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

COO-

HOCHc

COO-

HOCHc

N CH3 4-pyridoxic acid

N CH3 4-pyridoxic acid oxidase

CH2OH HOCH2, OH

'N CH3 pyridoxine

HC=O

HOCH2

HC=O

HOCH2

pyridoxal

pyridoxal

kinase phosphatase

'N CH3 pyridoxine kinase

CH2OH

CH2OH

"N CH3 pyridoxine phosphate

"N CH3 pyridoxine phosphate oxidase phosphatase

HC=O

HC=O

'N CH3 pyridoxal phosphate

'N CH3 pyridoxal phosphate ch2nh+

'N CH3 pyridoxamine

'N CH3 pyridoxamine

Figure 1 Metabolism of vitamin B6

kinase phosphatase transaminases

oxidase

CH2NH+

CH2NH+

'N CH3 pyridoxamine phosphate nuclear binding, where again it is the carbonyl group that is important.

The Role of Pyridoxal Phosphate in Amino Acid Metabolism

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, amino-transfer, racemization, and 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.

«-Decarboxylation If the electron-withdrawing effect of the ring nitrogen is primarily centered on the a-carbon-carboxyl bond, the result is decarbo-xylation 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 catecho-lamines dopamine, noradrenaline, and adrenaline, and histamine and 7-aminobutyrate (GABA)—and as the diamines and polyamines involved in the regulation of DNA metabolism. The decarboxyla-tion of phosphatidylserine to phosphatidylethanola-mine is important in phospholipid metabolism.

amino acid substrate pyridoxal phosphate internal aldimine (Schiff base)

O"

Figure 2 Roles of vitamin B6 in amino acid metabolism.

lysine

N XH3 substrate aldimine

O"

decarboxylation / I

O"

N XH3 substrate aldimine

R—CH2—NH3 product amine tautomerization

transamination (aminotransfer)

N CH3 substrate ketimine transamination (aminotransfer)

N CH3 substrate ketimine

'N "CH3 pyridoxamine phosphate

product oxo-acid (keto-acid)

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 have long been known to be important in bacterial metabolism since several D-amino acids are required for the synthesis of cell wall mucopolysaccharides. D-Serine is found in relatively large amounts in mammalian brain, where it acts as an agonist of the N-methyl-D-aspartate (NMDA) type of glutamate receptor. Serine race-mase has been purified from rat brain and cloned from human brain.

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. 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 3, releasing the amino acid corresponding to this second substrate after displacement from the

total oxidation and synthesis of the nicotinamide ring of NAD

Figure 3 Tryptophan metabolism, the basis of the tryptophan load test for vitamin B6 status.

aldimine by the reactive lysine residue to reform the internal Schiff base.

Transamination is of central importance in amino acid metabolism, providing pathways for catabolism of most amino acids as well as the synthesis of those amino acids for which there is a source of the oxo-acid other than from the amino acid itself—the nonessential amino acids.

The Role of Pyridoxal Phosphate in Steroid Hormone Action

Pyridoxal phosphate has a role in controlling the action of hormones that act by binding to a nuclear receptor protein and modulating gene expression. Such hormones include androgens, estrogens, progesterone, glucocorticoids, calcitriol (the active metabolite of vitamin D), retinoic acid and other retinoids, and thyroid hormone. Pyridoxal phosphate reacts with a lysine residue in the receptor protein and displaces the hormone-receptor complex from DNA binding, so terminating the hormone action.

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. In a variety of cells in culture that have been transfected with a gluco-corticoid, estrogen or progesterone response element linked to a reporter gene, acute vitamin B6 depletion (by incubation with 4-deoxypyridoxine) leads to a 2-fold 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.

Assessment of Vitamin B6 Nutritional Status

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, as is the urinary excretion of 4-pyridoxic acid. The generally accepted criteria of adequacy are shown in Table 1.

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 coen-zyme. This can be determined by measuring the activity of the enzyme before and after the

Table 1 Indices of vitamin B6 nutritional status

Index

Adequate status

Plasma total vitamin B6 Plasma pyridoxal phosphate Erythrocyte alanine aminotransferase activation coefficient Erythrocyte aspartate aminotransferase activation coefficient Erythrocyte aspartate aminotransferase Urine 4-pyridoxic acid

Urine total vitamin B6

Urine xanthurenic acid after

2 g tryptophan load Urine cystathionine after 3g methionine load

>40nmol (10 mg)/l >30nmol (7.5 mg)/l <1.25

>3.0 mmol/24 h >1.3mmol/mol creatinine >0.5 mmol/24 h >0.2mmol/mol creatinine <65 mmol/24h increase

<350 mmol/24h increase

Data from Bitsch R (1993) Vitamin B6. International Journal of Vitamin and Nutrition Research 63: 278-282; Leklem JE (1990) Vitamin B-6: A status report. Journal of Nutrition 120(supplement 11): 1503-1507; McChrisley B, Thye FW, McNair HM and Driskell JA (1988) Plasma B6 vitamer and 4-pyridoxic acid concentrations of men fed controlled diets. Journal of Chromatography 428: 35-42.

activation of any apoenzyme present in the sample by incubation with pyridoxal phosphate added in vitro. Erythrocyte aspartate and alanine transami-nases are both commonly used; the results are usually expressed as an activation coefficient— the ratio of activity with added coenzyme to that without.

It seems to be normal for a proportion of pyri-doxal phosphate-dependent enzymes to be present as inactive apoenzyme, without coenzyme. This may be a mechanism for metabolic regulation. It is possible that increasing the intake of vitamin B6, so as to ensure complete saturation of pyridoxal phosphate-dependent enzymes, may not be desirable.

Tryptophan Load Test

The oxidative pathway of tryptophan metabolism is shown in Figure 3. Kynureninase is a pyridoxal phosphate-dependent enzyme, and in deficiency its activity is lower than that of tryptophan dioxygen-ase, so that there is an accumulation of hydroxy-kynurenine and kynurenine, resulting in greater metabolic flux through kynurenine transaminase and increased formation of kynurenic and xanthurenic acids. Kynureninase is exquisitely sensitive to vitamin B6 deficiency because it undergoes a slow inactivation as a result of catalysing the half-reaction of transamination instead of 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.

The ability to metabolise a test dose of tryptophan has been widely adopted as a convenient and sensitive index of vitamin B6 nutritional status. However, induction of tryptophan dioxygenase by glucocorti-coid 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 glu-cocorticoid 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. This has been widely, but incorrectly, interpreted as estrogen-induced vitamin B6 deficiency: it is in fact simple competitive inhibition of the enzyme that is the basis of the tryptophan load test by estrogen metabolites.

While the tryptophan load test is a useful index of status in controlled depletion/repletion studies to determine vitamin B6 requirements, it is not an appropriate index of status in population studies.

Methionine Loading Test

The metabolism of methionine, shown in Figure 4, includes two pyridoxal phosphate-dependent steps, catalysed by cystathionine synthetase and cystathio-nase. In vitamin B6 deficiency there is an increase in the plasma concentration of homocysteine, and increased urinary excretion of cystathionine and homocysteine, both after a loading dose of methio-nine and under basal conditions. The ability to metabolize a test dose of methionine therefore provides an index of vitamin B6 nutritional status.

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 polymorphisms in the gene for methylenetetrahydrofolate reductase. 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

cysteine

Figure 4 Methionine metabolism, the basis of the methionine load test for vitamin B6 status.

cysteine

Figure 4 Methionine metabolism, the basis of the methionine load test for vitamin B6 status.

controlled depletion/repletion studies to determine vitamin B6 requirements, but not in population studies.

Requirements and Reference Intakes

The total body pool of vitamin B6 is of the order of 15 mmol (3.7 mg) per kilogram body weight. Isotope tracer studies suggest there is turnover of about 0.13% per day, and hence a minimum requirement for replacement of 0.02 mmol (5 mg) per kilogram body weight—some 350 mg per day for a 70 kg adult. However, depletion/repletion studies suggest that requirements are higher than this.

Most studies of vitamin B6 requirements have followed the development of abnormalities of tryp-tophan (and sometimes also methionine) metabolism during depletion and normalization during repletion with graded intakes of the vitamin.

Although some 80% of the total body pool of vitamin B6 is associated with muscle glycogen phos-phorylase, this pool 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 B6-deficient 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, tryp-tophan and methionine metabolism and blood vitamin B6 are normalized faster at low than at high levels of protein intake.

These studies suggest a mean requirement of 13 mg of vitamin B6 per gram of dietary protein; reference intakes are based on 15-16 mg per gram 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 depletion/repletion studies, using more sensitive indices of status, in which subjects were repleted with either a constant intake of vitamin B6 and varying amounts of protein or a constant amount of protein and varying amounts of vitamin B6, have shown average requirements of 15-16 mg/g of dietary protein, suggesting a reference intake of 18-20 mg/g protein.

In 1998 the reference intake in the United States and Canada was reduced from the previous RDA of 2mg/day for men and 1.6mg/day for women to 1.3mg/day for both, compared with the UK RNI of 1.2 mg for women and 1.4 mg for men. The report cites six studies that demonstrated that this level of intake would maintain a plasma concentration of pyridoxal phosphate at least 20 nmol/l, although, as shown in Table 1, the more generally accepted criterion of adequacy is 30 nmol/l.

Possible Benefits of Higher Levels of Intake

The identification of hyperhomocysteinaemia as an independent risk factor in atherosclerosis and coronary heart disease has led to suggestions that higher intakes of vitamin B6 may be beneficial. As shown in Figure 4, homocysteine may undergo either of two metabolic fates: remethylation to methionine (a reaction that is dependent on vitamin B12 and folate) or vitamin B6-dependent trans-sulfuration to yield cysteine.

A number of studies have shown that while folate supplements lower fasting homocysteine in moderately hyperhomocysteinemic subjects, 10mg/day vitamin B6 has no effect, although they do reduce the peak plasma concentration of homocysteine following a test dose of methionine.

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 per liter, 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 per day there was an incidence of convulsions of 0.3%. Provision of 260 mg per day prevented or cured convulsions, but 300 mg per day was required to normalize tryptophan metabolism. This is almost certainly a considerable overestimate of requirements since pyr-idoxyllysine, 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 (3.7mg) of vitamin B6 per kilogram 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 (417nmol) per day to establish tissue reserves.

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