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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., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Water-soluble vitamins like B3 (nicotinamide), B6 (pyridoxine), and B9 (folic acid) are of utmost importance in human health and disease, as they are involved in numerous critical metabolic reactions. Not surprisingly, deficiencies of these vitamins have been linked to various disease states. Unfortunately, not much is known about the physiological levels of B6 vitamers and vitamin B3 in an ethnically isolated group (such as an Emirati population), as well as their relationship with obesity. The aim of the present study was to quantify various B6 vitamers, as well as B3, in the plasma of obese and healthy Emirati populations and to examine their correlation with obesity. A sensitive and robust HPLC-MS/MS-based method was developed for the simultaneous quantitation of five physiologically relevant forms of vitamin B6, namely pyridoxal, pyridoxine, pyridoxamine, pyridoxamine phosphate, and pyridoxal phosphate, as well as nicotinamide, in human plasma. This method was used to quantify the concentrations of these vitamers in the plasma of 57 healthy and 57 obese Emirati volunteers. Our analysis showed that the plasma concentrations of nicotinamide, pyridoxal, and pyridoxamine phosphate in the obese Emirati population were significantly higher than those in healthy volunteers ( p < 0.0001, p = 0.0006, and p = 0.002, respectively). No significant differences were observed for the plasma concentrations of pyridoxine and pyridoxal phosphate. Furthermore, the concentrations of some of these vitamers in healthy Emirati volunteers were significantly different than those published in the literature for Western populations, such as American and European volunteers. This initial study underscores the need to quantify micronutrients in distinct ethnic groups, as well as people suffering from chronic metabolic disorders.

We report the development of a rapid, simple, and robust LC-MS/MS-based enzyme assay using dried blood spots (DBS) for the diagnosis of pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency (OMIM 610090). PNPO deficiency leads to potentially fatal early infantile epileptic encephalopathy, severe developmental delay, and other features of neurological dysfunction. However, upon prompt treatment with high doses of vitamin B 6 , affected patients can have a normal developmental outcome. Prognosis of these patients is therefore reliant upon a rapid diagnosis. PNPO activity was quantified by measuring pyridoxal 5'-phosphate (PLP) concentrations in a DBS before and after a 30 min incubation with pyridoxine 5'-phosphate (PNP). Samples from 18 PNPO deficient patients (1 day-25 years), 13 children with other seizure disorders receiving B 6 supplementation (1 month-16 years), and 37 child hospital controls (5 days-15 years) were analyzed. DBS from the PNPO-deficient samples showed enzyme activity levels lower than all samples from these two other groups as well as seven adult controls; no false positives or negatives were identified. The method was fully validated and is suitable for translation into the clinical diagnostic arena.

Pyridorin ® , a naturally occurring metabolite of vitamin B6 that inhibits and scavenges reactive oxygen species, is being developed as a potential therapeutic for acute kidney injury. An investigational new drug application (IND) was opened for Pyridorin in support of its ongoing oral drug clinical development program. Currently, a Pyridorin intravenous (IV) formulation is being developed for use in surgical patients. To support the IND for Pyridorin, a full battery of nonclinical Good Laboratory Practice compliant studies was performed with no neurological or behavioral signs of toxicity seen following oral or IV administration of pyridoxine dihydrochloride (the active ingredient in Pyridorin). However, excessive ingestion of vitamin B6 has been reported to cause neurotoxic syndrome in humans. Therefore, under Food and Drug Administration recommendation, a 7-day IV study in rats was conducted to further evaluate the drug's potential to cause neurotoxicity. Blood plasma samples indicated that exposure to pyridoxamine dihydrochloride and its metabolites, pyridoxal, pyridoxine, and 4-pyridoxic acid was linearly dose proportional and independent of gender. At doses of up to 200 mg/kg/day pyridoxine dihydrochloride, no treatment-related effects were seen in rats, providing further evidence for the absence of pyridoxine dihydrochloride-related changes in the nervous system. A no observed adverse effect level of 200 mg/kg/day was identified for this study.

Pyridoxamine, a reactive carbonyl (RCO) scavenger, can ameliorate peritoneal deterioration in uremic peritoneal dialysis (PD) rats when given via dialysate. We examined the effects of scavenging circulating RCOs by oral pyridoxamine. Rats underwent nephrectomy and 3 weeks of twice daily PD either alone or with once daily oral pyridoxamine. PD solution was supplemented with methylglyoxal, a major glucose-derived RCO, to quench intraperitoneal pyridoxamine. Oral pyridoxamine achieved comparable blood and dialysate pyridoxamine concentrations, suppressed pentosidine accumulation in the blood but not in the mesenterium or dialysate, and reduced the increases in small solute transport and mesenteric vessel densities, with no effects on submesothelial matrix layer thickening or serum creatinine. Thus, reducing circulating RCOs by giving oral pyridoxamine with PD provides limited peritoneal protection. However, orally given pyridoxamine efficiently reaches the peritoneal cavity and would eliminate intraperitoneal RCOs. Oral pyridoxamine is more clinically favorable and may be as protective as intraperitoneal administration., (© 2016 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.)

Background: Recent decades have unravelled the molecular background of a number of inborn errors of metabolism (IEM) causing vitamin B6-dependent epilepsy. As these defects interfere with vitamin B6 metabolism by different mechanisms, the plasma vitamin B6 profile can give important clues for further molecular work-up. This has so far been investigated in only a small number of patients., Methods: We evaluated the vitamin B6 vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN) and the catabolite pyridoxic acid (PA) in the so far largest patient cohort: reference (n = 50); pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency (n = 6); antiquitin (ATQ) deficiency (n = 21); tissue non-specific alkaline phosphatase (TNSALP) deficiency (n = 2) and epileptic encephalopathy (EE) of unknown etiology tested negative for ATQ and PNPO deficiency (n = 64)., Results: High plasma PM concentration was found in all patients with PNPO deficiency irrespective of vitamin B6 supplementation. Their PM concentration and the PM/PA ratio was significantly higher (p < 0.0001), compared to any other patients analysed. One patient with TNSALP deficiency and sampling prior to PN supplementation had markedly elevated plasma PLP concentration. On PN supplementation, patients with TNSALP deficiency, ATQ deficiency and patients of the EE cohort had similar plasma vitamin B6 profiles that merely reflect the intake of supra-physiological doses of vitamin B6. The interval of sampling to the last PN intake strongly affected the plasma concentrations of PN, PL and PA., Conclusions: PM concentrations and the PM/PA ratio clearly separated PNPO-deficient patients from the other cohorts. The plasma PM/PA ratio thus represents a robust biomarker for the selective screening of PNPO deficiency.

Acute kidney injury (AKI) is a common and independent risk factor for death and chronic kidney disease (CKD). Despite promising preclinical data, there is no evidence that antioxidants reduce the severity of injury, increase recovery, or prevent CKD in patients with AKI. Pyridoxamine (PM) is a structural analog of vitamin B6 that interferes with oxidative macromolecular damage via a number of different mechanisms and is in a phase 3 clinical efficacy trial to delay CKD progression in patients with diabetic kidney disease. Because oxidative stress is implicated as one of the main drivers of renal injury after AKI, the ability of PM to interfere with multiple aspects of oxidative damage may be favorable for AKI treatment. In these studies we therefore evaluated PM treatment in a mouse model of AKI. Pretreatment with PM caused a dose-dependent reduction in acute tubular injury, long-term postinjury fibrosis, as well as improved functional recovery after ischemia-reperfusion AKI (IR-AKI). This was associated with a dose-dependent reduction in the oxidative stress marker isofuran-to-F2-isoprostane ratio, indicating that PM reduces renal oxidative damage post-AKI. PM also reduced postinjury fibrosis when administered 24 h after the initiating injury, but this was not associated with improvement in functional recovery after IR-AKI. This is the first report showing that treatment with PM reduces short- and long-term injury, fibrosis, and renal functional recovery after IR-AKI. These preclinical findings suggest that PM, which has a favorable clinical safety profile, holds therapeutic promise for AKI and, most importantly, for prevention of adverse long-term outcomes after AKI., (Copyright © 2016 the American Physiological Society.)

Pyridox(am)ine phosphate oxidase (PNPO) deficiency causes severe early infantile epileptic encephalopathy and has been characterized as responding to pyridoxal-5'-phosphate but not to pyridoxine. Two males with PNPO deficiency and novel PNPO mutations are reported and their clinical, metabolic, and video-electroencephalographic (EEG) findings described. The first child showed electro-clinical responses to pyridoxine and deterioration when pyridoxine was withheld. At last review, he has well-controlled epilepsy with pyridoxal-5'-phosphate monotherapy and an autism spectrum disorder. The second child had a perinatal middle cerebral artery infarct and a myoclonic encephalopathy. He failed to respond to pyridoxine but responded well to pyridoxal-5'-phosphate. At the age of 21 months he has global developmental delay and hemiparesis but is seizure-free with pyridoxal-5'-phosphate monotherapy. Plasma and cerebrospinal fluid pyridoxamine levels were increased in both children during treatment with pyridoxine or pyridoxal-5'-phosphate. These observations indicate that differential responses to pyridoxine and pyridoxal-5'-phosphate treatment cannot be relied upon to diagnose PNPO deficiency., (© 2013 Mac Keith Press.)

The benefits of antioxidant therapy for treating age-related macular degeneration, a devastating retinal disease, are limited. Perhaps species other than reactive oxygen intermediates should be considered as therapeutic targets. These could be lipid peroxidation products, including isolevuglandins (isoLGs), prototypical and extraordinarily reactive γ-ketoaldehydes that avidly bind to proteins, phospholipids, and DNA and modulate the properties of these biomolecules. We found isoLG adducts in aged human retina but not in the retina of mice kept under dim lighting. Hence, to test whether scavenging of isoLGs could complement or supplant antioxidant therapy, we exposed mice to bright light and found that this insult leads to retinal isoLG-adduct formation. We then pretreated mice with pyridoxamine, a B6 vitamer and efficient scavenger of γ-ketoaldehydes, and found that the levels of retinal isoLG adducts are decreased, and morphological changes in photoreceptor mitochondria are not as pronounced as in untreated animals. Our study demonstrates that preventing the damage to biomolecules by lipid peroxidation products, a novel concept in vision research, is a viable strategy to combat oxidative stress in the retina.

Objective: The study aimed to determine vitamin B6 status in elderly (age ≥ 60 years) and younger (age <60 years) recipients of allogeneic kidney graft and to investigate associations between vitamin B6 status and immunity markers., Design: A retrospective observational study., Setting: The study was conducted at the Medical University of Gdańsk, Poland., Subjects: We recruited 34 kidney allograft recipients (17 males and 17 females) and allocated them into 2 groups: patients aged ≥ 60 years (18 patients) and those aged <60 years (16 patients). Exclusion criteria included patients receiving vitamin B6 supplementation or drugs known to influence vitamin B6 metabolism., Main Outcome Measure: Plasma levels of pyridoxal 5'-phosphate (PLP), pyridoxal, pyridoxine, pyridoxamine, pyridoxamine 5'-phosphate, and 4 pyridoxic acid were determined by high-performance liquid chromatography. Measured immunity markers were serum cytokines (interleukin-6, interleukin-10, and transforming growth factor-β), levels of T-lymphocyte subsets, and the proliferative ability of peripheral blood mononuclear cells., Results: Concentrations of all vitamin B6 vitamers in plasma (PLP, pyridoxal, pyridoxamine 5'-phosphate, pyridoxamine, pyridoxine, 4 pyridoxic acid) were comparable in the 2 studied groups. There were no cases of PLP deficiency in the study population, but 29% of patients had PLP concentrations more than the upper reference limit. Vitamin B6 vitamer concentrations were not influenced by gender, estimated glomerular filtration rate, and circulating phosphate concentration. There was no difference in immunity markers according to age. However, the plasma concentrations of vitamin B6 vitamers were inversely associated with levels of CD28(+) lymphocyte subsets, as well as with the proliferative response of peripheral blood mononuclear cells in both groups., Conclusions: No cases of vitamin B6 deficiency were found among kidney allograft recipients, and we report inverse links between vitamin B6 vitamer concentrations and markers of cellular immunity, suggesting that bioactive vitamin B6 concentration in kidney allograft recipients merits further investigation., (Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

To examine the responses of the levels of B₆-vitamers in several tissues to the dietary level of pyridoxine (PN), mice were fed diets containing 0, 1, 7 (the recommended level) or 35 mg PN HCl/kg diet for 5 wk. Compared with the 0 mg PN HCl/kg diet, the 35 mg PN HCl/kg diet caused the highest elevation in the concentration of pyridoxal 5'-phosphate (PLP) in small intestine and epididymal adipose tissue, moderate elevation in colon, lung, spleen and stomach, slight elevation in brain, kidney and liver (p<0.05), and no elevation in heart and gastrocnemius muscle. In general, the alterations in PLP level in many tissues and serum were remarkable for diets between 1 mg and 7 mg PN HCl/kg diets. Compared to the 7 mg PN HCl/kg diet, the 35 mg PN HCl/kg diet further elevated the PLP level in adipose tissue, spleen and stomach (p<0.05). Dietary supplemental PN elevated the level of PN in small intestine and colon in a dose-dependent manner (p<0.05), but not in other tissues. There was a significant correlation between the PN and PLP levels in small intestine and colon (p<0.05), implying that PN absorbed from the diet can be at least in part metabolized to PLP within the absorptive intestinal cells. The results suggest that the responses of concentrations of B₆-vitamers to dietary level of PN greatly differ among several tissues.

Background: Vitamin deficiency is common in chronic kidney disease (CKD). Data on B(6) supply and possible relationships to cardiovascular events (CVE) in CKD are rare. Pyridoxamine exerts inhibitory effects on the formation of advanced glycation endproducts (AGE) implicated in the pathogenesis of CKD and atherosclerosis., Methods: In 48 CKD patients at stage 2-4, 72 hemodialysis patients (HD), 38 renal transplant recipients (RTR) and 141 healthy controls (mean age 58 +/- 13, 61 +/- 12, 50 +/- 12 and 54 +/- 16 years, respectively), plasma and red blood cell (RBC) concentrations of pyridoxal-5'-phosphate (PLP), pyridoxal (PL), 4-pyridoxic acid (PA), pyridoxamine-5'-phosphate (PMP) and of the AGE pentosidine were measured by high-performance liquid chromatography, N(epsilon)-(carboxymethyl)lysine and imidazolone by an ELISA, and total homocysteine and cystathionine by gas chromatography-mass spectrometry., Results: Despite routine low-dose vitamin supplementation in HD, plasma PLP was decreased in HD (79 +/- 69 nmol/l) compared with CKD stage 2-4 patients (497 +/- 944 nmol/l), RTR (416 +/- 604 nmol/l) and controls (159 +/- 230 nmol/l; p < 0.001). Plasma PA was significantly increased in HD (11,667 +/- 17,871 nmol/l) in comparison with CKD stage 2-4 (435 +/- 441 nmol/l), RTR (583 +/- 668 nmol/l) and controls (46 +/- 49 nmol/l; p < 0.001). B(6) forms were significantly affected by renal function (R = 0.792, p < 0.001 for CKD stage 2-4). There was no relation of vitamers with a history of CVE. Relationships between B(6) forms and AGE (RBC-PMP with pentosidine in CKD stage 2-4: R = -0.351, p < 0.05) were found., Conclusion: HD patients showed a deficiency in PLP in plasma but not in RBC. Prospective trials are needed to elucidate the potential role of elevated PA on cardiovascular and renal outcome in CKD. Vitamin B(6) supplementation might be successful in preventing AGE-related pathologies., (Copyright 2009 S. Karger AG, Basel.)

Plasma concentrations of B-6 vitamers and homocysteine as well as erythrocyte alanine aminotransferase activity coefficients and vitamin B-6 (dietary + supplement) intakes of apparently healthy young Latino children of immigrant parents living in rural Nebraska were determined and differences determined by gender. Thirty-five Latino children (16 males and 19 females), aged 4-8 years, were included in the study. Nutrient intake information was obtained from the children's parents utilizing two nonconsecutive 24-hour food recalls. No differences were observed by gender with regard to vitamin B-6 intakes, plasma concentrations of B-6 vitamers and homocysteine, and erythrocyte alanine aminotransferase activity coefficients. The intakes of all children met the Recommended Dietary Allowance for vitamin B-6. Plasma pyridoxal 5'-phosphate concentrations, plasma homocysteine concentrations, and erythrocyte alanine aminotransferase activity coefficients of the children were (mean +/- SD) 83.71 +/- 37.35 nmol/L, 6.81 +/- 1.63 micromol/L, and 1.08 +/- 0.06, respectively. All the Latino children of immigrant parents in this study had values indicative of adequate vitamin B-6 status.

A column packed with red blood cells (RBCs) was prepared for electrochromatography as a separation and reaction column. RBCs were kept inside a piece of fused silica capillary tubing with 2% agarose gel. In the column, RBCs were uniformly distributed in the agarose gel matrix and their electrophoretic movements due to an applied voltage were suppressed well. The durability of the biological function of the column under applied voltage was about 1 h, although it could remain for 2-3 days without applied voltage. The column could not be used when hemolysis of the RBCs was observed in the column. When the developed "RBC-gel column" was used, both pyridoxamine and serotonin were converted to other compounds through their direct contact with RBCs.

Background: A micellar reversed-phase liquid chromatographic procedure was developed for the determination of B6 group vitamins, i.e. pyridoxine, pyridoxal and pyridoxamine, in human serum., Methods: Chromatographic conditions used were a C18 column, isocratic mode, flow-rate of 1 ml/min and UV-detection at 290 nm. Optimization of the composition of the mobile phase was performed using an interpretative strategy., Results: After modeling, the composition of the selected mobile phase was 150 mM sodium dodecyl sulphate (SDS)--2% (v/v) pentanol-dihydrogenphosphate buffer 10 mM at pH 3. In this mobile phase, serum samples were injected without pretreatment and analysis time was below 14 min. Calibrations for the three vitamins were linear, with coefficient regression better than 0.999, and intra- and inter-day precision, achieved according to ICH, offering values below 4.3% and 3.2%, respectively. The method was applied to the determination of the B6 vitamins in spiked serum samples, with recoveries around 100%, and in the pharmacokinetic determination of pyridoxine half-life in serum, which was found to be 47.5 +/- 3.2 min (n = 5). The procedure was also applied for the analysis of pyridoxine in human serum spiked with several pharmaceutical preparations that contain other drugs which do not produce any kind of interference. Finally, solutions of B6 vitamins kept at -201 degrees C are stable for up to 3 months., Conclusions: Using the method proposed here, with an SDS-pentanol mobile phase, it is possible to carry out the fast sensitive determination of B6 vitamins in serum following direct injection, without sample pretreatment.

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.

The effect of vitamin B6-deficiency on the B6-vitamer concentrations, level of S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were studied in rat tissues. The plasma pyridoxal 5'-phosphate (PLP) and, pyridoxal (PL) levels were lower in the B6-deficient group compared to the control group. After 5 weeks of feeding the experimental diets, tissue PLP, pyridoxamine 5'-phosphate (PMP) and PL concentrations were significantly lower in the B6-deficient group compared to the control and the pair-fed control groups. Thymus PLP and PL levels were lower in the B6-deficient group. The concentration of SAM in the B6-deficient group decreased to approximately 50% and 25% in liver and thymus, respectively. However SAH concentration was 3.5 and 2 fold higher compared to the control and the pair-fed control groups. Thus, the ratio of SAM/SAH was significantly decreased in the B6-deficient group compared to the control or the pair fed-control group. In addition, the S-adenosylhomocysteine hydrolase (EC 3.3.1.1) activity increased by 45% and 15% in liver and thymus, respectively, in the B6-deficient group compared to the pair-fed control and the control groups. However, the activity of L-methionine S-adenosyltransferase (EC 2.5.1.6) was also unaffected. Concentrations of SAH and SAM, SAM/SAH ratio and activities of S-adenosylhomocysteine hydrolase and L-methionine S-adenosyltrasferase in rat brain were not affected by the B6-deficiency. We infer that the alteration of B6 metabolism, especially the reduction of PLP contents in liver and thymus, caused by the B6 deficiency, resulted in accumulation of SAH as well as reduction of SAM and the SAM/SAH ratio. The reduction of the SAM/SAH ratio was due to a block in the catabolism of methionine via the trans-sulfuration pathway. These may lead to inhibition of transmethylation reaction of DNA, RNA and protein, the synthesis and function of thymic lymphocyte and result in damage to tissues.

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.

The vitamin B-6 intakes and plasma B-6 vitamer levels of healthy nonsupplemented men and women, 19-24 and 25-50 years, were compared. The subjects did not take nutrient supplements or medications or use tobacco products. Subjects were grouped as follows: eight, 19-24 y men; nine, 25-50 y men; 11, 19-24 y women; and 13, 25-50 y women. The estimated vitamin B-6 intakes, obtained via 24-h recalls followed by 2-d food records, of the two groups of men were significantly higher (P < 0.05) than those of the two groups of women. Thirty-five percent of the women reported consuming less than the Estimated Average Requirement for vitamin B-6. The four gender: age groups had similar B-6 vitamer concentrations of plasma pyridoxal-5'-phosphate, 4-pyridoxic acid, pyridoxine, pyridoxamine, and pyridoxamine-5'-phosphate. Males 25-50 y had significantly higher (P < 0.05) plasma pyridoxal concentrations than the two groups of females. All subjects had pyridoxal-5'-phosphate concentrations indicative of vitamin B-6 adequacy. Generally the plasma B-6 vitamer concentrations of these men and women, 19-24 and 25-50 years of age, all having adequate vitamin B-6 status, were similar.

Total activity [pyridoxal 5'-phosphate (PLP) added in the assay] of hepatic tyrosine aminotransferase (TAT) measured in cats at 0300, 0900, 1500 and 2100h was 10.3 +/-1.1, 14.0 +/- 0.7, 9.8 +/- 1.3 and 11.0 +/- 0.7 nkat/g liver, indicating little diurnal variation. Activity after 18 h of food deprivation was 10.0 +/- 0.3 nkat/g liver, also not different from cats that were eating ad libitum. These findings support the idea that cats have only limited changes in the activity of hepatic TAT compared with rats. Total TAT activity was measured in cats fed high protein (550 g/kg) and low protein (180 g/kg) diets for 4 wk. Cats fed a high protein diet had activities significantly higher (about twice) than cats fed the low protein diet. Hepatic TAT activity of vitamin B-6-deficient cats (diet without pyridoxine for 9 wk) was compared with cats given the same diet with 8 mg pyridoxine/kg. Total hepatic TAT activity in deficient cats was significantly (P < 0.05) lower per gram soluble or total protein (but not per gram liver) than control cats; holoenzyme activity and percentage of active enzyme in deficient cats were also significantly lower by 75 and 64%, respectively. The apparent Km of TAT from cats for tyrosine (2.1 mmol/L) was similar to that for rats (1.9 mmol/L), but higher for PLP in cats (0.16 micromol/L) than rats (0.034 micromol/L). Part of the reason for the higher plasma tyrosine in vitamin B-6-deficient cats than rats is the higher Km of TAT for PLP in cats than rats.

Erythrocyte and plasma B-6 vitamer concentrations were determined in males aged 25-55 y who were long-term smokers, chewers, or nonusers. Tobacco users had either smoked (n = 23) or chewed (n = 11) for > 15 y; nonusers (n = 11) had never smoked or chewed. All subjects had normal hematocrit values. Food energy, protein, and vitamin B-6 intakes of the three groups of subjects were not significantly different. All subjects had fasting plasma pyridoxal-5'-phosphate (PLP) concentrations indicative of adequacy. Erythrocyte B-6 vitamer and 4-pyridoxic acid (4-PA) concentrations of all three groups were not significantly different. Nonusers had significantly higher plasma PLP concentrations than did smokers, whereas PLP concentrations of chewers were intermediate between the two groups. Chewers had significantly higher concentrations of plasma pyridoxamine-5'-phosphate (PMP) than other groups. Plasma pyridoxal, pyridoxine, pyridoxamine, and 4-PA concentrations of the three groups were not significantly different. Differences in some B-6 vitamer concentrations in plasma but not in erythrocytes were observed between tobacco users and nonusers.

The utilization of a usual dose of intravenously infused pyridoxine (100 mg pyridoxine hydrochloride) was investigated in ten healthy males. Blood plasma and erythrocytes were investigated by means of high-performance liquid chromatography. Detectable metabolites in blood plasma were pyridoxine, pyridoxal 5'-phosphate, pyridoxal and 4-pyridoxic acid. In erythrocytes pyridoxine, pyridoxal 5'-phosphate, pyridoxal, and pyridoxamine 5'-phosphate were found. From their concentration-time curves rate constants of elimination and invasion, volume of distribution (pyridoxine) and the areas under the curves were calculated. Values for concmax and tmax are reported. A comparison with earlier results of oral pyridoxine administration revealed a better utilization after intravenous than after oral application, i.e. a greater build-up of coenzyme forms. A regulatory phenomenon in erythrocytes caused by high doses of pyridoxine is described. In view of the potential toxicity of pyridoxine the doses used in parenteral nutrition are called into question.

By using an HPLC method which we have developed, concentration changes of vitamin B-6 derivatives in blood of pregnant and lactating rats were studied. PLP and PL were the main derivatives in plasma and erythrocytes, and occasionally, PMP was found in the plasma of rats fed a normal solid diet which contained 8.3 mg PN.HCl/kg diet. Upon pregnancy, the plasma PLP concentration decreased significantly (p < 0.01), whereas plasma PL tended to increase (p < 0.05). PLP concentration in erythrocytes tended to increase upon pregnancy. These results suggest that metabolism or utilization of vitamin B-6 is altered upon pregnancy and that plasma PLP concentrations alone may not be a good indicator of nutritional status. Further confirmation was needed for so from erythrocyte samples under our analytical conditions was not so from erythrocyte samples under our analytical conditions was not converted to PIC-P via PLP, after the sample was treated with glyoxylic acid and then with KCN.

Pyridoxamine 5'-phosphate in 18 microliters of human capillary blood plasma is determined by catalytic amplification using the apoenzyme of aspartate aminotransferase. Prior isolation from interfering substances is accomplished by employment of a cation exchange resin in batch operation. The procedure consists of the following stages. Stage I, denaturation of proteins. Trichloroacetic acid is used to precipitate plasma proteins and liberate any bound coenzyme. Dilute NaCl is added to expand the volume thus minimizing coenzyme entrapment in the precipitate. Stage II, isolation of the coenzyme. A sulfonated polystyrene ion exchange resin is used inside a centrifugal filter. Pyridoxamine 5'-phosphate in the supernatant from Stage I adsorbs to the resin. Pyridoxal 5'-phosphate, other organic phosphates, and Pi are removed by centrifugation. Rinsing with dilute NaBH4 destroys traces of pyridoxal 5'-phosphate and washes off residual inhibitors. Pyridoxamine 5'-phosphate is then desorbed with NaOH and Tris buffer and recovered by centrifugation. Stage III, reconstitution and assay. The desorbate from Stage II is incubated with excess apoenzyme. Specific activity of the reconstituted enzyme is measured. Interpolation from a standard curve relating enzyme specific activity and pyridoxamine 5'-phosphate concentration yields the plasma level of the cofactor. Approximately 3 h are required to carry out the procedure. Much of the coenzyme was found not be assayable if plasma was refrigerated overnight or if whole blood was left standing at room temperature for a few hours. The degradation was arrested with freezing at -80 degrees C. In a 13-day experiment involving a healthy subject, sharp rises of plasma pyridoxamine 5'-phosphate were found to occur in response to small doses of oral vitamin B6.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma B-6 vitamer and plasma and urinary 4-pyridoxic acid concentrations of 21 young white women, 21-27 years, having radiomonitored pyridoxal 5'-phosphate and coenzyme stimulation of erythrocyte alanine aminotransferase activities indicative of adequate vitamin B-6 status were determined in an effort to establish normal ranges for plasma B-6 vitamers. B-6 vitamers and 4-pyridoxic acid were quantitated using reversed phase high performance liquid chromatography with fluorometric and ultraviolet detection. Pyridoxal phosphate values obtained by radioenzymatic and chromatographic, fluorometric and ultraviolet, assays were highly correlated as were pyridoxine phosphate values determined using both detectors. The B-6 vitamer and 4-pyridoxic acid values of these subjects should be of use in the establishment of normal ranges of these congeners in women.

The vitamin B6 status of seemingly healthy adolescent girls was determined using several accepted and proposed parameters in an effort to establish guidelines for status evaluation. High-performance liquid chromatography-derived plasma B6 vitamers (pyridoxal phosphate, PLP; pyridoxine phosphate. PNP; pyridoxamine phosphate, PMP; pyridoxal, PL; pyridoxine, PN; and pyridoxamine, PM) and 4-pyridoxic acid (4-PA) concentrations and urinary 4-PA levels of 28 white adolescent females, 12-15 years, having radiomonitored plasma PLP concentrations and coenzyme stimulation of erythrocyte alanine aminotransferase activities indicative of adequate status were determined. Mean vitamin B6 and protein intakes were 1.48 mg and 78.3 g. Ranges for plasma B6 vitamer and 4-PA concentrations (nmol/l) were: PLP, 40.9-122.2; PNP, non-detectable (ND)-16.1; PMP, ND-8.1; PL, ND-15; PN, ND-21.9; PM, ND-17.8; and 4-PA, ND-55.7. PLP was the only vitamer found in plasma of all subjects. Urinary 4-PA concentrations ranged from 0.11 to 2.50 mumol/mmol of creatinine. B6 vitamer values of these girls should be of use in the establishment of normal ranges for vitamin B6 status parameters.

Plasma pyridoxal 5'-phosphate (PLP) concentrations are considered to be the most reliable single indicator of vitamin B-6 nutritional status and are thought to reflect tissue PLP and pyridoxamine 5'-phosphate (PMP) levels. We investigated the relationship between dietary level of pyridoxine hydrochloride (PN-HCl) and concentrations of PLP in blood and PLP and PMP in liver and brain of mice. Female heterogeneous stock mice, 60 to 90 d old, were fed purified diets containing 0.5, 1.0, 2.0, 3.0, 5.0, or 7.0 mg PN-HCl/kg diet for 5 wk. PLP and PMP concentrations were determined by a spectrophotometric apotryptophanase assay. PLP content of plasma, erythrocytes, whole blood, liver and brain and PMP levels in liver and brain were highly correlated with dietary level of PN-HCl (r values ranged from 0.81 to 0.94, n per correlation = 32 to 43). By using the entire range of dietary levels of PN-HCl, both plasma and erythrocyte PLP were found to be significantly correlated with tissue PLP and PMP concentrations. For any one dietary level, however, correlations between plasma or erythrocyte PLP and tissue PLP and PMP concentrations were low and nonsignificant. These results suggest that plasma PLP levels may be suitable to determine vitamin B-6 status of populations, but not to reliably predict tissue concentrations of PLP or PMP in individuals.

The effects of chronic ethanol administration on vitamin B-6 metabolism were studied in female Long-Sleep (LS) and Short-Sleep (SS) mice. Animals were fed an ethanol containing liquid diet (AIN-76) for four weeks. Concentration of ethanol in the diet increased from 10 to 25% ethanol-derived calories (EDC) during weeks 1-3 and was maintained at 30% EDC for 1 additional week. We measured concentrations of pyridoxal 5'-phosphate (PLP) in plasma, erythrocytes and whole blood, and liver and brain PLP and pyridoxamine 5'-phosphate (PMP) in ethanol-fed and pair-fed control mice. Chronic ethanol administration significantly increased PMP and total (PLP + PMP) levels in the liver of SS mice. In LS mice ethanol feeding significantly decreased PMP and total (PLP + PMP) levels in the brain, but these values were still within normal limits. These results suggest that both control and ethanol-containing liquid diets are nutritionally adequate with respect to vitamin B-6, and that chronic ethanol administration does not adversely affect vitamin B-6 metabolism in adult mice.

A method for the separation and assay of all B6 vitamers from a single sample of serum is reported. A new reagent system containing sodium glyoxalate, potassium aluminum, sulfate and manganese dioxide was used for quantitative conversion of pyridoxamine and its phosphate to the corresponding aldehydes by a combination of transamination and oxidation reactions.

The plasma content of B6 vitamers is governed by, among other factors, dietary supply and metabolic interconversion. This study examines the effect of pyridoxine supplementation on the plasma content of B6 vitamers and pyridoxic acid in man, and the metabolic conversion and release of B6 compounds in isolated rat hepatocytes. Six healthy human subjects were given 100 mg pyridoxine-HCl/d orally for 1--3 wk. Before pyridoxine supplementation, the mean total plasma level of B6 vitamers was 114 +/- 9 nM; and pyridoxal-P, pyridoxamine-P, pyridoxal, pyridoxine, and pyridoxamine accounted for 54, 3, 11, 27, and 5%, respectively. Plasma level of pyridoxic acid was 40 +/- 7 nM. Thus, pyridoxal-P is the principal B6 vitamer in plasma. During pyridoxine supplementation, mean plasma levels of the B6 vitamers and pyridoxic acid increased to 655 +/- 122 and 222 +/- 55 nM, respectively. The plasma content of pyridoxal-P and pyridoxic acid increased 6--7-fold and that of pyridoxal, 12-fold, but the pyridoxine level did not increase. Isolated hepatocytes, 1 g/15 ml, were incubated for 2 h with 3.33 microM [14C]pyridoxine (6 microCi/mumol). At zero time, the cells contained about 35 nmol pyridoxal-P and 25 nmol pyridoxamine-P. After 2 h incubation, the cellular content of pyridoxal-P and pyridoxamine-P did not change significantly, but the medium contained 5.9 nmol pyridoxal-P, 0.3 nmol pyridoxamine-P, 7.2 nmol pyridoxal, 26.6 nmol pyridoxine, 0.3 nmol pyridoxamine, and 7.5 nmol pyridoxic acid. Whereas the specific radioactivity of pyridoxal-P, pyridoxal, and pyridoxic acid in the medium approached that of [14C]pyridoxine, the specific radioactivity of cellular pyridoxal-P and pyridoxamine-P was only 20% of that of pyridoxine. Thus, newly synthesized pyridoxal-P is not freely exchangeable with endogenous pyridoxal-P, but is preferentially released or degraded to pyridoxal and pyridoxic acid. The latter B6 compounds are also released. These results suggest that orally ingested pyridoxine is rapidly metabolized in liver and its products are released into the circulation in the form of pyridoxal-P, pyridoxal, and pyridoxic acid.

A decrease in plasma pyridoxal-5'-phosphate (PLP) occurs during pregnancy in humans and experimental animals for reasons that are not known. To determine if mice also develop decreased plasma PLP concentrations during pregnancy, and if plasma PLP levels in pregnancy reflect tissue levels of PLP and pyridoxamine-5'-phosphate (PMP), we measured PLP concentrations in plasma, erythrocytes and whole blood, and liver and brain PLP and PMP in control and pregnant mice. Mice were fed a nonpurified diet containing 8.13 mg pyridoxine-HCl/kg. The PLP analyses were performed in our newly developed apotryptophanase method in which the substrate S-benzyl-L-cysteine is hydrolyzed to benzyl mercaptan, reacted with Ellman's reagent and measured spectrophotometrically. During pregnancy, plasma PLP levels decreased 50% below control levels, but erythrocyte and whole blood PLP levels increased 2.9- and 1.6-fold, respectively. Liver PLP and PMP decreased 25%, in parallel with plasma PLP, but brain PLP and PMP concentrations were unchanged during pregnancy. These results suggest that metabolism or utilization of vitamin B-6 is altered in pregnancy, and that plasma PLP concentrations alone may not be a good indicator of nutritional status in pregnancy.

Conflicting results using erythrocyte aminotransferase (eAST) stimulation to assess vitamin B6 nutritional status in patients with less severe B6 deficiencies are common. It has been claimed that the presence of different B6 vitamers may modify the activation of eAST by pyridoxal-5'-phosphate (PLP) leading to stimulatory or even inhibitory effects. To investigate the possible role of this phenomenon in producing inconsistent AST stimulations, aliquots of whole blood were incubated with equivalent amounts of different B6 vitamers, and the AST stimulation was correlated with the concentrations of PLP, measured by high-performance liquid chromatography. At the end of the incubation period the erythrocytes and plasma were separately analyzed. The conversion of non-PLP B6 vitamers to PLP, by the erythrocytes, was similar (approximately 70%) for all B6 vitamers used in the incubation experiments. The newly formed PLP accumulated in the erythrocytes, but the percentage activation of AST did not change significantly from the basal levels, in spite of the presence of increased levels of PLP and other B6 vitamers used for incubation. When PLP was used in the incubation studies, all of it was retained by the plasma and was associated with a marked suppression of plasma AST stimulation. To determine the degree to which plasma and erythrocyte AST was dose-dependent, plasma and haemolysates were incubated with increasing concentrations of PLP. A very significant inverse relationship was obtained in plasma between AST stimulation and PLP even at modest PLP levels, while haemolysates required incubation with much higher PLP concentrations to demonstrate the same effect. Since plasma PLP is considered to be the most reliable indicator of B6 nutritional status in man, our findings suggest that plasma percentage AST stimulation more closely reflects the B6 nutritional status than erythrocyte AST stimulation test which may reflect B6 status only in severe, longstanding B6 deficiencies. Conflicting results using erythrocyte AST stimulations may be attributed to the insensitivity of red cell AST to changes in PLP content. It is unlikely that the presence of non-PLP B6 vitamers in haemolysate may affect the percentage stimulation of aminotransferase enzymes by PLP.

An extraction procedure that can be used successfully with a high performance liquid chromatography sample clean-up and analytical method is outlined for the determination of B6 vitamer and pyridoxic acid concentrations in human plasma. The method is a modification of ones developed for food and animal tissue investigations. Chromatographic traces, relatively clean of interfering compounds are obtained. The method has the advantage of speed and reproducibility and yields recoveries of from 90 to 100% for all vitamers and pyridoxic acid.