Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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reduced at the upstream (or generator) electrode to

the quinol, which is then reoxidized at the down-

stream (or detector) electrode: the current generated

in the second reoxidation step is measured by the

detector and is proportional to the amount of vitamin

K passing through the electrochemical cell. This

method is capable of detecting 50 pg or less of phyl-

loquinone. Fluorescence detection again requires the

postcolumn generation of the quinols after the chro-

matographic separation of their respective quinones.

Reduction can be achieved by chemical, photochem-

ical, or electrochemical techniques, with fluorescence

detection providing similar sensitivity to redox

electrochemical detection (c.25–50 pg). Currently,

the most commonly employed reduction technique

involves a short dry-packed zinc or platinum oxide

column, configured between the separation column

and detector. Examples of chromatography achieved

for a representative infant formula and margarine are

illustrated in Figure 4, utilizing a C18 column and

fluorescence detection.

0036Further theoretical and practical details are pro-

vided in the methodological references listed in the

Further Reading

Ball GFM (1988) Fat-soluble Vitamin Assays in Food

Analysis: A Comprehensive review. London: Elsevier.

Bolton-Smith C, Price RJG, Fenton ST, Harrington DJ and

Shearer MJ (2000) Compilation of a provisional data-

base for the phylloquinone (vitamin K

) content of

foods. British Journal of Nutrition 83: 389–399.

Booth SL, McKeown NM, Lichtenstein AH et al. (2000) A

hydrogenated form of vitamin K: its relative bioavail-

ability and presence in the food supply. Journal of Food

Composition and Analysis 13: 311–317.

Booth SL and Sadowski JA (1997) Determination of phyl-

loquinone in foods by high-performance liquid chroma-

tography. Methods in Enzymology 282: 446–456.

Davidson RT, Foley AL, Engelke JA and Suttie JW (1998)

Conversion of dietary phylloquinone to tissue mena-

quinone-4 in rats is not dependent on gut bacteria.

Journal of Nutrition 128: 220–223.

Eitenmiller RR and Landon WO (1999) Vitamin K. In:

Eitenmiller RR and Landon WO (eds) Vitamin Analysis

for the Health and Food Sciences, pp. 149–184. Boca

Raton: CRC Press.

Indyk HE and Woollard DC (2000) Determination of vita-

min K in milk and infant formulas by liquid chromatog-

raphy: Collaborative study. Journal of the Association of

Analytical Chemists International 83: 121–130.

Piironen V and Koivu T (2000) Quality of vitamin K analy-

sis and food composition data in Finland. Food Chemis-

try 68: 223–226.

Schurgers LJ, Geleijnse JM, Grobbee DE et al. (1999) Nu-

tritional intake of vitamins K

(phylloquinone) and K

(menaquinone) in The Netherlands. Journal of Nutrition

and Environmental Medicine 9: 115–122.

Shearer MJ (1992) Vitamin K metabolism and Nutriture.

Blood Reviews 6: 92–104.

Shearer MJ, Bach A and Kohlmeier M (1996) Chemistry,

nutritional sources, tissue distribution and metabolism

of vitamin K with special reference to bone health. Jour-

nal of Nutrition 126: 1181S–1186S.

Suttie JW (1991) Vitamin K. In: Machlin LJ (ed.) Hand-

book of Vitamins, 2nd edn. pp. 145–194. New York:

Marcel Dekker.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

2345

Minutes

Volts

MK-4

dihydro-K

Volts

678910

012345

Minutes

678910

0.0

0.1

0.2

0.3

0.4

(a)

(b)

fig0004 Figure 4 Liquid chromatography obtained for (a) infant formula

and (b) margarine. C18 column, isocratic elution, postcolumn

reduction, and fluorescence detection.

6038 VITAMIN K/Properties and Determination

Physiology

M J Shearer, St Thomas’s Hospital, London, UK

Background

0001 From the 1930s until the 1970s, the only known role

of vitamin K was as an essential antihemorrhagic

factor, which initially had been shown to promote

the synthesis of active prothrombin (factor II), but

later also of factors VII, IX, and X. Collectively,

these four factors, all synthesized in the liver and

then released into the circulation, became known

as the classical vitamin K-dependent coagulation

factors. In 1974, it was shown that vitamin K was

required as a cofactor for the enzymic conversion of

specific glutamate residues in the prothrombin mol-

ecule to g-carboxyglutamate (Gla) residues. The elu-

cidation of this biochemical pathway leading to the

synthesis of a new amino acid was the impetus that led

to the discovery of many more vitamin K-dependent

proteins. These proteins, also known as Gla proteins,

are now known to have a widespread tissue distribu-

tion. They include further coagulation proteins (pro-

teins C, S, and Z) and others with putative roles in the

regulation of bone turnover (osteocalcin), and bone

calcification (matrix Gla protein (MGP)). New evi-

dence suggests that MGP is also a powerful inhibitor

of vascular calcification. Other suspected functions of

Gla proteins are in vascular repair processes (Protein

S), cell-cycle regulation, cell–cell adhesion (Gas6),

and signal transduction (Gas6 and proline-rich Gla

proteins). For the most part, the precise physiological

function of these new Gla proteins is poorly under-

stood. However, nutritional and epidemiological

studies in humans suggest that optimal intakes of

vitamin K are necessary to maintain skeletal and car-

diovascular health. Despite the possibility of these

new health roles, the only clearly defined deficiency

syndrome is bleeding due to a lack of functional

molecules of the vitamin K-dependent coagulation

factors II, VII, IX, and X. Bleeding due to vitamin K

deficiency is rare in adults but is more common in

infants during the first 6 months of life. The potential

risk of serious life-threatening bleeding in infancy

means that most countries recommend that all new-

borns should receive vitamin K prophylaxis.

Intestinal Absorption and Bioavailability

0002 Dietary vitamin K, mainly as phylloquinone, is ab-

sorbed chemically unchanged from the proximal in-

testine after solubilization into mixed micelles

composed of bile salts and the products of pancreatic

lipolysis. In healthy adults, the efficiency of absorp-

tion of phylloquinone in its free form is about 80%.

The bioavailability of vitamin K from foods varies

according to food type and the associated matrix.

Thus, the efficiency of absorption of phylloquinone

may be as low as 10% from green leafy vegetables

(e.g., spinach) in which the vitamin is tightly associ-

ated with chloroplast membranes. Less is known

about the absorption of dietary menaquinones, but

the efficiency of absorption decreases with increasing

length of the side-chain.

0003Menaquinones (MKs) are also synthesized by the

intestinal microflora, predominately in the colon.

Major forms are MK-10 and MK-11 synthesized by

Bacteroides species, MK-8 by Enterobacteria, MK-7

by Veillonella, and MK-6 by Eubacterium lentum.It

is commonly held that microfloral synthesis provides

an important nutritional source of vitamin K for

humans, but direct evidence is lacking. A microfloral

contribution of vitamin K is supported by the findings

that very-long-chain MKs 10–13 are rare in foods but

make up the majority of hepatic stores. However, all

experimental evidence suggests that the absorption of

menaquinones from the large intestine is an extremely

inefficient process.

Transport in Plasma and Tissue

Distribution

0004After intestinal absorption, vitamin K is incorporated

into chylomicrons, secreted into the lymph, and

enters the blood via the lacteals. Once in the

circulation, phylloquinone is rapidly cleared at a

rate consistent with its continuing association with

chylomicrons and the chylomicron remnants that are

produced by lipoprotein lipase hydrolysis at the sur-

face of capillary endothelial cells. After an overnight

fast, more than half of the circulating phylloquinone

is still associated with triglyceride-rich lipoproteins,

with the remainder being equally distributed between

low-density and high-density lipoproteins. Phyllo-

quinone is the major circulating form of vitamin K,

but menaquinones MK-7 and MK-8 have been

detected at lower concentrations.

0005Dietary vitamin K is delivered to the liver and

possibly other tissues, including bone marrow, in the

form of chylomicron remnants. The cellular uptake of

vitamin K is mediated by apolipoprotein E in the

chylomicron remnants and heparin sulfate proteogly-

cans on the cell surface. The liver, the site of synthesis

of the vitamin K-dependent coagulation proteins, has

a limited capacity for storing phylloquinone. Thus,

the hepatic reserves of phylloquinone are several

thousand-fold lower than those of vitamin A, despite

the fact that dietary intakes of phylloquinone are

VITAMIN K/Physiology 6039

only about 10-fold lower. The distribution of molecu-

lar forms of vitamin K in the liver is quite different

from that in plasma in that the major transport form,

phylloquinone, represents only about 10% of total

stores; the remainder comprise bacterial menaquin-

ones, mainly MKs 7–13. Long-chain menaquinones

are also present in other nonhepatic tissues but at

lower concentrations than the liver. Some tissues

such as the brain and pancreas seem to have the

capacity to selectively convert phylloquinone to

MK-4, but little is known about the biochemistry or

physiological significance of this enrichment.

Catabolism and Turnover

0006 The metabolic transformations leading to the elimin-

ation of vitamin K from the body occur exclusively in

the liver. Phylloquinone is rapidly degraded with

about 20% of a single dose being excreted in the

urine within 3 days and about 40–50% excreted in

the feces via the bile. The hepatic reserves of individ-

uals on a low-phylloquinone diet decline rapidly. The

overall picture is that phylloquinone has a fast turn-

over time, suggesting that the body stores are being

constantly replenished.

0007 The route of hepatic catabolism leading to the

urinary excretion of vitamin K proceeds by oxidative

degradation of the phytyl side-chain, probably in-

volving the same enzymes used for the o-methyl and

b-oxidation of fatty acids, steroids, and prosta-

glandins. The end products of this pathway are

shown in Figure 1. Two major aglycone metabolites

have been identified; they are carboxylic acids with 5

and 7 carbon atom side-chains that are excreted in the

urine as glucuronide conjugates. The biliary metabol-

ites have not been clearly identified but are initially

excreted as water-soluble conjugates and become

lipid-soluble during their passage through the gut,

probably through deconjugation by the intestinal

microflora. There is no evidence that body stores of

vitamin K can be conserved by an enterohepatic cir-

culation, since vitamin K itself is not excreted in bile,

and the side-chain shortened carboxylic acid metab-

olites are not biologically active.

Cofactor Role of Vitamin K

Vitamin K-dependent Carboxylation of Glutamate

Residues

0008The only unequivocal biochemical role for vitamin K

is as a cofactor for a unique posttranslational

chemical modification confined to a small group

of unusual calcium-binding proteins. The vitamin

K-dependent step is a carboxylation reaction that

transforms selective glutamic (Glu) residues in the

protein precursor into Gla. The reaction shown in

Figure 2 is catalyzed by a microsomal enzyme called

vitamin K-dependent (g-glutamyl) carboxylase and

requires molecular oxygen and carbon dioxide. The

form of vitamin K required as the cofactor is not

the stable quinone structure found in the diet but the

reduced quinol (or hydroquinone) structure (KH

The subsequent oxidation of vitamin KH

to vitamin

K 2,3 epoxide is coupled to the abstraction of a

proton from the g-carbon of the Glu residue to gener-

ate a carbanion that reacts with CO

to yield the final

Gla product. The biological relevance of the Gla

structure is that it forms a cage structure to which

divalent metal ions such as calcium may be bound.

In the case of the vitamin K-dependent procoagu-

lants, this induces the conformational change and

COOH

III

fig0001 Figure 1 Simplified pathway and chemical structures of the

two major urinary metabolites of phylloquinone in their

aglycone forms (structures I and II). In acidic conditions, metab-

olite I may cyclize to produce phylloquinone g-lactone (structure

III). (I) 2-Methyl-3-(5

-carboxy-3

-methyl-2

-pentenyl)-1,4-naphtho-

quinone; (II) 2-methyl-3-(3

-carboxy-3

-methylpropyl)-1,4-

naphthoquinone;(III) 2-methyl-3-(5

-carboxy-3

-hydroxy-3

methylpentyl)-1,4-naphthoquinone lactone.

Protein

precursors

Glutamyl

residues

Vitamin K

COOH

Completed

proteins

γ-Carboxyglutamyl

residues

COOH

fig0002Figure 2 Posttranslational vitamin K-dependent carboxylation

reaction showing the conversion of protein-bound glutamyl resi-

dues (Glu) to g-carboxyglutamate (Gla) residues.

6040 VITAMIN K/Physiology

membrane-binding properties that are essential for

their biological activity.

Vitamin K Epoxide Cycle

0009 In all tissues and cells that synthesize Gla proteins,

vitamin K-dependent carboxylation is intimately

linked to a metabolic sequence known as the vita-

min K-epoxide cycle. This cycle and the associated

enzyme activities are shown in Figure 3. During g -

glutamyl carboxylation, vitamin KH

is transformed

to vitamin K epoxide (enzyme activity E

). The epox-

ide product is recycled in two steps; first by a vitamin

K epoxide reductase activity (E

) to produce vitamin

K quinone, and second by a quinone reductase activ-

ity (E

) to produce the coenzyme vitamin KH

. Both

these activities are thiol-dependent and are probably

effected by the same enzyme.

0010 An important property of the dithiol-dependent

epoxide and quinone reductase activities is their sen-

sitivity to certain antagonists, especially those based

on 4-hydroxycoumarin (e.g., warfarin) or indandione

structures. Drugs based on these structures have long

been used as oral anticoagulants, and it is now clear

that their anticoagulant action is based on their abil-

ity to inhibit the epoxide reductase activity (E

) and

block the recycling of the vitamin. The dithiol-

dependent quinone reductase (E

) is also sensitive to

warfarin, but there is a second quinone reductase

activity catalyzed by an NAD(P)H-dependent enzyme

activity (E

) that is less sensitive to warfarin inhib-

ition, and this provides an alternative pathway for the

reduction of vitamin K to KH

in the presence of

warfarin or other oral anticoagulant drugs. Thera-

peutic anticoagulation is dependent on a balance

being achieved between the inhibition of the recycling

enzymes and the amount of dietary vitamin K that

can enter the cycle to support carboxylation at a

reduced efficiency.

Vitamin K-dependent (Gla) Proteins

Distribution and Structure

0011g-Glutamyl carboxylase activity is present in a wide

variety of extrahepatic tissues such as bone, kidney,

placenta, pancreas, skin, spleen, lung, testis, and the

vessel wall. New Gla proteins continue to be dis-

covered; the present list together with their tissue

distribution is shown in Table 1. Some of these Gla

proteins are found in only one major tissue (e.g., pro-

coagulant factors in liver and osteocalcin in bone),

while others (e.g., matrix Gla protein and protein S)

are more widely distributed.

0012All Gla-containing proteins are secretory proteins

and are characterized by an intracellular precursor

form possessing a leader sequence that facilitates spe-

cific posttranslational events and is then normally

cleaved before secretion. Within the leader sequence,

a hydrophobic presequence facilitates the transloca-

tion of the precursor Gla proteins across the endo-

plasmic reticulum, while a second prosequence serves

as a recognition signal for the vitamin K-dependent

carboxylase. With the exception of matrix-Gla pro-

tein, the prosequence of currently characterized Gla

proteins is located immediately before the N-terminal

of the mature protein. In matrix-Gla protein, the

carboxylation recognition site is internal and is

retained in the mature protein. The number of

Gla residues varies: the four classical vitamin K-

dependent clotting factors contain 10–12 Gla resi-

dues, whereas the much smaller MGP and osteocalcin

contain five and three Gla residues, respectively.

Role of Gla proteins

0013Hemostasis (procoagulants and anticoagulants) The

vitamin K-dependent coagulation proteins comprise

four plasma procoagulants (factors II, VII, IX, and X)

and two feedback anticoagulants (proteins C and S).

The physiological function of a seventh plasma pro-

tein (protein Z) is less established but seems to play a

role in dampening coagulation by acting as a cofactor

for the inhibition of factor Xa. The four vitamin

K-dependent procoagulants have long been known

to play a central role in the blood clotting cascade

leading to the formation of a fibrin clot. Circulating

Warfarin Warfarin

Quinone

Quinol

Epoxide

NADH + H

NAD

Glu Gla

X-(SH)

X-S

fig0003 Figure 3 Scheme showing the reactions and enzyme activities

(denoted by E

, and E

) of the vitamin K-epoxide cycle. The

active form of vitamin K needed for the conversion of glutamate

residues (Glu) to g-carboxyglutamate (Gla) residues is the re-

duced form, vitamin K quinol (K quinol). The carboxylation reac-

tion is driven by a vitamin K-dependent carboxylase activity (E

which simultaneously converts K quinol to vitamin K 2,3-epoxide

(K epoxide). K epoxide is reduced back to the quinone (K quin-

one) by vitamin K epoxide reductase (E

). The cycle is completed

by the reduction of recycled K quinone by a vitamin K reductase

activity (E

). Both E

and E

reductase activities are dithiol-

dependent (X-(SH

) and X-S

denote reduced and oxidized

dithiols) and are inhibited by coumarin anticoagulants such as

warfarin. Dietary vitamin K (K quinone) may enter the cycle via an

NAD(P)H-dependent vitamin K reductase activity (E

), which is

not inhibited by warfarin.

VITAMIN K/Physiology 6041

as inactive forms (or zymogens) of serine proteases,

their biological activity is dependent on their ability

to bind to anionic phospholipid surfaces where cleav-

age of the zymogens yields the active protease clotting

factors. The Gla residues of these clotting factors

provide an efficient chelating site for calcium ions

that enables ion bridges to be made between the

factor and the surface phospholipids of platelets and

endothelial cells where, together with other cofactors,

they form membrane-bound enzyme complexes. The

intricate control of coagulation is illustrated by the

fact that another Gla protein (protein C), once acti-

vated, performs an anticoagulant role by specifically

degrading phospholipid-bound activated factors V

and VIII in the presence of calcium. This anticoagu-

lant activity of activated protein C is dependent on yet

another Gla protein (protein S) that acts as a syner-

gistic cofactor by enhancing the binding of activated

protein C to negatively charged phospholipids.

0014 Bone metabolism The discovery in 1975 that bone

tissue contains a Gla protein, which is also one of the

most abundant proteins in the body, has revolution-

ized earlier thinking that vitamin K is only needed for

coagulation. This protein, called osteocalcin or bone

Gla protein, accounts for about 15% of the noncol-

lagenous proteins in the bone of most vertebrate

species, although this proportion is less in humans.

The three Gla residues of osteocalcin project away

from the Gla helix, and their periodic spacing com-

plements the interatomic spacing of the calcium ions

of the hydroxyapatite lattice; this is thought to ac-

count for the strong mineral-binding characteristics

of the fully carboxylated form of osteocalcin. The

adsorption affinity of osteocalcin for hydroxyapatite

and other features such as its high degree of conser-

vation throughout evolution, its regulation by vita-

min D, and its chemotractant properties suggest an

important role for osteocalcin in bone metabolism,

although the nature of this role still remains

unknown. Osteocalcin is specifically synthesized by

osteoblasts, and since its concentration in blood

reflects osteoblastic activity, measurements of total

osteocalcin have become clinically important in the

diagnosis of bone disease.

0015Besides osteocalcin, bone tissue contains lower

concentrations of other Gla proteins; matrix Gla-

protein (MGP), protein S, and Gas6 (Table 1). MGP

is a 10-kDa secretory protein, with five residues of

Gla. It was first isolated from demineralized extracts

of bone by Price in 1983 but is expressed by a wide

variety of tissues and cell types. Its function in bone is

unclear but is probably related to its capacity to

inhibit localized calcification as recently shown

for arteries. Transgenic MGP-deficient mice show

inappropriate calcification of the cartilage, including

the growth plate, and a phenotype characterized by

short stature, osteopenia, and fractures. This function

is consistent with the effects of the vitamin K antag-

onist warfarin, which, when given in utero or to

young animals, results in excessive mineralization

and premature closure of the epiphyseal growth

plate. An important role for Gla proteins during

human fetal bone development is suggested by the

bone defects often seen in infants born to mothers

who had received vitamin K antagonists such as war-

farin during the first trimester of pregnancy. Evidence

that warfarin embryopathy is mediated by a dysfunc-

tional MGP comes from the demonstration that mu-

tations encoding MGP are responsible for a rare

inherited disorder called Keutal syndrome, which is

characterized by a similar abnormal calcification of

cartilage. The functions of protein S and Gas6 in bone

are presently unknown.

0016Vascular calcification A series of remarkable studies

since 1997 have firmly established MGP as a potent

inhibitor of calcification of arteries and cartilage.

Thus, targeted deletion of the MGP gene in mice

resulted in rapid focal calcification of the elastic

tbl0001 Table 1 Distribution and roles of vitamin K-dependent (Gla) proteins

Gla protein Tissue Role

Prothrombin (factor II), factors

VII, IX, and X

Liver (then plasma) Blood clotting (procoagulants)

Protein C Liver (then plasma) Blood clotting (anticoagulant)

Protein S Liver (then plasma),

endothelium, bone

Anticoagulant role (cofactor for protein C); role in bone unknown

Osteocalcin (or bone Gla

protein)

Bone Implicated in bone remodeling

Matrix Gla protein Most tissues Inhibits calcification of arteries and cartilage

Gas 6 Most tissues Implicated in cell-cycle regulation, cell survival, cell proliferation,

cell adhesion, platelet signaling

Proline-rich Gla proteins Most tissues Unknown, but protein structures suggest roles as cell-surface

receptors

6042 VITAMIN K/Physiology

lamellae of the arterial media but not of the arterioles,

capillaries, or veins. This process begins at birth and

by 3–6 weeks is sufficiently severe to cause the arter-

ies to become rigid tubes that fracture, causing death

from bleeding. Most of the phenotypic features of

arterial calcification seen in MGP-deficient mice can

be mimicked, albeit to a somewhat muted degree, by

the vitamin K antagonist warfarin. This effect of war-

farin points to the need for Gla residues, and hence

vitamin K, to the function of MGP. Overall, the

dramatic features of both genetic and biochemi-

cal models of MGP depletion strongly suggest that

MGP is central to the process by which calcification

of arteries is normally inhibited in vivo and that there

is no effective alternative inhibitor at aortal sites.

Assessment of Vitamin K Status

0017 The traditional criterion for vitamin K sufficiency is

the maintenance of normal plasma concentrations of

fully g-carboxylated vitamin K-dependent procoagu-

lants II, VII, IX, and X. The presence of a clinically

significant vitamin K-deficient state is usually investi-

gated by global coagulation assays such as the one-

stage prothrombin time. Such assays are insensitive to

small changes in g-carboxylation of the vitamin

K-dependent coagulants and, while appropriate for

detecting overt vitamin K deficiency, are not useful

for picking up suboptimal states of vitamin K

deficiency.

0018 Modern assessments of vitamin K status need to

take into account both coagulation and noncoagula-

tion roles of Gla proteins and their different sites of

synthesis. When vitamin K is in short supply, or its

action is blocked by an oral anticoagulant such as

warfarin, undercarboxylated (des-g -carboxy) species

of Gla proteins are produced at the tissue site of

synthesis and, in some species, including humans,

are secreted into the circulation. Collectively, these

undercarboxylated species are known as proteins in-

duced by vitamin Kabsence or antagonism (PIVKA).

In the case of the coagulation protein prothrombin,

specific and sensitive immunoassays for undercar-

boxylated prothrombin (PIVKA-II) provide a power-

ful marker of the coagulation status of vitamin K

because they can detect small decreases in the Gla

content of prothrombin well before any changes

occur in conventional coagulation tests. Similarly,

the assay of circulating undercarboxylated species of

osteocalcin provides a specific functional marker to

assess the sufficiency of vitamin K for the carboxyla-

tion of bone Gla proteins. Since prothrombin and

osteocalcin are synthesized in the liver and bone,

respectively, the detection of their respective under-

carboxylated species allows the vitamin K status of

these tissues to be assessed independently. Such stud-

ies have shown that the carboxylation of osteocalcin

in bone is more susceptible to reduced dietary intakes

of vitamin K than the carboxylation of the classical

coagulation proteins. Measurements of undercar-

boxylated osteocalcin are being increasingly used to

assess the importance of optimal vitamin K intakes

with respect to bone health. The excretion of free Gla

in urine represents the final catabolic product of all

Gla proteins, and its measurement has also been

shown to reflect dietary intakes and status. Another

method for assessing vitamin K status is from direct

serum assays of phylloquinone, the predominant

transport form of vitamin K. A low serum phyllo-

quinone is a good indicator of a poor overall vitamin

K status.

Vitamin K and Health

Role in Hemostasis

0019Overt vitamin K deficiency in adults, resulting in

clinical bleeding, is almost unknown except as a con-

sequence of underlying disease, most commonly

resulting from hepatointestinal disorders that disturb

the absorption or utilization of the vitamin. However,

oral coumarin anticoagulant drugs, such as warfarin,

are widely used in the prevention and management of

thromboembolic disease. These vitamin K antagon-

ists are used to induce an artifical but controlled state

of vitamin K deficiency, the aim being to reduce

the circulating levels of the vitamin K-dependent clot-

ting factors to within a predetermined target range

depending on the clinical condition.

0020In contrast to adults, bleeding due to vitamin K

deficiency may occur spontaneously in the first few

months of life. The resulting bleeding syndrome is

traditionally known as hemorrhagic disease of the

newborn, in recognition of the Boston physician

Townsend, who is credited with the first description

of the syndrome in 1894. The modern name is vita-

min K deficiency bleeding (VKDB) of early infancy.

Until the late 1960s, VKDB was considered to be

solely a problem of the first week of life (classical

VKDB), but two other forms (early and late VKDB)

have now been recognized (Table 2). Late VKDB,

peaking between the third and sixth weeks of life, is

the most common syndrome and is still a significant

worldwide cause of infant morbidity and mortality. In

different parts of the world, some two to 100 cases

per 100 000 births of late VKDB have been reported,

with a particularly high incidence in the Far East.

Unlike the classical form, late VKDB has a high inci-

dence of intracranial hemorrhage, resulting in

death or severe and permanent brain damage in

VITAMIN K/Physiology 6043

around 50% of cases. One major risk factor, exclusive

breast-feeding, is common to both classical and late

syndromes and is probably related to the relatively

low concentrations of vitamin K in breast milk

(1–2 mgl

1

) compared to about 50 mgl

1

in formula

feeds. Another probable etiological factor is the pre-

carious hepatic stores of vitamin K in newborns be-

cause of poor placental transfer of phylloquinone and

the lack of neonatal stores of bacterial menaquinones,

which only build up slowly after bacterial coloniza-

tion of the intestine. Some infants who develop late

VKDB have abnormalities of liver function, leading

to cholestasis and an impaired absorption efficiency

of vitamin K.

0021 By the early 1980s, reports in the literature were

suggesting a worldwide rise in the incidence of late

VKDB. This resurgence may have been related to

both a decline in routine vitamin K prophylaxis and

the promotion of exclusive breast-feeding. In recent

years, many countries have reemphasized the need

to give all babies extra vitamin K as a public-health

measure. Often, this was given as a single intramus-

cular dose at birth, but since 1992, concerns about an

epidemiological association between the intramuscu-

lar route and later childhood cancer has seen a shift in

some countries towards oral prophylaxis. However,

there is evidence that even multidose oral regimens

fail to give full protection against late VKDB and that

this is because there is an underlying reservoir of

infants who have a reduced efficiency of absorption

due to underlying and unrecognized cholestasis. Sev-

eral epidemiological studies conducted since 1992

have not substantiated the link between vitamin K

prophylaxis and cancer.

Role in Bone Health

0022 Despite mechanistic gaps in our knowledge, a picture

is emerging that a suboptimal vitamin K status may

play a role in the pathogenesis of osteoporosis. Much

of this evidence is still circumstantial, being based on

associations of fracture risk and bone mineral density

with indices of poor vitamin K status and low dietary

intakes. Of particular note is that undercarboxylated

osteocalcin is an independent risk factor of both

hip-fracture risk and bone mineral density. However,

osteoporosis is a complex disease, and without a

fuller understanding of the biological function of

osteocalcin, the interpretation of these associations

with vitamin K status remains uncertain.

0023In the near future, the results from a number of

long-term intervention studies on the effects of vita-

min K supplementation on bone health will become

available. These may help to answer the question of

whether extra intakes of vitamin K can influence bone

mineral density and bone turnover. Most of these

studies have used phylloquinone because this is the

major dietary form, but in Japan, the effects of very

large doses of MK-4 (menatetranone) on bone health

are being studied. Both phylloquinone and MK-4 are

known to promote the g-carboxylation of osteocal-

cin, but these forms differ in other important respects.

Thus, there is evidence that tissues can convert phyl-

loquinone into MK-4 in vivo (but not vice versa) and

that MK-4 may have a direct inhibitory effect on bone

resorption. In an intervention study in which Japan-

ese women with osteoporosis were treated with a

high-dose MK-4 regimen, the incidence of fractures

was significantly reduced, and the rate of bone loss

was decreased.

Dietary Intakes and Requirements

0024The major dietary source of vitamin K is phylloquin-

one. In Western societies, the majority of phylloquin-

one intakes (up to 50%) comes from green leafy

vegetables followed by certain vegetable oils such as

soybean, canola (rapeseed), and olive oils. Mixed

dishes and meals contribute about 15% of total phyl-

loquinone intake, largely because of their oil content.

The wide range of the vitamin K content of different

food items means that individual daily intakes may

vary widely, but average adult intakes mostly range

from 60 to 120 mg per day. Intakes of this order are

more than sufficient to maintain normal hemostasis,

and even intakes as low as 10 mg per day for a few

weeks have no effect on global coagulation assays.

tbl0002 Table 2 Classification of vitamin K deficiency in the newborn (VKDB)

VKDB syndrome Time of

presentation

Commonbleedingsites Etiological factors

Early 0–24 h Cephalohematoma, intracranial,

intrathoracic, intraabdominal

Maternal drugs (e.g., warfarin, anticonvulsants)

Classical 1–7 days Gastrointestinal, skin, nasal,

circumcision

Mainly idiopathic, breast-feeding (may be related to

low milk intakes)

Late 2–26 weeks Intracranial, skin, gastrointestinal Mainly idiopathic, breast feeding, some degree of

cholestasis often present;

May reflect underlying disease (e.g., biliary

atresia, a-1-antitrypsin deficiency, cystic fibrosis)

6044 VITAMIN K/Physiology

However, more sensitive measures of vitamin K status

(e.g., undercarboxylation of Gla proteins, urinary Gla

excretion, and serum phylloquinone) respond readily

to moderate changes in dietary intakes. One conclu-

sion from nutritional depletion/repletion studies is

that the daily intakes of vitamin K needed to sustain

the posttranslational g-carboxylation of extrahepatic

Gla proteins such as osteocalcin are higher than those

needed for the coagulation Gla proteins synthesized

in the liver. Therefore, previous recommendations of

a daily intake of 1 mg per kilogram of body weight,

which have been based on the coagulation role of

vitamin K, may need to be revised upwards if evi-

dence continues to harden on other potential health

benefits such as the amelioration of osteoporosis and

arterial calcification. Studies in the UK and USA al-

ready suggest that a substantial proportion of their

populations have intakes below 1 mg per kilogram of

body weight. What this optimal intake should be

must await further research on the physiological

function of Gla proteins, the further delineation of

functional markers of vitamin K status, and studies of

the health consequences of different intakes.

See also: Bone; Calcium: Physiology; Dietary Reference

Values; Dietary Requirements of Adults; Dietary

Surveys: Surveys of National Food Intake; Surveys of

Food Intakes in Groups and Individuals; Infants:

Nutritional Requirements; Lipoproteins; Microflora of

the Intestine: Role and Effects; Osteoporosis; Vitamins:

Overview

Further Reading

Angelillo-Scherrer A, Garcia de Frutos P, Aparicio C et al.

(2001) Deficiency or inhibition of Gas6 causes platelet

dysfunction and protects mice against thrombosis.

Nature Medicine 7: 215–221.

Benzakour O and Kanthou C (2000) The anticoagulant

factor, protein S, is produced by cultured human vascu-

lar smooth muscle cells and its expression is up-regu-

lated by thrombin. Blood 95: 2008–2014.

Booth SL and Suttie JW (1998) Dietary intake and ad-

equacy of vitamin K. Journal of Nutrition 128: 785–788.

Broze GJ, Jr. (2001) Protein Z-dependent regulation of

coagulation. Thrombosis and Haemostasis 86: 8–13.

Furie B, Bouchard BA and Furie BC (1999) Vitamin

K-dependent biosynthesis of g-carboxyglutamic acid.

Blood 93: 1798–1808.

Institute of Medicine (2001) Dietary Reference Intakes for

Vitamin A, Vitamin K, Arsenic, Boron, Chromium,

Copper, Iodine, Iron, Manganese, Molybdenum, Nickel,

Silicon, Vanadium, and Zinc, pp. 162–196. Washington,

DC: National Academy Press.

Kulman JD, Harris JE, Xie L and Davie EW (2001) Identi-

fication of two novel transmembrane g-carboxyglutamic

acid proteins expressed broadly in fetal and adult tissues.

Proceedings of the National Academy of Sciences 98:

1370–1375.

Luo G, Ducy P, McKee MD et al. (1997) Spontaneous

calcification of arteries and cartilage in mice lacking

matrix GLA protein. Nature 386: 78–81.

Nelsestuen GL, Shah AM and Harvey SB (2000) Vitamin K-

dependent proteins. Vitamins and Hormones 58:

355–389.

Newman P and Shearer MJ (1998) Vitamin K metabolism.

In: Quinn PJ and Kagan VE, (eds) Subcellular Biochem-

istry, Volume 30, Fat-soluble Vitamins, pp. 455–488.

New York: Plenum Press.

Olson RE (1999) Vitamin K. In: Shils ME, Olson JA, Shike

M and Ross AC (eds) Modern Nutrition in Health and

Disease 9th edn, pp. 363–380. Baltimore, MD: Lippin-

cott, Williams & Wilkins.

Shanahan CM, Proudfoot D, Farznaneh-Far A and Weiss-

berg PL (1998) The role of Gla proteins in vascular

calcification. Critical Reviews in Eukaryotic Gene

Expression 8: 357–375.

Shearer MJ (1997) The roles of vitamins D and K in bone

health and osteoporosis prevention. Proceedings of the

Nutrition Society 56: 915–937.

Shearer MJ (2000) Role of vitamin K and Gla proteins in

the pathophysiology of osteoporosis and vascular calci-

fication. Current Opinion in Clinical Nutrition and

Metabolic Care 3: 433–438.

Tsaioun KI (1999) Vitamin K-dependent proteins in the

developing and aging nervous system. Nutrition

Reviews 57: 231–240.

VITAMIN K/Physiology 6045

VITAMINS

Contents

Overview

Determination

Overview

P M Finglas, Institute of Food Research, Norwich

Research Park, Norwich, UK

Overview

0001 This article covers the definition, a brief historical

account of the discovery, classification and nutri-

tional role, forms found in food, and effects of

processing on vitamin stability.

0002 Vitamins are defined as a group of complex organic

micronutrients present in small amounts in food or

nutritional supplements that the body requires for its

normal metabolism and function. They cannot be

synthesized by humans in sufficient quantity for

normal metabolic requirements. Certain substances

that are considered vitamins can be synthesized

in small amounts by intestinal-tract bacteria (e.g.,

B-groups). Each vitamin consists of a mixed group

of compounds, which may not be related to each

other chemically and are therefore classified by func-

tion. (See Microflora of the Intestine: Role and Effects.)

History

0003 It was not until the early 1900s that the existence of

the ‘accessory food factors’ was recognized. Indeed,

the term ‘vitamine’ was first used by a Polish bio-

chemist, named Funk, to describe an ‘accessory food

factor.’ However, deficiency diseases were well

known many centuries earlier, the earliest docu-

mented disease being beriberi, which was recorded

in China around 2600 bc. Yet, it was not until the

end of the eighteenth century that the nutritional

etiology of the disease was established. In 1885, a

Japanese physician named Takaki succeeded in

reducing the effect of beriberi among Japanese sailors

by feeding them a diet containing more meat and

vegetables, and less rice. Then, in 1890, a Dutch

doctor named Eijkman reproduced symptoms of the

disease in chickens fed exclusively with shelled rice.

His collaborator, Grijns, showed that these symptoms

disappeared by adding rice husks to the chicken diet.

Finally, in 1912, Funk isolated the antiberriberri

factor from the cuticle of the rice and named it ‘vita-

mine,’ the ‘life-essential amine.’ This term was

adopted to name all the nutritional factors respon-

sible for deficiency diseases.

0004Similarly, scurvy and night blindness were de-

scribed in ancient Egypt around 1500 bc. During

the period ad 1400–1600, the explorers Vasco de

Gamma and Magellan described in detail the

scurvy and night blindness that had devastated

their ships’ crew. A century earlier, Jacques Cartier

had succeeded in saving a good many sailors thanks

to an Indian medicine based on pine needles. Finally,

in 1747, James Lind found the preventive effect of

citrus fruit using controlled experiments at sea, and

this was shortly followed by the demonstration,

during the voyages of Captain Cook in the period

1768–1771, that prolonged sea voyages were possible

without the ravages of scurvy. (See Hypovitaminosis

A; Scurvy.)

0005Thirteen vitamins were isolated, identified, and

synthesized between 1929 and 1975. The last vitamin

to be discovered was vitamin B

in 1948. Many

researchers who contributed to the fundamental dis-

coveries have been honored by the Nobel Prizes Com-

mittee, among them: Szent-Gyorgyi and King for the

simultaneous discovery of ascorbic acid in 1928; Kar-

rer for his work on vitamin A and b-carotene between

1928 and 1933; Dam and Doisy for the discovery and

chemical nature of vitamin K in 1943; and Hodgkin

for the structural determination of vitamin B

1964. (See Carotenoids: Physiology.)

0006Nowadays, vitamin research is still very active. The

understanding of the precise, complex role of vita-

mins has made remarkable progress owing to the

study of vitamin-dependent metabolite diseases of

genetic origin. More recently, because of their anti-

oxidant properties, some fat-soluble vitamins,

notably vitamins A, E, and C, and provitamin A

compounds (carotenoids; including b-carotene,

lutein, and lycopene), are also considered to play an

important role in the prevention against chronic

degenerative diseases and cancers. Also, it has been

shown that folic acid can reduce significantly the

incidence of neural tube defects (such as spina bifida),

and folates, in general, may protect against vascular

diseases and some cancers, notably colonic cancer.

6046 VITAMINS/Overview

(See Antioxidants: Natural Antioxidants; Synthetic

Antioxidants; Synthetic Antioxidants, Characteriza-

tion and Analysis; Role of Antioxidant Nutrients in

Defence Systems; Cancer: Epidemiology; Carcino-

gens in the Food Chain; Coronary Heart Disease:

Etiology and Risk Factor; Pregnancy: Maternal Diet,

Vitamins, and Neural Tube Defects; Vitamin B

Properties, and Determination.)

Classification and properties

0007 A number of factors can make the identification and

classification of vitamins difficult: (1) a vitamin can

exist in a number of different structures with similar,

or different, biological activities; (2) the biological

activity is organism-dependent; (3) the physiologic-

ally or metabolically active form of a vitamin

in vivo may be different from that found in food

(e.g., B vitamins are active in coenzyme forms; 1,25-

dihydroxycholecalciferol is the active form of vitamin

; (4) several forms of the same vitamin can inter-

convert during extraction (e.g., pyridoxine, pyri-

doxamine, pyridoxal, and their phosphorylated

derivatives; and (5) synthetic forms of a vitamin are

often available commercially in a more stable form,

e.g., a-tocopherol acetate and retinol acetate are more

stable compared with the free vitamins and are con-

verted to the letter in the body.

0008 Vitamins are generally classified by their solubility

characteristics into fat-soluble and water-soluble

groups. A full list of vitamins, forms, functions, and

health benefits is given in Table 1. The fat-soluble

vitamins (A, E, D

, and K, and carotenoids) are de-

rived from the isoprenoid pathway, whereas the syn-

thesis of the water-soluble vitamins (B

C, biotin, niacin, and pantothenic acid) is much less

connected, and several of them have multiple bio-

synthetic pathways. Other ‘vitamin-like’ compounds

are sometimes associated with the classical vitamin

compounds. These include vitamins F (essential fatty

acids), L (o-aminobenzoic acid), P (bioflavonoids),

U(l-methioninylmethylsulfonium chloride), and

(orotic acid), choline, a-lipoic acid, myoinositol,

plastoquinone, pyrroloquinoline quinone, and ubi-

quinones. Refer to individual vitamins.

0009 During the early years of vitamin discovery, their

chemical composition was largely unknown and were

each assigned a letter of the alphabet for convenience.

This was further complicated when it was found that

the activity attributed to a single vitamin was, in some

instances, the result of a combination of several vita-

mins, e.g., B-complex vitamins. Once a vitamin had

been isolated from a food and its chemical structure

determined, variations in the structure were found

within compounds having the same vitamin activity

but in different species. A system of numerical sub-

scripts was therefore adopted to overcome this prob-

lem, e.g., D

and D

0010In order to simplify the system, letters were some-

times replaced with names, e.g., thiamin (vitamin B

riboflavin (B

), pantothenic acid (B

) and biotin (vita-

min H), based on the chemical structure of a specific

function or source. ‘Thiamin’ contains the prefix ‘thi,’

which is derived from the Greek word for sulfur, and

refers to its sulfur content. ‘Riboflavin’ indicates the

chemical structure and is derived from the chemical

names ‘ribose’ (a pentose sugar) and ‘flavin’ (a het-

erocyclic ketone). ‘Pantothenic acid’ is derived from

the Greek word pantos, meaning ‘found everywhere.’

‘Biotin’ originates from the German word haut,

meaning skin, as this vitamin protects the skin.

Occurrence in Food

0011One of the main differences between fat- and water-

soluble vitamins is their distribution in biological

tissues. The water-soluble vitamins are found in all

living tissues, whereas the fat-soluble vitamins are

completely absent from some tissues. Vitamins A

and D occur widely in plant tissues in the form of a

provitamin. Similarly, the amino acid tryptophan can

be converted into nicotinic acid in the body. Although

it is generally assumed that sufficient amounts of

vitamins can be absorbed from a balanced diet for

normal requirements, the use of vitamin supplements,

either as single compounds or in multivitamin prod-

ucts, is widely consumed. The major vitamin forms

found in foods, some commercial forms, and some

examples of rich food sources, are given in Table 2.

Nutritional Aspects

0012Vitamins are required in trace amounts in the diet for

the maintenance of optimal health, growth, and re-

production. Deficiency symptoms will occur if a

single vitamin is omitted from a diet of a species

that requires it. Many of the vitamins act as coen-

zymes, whereas others have no single role but

perform certain essential functions. A list of known

functions for each vitamin is given in Table 1. Vita-

min requirements based on metabolic needs are simi-

lar for animals and humans, but dietary needs widely

among species. (See Coenzymes.)

0013The recommended intakes of nutrients, including

vitamins, are intended as daily intake guidelines for

preventing deficiencies and maintaining body re-

serves among healthy individuals. The values are de-

rived from studies on volunteers on nutrient balance

studies, measures of tissue saturation, normal vitamin

intakes in the general population, and extrapolation

VITAMINS/Overview 6047