Development of simultaneous enzymatic assay method for all six individual vitamin B6 forms and pyridoxine-beta-glucoside.

A method for determining all of the six natural vitamin B(6) compounds and pyridoxine-beta-glucoside in urine from humans consuming their usual diet was developed. These compounds were specifically converted with 5 enzymes into a high fluorescent 4-pyridoxolactone which was supersensitively determined by an isocratic HPLC. All of the compounds in urine from humans consuming their usual diets were for the first time determined together. The preparation procedure for urine samples was easy without HCl-hydrolysis, and the enzyme reactions took only 2 or 3 h. It required only 5 microL of the urine sample for analysis of one of the compounds. Urine samples from five young Japanese males consuming their usual diet contained pyridoxal, pyridoxamine, and pyridoxine-beta-glucoside but not pyridoxine or phosphoester forms. The contents of 4-pyridoxic acid and pyridoxal correlate well with a correlation coefficient of 0.98. On the other hand, the content of pyridoxamine did not correlate with that of 4-pyridoxic acid.

[Effect of protein content in rat diet on water-soluble vitamin metabolism in streptozotocin-induced diabetes]. / Vliianie urovnia belka v ratsione krys na obmen vodorastvorimykh vitaminov pri streptozototsinovom diabete.

Water-soluble group B vitamins metabolism was studied over the course of streptozotocin-induced diabetes mellitus in rats fed semisynthetic isocaloric diets containing 18 and 50% of protein. A high-protein diet in diabetes mellitus does not influence riboflavin metabolism disordered in this disease but reduced 4-pyridoxyl acid excretion to the level characteristic of healthy animals. The observed trend to an increase of liver nicotinamide coenzymes levels and of 1-methylnicotinamide urinary excretion reflects increased niacin synthesis from the diet protein tryptophan, for niacin level is reduced in diabetes.

Assay of pyridoxal-5'-phosphate, pyridoxal and pyridoxic acid in biological material.

The two vitamin B6-vitamers having an aldehyde function are oxidised to the corresponding acids and subjected to an HPLC separation on an RP 18 phase with a solvent consisting of 5% methanol in phosphate buffer at pH 3.5. The detection is carried out by fluorometry with excitation at 318 nm and emission at 418 nm. The peaks obtained correspond to pyridoxic acid 5'-phosphate and pyridoxic acid. Pyridoxal-5'-phosphate is determined as pyridoxic acid 5'-phosphate. Pyridoxal is determined as pyridoxic acid by subtracting the amount of pyridoxic acid already existing before oxidation.

Identification of pyridoxine 3-sulfate, pyridoxal 3-sulfate, and N-methylpyridoxine as major urinary metabolites of vitamin B6 in domestic cats.

Preliminary studies of vitamin B6 metabolism in three adult domestic cats detected very little pyridoxic acid in the urine. At oral doses of 49 to 490 mumol of [14C]pyridoxine hydrochloride, 50% of the excreted dose occurred as pyridoxine 3-sulfate and 25% as N-methylpyridoxine. The identity of these two metabolites was confirmed by isolation from urine and comparison with known compounds. A third compound was identified as pyridoxal 3-sulfate on the basis of chromatographic behavior and fluorescent properties before and after hydrolysis. At pyridoxine intakes of 0.97 mumol/day, the concentration of pyridoxal 3-sulfate in the urine sometimes exceeded the concentration of pyridoxine 3-sulfate. Pyridoxic acid remained a minor urinary metabolite at pyridoxine intakes ranging from 0.97 to 490 mumol/day. Although sulfation of phenol groups and methylation of the ring nitrogen are well-known detoxication reactions, this appears to be the first time such reactions have been observed in normal metabolism of vitamin B6. These observations provide further evidence of the diversity of vitamin B6 metabolism between species. While such diversity complicates the extrapolation of data from animal studies to humans, it does provide a variety of models for examining the influences of various factors on vitamin B6 metabolism.

Relationship between body store of vitamin B6 and plasma pyridoxal-P clearance: metabolic balance studies in humans.

Factors that regulate the clearance of plasma pyridoxal-P (PLP) are unknown. Four volunteers were given a diet supplying approximately 12 mumol pyridoxine (PN) per day. The pharmacokinetics of plasma PLP clearance were measured in these subjects before and after 4 weeks of intravenous PN supplementation (122 mumol/day). Urinary B6 excretion, mainly as 4-pyridoxic acid (4-PA), increased progressively after initiation of PN supplementation until a new steady state was reached on day 10 of supplementation, whereupon greater than 93% of the daily injected PN could be recovered in the urine. Hence, urinary excretion is almost the sole route for vitamin B6 elimination. Fasting plasma PLP concentration increased with supplementation and also reached a new steady state at this time. When supplementation was terminated, urinary B6 excretion decreased in 5 days to an amount only slightly higher than that before supplementation. This amount was maintained for 2 months. By comparison, plasma PLP decreased more slowly and remained considerably higher than the presupplementation level for the rest of the study. These data confirm that urinary 4-PA excretion is a better indicator of B6 intake than is plasma PLP content, whereas plasma PLP content is a better indicator of the body store of the vitamin. Plasma clearance and volume of distribution of PLP decreased significantly after supplementation, but half-life t 1/2 did not change. Plasma clearance of PLP, therefore, is dependent on the vitamin B6 status of an individual.

Polyamine-pyridoxal Schiff bases in urine.

Schiff bases of the diamines 1,3-diaminopropane, putrescine, and cadaverine and the polyamines spermidine and spermine with pyridoxal or pyridoxal phosphate occur in human urine, as shown by gas chromatographic/mass spectrometric selected ion-monitoring techniques. By use of synthetic standards, procedures were devised for conversion of the Schiff bases to stable derivatives amenable to gas chromatographic/mass spectrometric analysis. These procedures involve borohydride reduction of the C = N double bond, hydrolytic removal of the phosphate group, chromatographic separation from the bulk of urinary constituents, and trifluoroacetylation of polar functional groups. The levels of the polyamine-pyridoxal Schiff bases were estimated to be in the range of pmol/ml or urine.

Effects of penicillamine on distribution of B6 vitamens in rat urine.

The antivitamin B6 effect of DL- and D-penicillamine has been studied in rats. A considerable elevation in the urinary excretion of vitamin B6 activity (pyridoxal and its thiazolidine derivative) has been shown as a parameter of B6 antagonism. Both DL- and D-pencillamine have been shown to have an antivitamin B6 effect in rats, although that induced by the DL-form is considerably greater, as would be expected from previous studies. We suggest that B6 supplementation should be included in any long term penicillamine therapy, regardless of the isomer that is employed.

New pathway of conversion of pyridoxal to 4-pyridoxic acid.

The only enzyme demonstrated so far to catalyze the formation of 4-pyridoxic acid, the final excretory product of vitamin B6, was aldehyde oxidase. In this paper we have presented an evidence that another enzyme, NAD+-dependent aldehyde dehydrogenase, is capable of catalyzing this reaction. Rat mutants with high, low and no aldehyde oxidase activity excrete the same amount of isotope after a single injection of 3H-pyridoxol in a 12-day experiment; 4-pyridoxic acid is also present in the urine of animals without aldehyde oxidase activity. There is no correlation between the proportion of the excreted acid and the amount of pyridoxal excreted, after the injection of a single dose of the aldehyde form. The Km values for pyridoxal and NAD+, at pH 9.6, have been found to be 75 and 260 mumol/l, respectively. NAD+-dependent pyridoxal dehydrogenase was partially purified from the rat tissues, and the following specific enzyme activities (nmol-min-1-mg-1 protein) were found: red blood cell 71.5 +/- 3.0, intestine 64.9 +/- 9.0, muscle 61.4 +/- 1.6, brain 39.5 +/- 6.0, liver 34.4 +/- 3.3, kidney 21.1 +/- 5.6, heart 18.8 +/- 0.9, and lung 6.5 +/- 2.0. This is believed to be the first report demonstrating pyridoxal oxidation, catalyzed by an NAD+-dependent aldehyde dehydrogenase.