High-performance liquid chromatographic method for profiling 2-oxo acids in urine and its application in evaluating vitamin status in rats.

B-group vitamins are involved in the catabolism of 2-oxo acids. To identify the functional biomarkers of B-group vitamins, we developed a high-performance liquid chromatographic method for profiling 2-oxo acids in urine and applied this method to urine samples from rats deficient in vitamins B1 and B6 and pantothenic acid. 2-Oxo acids were reacted with 1,2-diamino-4,5-methylenebenzene to produce fluorescent derivatives, which were then separated using a TSKgel ODS-80Ts column with 30 mmol/L of KH2PO4 (pH 3.0):acetonitrile (7:3) at a flow rate of 1.0 mL/min. Vitamin B1 deficiency increased urinary levels of all 2-oxo acids, while vitamin B6 deficiency only increased levels of sum of 2-oxaloacetic acid and pyruvic acid, and pantothenic acid deficiency only increased levels of 2-oxoisovaleric acid. Profiles of 2-oxo acids in urine samples might be a non-invasive way of clarifying the functional biomarker of B-group vitamins.

Direct and Functional Biomarkers of Vitamin B6 Status.

Measures of B6 status are categorized as direct biomarkers and as functional biomarkers. Direct biomarkers measure B6 vitamers in plasma/serum, urine and erythrocytes, and among these plasma pyridoxal 5'-phosphate (PLP) is most commonly used. Functional biomarkers include erythrocyte transaminase activities and, more recently, plasma levels of metabolites involved in PLP-dependent reactions, such as the kynurenine pathway, one-carbon metabolism, transsulfuration (cystathionine), and glycine decarboxylation (serine and glycine). Vitamin B6 status is best assessed by using a combination of biomarkers because of the influence of potential confounders, such as inflammation, alkaline phosphatase activity, low serum albumin, renal function, and inorganic phosphate. Ratios between substrate-products pairs have recently been investigated as a strategy to attenuate such influence. These efforts have provided promising new markers such as the PAr index, the 3-hydroxykynurenine:xanthurenic acid ratio, and the oxoglutarate:glutamate ratio. Targeted metabolic profiling or untargeted metabolomics based on mass spectrometry allow the simultaneous quantification of a large number of metabolites, which are currently evaluated as functional biomarkers, using data reduction statistics.

A mathematical model of tryptophan metabolism via the kynurenine pathway provides insights into the effects of vitamin B-6 deficiency, tryptophan loading, and induction of tryptophan 2,3-dioxygenase on tryptophan metabolites.

Vitamin B-6 deficiency is associated with impaired tryptophan metabolism because of the coenzyme role of pyridoxal 5'-phosphate (PLP) for kynureninase and kynurenine aminotransferase. To investigate the underlying mechanism, we developed a mathematical model of tryptophan metabolism via the kynurenine pathway. The model includes mammalian data on enzyme kinetics and tryptophan transport from the intestinal lumen to liver, muscle, and brain. Regulatory mechanisms and inhibition of relevant enzymes were included. We simulated the effects of graded reduction in cellular PLP concentration, tryptophan loads and induction of tryptophan 2,3-dioxygenase (TDO) on metabolite profiles and urinary excretion. The model predictions matched experimental data and provided clarification of the response of metabolites in various extents of vitamin B-6 deficiency. We found that moderate deficiency yielded increased 3-hydroxykynurenine and a decrease in kynurenic acid and anthranilic acid. More severe deficiency also yielded an increase in kynurenine and xanthurenic acid and more pronounced effects on the other metabolites. Tryptophan load simulations with and without vitamin B-6 deficiency showed altered metabolite concentrations consistent with published data. Induction of TDO caused an increase in all metabolites, and TDO induction together with a simulated vitamin B-6 deficiency, as has been reported in oral contraceptive users, yielded increases in kynurenine, 3-hydroxykynurenine, and xanthurenic acid and decreases in kynurenic acid and anthranilic acid. These results show that the model successfully simulated tryptophan metabolism via the kynurenine pathway and can be used to complement experimental investigations.

Type 1 diabetes impairs vitamin B(6) metabolism at an early stage of women's adulthood.

Vitamin B(6) (pyridoxine) metabolism in diabetes has never been investigated except for a few reports on plasma pyridoxal 5'-phosphate (PLP). These studies indicated that this most active (coenzyme) vitamer can be reduced. The present clinical investigation aimed to measure all vitamers in blood and urine by high performance liquid chromatography as well as important related factors, in women during active reproductive years. Thirty-two insulin-treated type 1 diabetic (T1D) patients, without renal complication, and 27 well-matched healthy controls, aged 30 to 40 years old, were recruited using rigorous criteria. Both groups had normal hemoglobin and serum albumin levels. Plasma PLP and pyridoxal (PL) did not differ significantly in the T1D group but alkaline phosphatase (ALP) activity was greater (p < 0.01). This produced a shift in plasma PLP-PL profile, as evidenced by a lower plasma PLP/PL ratio (p < 0.05). Enhanced ALP activity meant more PLP being dephosphorylated to PL (the membrane transfer form), with more ending up in erythrocytes to be rephosphorylated in its active form, as suggested by the significant positive correlation (p < 0.001) between plasma PL and erythrocyte PLP. More PL into blood circulation also means more oxidation of this vitamer to 4'-pyridoxic acid in kidneys, as confirmed by the positive correlation between plasma PL and urinary 4'-pyridoxic acid (p < 0.001). The positive correlation (p < 0.001) between ALP activity and glycosylated hemoglobin indicated a direct effect of the disease. The T1D-induced alteration in vitamin B(6) metabolism, consecutive to enhanced ALP activity, may put patients at greater risk of vitamin B(6) deficiency and diabetic complications.

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration.

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration. Am J Physiol Endocrinol Metab 301: E000-E000, 2011. First published September 20, 2011; 10.1152/ajpendo.00345.2011.-We carried out a (1)H-NMR metabolomic analysis of sera from vitamin B(12)-deficient rats. In addition to the expected increases in methylmalonate and homocysteine (Hcy), we observed an approximately sevenfold increase in formate levels, from 64 µM in control rats to 402 µM in vitamin B(12)-deficient rats. Urinary formate was also elevated. This elevation of formate could be attributed to impaired one-carbon metabolism since formate is assimilated into the one-carbon pool by incorporation into 10-formyl-THF via the enzyme 10-formyl-THF synthase. Both plasma and urinary formate were also increased in folate-deficient rats. Hcy was elevated in both the vitamin B(12)- and folate-deficient rats. Although plasma Hcy was also elevated, plasma formate was unaffected in vitamin B(6)-deficient rats (impaired transsulfuration pathway). These results were in accord with a mathematical model of folate metabolism, which predicted that reduction in methionine synthase activity would cause increased formate levels, whereas reduced cystathionine ß-synthase activity would not. Our data indicate that formate provides a novel window into cellular folate metabolism, that elevated formate can be a useful indicator of deranged one-carbon metabolism and can be used to discriminate between the hyperhomocysteinemia caused by defects in the remethylation and transsulfuration pathways.

Vitamin B6 deficiency augments endogenous oxalogenesis after intravenous L-hydroxyproline loading in rats.

The effects of an intravenous hydroxyproline load on endogenous oxalogenesis were compared in rats fed a standard diet or a vitamin B6-deficient diet. Twelve male Wistar rats were randomized to two groups and were fed either a standard diet (control group) or a vitamin B6-deficient diet for 3 weeks. Then the animals were intravenously administered 100 mg (762.6 micromol)/ml hydroxyproline. In the control group, infusion of hydroxyproline increased the 5-h urinary oxalate and glycolate excretion above baseline to 0.27% (2.02 +/- 1.11 micromol) and 0.32% (2.43 +/- 1.60 micromol) of the administered dose (mol/mol), while it was respectively 2.01% (15.24 +/- 2.13 micromol) and 0.00% (-0.02 +/- 0.19 micromol) of the dose in the vitamin B6-deficient group. Therefore, vitamin B6 deficiency augmented endogenous synthesis of oxalate from hydroxyproline by 7.56-fold (15.24/2.02) compared with that in the control group. Urinary citrate excretion was significantly lower at baseline and all other times in the vitamin B6-deficient group compared with the control group. In conclusions, L-hydroxyproline loading augmented endogenous oxalogenesis in the vitamin B6-deficient group without causing hyperglycolic aciduria, and also led to significant hypocitraturia. These findings suggest that hydroxyproline is not metabolized to oxalate via glycolate, but rather via the 4-hydroxyglutamate to glyoxylate pathway (usually requiring vitamin B6-dependent enzymes) even in the presence of vitamin B6 deficiency.

Pyridoxic acid excretion during low vitamin B-6 intake, total fasting, and bed rest.

Vitamin B-6 metabolism in 10 volunteers during 21 d of total fasting was compared with results from 10 men consuming a diet low only in vitamin B-6 (1.76 mumol/d) and with men consuming a normal diet during bed rest. At the end of the fast mean plasma concentrations of vitamin B-6 metabolites and urinary excretion of 4-pyridoxic acid tended to be higher in the fasting subjects than in the low-vitamin B-6 group. The fasting subjects lost approximately 10% of their total vitamin B-6 pool and approximately 13% of their body weight. The low-vitamin B-6 group lost only approximately 4% of their vitamin B-6 pool. Compared with baseline, urinary excretion of pyridoxic acid was significantly increased during 17 wk of bed rest. There was no increase in pyridoxic acid excretion during a second 15-d bed rest study. These data suggest the possibility of complex interactions between diet and muscle metabolism that may influence indexes that are frequently used to assess vitamin B-6 status.

Effects of vitamin B6 deficiency on the conversion ratio of tryptophan to niacin.

To investigate how vitamin B6 (B6) deficiency affects the whole metabolism of tryptophan-niacin, rats were fed for 19 days with each of the following four kinds of diets; a complete 20% casein diet (control diet), the control diet without B6, the control diet without nicotinic acid, and the control diet without nicotinic acid and B6, and the urinary excretion of such tryptophan metabolites as kynurenic acid, xanthurenic acid, nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide each and the enzyme activities involved in tryptophan-niacin pathway were measured. The urinary excretion of kynurenic acid decreased while that of xanthurenic acid increased drastically in the two B6-deficient groups, when compared with the B6-containing groups. These results indicate that the rats fed with the B6-free diets were in the vitamin-deficient state. The conversion ratio was calculated from the ratio of the urinary excretion of sum of nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, to the Trp intake. The ratio was statistically lower in the B6-free diet than in the B6-containing diet under the niacin-free conditions.

Thiamin, riboflavin and vitamin B6: impact of restricted intake on physical performance in man.

OBJECTIVE: A combined marginally deficient status of thiamin, riboflavin, vitamin B6 and vitamin C may affect physical performance, but the relative contribution of each vitamin can only be speculated. In a previous study we did not find any effect of restricted intake of vitamin C individually. Therefore, the functional effect of restriction of thiamin, riboflavin or vitamin B6, individually or in conjunction, was investigated. METHODS: A double-blind, 2 x 2 x 2 complete factorial experiment on the effects of thiamin, riboflavin and vitamin B6 restriction on physical performance was executed with 24 healthy men. During 11 weeks of low vitamin intake, the subjects were given a daily diet of regular food products providing no more than 55% of the Dutch Recommended Dietary Allowances (RDA) for thiamin, riboflavin and vitamin B6. Other vitamins were supplemented at twice the RDA level. RESULTS: In vitamin-restricted subjects, blood vitamin levels, erythrocytic enzyme activities and urinary vitamin excretion decreased and in vitro erythrocytic enzyme stimulation increased. Short-time vitamin restriction had no harmful effects on health. A significant overall decrease was observed in aerobic power (VO2-max; 11.6%), onset of blood lactate accumulation (OBLA; 7.0%) and oxygen consumption at this power output (VO2-OBLA; 12.0%), peak power (9.3%), mean power (6.9%) and related variables (p < 0.01). However, the observed performance decrements could not be attributed to marginal deficiency for any of the vitamins studied. CONCLUSION: The absence of vitamin-specific effects on performance decrements due to thiamin, riboflavin and vitamin B6 restriction suggests quantitatively similar but non-additive effects of these B-vitamins on mitochondrial metabolism.

Accumulation of 3-hydroxy-L-kynurenine sulfate and ethanolamine in urine of the rat injected with 1-aminoproline.

By intraperitoneal injection of 1-amino-L-proline, a vitamin B6 antagonist, two unidentified ninhydrin-positive compounds were excreted abnormally in rat urine. One of them was definitely identified as 3-hydroxy-L-kynurenine sulfate by chromatographic and spectroscopic analyses. The other was proved to be ethanolamine. This is the first report on the abnormal excretion of ethanolamine in urine of the vitamin B6-deficient rats.

Tryptophan metabolism in vitamin B6-deficient mice.

Vitamin B6 deficiency was induced in mice by maintenance for 4 weeks on a vitamin B6-free diet. Tryptophan metabolism was assessed by determining the urinary excretion of tryptophan metabolites, the metabolism of [14C]tryptophan in vivo and the formation of tryptophan and niacin metabolites by isolated hepatocytes. The vitamin B6-deficient animals excreted more xanthurenic acid and 3-hydroxykynurenine, and less of the niacin metabolites N1-methyl nicotinamide and methyl-2-pyridone-4-carboxamide, than did control animals maintained on the same diet supplemented with 5 mg vitamin B6/kg. After intraperitoneal injection of [14C]tryptophan, vitamin B6-deficient mice showed lower liberation of 14CO2 from [methylene-14C]tryptophan and [U-14C]tryptophan than did controls, indicating impairment of kynureninase (EC 3.7.1.3) activity. There was no difference between the two groups of animals in the metabolism of [ring-2-14C]tryptophan. Hepatocytes isolated from the vitamin B6-deficient animals formed more 3-hydroxykynurenine and xanthurenic acid than did cells from control animals, but also formed more NADP and free niacin.

[The use of PALP-effect as an index of vitamin B6 supply]. / Ob ispol'zovanii PALF-effekta kak pokazatelia obespechennosti vitaminom B6.

Various methods used for estimation of a body supplying with vitamin B6 as well as correspondence between the data obtained by means of these methods were studied. The factors affecting the values of conventional patterns of the vitamin requirements were also studied: PALP-effect and content of 4-pyridoxilic acid in urine. A necessity was shown in standardization of the methods for estimation of a body supplying with vitamin B6 with regard to these factors.

[Studies on tryptophan metabolism in calcium oxalate urolithiasis]. / Tryptophanstoffwechselstudien bei Calciumoxalat-Urolithiasis.

In 90 patients with calcium oxalate urolithiasis an oral tryptophan-loading test with 5 g L-tryptophan was performed and the 24-hour urinary excretion of xanthurenic acid and kynurenine was measured. In 10 cases pathological deviations and an excretion pattern of tryptophan metabolites via kynurenine similar as in the hereditary vitamin-B6-dependent xanthurenic aciduria in homozygous or heterozygous from were found. Correlations between the oxalate excretion and the tryptophan metabolism do not exist. A 2-year therapy with 60 mg vitamin B6 was favourable in patients with an excretion of more than 300 mumol XA after a tryptophan load.

Growth inhibitor of calcium oxalate monohydrate crystal in vitamin B6-deficient rat urine and kidney.

Urinary and renal inhibitory activities of calcium oxalate monohydrate crystal growth in normal or vitamin B6-deficient rats were investigated. Renal inhibitory activity in vitamin B6-deficient rats was lower than that in normal rats. Renal inhibitory activity in vitamin B6-deficient rats was about 74% of normal level. Urinary inhibitory activity did not show a significant difference between normal and vitamin B6-deficient rats.

Taurine levels in blood and urine of vitamin B6-deficient and estrogen-treated rats.

The objects of the present study were to determine whether a L-methionine or a L-cysteine test-load was more effective in evaluating the effects of estrogen on vitamin B6 deficiency in rats, and to determine whether urine or blood was the better test fluid to measure taurine levels. In animals fed a vitamin B6-supplemented diet, urinary excretion of taurine was twofold higher after administration of a L-cysteine test-load compared to a L-methionine test-load. Greater percentage reductions in the quantity of excreted taurine were observed in the vitamin B6-deficient rats when L-cysteine was utilized as the test-load than when L-methionine (2 mmole) was utilized. Treatment with estrogen demonstrated no significant differences in urinary taurine excretion. Vitamin B6 deficiency without estrogen treatment raised the levels of taurine in the blood of the animals treated with a L-methionine test-load. No changes in the blood taurine levels were observed in the animals administered a L-cysteine test-load.

Inhibition of calcium oxalate monohydrate (COM) crystal growth by pyrophosphate, citrate and rat urine.

An assay system for the measurement of the rate of Calcium Oxalate Monohydrate (COM) seed crystal growth in a metastable solution of calcium chloride and sodium oxalate containing traces of 14C-oxalic acid was used to assess the inhibitory activity of pyrophosphate (10(-5) M-10(-4) M), citrate (10(-4) M-10(-3) M) and urines of normal and pyridoxine deficient rats. Both pyrophosphate and citrate were strong inhibitors of COM crystal growth and caused a 50% decrease in crystal growth rate at 1.50 X 10(-5) M and 2.85 X 10(-4) M respectively. Normal rat urine strongly inhibited the COM crystal growth, while pyridoxine deficient animals showed a significant (p less than 0.01) decrease in mean inhibitory activity as compared to pair-fed controls. A lowered urinary inhibitory potential accompanied with hyperoxaluria and hypercalciuria, which is known to be associated with pyridoxine deficiency, may be a contributory risk of calcium oxalate crystallization and stone formation.

[Comparative study of the vitamin activity of pyridoxal phosphate and pyridoxine used cutaneously]. / Sravnitel'noe izuchenie vitaminnoi aktivnosti piridoksal'-fosfata i piridoksina pri ikh nakozhnom primenenii.

In white rats with B6-avitaminosis, the B6-vitamin activity of pyridoxal-phosphate and of pyridoxine was studied in their epicutaneous and peroral application. It is revealed that pyridoxal-phosphate in epicutaneous application displays more activity than pyridoxine and pyridoxal-phosphate applied perorally. Pyridoxine in epicutaneous application does not reveal the B6-vitamin activity.

Urinary 4-pyridoxic acid excretion in 24-hour versus random urine samples as a measurement of vitamin B6 status in humans.

Urinary excretion of 4-pyridoxic acid (4PA) in 19 men (n = 5) and women (n = 14) was measured to evaluate the validity of determining the 4PA/creatinine ratio in random urine samples as an alternative to total 24-h 4PA excretion in assessing vitamin B6 nutritional status. The relationships among dietary vitamin B6 intake, 4PA excretion, plasma pyridoxal 5'-phosphate levels, and erythrocyte aspartate aminotransferase activity and in vitro stimulation by added plasma pyridoxal 5'-phosphate were examined. The subjects consumed all meals for 3 days in a metabolic unit, and protein intake was kept constant. Plasma pyridoxal 5'-phosphate concentration was positively correlated with vitamin B6 intake of the previous day (r = 0.61, p less than 0.01). There was a positive correlation (r = 0.59, p less than 0.01) between total 4PA and 4PA/creatinine in the 24-h urine samples. No difference (p greater than 0.05) in 4PA/creatinine between the 24-h samples and either morning or afternoon random samples taken the next day was found. These findings support the use of the 4PA/creatinine ratio in random urine samples as an alternative to 24-h urinary 4PA excretion.

The assessment of the vitamin B6 status among Egyptian school children by measuring the urinary cystathionine excretion.

A metabolic study was carried out on 19 school boys and girls (age 7-13 years) to assess their vitamin B6 status. The metabolic study was divided in five different periods, basal (A), post-methionine loading (B), one (C) and two (D) week after oral vitamin B6 therapy (50 mg/day) and post-methionine. Mean cystathionine excretion (beta mol/24 h) increased significantly (p less than 0.01) at stage B compared to mean values obtained at stage A reflecting a deficit in the vitamin B6 nutriture. Vitamin B6 therapy corrected the deficiency and the utilization of administered methionine.