Quantification of the B6 vitamers in human plasma and urine in a study with pyridoxamine as an oral supplement; pyridoxamine as an alternative for pyridoxine.

BACKGROUND AND AIMS: Vitamin B6 is involved in a large spectrum of physiological processes and comprises of the vitamers pyridoxamine (PM), pyridoxal (PL), pyridoxine (PN), and their phosphorylated derivatives including the biological active pyridoxal 5'-phosphate (PLP). While PN toxicity is known to complicate several treatments, PM has shown promise in relation to the treatment of metabolic and age-related diseases by blocking oxidative degradation and scavenging toxic dicarbonyl compounds and reactive oxygen species. We aimed to assess the metabolization of oral PM supplements in a single and three daily dose. MATERIALS AND METHODS: We optimized and validated a method for the quantification of the B6 vitamers in plasma and urine using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Five healthy volunteers were recruited to study PM metabolization after a single oral dose of 200 mg PM or a three daily dose of 67 mg PM. A third protocol was implemented as control for dietary intake. Venous blood samples, 24 h urine and fasted second void urine samples were collected. RESULTS: After a single oral dose of 200 mg PM, plasma PM increased in the first 3 h to a maximum of 2324 ± 266 nmol/L. While plasma PM levels returned to baseline after ~10 h of PM intake, PLP increased to a maximum of 2787 ± 329 nmol/L and reached a plateau. We found a small increase of PN to a maximum of 13.5 ± 2.1 nmol/L; it was nearly undetectable after ~12 h. With a three daily dose of 67 mg PM we observed an increase and decline of plasma PM, PL, and PN concentrations after each PM intake. PLP showed a similar increase as in the single dose protocol and accumulated over time. CONCLUSION: In this study we showed high plasma levels of PM after oral PM supplementation. We found steadily increasing levels of the biologically active PLP, with minimal formation of PN. The B6 vitamer PM is an interesting supplement as an inhibitor of harmful processes in metabolic diseases and for the treatment of vitamin B6 deficiency. CLINICAL TRIAL REGISTRY: The study was approved by the Medical Ethics Committee of Maastricht University (NL) and was registered at ClinicalTrials.gov as NCT02954588.

Identification of urinary metabolites with potential blood pressure-lowering effects in lentil-fed spontaneously hypertensive rats.

PURPOSE: Urine samples were obtained from a previously completed study that showed lentil consumption attenuates the increase in blood pressure that occurs over time in spontaneously hypertensive rats (SHRs). The objective of the present study was to compare the metabolite profile of the urine samples from control and lentil-fed SHR in relation to the compounds present in lentils but not in other pulses. METHODS: The urine samples were from 17-week-old, male SHR fed semi-purified diet prepared with powder (30 %, w/w) from cooked whole pulses or a pulse-free control diet (n = 8/group) for 4 weeks. Pulse powders, control diet and urine samples were extracted using acetonitrile and analyzed by a high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). RESULTS: Twenty-seven metabolites were significantly different in urine samples from lentil-fed SHR compared to SHR fed control diet, but only 7 were not present in the urine of SHR fed other pulses. Of these metabolites, only citrulline is linked to blood pressure regulation via production of the vasodilator nitric oxide (NO). Several arginine-related compounds that are NO synthase substrates or inhibitors were detected in lentils but not the control diet or other pulse powders. CONCLUSIONS: Consumption of lentils increases the availability of arginine and several related compounds that could potentially elevate production of NO and contribute to the blood pressure-lowering effects of lentil-rich diets.

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.

N-methylpyridoxamine: novel canine vitamin B6 urine metabolite.

Cation-exchange HPLC analysis of urine from dogs given large daily doses of pyridoxamine revealed an unidentified metabolite hypothesized to be N-methylpyridoxamine. Identity was established by N-methylpyridoxamine synthesis and HPLC comparison to the canine metabolite. Compound synthesis was confirmed by IR, NMR, UV-vis and emission spectroscopy. It seems to have less fluorescent character than other routinely-measured vitamin B(6) metabolites. Upon administration of substantial pyridoxamine doses, N-methylpyridoxamine appears to be a quantifiable canine urine metabolite, although, at either pharmacological or dietary pyridoxamine intakes, its relevance to vitamin B(6) metabolism in other species, including humans, is not yet determined.

Pyridoxamine traps intermediates in lipid peroxidation reactions in vivo: evidence on the role of lipids in chemical modification of protein and development of diabetic complications.

Maillard or browning reactions between reducing sugars and protein lead to formation of advanced glycation end products (AGEs) and are thought to contribute to the pathogenesis of diabetic complications. AGE inhibitors such as aminoguanidine and pyridoxamine (PM) inhibit both the formation of AGEs and development of complications in animal models of diabetes. PM also inhibits the chemical modification of protein by advanced lipoxidation end products (ALEs) during lipid peroxidation reactions in vitro. We show here that several PM adducts, formed in incubations of PM with linoleate and arachidonate in vitro, are also excreted in the urine of PM-treated animals. The PM adducts N-nonanedioyl-PM (derived from linoleate), N-pentanedioyl-PM, N-pyrrolo-PM, and N-(2-formyl)-pyrrolo-PM (derived from arachidonate), and N-formyl-PM and N-hexanoyl-PM (derived from both fatty acids) were quantified by liquid chromatography-mass spectrometry analysis of rat urine. Levels of these adducts were increased 5-10-fold in the urine of PM-treated diabetic and hyperlipidemic rats, compared with control animals. We conclude that the PM functions, at least in part, by trapping intermediates in AGE/ALE formation and propose a mechanism for PM inhibition of AGE/ALE formation involving cleavage of alpha-dicarbonyl intermediates in glycoxidation and lipoxidation reactions. We also conclude that ALEs derived from polyunsaturated fatty acids are increased in diabetes and hyperlipidemia and may contribute to development of long term renal and vascular pathology in these diseases.

Pyridoxamine inhibits early renal disease and dyslipidemia in the streptozotocin-diabetic rat.

BACKGROUND: Nonenzymatic reactions between sugars or lipids and protein and formation of advanced glycation and lipoxidation end products (AGE/ALEs) contribute to the chemical modification and cross-linking of tissue proteins with age. Accelerated formation of AGE/ALEs during hyperglycemia is implicated in the development of diabetic complications. In this study, we examined the effect of the AGE/ALE inhibitor pyridoxamine on chemical modification and cross-linking of collagen and development of renal disease in the streptozotocin-diabetic rat. METHODS: Diabetic rats were treated with pyridoxamine; parallel experiments were conducted with aminoguanidine, the prototype AGE inhibitor. Progression of renal disease was evaluated by measurements of albuminuria and plasma creatinine concentration. Plasma triglycerides, cholesterol, lactate and pyruvate were measured by enzymatic assays, and AGE/ALEs in skin collagen by HPLC and GC-MS assays. RESULTS: Pyridoxamine significantly inhibited the increase in albuminuria, plasma creatinine, hyperlipidemia and plasma lactate/pyruvate ratio in diabetic rats, without an effect on blood glucose or glycated hemoglobin. AGE/ALEs, fluorescence and cross-linking of skin collagen increased approximately twofold in diabetic versus control rats after seven months of diabetes. Pyridoxamine caused a significant (25 to 50%) decrease the AGE/ALEs, carboxymethyllysine and carboxyethyllysine, cross-linking and fluorescence in skin collagen of diabetic rats, but did not affect pentosidine. CONCLUSIONS: Pyridoxamine inhibits the progression of renal disease, and decreases hyperlipidemia and apparent redox imbalances in diabetic rats. Pyridoxamine and aminoguanidine had similar effects on parameters measured, supporting a mechanism of action involving AGE/ALE inhibition.

Assessment of vitamin B-6 status in young women consuming a controlled diet containing four levels of vitamin B-6 provides an estimated average requirement and recommended dietary allowance.

The Recommended Dietary Allowance (RDA) of vitamin B-6 for young women was recently reduced from 1.6 to 1.3 mg/d based on an adequate plasma pyridoxal phosphate (PLP) concentration of 20 nmol/L. To assess vitamin B-6 requirements and suggest recommendations for intake, seven healthy young women consumed a controlled diet providing 1.2 g protein/kg body weight for a 7-d adjustment period (1.0 mg vitamin B-6/d) and three successive 14-d experimental periods (1.5, 2.1 and 2.7 mg/d, respectively). Direct and indirect vitamin B-6 status indicators were measured in plasma, erythrocytes and urine. Indicators most strongly correlated with vitamin B-6 intake [i.e., plasma and erythrocyte PLP, urinary 4-pyridoxic acid (4-PA) and total vitamin B-6] were regressed on vitamin B-6 intake and the dietary vitamin B-6 to protein ratio. Inverse prediction using adequate and baseline values estimated vitamin B-6 requirement. Adequate values were determined for plasma PLP and urinary 4-PA from baseline values of 60 previous subjects, using the statistical method suggested by Sauberlich. The current study suggests a vitamin B-6 Estimated Average Requirement (EAR) for young women of 1.1 mg/d or 0.016 mg/g protein, and a RDA of 1.5 mg/d or 0.020 mg/g protein. When results from this study are combined with data from four other recent studies, the combined data predict an EAR of 1.2 mg/d or 0.015 mg/g protein, and a RDA of 1.7 mg/d or 0.018 mg/g protein. This study suggests that the current vitamin B-6 RDA may not be adequate.

Determination of pyridoxamine in urine by matrix isopotential synchronous fluorescence spectrometry.

A spectrofluorimetric technique was applied to the determination of compounds in samples with unknown background fluorescence, consisting of performing synchronous scans through a trajectory joining points of equal intensity of a fluorescence matrix three-dimensional spectrum, which is called 'matrix isopotential synchronous fluorescence'. This technique can be improved by application of derivatives. The determination of pyridoxamine in urine was performed using this technique and the validity, applicability and simplicity of the method were demonstrated. With first-derivative matrix isopotential synchronous fluorescence the maximum sensitivity was 12.5 mg l-1 for pyridoxamine in real urine samples. The measurements were performed in aqueous medium at pH 7.0, adjusted by adding 0.05 mol l-1 phosphate buffer, and without any previous derivatization reactions. A complete statistical analysis of the experimental data was performed. Pyridoxamine in urine was determined by this method with good results and without the need for tedious prior separation.

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.