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indices of vitamin B

nutritional status in field

studies, under the controlled conditions of deple-

tion/repletion studies they do indeed provide a useful

indication of the state of vitamin B

nutrition.

0049 Although some 80% of the total body pool of

vitamin B

is associated with muscle glycogen phos-

phorylase, this pools turns over relatively slowly. The

major metabolic role of the remaining 20% of total

body vitamin B

, which turns over considerably more

rapidly, is in amino acid metabolism. Therefore, a

priori, it seems likely that protein intake will affect

vitamin B

requirements.

0050 People maintained on (experimental) vitamin B

deficient diets develop abnormalities of tryptophan

and methionine metabolism faster, and their blood

vitamin B

falls more rapidly, when their protein

intake is high. Similarly, during repletion of deficient

subjects, tryptophan and methionine metabolism

and blood vitamin B

are normalized faster at

low than at high levels of protein intake. However,

the relevance of these studies to normal nutrition is

unclear – they were conducted using extremes of

protein intake: 40 g day

1

for the low protein intake,

which is barely adequate to maintain nitrogen

balance, and 150 g day

1

for the high intake, which

is considerably higher than average western intakes

of protein.

0051 These studies suggest a mean requirement of 13 mg

of vitamin B

per gram of dietary protein; recom-

mended dietary allowances (RDA) are based on 15–

16 mg per g of protein. At average intakes of about

100 g of protein per day, this gives an RDA of 1.4–

1.6 mg of vitamin B

0052 More recent studies of women’s requirements have

suggested an RDA of 20 mgg

1

protein intake. It is

not clear whether this reflects a gender difference

(most of the earlier studies were conducted using

men) or the use of more sensitive criteria of adequacy

than were used previously.

Possible Benefits of Higher Levels of Intake

0053 The identification of hyperhomocysteinemia as an

independent risk factor in atherosclerosis and coron-

ary heart disease has led to suggestions that higher

intakes of vitamin B

than are currently considered

adequate to meet requirements may be desirable. As

shown in Figure 5, homocysteine is an intermediate in

methionine metabolism, and may undergo one of two

metabolic fates: remethylation to methionine (a reac-

tion that is dependent on vitamin B

and folic acid),

or onward metabolism leading to the synthesis of

cysteine (the vitamin B

-dependent transsulfuration

pathway).

0054 Among elderly survivors of the Framingham study

cohort (aged 67–96), hyperhomocysteinemia was

most significantly correlated with low folate

status, but there was also a significant association

with low vitamin B

status. However, a number of

studies have shown that, while folate supplements

lower fasting homocysteine in moderately hyperho-

mocysteinemic subjects, 10 mg day

1

vitamin B

has

no effect.

0055Vitamin B

supplements do, however, reduce the

peak plasma concentration of homocysteine

following a test dose of methionine. This can prob-

ably be explained by the kinetics of the enzymes

involved. The K

of cystathionine synthetase is 10-

fold higher than that of methionine synthetase. Under

basal conditions, little homocysteine is metabolized

by way of the transsulfuration pathway. It is only

after a loading dose of methionine, when homocys-

teine rises to relatively high levels, that the activity of

cystathionine synthetase, rather than the availability

of its substrate, is limiting or transsulfuration.

0056It is thus unlikely that intakes of vitamin B

above

amounts that are adequate to prevent metabolic signs

of deficiency will be beneficial in lowering plasma

concentrations of homocysteine.

Vitamin B

Requirements of Infants

0057Estimation of the RDA for vitamin B

of infants

presents a problem, and there is a clear need for

further research to achieve a realistic estimate of

infants’ requirements. Human milk, which must be

assumed to be adequate for infant nutrition, provides

only some 40–100 mgl

1

,or3–8 mg of vitamin B

per

gram of protein – very much lower than the apparent

requirement for adults. There is no reason why

infants should have a lower requirement than adults,

and indeed since they must increase their total body

pool of the vitamin as they grow, they might be

expected to have a proportionally higher requirement

than adults.

0058A first approximation of the vitamin B

needs of

infants came from studies of those who convulsed as a

result of gross deficiency caused by overheated infant

milk formula in the 1950s. At intakes of 60 mgday

1

there was an incidence of convulsions of 0.3%. Pro-

vision of 260 mg day

1

prevented or cured convul-

sions, but 300 mg day

1

was required to normalize

tryptophan metabolism. This is almost certainly a

considerable overestimate of requirements, since

pyridoxyllysine, formed by heating the vitamin with

proteins, has antivitamin activity, and would there-

fore result in a higher apparent requirement.

0059Based on the body content of 15 mmol (3.7 mg) of

vitamin B

per kg of body weight, and the rate of

weight gain, the minimum requirement for infants

over the first 6 months of life would appear to be

100 mg (417 nmol) day

1

to establish tissue reserves.

6028 VITAMIN B

/Physiology

Pharmacological Uses and Toxicity of

Vitamin B

Supplements

0060 Supplements of vitamin B

ranging from 25 to

500 mg day

1

, and sometimes higher, have been rec-

ommended for the treatment of a variety of condi-

tions in which there is an underlying physiological or

biochemical mechanism to justify the use of supple-

ments, although in most cases there is little evidence

of efficacy. Such conditions include postnatal depres-

sion, depression and other side-effects associated with

oral contraceptives, hyperemesis of pregnancy, and

the premenstrual syndrome.

0061 Supplements have also been used empirically, with

little or no rational basis, and little or no evidence of

efficacy, in the treatment of a variety of conditions,

including acute alcohol intoxication, atopic derma-

titis, autism, dental caries, diabetic neuropathy,

Down’s syndrome, Huntington’s chorea, schizophre-

nia, and steroid-dependent asthma.

0062 Doses of 100 mg day

1

have been reported to be

beneficial in the treatment of the carpal tunnel syn-

drome, or what has been called tenosynovitis. How-

ever, most of the reports originate from one center,

and there appears to be little or no independent con-

firmation of the usefulness of the vitamin in this

condition.

0063 Vitamin B

has been reported to be effective in

suppression of lactation, although other reports

have shown no difference from placebo. Because the

vitamin suppresses the increase in prolactin induced

by treatment with the dopamine receptor antagonist

pimozide, and because lactation is also suppressed by

the dopamine agonist bromocriptine, it has been sug-

gested that it acts to stimulate dopaminergic activity

in the hypothalamus. However, it is more likely that

its action is by reduction in target tissue responsive-

ness to the steroid hormones that stimulate prolactin

secretion.

0064 Doses of 50–200 mg day

1

have an antiemetic

effect, and the vitamin is widely used, alone or in

conjunction with other antiemetics, to minimize the

nausea associated with radiotherapy and to treat

pregnancy sickness. There is no evidence that vitamin

has any beneficial effect in pregnancy sickness, nor

that women who suffer from morning sickness have

lower vitamin B

nutritional status than other preg-

nant women. There have been reports of a teratogenic

effect of vitamin B

used to treat morning sickness,

but all of these involved use of the vitamin together

with the sedative Debendox, and there is no evidence

that vitamin B

itself is teratogenic. However, since it

downregulates responsiveness to steroid hormones

and retinoids, it is possible that high levels of vitamin

intake may affect embryonic or fetal development.

Vitamin B

and the Side-Effects of Oral

Contraceptives

0065Although estrogens do not cause vitamin B

defi-

ciency, the administration of vitamin B

supplements

has beneficial effects on some of the side-effects of

both administered and endogenous estrogens. The

supplements act in two main areas: in normalizing

glucose tolerance and as an antidepressant.

0066Impairment of glucose tolerance is common in

pregnancy, and may indeed be severe enough to be

classified as gestational diabetes mellitus, which gen-

erally resolves at parturition, although in some sub-

jects it may persist, pregnancy having been the trigger

for the development of maturity-onset diabetes.

High-estrogen oral contraceptives may also cause

impaired glucose tolerance. This seems to be the

result of increased tissue and blood concentrations

of xanthurenic acid, because of the inhibition of

kynureninase by estrogen metabolites. Xanthurenic

acid forms a complex with insulin which has little or

no hormonal activity. Vitamin B

supplements may

have a beneficial effect on glucose tolerance by acti-

vating apokynureninase or kynureninase that has

been inactivated by undergoing transamination.

0067One of the relatively common side-effects of estro-

genic oral contraceptives is depression, affecting

about 6% of women in some studies. This frequently

responds well to the administration of relatively large

amounts of vitamin B

(generally in excess of 40 mg

day

1

). Postnatal depression also responds to similar

supplements in some studies.

0068Again, this does not seem to be due to correction of

vitamin B

deficiency, but rather to a direct effect of

pyridoxal phosphate on the metabolism of trypto-

phan. High concentrations of pyridoxal phosphate

attenuate the response to glucocorticoid hormones;

tryptophan dioxygenase is a glucocorticoid-induced

enzyme, and thus its synthesis and activity will

be reduced by high intakes of vitamin B

. This

reduces the oxidative metabolism of tryptophan,

and increases the amount available for synthesis

of 5-hydroxytryptamine in the brain. Increased

brain 5-hydroxytryptamine synthesis has a mood-

elevating effect.

Vitamin B

in the Premenstrual Syndrome

0069The studies showing a beneficial action of vitamin B

in overcoming depression associated with oral con-

traceptives have led to the use of the vitamin in

depression and other pathology associated with en-

dogenous estrogens, in the premenstrual syndrome.

There is no evidence of poorer vitamin B

nutritional

status in women who suffer from the premenstrual

syndrome.

VITAMIN B

/Physiology 6029

0070 There are few well-controlled studies of the

effects of vitamin B

in premenstrual syndrome. In

general, those that have been properly controlled

report little benefit from doses between 50 and

200 mg day

1

compared with placebo, although

some studies do claim a beneficial effect. Interest-

ingly, meta-analysis of controlled cross-over trials

shows that whichever treatment is used second,

active vitamin or placebo is (marginally) more

effective. There is no obvious explanation for this

observation.

0071 Despite the lack of evidence of efficacy, vitamin B

is widely prescribed (and self-prescribed) for the

treatment of the premenstrual syndrome.

Toxicity of Vitamin B

0072 Animal studies have demonstrated the development

of signs of peripheral neuropathy, with ataxia, muscle

weakness and loss of balance, in dogs given 200 mg

pyridoxine HCl per kg of body weight for 40–75

days, and the development of a swaying gait and

ataxia within 9 days at a dose of 300 mg per kg of

body weight. At a dose of 50 mg per kg of body

weight, there are no clinical signs of toxicity, but

histologically there is a loss of myelin in dorsal

nerve roots. At higher doses there is more widespread

neuronal damage, with loss of myelin and degener-

ation of sensory fibers in peripheral nerves, the dorsal

columns of the spinal cord, and the descending spinal

tract of the trigeminal nerve. The clinical signs of

vitamin B

toxicity in animals regress after with-

drawal of these massive doses, but sensory nerve

conduction velocity, which decreases during the de-

velopment of the neuropathy, does not recover fully.

The mechanism of the neurotoxic action of vitamin

is unknown.

0073 The development of sensory neuropathy has been

reported in patients taking 2–7 g of pyridoxine HCl

day

1

. Although there was residual damage in some

patients, withdrawal of these extremely high doses

resulted in a considerable recovery of sensory nerve

function.

0074 Other reports have suggested that intakes as low as

50 mg day

1

are associated with neurological damage,

although these have been based on patients reporting

symptoms rather than on detailed neurological exam-

ination. Nevertheless, this led to a proposal in the UK

in 1997 to regulate the sale of vitamin B

supple-

ments, with 50 mg day

1

being available only on

prescription, and between 10 and 50 mg day

1

only

available from qualified pharmacists. The proposals

were put in abeyance in 1998, pending further studies

and reexamination of the evidence of toxicity at

intakes below 100–200 mg day

1

Vitamin B

Deficiency

0075Gross clinical deficiency of vitamin B

is more or less

unknown. The vitamin is widely distributed in

foods, and intestinal flora synthesize relatively large

amounts, at least some of which is believed to be

absorbed and hence available.

0076In vitamin B

-deficient experimental animals there

are more or less specific skin lesions (e.g., acrodynia

in the rat) and fissures or ulceration at the corners of

the mouth and over the tongue, as well as a number

of endocrine abnormalities, defects in the metabolism

of tryptophan, methionine and other amino acids,

hypochromic microcytic anemia (the first step of

heme biosynthesis is a pyridoxal phosphate-depend-

ent reaction), changes in leukocyte count and activity,

a tendency to epileptiform convulsions, and periph-

eral nervous system damage resulting in ataxia and

sensory neuropathy.

0077Much of our knowledge of human vitamin B

defi-

ciency is derived from an outbreak in the early 1950s,

which resulted from an infant milk preparation which

had undergone severe heating in manufacture. The

probable result of this was the formation of pyridox-

yllysine by reaction between pyridoxal phosphate

and the e-amino groups of lysine in proteins. In add-

ition to a number of metabolic abnormalities, many

of the affected infants convulsed. They responded to

the administration of vitamin B

supplements.

0078Investigation of the neurochemical basis of the

convulsions in vitamin B

deficiency helped to eluci-

date the role of GABA as a neurotransmitter; GABA

is synthesized by the decarboxylation of glutamate.

More recent studies have suggested that the accumu-

lation of hydroxykynurenine in the brain may be the

critical factor precipitating convulsions in deficiency;

GABA is depleted in the brains of deficient adult and

neonate animals, while hydroxykynurenine accumu-

lation is considerably more marked in neonates than

adults – only neonates convulse in vitamin B

defi-

ciency. GABA depletion may be a necessary but not

sufficient condition for convulsions in vitamin B

deficiency.

Vitamin B

Dependency Syndromes

0079A small number of cases have been reported of pa-

tients with genetic defects which result in an abnor-

mally high requirement for vitamin B

in order

to maintain the activity of the affected enzyme

(Table 2). Such vitamin B

dependency syndromes

have been reported in cases of xanthurenic aciduria,

homocystinuria, hypochromic sideroblastic anemia,

ornithinemia, and infantile convulsions. The mole-

cular basis of the defects appears to be a severely

impaired affinity of the affected enzyme for its

6030 VITAMIN B

/Physiology

cofactor, and patients respond well to doses of 500–

1000 mg of vitamin B

per day. Apart from the

affected enzyme, other biochemical indices of vitamin

nutritional status are normal in these patients.

Interestingly, there are few reports of peripheral neur-

opathy among such patients treated with high doses

of vitamin B

for many years.

Groups at Risk of Deficiency

0080 A number of studies have shown that between 10 and

20% of the apparently healthy population have low

plasma concentrations of pyridoxal phosphate or

abnormal erythrocyte transaminase activation coeffi-

cient, suggesting vitamin B

inadequacy or deficiency.

In most studies, only one of these indices of vitamin

nutritional status has been assessed. Where both

have been assessed, while each shows some 10% of

the population apparently inadequately provided

with vitamin B

, few of the subjects show inadequacy

by both criteria.

008 1 There is a decrease in the plasma concentration of

vitamin B

with increasing age, and some studies have

shown a high prevalence of abnormal transaminase

activation coefficient in elderly subjects, suggesting

that the elderly may be at risk of vitamin B

defi-

ciency. It is not known whether this reflects an inad-

equate intake, a greater requirement, or changes in

the tissue distribution and metabolism of the vitamin

with increasing age.

0082 Drug-induced vitamin B

deficiency A number of

drugs which react with carbonyl compounds are

capable of causing vitamin B

deficiency on pro-

longed use. These include the antituberculosis drug

isoniazid (iso-nicotinic acid hydrazide), penicilla-

mine, and the antiparkinsonian drugs Benserazide

and Carbidopa. In general, the main effect is impair-

ment of tryptophan metabolism by inhibition of

kynureninase, and hence the development of the

niacin deficiency disease pellagra. The condition

therefore responds to the administration of either

vitamin B

or niacin. Isoniazid also causes peripheral

neuropathy, which responds to vitamin B

supple-

ments, but not to niacin.

0083Estrogens and vitamin B

nutritional status There

have been many reports of abnormal tryptophan me-

tabolism in women taking estrogens as oral contra-

ceptives and menopausal hormone replacement

therapy. These have been widely interpreted as evi-

dence of estrogen-induced vitamin B

deficiency.

However, as discussed above, this is the result of

inhibition of kynureninase by estrogen metabolites,

not estrogen-induced deficiency of the vitamin. Where

other indices of vitamin B

status have been reported,

they have been generally unaffected by contraceptive

use, again suggesting an effect on tryptophan metab-

olism per se, rather than on vitamin B

nutritional

status.

0084In many cases the metabolism of tryptophan has

been normalized by the administration of vitamin B

supplements of the order of 20–50 mg day

1

,com-

pared with an RDA of 1.4–1.6mg day

1

. It was

noted above that there is an apparent excess of apo-

kynureninase in the liver and therefore the adminis-

tration of vitamin B

supplements will increase

kynurenine metabolism, even when there is no pre-

existing deficiency.

Seealso: Amino Acids:Metabolism; Contraceptives:

Nutritional Aspects; Glycogen; Hormones:Steroid

Hormones; Infants:NutritionalRequirements;

Premenstrual Syndrome: Nutritional Aspects; Vitamin

:PropertiesandDetermination

Further Reading

Bender DA (1987) Oestrogens and vitamin B

– actions and

interactions. World Review of Nutrition and Dietetics

51: 140–188.

Bender DA (1992) Nutritional Biochemistry of the Vita-

mins. Cambridge: Cambridge University Press.

Bender DA (1999) Non-nutritional uses of vitamin B

British Journal of Nutrition 81: 7–20.

Bender DA (2003) Nutritional Biochemistry of the Vita-

mins, 2nd edn. New York, Cambridge University Press.

Coburn SP (1996) Modelling vitamin B

metabolism.

Advances in Food and Nutrition Research 40: 107–132.

Fasella PM (1967) Pyridoxal phosphate. Annual Review of

Biochemistry 36: 185–210.

Hayashi H, Wada H, Yoshimura T, Esaki N and Soda K

(1990) Recent topics in pyridoxal 5

-phosphate enzyme

studies. Annual Review of Biochemistry 59: 87–110.

Ink SL and Henderson LM (1984) Vitamin B

metabolism.

Annual Review of Nutrition 4: 455–470.

Lumeng L, Lui A and Li T-K (1980) Plasma content of B

vitamers and its relationship to hepatic vitamin B

metabolism. Journal of Clinical Investigation 66:

688–695.

tbl0002 Table 2 Vitamin B

-responsive inborn errors of metabolism

Enzyme affected

Convulsions of the newborn Enzyme defect not known

CystathioninuriaCystathionase(Figure 5 )

Gyrate atrophy with ornithinuria Ornithine-d-aminotransferase

Homocystinuria Cystathionine synthase

(Figure 5 )

Primary hyperoxaluria, type 1 Peroxisomal alanine-

glyoxylate transaminase

Sideroblastic anemia d-Aminolevulinate synthase

(#hemesynthesis)

XanthurenicaciduriaKynureninase(Figure 4 )

VITAMIN B

/Physiology 6031

Martell AE (1982) Reaction pathways and mechanisms

of pyridoxal catalysis. Advances in Enzymology 53:

163–169.

Merrill AH and Henderson LM (1987) Diseases associated

with defects in vitamin B

metabolism of utilization.

Annual Review of Nutrition 7: 137–156.

Wiss O and Weber F (1964) Biochemical pathology of

vitamin B

deficiency. Vitamins and Hormones 22:

495–501.

Vitamin C See Ascorbic Acid: Properties and Determination; Physiology

Vitamin D See Cholecalciferol: Properties and Determination; Physiology

Vitamin E See Tocopherols: Properties and Determination; Physiology

VITAMIN K

Contents

Properties and Determination

Physiology

Properties and Determination

H E Indyk, Anchor Products, NZMP, Waitoa, New

Zealand

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

D C Woollard, Agriquality NZ, Auckland 1,

New Zealand

Background

0001 Vitamin K is a fat-soluble vitamin widely distributed

in nature and comprising several molecular forms.

It was discovered as an essential antihemorrhagic

factor in the 1930s by the Danish scientist Henrik

Dam, who named it Koagulationsvitamin. This

article reviews the properties and analysis of

vitamin K, with special reference to its measurement

in foods. Such measurements have proved difficult,

and reliable methods have only become available

since the 1980s with the development of phy-

sicochemical assays based on high-performance

liquid chromatography (HPLC). A subsequent

article deals with the physiology of vitamin K,

where the introduction of new assay techniques for

vitamin K and its dependent proteins has led to a

greater understanding of its nutritional role.

According to recent evidence, this includes the

probability that, in addition to its well-defined role

in blood coagulation, vitamin K is needed for a var-

iety of physiological processes related to a number

of extrahepatic vitamin K-dependent proteins. (See

Vitamins: Overview.)

Chemical Structures and Nomenclature

0002Naturally occurring compounds with vitamin K activity

possess a common 2-methyl-1,4-naphthoquinone

6032 VITAMIN K/Properties and Determination

nucleus, but differ in the structures of the side-chain

at the 3 position (Figure 1).

0003 In traditional nomenclature (Table 1), the vitamin

K found in plants (of which there is only one major

chemical form) is known as vitamin K

and the mul-

tiple forms synthesized by bacteria as vitamin K

where the number of carbon atoms (n) in the side

chain is indicated as vitamin K

2(n)

. This nomenclature

was revised by the International Union of Pure and

Applied Chemistry – International Union of Biochem-

istry (IUPAC–IUB) Subcommittee on Nomenclature

of Quinones, who recommended that vitamins K

and K

be called phylloquinone and menaquinones,

respectively. Recommended current abbreviations are

for phylloquinone and MK-n for menaquinones.

Table 1 also shows a nomenclature that the Inter-

national Union of Nutritional Sciences (IUNS) recom-

mended should replace the IUPAC system. This new

system, however, has not found favor among most

workers in this field. Consequently, the IUPAC-IUB

nomenclature is still the one most generally accepted

at the present time and will be used throughout this

article.

0004The parent compound of the vitamin K group of

vitamers is 2-methyl-1,4-naphthoquinone. This

structure is not found in nature but, because of its

historical use as a synthetic derivative, was originally

designated vitamin K

. Under the IUPAC system, this

artificial vitamin K is called menadione.

0005Phylloquinone, which has the same phytyl side-

chain as chlorophyl, is found throughout the plant

kingdom and in cyanobacteria (blue–green algae). In

higher plants, the synthesis of phylloquinone is asso-

ciated with the chloroplast, while the unusual finding

of phylloquinone in cyanobacteria has been cited as

supportive evidence for the possible bacterial origin

of the chloroplast in evolution.

0006The menaquinones represent a highly diverse

family of compounds all synthesized by bacteria.

The side-chain of menaquinones is based on a number

of repeating unsaturated five-carbon (prenyl) units.

The major forms are designated menaquinone-n

(MK-n), with n denoting the number of prenyl units.

Some bacteria also synthesize menaquinones in which

one or more of the multiprenyl side-chain double

bonds are saturated. In such cases, the additional

hydrogen atoms are indicated by the prefixes dihydro,

tetrahydro, hexahydro, etc., and these may be abbre-

viated to MK-n(H

), MK-n(H

), etc. The

division between the plant-form phylloquinone and

the bacterial menaquinones is somewhat artificial,

since phylloquinone may be regarded as a partially

saturated form of menaquinone-4, namely MK-

4(H

), but the distinction serves to emphasize their

different origins.

0007Carbon atoms in the side-chain are designated C1

etc., and the presence of double bonds at C2

phylloquinone and at C2

,10

, etc. in menaquin-

ones indicates that cis–trans isomerism is possible.

Natural forms of phylloquinone and menaquinones

exist as trans isomers, but synthetic forms contain

both trans and cis isomers.

Biological Activities

0008The biological activity of different vitamin K com-

pounds has been assessed in whole animal systems

(in vivo), or as a cofactor to the vitamin K-dependent

g-glutamyl carboxylase in isolated enzyme systems

Phylloquinone

(a)

Menaquinone

(b)

fig0001 Figure 1 Chemical structures of naturally occurring K vita-

mins: (a) phylloquinone (vitamin K

) and (b) menaquinones

(vitamin K

tbl0001 Table 1 Nomenclature of K vitamins

Chemicalname Oldname IUPAC^IUB (abbreviation) IUNS (abbreviation)

2-Methyl-1,4-naphthoquinone Vitamin K

Menadione Menaquinone

2-Methyl-3-phytyl-1,4-naphthoquinone Vitamin K

Phylloquinone (K) Phytylmenaquinone (PMQ)

2-Methyl-3-multiprenyl-1,4-naphthoquinone (class) Vitamin K

Menaquinone-n (MK-n) Prenylmenaquinone-n (MQ-n)

2-Methyl-3-farnesylfarnesyl-1,4-naphthoquinone Vitamin K

2(30)

Menaquinone-6 (MK-6) Prenylmenaquinone-6 (MQ-7)

VITAMIN K/Properties and Determination 6033

(in vitro). g-Glutamyl carboxylase is an integral

membrane glycoprotein that requires vitamin K to

activate posttranslational modification of glutamate

(Glu) to g-carboxyl glutamate (Gla) in specific vitamin

K-dependent proteins. The in vivo systems are nor-

mally based on the correction of blood clotting time

in chicks induced by vitamin K deficiency, or through

measurement of undercarboxylated vitamin K-

dependent proteins, and their interpretation needs to

take account of the route of administration and other

metabolic considerations, such as absorption and inter-

conversion to other chemical forms. (See Coenzymes.)

0009 It has been established that besides the 1,4-

naphthoquinone nucleus, a methyl group at the C2

position is essential for both in vivo and in vitro

activity. The activity of menadione in vivo is due to

the presence of tissue alkylating enzymes that can

transform this compound to MK-4. Recent evidence

is accumulating that suggests an alternative in vivo

route to MK-4 via tissue-mediated conversion of

phylloquinone. The essential requirement for in vitro

activity is for a 2-methyl-3-phytyl (or polyisoprenyl)-

1,4-naphthoquinone structure, which can be reduced

to the corresponding hydroquinone. The biological

activity of K

is reduced by saturation of the phytyl

double bond (2

-dihydrophylloquinone), whereas

the desmethyl or cis isomers have no appreciable

activity in vitro, although some conversion of the cis

form to the active trans form may occur in vivo.

Physicochemical Properties

Appearance, Solubility, and Stability

0010 Phylloquinone and menaquinones are golden yellow

oils at ambient temperature, though the higher mena-

quinones may also be obtained as fine, yellow crystals.

0011 All K vitamins are insoluble in water but are soluble

in normal lipid solvents such as hexane, chloroform,

diethyl ether, and acetone, but are less soluble in etha-

nol. They are only sparingly soluble in methanol, and

the degree of solubility depends on their hydrophobi-

city (i.e., menaquinones with long side-chains are less

soluble than those with short side-chains).

0012 Vitamin K compounds are reasonably stable to-

wards heat, oxygen, and mild acidic conditions, but

are degraded by UV light, alkalis, and strong acids.

Spectroscopy

0013 The spectroscopic properties of K vitamins are im-

portant for their detection, identification, and quan-

tification in assays. All vitamin K compounds possess

the same characteristic ultraviolet spectrum, which

is a summation of the benzenoid and 1,4-quinone

components. Naturally occurring phylloquinone and

menaquinones exhibit typical benzenoid bands with

maxima at 242, 248, and 238 nm (shoulder), whereas

the quinone contributions appear as distinct bands

with absorption maxima at 260 and 269 nm and a

higher-wavelength maximum at 326 nm. Ultraviolet

spectroscopy is particularly useful for the quanti-

fication of standard solutions; the molar absorption

coefficient (e) is the same for both phylloquinone

and menaquinones and, at 248 nm (the wavelength

of highest absorption), has a value of 18 900. (See

Spectroscopy: Overview.)

0014Vitamin K compounds, in their stable quinone

forms, do not possess native fluorescence, but they

can be readily reduced to the corresponding quinols,

whose fluorescent properties have been exploited for

the assay of K vitamins in foods and other biological

tissues. (See Spectroscopy: Fluorescence.)

0015The K vitamers also exhibit infrared and nuclear

magnetic resonance absorption spectra distinctive of

the naphthoquinone nucleus, and may be further

characterized by mass spectroscopy. (See Mass

Spectrometry: Principles and Instrumentation; Appli-

cations.)

Occurrence and Forms in Foods

0016Vitamin K is largely found in the membranous frac-

tions of cells. Thus, in plants, phylloquinone is con-

centrated in the chloroplast lamellae, and in bacteria,

menaquinones are located in the plasma membrane.

In animals, the vitamin is found in various cellular

membranes, especially those of the microsomes and

mitochondria.

0017Recent progress in analytical techniques for this

vitamin, as well as increased emphasis on its nutri-

tional significance, is responsible for the current

development of a comprehensive database of the vita-

min K content of foods. Whereas, before 1980, most

published values for the vitamin K content of foods

originated from bioassays, contemporary data derive

from the highly specific and accurate HPLC methods.

These techniques have focused predominantly on

phylloquinone ubiquitously distributed in foods,

although more recently, increasing amounts of data

are becoming available for the menaquinones.

0018Table 2 lists some values of the phylloquinone

content of various foods arranged into four concen-

tration ranges to illustrate the wide distribution of the

vitamin in the diet.

0019In general, the relative values confirm the known

association of phylloquinone with photosynthetic

tissues, with the highest values (400–600 mg per

100 g) being found in green, leafy vegetables.

Nutritionally significant amounts of phylloquinone

are, however, present in many nonleafy vegetables,

6034 VITAMIN K/Properties and Determination

as well as in fruits, oils, dairy products, eggs, and

some meat products. With modern HPLC techniques,

naturally occurring phylloquinone can be detected in

most foods. Vegetable oils show a high degree of

variability, ranging from < 5 mg per 100 g in coconut,

corn, groundnut, and safflower oils, to 100–200 mg

per 100 g in soya bean and rapeseed oils. This leads to

a similar variation in the phylloquinone content of

margarines according to the oil base. The phylloquin-

one content of lean meat and fish is generally low

(< 1 mg per 100 g), but higher amounts are found in

animal livers; this organ acts as a storage site of the

vitamin. Please refer to individual foods.

0020 There is less information on the menaquinone con-

tent of foods. It is known that animal liver contains

substantial concentrations of menaquinones, which

normally greatly exceed the phylloquinone content.

The livers of ruminants contain particularly

large concentrations of long-chain menaquinones

(MK-6–13), which derive from bacterial fermenta-

tion in the rumen. The only other known sources of

significant amounts of menaquinones in the Western

diet are fermented foods, such as cheese and yogurt.

All cheeses contain relatively high concentrations

(20 mg per 100 g) of MK-8 and MK-9, with the

latter predominating. Natto, the Japanese fermented

soya bean food, contains substantial amounts of

MK-7. Whereas menaquinones are detectable in

cow’s milk at much lower concentrations than phyllo-

quinone, MK-4 has been confirmed to be present at

comparable, or higher, levels than phylloquinone in

the milk of several mammalian species.

0021Foods containing hydrogenated vitamin K-rich oils

will also include a contribution from 2

-dihydro-

phylloquinone. The biological activity of this K-

vitamer is currently under investigation, and hence

its abundance in the diet is of uncertain nutritional

significance.

tbl0002 Table 2 Vitamin K

content of common foods determined by HPLC (mg per 100 g)

0.1^1.0 1^10 10^100 100^1000

Bananas Apples Avocado Broccoli tops

Beef, steak Aubergines Beans, runner Brussels sprouts

Bread, white Baked beans Beans, French Cabbage, green

Chicken, thigh Barley Beans, broad Kale

Coconut oil Beef, minced (1–30) Cabbage, red Lettuce

Cod, fillet (0.1–2) Bilberries Cauliflower Margarine

Cornflakes Bran, wheat Chick peas Parsley

Flour, white Bread, wholemeal Cucumber Rapeseed oil

Grapefruit Butter Greengages Soybean oil

Ham, tinned Carrots Kiwi fruit Spinach

Icecream Celery Margarine Water cress

Maize Cheeses (1–80) Mustard cress

Mangoes Chocolate, plain Natto (900–1200)

Melon, yellow Corn oil Olive oil, ex. Virgin

Melon, water Courgettes Peas

Milk, cows Cranberries

Mushrooms Cream, double

Onion Dates, fresh

Oranges Eggs (10–25)

Parsnips Figs, fresh

Peanuts, roast Grapes, black

Pilchards, brine (0.1–2) Grapes, green

Pineapple Infant Formula

Pork chop, lean Leeks

Potatoes Liver, lamb

Rice white Liver, ox (10–20)

Rice brown Nectarines

Salmon, brine Oats

Sausages, pork/beef Palm oil

Spaghetti Peaches, fresh

Trout, Rainbow (0.1–3) Pears

Tuna, brine Peppers, green

Turnips Peppers, red

Yogurt (0.1–0.4) Plums, red

Raisins

Rhubarb

MK-n ranges are shown in parentheses.

VITAMIN K/Properties and Determination 6035

0022 The processing of foods can have a variable effect

on retention of vitamin K, with conventional cooking,

canning, g-irradiation, and freezing practices having a

relatively minor influence, whereas significant and

rapid loss occurs on exposure to UV light.

Use in Food Fortification

0023 Since the requirements for vitamin K are low, fortifi-

cation of foods with vitamin K is not common, with

the exception of infant formula feeds to which syn-

thetic preparations of phylloquinone are invariably

added. Depending on the method of chemical synthe-

sis, the ratio of the natural trans to the inactive cis

form will vary, but is typically c. 7:1. (See Food For-

tification; Infant Foods: Milk Formulas.)

0024 Although national guidelines for fortification of

infant formulas differ, their common aim is to protect

against rare, but life-threatening, bleeding due to

deficiency of vitamin K in early life. It is now known

that the typical fortification levels of c.50mgl

1

(as

fed) are several times higher than human breast

milk concentrations of phylloquinone, which average

c.2mgl

1

. Although the reasons for adopting such

high levels of fortification are somewhat empirical

and based on the erroneously high estimates of vita-

min K in breast milk by early bioassays, there has

been little impetus for change because the low con-

centrations in breast milk are an established risk

factor for neonatal vitamin K deficiency.

0025 The synthetic vitamin K derivative menadione is no

longer used to fortify infant formulae because of its

association with neonatal hemolytic anemia and liver

damage. In fact, the vitamin K activity of menadione

is dependent on its in vivo conversion to biologically

active MK-4. Nevertheless, because menadione is

much cheaper to manufacture than phylloquinone,

menadione derivatives are widely used in animal hus-

bandry, particularly in poultry feeds, since poultry are

very prone to bleeding due to dietary deficiency of

vitamin K. In the poultry industry, the birds’ dietary

requirements may be further increased by the use

of antibiotics, the effect of which may be either

to inhibit the growth of intestinal microorganisms

that synthesize menaquinones or to interfere directly

with vitamin K metabolism. Consequently, stabilized

water-soluble derivatives of menadione, such as crys-

talline menadione sodium bisulfite complex, are used

universally as poultry feed supplements.

Measurement of Vitamin K in Food

0026 In the 1930s, shortly after the discovery of vitamin K,

Dam and colleagues developed a curative bioassay

that involved the feeding of substances to vitamin

K-deficient chicks and measuring the response on

blood clotting. Although values that have been

derived from this early work are often given in text-

books and handbooks of nutrition, a closer evalu-

ation of the methodology shows that attempts to

use these results as the basis for absolute food values

are unjustified. Although such bioassays were greatly

improved in the 1960s, little further work has

emerged on their routine use and validation for food

analysis.

0027The introduction of modern HPLC techniques has

revolutionized the measurement of vitamin K in

tissues. This powerful technique has met the criteria

of high selectivity and sensitivity, which are also ne-

cessary for measurement of the low concentrations in

most foods. Although several diverse protocols have

been developed for phylloquinone, only one proced-

ure has been validated by collaborative study. A range

of sample extract purification strategies may be im-

plemented prior to the final chromatographic analy-

sis, which is usually accomplished by analytical

reversed-phase HPLC. Due to the instability of

vitamin K at high pH, saponification techniques

commonly utilized for other fat-soluble vitamins are

inappropriate for vitamin K analysis. The need for

preliminary enhancement techniques is dependent

principally on food type, analyte level, and final

detection mode and may incorporate one or

more techniques including (1) lipid extraction, (2)

enzymatic lipid digestion, (3) solid-phase extraction

(SPE) and (4) semipreparative normal-phase HPLC.

(See Chromatography: High-performance Liquid

Chromatography.)

Extraction and Clean-up

0028No universal extraction and purification procedure

exists for vitamin K analysis in all foods. Nonpolar

lipid solvents including hexane, isopropanol, dichlor-

omethane, acetone, diethyl ether, and methanol, used

either singly or in combination, have been used for

initial total lipid extraction depending on the food

type. For certain high-fat foods (fats, oils and milk),

bulk lipid may be effectively removed with enzymatic

digestion prior to hexane extraction of triglyceride-

depleted lipid. For low-fat foods (vegetables, fruit,

cereals, meats, and fish), acetone is an effective solv-

ent, whereby, after the addition of water and hexane,

the K vitamins partition entirely in the upper hexane

phase, leaving polar lipids in the acetone–water

phase. Alternatively, hexane:isopropanol or chloro-

form:methanol binary solvents are commonly used.

0029For protocols relying on a preliminary total lipid

extraction, further purification is required in view of

the overwhelming proportion of triglycerides and

their incompatibility with reversed-phase HPLC.

6036 VITAMIN K/Properties and Determination

Various combinations of lipase digestion, SPE and

semipreparative normal-phase HPLC techniques are

commonly recommended.

0030 The extraction stage may also incorporate the add-

ition of an internal standard, which, in general terms,

compensates for procedural losses of the target ana-

lyte. This technique is essential when multidimen-

sional protocols for vitamin K are implemented. The

internal standard is chosen on the basis of several

criteria that include its similar physical, chemical,

and chromatographic properties to the analyte.

Depending on the sample matrix, several vitamin K

compounds and analogues (including radiolabelled

compounds) have been found to be suitable internal

standards for vitamin K assays.

0031 A general scheme outlining commonly applied ex-

traction and clean-up options is illustrated in Figure 2.

Purification techniques usually comprise two succes-

sive stages based on the principles of adsorption

chromatography, which facilitate the separation of

K vitamers from most other potentially interfering

lipid classes (e.g., hydrocarbons, sterols, fatty acids).

If required, semipreparative normal-phase HPLC can

also be used to isolate both K

and MK fractions

and to separate the cis and trans isomers of phyllo-

quinone.

0032 The fraction(s) collected at the semipreparative

HPLC stage may then be analyzed by reversed-phase

HPLC using a variety of detection methods. For cer-

tain foods, a lipase digestion and hexane extraction

scheme has been found to be sufficient to facilitate a

direct analytical HPLC quantitation, without the

need for additional purification steps.

Detection and Quantification

0033The final analytical stage of HPLC is almost exclu-

sively based on the principles of partition chromatog-

raphy in the reversed-phase mode. This robust mode

of liquid chromatography resolves the individual

K vitamins from each other, while supporting the

mobile phase additives necessary for the most sensi-

tive detection techniques currently utilized. Whereas

isocratic systems are generally used for the analysis of

vitamin K

, gradient elution is preferable when pro-

filing the menaquinones, in view of their very wide

hydrophobic range. The C18 functionality has most

commonly been employed, although the recently de-

veloped C30 phase has been shown to allow reso-

lution of the cis and trans isomers of phylloquinone,

previously only accomplished under normal phase

conditions.

0034The properties of K vitamins offer several avenues

of approach to their detection by HPLC, and the most

common options are summarized in Figure 3. The

parent quinone form, in which the vitamin is iso-

lated, may be detected with reasonable sensitivity

(c. 500 pg) by ultraviolet absorbance detection, but

this lacks selectivity and may be subject to interfer-

ences from other lipids. This detection method does,

however, give reliable results for green vegetables, but

is less satisfactory for animal tissues.

0035Alternatively, the more recently developed strat-

egies exploit the reversible reduction of the quinone

to the quinol, which may then be detected by either

electrochemical or fluorescent techniques. For elec-

trochemical detection, the greatest sensitivity and se-

lectivity is obtained by employing a redox technique

in which the detector has a dual-electrode cell with

the electrodes arranged in series. Vitamin K is first

Partition into hexane

SPE

Semipreparative NP-HPLC

Analytical RP-HPLC

Lipid extraction Lipase digestion

Food

fig0002 Figure 2 Flow chart summarizing extraction and purification of

vitamin K from foods.

detection

(~500 pg) (< 50 pg)

(< 50 pg)

Fluorescence

detection

Quinone Quinol

Electrochemical

detection

Reduction

fig0003Figure 3 Detection options for vitamin K.

VITAMIN K/Properties and Determination 6037