405, 442–443, 450,
515
Adipose tissue, tocopherol content, 170
Adrenal:
biotin, 408
pantothenic acid, 327, 331
riboflavin, 258
vitamin C, 537
vitamin E, 170
vitamin K, 130
Adriamycin (see also Anthracycline), 258
Aged (see Elderly)
Aging (see also Elderly), 134, 268, 288,
576
Air pollution:
vitamin D, 84
vitamin E, 577
Alanine aminotransferase (ALAT, GPT),
361
Alcohol:
biotin, 400, 403, 411
choline, 519
folic acid, 454
niacin, 217, 219, 235
riboflavin, 259, 262
thiamine, 283, 285, 294, 299, 308
vitamin C, 542
vitamin D, 91
Aldehyde:
oxidase, 353
peroxidation, 558, 575
Alkaline phosphatase, 350–352
Alopecia, 259
biotin deficiency, 397, 410, 414
?-Ketoacids, 288
Amethopterin (see Methotrexate)
Amino acid metabolism, 204, 367, 410,
437–444
p-Aminobenzoic acid (PABA), 429
6-Aminonicotanimide, niacin antagonist,
219
Aminotransferase, 205
Amprolium, 278, 280
Amygdalin (see Laetrile)
590 Index
Anemia:
hemolytic, 150, 174, 261, 308
megaloblastic, macrocytic, 307, 309, 427,
447–449, 483–484, 488
normocytic, hypochromic, 363
niacin deficiency, 236
pantothenic acid, 331
pernicious, 448, 488
vitamin B6 responsive, 377
vitamin E, 174, 583
Anorexia, 280
Antibody production, 330
Antibiotics, 148
Anticoagulants, 120–123, 142–144
Anticoccidial compounds, 278, 280
Anticonvulsant drugs, 91, 409, 410, 454
Antihemorrhagic, 148–149, 115
Antioxidant, 37, 537, 558
ascorbic acid, 539
bioflavanoids, 578
hypothesis, 539
riboflavin, 263
vitamin E, 166, 177, 180
Antithiamin factors, 220
Appetite, 23
Aqua mephyton (see Phylloquinine)
Aquacobalamin, 466
Arachidonic acid, 177, 577
metabolism, 365–366
d-Araboascorbic acid (see d-Erythorbic acid)
Arteriosclerosis, 366
Ascorbate free radical (see also Semidehydro-
l-ascorbate free radical), 530, 539
structure, 531
Ascorbic acid:
antioxidant role, 530, 537–538
gene expression, 557–561
interaction with vitamin E, 131, 179–181,
535, 543
radical, 530, 539
regeneration of tocopherol, 182, 543
riboflavin, 262
thiamin-sparing effect, 284
Ascorbic acid-2-hydrogen sulfate (see also
l-Ascorbic acid sulfate), 536
structure, 536
Aspartate amino transferase (GOT), 353
Aspirin, 544
Ataxia, 301, 410, 412, 583
Atherosclerosis, 240, 543, 572
ATPase, 51
Avidin, 399, 401, 410, 414
Bacterial synthesis (see also Intestinal synthesis):
biotin, 397, 400, 416
thiamin, 276
vitamin K, 131, 133, 145, 147
Bacteriorhodopsin, 28–29
Beriberi, 275, 297, 299
dry, 298
wet, 298–299, 308
Betel nuts, antithiamin, 284–285, 293
Betaine, 514
Bile salts:
riboflavin, 262
vitamin A, 12–13
vitamin D, 60
vitamin E, 170
vitamin K, 128–129, 147
Biliary atresia, 175
Biliary cirrhosis, 89, 175
Biliary excretion, of vitamin K, 133–134
Biliary obstruction, 175
Binding proteins:
see Cellular retinoic acid-binding protein
see Cellular retinol-binding protein
see Cytosolic binding protein
see Folic acid, binding proteins
see Intrinsic factor
see Retinal binding protein
see Retinoic acid
see Retinol binding protein
see Riboflavin binding protein
see Thiamin binding protein
see Tocopherol, binding proteins
see Vitamin D, binding protein
Biocytin, 397, 399, 400, 408
Bioflavanoids, 576, 579
Bios, 397
Biotin:
carboxylases, 402
operon, 404
recycling, 408
sulfone, 400
sulfoxide, 398, 403, 408, 417
Biotinidase, 399, 401, 408, 414
Bisnorbiotin, 398, 403, 409, 417
Bitot’s spots, 9
Black tongue, 214
Blindness, vitamin A, 9, 40
Blood-brain barrier, 280, 286, 402
Bone, 53, 71–72
calcium mobilization, 71–72, 79
choline, 519
Index 591
[Bone]
demineralization, 72, 86
Gla-containing protein, 72, 140–142
growth, 86
growth, effected by biotin, 415
mineralization, 63, 52, 141, 135–138
pain, 32
vitamin C, 544
Bone marrow:
niacin, 236
vitamin K and, 130
Boric acid, 263
Butylated hydroxy toluene (BHT), 148
Burning hands and feet, 242
CaBP (see Calcium binding protein)
Caffeic acid, 581
antithiamin, 284
Caffeine, 224, 262
Calbindin D (see Calcium binding protein)
Calcitonin, 51, 88, 538
Calcium, 51–52, 54, 87, 91
absorption, 70–71, 78–79
mobilization, 72–73, 79, 224
Calcium binding protein (CaBP), 70–72, 79
Cancer:
ascorbic acid, 539, 544
carotenoids, 38–40
choline, 513
esophageal, 235
folic acid, 450
riboflavin, 266
vitamin A, 30–31
vitamin B6, 373
vitamin E, 187
Carbohydrate metabolic index (CMI), 305
2-Carboxyglutamic acid, 136–137
Carboxylase:
biotin dependent, 404–409
vitamin K dependent, 135–138
Carcinogenesis, 187, 232, 264, 513, 520
Cardiovascular disease, 40, 187, 219, 241,
265, 375–376, 543
Carnitine:
acyl transferase, 319, 320–330
biosynthesis, 516, 540
?-Carotene, 2, 5, 18
?-Carotene oxygenase, 18
Carotenoids, 2, 4, 5, 9, 12–13, 18, 37–41,
180–183
Carpal tunnel syndrome, treatment with vitamin
B6, 378
Cartilage, 556
Casall’s collar, 213
Catalase, 179–180, 579
Cataracts, 41, 259, 261, 544
Catecholamine, biosynthesis, 292
Celiac disease (see also Sprue), 128, 174, 455
Cell differentiation, 3, 23–25, 233
Cellular retinoic acid-binding protein
(CRABP), 19
Cellular retinol-binding proteins (CRBP),
16–17, 19
Chediak-Higashi Syndrome, 543
Cheilosis, 261
Chemotaxis, vitamin C and, 543, 562
Chemotherapy, 236, 243, 450
Chlorpromazine, 258, 259, 263
Cholecalciferol, 51, 54
Cholestatic liver disease (see also Biliary
atresia), 174
Cholesterol:
relation to plasma tocopherol, 186–187
niacin, lowering effect, 237, 242
vitamin B6 deficiency, 366
vitamin D synthesis, 59
Choline acetyl transferase (CAT), 514
Chromanol, 166, 167
Chronic granulomatous disease, 543
Chylomicra:
vitamin A, 13
vitamin D, 60–61
vitamin E, 170, 171, 581
vitamin K, 129
Cirrhosis:
biliary, 89
liver, 91
Cis-trans isomerization, 20–21
Cleft palate, 415
Clofibrate, vitamin K, 149
Clotting factors, vitamin K dependent, 134–
140, 142
Clotting time, 115, 144, 147, 175
Cobalamin:
binding proteins, 470–472
ileal receptors for, 472
Cobalaphilin, 471
Cod liver oil, 1, 53, 85
Coenzyme A, 206, 318, 398
Coenzyme, definition, 199
Cofactor, definition, 199
Coffee, antithiamin, 285
Cold, common, 542
Colitis, ulcerative, 175
592 Index
Collagen biosynthesis, 16, 72, 556, 560
Complement component (Clq), 543
Congenital malformations (see also Teratogens),
33
Conjunctivitis, 410
Conjunctival xerosis, 9
Convulsions in infants, 364
Copper, 222, 262, 538
Coprophagy, 145, 448
Corn, bound niacin, 214
Cornea:
vascularization, 261
vitamin A, 9
xerosis, 9
Corrinoids, 465–466
Coumarin (see also Hydroxy coumarin)
120–123
Corticosteroids:
biosynthesis, 538
pantothenate deficiency, 320
CRABP (see Cellular retinoic acid-binding
protein)
CRBP (see Cellular retinol-binding proteins)
Creatinine, 304, 404, 535
Crohn’s disease, 417, 457
Cyanide:
metabolism, 482–483
toxicity, effect of hydroxocobalamin,
511
toxicity from cyanogenic glucosides, 583
toxicity and cobalamin, 494
Cyanocobalamin, 211, 464–466
Cystic fibrosis, 171, 175
Cytochrome P450, 62–63, 91, 257, 263, 267
Cytochrome reductase, 173, 202, 441
Cytosolic binding protein, vitamin E, 170–
172
Dark adaptation, 9
d-Araboascorbic acid (see d-Erythorbic
acid)
Decarboxylation, 363
Dehydroascorbic acid, 530, 531, 537
7-Dehydrocholesterol, 51, 55, 58–60
Dehydroretinol, 3, 15
Dementia, 216, 239
Deoxyuridine suppression test, 456
5?-deoxyadenosylcobalamin, 211
Depression, mental:
biotin, 411
pantothenic acid, 322
in pellagra, 216
Dermatitis:
biotin, 410–414
niacin, 214–216, 239
pantothenic acid, 317, 331
riboflavin, 257, 259
seborrheic, 410–412
vitamin A, 30
Dermatology, (see also Skin, Skin diseases), 30
Dethiobiotin, 404
Detoxification, 569
Diabetes mellitus:
niacin, 234, 241, 243–245
thiamine, 305
vitamin C, 540, 541, 561–562
Diarrhea, 150, 214, 239
Dicumarol, 120
Difenacoum, 121
Differentiation, 23–25
Dihydrolipoic acid, 182
1,25 Dihydroxy vitamin D3, 51–54
metabolism, 63–65
structure, 55
synthesis, 58–60
transport, 61
Diphenylhydantoin:
folic acid, 454
vitamin D, 91
vitamin K, 148
Diphenyl-p-phenylene diamine (DPPD),
Diverticulosis, 88
DNA synthesis, 408, 448, 493, 490, 520, 539
Dopa (dihydroxyphenylalanine), 364
Dopamine, 327, 364, 537
?-hydroxylase, 537
Drugs:
biotin, 410
niacin, 236, 241
pantothenate, 330
riboflavin, 258–263, 267
thiamin, 278
vitamin A, 29
vitamin B6, 378–379
vitamin D, 91
Duchenne’s disease, 330
Edema, 298–299, 305, 542
Egg:
fertility, 63
hatchablity, 63, 87, 262
production, 63, 87, 262
shell, 87
white, raw, injury, 397, 409, 416
Index 593
Elastin biosynthesis, 556
Elderly:
riboflavin, 268
vitamin B 61
vitamin D, 91
vitamin K, 148
Electroencephalographic abnormalities, 364
Electron transport, 571, 576
niacin, 216, 238
pantothenic acid, 330
riboflavin, 202, 259
thiamin, 291
vitamin E, 169
Embryogenesis, 25–26
Encephalomalacia, 174
Enolization, 206
Eosinophilia, 175, 181
Eosinophilic enteritis, 175, 181
Epileptics, 454
Epinephrine, 205, 364
Epoxidase, vitamin K, 137–140
Ergocalciferol, 51, 54
Ergosterol, 51, 58
d-Erythorbic acid (see also d-Araboascorbic
acid), 532
Erythrocyte:
folate status, 434
glutathione reductase, 267
hemolysis (see also Peroxidative hemolysis),
185
vitamin B6, 352, 362–363
Erythropoiesis, 363
Estrogen, 149, 286, 367
Exercise:
pantothenic acid, 331
riboflavin, 268
thiamin, 296, 309
vitamin C, 542
vitamin E, 179
Extracellular matrix, 555–557
Exudative diathesis, 174
Eyes, 20–22
FAD (see Flavin adenine dinucleotide)
FAD pyrophosphatase, 257
Familial isolate vitamin E deficiency syndrome,
174
Fat, dietary (see also Lipid):
effect on pantothenate deficiency, 330
effect on riboflavin requirement, 259
metabolism, 319
thiamin-sparing effect, 291
Fatty acid synthesis, 492–493
Fatty livers:
choline deficiency, 513
and kidney syndrome (FLKS), 410
Ferredoxin, 63
Ferritin, 178, 182
Fertility, 63
Fetal abnormalities, 25–27
Fetus, 261, 403
Fever, thiamin, 296
Fiber, effect on vitamin A absorption, 13
Fibrin, 134, 144
Fibrinogen, 115, 134
Fish oil (see also Cod liver oil), 2
Fish, raw:
antithiamin, 284, 293
source of anti-B12 tapeworm, 483
Flavin adenine dinucleotide (FAD), 202,
255, 258, 263–267
Flavinoids (see Bioflavanoids)
Flavocoenzymes, 201–203
Flavokinase, 257, 263
Flavoproteins, 201, 209, 255–256
FMN (see Riboflavin-5?-phosphate)
Folic acid:
binding proteins, 433–435
and cobalamin deficiency, 489, 491
need for riboflavin, 261, 265
role in choline synthesis, 514–515
thiamin, 285, 307
Formaldehyde, 209
Formate, 209
Fortification of foods:
vitamin A, 4, 30
vitamin B6, 347
vitamin D, 85
Free radical, 177, 539, 569
and aging, 576
generation of, 480
in choline deficiency, 520
GABA (see Gamma amino butyric acid)
Gamma aminobutyric acid, 205, 292, 329,
330
Gastrectomy, 174, 483
Gastric mucosa, atrophy of, 483
Genetics:
biotin, 399, 408
riboflavin, 262
vitamin D, 90–91
vitamin E, 171, 585
Geographic tongue, 261
594 Index
Glossitis, 257
Glucocorticoids, 64, 89
Glucose-6-phosphate dehydrogenase defi-
ciency, 546
Glucuronic acid pathway, for synthesis of
ascorbic acid, 534, 537
Glutamate mutase, 476–477
Glutathione, 178–182, 223, 258, 264, 537
Glutathione peroxidase, 178–182, 264, 579
Glutathione reductase (see also Erythrocyte
glutathione reductase), 180, 258, 264, 268
Glutathione synthesis deficiency, 537
Glycogen phosphorylase, 205
Goblet cells, 9
Growth:
biotin, 409, 411
choline, 519
folic acid, 428
riboflavin, 259
thiamin, 296
vitamin A, 1, 22–23
vitamin D, 79
Growth hormone, 64, 538
Gulonic acid, 533
l-Gulono-?-lactone, 533, 561
l-Gulono-?-lactone oxidase, 256, 533, 535, 561
Hair loss (see Alopecia)
Haptocorrin, 472–474
Hartnup disease, 222, 244
skin pigmentation, 242
Heart:
disease, 241, 375–376, 451–452, 543
failure, 298
hypertrophy, 298
Heavy metals:
ascorbic acid, 530
riboflavin, 262
Heme protein synthesis, 205, 331
Hemochromatosis, 183
Hemodialysis, 411
Hemolytic anemias, 150, 165, 173, 175, 268,
546
Hemorrhage, 115
disease of cattle, 120
disease of chicken, 174
disease of newborn, 148
pantothenic acid, 326
in scurvy, 542
Hepatotoxicity (see also Liver necrosis,
Liver degeneration), 242
Histamine, 542–544
Histidine load test, 456
Histones, 227, 231, 321
History:
ascorbic acid, 527
biotin, 397
folic acid, 427
niacin, 213
riboflavin, 356
thiamine, 275
vitamin A, 1
vitamin B12, 463
vitamin D, 52
vitamin K, 115
Homocysteine:
cobalamin, 486
choline, 514
heart disease, 375–376
riboflavin, 265
synthesis, 442
Homocystinuria, 375–376, 451
Hydrazides, 378
Hydrogen peroxide, 177–181, 203, 262, 514,
535
Hydroperoxide, 177, 181, 183, 557, 573, 579
Hydroxycobalamin, 464
effect on cyanide toxicity, 482–483
4-Hydroxy coumarins, 120–123, 133, 142–144
Hydroxylase, 62–64, 72, 557
Hydroxylation:
in formation of corticosteroids, 538
of lysine and proline, 556–557
Hydroxyl radical, 180, 182, 574
25-Hydroxy vitamin D3, 62–63
Hypercalciuria, 88
Hypercholesterolemia, 539
Hyperlipidemia, 241–242
Hyperoxaluria, 545
Hyperpigmentation, 214
Hyperparathyrodism, vitamin D and, 76, 86, 90
Hypertension, choline deficiency, 519
Hyperthyroidism, 268
Hyperuricemia, 242
Hyperuricosuria, 545
Hypervitaminosis A, 31–33
Hypervitaminosis D, 88
Hypoglycemia, 266, 330–332, 410
Hypoparathyroidism, 90, 93
Hypophosphatemia, 90
Hypoprothombinemia, 123, 126, 147–148
Hypothyroidism:
riboflavin, 268
vitamin K, 149
Index 595
Immune response:
pantothenic acid, 328–330
vitamin A, 27–28
vitamin C, 542
vitamin D, 74–75
vitamin E, 177, 186
Infant formula
biotin, 413
choline, 514
vitamin K, 126
Infants, 185, 219, 268, 295, 519
beri-beri, 297
biotin, 410–412
Infection, 296, 309
Inflammation, 577
Inositol, 224, 515
Insomnia, 331
Insulin, 243, 326, 331, 513, 519
Intestinal calcium absorption (see also Calcium
absorption), 78
Intestinal disorders, 88
Intestinal microflora (see Bacterial synthesis)
Intestinal synthesis (see also Bacterial synthesis):
biotin, 8
riboflavin, 8
vitamin B12 (cobalamins), 470
of vitamin K, 145–146
Intrinsic factor, 471
absorption of cobalamins, 471–472
Iodopsin, 3, 20
Iron:
biotin, 405
mobilization, ascorbic acid, 546
niacin, 222
overload, 183, 546, 571
riboflavin, 259, 262
thiamin, 278, 307
vitamin E, 182, 183
Irradiation of food meat, 54
Isoalloxazine ring, 201, 203, 255, 263
Isoprene, 119
Isoprenoid, 320, 325
Jaundice, obstructive, 89, 115, 128, 150,
257
Kappadione (see also Menadiol sodium),
119–120
Kernicterus, 150
Kidney:
biotin, 408, 410
stones, 543
[Kidney]
vitamin D, 54
vitamin K, 130
Kidney dysfunction:
vitamin A, 15
vitamin B6, 368
vitamin D, 90, 93
Konakion (see also Phylloquinine), 120
Lactation, 29, 296
Lactic acid dehydrogenase, 406
Lactic acidosis, 406, 414
Lecithin, 513, 518–519
Leigh’s disease (see Subacute necrotizing
encephalomyelopathy)
Leiner’s disease, 410
Leukemia, 236, 308
Leukopenia, 236, 448
Light, destruction of riboflavin (see also Radiation),
261
Linoleic acid (see Polyunsaturated fatty
acids)
Lipid, metabolism, 205, 263, 365, 414
Lipodystrophy (see Fatty livers)
Lipoic acid, 207, 288, 408
Lipoprotein, 129, 539, 541
relation to vitamin A, 13, 15
relation to vitamin E, 170–172
relation to choline, 517
Lipotropic activity, choline, 513
Lipoxygenase, 178
Lipoyl, 200, 207, 208
Liver:
cirrhosis, 91, 307, 519
disease, 89, 147, 181, 519
fatty, 365–366, 519
Liver storage:
riboflavin, 261
vitamin A, 15–17
vitamin D, 62
vitamin E, 170
vitamin K, 130–131
Long-chain fatty acids:
pantothenic acid, 330
vitamin E, 330
Longevity, 10
Lumichrome, 257
Lumiflavin, 257, 263, 266
Lumisterol, 59–60
Lung:
cancer, 38
injury, 237
596 Index
[Lung]
niacin, 237
vitamin E, deposition, 171
vitamin K, deposition, 130
Lymphatic system:
vitamin A, 13
vitamin D, 60
vitamin E, 169
Lymph nodes, 130
Magnesium, 202, 222
Malabsorption:
biotin, 409
folic acid, 447
riboflavin, 259
thiamin, 295
vitamin D, 88
vitamin E, 170, 175
vitamin K, 150
Malaria parasitemia, 264
Marasmus, 214
Medium chain triglycerides, 170
Membrane, cellular:
vitamin E concentration, 171, 181
vitamin K, 130
Memory, effect of choline, 521
Menadiol sodium diphosphate, 120
Menadione (see also Menaquinone), 146
alkylation of, 118
safety, 150
Menadione pyridnol bisulfite (MPB), 120
Menadione sodium bisulfite (MSB), 119
complex, 120
Menaquinone (see also Menadione), 117–
118
Mental disturbances (see Dementia, Depression,
Hysteria)
Mephyton (see also Phylloquinone), 120
Messenger RNA, 66–70, 557
Methionine:
conversion of pantothenate to CoA, 319
load test, 375–376
role in choline synthesis, 514–515
synthetase, 440–443, 481–482
Methotrexate, 209, 435–436, 450–451
Methylation, role of choline, 514–517, 520
Methylcobalamin, 211, 467
reactions requiring, 481–482
Methylnicotinamide:
analysis of, 219–220
assessment of niacin nutrition, 220
excretion, 222–223
Methylmalonate, 485–486
Micelles:
vitamin A, 13
vitamin E, 170
vitamin K, 128
Microsomes:
riboflavin, 256
vitamin E content, 178
vitamin K, 130–131
Milk:
biotin, 403
choline, 514, 521
pantothenic acid, 319
riboflavin, 261
thiamin, 282, 287, 293, 296
vitamin B6, 347
vitamin E, 183
vitamin K, 126
Mineral oil, 149
Mitochondria:
biotin, 402
folic acid, 447
function, 568, 577
riboflavin, 259
vitamin K content, 130–131
Mixed function oxidase, 63–64
Monodehydroascorbic acid, iron-pyridine nucleotide
interrelationship, 258
Muscle:
vitamin D, 73
vitamin E disposition, 111–112, 171
weakness, 88, 292
Muscular degeneration (see Necrotizing myopathy)
Muscular dystrophy (see also Necrotizing
myopathy), 183
Mutagenesis, 34–35, 575
Myelin degeneration, 331
Myoinositol (see Inositol)
Myopathy (see Necrotizing myopathy)
Myristoylation, 325
NAD (see Nicotinamide adenine dinucleotide)
NADP (see Nicotinamide adenine dinucleotide
phosphate)
Napthoquinone, 116, 123, 125, 131
Necrotizing myopathy (see also Muscular
dystrophy, Muscular degeneration),
174
Neonates, 36–37, 408
Nervous systems, niacin, 224
Index 597
Nerve conduction, 292
effect of inositol, 224
Neuromuscular defect, 176
Neuropathy (see also Neuromuscular deficits,
Axonal dystrophy):
cobalamin, 490–494
excess vitamin B6, 379
niacin, 239
pantothenic acid, 329
peripheral, 411
riboflavin, 261
thiamin, 292, 298, 308
vitamin C, 542
vitamin E, 174, 581
Neurotransmitters, 291–292, 320, 329
Neutrophils, hypersegmentation:
choline, 518
niacin, 232
pantothenic acid, 331
vitamin C, 540
Niacin (see Nicotinic acid)
Niacin equivalents, 219–221
Nicotinamide, 218
Nicotinamide adenine dinucleotide (NAD):
metabolism, 220–222
tissue concentration, 219, 222–223
Nicotinamide dinucleotide phosphate
(NADP):
metabolism, 220
tissue concentration, 219, 222–223
Nicotinic acid adenine dinucleotide phosphate
(NAADP), 218, 226–227
Nicotinic acid (niacin), 213
bound forms, 213
conversion from tryptophan, 220–222,
363
need for riboflavin, 261, 262
Night blindness, 1, 9
Nitrocobalamin, 466
Nonketotic hyperglycemia, 439
Norepinephrine, 205, 292, 364, 537
Obstructive jaundice, 89, 128, 150
Odd-chain fatty acids, 406, 413
One-carbon metabolism, 209, 212,
512
Optic nerve, 410
Oral contraceptives, 369
Oral lesions, 542
Osteocalcin, 72, 141–142
Osteomalacia, 53, 86–87, 141
Osteoporosis, 86–87, 141
Oxalosis ((see Kidney stone)
Oxalic acid, from oxidation of ascorbic acid,
531, 545
Oxidant response elements, 576
Oxidation, 570
Oxidation-reduction reaction, 200–203, 223,
263
Oxidative stress, 571–573
Oxythiamin, 278–280
Ozone, 539
Pancreas, 74
Pancreatic insufficiency, 128, 150
effect on cobalamin absorption, 472
Pancreatic juice:
biotin, 399
vitamin K, 128
Pancreatitis, 174
Panthenol, 319
Parasites, 262, 483
Parathyroid, 51, 71
Parathyroid hormone (PTH), 51, 63, 73
Parenteral nutrition (see also Total parenteral
nutrition), 88, 294, 312, 409, 413, 416,
530
Paresthesia, 411
Pellagra, 214
Pentose phosphate shunt pathway, 183, 204,
223, 264, 271
Pernicious anemia, 307, 463, 483
Peroxidation, 177, 259, 266, 330, 520, 557
Peroxidative hemolysis (see Erythrocyte
hemolysis)
Peroxides (see also Hydroperoxide), 570–
571
Peroxyl radicals, 167, 169, 177–180, 539,
570
Phagocytosis
vitamin C and, 542
Phenobarbital
biotin, 410
vitamin D, 91
vitamin K, 148
Phenolic compounds, 572, 580
Phosphatidylcholine (see Lecithin)
Phosphatidylinositol, 518
Phosphopantetheine coenzymes, 206
Phosphorous, 51–52, 54, 71
Photoisomerization, 58
Phototherapy, 266
Phylloquinine, 117
safety, 150
598 Index
Phytol side chain, 167
Phytonadione, 117
Phytylmenaquinone (PMQ), 118
Phytyl side chain, 166–167, 172, 176
Pigmentation, vitamin E toxicity, 175
Pigment epithelium, 15
Pituitary, 94
Placenta, 263, 286, 295, 409, 514, 521
Plant synthesis of thiamin, 276
Platelets:
aggregation, 187, 515, 575
vitamin E content, 185
PLP (see Pyridoxal-5-phosphate)
Polar bear liver, 9
Poly (ADP-ribose) polymerase (PARP),
229–234, 242
Polyglutamates (folate), 428, 434–436
Polyneuritis, 275
Polyphenols, antithiamin, 284
Polyunsaturated fatty acids (PUFA), 169, 174,
177–178, 180–181, 185, 414, 539, 573
Pregnancy, 35–36
Pre-RBP, 13
Propionic acidemia, 408
Propionyl-CoA carboxylase, 208
Prostaglandin, 177, 576
metabolism, 186, 242
synthesis, 415
Pro-oxidant, 178, 539–576
Protein:
effect on riboflavin requirement, 259
effect on vitamin C, 540
effect on vitamin B6 requirement, 367–368
sparing effect on pantothenic acid, 320
Protein calorie malnutrition:
biotin, 410
niacin, 235
riboflavin, 268
thiamin, 285
vitamin A, 15
vitamin E, 175
Protein deficiency, 285
Prothrombin, 115
assay, 147
synthesis, 134–136
Provitamin A, 8, 12
Pseudovitamin B12,
Psoriasis, 4, 73
Pterin, 208
PTH (see Parathyroid hormone)
Pyridine nucleotides, cycle, 220
Pyridoxamine, 342
Pyridoxal-5-phosphate (PLP), 204, 342,
354–360, 438, 516
Pyridoxamine, 342
Pyridoxamine-5-phosphate, 204, 342
Pyridoxic acid, 342, 360
Pyridoxine, 340–342
glycosylated forms, 345–347, 349
metabolism, need for riboflavin, 259, 350–351
metabolism, need for niacin, 222
Pyrithiamin, 278–280
Pyruvate:
carboxylase, 208, 304, 405
decarboxylase, 305
dehydrogenase complex, 288, 408
metabolism, 276, 305
Pyruvate kinase, 176
Quercetin, 580
Quinones, 116–117, 202
antithiamin, 284
Racemizations, 205
Radiation, destruction of thiamin, 282, 283
RBP (see Retinol binding protein)
Receptors, 327
Red blood cell (see Erythrocyte)
Regional enteritis, 88
Renal disease (see also Kidney dysfunction),
14, 90, 519
Renal osteodystrophy, 90
Renal reabsorption, 86
Reproduction, 259, 452–454
Retinal (retinaldehyde), 2–3
Retinoic acid, 3–4, 15, 18, 22–23
Retinoid receptors, 3–4, 23–26
Retinol, 2–3, 7, 16
transport, 13–15
Retinol binding protein (RBP), 9, 13–19
Retinyl acetate, 7
Retinyl palmitate, 4
Retro-retinol, 28
Rhodopsin, 2–3, 20–22, 327
Riboflavin binding protein, 263, 267
Ribo-d-Galactoflavin, 263
Riboflavin-5?-phosphate (FMN), 255–257,
258, 263–265
Riboflavinuria, renal, 262
Ribonucleotide reductase, 479–480
Ribosylation, 214, 226–237, 560
Rice bran, 281
loss of thiamin, 282–283
polished, 275, 296
Index 599
Rickets, 52, 84, 86–87, 90–91, 93
hypophosphatemic, 90
vitamin D-resistant, 90
R protein, affinity of cobalamins for, 472–
476
Rodenticide, 121–123
Rose hips, source of ascorbic acid, 534
Saccharin, 262
Sarcoidosis, 89
Schiff base, formation of, 342
Schilling test, 487–488
Schizophrenics, niacin deficiency, 216, 241,
243
Scurvy, 542, 561
rebound, 545
Seafood, antithiamin (see also Fish), 284, 293
Selenium, 174, 180, 181
Semidehydro-l-ascorbate free radical (see
also Ascorbate radical), 531
Serine transhydroxy methylase, 361
Serotonin, 205, 292
Shoshin, 298
Sickle cell anemia, vitamin B6, 362
Singlet oxygen, 4, 37, 178, 179, 182, 539
Skin (see also Dermatitis, Dermatology), 238
cancer, 237
diseases, 30, 73
pigmentation, 30, 84, 242, 266
Smoking, 235, 491, 494, 539, 542–543, 572
Species differences:
riboflavin, 259, 261, 263
thiamin, 276
vitamin E, 165
Sphingomyelin, 518–519
Sphinogolipids, 320, 519
Spinocerebellar dysfunction, 172
Sprue:
folic acid, 457
nontropical, 175
tropical, 88
Steatorrhea (see Malabsorption)
Sterility, 519
Stomatitis, 257, 261
Stress, 531
Stroke, 234
Subacute necrotizing encephalomyelopathy,
309
Succinic dehydrogenase, 256
Sudden Infant Death Syndrome (SIDS), 410
Sulfa drugs, 451
vitamin K, 148
Sulfur amino acids, 178, 183
Sunlight, 52, 58, 83
Sun screens, 84
Superoxide radical, 169, 177, 539
Superoxide dismutase, 179–180, 182, 579
Synkayvite (see also Menadiol sodium diphosphate),
120
Tannic acid, antithiamin, 278, 284, 285
Tannins, 581
Tapeworm, competitor for vitamin B12, 483
Tardive dyskinesia, 331
Tea, antithiamin, 284, 285
Teratogens, 33–35, 261, 411
Tetany, 87
Tetrahydrofurfuryldisulfide, 278, 300
Thiamin alkyl disulfides, 278
Thiaminase, 284
Thiamin binding protein, 285–286
Thiamin monophosphate, 277, 286, 287, 304
Thiamin propyl disulfide, 278, 300
Thiamin pyrophosphate, 203, 276–278, 286–
290, 295, 305
Thiamin pyrophosphokinase, 277
Thiamin triphosphate, 277, 286, 287
TTP effect, 295, 298, 302, 305–308
Thiazole ring, 203, 276, 279, 287
Thiochrome, 278, 282, 283
Thiol, 206, 578
Thiopene ring, 401, 405
Thrombin, 115–116
Thymidylate synthase, 210, 444
Thyroid, 268
Thyroid hormone, 256, 258
Tocopherol, 165, 583
binding proteins, 171–175, 582
Tocopherol equivalents, 176–178, 185
?-Tocopheronic acid, 173
Tocopheroxy radical, 178
Tocopheryl acetate, 170, 176–177
Tocopheryl hydroquinone, 173
Tocopheryl palmitate, 170
Tocopheryl quinone, 167
Tocopheryl succinate, 170, 177
Tocols, 166–167
Tocotrienols, 166–169, 583
Tongue (see Geographic tongue)
Total parenteral nutrition, 88
choline, 522
TPN (see Total parenteral nutrition)
Transaminase, in determining vitamin B6
status, 360
600 Index
Transamination, 204, 342
Transcobalamins, 472–474
hereditary deficiency, 494, 534
Transketolase, 204, 291–295, 300, 305,
306
Transmethylation, 212
Transthyretin (prealbumin), 13–15
Tropical sprue, 88, 455
Tryptophan:
conversion to niacin, 214, 219, 220–
222, 262
metabolism, 266, 375
Tubulin, 323, 411
Tumors, growth, 326
Ulcer, gastric, 562
Ultraviolet light, 54, 58, 261, 574
Ureido group, 397, 400
Uric acid, antioxidant role, 543–545,
578
Valeric acid, 400, 404, 408, 410
Vasodilator, niacin, 242
Vegetarian, vitamin B12 deficiency in,
483
Vision, 20–22
Visual pigments, 20
Vitamin B12:
interaction with folic acid, 449
role in choline synthesis, 515
role of thiamin, 308
vitamin C, 546
[Vitamin]
D:
binding protein (DBP), 61, 89
ligand binding domain, 93–94
receptor, 66, 69–70, 73–74, 93
E, prevention of vitamin A toxicity, 182
K:
dependent carboxylase, 137–140
dependent proteins, 134–137, 140– 142
epoxide reductase, 142–143
vitamin A, 149
vitamin E, 149, 175
K oxide (epoxide), 133, 138–144
Wafarin, 120–121, 133, 142
Wernicke’s encephalopathy, 293–296, 298, 308
Wernicke-Korsakoff syndrome, 299–300, 308
Work (see Exercise)
Wound healing, 331, 544, 561
Xanthine oxidase, 178–179
Xanthurenic acid, 360–361, 365
Xenobiotic, 577–580
Xerophthalmia, 9
Yellow enzyme, 255
Ylid, 203
Zinc:
folic acid, 441
niacin, 229
riboflavin,