Lennarz W.J., Lane M.D. (eds.) Encyclopedia of Biological Chemistry. Four-Volume Set . V. 4

Подождите немного. Документ загружается.

Dovonex A drug marketed by Leo Pharmaceuticals containing an

analogue of 1,25-(OH)

and used to treat psoriasis s by topical

administration.

hepatocytes Cells comprising , 30% of the liver that carry out many

of the liver’s functions.

keratinocytes Cells of the skin that produce the keratin proteins and

contribute to the skin barrier.

macrophages Cells of the immune system that are responsible for

destroying foreign materials.

nongenomic Referring to or describing activities that are carried out

not involving gene expression.

osteoblasts Cells of the skeleton that carry out synthesis of new bone

and that mediate regulation of bone activities.

previtamin D An isomer of vitamin D formed during the irradia-

tion process for producing vitamin D. It is in equilibrium with

vitamin D.

proximal convoluted tubule cells Cells lining the proximal convo-

luted tubule of the kidney.

RANKL Receptor activator of NF-Kappab ligand, a signal protein

produced by osteoblasts and stromal cells in response to such

stimulants as 1,25-(OH)

and parathyroid hormone.

transcription factor A protein usually found in the nucleus that has a

marked effect or role in the transcription of genes.

FURTHER READING

Darwish, H. M., and DeLuca, H. F. (1996). Recent advances in the

molecular biology of vitamin D action. In Progress in Nucleic Acid

Research and Molecular Biology (W. E. Cohn and K. Moldave,

eds.) pp. 321 –344. Academic Press, San Diego, CA.

DeLuca, H. F. (1974). Vitamin D: The vitamin and the hormone. Fed.

Proc. 33, 2211 –2219.

DeLuca, H. F. (1997). Historical overview. In Vitamin D (D. Feldman,

F. H. Glorieux and J. W. Pike, eds.) Vol 1, pp. 3–11. Academic

Press, San Diego, CA.

Feldman, D., Glorieux, F. H., and Pike, J. W. (1997). Vitamin D.

Academic Press, San Diego, CA, 1285pp.

Jones, G., Strugnell, S. A., and DeLuca, H. F. (1998). Current

understanding of the molecular actions of vitamin D. Physiol.

Rev. 78, 1193–1231.

BIOGRAPHY

Hector F. DeLuca is Steenbock Research Professor and Chairman of

the Department of Biochemistry at the University of Wisconsin-

Madison. He has been a major contributor to the current under-

standing of the metabolism and mechanism of action of vitamin D. He

has published over 2000 papers and patents. He has trained well over

250 students in vitamin D, vitamin A, bone and calcium ﬁelds. He is a

member of the National Academy of Sciences, the American Academy

of Arts and Sciences, a Fellow of the AAAS and has won many awards

for his work on vitamin D.

Margaret Clagett-Dame is a Professor of Biochemistry and Pharma-

ceutical Sciences in the Department of Biochemistry at the University of

Wisconsin-Madison and is best known for her work in the develop-

mental role of the retinoids. She has published some 48 papers and

6 patents.

VITAMIN D 377

Vitamin E

Ute C. Obermuller-Jevic

BASF Aktiengesellschaft, Ludwigshafen, Germany

Lester Packer

University of Southern California, Los Angeles, California, USA

Vitamine E is the name for a group of biologically active

substances including tocopherols and tocotrienols. Alpha-

tocopherol shows the highest biological activity of all vitamin E

forms and is the most common form found in the human body.

Vitamin E was discovered in 1922 as essential micronutrient

for reproduction. In the 1950s it was recognized that vitamin E

is the body’s major lipid-soluble antioxidant protecting

lipoproteins and membranes against oxidative damage. The

biological role of vitamin E goes, however, beyond its

antioxidant function. Studies on the effects of vitamin E on

gene expression have revealed many genes and signal

transduction pathways which are inﬂuenced by vitamin E.

All these actions of vitamin E help to explain the observed

beneﬁcial effects of vitamin E in chronic and degenerative

diseases. A well-balanced diet rich in vitamin E seems a basic

requirement for human health. Intake of supplementary

vitamin E may be advisable for individuals who do not get

adequate levels from diet, or who are at high risk for, or who

are already suffering from, chronic and degenerative diseases.

Molecular Structure of Vitamin E

Vitamin E is the generic name for a group of eight

plant-derived, lipid-soluble substances (tocols) including

four tocopherols and four tocotrienols (Figure 1).

The molecular structure of vitamin E is comprised of a

chromanol ring with a side chain located at the C-2

position. Tocopherols have a saturated phytyl side chain

and tocotrienols have an unsaturated isoprenoid side

chain. The number and position of methyl groups

located around the chromanol ring varies among the

different tocopherols and tocotrienols, and accounts for

the designation as alpha-, beta-, gamma-, or delta-forms.

Natural and natural-source alpha-tocopherol occur in

RRR-conﬁguration only (formerly designated as

d-alpha-tocopherol). In contrast, synthetic alpha-toco-

pherol consists of an equal racemic mixture of eight

stereoisomers (RRR, RSR, RRS, RSS, SRR, SSR, SRS,

SSS) arising from the three chiral centers of the molecule

at positions C2, C4

and C8

and designated as all-rac-

alpha-tocopherol (or dl-alpha-tocopherol).

Biological Activity of Vitamin E

The various forms of vitamin E differ in their biological

activities. Alpha-tocopherol is the most common form of

vitamin E occurring in human blood and tissues and it has

the highest biological activity among all tocopherols and

tocotrienols. Moreover, the human body preferentially

accumulates the RRR-or2R-forms of vitamin E. Thus

natural RRR-alpha-tocopherol has a higher bioavail-

ability and “biological activity” than synthetic all rac-

alpha-tocopherol. According to the Food and Nutrition

Board (FNB) of the US National Academy of Sciences,

which publishes recommendations for dietary intake of

vitamin E, only RRR-alpha-tocopherol itself, or the

RRR- and 2R-stereoisomers of all rac-alpha-tocopherol

meet the vitamin E requirements in humans. The FNB

suggests a 2-fold higher potency of natural versus

synthetic alpha-tocopherol sources, however, this value

has been challenged. The biological activity of vitamin E

in dietary supplements is usually expressed as inter-

national units (IU). As published in the United States

Pharmacopeia (USP), 1 IU is deﬁned as the biological

activity of 1 mg all rac-alpha-tocopheryl acetate. Other

biological activities of vitamin E are 1 mg all rac-alpha-

tocopherol ¼ 1.1 IU; 1 mg RRR-alpha-tocopherol ¼

1.49 IU; 1 mg gamma-tocopherol ¼ 0.15 IU.

Dietary Sources and Recommended

Intake of Vitamin E

Vegetable oils and lipid-rich plant products (e.g., nuts,

seeds, grains) are the main dietary sources of vitamin

E. In Western diets, vitamin E intake mainly derives from

fats and oils as found in margarine, mayonnaise, salad

dressing, and desserts, and increasingly also from

fortiﬁed food (e.g., breakfast cereals, milk, fruit juices).

Noteworthy, the diet of Americans contains large

amounts of gamma-tocopherol when compared to

populations in other Western countries, which is a result

of the high consumption of soybean and corn oil

containing more gamma- than alpha-tocopherol. Vita-

min E used for food fortiﬁcation or dietary supplements

consists mainly of alpha-tocopherol, either derived from

natural sources (i.e., methylated gamma-tocopherol

from vegetable oil) or from synthetic production and it

is usually esteriﬁed to increase stability.

The FNB recommends for adults a daily intake

(recommended dietary allowance, or RDA) of 15 mg

vitamin E including food, fortiﬁed food, and sup-

plements. The recommendations are largely based on

in vitro studies of M. Horwitt dating back to 1960,

which are still considered the most adequate data for

deﬁning the physiological status and a health beneﬁt of

vitamin E in humans. In these studies, prevention of

-induced erythrocyte hemolysis was used as test

system. However, except when vitamin E is clearly

deﬁcient the erythrocyte hemolysis test is not a useful

indicator of vitamin E functional status. Thus, the

guidelines of the FNB were critically discussed in the

literature and it was strongly agreed upon that other

assays and new biomarkers need to be identiﬁed to

deﬁne the physiologic role and beneﬁcial potential of

vitamin E in humans.

The recommended daily intake of 15 mg vitamin E is

considered rather unlikely to be achieved by North

Americans and other Western countries through

diet alone. Although universal dietary supplementation

has not been recommended, a dose of 200 mg/day

vitamin E (alpha-tocopherol) has been suggested to

saturate plasma levels and elevate tissue levels which

may have possible health effects in the long term.

As an upper limit for supplemental vitamin E intake

the FNB has published for adults a dose of 1 g/day

alpha-tocopherol (i.e., 1 500 IU RRR- or 1 100 IU all

rac-alpha-tocopherol) showing no apparent side effects,

which is considered safe. In human intervention studies,

various doses of vitamin E up to 3 600 IU/day have been

used. Nevertheless, conclusive evidence from long-term

studies regarding biological effects and safety of chronic

intake of pharmacologic doses of vitamin E are lacking.

In the Alpha-Tocopherol, Beta Carotene (ATBC) Cancer

Prevention Study with Finnish smokers consuming

50 IU/day vitamin E for 6 years, an increase in mortality

from hemorrhagic stroke was observed, however, other

intervention studies did not report such an adverse

effect. It was suggested that pharmacologic doses of

vitamin E are contraindicated in persons with blood

coagulation disorders as vitamin E might exacerbate

defects in the blood coagulation system based on its

inhibitory effects on platelet aggregation.

Uptake, Distribution, and

Metabolism of Vitamin E

Together with dietary fat, alpha-tocopherol and all

other forms of vitamin E are absorbed in the digestive

tract, incorporated into chylomicrons and transported

in the lymphatic system. Part of the absorbed stereo-

isomers are taken up into extrahepatic tissues by the

action of lipoprotein lipase and the remainder is

delivered in chylomicron remnants to the liver. Distribu-

tion of vitamin E into the circulation is regulated by a

cytosolic alpha-tocopherol transfer protein (alpha-TTP)

in the liver, which is selective for alpha-tocopherol in its

RRR-or2R-forms. The other stereoisomers of alpha-

tocopherol as well as the other tocopherols and

tocotrienols exert much less afﬁnity for alpha-TTP.

Alpha-TTP plays an important role in maintaining

plasma levels of vitamin E.

In the liver, vitamin E is incorporated into very low

density lipoproteins (VLDLs) and released to the

systemic blood circulation. Excess amounts of alpha-

tocopherol along with the other absorbed forms of

tocopherols and tocotrienols are metabolized or elimi-

nated by the biliary tract. VLDLs are converted into

low-density lipoproteins (LDLs) and excess surface

components including alpha-tocopherol are transferred

Phytyl chain

Isoprenoid chain

8´

4´

The four isoforms of tocopherols and tocotrienols:

a-: R

= CH

b-: R

= CH

= H R

= CH

g -: R

= H R

= CH

d -: R

= H R

= CH

Tocopherol

Tocotrienol

FIGURE 1 The molecular structure of vitamin E. Tocopherols and

Tocotrienols exist in four different isoforms (

-, and

-forms).

Tocopherol is shown in its naturally occurring RRR-conﬁguration.

(Adapted from Food and Nutrition Board, 2000.)

VITAMIN E 385

to high-density lipoproteins (HDLs). Delivery of alpha-

tocopherol to peripheral tissues takes place via binding

of LDL to LDL receptors and subsequent cellular

uptake. Vitamin E preferably accumulates in adipose

tissues. Cytosolic tocopherol-associated proteins (TAP)

have been reported showing alpha-tocopherol-speciﬁc

binding characteristics and also nuclear translocation

and transcriptional activation in various mammalian

cell types and organs. TAP seems to play an important

role in vitamin E-induced gene expression, however, its

biological functions are not widely known. A further

cytosolic vitamin E regulatory protein, i.e., tocopherol-

binding protein (TBP) has been reported whose func-

tions are not fully understood.

The lowest acceptable vitamin E level in plasma is

11.6 mmol L

(0.5 mg dL

) and a ratio of vitamin

E:cholesterol of 2.25 mmol mmol

. Serum levels of

vitamin E correlate with cholesterol levels and hence

do not necessarily correlate with vitamin E intake.

Except for non- or poorly responding subjects, serum

levels of vitamin E can usually be increased up to

threefold by intake of dietary supplements reaching a

saturation level.

Biological Importance of Vitamin E

in Reproduction and Essentiality

Vitamin E was discovered in 1922 by H. Evans and

K. Bishop as essential micronutrient needed to ensure

normal reproduction in rats. It was named according to a

consecutive alphabetical order preceded by the discovery

of vitamins A to D. Later vitamin E was called alpha-

tocopherol, according to the greek term “tokos” child-

birth, “phero” to bear, and -ol, indicating an alcohol.

Rats on a diet low in vitamin E showed reduced fertility

and a high rate of fetal resorption. However, when

animals were fed a lipophilic fraction from lettuce or, as

later shown, wheat germ oil, their fertility was retained

and a successful implantation of the fetus was observed.

The biological activity of vitamin E is based on this so-

called rat resorption-gestation assay. The family of

natural vitamin E molecules as well as the stereoisomers

of all rac-alpha-tocopherol all exhibit to varying degrees

vitamin E activity in this bioassay. Unfortunately, this

assay of reproductive activity in pregnant rats may bear

limited relevance to human health.

Unique Role of Vitamin E

as a Lipophilic Antioxidant in

Lipoproteins and Cell Membranes

In the 1950s it was recognized under leadership of

A. L. Tappel’s group that vitamin E is the body’s major

lipid soluble antioxidant protecting lipoproteins and

membranes where it resides against free radical-

mediated lipid peroxidation which, if not prevented or

interrupted by vitamin E, causes widespread oxidative

molecular damage and pathology. All natural isoforms

and synthetic stereoisomers of vitamin E exhibit to

varying degrees the ability to inhibit lipid peroxidation

as a “chain-breaking” antioxidant. Vitamin E primarily

destroys peroxyl radicals and thus protects unsaturated

fatty acids from oxidation. Additionally, vitamin E

scavenges a variety of oxygen-derived free radicals

including alkoxyl radicals, superoxide, and other

reactive oxygen species (ROS) such as singlet oxygen

and ozone, and it reacts with nitrogen species. Vitamin E

participates in an antioxidant network (a concept

advanced by L. Packer’s group) and thus vitamin E

radicals can be recycled or regenerated back to their

native form, e.g., by vitamin C. The antioxidant

network is strengthened by bioﬂavonoids (recycle

vitamin C) and by carotenoids (free radical traps sparing

vitamin E) (Figure 2).

Effects of Vitamin E on Cell

Signaling and Gene Expression

In the early 1990s, inhibition of protein kinase C (PKC)

activity by vitamin E was suggested by A. Azzi’s group as

the crucial factor for inhibition of cell proliferation in

smooth muscle cells. PKC activity is an important factor

contributing to disorders such as vascular disease,

cancer, diabetes, and other age-related degenerative

diseases. Vitamin E was found to inhibit PKC activity

in many cell types including smooth muscle cells,

monocytes, macrophages, neutrophils, ﬁbroblasts, and

mesangial cells and the effects were repeatedly con-

ﬁrmed in animal studies. Inhibition of PKC activity by

vitamin E occurs indirectly via activation of a phospha-

tase that cleaves the active, phosphorylated form of

PKC, or by modulating diacylglycerol kinase activity.

What is novel, is that inhibition of PKC is apparently

independent of antioxidant activity and rather is the

result of inhibition due to the speciﬁc molecular

structure of the RRR stereoisomer of alpha tocpopherol.

Hence the biological role of vitamin E goes beyond its

antioxidant function.

Recent advances in molecular biology and availability

of microarray techniques for studying effects of vitamin E

on gene expression have revealed novel vitamin

E-sensitive genes and signal transduction pathways.

Vitamin E regulates at the transcriptional level the

expression of several genes including collagen alpha-1

and alpha-TTP in liver, collagenase in skin, adhesion

molecules, and chemokines such as VCAM-1 and MCP-1

in endothelial cells, different integrins in erythroleukemia

386

VITAMIN E

cells, alpha-tropomyosin in smooth muscle cells, and

scavenger receptors class A (SR-A) and CD 36 in

macrophages and smooth muscle cells. At the posttran-

slational level, vitamin E regulates the expression of

cyclooxygenase in monocytes leading to a decrease in

prostaglandin E

levels. As this latter effect was also

observed using other vitamin E homologues compatible

with antioxidant activity this function of vitamin E may

involve redox-signaling. In alpha-TTP knockout mice

many genes associated with functions of liver or brain are

remarkably modulated by deﬁciency in vitamin E,

whereas the majority of genes over a broad range

of expression levels show no change. This indicates

that vitamin E regulates tissue-speciﬁc gene expression.

These newly discovered actions of vitamin E help to

explain the observed beneﬁcial effects of vitamin E in

chronic and degenerative diseases of aging.

Recognition of Vitamin E

Deﬁciency Diseases and the

Beneﬁcial Effects of Vitamin E

Supplements in Human Health

and Disease Prevention

Clinically manifest vitamin E deﬁciency in humans is

rare. Clear indications of vitamin E deﬁciency have been

found in premature babies and in children suffering from

an abnormal ability to absorb vitamin E as in

abetalipoproteinemia, chronic cholestatic liver disease,

cystic ﬁbrosis, and in short-bowel syndrome.

A mutation in the alpha-TTP gene as found in

“familial isolated vitamin E deﬁciency” results in a

failure to deliver alpha-tocopherol to the systemic

circulation. In these cases clinical vitamin E deﬁciency

is recognized in babies showing symptoms of retrolental

ﬁblasia and intraventricular hemorrhage, and in children

suffering from muscular dystrophy, ataxia, and other

disorders. High-dose vitamin E supplementation can

reduce or eliminate clinical symptoms in these patients,

however, to achieve amelioration of neurological symp-

toms early diagnosis and early start of treatment

are crucial.

More frequently, a chronic sub-optimal supply in

vitamin E (i.e., theoretically an intake below RDA

levels) occurs population-wide and in all age groups.

This may cause impaired defense against oxidative stress

and increased susceptibility to oxidative injury and

adverse health effects. In most diseases that have been

examined with any degree of scrutiny, evidence for

“oxidative stress” and oxidative damage has been

observed in some stage of disease initiation or pro-

gression. Therefore it seems obvious that vitamin E like

other antioxidants may prevent or delay disease

progression. In numerous epidemiologic studies it has

been observed that high intake of vitamin E is associated

with a lower risk of age-related and chronic diseases,

and experimental studies have suggested substantial

a-Tocopheroxyl

radical

a-Tocopherol

Vitamin E

cycle

Ascorbyl

Radical

Ascorbate

Vitamin C

cycle

THIOL

cycle

Unsaturated fatty acid

Metabolism, strenuous exercise

Cigarette smoke

Ultraviolet irradiation (sunlight)

Lipid

radical

Lipid Water

Inter face

Carotenoids

free radical trap

Most reactive oxygen

species destroyed

a-Lipoic acid

(red)

glutathione,

thioredoxin

(red)

a-Lipoic acid,

glutathione,

disulfide

thioredoxin

(ox)

NAD(P)H + H

NAD(P)

Bioflavonoid

Bioflavonoid radical

NAD(P)H

cycle

–

Lipid

–

FIGURE 2 The antioxidant network concept.

VITAMIN E 387

health beneﬁts from vitamin E in disease prevention and

therapy. Most large-scale human intervention trials on

the prevention of cardiovascular disease, the main cause

of morbidity and mortality in the Western world,

however have failed to convincingly show that (rela-

tively short-term) supplementation with vitamin E

lowers the incidence of cardiovascular events or the

rate of mortality from heart disease or stroke. Interes-

tingly, a plethora of small clinical trials report a

signiﬁcant improvement of health status and/or retar-

dation of disease progression by vitamin E supplemen-

tation in patients suffering from cardiovascular disease

or other chronic and age-related diseases. Vitamin E

appears to have important health beneﬁts such as skin

health, in reproductive diseases (pre-eclampsia), age-

related eye diseases (cataract, AMD), metabolic dis-

orders (Diabetes mellitus), neurodegenerative disorders

(Alzheimer’s and Parkinson’s disease), skin aging

(photoaging), and healthy aging.

Conclusion

An adequate dietary intake of vitamin E seems of crucial

importance to guarantee throughout life normal func-

tion of physiologic processes in the body as well as

adequate defense against oxidants generated by

endogenous sources or from exposure to environmental

stress. Vitamin E plays a unique role in the prevention of

chronic and degenerative diseases, and thus in healthy

aging. A well-balanced diet rich in vitamin E as part of a

healthy lifestyle seems a basic requirement for human

health. Moreover, intake of supplementary vitamin E

may be advisable for individuals who are unable to

sustain adequate levels, or who are at high risk for, or

who are already suffering from chronic and degenerative

diseases.

FURTHER READING

Brigelius-Flohe, R., and Traber, M. G. (1999). Vitamin E: Function and

metabolism. FASEB J. 13, 1145–1155.

Food and Nutrition Board (2000). Dietary Reference Intakes for

Vitamin C, Vitamin E, Selenium, and Carotenoids, pp. 186–283.

National Academy Press, Washington, DC.

Gordon, N. (2001). Hereditary vitamin-E deﬁciency. Dev. Med. Child

Neurol. 43, 133–135.

Hoppe, P., and Krennrich, G. (2000). Bioavailability and potency

of natural-source and all-racemic alpha-tocopherol in the human:

A dispute. Eur. J. Nutr. 39, 183– 193.

Packer, L., and Obermu

ller-Jevic, U. (2002). Vitamin E in disease

prevention and therapy: Future perspectives. In The Antioxidant

Vitamins C and E (L. Packer, M. G. Traber, K. Kra

mer and B. Frei,

eds.) AOCS Press, Champaign.

Ricciarelli, R., Zingg, J. M., and Azzi, A. (2002). The 80th anniversary

of vitamin E: Beyond its antioxidant properties. Biol. Chem. 383,

457–465.

Rimbach, G., Minihane, A., Majewicz, J., Fischer, A., Pallauf, J.,

Virgli, F., and Weinberg, P. (2002). Regulation of cell signaling by

vitamin E. Prog. Nutr. Soc. 61, 415–425.

BIOGRAPHY

Ute Obermu

ller-Jevic Ph.D. is a Nutrition Scientist holding a doctoral

degree in the ﬁeld of antioxidant nutrients. Currently she is a scientiﬁc

affairs manager for BASF Aktiengesellschaft, Ludwigshafen, focusing

on the health beneﬁts of vitamins and carotenoids.

Lester Packer received his Ph.D. from Yale University in 1956 and in

1961 joined the University of California at Berkeley where he was

Professor of Molecular and Cell Biology and since 2000 is Adjunct

Professor in the Department of Molecular Pharmacology and

Toxicology, University of Southern California Health Sciences Center.

Dr. Packer is a leading researcher on Antioxidants who has published

over 800 articles, edited more than 90 books, is a member of numerous

professional societies and editorial boards. He was Founder and

Honorary President of The Oxygen Club of California, Past President

of the Society for Free Radical Research International: he has received

numerous awards including three Honorary Doctoral degrees.

388 VITAMIN E

Vitamin K: Biochemistry, Metabolism,

and Nutritional Aspects

J. W. Suttie

University of Wisconsin-Madison, Wisconsin, USA

The term vitamin K refers to a number of compounds

containing a 2-methyl-1,4-naphthoquinone ring with a hydro-

phobic side chain constituted at the 3-position (Figure 1). The

biochemical role of the vitamin was not established until the

mid-1970s, when it was shown that it was a substrate for a

microsomal enzyme that converted protein bound glutamyl

residues to

-carboxyglutamyl (Gla) residues. This posttrans-

lational modiﬁcation has been found in relatively few proteins.

Vitamin K-Dependent Proteins

Prothrombin (clotting factor II), the zymogen of the

plasma procoagulant thrombin, was the ﬁrst protein

shown to be dependent to vitamin K for its synthesis, and

was also the ﬁrst protein demonstrated to contain

-carboxylglutamyl (Gla) residues. Plasma clotting

factors VII, IX, and X were all initially identiﬁed in

patients with hereditary bleeding disorders and were

subsequently shown to be vitamin K dependent. Until the

mid-1970s, these four vitamin K-dependent clotting fac-

tors were the only proteins known to require this vitamin

for their synthesis. The amino-terminal, Gla domains of

these four vitamin K-dependent procoagulants are very

homologous, and the 10–13 Gla residues in each are

in essentially the same position as in prothrombin.

Following the discovery of Gla, three more Gla-

containing plasma proteins with similar homology were

discovered. Protein C and protein S are involved in an

anticoagulant rather than a procoagulant role in normal

hemostasis, and the seventh Gla-containing plasma

protein (protein Z) also has an anticoagulant function

under some conditions. As these proteins play a critical

role in hemostasis, they have been extensively studied,

and the cDNA and genomic organization of each of

them is well documented.

The discovery of Gla residues in the plasma vitamin K-

dependent proteins led to a search for other proteins with

this modiﬁcation, and a 49 residue protein that contained

three Gla residues, called osteocalcin (OC), was isolated

from bone. It has little structural homology to the

vitamin K-dependent plasma proteins. Although it is the

second most abundant protein in bone, its function is

not clearly deﬁned. A second low-molecular-weight (79

residue) protein, matrix Gla protein (MGP), contains ﬁve

Gla residues and was also ﬁrst isolated from bone, but is

also synthesized in cartilage and many other soft tissues.

The function of these two proteins has not been clearly

deﬁned, but it has been shown that osteocalcin gene

knockout mice develop more dense bones, and MGP

knockout mice die from spontaneous calciﬁcation of

arteries and cartilage. A limited number of other

mammalian proteins have been found to contain Gla

residues: Gas 6, a ligand for the tyrosine kinase Ax1, two

proline rich integral membrane Gla proteins (PRGP-1,

PRGP-2), and two additional Gla proteins (TMG-3 and

TMG-4). A large number of Gla-containing toxic venom

peptides are secreted by marine Conus snails and are

found in some snake venoms. The vitamin K-dependent

carboxylase has been cloned from a number of ver-

tebrates, the Conus snail, a tunicate, and Drosophila,

indicating the ancient evolutionary origin of this post-

translational modiﬁcation.

Biochemical Role of Vitamin K

Beginning in the early 1960s, studies of prothrombin

production in humans and experimental animals led to

an understanding of the metabolic role of vitamin K.

A biologically inactive form of prothrombin was demon-

strated to be present in the plasma of patients treated

with oral anticoagulants, and studies with hypopro-

thrombinemic rats were consistent with the presence of a

hepatic precursor protein pool that was rapidly being

synthesized and which could be converted to prothrom-

bin by a posttranslational modiﬁcation. Characteriz-

ation of the abnormal prothrombin isolated from the

plasma of cows fed the anticoagulant dicoumarol led

directly to an understanding of the metabolic

role of vitamin K. This protein lacked the speciﬁc

calcium-binding sites present in normal prothrombin.

Acidic peptides obtained by proteolytic enzyme diges-

tion of prothrombin were shown to contain Gla

residues, but Gla residues could not be obtained by

proteolysis of the abnormal prothrombin.

THE VITAMIN K-DEPENDENT

CARBOXYLASE

In 1975, it was demonstrated that crude rat liver

microsomal preparation contained an enzymatic activity

(the vitamin K-dependent carboxylase) that promoted

a vitamin K-dependent incorporation of H

into

Gla residues of endogenous precursors of vitamin

K-dependent proteins present in these preparations.

Detergent-solubilized microsomal preparations retained

this activity, and small peptides containing adjacent

Glu–Glu sequences were found to be substrates for the

enzyme. A general understanding of the properties of

this unique enzyme was gained from studies utilizing this

crude enzyme preparation, and these data have been

adequately reviewed. The vitamin K-dependent carbox-

ylation reaction does not require ATP, and the energy to

drive this carboxylation reaction is derived from the

oxidation of the reduced, hydronaphthoquinone, form

of vitamin K (vitamin KH

)byO

to form vitamin

K-2,3-epoxide (Figure 2). The lack of a requirement for

biotin and studies of the CO

=HCO

requirement

indicate that carbon dioxide rather than HCO

is the

active species in the carboxylation reaction. Active

substrates for the enzyme are 2-methyl-1,4-naphthoqui-

nones substituted at the 3-position with a rather

hydrophobic group. Although some differences in

biological activity can be measured, phylloquinone, the

plant form of the vitamin, and the predominant bacterial

menaquinones are all effective substrates (Figure 1).

Only a small fraction of the proteins secreted by the

liver to the plasma are vitamin K dependent, so an

efﬁcient mechanism for recognizing the precursors of the

vitamin K-dependent proteins is essential. The primary

gene products of the vitamin K-dependent proteins

contain a very homologous domain between the amino

terminus of the mature protein and the signal sequence.

This propeptide region appears to be both a docking or

recognition site for the enzyme and a modulator of the

activity of the enzyme by decreasing the apparent K

the Glu site substrate. The role of vitamin K in the

overall reaction catalyzed by the enzyme is to abstract

the hydrogen on the

-carbon of the glutamyl residue to

allow attack of CO

at this position. A number of studies

that utilized substrates tritiated at the

-carbon of each

Glu residue have been used to deﬁne the action and

stoichiometry involved and have shown that it was an

apparent equivalent stoichiometry between vitamin

K-2,3-epoxide formation and Gla formation. The

mechanism by which epoxide formation is coupled to

-hydrogen abstraction is key to a complete under-

standing of the role of vitamin K. The association

between epoxide formation, Gla formation, and

-C–H

bond cleavage has been studied, and the reaction

efﬁciency deﬁned as the ratio of Gla residues formed to

-C–H bonds cleaved has been shown to be independent

of Glu substrate concentrations, and to approach unity

at high CO

concentrations. Identiﬁcation of an

intermediate chemical form of vitamin K which could

be sufﬁciently basic to abstract the

-hydrogen of the

glutamyl residue has been a challenge. A possible

candidate is that ﬁrst proposed by Dowd, who suggested

that an initial attack of O

at the naphthoquinone

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

-methylene

hydrogen and leaves a carbanion that can interact with

. This pathway leads to the possibility that a second

atom of molecular oxygen can be incorporated into the

carbonyl group of the epoxide product as well as the

Phylloquinone

Menaquinone-9

Menaquinone-4

Menadione

FIGURE 1 Structures of vitamin K active compounds. Phylloquinone (vitamin K

) synthesized in plants is the main dietary form of vitamin K.

Menaquinone-9 is a prominent member of a series of menaquinones (vitamin K

) produced by intestinal bacteria, and menadione, vitamin K

,is

a synthetic compound that can be converted to menaquinone-4 by animal tissues.

390 VITAMIN K: BIOCHEMISTRY, METABOLISM, AND NUTRITIONAL ASPECTS

epoxide oxygen, and this partial dioxygenase activity

has been demonstrated. Although the general scheme

shown in Figure 2 is consistent with all of the available

data, the mechanism remains a hypothesis at this time.

Progress in purifying this enzyme was slow. Utilization

of bound propeptide as an afﬁnity column aided progress

in this ﬁeld, and the enzyme was eventually puriﬁed to

near homogeneity and cloned. The carboxylase is a

unique 758 amino acid residue protein with a sequence

suggestive of an integral membrane protein with a

number of membrane-spanning domains in the N termi-

nus, and a C-terminal domain located in the lumen of the

endoplasmic reticulum. It has been demonstrated that

the multiple Glu sites on the substrate for this enzyme are

carboxylated processively as they are bound to the

enzyme via their propeptide, while the Gla domain

undergoes intramolecular movement to reposition each

Glu for catalylsis, and that release of the carboxylated

substrate is the rate-limiting step in the reaction.

Metabolic Interconversion

of Vitamin K

Gla residues are not metabolized but are excreted in the

urine. The amount of Gla excreted by an adult human is

in the range of 50 mmol per day, so a similar amount

must be formed each day. As the dietary requirement of

vitamin K is only , 0.2 mmol per day, and tissue stores

are very low, it is clear that the vitamin K 2,3-epoxide

generated by the carboxylase can be actively recycled by

the pathway shown in Figure 3. The vitamin K-epoxide

reductase is capable of reducing both the epoxide to the

naphthoquinone, and naphthoquinone to the hydro-

naphthoquinone, and this is the enzyme which is

blocked by the common oral anticoagulant warfarin.

In addition to the epoxide reductase, the quinone and

hydronaphthoquinone forms of the vitamin can also be

interconverted by a number of NAD(P)H-linked

reductases including one that appears to be a micro-

somal-bound form of the extensively studied liver

DT-diaphorase activity. The epoxide reductase utilizes

a sulfhydryl compound as a reductant in vitro, but the

physiological reductant has not been identiﬁed.

Efforts to purify and characterize this enzyme have

not yet been successful.

A second metabolic pathway utilizing phylloqui-

none has been discovered more recently. Chick liver

contains a high concentration of menaquinone-4

(MK-4), and a number of extrahepatic tissues such as

brain, salivary gland, and pancreas of rats and humans

fed phylloquinone also contained a much higher

–

Gla

–

Vitamin K epoxide

–

Vitamin KH

–

Glu

FIGURE 2 The vitamin K-dependent,

-glutamyl carboxylase. The available data support an interaction of O

with vitamin KH

, the reduced

(hydronaphthoquinone) form of vitamin K, to form an oxygenated intermediate which is sufﬁciently basic to abstract the

-hydrogen of the

glutamyl residue. The products of this reaction are vitamin K-2,3-epoxide and a glutamyl carbanion. Attack of CO

on the carbanion leads to the

formation of a Gla residue. The bracketed peroxy, dioxetane, and alkoxide intermediates have not been identiﬁed in the enzyme catalyzed reaction

but are postulated based on model organic reactions, and the available data are consistent with their presence.

VITAMIN K: BIOCHEMISTRY, METABOLISM, AND NUTRITIONAL ASPECTS 391

concentration of MK-4 than of phylloquinone. Tissue

formation of MK-4 from phylloquinone fed to germ-

free rats and the observation that cultured kidney cells

can convert phylloquinone to MK-4 have now

demonstrated that bacterial action is not involved in

the conversion and that numerous cell types have the

ability to carry out this transformation. Details of the

mechanism of this conversion are lacking, but it seems

unlikely that a metabolic pathway leading to MK-4

has evolved unless there is some speciﬁc role for

this vitamin. This role is unlikely to involve the

vitamin K-dependent carboxylase, as phylloquinone

and MK-4 have similar activity as a substrate for this

enzymatic activity.

Nutritional Aspects of Vitamin K

Reports of uncomplicated adult deﬁciencies of vitamin

K are extremely rare, and most diets contain an

adequate amount (, 100 mg per day) of vitamin

K. Low lipid intake or the impaired lipid absorption

resulting from the lack of bile salts will adversely

affect vitamin K absorption, and depression of the

vitamin K-dependent coagulation factors has fre-

quently been reported in various malabsorption syn-

dromes. This classic example of a human vitamin K

deﬁciency is that of early vitamin K deﬁciency bleeding

occurring during the ﬁrst week of life in healthy

appearing neonates. Low placental transfer of phyllo-

quinone, low clotting factor levels, a sterile gut, and

the low vitamin K content of breast milk all contribute

to the disease. Although the incidence is low, the

mortality rate from intracranial bleeding is high, and

prevention by oral or parenteral administration of

vitamin K immediately following birth is the standard

cure. The presence of large amounts of osteocalcin in

bone and reports that low vitamin K intake or

increased amounts of circulating under-

-carboxylated

osteocalcin are correlated with low bone mineral

density or fracture rate have focused signiﬁcant

attention on a possible role for vitamin K in bone

health. A clear understanding of the signiﬁcance of

these reports is not yet available.

HCH

COOH

HC–COOH

COOH

NAD(P)

NAD(P)H

WarfarinWarfarin

SH SH

FIGURE 3 Tissue metabolism of vitamin K. Vitamin K epoxide formed in the carboxylation reaction is reduced to the quinone form of the

vitamin by a warfarin-sensitive pathway, vitamin K epoxide reductase, that is driven by a reduced dithiol. The naphthoquinone form of the vitamin

can be reduced to the hydronaphthoquinone form either by the same warfarin-sensitive dithiol-driven reductase or by one or more of the hepatic

NADH or NADPH-lined quinone reductases which are less sensitive to warfarin.

392 VITAMIN K: BIOCHEMISTRY, METABOLISM, AND NUTRITIONAL ASPECTS