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VITAMIN B6 | Physiology
6020 VITAMIN B6/Physiology Yuan X, Marchello MJ and Driskell JA (1999) Selected vitamin contents and retentions in bison patties as related to cooking method. Journal of Food Science 64: 462–464.
Physiology D A Bender, University College London, London, UK Copyright 2003, Elsevier Science Ltd. All Rights Reserved.
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Pyridoxal phosphate bound as a Schiff base to lysine in dietary proteins is released on digestion of the protein. The phosphorylated vitamers are dephosphorylated by membrane-bound alkaline phosphatase in the intestinal mucosa; pyridoxal, pyridoxamine, and pyridoxine are all absorbed rapidly by passive diffusion. Intestinal mucosal cells have pyridoxine kinase, and pyridoxine phosphate oxidase so that there is net accumulation by metabolic trapping. Much of the ingested pyridoxine is released into the portal circulation as pyridoxal, after dephosphorylation at the serosal surface.
Introduction
Metabolism and Transport
Vitamin B6 has a central role in amino acid metabolism, as the coenzyme for a variety of reactions, including transamination and decarboxylation. It is also the coenzyme of glycogen phosphorylase, and acts to modulate the activity of steroid and other hormones (including retinoids and vitamin D) which act by regulation of gene expression.
Most of the absorbed vitamin is taken up by the liver, although other tissues can also take up the unphosphorylated vitamers from the circulation. Uptake is by carrier-mediated diffusion, followed by metabolic trapping as phosphate esters. Pyridoxine and pyridoxamine phosphates are oxidized to pyridoxal phosphate. All tissues have pyridoxine kinase activity, but pyridoxine phosphate oxidase is only found in liver, kidney, and brain (Figure 1). Pyridoxine phosphate oxidase is a flavoprotein, and its activity falls markedly in riboflavin deficiency. Despite this central role of riboflavin in vitamin B6 metabolism, blood and tissue concentrations of pyridoxal phosphate are not affected by riboflavin deficiency, and riboflavin nutrition appears to have no effect on vitamin B6 nutritional status. Pyridoxine phosphate oxidase is inhibited by its product, pyridoxal phosphate. This is not simple product inhibition, but involves binding at a specific inhibitor site on the enzyme. The normal intracellular concentration of free pyridoxal phosphate gives significant inhibition, which indicates that this is a physiologically important mechanism in the control of tissue pyridoxal phosphate. Pyridoxine is phosphorylated rapidly in liver and other tissues. Pyridoxal phosphate does not cross cell membranes, and uptake and efflux of the vitamin in most tissues are as pyridoxal. Pyridoxal phosphate is exported from the liver bound to albumin. Much of the free pyridoxal phosphate in the liver is hydrolyzed to pyridoxal, which is also exported, and circulates bound both to albumin and to hemoglobin in erythrocytes. Free pyridoxal remaining in the liver is rapidly oxidized to 4-pyridoxic acid, which is the main excretory product of the vitamin. Extrahepatic tissues take up pyridoxal from the plasma. Pyridoxal phosphate is hydrolyzed to pyridoxal, which can cross cell membranes, by
Dietary Forms, Biological Availability, and Metabolism 0002
Digestion and Absorption
The main form of vitamin B6 in foods is pyridoxal phosphate, bound to enzymes. There is also a small amount of pyridoxamine phosphate. In plant foods a significant amount of the vitamin is present as pyridoxine. A number of plants contain relatively large amounts of pyridoxine glycosides, which are not biologically available, since they are not substrates for mammalian glycosidases. They are absorbed (passively) from the intestinal lumen and are excreted more or less quantitatively in the urine. Between 5% and 50% of the total vitamin B6 in some foods may be present as pyridoxine glycosides. A proportion of the vitamin B6 in foods may be biologically unavailable, especially after heating, as a result of the formation of (phospho)pyridoxyllysine by reduction of the aldimine (Schiff base), by which pyridoxal phosphate is bound to the e-amino groups of lysine residues in proteins. While some of this pyridoxyllysine may be useable, since it is a substrate for pyridoxine phosphate oxidase, it is also a vitamin B6 antimetabolite, and even at relatively low concentrations can accelerate the development of deficiency in experimental animals maintained on deficient diets. In the 1950s there was an outbreak of vitamin B6 deficiency among infants fed on formula that had been overheated in manufacture, resulting in the formation of relatively large amounts of pyridoxyllysine.
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VITAMIN B6/Physiology OH
CH2OH HOCH2
O
Kinase
OH
P
CH2OH O
CH2
OH
OH
Phosphatase CH3
N
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CH3
N
Pyridoxine
Pyridoxine phosphate Oxidase
HC
COOH HOCH2
OH
O
HOCH2
OH
OH
O
Kinase
Oxidase
HC
P
O
O
CH2
OH
OH Phosphatase
N
CH3
CH3
N
4-Pyridoxic acid
Pyridoxal phosphate
Pyridoxal
Transaminases
CH2NH2 HOCH2
OH
N
CH3
Pyridoxamine fig0001
0011
0012
CH3
N
Oxidase
CH2NH2
OH Kinase Phosphatase
O
P
O
CH2
OH
OH
N
CH3
Pyridoxamine phosphate
Figure 1 Metabolism of vitamin B6.
extracellular alkaline phosphatase, then trapped intracellularly by phosphorylation. Tissue concentrations of pyridoxal phosphate are controlled by the balance between phosphorylation and dephosphorylation. The activity of phosphatases acting on pyridoxal phosphate is greater than that of the kinase in most tissues. This means that pyridoxal phosphate that is not bound to enzymes will be dephosphorylated and hence leave the cell by diffusion. Thus there is little accumulation of pyridoxal phosphate in tissues, other than that which is bound to enzymes and other proteins (e.g., hormone receptors). Free pyridoxal either leaves the cell or is oxidized to 4-pyridoxic acid by aldehyde dehydrogenase, which is present in all tissues, and also by hepatic and renal aldehyde oxidase. 4-Pyridoxic acid is the main excretory product of vitamin B6, and its excretion reflects recent intake more than the state of underlying tissue reserves of the vitamin. Small amounts of pyridoxal and pyridoxamine are also
excreted in the urine, although much of the active vitamin B6 which is filtered at the glomerulus is resorbed in the kidney tubules. Storage and Body Reserves
There is no specific storage of vitamin B6 in the body; as discussed above, pyridoxal phosphate that is not bound to enzymes is rapidly dephosphorylated, oxidized to 4-pyridoxic acid, and excreted. The total body pool of vitamin B6 is of the order of 60 mmol (250 mg); 15 mmol (3.7 mg) per kg body weight. About 80% of this is in muscle, associated with glycogen phosphorylase. This does not seem to function as a true reserve of the vitamin and is not released from muscle in times of deficiency. Muscle pyridoxal phosphate is released into the circulation (as pyridoxal) in starvation, as muscle glycogen reserves are exhausted, and there is less requirement for glycogen phosphorylase activity. Under these conditions it is available for redistribution to other tissues, especially liver and kidney, to
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C H2N
Lysine NH
H C N OH O
P O CH2
Lysine
HC
NH
O
H
R C COOH NH2 Amino acid substrate
OH
O
O
N
N OH
CO2 O OH Decarboxylation
HC
P O CH2 OH
OH
C
R C COOH
CH3 N Substrate aldimine
CH3 N Pyridoxal phosphate internal aldimine (Schiff base)
OH
HC
O
Lysine NH
P O CH2
OH
OH N
CH3
R−CH2−NH2 Product amine
Tautomerization H R C COOH N OH O
HC
P O CH2 OH N
Transamination (amino transfer) OH H2C NH2 H2O O P O CH2 OH OH OH CH3
Substrate ketimine
fig0002
Metabolic Functions of Vitamin B6 The metabolically active vitamer is pyridoxal phosphate, which is involved in many reactions of amino acid metabolism, where the carbonyl group is the reactive moiety, in glycogen phosphorylase, where it is the phosphate group that is important in catalysis, and in the release of hormone receptors from tight nuclear binding, where again it is the carbonyl group that is important. The Role of Pyridoxal Phosphate in Amino Acid Metabolism 0017
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CH3 N Pyridoxamine phosphate
O Product oxo-acid (keto-acid)
Figure 2 Roles of vitamin B6 in amino acid metabolism.
meet the increased requirement of transamination of amino acids for gluconeogenesis.
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R C COOH
The various reactions of pyridoxal phosphate in amino acid metabolism (Figure 2) all depend on the same chemical principle – the ability to stabilize amino acid carbanions, and hence to weaken bonds about the a-carbon of the substrate. This is achieved by reaction of the a-amino group with the carbonyl group of the coenzyme to form a Schiff base (aldimine). Pyridoxal phosphate is bound to enzymes, in the absence of the substrate, by the formation of an
internal Schiff base to the e-amino group of a lysine residue at the active site. Thus the first reaction between the substrate and the coenzyme is transfer of the aldimine linkage from this e-amino group to the a-amino group of the substrate. The ring nitrogen of pyridoxal phosphate exerts a strong electron-withdrawing effect on the aldimine, and this leads to weakening of all three bonds about the a-carbon of the substrate. In nonenzymic model systems, all the possible pyridoxal-catalyzed reactions are observed: a-decarboxylation, aminotransfer, racemization, and relevant side-chain elimination and replacement reactions. By contrast, enzymes show specificity for the reaction pathway followed; which bond is cleaved will depend on the orientation of the Schiff base relative to reactive groups of the catalytic site. However, a number of decarboxylases and enzymes that catalyze side-chain elimination reactions of amino acids undergo gradual inactivation as a result of catalyzing the half-reaction of transamination, leaving (catalytically inactive) pyridoxamine phosphate at the catalytic site. a-Decarboxylation If the electron-withdrawing effect of the ring nitrogen is primarily centered on
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the a-carbon-carboxyl bond, the result is decarboxylation of the amino acid with the release of carbon dioxide. The resultant carbanion is then protonated, and the primary amine corresponding to the amino acid is displaced by the lysine residue at the active site, with reformation of the internal Schiff base. A number of the products of the decarboxylation of amino acids are important as neurotransmitters and hormones: 5-hydroxytryptamine, the catecholamines dopamine, norepinephrine (noradrenaline), and epinephrine (adrenaline), histamine and g-aminobutyrate (GABA), and as the diamines and polyamines involved in the regulation of DNA metabolism. The decarboxylation of phosphatidylserine to phosphatidylethanolamine is important in phospholipid metabolism.
Transamination Hydrolysis of the a-carbon-amino bond of the ketimine formed by deprotonation of the a-carbon of the amino acid results in the release of the 2-oxo-acid corresponding to the amino acid substrate, and leaves pyridoxamine phosphate at the catalytic site of the enzyme. In this case there is no reformation of the internal Schiff base to the reactive lysine residue. This is the half-reaction of transamination. The process is completed by reaction of pyridoxamine phosphate with a second oxo-acid substrate, forming an intermediate ketimine, followed by the reverse of the reaction sequence shown in Figure 2, releasing the amino acid corresponding to this second substrate after displacement from the aldimine by the reactive lysine residue to reform the internal Schiff base. Transamination (Figure 3) is of central importance in amino acid metabolism, providing pathways for the catabolism of all of the amino acids except lysine, which does not undergo transamination. Many of these reactions are linked to the amination of 2-oxoglutarate to glutamate or glyoxylate to glycine, which are substrates for oxidative deamination, reforming the oxo-acids. Transamination reactions also provide a pathway for the synthesis of those amino acids for which there is an alternative source of the oxo-acid (the nonessential amino acids). Indeed, the nonessential amino acids can be defined as those whose oxoacids can be formed other than from the amino acid itself.
Racemization of Amino Acids Deprotonation of the a-carbon of the amino acid leads to tautomerization of the Schiff base to yield a quinonoid ketimine. The simplest reaction that the ketimine can undergo is reprotonation at the now symmetrical a-carbon. Displacement of the substrate by the reactive lysine residue results in the racemic mixture of d- and l-amino acid. Amino acid racemases are important in bacterial metabolism, since several d-amino acids are required for the synthesis of cell wall mucopolysaccharides. There is no evidence that there are any mammalian amino acid racemases; such utilization of d-amino acids as occurs is probably due to the action of renal d-amino acids oxidase to form the symmetrical 2-oxo-acid which is then a substrate for transamination.
H R1
C
O
P
Side-chain elimination and replacement reactions The third bond in the Schiff base aldimine that can be labilized by the electron-withdrawing H
OH O
C
O
NH2
R2
N Pyridoxal phosphate
COOH
O Product oxo-acid (keto-acid)
fig0003
COOH
Product amino acid
CH2
OH C
C NH2
CH3
Substrate amino acid Amino donor
R1
H
OH
CH2
OH
COOH
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O
P
O
CH2
NH2 OH
OH N Pyridoxamine phosphate
Figure 3 The role of vitamin B6 in transamination reactions.
R2
C
COOH
O CH3
Substrate oxo-acid (keto-acid) Amino acceptor
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effect of the ring nitrogen of pyridoxal phosphate is that between the a-carbon and the side-chain of the amino acids, resulting in a variety of a–b elimination and b–g replacement reactions. The Role of Pyridoxal Phosphate in Glycogen Phosphorylase 0027
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Glycogen phosphorylase catalyzes the sequential phosphorolysis of glycogen to release glucose 1-phosphate; it is thus the key enzyme in the utilization of muscle and liver reserves of glycogen. Unlike other pyridoxal phosphate-dependent enzymes, in which the carbonyl group is essential for catalysis, the internal Schiff base between pyridoxal phosphate and lysine in glycogen phosphorylase is not broken during the reaction. The catalytic region of the coenzyme is the 50 -phosphate group. The initial stage in the phosphorolysis of glycogen is protonation of the glycosidic oxygen of the polysaccharide by inorganic phosphate. The resultant oxycarbonium ion is stabilized by the inorganic phosphate. The role of pyridoxal phosphate is as a proton shuttle or buffer to stabilize the oxycarboniumphosphate ion pair, permitting covalent binding of the phosphate to the oxycarbonium ion, to form glucose 1-phosphate. The Role of Pyridoxal Phosphate in Steroid Hormone Action
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Pyridoxal phosphate has a role in modulating the action of those hormones which act by binding to a nuclear receptor protein, inducing transcription of DNA, and hence regulating gene expression, leading to new protein synthesis. Such hormones include androgens, estrogens, progesterone, glucocorticoids, calcitriol (the active metabolite of vitamin D), retinoic acid and other retinoids, and thyroid hormone. Target-tissue specificity of hormone action is insured by the presence of characteristic receptor proteins with a zinc finger motif, which are responsible for both nuclear uptake of the hormone and the interaction with DNA and nucleoproteins to initiate gene expression. Pyridoxal phosphate reacts with a lysine residue in the receptor protein, and displaces the hormone– receptor complex from tight nuclear binding. In vitro, reaction with pyridoxal phosphate also inhibits the binding of receptor protein to isolated DNA and chromatin. The effect is specific for the phosphorylated vitamer, suggesting that there may be a specific pyridoxal-phosphate binding site on the receptor proteins, and it occurs at low concentrations of pyridoxal phosphate, of the same order of magnitude as occur in tissues under normal conditions.
This suggests that pyridoxal phosphate acts as a cofactor in the release of hormone–receptor complexes from tight nuclear binding, resulting in release of the receptor from the nucleus, termination of hormone action, and recycling receptor protein for further uptake of hormone. In experimental animals, vitamin B6 deficiency results in increased and prolonged nuclear uptake and retention of steroid hormones in target tissues, and there is enhanced sensitivity to hormone action. Deficient animals show greater induction of uterine peroxidase, and considerably greater suppression of the hypothalamic secretion of luteinizing hormone, by estrogens than do vitamin B6-supplemented controls. In vitamin B6-deficient male animals there is an increased mitotic response of the prostate to low doses of testosterone. Deficient male animals have a higher activity of ornithine decarboxylase (an androgen-induced enzyme) in the liver, and deficient females have higher renal ornithine transaminase (an estrogen-induced enzyme). The induction of hepatic tyrosine transaminase and tryptophan dioxygenase by glucocorticoids is also enhanced in vitamin B6-deficient animals. In a variety of cells in culture that have been transfected with a glucocorticoid, estrogen, or progesterone response element linked to a reporter gene, acute vitamin B6 depletion (by incubation with 4-deoxypyridoxine) leads to a twofold increase in expression of the reporter gene in response to hormone action. Conversely, incubation of these cells with high concentrations of pyridoxal, leading to a high intracellular concentration of pyridoxal phosphate, results in a halving of the expression of the reporter gene in response to hormone stimulation.
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Criteria of Adequacy and Assessment of Nutritional Status Plasma Concentrations of the Vitamin
The fasting plasma concentration of either total vitamin B6 or, more specifically, pyridoxal phosphate, is widely used as an index of vitamin B6 nutritional status. The generally accepted criteria of adequacy are shown in Table 1. Conditions that involve increased plasma activity of alkaline phosphatase may result in reduced plasma pyridoxal phosphate, without affecting tissue concentrations of pyridoxal phosphate or vitamin B6 nutritional status, as assessed by other criteria. There is a compensatory increase in the circulating concentration of pyridoxal, which is the main form for tissue uptake of vitamin B6. Despite the fall in plasma pyridoxal phosphate in pregnancy, which has been
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VITAMIN B6/Physiology tbl0001
Table 1 Indices of vitamin B6 nutritional status Adequate status Plasma total vitamin B6 Plasma pyridoxal phosphate Erythrocyte alanine aminotransferase activation coefficient Erythrocyte asparate aminotransferase activation coefficient Erythrocyte asparate aminotransferase Urine 4-pyridoxic acid
Urine total vitamin B6
Urine xanthurenic acid after 2 g tryptophan load Urine cystathionine after 3 g methionine load
> 40 nmol (10 mg) l1 >30 nmol (7.5 mg) l1 <1.25 <1.80 >0.13 units (8.4 mkat) l1 >3.0 mmol 24 h1 >1.3 mmol mol1 creatinine >0.5 mmol 24 h1 >0.2 mmol mol1 creatinine <65 mmol 24 h1 increase <350 mmol 24 h1 increase
widely interpreted as indicating vitamin B6 depletion, the plasma concentration of (pyridoxal phosphate plus pyridoxal) is unchanged. This suggests that determination of plasma pyridoxal phosphate alone may not be a reliable index of vitamin B6 nutritional status. Urinary Excretion of 4-Pyridoxic Acid 0036
About half of the normal dietary intake of vitamin B6 is excreted as 4-pyridoxic acid. Urinary excretion of 4-pyridoxic acid will largely reflect recent intake of the vitamin rather than underlying nutritional status; the criteria for assessment of 4-pyridoxic acid excretion are shown in Table 1. Coenzyme Saturation of Transaminases
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Various pyridoxal phosphate-dependent enzymes compete with each other for the available pool of coenzyme. Thus the extent to which an enzyme is saturated with its coenzyme provides a means of assessing the adequacy of the body pool of coenzyme. This can be determined by measuring the activity of the enzyme before and after the activation of any apoenzyme present in the sample by incubation with pyridoxal phosphate added in vitro. Erythrocyte aspartate and alanine transaminases are both commonly used; the results are expressed as either the percentage stimulation of activity by added pyridoxal phosphate, or the activation coefficient – the ratio of activity with added coenzyme to that without added coenzyme. It seems to be normal for a proportion of pyridoxal phosphate-dependent enzymes to be present as inactive apoenzyme, without coenzyme. This may be a
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mechanism for metabolic regulation. It is possible that increasing the intake of vitamin B6, so as to insure complete saturation of pyridoxal phosphatedependent enzymes, may not be desirable. Metabolic Loading Tests
A direct test of the adequacy of an individual’s intake to meet his or her idiosyncratic metabolic requirement is the ability to metabolize a test dose of a substrate whose metabolism is dependent on the vitamin. For vitamin B6, two metabolic loading tests can be used, although neither can be considered to be reliable for population studies of vitamin B6 status. The Tryptophan Load Test The oxidative pathway of tryptophan metabolism is shown in Figure 4. Kynureninase is a pyridoxal phosphate-dependent enzyme, and in vitamin B6 deficiency its activity is lower than that of tryptophan dioxygenase. This means that there is a considerable accumulation of both hydroxykynurenine and kynurenine, sufficient to permit greater than usual metabolic flux through kynurenine transaminase, resulting in increased formation of kynurenic and xanthurenic acids. Although kynurenine transaminase is also pyridoxal phosphate-dependent, it is relatively unaffected in vitamin B6 deficiency. Kynureninase is exquisitely sensitive to vitamin B6 deficiency because it undergoes a slow inactivation as a result of catalyzing the half-reaction of transamination in addition to its normal reaction. The resultant enzyme with pyridoxamine phosphate at the catalytic site is catalytically inactive, and can only be reactivated if there is an adequate concentration of pyridoxal phosphate to displace the pyridoxamine phosphate. Xanthurenic and kynurenic acids are easy to measure in urine, so that the ability to metabolize a test dose of 2 or 5 g of tryptophan has been widely adopted as a convenient and sensitive index of vitamin B6 nutritional status. However, induction of tryptophan dioxygenase by glucocorticoid hormones will result in a greater rate of formation of kynurenine and hydroxykynurenine than the capacity of kynureninase, and will thus lead to increased formation of kynurenic and xanthurenic acids – an effect similar to that seen in vitamin B6 deficiency. Such results may be erroneously interpreted as indicating vitamin B6 deficiency in a variety of subjects whose problem is increased glucocorticoid secretion as a result of stress or illness, not vitamin B6 deficiency. Inhibition of kynureninase, e.g., by estrogen metabolites, also results in accumulation of kynurenine and hydroxykynurenine, and hence increased formation of kynurenic and xanthurenic acids, again giving results which falsely suggest vitamin B6 deficiency.
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6026 VITAMIN B6/Physiology COOH CH2
Tryptophan
CH
NH2
N H
Tryptophan dioxygenase and formylkynurenine hydroxylase O C
OH CH2
CH
COOH
NH2 Kynurenine aminotransferase
NH2 Kynurenine
COOH N Kynurenic acid
Kynurenine hydroxylase O C
OH CH2
CH
COOH
NH2 NH2
Kynurenine aminotransferase
OH 3-Hydroxykynurenine
COOH
N OH
Xanthurenic acid
Kynureninase (vitamin B6-dependent) Alanine CO2
Acetyl CoA COOH COOH
Nicotinamide
NH2 OH 3-Hydroxyanthranilic acid fig0004
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N Quinolinic acid
COOH
Figure 4 The oxidative pathway of tryptophan metabolism – the basis of the tryptophan load test for vitamin B6 status.
This has been widely, but incorrectly, interpreted as estrogen-induced vitamin B6 deficiency – it is in fact simple competitive inhibition by estrogen metabolites of the enzyme that is the basis of the tryptophan load test. There is normally a considerable excess of either apokynureninase or kynureninase that has undergone transamination, and has pyridoxamine phosphate at the catalytic site, in the liver. This can be activated by (relatively high concentrations of) pyridoxal phosphate. The abnormalities of tryptophan metabolism associated with increased activity of tryptophan dioxygenase, or inhibition of kynureninase by estrogen metabolites, are thus corrected by the administration
of high doses of vitamin B6, although they are not in fact due to deficiency. This means that, while the tryptophan load test may be an appropriate index of status in controlled depletion/repletion studies to determine vitamin B6 requirements, it is not an appropriate index of status in population studies. The methionine loading test The metabolism of methionine, shown in Figure 5, includes two pyridoxal phosphate-dependent steps, catalyzed by cystathionine synthetase and cystathionase. In vitamin B6 deficiency there is an increase in the plasma concentration of homocysteine, and increased urinary
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VITAMIN B6/Physiology
Acceptor
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Methylated product S -adenosylhomocysteine
S-adenosylmethionine Methyltransferase
PPi, Pi
Methionine adenosyltransferase ATP
Adenosine
CH3 SH
S Tetrahydrofolate
CH2
Methyl tetrahydrofolate
CH2 HC
NH2
COOH
CH2
CH2
CH2
CH2
HC Methionine synthetase (vitamin B12-dependent)
Methionine
NH2
HC Nonenzymic
S
S
CH2 CH2
NH2
HC
COOH
COOH
Homocysteine
Homocystine
NH2
COOH
Serine Cystathionine synthetase (vitamin B6-dependent) H 2O
HC
CH2 HC
NH2
COOH Cystathionine fig0005
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Cystathionase (vitamin B6-dependent)
CH2
S
CH2
CH2
NH2
COOH
HC
H2O
NH4+ + α-Ketobutyrate
SH NH2
COOH Cysteine
Figure 5 The pathway of methionine metabolism – the basis of the methionine load test for vitamin B6 status.
excretion of cystathionine and homocysteine, both after a loading dose of methionine and under basal conditions. The ability to metabolize a test dose of methionine therefore provides an index of vitamin B6 nutritional status. Because measurement of homocysteine and cystathionine is technically less easy than measurement of xanthurenic and kynurenic acids, the methionine load test has been less widely used than the tryptophan load test. Some 10–25% of the population have a genetic predisposition to hyperhomocysteinemia, which is a risk factor for atherosclerosis and coronary heart disease, as a result of polymorphism in the gene for methylenetetrahydrofolate reductase. As discussed below, there is no evidence that supplements of vitamin B6 reduce fasting plasma homocysteine in these subjects and, like the tryptophan load test, the methionine load test may be an appropriate index
of status in controlled depletion/repletion studies to determine vitamin B6 requirements, but not in population studies.
Requirements and Recommendations The total body pool of vitamin B6 is of the order of 15 mmol (3.7 mg) per kg of body weight. Isotope tracer studies suggest that there is a turnover of about 0.13% per day, and hence a minimum requirement for replacement of 0.02 mmol (5 mg) per kg of body weight – some 350 mg day1 for a 70-kg adult. Most studies of vitamin B6 requirements have followed the development of abnormalities of tryptophan (and sometimes also methionine) metabolism during depletion, and normalization during repletion with graded intakes of the vitamin. While the tryptophan and methionine loading tests are unreliable as
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indices of vitamin B6 nutritional status in field studies, under the controlled conditions of depletion/repletion studies they do indeed provide a useful indication of the state of vitamin B6 nutrition. Although some 80% of the total body pool of vitamin B6 is associated with muscle glycogen phosphorylase, this pools turns over relatively slowly. The major metabolic role of the remaining 20% of total body vitamin B6, which turns over considerably more rapidly, is in amino acid metabolism. Therefore, a priori, it seems likely that protein intake will affect vitamin B6 requirements. People maintained on (experimental) vitamin B6deficient diets develop abnormalities of tryptophan and methionine metabolism faster, and their blood vitamin B6 falls more rapidly, when their protein intake is high. Similarly, during repletion of deficient subjects, tryptophan and methionine metabolism and blood vitamin B6 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 day1 for the low protein intake, which is barely adequate to maintain nitrogen balance, and 150 g day1 for the high intake, which is considerably higher than average western intakes of protein. These studies suggest a mean requirement of 13 mg of vitamin B6 per gram of dietary protein; recommended 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 B6. More recent studies of women’s requirements have suggested an RDA of 20 mg g1 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
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The identification of hyperhomocysteinemia as an independent risk factor in atherosclerosis and coronary heart disease has led to suggestions that higher intakes of vitamin B6 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 reaction that is dependent on vitamin B12 and folic acid), or onward metabolism leading to the synthesis of cysteine (the vitamin B6-dependent transsulfuration pathway). 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 B6 status. However, a number of studies have shown that, while folate supplements lower fasting homocysteine in moderately hyperhomocysteinemic subjects, 10 mg day1 vitamin B6 has no effect. Vitamin B6 supplements do, however, reduce the peak plasma concentration of homocysteine following a test dose of methionine. This can probably be explained by the kinetics of the enzymes involved. The Km of cystathionine synthetase is 10fold 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 homocysteine rises to relatively high levels, that the activity of cystathionine synthetase, rather than the availability of its substrate, is limiting or transsulfuration. It is thus unlikely that intakes of vitamin B6 above amounts that are adequate to prevent metabolic signs of deficiency will be beneficial in lowering plasma concentrations of homocysteine.
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Vitamin B6 Requirements of Infants
Estimation of the RDA for vitamin B6 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 mg l1, or 3–8 mg of vitamin B6 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. A first approximation of the vitamin B6 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 mg day1 there was an incidence of convulsions of 0.3%. Provision of 260 mg day1 prevented or cured convulsions, but 300 mg day1 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 therefore result in a higher apparent requirement. Based on the body content of 15 mmol (3.7 mg) of vitamin B6 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) day1 to establish tissue reserves.
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Pharmacological Uses and Toxicity of Vitamin B6 Supplements
Vitamin B6 and the Side-Effects of Oral Contraceptives
Supplements of vitamin B6 ranging from 25 to 500 mg day1, and sometimes higher, have been recommended for the treatment of a variety of conditions in which there is an underlying physiological or biochemical mechanism to justify the use of supplements, although in most cases there is little evidence of efficacy. Such conditions include postnatal depression, depression and other side-effects associated with oral contraceptives, hyperemesis of pregnancy, and the premenstrual syndrome. 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 dermatitis, autism, dental caries, diabetic neuropathy, Down’s syndrome, Huntington’s chorea, schizophrenia, and steroid-dependent asthma. Doses of 100 mg day1 have been reported to be beneficial in the treatment of the carpal tunnel syndrome, or what has been called tenosynovitis. However, most of the reports originate from one center, and there appears to be little or no independent confirmation of the usefulness of the vitamin in this condition. Vitamin B6 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 suggested that it acts to stimulate dopaminergic activity in the hypothalamus. However, it is more likely that its action is by reduction in target tissue responsiveness to the steroid hormones that stimulate prolactin secretion. Doses of 50–200 mg day1 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 B6 has any beneficial effect in pregnancy sickness, nor that women who suffer from morning sickness have lower vitamin B6 nutritional status than other pregnant women. There have been reports of a teratogenic effect of vitamin B6 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 B6 itself is teratogenic. However, since it downregulates responsiveness to steroid hormones and retinoids, it is possible that high levels of vitamin B6 intake may affect embryonic or fetal development.
Although estrogens do not cause vitamin B6 deficiency, the administration of vitamin B6 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. Impairment of glucose tolerance is common in pregnancy, and may indeed be severe enough to be classified as gestational diabetes mellitus, which generally resolves at parturition, although in some subjects 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 B6 supplements may have a beneficial effect on glucose tolerance by activating apokynureninase or kynureninase that has been inactivated by undergoing transamination. One of the relatively common side-effects of estrogenic 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 B6 (generally in excess of 40 mg day1). Postnatal depression also responds to similar supplements in some studies. Again, this does not seem to be due to correction of vitamin B6 deficiency, but rather to a direct effect of pyridoxal phosphate on the metabolism of tryptophan. 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 B6. 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 moodelevating effect.
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Vitamin B6 in the Premenstrual Syndrome
The studies showing a beneficial action of vitamin B6 in overcoming depression associated with oral contraceptives have led to the use of the vitamin in depression and other pathology associated with endogenous estrogens, in the premenstrual syndrome. There is no evidence of poorer vitamin B6 nutritional status in women who suffer from the premenstrual syndrome.
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There are few well-controlled studies of the effects of vitamin B6 in premenstrual syndrome. In general, those that have been properly controlled report little benefit from doses between 50 and 200 mg day1 compared with placebo, although some studies do claim a beneficial effect. Interestingly, 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. Despite the lack of evidence of efficacy, vitamin B6 is widely prescribed (and self-prescribed) for the treatment of the premenstrual syndrome. Toxicity of Vitamin B6
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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 degeneration 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 B6 toxicity in animals regress after withdrawal of these massive doses, but sensory nerve conduction velocity, which decreases during the development of the neuropathy, does not recover fully. The mechanism of the neurotoxic action of vitamin B6 is unknown. The development of sensory neuropathy has been reported in patients taking 2–7 g of pyridoxine HCl day1. Although there was residual damage in some patients, withdrawal of these extremely high doses resulted in a considerable recovery of sensory nerve function. Other reports have suggested that intakes as low as 50 mg day1 are associated with neurological damage, although these have been based on patients reporting symptoms rather than on detailed neurological examination. Nevertheless, this led to a proposal in the UK in 1997 to regulate the sale of vitamin B6 supplements, with 50 mg day1 being available only on prescription, and between 10 and 50 mg day1 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 day1.
Vitamin B6 Deficiency Gross clinical deficiency of vitamin B6 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. In vitamin B6-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-dependent reaction), changes in leukocyte count and activity, a tendency to epileptiform convulsions, and peripheral nervous system damage resulting in ataxia and sensory neuropathy. Much of our knowledge of human vitamin B6 deficiency 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 pyridoxyllysine by reaction between pyridoxal phosphate and the e-amino groups of lysine in proteins. In addition to a number of metabolic abnormalities, many of the affected infants convulsed. They responded to the administration of vitamin B6 supplements. Investigation of the neurochemical basis of the convulsions in vitamin B6 deficiency helped to elucidate the role of GABA as a neurotransmitter; GABA is synthesized by the decarboxylation of glutamate. More recent studies have suggested that the accumulation 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 accumulation is considerably more marked in neonates than adults – only neonates convulse in vitamin B6 deficiency. GABA depletion may be a necessary but not sufficient condition for convulsions in vitamin B6 deficiency.
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Vitamin B6 Dependency Syndromes
A small number of cases have been reported of patients with genetic defects which result in an abnormally high requirement for vitamin B6 in order to maintain the activity of the affected enzyme (Table 2). Such vitamin B6 dependency syndromes have been reported in cases of xanthurenic aciduria, homocystinuria, hypochromic sideroblastic anemia, ornithinemia, and infantile convulsions. The molecular basis of the defects appears to be a severely impaired affinity of the affected enzyme for its
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VITAMIN B6/Physiology tbl0002
Table 2 Vitamin B6-responsive inborn errors of metabolism Enzyme affected Convulsions of the newborn Cystathioninuria Gyrate atrophy with ornithinuria Homocystinuria Primary hyperoxaluria, type 1 Sideroblastic anemia Xanthurenic aciduria
Enzyme defect not known Cystathionase (Figure 5 ) Ornithine-d-aminotransferase Cystathionine synthase (Figure 5 ) Peroxisomal alanineglyoxylate transaminase d-Aminolevulinate synthase (# heme synthesis) Kynureninase (Figure 4 )
cofactor, and patients respond well to doses of 500– 1000 mg of vitamin B6 per day. Apart from the affected enzyme, other biochemical indices of vitamin B6 nutritional status are normal in these patients. Interestingly, there are few reports of peripheral neuropathy among such patients treated with high doses of vitamin B6 for many years. Groups at Risk of Deficiency 0080
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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 coefficient, suggesting vitamin B6 inadequacy or deficiency. In most studies, only one of these indices of vitamin B6 nutritional status has been assessed. Where both have been assessed, while each shows some 10% of the population apparently inadequately provided with vitamin B6, few of the subjects show inadequacy by both criteria. There is a decrease in the plasma concentration of vitamin B6 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 B6 deficiency. It is not known whether this reflects an inadequate intake, a greater requirement, or changes in the tissue distribution and metabolism of the vitamin with increasing age. Drug-induced vitamin B6 deficiency A number of drugs which react with carbonyl compounds are capable of causing vitamin B6 deficiency on prolonged use. These include the antituberculosis drug isoniazid (iso-nicotinic acid hydrazide), penicillamine, and the antiparkinsonian drugs Benserazide and Carbidopa. In general, the main effect is impairment 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 B6 or niacin. Isoniazid also causes peripheral
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neuropathy, which responds to vitamin B6 supplements, but not to niacin. Estrogens and vitamin B6 nutritional status There have been many reports of abnormal tryptophan metabolism in women taking estrogens as oral contraceptives and menopausal hormone replacement therapy. These have been widely interpreted as evidence of estrogen-induced vitamin B6 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 B6 status have been reported, they have been generally unaffected by contraceptive use, again suggesting an effect on tryptophan metabolism per se, rather than on vitamin B6 nutritional status. In many cases the metabolism of tryptophan has been normalized by the administration of vitamin B6 supplements of the order of 20–50 mg day1, compared with an RDA of 1.4–1.6 mg day1. It was noted above that there is an apparent excess of apokynureninase in the liver and therefore the administration of vitamin B6 supplements will increase kynurenine metabolism, even when there is no preexisting deficiency. See also: Amino Acids: Metabolism; Contraceptives: Nutritional Aspects; Glycogen; Hormones: Steroid Hormones; Infants: Nutritional Requirements; Premenstrual Syndrome: Nutritional Aspects; Vitamin B6: Properties and Determination
Further Reading Bender DA (1987) Oestrogens and vitamin B6 – actions and interactions. World Review of Nutrition and Dietetics 51: 140–188. Bender DA (1992) Nutritional Biochemistry of the Vitamins. Cambridge: Cambridge University Press. Bender DA (1999) Non-nutritional uses of vitamin B6. British Journal of Nutrition 81: 7–20. Bender DA (2003) Nutritional Biochemistry of the Vitamins, 2nd edn. New York, Cambridge University Press. Coburn SP (1996) Modelling vitamin B6 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 50 -phosphate enzyme studies. Annual Review of Biochemistry 59: 87–110. Ink SL and Henderson LM (1984) Vitamin B6 metabolism. Annual Review of Nutrition 4: 455–470. Lumeng L, Lui A and Li T-K (1980) Plasma content of B6 vitamers and its relationship to hepatic vitamin B6 metabolism. Journal of Clinical Investigation 66: 688–695.
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6032 VITAMIN K/Properties and Determination 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 B6 metabolism of utilization. Annual Review of Nutrition 7: 137–156.
Wiss O and Weber F (1964) Biochemical pathology of vitamin B6 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 Copyright 2003, Elsevier Science Ltd. All Rights Reserved.
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 physicochemical 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 variety of physiological processes related to a number of extrahepatic vitamin K-dependent proteins. (See Vitamins: Overview.)
Chemical Structures and Nomenclature Naturally occurring compounds with vitamin K activity possess a common 2-methyl-1,4-naphthoquinone
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Physiology D A Bender, University College London, London, UK Copyright 2003, Elsevier Science Ltd. All Rights Reserved.
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Pyridoxal phosphate bound as a Schiff base to lysine in dietary proteins is released on digestion of the protein. The phosphorylated vitamers are dephosphorylated by membrane-bound alkaline phosphatase in the intestinal mucosa; pyridoxal, pyridoxamine, and pyridoxine are all absorbed rapidly by passive diffusion. Intestinal mucosal cells have pyridoxine kinase, and pyridoxine phosphate oxidase so that there is net accumulation by metabolic trapping. Much of the ingested pyridoxine is released into the portal circulation as pyridoxal, after dephosphorylation at the serosal surface.
Introduction
Metabolism and Transport
Vitamin B6 has a central role in amino acid metabolism, as the coenzyme for a variety of reactions, including transamination and decarboxylation. It is also the coenzyme of glycogen phosphorylase, and acts to modulate the activity of steroid and other hormones (including retinoids and vitamin D) which act by regulation of gene expression.
Most of the absorbed vitamin is taken up by the liver, although other tissues can also take up the unphosphorylated vitamers from the circulation. Uptake is by carrier-mediated diffusion, followed by metabolic trapping as phosphate esters. Pyridoxine and pyridoxamine phosphates are oxidized to pyridoxal phosphate. All tissues have pyridoxine kinase activity, but pyridoxine phosphate oxidase is only found in liver, kidney, and brain (Figure 1). Pyridoxine phosphate oxidase is a flavoprotein, and its activity falls markedly in riboflavin deficiency. Despite this central role of riboflavin in vitamin B6 metabolism, blood and tissue concentrations of pyridoxal phosphate are not affected by riboflavin deficiency, and riboflavin nutrition appears to have no effect on vitamin B6 nutritional status. Pyridoxine phosphate oxidase is inhibited by its product, pyridoxal phosphate. This is not simple product inhibition, but involves binding at a specific inhibitor site on the enzyme. The normal intracellular concentration of free pyridoxal phosphate gives significant inhibition, which indicates that this is a physiologically important mechanism in the control of tissue pyridoxal phosphate. Pyridoxine is phosphorylated rapidly in liver and other tissues. Pyridoxal phosphate does not cross cell membranes, and uptake and efflux of the vitamin in most tissues are as pyridoxal. Pyridoxal phosphate is exported from the liver bound to albumin. Much of the free pyridoxal phosphate in the liver is hydrolyzed to pyridoxal, which is also exported, and circulates bound both to albumin and to hemoglobin in erythrocytes. Free pyridoxal remaining in the liver is rapidly oxidized to 4-pyridoxic acid, which is the main excretory product of the vitamin. Extrahepatic tissues take up pyridoxal from the plasma. Pyridoxal phosphate is hydrolyzed to pyridoxal, which can cross cell membranes, by
Dietary Forms, Biological Availability, and Metabolism 0002
Digestion and Absorption
The main form of vitamin B6 in foods is pyridoxal phosphate, bound to enzymes. There is also a small amount of pyridoxamine phosphate. In plant foods a significant amount of the vitamin is present as pyridoxine. A number of plants contain relatively large amounts of pyridoxine glycosides, which are not biologically available, since they are not substrates for mammalian glycosidases. They are absorbed (passively) from the intestinal lumen and are excreted more or less quantitatively in the urine. Between 5% and 50% of the total vitamin B6 in some foods may be present as pyridoxine glycosides. A proportion of the vitamin B6 in foods may be biologically unavailable, especially after heating, as a result of the formation of (phospho)pyridoxyllysine by reduction of the aldimine (Schiff base), by which pyridoxal phosphate is bound to the e-amino groups of lysine residues in proteins. While some of this pyridoxyllysine may be useable, since it is a substrate for pyridoxine phosphate oxidase, it is also a vitamin B6 antimetabolite, and even at relatively low concentrations can accelerate the development of deficiency in experimental animals maintained on deficient diets. In the 1950s there was an outbreak of vitamin B6 deficiency among infants fed on formula that had been overheated in manufacture, resulting in the formation of relatively large amounts of pyridoxyllysine.
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VITAMIN B6/Physiology OH
CH2OH HOCH2
O
Kinase
OH
P
CH2OH O
CH2
OH
OH
Phosphatase CH3
N
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CH3
N
Pyridoxine
Pyridoxine phosphate Oxidase
HC
COOH HOCH2
OH
O
HOCH2
OH
OH
O
Kinase
Oxidase
HC
P
O
O
CH2
OH
OH Phosphatase
N
CH3
CH3
N
4-Pyridoxic acid
Pyridoxal phosphate
Pyridoxal
Transaminases
CH2NH2 HOCH2
OH
N
CH3
Pyridoxamine fig0001
0011
0012
CH3
N
Oxidase
CH2NH2
OH Kinase Phosphatase
O
P
O
CH2
OH
OH
N
CH3
Pyridoxamine phosphate
Figure 1 Metabolism of vitamin B6.
extracellular alkaline phosphatase, then trapped intracellularly by phosphorylation. Tissue concentrations of pyridoxal phosphate are controlled by the balance between phosphorylation and dephosphorylation. The activity of phosphatases acting on pyridoxal phosphate is greater than that of the kinase in most tissues. This means that pyridoxal phosphate that is not bound to enzymes will be dephosphorylated and hence leave the cell by diffusion. Thus there is little accumulation of pyridoxal phosphate in tissues, other than that which is bound to enzymes and other proteins (e.g., hormone receptors). Free pyridoxal either leaves the cell or is oxidized to 4-pyridoxic acid by aldehyde dehydrogenase, which is present in all tissues, and also by hepatic and renal aldehyde oxidase. 4-Pyridoxic acid is the main excretory product of vitamin B6, and its excretion reflects recent intake more than the state of underlying tissue reserves of the vitamin. Small amounts of pyridoxal and pyridoxamine are also
excreted in the urine, although much of the active vitamin B6 which is filtered at the glomerulus is resorbed in the kidney tubules. Storage and Body Reserves
There is no specific storage of vitamin B6 in the body; as discussed above, pyridoxal phosphate that is not bound to enzymes is rapidly dephosphorylated, oxidized to 4-pyridoxic acid, and excreted. The total body pool of vitamin B6 is of the order of 60 mmol (250 mg); 15 mmol (3.7 mg) per kg body weight. About 80% of this is in muscle, associated with glycogen phosphorylase. This does not seem to function as a true reserve of the vitamin and is not released from muscle in times of deficiency. Muscle pyridoxal phosphate is released into the circulation (as pyridoxal) in starvation, as muscle glycogen reserves are exhausted, and there is less requirement for glycogen phosphorylase activity. Under these conditions it is available for redistribution to other tissues, especially liver and kidney, to
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C H2N
Lysine NH
H C N OH O
P O CH2
Lysine
HC
NH
O
H
R C COOH NH2 Amino acid substrate
OH
O
O
N
N OH
CO2 O OH Decarboxylation
HC
P O CH2 OH
OH
C
R C COOH
CH3 N Substrate aldimine
CH3 N Pyridoxal phosphate internal aldimine (Schiff base)
OH
HC
O
Lysine NH
P O CH2
OH
OH N
CH3
R−CH2−NH2 Product amine
Tautomerization H R C COOH N OH O
HC
P O CH2 OH N
Transamination (amino transfer) OH H2C NH2 H2O O P O CH2 OH OH OH CH3
Substrate ketimine
fig0002
Metabolic Functions of Vitamin B6 The metabolically active vitamer is pyridoxal phosphate, which is involved in many reactions of amino acid metabolism, where the carbonyl group is the reactive moiety, in glycogen phosphorylase, where it is the phosphate group that is important in catalysis, and in the release of hormone receptors from tight nuclear binding, where again it is the carbonyl group that is important. The Role of Pyridoxal Phosphate in Amino Acid Metabolism 0017
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CH3 N Pyridoxamine phosphate
O Product oxo-acid (keto-acid)
Figure 2 Roles of vitamin B6 in amino acid metabolism.
meet the increased requirement of transamination of amino acids for gluconeogenesis.
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R C COOH
The various reactions of pyridoxal phosphate in amino acid metabolism (Figure 2) all depend on the same chemical principle – the ability to stabilize amino acid carbanions, and hence to weaken bonds about the a-carbon of the substrate. This is achieved by reaction of the a-amino group with the carbonyl group of the coenzyme to form a Schiff base (aldimine). Pyridoxal phosphate is bound to enzymes, in the absence of the substrate, by the formation of an
internal Schiff base to the e-amino group of a lysine residue at the active site. Thus the first reaction between the substrate and the coenzyme is transfer of the aldimine linkage from this e-amino group to the a-amino group of the substrate. The ring nitrogen of pyridoxal phosphate exerts a strong electron-withdrawing effect on the aldimine, and this leads to weakening of all three bonds about the a-carbon of the substrate. In nonenzymic model systems, all the possible pyridoxal-catalyzed reactions are observed: a-decarboxylation, aminotransfer, racemization, and relevant side-chain elimination and replacement reactions. By contrast, enzymes show specificity for the reaction pathway followed; which bond is cleaved will depend on the orientation of the Schiff base relative to reactive groups of the catalytic site. However, a number of decarboxylases and enzymes that catalyze side-chain elimination reactions of amino acids undergo gradual inactivation as a result of catalyzing the half-reaction of transamination, leaving (catalytically inactive) pyridoxamine phosphate at the catalytic site. a-Decarboxylation If the electron-withdrawing effect of the ring nitrogen is primarily centered on
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the a-carbon-carboxyl bond, the result is decarboxylation of the amino acid with the release of carbon dioxide. The resultant carbanion is then protonated, and the primary amine corresponding to the amino acid is displaced by the lysine residue at the active site, with reformation of the internal Schiff base. A number of the products of the decarboxylation of amino acids are important as neurotransmitters and hormones: 5-hydroxytryptamine, the catecholamines dopamine, norepinephrine (noradrenaline), and epinephrine (adrenaline), histamine and g-aminobutyrate (GABA), and as the diamines and polyamines involved in the regulation of DNA metabolism. The decarboxylation of phosphatidylserine to phosphatidylethanolamine is important in phospholipid metabolism.
Transamination Hydrolysis of the a-carbon-amino bond of the ketimine formed by deprotonation of the a-carbon of the amino acid results in the release of the 2-oxo-acid corresponding to the amino acid substrate, and leaves pyridoxamine phosphate at the catalytic site of the enzyme. In this case there is no reformation of the internal Schiff base to the reactive lysine residue. This is the half-reaction of transamination. The process is completed by reaction of pyridoxamine phosphate with a second oxo-acid substrate, forming an intermediate ketimine, followed by the reverse of the reaction sequence shown in Figure 2, releasing the amino acid corresponding to this second substrate after displacement from the aldimine by the reactive lysine residue to reform the internal Schiff base. Transamination (Figure 3) is of central importance in amino acid metabolism, providing pathways for the catabolism of all of the amino acids except lysine, which does not undergo transamination. Many of these reactions are linked to the amination of 2-oxoglutarate to glutamate or glyoxylate to glycine, which are substrates for oxidative deamination, reforming the oxo-acids. Transamination reactions also provide a pathway for the synthesis of those amino acids for which there is an alternative source of the oxo-acid (the nonessential amino acids). Indeed, the nonessential amino acids can be defined as those whose oxoacids can be formed other than from the amino acid itself.
Racemization of Amino Acids Deprotonation of the a-carbon of the amino acid leads to tautomerization of the Schiff base to yield a quinonoid ketimine. The simplest reaction that the ketimine can undergo is reprotonation at the now symmetrical a-carbon. Displacement of the substrate by the reactive lysine residue results in the racemic mixture of d- and l-amino acid. Amino acid racemases are important in bacterial metabolism, since several d-amino acids are required for the synthesis of cell wall mucopolysaccharides. There is no evidence that there are any mammalian amino acid racemases; such utilization of d-amino acids as occurs is probably due to the action of renal d-amino acids oxidase to form the symmetrical 2-oxo-acid which is then a substrate for transamination.
H R1
C
O
P
Side-chain elimination and replacement reactions The third bond in the Schiff base aldimine that can be labilized by the electron-withdrawing H
OH O
C
O
NH2
R2
N Pyridoxal phosphate
COOH
O Product oxo-acid (keto-acid)
fig0003
COOH
Product amino acid
CH2
OH C
C NH2
CH3
Substrate amino acid Amino donor
R1
H
OH
CH2
OH
COOH
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O
P
O
CH2
NH2 OH
OH N Pyridoxamine phosphate
Figure 3 The role of vitamin B6 in transamination reactions.
R2
C
COOH
O CH3
Substrate oxo-acid (keto-acid) Amino acceptor
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effect of the ring nitrogen of pyridoxal phosphate is that between the a-carbon and the side-chain of the amino acids, resulting in a variety of a–b elimination and b–g replacement reactions. The Role of Pyridoxal Phosphate in Glycogen Phosphorylase 0027
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Glycogen phosphorylase catalyzes the sequential phosphorolysis of glycogen to release glucose 1-phosphate; it is thus the key enzyme in the utilization of muscle and liver reserves of glycogen. Unlike other pyridoxal phosphate-dependent enzymes, in which the carbonyl group is essential for catalysis, the internal Schiff base between pyridoxal phosphate and lysine in glycogen phosphorylase is not broken during the reaction. The catalytic region of the coenzyme is the 50 -phosphate group. The initial stage in the phosphorolysis of glycogen is protonation of the glycosidic oxygen of the polysaccharide by inorganic phosphate. The resultant oxycarbonium ion is stabilized by the inorganic phosphate. The role of pyridoxal phosphate is as a proton shuttle or buffer to stabilize the oxycarboniumphosphate ion pair, permitting covalent binding of the phosphate to the oxycarbonium ion, to form glucose 1-phosphate. The Role of Pyridoxal Phosphate in Steroid Hormone Action
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Pyridoxal phosphate has a role in modulating the action of those hormones which act by binding to a nuclear receptor protein, inducing transcription of DNA, and hence regulating gene expression, leading to new protein synthesis. Such hormones include androgens, estrogens, progesterone, glucocorticoids, calcitriol (the active metabolite of vitamin D), retinoic acid and other retinoids, and thyroid hormone. Target-tissue specificity of hormone action is insured by the presence of characteristic receptor proteins with a zinc finger motif, which are responsible for both nuclear uptake of the hormone and the interaction with DNA and nucleoproteins to initiate gene expression. Pyridoxal phosphate reacts with a lysine residue in the receptor protein, and displaces the hormone– receptor complex from tight nuclear binding. In vitro, reaction with pyridoxal phosphate also inhibits the binding of receptor protein to isolated DNA and chromatin. The effect is specific for the phosphorylated vitamer, suggesting that there may be a specific pyridoxal-phosphate binding site on the receptor proteins, and it occurs at low concentrations of pyridoxal phosphate, of the same order of magnitude as occur in tissues under normal conditions.
This suggests that pyridoxal phosphate acts as a cofactor in the release of hormone–receptor complexes from tight nuclear binding, resulting in release of the receptor from the nucleus, termination of hormone action, and recycling receptor protein for further uptake of hormone. In experimental animals, vitamin B6 deficiency results in increased and prolonged nuclear uptake and retention of steroid hormones in target tissues, and there is enhanced sensitivity to hormone action. Deficient animals show greater induction of uterine peroxidase, and considerably greater suppression of the hypothalamic secretion of luteinizing hormone, by estrogens than do vitamin B6-supplemented controls. In vitamin B6-deficient male animals there is an increased mitotic response of the prostate to low doses of testosterone. Deficient male animals have a higher activity of ornithine decarboxylase (an androgen-induced enzyme) in the liver, and deficient females have higher renal ornithine transaminase (an estrogen-induced enzyme). The induction of hepatic tyrosine transaminase and tryptophan dioxygenase by glucocorticoids is also enhanced in vitamin B6-deficient animals. In a variety of cells in culture that have been transfected with a glucocorticoid, estrogen, or progesterone response element linked to a reporter gene, acute vitamin B6 depletion (by incubation with 4-deoxypyridoxine) leads to a twofold increase in expression of the reporter gene in response to hormone action. Conversely, incubation of these cells with high concentrations of pyridoxal, leading to a high intracellular concentration of pyridoxal phosphate, results in a halving of the expression of the reporter gene in response to hormone stimulation.
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Criteria of Adequacy and Assessment of Nutritional Status Plasma Concentrations of the Vitamin
The fasting plasma concentration of either total vitamin B6 or, more specifically, pyridoxal phosphate, is widely used as an index of vitamin B6 nutritional status. The generally accepted criteria of adequacy are shown in Table 1. Conditions that involve increased plasma activity of alkaline phosphatase may result in reduced plasma pyridoxal phosphate, without affecting tissue concentrations of pyridoxal phosphate or vitamin B6 nutritional status, as assessed by other criteria. There is a compensatory increase in the circulating concentration of pyridoxal, which is the main form for tissue uptake of vitamin B6. Despite the fall in plasma pyridoxal phosphate in pregnancy, which has been
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VITAMIN B6/Physiology tbl0001
Table 1 Indices of vitamin B6 nutritional status Adequate status Plasma total vitamin B6 Plasma pyridoxal phosphate Erythrocyte alanine aminotransferase activation coefficient Erythrocyte asparate aminotransferase activation coefficient Erythrocyte asparate aminotransferase Urine 4-pyridoxic acid
Urine total vitamin B6
Urine xanthurenic acid after 2 g tryptophan load Urine cystathionine after 3 g methionine load
> 40 nmol (10 mg) l1 >30 nmol (7.5 mg) l1 <1.25 <1.80 >0.13 units (8.4 mkat) l1 >3.0 mmol 24 h1 >1.3 mmol mol1 creatinine >0.5 mmol 24 h1 >0.2 mmol mol1 creatinine <65 mmol 24 h1 increase <350 mmol 24 h1 increase
widely interpreted as indicating vitamin B6 depletion, the plasma concentration of (pyridoxal phosphate plus pyridoxal) is unchanged. This suggests that determination of plasma pyridoxal phosphate alone may not be a reliable index of vitamin B6 nutritional status. Urinary Excretion of 4-Pyridoxic Acid 0036
About half of the normal dietary intake of vitamin B6 is excreted as 4-pyridoxic acid. Urinary excretion of 4-pyridoxic acid will largely reflect recent intake of the vitamin rather than underlying nutritional status; the criteria for assessment of 4-pyridoxic acid excretion are shown in Table 1. Coenzyme Saturation of Transaminases
0037
0038
Various pyridoxal phosphate-dependent enzymes compete with each other for the available pool of coenzyme. Thus the extent to which an enzyme is saturated with its coenzyme provides a means of assessing the adequacy of the body pool of coenzyme. This can be determined by measuring the activity of the enzyme before and after the activation of any apoenzyme present in the sample by incubation with pyridoxal phosphate added in vitro. Erythrocyte aspartate and alanine transaminases are both commonly used; the results are expressed as either the percentage stimulation of activity by added pyridoxal phosphate, or the activation coefficient – the ratio of activity with added coenzyme to that without added coenzyme. It seems to be normal for a proportion of pyridoxal phosphate-dependent enzymes to be present as inactive apoenzyme, without coenzyme. This may be a
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mechanism for metabolic regulation. It is possible that increasing the intake of vitamin B6, so as to insure complete saturation of pyridoxal phosphatedependent enzymes, may not be desirable. Metabolic Loading Tests
A direct test of the adequacy of an individual’s intake to meet his or her idiosyncratic metabolic requirement is the ability to metabolize a test dose of a substrate whose metabolism is dependent on the vitamin. For vitamin B6, two metabolic loading tests can be used, although neither can be considered to be reliable for population studies of vitamin B6 status. The Tryptophan Load Test The oxidative pathway of tryptophan metabolism is shown in Figure 4. Kynureninase is a pyridoxal phosphate-dependent enzyme, and in vitamin B6 deficiency its activity is lower than that of tryptophan dioxygenase. This means that there is a considerable accumulation of both hydroxykynurenine and kynurenine, sufficient to permit greater than usual metabolic flux through kynurenine transaminase, resulting in increased formation of kynurenic and xanthurenic acids. Although kynurenine transaminase is also pyridoxal phosphate-dependent, it is relatively unaffected in vitamin B6 deficiency. Kynureninase is exquisitely sensitive to vitamin B6 deficiency because it undergoes a slow inactivation as a result of catalyzing the half-reaction of transamination in addition to its normal reaction. The resultant enzyme with pyridoxamine phosphate at the catalytic site is catalytically inactive, and can only be reactivated if there is an adequate concentration of pyridoxal phosphate to displace the pyridoxamine phosphate. Xanthurenic and kynurenic acids are easy to measure in urine, so that the ability to metabolize a test dose of 2 or 5 g of tryptophan has been widely adopted as a convenient and sensitive index of vitamin B6 nutritional status. However, induction of tryptophan dioxygenase by glucocorticoid hormones will result in a greater rate of formation of kynurenine and hydroxykynurenine than the capacity of kynureninase, and will thus lead to increased formation of kynurenic and xanthurenic acids – an effect similar to that seen in vitamin B6 deficiency. Such results may be erroneously interpreted as indicating vitamin B6 deficiency in a variety of subjects whose problem is increased glucocorticoid secretion as a result of stress or illness, not vitamin B6 deficiency. Inhibition of kynureninase, e.g., by estrogen metabolites, also results in accumulation of kynurenine and hydroxykynurenine, and hence increased formation of kynurenic and xanthurenic acids, again giving results which falsely suggest vitamin B6 deficiency.
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6026 VITAMIN B6/Physiology COOH CH2
Tryptophan
CH
NH2
N H
Tryptophan dioxygenase and formylkynurenine hydroxylase O C
OH CH2
CH
COOH
NH2 Kynurenine aminotransferase
NH2 Kynurenine
COOH N Kynurenic acid
Kynurenine hydroxylase O C
OH CH2
CH
COOH
NH2 NH2
Kynurenine aminotransferase
OH 3-Hydroxykynurenine
COOH
N OH
Xanthurenic acid
Kynureninase (vitamin B6-dependent) Alanine CO2
Acetyl CoA COOH COOH
Nicotinamide
NH2 OH 3-Hydroxyanthranilic acid fig0004
0043
N Quinolinic acid
COOH
Figure 4 The oxidative pathway of tryptophan metabolism – the basis of the tryptophan load test for vitamin B6 status.
This has been widely, but incorrectly, interpreted as estrogen-induced vitamin B6 deficiency – it is in fact simple competitive inhibition by estrogen metabolites of the enzyme that is the basis of the tryptophan load test. There is normally a considerable excess of either apokynureninase or kynureninase that has undergone transamination, and has pyridoxamine phosphate at the catalytic site, in the liver. This can be activated by (relatively high concentrations of) pyridoxal phosphate. The abnormalities of tryptophan metabolism associated with increased activity of tryptophan dioxygenase, or inhibition of kynureninase by estrogen metabolites, are thus corrected by the administration
of high doses of vitamin B6, although they are not in fact due to deficiency. This means that, while the tryptophan load test may be an appropriate index of status in controlled depletion/repletion studies to determine vitamin B6 requirements, it is not an appropriate index of status in population studies. The methionine loading test The metabolism of methionine, shown in Figure 5, includes two pyridoxal phosphate-dependent steps, catalyzed by cystathionine synthetase and cystathionase. In vitamin B6 deficiency there is an increase in the plasma concentration of homocysteine, and increased urinary
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VITAMIN B6/Physiology
Acceptor
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Methylated product S -adenosylhomocysteine
S-adenosylmethionine Methyltransferase
PPi, Pi
Methionine adenosyltransferase ATP
Adenosine
CH3 SH
S Tetrahydrofolate
CH2
Methyl tetrahydrofolate
CH2 HC
NH2
COOH
CH2
CH2
CH2
CH2
HC Methionine synthetase (vitamin B12-dependent)
Methionine
NH2
HC Nonenzymic
S
S
CH2 CH2
NH2
HC
COOH
COOH
Homocysteine
Homocystine
NH2
COOH
Serine Cystathionine synthetase (vitamin B6-dependent) H 2O
HC
CH2 HC
NH2
COOH Cystathionine fig0005
0046
Cystathionase (vitamin B6-dependent)
CH2
S
CH2
CH2
NH2
COOH
HC
H2O
NH4+ + α-Ketobutyrate
SH NH2
COOH Cysteine
Figure 5 The pathway of methionine metabolism – the basis of the methionine load test for vitamin B6 status.
excretion of cystathionine and homocysteine, both after a loading dose of methionine and under basal conditions. The ability to metabolize a test dose of methionine therefore provides an index of vitamin B6 nutritional status. Because measurement of homocysteine and cystathionine is technically less easy than measurement of xanthurenic and kynurenic acids, the methionine load test has been less widely used than the tryptophan load test. Some 10–25% of the population have a genetic predisposition to hyperhomocysteinemia, which is a risk factor for atherosclerosis and coronary heart disease, as a result of polymorphism in the gene for methylenetetrahydrofolate reductase. As discussed below, there is no evidence that supplements of vitamin B6 reduce fasting plasma homocysteine in these subjects and, like the tryptophan load test, the methionine load test may be an appropriate index
of status in controlled depletion/repletion studies to determine vitamin B6 requirements, but not in population studies.
Requirements and Recommendations The total body pool of vitamin B6 is of the order of 15 mmol (3.7 mg) per kg of body weight. Isotope tracer studies suggest that there is a turnover of about 0.13% per day, and hence a minimum requirement for replacement of 0.02 mmol (5 mg) per kg of body weight – some 350 mg day1 for a 70-kg adult. Most studies of vitamin B6 requirements have followed the development of abnormalities of tryptophan (and sometimes also methionine) metabolism during depletion, and normalization during repletion with graded intakes of the vitamin. While the tryptophan and methionine loading tests are unreliable as
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6028 VITAMIN B6/Physiology
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indices of vitamin B6 nutritional status in field studies, under the controlled conditions of depletion/repletion studies they do indeed provide a useful indication of the state of vitamin B6 nutrition. Although some 80% of the total body pool of vitamin B6 is associated with muscle glycogen phosphorylase, this pools turns over relatively slowly. The major metabolic role of the remaining 20% of total body vitamin B6, which turns over considerably more rapidly, is in amino acid metabolism. Therefore, a priori, it seems likely that protein intake will affect vitamin B6 requirements. People maintained on (experimental) vitamin B6deficient diets develop abnormalities of tryptophan and methionine metabolism faster, and their blood vitamin B6 falls more rapidly, when their protein intake is high. Similarly, during repletion of deficient subjects, tryptophan and methionine metabolism and blood vitamin B6 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 day1 for the low protein intake, which is barely adequate to maintain nitrogen balance, and 150 g day1 for the high intake, which is considerably higher than average western intakes of protein. These studies suggest a mean requirement of 13 mg of vitamin B6 per gram of dietary protein; recommended 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 B6. More recent studies of women’s requirements have suggested an RDA of 20 mg g1 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
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The identification of hyperhomocysteinemia as an independent risk factor in atherosclerosis and coronary heart disease has led to suggestions that higher intakes of vitamin B6 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 reaction that is dependent on vitamin B12 and folic acid), or onward metabolism leading to the synthesis of cysteine (the vitamin B6-dependent transsulfuration pathway). 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 B6 status. However, a number of studies have shown that, while folate supplements lower fasting homocysteine in moderately hyperhomocysteinemic subjects, 10 mg day1 vitamin B6 has no effect. Vitamin B6 supplements do, however, reduce the peak plasma concentration of homocysteine following a test dose of methionine. This can probably be explained by the kinetics of the enzymes involved. The Km of cystathionine synthetase is 10fold 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 homocysteine rises to relatively high levels, that the activity of cystathionine synthetase, rather than the availability of its substrate, is limiting or transsulfuration. It is thus unlikely that intakes of vitamin B6 above amounts that are adequate to prevent metabolic signs of deficiency will be beneficial in lowering plasma concentrations of homocysteine.
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Vitamin B6 Requirements of Infants
Estimation of the RDA for vitamin B6 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 mg l1, or 3–8 mg of vitamin B6 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. A first approximation of the vitamin B6 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 mg day1 there was an incidence of convulsions of 0.3%. Provision of 260 mg day1 prevented or cured convulsions, but 300 mg day1 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 therefore result in a higher apparent requirement. Based on the body content of 15 mmol (3.7 mg) of vitamin B6 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) day1 to establish tissue reserves.
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VITAMIN B6/Physiology
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Pharmacological Uses and Toxicity of Vitamin B6 Supplements
Vitamin B6 and the Side-Effects of Oral Contraceptives
Supplements of vitamin B6 ranging from 25 to 500 mg day1, and sometimes higher, have been recommended for the treatment of a variety of conditions in which there is an underlying physiological or biochemical mechanism to justify the use of supplements, although in most cases there is little evidence of efficacy. Such conditions include postnatal depression, depression and other side-effects associated with oral contraceptives, hyperemesis of pregnancy, and the premenstrual syndrome. 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 dermatitis, autism, dental caries, diabetic neuropathy, Down’s syndrome, Huntington’s chorea, schizophrenia, and steroid-dependent asthma. Doses of 100 mg day1 have been reported to be beneficial in the treatment of the carpal tunnel syndrome, or what has been called tenosynovitis. However, most of the reports originate from one center, and there appears to be little or no independent confirmation of the usefulness of the vitamin in this condition. Vitamin B6 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 suggested that it acts to stimulate dopaminergic activity in the hypothalamus. However, it is more likely that its action is by reduction in target tissue responsiveness to the steroid hormones that stimulate prolactin secretion. Doses of 50–200 mg day1 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 B6 has any beneficial effect in pregnancy sickness, nor that women who suffer from morning sickness have lower vitamin B6 nutritional status than other pregnant women. There have been reports of a teratogenic effect of vitamin B6 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 B6 itself is teratogenic. However, since it downregulates responsiveness to steroid hormones and retinoids, it is possible that high levels of vitamin B6 intake may affect embryonic or fetal development.
Although estrogens do not cause vitamin B6 deficiency, the administration of vitamin B6 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. Impairment of glucose tolerance is common in pregnancy, and may indeed be severe enough to be classified as gestational diabetes mellitus, which generally resolves at parturition, although in some subjects 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 B6 supplements may have a beneficial effect on glucose tolerance by activating apokynureninase or kynureninase that has been inactivated by undergoing transamination. One of the relatively common side-effects of estrogenic 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 B6 (generally in excess of 40 mg day1). Postnatal depression also responds to similar supplements in some studies. Again, this does not seem to be due to correction of vitamin B6 deficiency, but rather to a direct effect of pyridoxal phosphate on the metabolism of tryptophan. 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 B6. 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 moodelevating effect.
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Vitamin B6 in the Premenstrual Syndrome
The studies showing a beneficial action of vitamin B6 in overcoming depression associated with oral contraceptives have led to the use of the vitamin in depression and other pathology associated with endogenous estrogens, in the premenstrual syndrome. There is no evidence of poorer vitamin B6 nutritional status in women who suffer from the premenstrual syndrome.
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6030 VITAMIN B6/Physiology 0070
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There are few well-controlled studies of the effects of vitamin B6 in premenstrual syndrome. In general, those that have been properly controlled report little benefit from doses between 50 and 200 mg day1 compared with placebo, although some studies do claim a beneficial effect. Interestingly, 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. Despite the lack of evidence of efficacy, vitamin B6 is widely prescribed (and self-prescribed) for the treatment of the premenstrual syndrome. Toxicity of Vitamin B6
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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 degeneration 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 B6 toxicity in animals regress after withdrawal of these massive doses, but sensory nerve conduction velocity, which decreases during the development of the neuropathy, does not recover fully. The mechanism of the neurotoxic action of vitamin B6 is unknown. The development of sensory neuropathy has been reported in patients taking 2–7 g of pyridoxine HCl day1. Although there was residual damage in some patients, withdrawal of these extremely high doses resulted in a considerable recovery of sensory nerve function. Other reports have suggested that intakes as low as 50 mg day1 are associated with neurological damage, although these have been based on patients reporting symptoms rather than on detailed neurological examination. Nevertheless, this led to a proposal in the UK in 1997 to regulate the sale of vitamin B6 supplements, with 50 mg day1 being available only on prescription, and between 10 and 50 mg day1 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 day1.
Vitamin B6 Deficiency Gross clinical deficiency of vitamin B6 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. In vitamin B6-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-dependent reaction), changes in leukocyte count and activity, a tendency to epileptiform convulsions, and peripheral nervous system damage resulting in ataxia and sensory neuropathy. Much of our knowledge of human vitamin B6 deficiency 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 pyridoxyllysine by reaction between pyridoxal phosphate and the e-amino groups of lysine in proteins. In addition to a number of metabolic abnormalities, many of the affected infants convulsed. They responded to the administration of vitamin B6 supplements. Investigation of the neurochemical basis of the convulsions in vitamin B6 deficiency helped to elucidate the role of GABA as a neurotransmitter; GABA is synthesized by the decarboxylation of glutamate. More recent studies have suggested that the accumulation 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 accumulation is considerably more marked in neonates than adults – only neonates convulse in vitamin B6 deficiency. GABA depletion may be a necessary but not sufficient condition for convulsions in vitamin B6 deficiency.
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Vitamin B6 Dependency Syndromes
A small number of cases have been reported of patients with genetic defects which result in an abnormally high requirement for vitamin B6 in order to maintain the activity of the affected enzyme (Table 2). Such vitamin B6 dependency syndromes have been reported in cases of xanthurenic aciduria, homocystinuria, hypochromic sideroblastic anemia, ornithinemia, and infantile convulsions. The molecular basis of the defects appears to be a severely impaired affinity of the affected enzyme for its
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VITAMIN B6/Physiology tbl0002
Table 2 Vitamin B6-responsive inborn errors of metabolism Enzyme affected Convulsions of the newborn Cystathioninuria Gyrate atrophy with ornithinuria Homocystinuria Primary hyperoxaluria, type 1 Sideroblastic anemia Xanthurenic aciduria
Enzyme defect not known Cystathionase (Figure 5 ) Ornithine-d-aminotransferase Cystathionine synthase (Figure 5 ) Peroxisomal alanineglyoxylate transaminase d-Aminolevulinate synthase (# heme synthesis) Kynureninase (Figure 4 )
cofactor, and patients respond well to doses of 500– 1000 mg of vitamin B6 per day. Apart from the affected enzyme, other biochemical indices of vitamin B6 nutritional status are normal in these patients. Interestingly, there are few reports of peripheral neuropathy among such patients treated with high doses of vitamin B6 for many years. Groups at Risk of Deficiency 0080
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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 coefficient, suggesting vitamin B6 inadequacy or deficiency. In most studies, only one of these indices of vitamin B6 nutritional status has been assessed. Where both have been assessed, while each shows some 10% of the population apparently inadequately provided with vitamin B6, few of the subjects show inadequacy by both criteria. There is a decrease in the plasma concentration of vitamin B6 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 B6 deficiency. It is not known whether this reflects an inadequate intake, a greater requirement, or changes in the tissue distribution and metabolism of the vitamin with increasing age. Drug-induced vitamin B6 deficiency A number of drugs which react with carbonyl compounds are capable of causing vitamin B6 deficiency on prolonged use. These include the antituberculosis drug isoniazid (iso-nicotinic acid hydrazide), penicillamine, and the antiparkinsonian drugs Benserazide and Carbidopa. In general, the main effect is impairment 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 B6 or niacin. Isoniazid also causes peripheral
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neuropathy, which responds to vitamin B6 supplements, but not to niacin. Estrogens and vitamin B6 nutritional status There have been many reports of abnormal tryptophan metabolism in women taking estrogens as oral contraceptives and menopausal hormone replacement therapy. These have been widely interpreted as evidence of estrogen-induced vitamin B6 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 B6 status have been reported, they have been generally unaffected by contraceptive use, again suggesting an effect on tryptophan metabolism per se, rather than on vitamin B6 nutritional status. In many cases the metabolism of tryptophan has been normalized by the administration of vitamin B6 supplements of the order of 20–50 mg day1, compared with an RDA of 1.4–1.6 mg day1. It was noted above that there is an apparent excess of apokynureninase in the liver and therefore the administration of vitamin B6 supplements will increase kynurenine metabolism, even when there is no preexisting deficiency. See also: Amino Acids: Metabolism; Contraceptives: Nutritional Aspects; Glycogen; Hormones: Steroid Hormones; Infants: Nutritional Requirements; Premenstrual Syndrome: Nutritional Aspects; Vitamin B6: Properties and Determination
Further Reading Bender DA (1987) Oestrogens and vitamin B6 – actions and interactions. World Review of Nutrition and Dietetics 51: 140–188. Bender DA (1992) Nutritional Biochemistry of the Vitamins. Cambridge: Cambridge University Press. Bender DA (1999) Non-nutritional uses of vitamin B6. British Journal of Nutrition 81: 7–20. Bender DA (2003) Nutritional Biochemistry of the Vitamins, 2nd edn. New York, Cambridge University Press. Coburn SP (1996) Modelling vitamin B6 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 50 -phosphate enzyme studies. Annual Review of Biochemistry 59: 87–110. Ink SL and Henderson LM (1984) Vitamin B6 metabolism. Annual Review of Nutrition 4: 455–470. Lumeng L, Lui A and Li T-K (1980) Plasma content of B6 vitamers and its relationship to hepatic vitamin B6 metabolism. Journal of Clinical Investigation 66: 688–695.
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6032 VITAMIN K/Properties and Determination 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 B6 metabolism of utilization. Annual Review of Nutrition 7: 137–156.
Wiss O and Weber F (1964) Biochemical pathology of vitamin B6 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 Copyright 2003, Elsevier Science Ltd. All Rights Reserved.
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 physicochemical 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 variety of physiological processes related to a number of extrahepatic vitamin K-dependent proteins. (See Vitamins: Overview.)
Chemical Structures and Nomenclature Naturally occurring compounds with vitamin K activity possess a common 2-methyl-1,4-naphthoquinone
0002