Pyridoxal Phosphate Concentrations Research Articles

A novel fully validated LC-MS/MS method for quantification of pyridoxal-5'-phosphate concentrations in samples of human whole blood.

Quantification of pyridoxal-5'-phosphate (PLP) in biological samples is challenging due to the presence of endogenous PLP in matrices used for preparation of calibrators and quality control samples (QCs). Hence, we have developed an LC-MS/MS method for accurate and precise measurement of the concentrations of PLP in samples (20μL) of human whole blood that addresses this issue by using a surrogate matrix and minimizing the matrix effect. We used a surrogate matrix comprising 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for making calibrators, QCs and the concentrations were adjusted to include the endogenous PLP concentrations in the surrogate matrix according to the method of standard addition. PLP was separated from the other components of the sample matrix using protein precipitation with trichloroacetic acid 10% w/v. After centrifugation, supernatant were injected directly into the LC-MS/MS system. Calibration curves were linear and recovery was >92%. QCs were accurate, precise, stable for four freeze-thaw cycles, and following storage at room temperature for 17h or at -80°C for 3 months. There was no significant matrix effect using 9 different individual human blood samples. Our novel LC-MS/MS method has satisfied all of the criteria specified in the 2012 EMEA guideline on bioanalytical method validation.

Common Variants at Putative Regulatory Sites of the Tissue Nonspecific Alkaline Phosphatase Gene Influence Circulating Pyridoxal 5′-Phosphate Concentration in Healthy Adults1–3

Background: Vitamin B-6 interconversion enzymes are important for supplying pyridoxal 5′-phosphate (PLP), the co-enzyme form, to tissues. Variants in the genes for these enzymes [tissue nonspecific alkaline phosphatase (ALPL), pyridoxamine 5′-phosphate oxidase, pyridoxal kinase, and pyridoxal phosphatase] could affect enzyme function and vitamin B-6 status.Objectives: We tested whether single-nucleotide polymorphisms (SNPs) in these genes influence vitamin B-6 status markers [plasma PLP, pyridoxal (PL), and 4-pyridoxic acid (PA)], and explored potential functional effects of the SNPs.Methods: Study subjects were young, healthy adults from Ireland (n = 2345). We measured plasma PLP, PL, and PA with liquid chromatography–tandem mass spectrometry and genotyped 66 tag SNPs in the 4 genes. We tested for associations with single SNPs in candidate genes and also performed genome-wide association study (GWAS) and gene-based analyses.Results: Seventeen SNPs in ALPL were associated with altered plasma PLP in candidate gene analyses (P < 1.89 × 10−4). In the GWAS, 5 additional ALPL SNPs were associated with altered plasma PLP (P < 5.0 × 10−8). Gene-based analyses that used the functional linear model β-spline (P = 4.04 × 10−15) and Fourier spline (P = 5.87 × 10−15) methods also showed associations between ALPL and altered plasma PLP. No SNPs in other genes were associated with plasma PLP. The association of the minor CC genotype of 1 ALPL SNP, rs1256341, with reduced ALPL expression in the HapMap Northern European ancestry population is consistent with the positive association between the CC genotype and plasma PLP in our study (P = 0.008). No SNP was associated with altered plasma PL or PA.Conclusions: In healthy adults, common variants in ALPL influence plasma PLP concentration, the most frequently used biomarker for vitamin B-6 status. Whether these associations are indicative of functional changes in vitamin B-6 status requires more investigation.

Vitamin B6 in plasma and cerebrospinal fluid of children.

BackgroundOver the past years, the essential role of vitamin B6 in brain development and functioning has been recognized and genetic metabolic disorders resulting in functional vitamin B6 deficiency have been identified. However, data on B6 vitamers in children are scarce.Materials and MethodsB6 vitamer concentrations in simultaneously sampled plasma and cerebrospinal fluid (CSF) of 70 children with intellectual disability were determined by ultra performance liquid chromatography-tandem mass spectrometry. For ethical reasons, CSF samples could not be obtained from healthy children. The influence of sex, age, epilepsy and treatment with anti-epileptic drugs, were investigated.ResultsThe B6 vitamer composition of plasma (pyridoxal phosphate (PLP) > pyridoxic acid > pyridoxal (PL)) differed from that of CSF (PL > PLP > pyridoxic acid > pyridoxamine). Strong correlations were found for B6 vitamers in and between plasma and CSF. Treatment with anti-epileptic drugs resulted in decreased concentrations of PL and PLP in CSF.ConclusionWe provide concentrations of all B6 vitamers in plasma and CSF of children with intellectual disability (±epilepsy), which can be used in the investigation of known and novel disorders associated with vitamin B6 metabolism as well as in monitoring of the biochemical effects of treatment with vitamin B6.

Biochemical characteristics of an alanine racemase from Aeromonas hydrophil HBNUAh01

We reveal that the genome of Aeromonas hydrophila HBNUAh01has an alanine racemase gene (alr-2); this gene encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain MB2795. The gene alr-2 was cloned and expressed in E. coli BL21 (DE3). The gene has an open reading frame (ORF) of 1230 bp encoding a protein of 369 amino acids with a calculated molecular mass of 40.39 kD. The amino acid sequence deduced revealed similarity of 98, 84 and 68% with alanine racemases from A. hydrophila ATCC7966, Aeromonas caviae_Ae398, Aeromonas vickers_b565, respectively. The optimal temperature and pH for enzyme activity was 37°C and pH 10. The enzyme had broad substrate specificity. The alanine racemase was shown to belong to the pyridoxal 5′-phosphate (PLP)-dependent family of enzymes that require a certain concentration of PLP for activity. The kinetic parameters Km and Vmax at 40°C of alanine racemase were 35.9 mM, 2898 units/mg for L-alanine and 15.36 mM, 1209 units/mg for D-alanine, respectively.

Enzyme assay for pyridoxal 5′-phosphate by apo-d-amino acid aminotransferase

D-amino acid aminotransferase (D-AAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme. D-AAT from thermophilic Bacillus sp. YM-1 was inactivated by ultraviolet irradiation. The activity was restored by the addition of PLP and showed a linear correlation with respect to PLP concentrations in the range of 10-40 nM.

Coenzyme Q10 and Pyridoxal Phosphate Deficiency Is a Common Feature in Mucopolysaccharidosis Type III.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by deficiencies of lysosomal enzymes catalyzing degradation of glycosaminoglycans (GAGs). Previously, we reported a secondary plasma coenzyme Q10 (CoQ) deficiency in MPS patients. For this study, nine MPS patients were recruited in the Hospital Sant Joan de Déu (HSJD, Barcelona) and two patients in the Neurometabolic Unit, National Hospital (NMU, London), to explore the nutritional status of MPS type III patients by analyzing several vitamins and micronutrients in blood and in cerebrospinal fluid. Plasma CoQ and plasma and cerebrospinal fluid pyridoxal phosphate (PLP) content were analyzed by high-pressure liquid chromatography (HPLC) with electrochemical and fluorescence detection, respectively. We found that most MPS-III patients disclosed low plasma pyridoxal phosphate (PLP) values (seven out of nine) and also low plasma CoQ concentrations (eight out of nine). We observed significantly lower median values of PLP, tocopherol, and CoQ (Mann-Whitney U test, p = 0.006, p = 0.004, and p = 0.001, respectively) in MPS patients when compared with age-matched controls. Chi-square test showed a significant association between the fact of having low plasma PLP and CoQ values in the whole cohort of patients. Cerebrospinal fluid PLP values were clearly deficient in the two patients studied. In conclusion, we report a combined CoQ and PLP deficiency in MPS-III patients. These observations could be related to the complexity of the physiopathology of the disease. If our results are confirmed in larger series of patients, CoQ and PLP therapy could be trialed as coadjuvant therapy with the current MPS treatments.

Vitamins B2 and B6 as determinants of kynurenines and related markers of interferon-γ-mediated immune activation in the community-based Hordaland Health Study.

Vitamins B2 and B6 are cofactors in the kynurenine pathway. Many of the kynurenines are neuroactive compounds with immunomodulatory effects. In the present study, we aimed to investigate plasma concentrations of vitamins B2 and B6 as determinants of kynurenines and two markers of interferon-γ-mediated immune activation (kynurenine:tryptophan ratio (KTR) and neopterin). We measured the concentrations of vitamins B2 and B6 vitamers, neopterin, tryptophan and six kynurenines (i.e. kynurenine, anthranilic acid, kynurenic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and xanthurenic acid) in plasma from 7051 individuals. Dietary intake of vitamins B2 and B6 was assessed using a validated FFQ. Associations were investigated using partial Spearman's correlations, generalised additive models, and segmented or multiple linear regression. The B2 vitamer, riboflavin, was positively associated with 3-hydroxyanthranilic acid and xanthurenic acid, with correlation coefficients, as obtained by segmented regression, of 0·20 (95% CI 0·16, 0·23) and 0·24 (95% CI 0·19, 0·28), at riboflavin concentrations below the median value (13·0nmol/l). The vitamin B6 vitamer, pyridoxal 5'-phosphate (PLP), was positively associated with most kynurenines at PLP concentrations <39·3-47·0nmol/l, and inversely associated with 3-hydroxykynurenine with the association being more prominent at PLP concentrations <18·9nmol/l. Riboflavin and PLP were associated with xanthurenic acid only at relatively low, but normal concentrations of both vitamers. Lastly, PLP was negatively correlated with neopterin and KTR. These results demonstrate the significant and complex determination of kynurenine metabolism by vitamin status. Future studies on B-vitamins and kynurenines in relation to chronic diseases should therefore integrate data on relevant biomarkers related to B-vitamins status and tryptophan metabolism.

Association of one‐carbon and tryptophan metabolites with vitamin B‐6 concentrations in oral contraceptive users (827.7)

Vitamin B‐6 in the form of pyridoxal 5’‐phosphate (PLP) serves as a coenzyme in 1C metabolism and tryptophan (trp) catabolism. In 1C metabolism, PLP acts a coenzyme for serine hydroxymethyltransferase and glycine decarboxylase (glycine‐cleavage system) involved in the provision of 1C units, as well as for the transsulfuration enzymes, cystathionine β‐synthase and cystathionine γ‐lyase. In trp catabolism, PLP is a cofactor for kynureninase and kynurenine aminotransferase. Associations between plasma PLP and changes in metabolites involved in these pathways have previously been reported. We investigated the associations between plasma PLP with 1C and trp metabolites in 165 oral contraceptive users (20‐40 y) from Gainesville, Florida. Plasma concentrations of PLP, homocysteine, cysteine, cysteinylglycine and glutathione were measured by HPLC. Plasma concentrations of other major 1C and trp metabolites were measured by LC‐MS/MS. Associations of PLP concentrations (14.4‐189 nmol/l) with 1C metabolite concentrations were explored by Pearson’s product‐moment correlation. Preliminary data showed a weak negative association of PLP with homocysteine, a moderate negative association of PLP with glutathione (P &lt; 0.05), and a weak positive association of PLP with cysteine and cysteinylglycine concentrations, separately (P &lt; 0.05).Grant Funding Source: Supported by NIH Grant DK072398 and NIH CTSA grant 1UL1RR029890.

Substrate product ratios of enzymes in the kynurenine pathway measured in plasma as indicators of functional vitamin B-6 status

Tryptophan metabolism through the kynurenine pathway includes 2 vitamin B-6 [pyridoxal 5'-phosphate (PLP)]-dependent enzymes. We recently showed that plasma 3-hydroxykynurenine (HK) was elevated at low PLP concentrations. We further evaluated and characterized kynurenine-based indexes as possible markers of functional B-vitamin status in plasma. Cross-sectional and longitudinal data were derived from the Western Norway B-vitamin Intervention Trial, including PLP, kynurenine, HK, kynurenic acid (KA), anthranilic acid, xanthurenic acid (XA), and 3-hydroxyanthranilic acid (HAA) measured in plasma at 2 time points. Partial Spearman's correlation, generalized additive models, and receiver operating characteristic (ROC) analysis were used to assess associations of kynurenines with PLP. Ratios HK:XA, HK:HAA, and HK:KA showed markedly stronger negative correlations with PLP than did HK alone (Spearman's ρ = -0.36, -0.29, and -0.31 compared with -0.18, respectively). All associations were nonlinear, with the strongest relation at low PLP. In the ROC analysis, areas under the curve for discriminating low PLP (less than the fifth percentile; 18.6 nmol/L) were 0.78, 0.78, and 0.74, respectively, compared with 0.65 for HK. Oral treatment with 40 mg pyridoxin hydrochloride for 28 d reduced the ratios by up to 60%, with strongest reductions for subjects with low plasma PLP at baseline. Whereas HK was associated with kidney function and several inflammatory markers, such associations were abolished or attenuated for the ratios. Plasma values of HK:XA and HK:HAA, which are substrate-product pairs for kynurenine transaminase and kynureninase, respectively, may reflect the intracellular availability of the cofactor (PLP) and, therefore, present as potential markers of functional vitamin B-6 status.

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.

Cellular pyridoxal phosphate concentration influences amino acid profiles and presence of hydrogen sulfide biomarkers in cultured HepG2 cells

Pyridoxal phosphate (PLP) functions as a coenzyme in cellular one carbon metabolism and many other roles in amino acid interconversion and catabolism. The PLP‐dependent transsulfuration enzymes, cystathionine beta‐synthase and cystathionine gamma‐lyase, have been implicated in hydrogen sulfide (H2S) production that is concurrent with the formation of lanthionine and homolanthionine. Thus, cellular PLP availability may influence the concentration of many amino acids including these H2S biomarkers. In this study, we examined amino acid profiles in HepG2 cells cultured six weeks in supraphysiological to deficient concentrations (2000, 200, 50, and 10 nM) of pyridoxal (PL). Fluorometric HPLC was used to quantify the amino acids as danysl derivatives. Assuming the 50 nM PL reflects nutritional adequacy, the deficient 10 nM cells showed higher concentrations of arginine, asparagine, glutamine, serine, threonine, glycine, alanine, methionine, valine, leucine, lanthionine, and lysine (P&lt;0.05). Results indicate that restriction of vitamin B6 can alter amino acid profiles, including biomarkers of H2S synthesis, in HepG2 cells.Grant Funding Source: NIH Grant DK072398

Vitamin B-6 restriction impairs fatty acid synthesis in cultured human hepatoma (HepG2) cells

Vitamin B-6 deficiency has been reported to alter n-6 and n-3 fatty acid profiles in plasma and tissue lipids; however, the mechanisms underlying such metabolic changes remain unclear. The objective of this study was to determine the effects of vitamin B-6 restriction on fatty acid profiles and fatty acid synthesis in HepG2 cells. Cells were cultured for 6 wk in media with four different vitamin B-6 concentrations (10, 20, 50, and 2,000 nM added pyridoxal, representing deficient, marginal, adequate, and supraphysiological conditions) that induced a range of steady-state cellular concentrations of pyridoxal phosphate. Total cellular lipid content was greatest in the deficient (10 nM pyridoxal) medium. The percentage of arachidonic acid and the ratio of arachidonic acid to linoleic acid in the total lipid fraction were ~15% lower in vitamin B-6-restricted cells, which suggests that vitamin B-6 restriction affects n-6 fatty acid interconversions. Metabolic flux studies indicated significantly lower fractional synthesis rate of oleic acid and arachidonic acid at 10, 20, and 50 nM pyridoxal, whereas that of eicosapentaenoic acid was lower in the cells cultured in 10 nM pyridoxal. Additionally, relative mRNA expressions of Δ5 and Δ6 desaturases were 40-50% lower in vitamin B-6-restricted cells. Overall, these findings suggest that vitamin B-6 restriction alters unsaturated fatty acid synthesis, particularly n-6 and n-3 polyunsaturated fatty acid synthesis. These results and observations of changes in human plasma fatty acid profiles caused by vitamin B-6 restriction suggest a mechanism by which vitamin B-6 inadequacy influences the cardiovascular risk.

Prediagnostic Plasma Pyridoxal 5′-Phosphate (Vitamin B6) Levels and Invasive Breast Carcinoma Risk: The Multiethnic Cohort

Evidence from experimental and epidemiologic studies suggests that vitamin B6 may reduce the risk of breast cancer. We examined the association of prediagnostic plasma concentrations of pyridoxal-5'-phosphate (PLP), an active form of vitamin B6, with postmenopausal breast cancer risk in a case-control study nested in the multiethnic cohort in Hawaii and Southern California, including 706 cases and 706 controls matched on date of birth, ethnicity, study site, date of blood draw, time of blood draw, hours of fasting before blood draw, and use of menopausal hormones. OR and 95% confidence intervals (CI) were calculated using conditional logistic regression models. Women with plasma PLP concentrations in the highest quartile had a 30% reduced risk of invasive breast cancer (CI: 0.50-0.98) as compared with the women in the lowest PLP quartile (P for trend = 0.02). The association seemed to be limited in cases with hormone receptor-positive tumors (P for heterogeneity = 0.04); and remained unchanged in the analysis restricted to women with blood samples collected more than one year before cancer diagnosis (OR = 0.69; CI: 0.48-0.99; P for trend = 0.03). These data suggest that higher circulating levels of vitamin B6 are associated with a reduced risk of invasive postmenopausal breast cancer. These results, in combination with information from two other prospective studies, suggest a role for vitamin B6 in the prevention of postmenopausal breast cancer. Additional studies are needed to further investigate potential heterogeneity of the vitamin B6 association with breast cancer risk by tumor hormone receptor status.

Allosteric Communication between the Pyridoxal 5′-Phosphate (PLP) and Heme Sites in the H2S Generator Human Cystathionine β-Synthase

Human cystathionine β-synthase (CBS) is a unique pyridoxal 5'-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ~2- to ~500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H(2)S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H(2)S-generating reactions catalyzed by CBS.

Vitamin B6 Vitamer Concentrations in Cerebrospinal Fluid Differ Between Preterm and Term Newborn Infants

Vitamin B(6) plays a pivotal role in brain development and functioning. Differences in vitamin B(6) homeostasis between preterm and term newborn infants have been reported. The authors sought to investigate whether B(6) vitamers in cerebrospinal fluid (CSF) of preterm and term newborn infants are different. B(6) vitamer concentrations were determined in 69 CSF samples of 36 newborn infants (26 born preterm and 10 born term) by ultra performance liquid chromatography-tandem mass spectrometry. CSF samples, taken from a subcutaneous intraventricular reservoir, were bedside frozen and protected from light. Concentrations of pyridoxal (PL), pyridoxal phosphate (PLP), pyridoxic acid (PA), and pyridoxamine (PM) in preterm newborns (postmenstrual age 30-37 weeks) were at least twice as high as in older newborns (postmenstrual age ≥ 42 weeks). Pyridoxine and pyridoxamine phosphate concentrations were below limits of quantification in all newborns. In CSF of 2 very preterm newborns (postmenstrual age <30 weeks), significant amounts of pyridoxine were present besides high concentrations of PL, PA, and PM, whereas PLP concentrations were relatively low. B(6) vitamers in CSF were positively correlated, especially PA, PLP, and PL. In CSF of newborn infants, PL, PLP, PA, and PM are present, and concentrations are strongly dependent on postmenstrual age. Our results indicate that vitamin B(6) homeostasis in brain differs between preterm and term newborns. These results should be taken into account for diagnosis and treatment of epilepsy and vitamin B(6) deficiency in newborn infants.

Plasma Pyridoxal-5-Phosphate Is Inversely Associated with Systemic Markers of Inflammation in a Population of U.S. Adults1–3

Low vitamin B-6 status, based on plasma concentrations of pyridoxal-5-phosphate (PLP), has been identified in inflammatory diseases, including cardiovascular disease, rheumatoid arthritis, inflammatory bowel disease, and diabetes. Our objective was to examine the association between plasma PLP and multiple markers of inflammation in a community-based cohort [n = 2229 participants (55% women, mean age 61 #x00B1; 9 y)]. We created an overall inflammation score (IS) as the sum of standardized values of 13 individual inflammatory markers. Multivariable-adjusted regression analysis was used to assess the associations between the IS and plasma PLP. Geometric mean plasma PLP concentrations were lower in the highest tertile category of IS relative to the lowest (61 vs. 80 nmol/L; P-trend < 0.0001). Similarly, the prevalence of PLP insufficiency was significantly higher for participants in the highest compared with the lowest tertiles for IS categories. These relationships persisted after accounting for vitamin B-6 intake. Also, there were significant inverse relationships between plasma PLP and 4 IS based on functionally related markers, including acute phase reactants, cytokines, adhesion molecules, and oxidative stress. In addition, secondary analyses revealed that many of the individual inflammatory markers were inversely associated with plasma PLP after adjusting for plasma C-reactive protein concentration. This study, in combination with past findings, further supports our hypothesis that inflammation is associated with a functional deficiency of vitamin B-6. We discuss 2 possible roles for PLP in the inflammatory process, including tryptophan metabolism and serine hydroxymethyltransferase activity.

Characterization of an acid phosphatase responsible for hydrolysis of pyridoxal 5′-phosphate in tobacco plants

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B(6), is an important cofactor for many enzymatic reactions. PLP is also a very reactive molecule, and the hydrolysis of PLP is crucial for controlling intracellular PLP concentrations. However, little is known about the enzymatic hydrolysis of PLP in plants. In this study, a novel acid phosphatase was purified from tobacco leaves and characterized by using PLP as a substrate. This phosphatase was purified 180-fold with a yield of 28% by ammonium sulfate precipitation and chromatography on DEAE-Sepharose FF, Sephadex G-100 and SP Sephadex C-25. Our data revealed that the purified enzyme was a dimer with a molecular mass of approximately 50 kDa. The purified phosphatase had maximum catalytic activity at pH 5.5, and displayed optimal activity at 50 °C. The enzyme required divalent metal ion for activity, and Mg(2+), among a few tested cations, was the most effective for catalysis under saturating substrate concentrations. The activity of the purified phosphatase was inhibited by molybdate, fluoride and EDTA, but was not inhibited by levamisole and tartrate. The phosphatase hydrolyzed a broad range of substrates at different rates, and the hydrolysis of PLP was competitively inhibited by ATP, pNPP, and by the reaction products, PL and inorganic phosphate. The phosphatase had a Km of 0.24 mM and a Vmax of 2.76 μmol/min/mg with PLP. When pyridoxamine 5'-phosphate or pyridoxine 5'-phosphate was tested as a substrate, the phosphatase activity was reduced by 50%. Our study suggests that the enzyme is a nonspecific acid phosphatase responsible for hydrolysis of all three phosphorylated B(6) vitamers in tobacco plants.

The Relationships Between Plasma and Red Cell Vitamin B2 and B6 Concentrations and the Systemic and Local Inflammatory Responses in Patients With Colorectal Cancer

B vitamins have been implicated in cancer pathogenesis. It is therefore of interest that plasma B6 falls as part of the systemic inflammatory response (SIR), whereas red cell concentrations do not. The modified Glasgow Prognostic Score (mGPS) is a validated inflammation-based prognostic score that consists of a combination of albumin and C-reactive protein concentrations. The aim of this study was to examine the relationships between the concentrations of plasma and red cell vitamin B concentrations, the local and systemic inflammatory response in patients with colorectal cancer. Preoperative venous blood of 108 patients with colorectal cancer were analyzed for C-reactive protein, albumin, flavin adenine dinucleotide (FAD), and pyridoxal phosphate (PLP), and lymphocyte counts. Pathological slides were retrieved for assessment of inflammatory cell infiltration. Increasing mGPS was associated with lower plasma PLP concentrations (P < 0.01) but not plasma and red cell FAD and red cell PLP concentrations. Increasing tumor stage was associated with the presence of venous invasion (P < 0.01) and low-grade inflammatory cell infiltrate (P < 0.05) but not the SIR, FAD, or PLP concentrations. A low-grade inflammatory cell infiltrate was not significantly associated with any other parameter. The presence of a SIR was associated with lower concentrations of plasma PLP but not red cell PLP concentrations in patients with colorectal cancer. Neither FAD and PLP were associated with the tumor inflammatory cell infiltrate.

Vitamin B6 and the Immunity in Kidney Transplant Recipients

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. A retrospective observational study. The study was conducted at the Medical University of Gdańsk, Poland. 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. 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. 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. 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.

Accumulation of γ-aminobutyric acid in germinated soybean (Glycine max L.) in relation to glutamate decarboxylase and diamine oxidase activity induced by additives under hypoxia

High levels of γ-aminobutyric acid (GABA) accumulate in plant tissues under various stresses and exogenous additives. The purpose of this research is to provide an effective finding that can prove a rapid accumulation of GABA in germinated soybean (Glycine max L.) in response to different additives under hypoxia. Hypoxia-induced GABA accumulation in soybean embryo resulted in part from polyamine oxidation. Response to different concentration of glutamate (Glu), pyridoxal phosphate, arginine, CuCl2, NaCl, and CaCl2, a significant difference including GABA accumulation, changes of Glutamate decarboxylase (GAD), and Diamine oxidase activity (DAO) activity in germinated soybean under hypoxia occurred (p < 0.05) and the maximum accumulation of GABA were 4.07, 3.02, 3.50, 3.26, 4.00, and 3.30 g kg−1 DW respectively, which were significantly higher than those germinated soybean under normal culture (CK) and hypoxia culture (CK0) (p < 0.05). The GAD and DAO have different distributions in cotyledon and embryo of germinated soybean, and the enzyme activity mainly located in embryo of germinated soybean. Germinated soybean is a good resource of GABA-rich food. Different additives have significant effects on GABA production, among which Glu and NaCl are ideal material for GABA accumulation.