Effect of vitamin B6 availability on serine hydroxymethyltransferase in MCF-7 cells

Folate-activated one-carbon units are derived from serine through the activity of the pyridoxal-phosphate (PLP)-dependent isozymes of serine hydroxymethyltransferase (SHMT). The effect of vitamin B(6) availability on the activity and expression of the human mitochondrial and cytoplasmic SHMT isozymes was investigated in human MCF-7 cells. Cells were cultured for 6 months in vitamin B(6) replete (4.9 microM pyridoxine) minimal essential medium (alphaMEM) or vitamin B(6)-deficient medium containing 49, 4.9 or 0.49 nM pyridoxine. Total cellular PLP levels and SHMT activity were reduced 72% and 7%, respectively, when medium pyridoxine was decreased from 4.9 microM to 49 nM. Cells cultured in medium containing 4.9 nM pyridoxine exhibited 75%, 27% and 60% reduced levels of PLP, SHMT activity and S-adenosylmethionine, respectively, compared to cells cultured in alphaMEM. Cytoplasmic SHMT activity and protein levels, but not mRNA levels, were decreased in cells cultured in vitamin B(6) deficient medium, whereas mitochondrial SHMT activity and protein levels were less sensitive to vitamin B(6) availability. PLP bound to cytoplasmic SHMT with a K(d)=850 nM, a value two orders of magnitude lower than previously reported for the bovine cytoplasmic SHMT isozyme. Collectively, these data indicate that vitamin B(6) restriction decreases the activity and stability of SHMT, and that the cytoplasmic isozyme is more sensitive to vitamin B(6) deficiency than the mitochondrial isozyme in MCF-7 cells.     

Correlation between pyridoxal-5'-phosphate levels and the percentage activation of aspartate aminotransferase enzyme in haemolysate and plasma during in vitro incubation studies with different B6 vitamers

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.     

Dietary vitamin B6 intake modulates colonic inflammation in the IL10−/− model of inflammatory bowel disease

Pyridoxal-5-phosphate, the biologically active form of vitamin B₆, is a cofactor for over 140 biochemical reactions. Although severe vitamin B₆ deficiency is rare, mild inadequacy [plasma pyridoxal 5’-phosphate (PLP) <20 nmol/L] is observed in 19–27% of the US population. Plasma PLP concentrations are inversely related to markers of inflammation such as C-reactive protein. Furthermore, plasma PLP is diminished in those with inflammatory conditions and, in the case of inflammatory bowel disease (IBD), more so in those with active versus quiescent disease. Restricting B₆ intake attenuates IBD pathology in mice; however, the effects of supplementation are unclear. We therefore sought to determine the effects of mild inadequacy and moderate supplementation of B6 on the severity of colonic inflammation. Weanling IL-10^?/? (positive for Helicobacter hepaticus) mice were fed diets containing 0.5 (deficient), 6.0 (replete) or 24 (supplemented) mg/kg pyridoxine HCl for 12 weeks and then assessed for histological and molecular markers of colonic inflammation. Both low and high plasma PLP were associated with a significant suppression of molecular (TNFα, IL-6, IFN-γ, COX-2 and iNOS expression) and histological markers of inflammation in the colon. PLP is required for the breakdown of sphingosine 1-phosphate (S1P), a chemotactic lipid, by S1P lyase. Colonic concentrations of S1P and PLP were significantly and inversely correlated. If confirmed, vitamin B6 supplementation may offer an additional tool for the management of IBD. Although B6 is required in dozens of reactions, its role in the breakdown of S1P may explain the biphasic relationship observed between PLP and inflammation.     

Clinical implications of the correlation between coenzyme Q10 and vitamin B6 status.

The endogenous biosynthesis of the quinone nucleus of coenzyme Q10 (CoQ10) from tyrosine is dependent on adequate vitamin B6 nutriture. Lowered blood and tissue levels of CoQ10 have been observed in a number of clinical conditions. Many of these clinical conditions are most prevalent among the elderly. Kalen et al. have shown that blood levels of CoQ10 decline with age. Similarly, Kant et al. have shown that indicators of vitamin B6 status also decline with age. Blood samples were collected from 29 patients who were not currently being supplemented with either CoQ10 or vitamin B6. Mean CoQ10 concentrations was 1.1 +/- 0.3 micrograms/ml of blood. Mean specific activities of EGOT was 0.30 +/- 0.13 mumol pyruvate/hr/10(8) erythrocytes and the mean percent saturation of EGOT with PLP was 78.2 +/- 13.9%. Means for all parameters were within normal ranges. Strong positive correlation was found between CoQ10 and the specific activity of EGOT (r = 0.5787, p < 0.001) and between CoQ10 and the percent saturation of EGOT with PLP (r = 0.4174, p < 0.024). Studies are currently in progress to determine the effect of supplementation with vitamin B6 of blood CoQ10 levels. It appears prudent to recommend that patients receiving supplemental CoQ10 be concurrently supplemented with vitamin B6 to provide for better endogenous synthesis of CoQ10 along with the exogenous CoQ10.     

Vitamin B6 in Plasma and Cerebrospinal Fluid of Children

Background

Over 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 Methods

B6 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.

Results

The 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.

Conclusion

We 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.     

Tat-mediated protein transduction of human brain pyridoxal kinase into PC12 cells

Pyridoxal kinase (PK) catalyses the phosphorylation of vitamin B6 to pyridoxal-5'-phosphate (PLP). A human brain PK gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat-PK fusion protein. The expressed and purified Tat-PK fusion proteins transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously in culture media. Once inside the cells, the transduced Tat-PK proteins showed catalytic activity and are stable for 48 h. The intracellular concentration of PLP, which is known as a biologically active form of vitamin B6, was increased by pre-treatment of Tat-PK to the PC12 cells. Those results suggest that the transduction of Tat-PK fusion protein can be one of the ways to regulate the PLP level and to replenish this enzyme in the various neurological disorders related to vitamin B6.     

Pyridoxal kinase activity and pyridoxal-P concentration in mammalian tissues under normal and experimental conditions]

The activity and the distribution of pyridoxal kinase in rat and mouse tissues are studied. The data obtained testify the presence of a relative excess of pyridoxal kinase in all the organs studied, which probably causes a high rate of pyridoxalphosphate (PLP) biosynthesis under comparatively low vitamin B6 concentration. A correlation between the level of pyridoxal kinase activity and the content of PLP in rat brain and liver during ontogenesis is observed. The activity of pyridoxal kinase and the content of PLP are shown to be sharply increased in liver of rats received a protein-rich diet. Bilateral adrenalectomy resulted in the decrease of absolute and specific enzyme activities in rat liver by 20--30%. The content of PLP in mouse brain and liver was sharply decreased under experimental B6-avitaminosis while the content of pyridoxal kinase practically did not change. The injection of vitamin B6 rapidly normalized the PLP content in mouse tissues. The data obtained show that under physiological conditions the functional activity of pyridoxal kinase may be regulated in tissues by enzyme and substate contents. Some aspects of vitamin B6 metabolism in mammals are considered. It is concluded that in body the pyridoxal catabolism connected with its phosphorylation by pyridoxal kinase and the formation of pyridoxalphosphate.     

Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism

BackgroundPyridoxal 5′-phosphate (PLP) is the active form of vitamin B6. Mammals cannot synthesize vitamin B6, so they rely on dietary uptake of the different B6 forms, and via the B6 salvage pathway they interconvert them into PLP. Humans possess three enzymes in this pathway: pyridoxal kinase, pyridox(am)ine phosphate oxidase and pyridoxal phosphatase. Besides these, a fourth enzyme has been described in plants and yeast but not in humans: pyridoxal reductase.MethodsWe analysed B6 vitamers in remnant CSF samples of PLP-treated patients and four mammalian cell lines (HepG2, Caco2, HEK293 and Neuro-2a) supplemented with PL as the sole source of vitamin B6.ResultsStrong accumulation of pyridoxine (PN) in CSF of PLP-treated patients was observed, suggesting the existence of a PN-forming enzyme. Our in vitro studies show that all cell lines reduce PL to PN in a time- and dose-dependent manner. We compared the amino acid sequences of known PL reductases to human sequences and found high homology for members of the voltage-gated potassium channel beta subunits and the human aldose reductases. Pharmacological inhibition and knockout of these proteins show that none of the candidates is solely responsible for PL reduction to PN.ConclusionsWe show evidence for the presence of PL reductase activity in humans. Further studies are needed to identify the responsible protein.General significanceThis study expands the number of enzymes with a role in B6 salvage pathway. We hypothesize a protective role of PL reductase(s) by limiting the intracellular amount of free PL and PLP.     

Impairments in pyridoxine-dependent sulphur amino acid metabolism are highly sensitive to the degree of vitamin B6 deficiency and repletion in the pig

The objectives of the current study included the characterization of the temporal changes in indices of sulphur amino acid metabolism in piglets in response to vitamin B6 deficiency and repletion with graded levels of pyridoxine hydrochloride. In Experiment 1, 12 piglets (average initial weight = 5.3 kg; n = 6 per group) were fed a semi-purified diet containing either 0 (deficiency group) or 3 mg (control group) pyridoxine·HCl/kg diet, using a pair-feeding design, for 6 weeks. Piglets consuming vitamin B6-deficient diets exhibited decreased average daily gains on the 4th week and feed conversion efficiency from the 4th week until the end of the trial (P < 0.05). Plasma pyridoxal-5'-phosphate (PLP), in pigs consuming vitamin B6-deficient diets, was significantly lower than controls throughout the experiment (P < 0.01), reaching a nadir of 14% of the control animals' value by the end of the trial. Indices of sulphur amino acid metabolism, including activities of hepatic cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CGL) and serine hydroxymethyltransferase, as well as hepatic-free cysteine concentrations were markedly decreased after 6 weeks of B6 deficiency (P < 0.05). Total hepatic mRNA expressions for CBS and CGL were not affected. Concurrently, hepatic-free homocysteine concentrations increased by more than eight-fold (P < 0.01) at the end of the trial. An examination of plasma total homocysteine and cysteine concentrations revealed significant (P < 0.05) differences between treatments, with evidence of an abrupt shift in concentrations at 3 weeks post-initiation of dietary treatments (>25-fold increase in homocysteine; halving of cysteine values). At the end of Experiment 1, vitamin B6 deficiency significantly increased plasma methionine and serine levels, but decreased plasma glycine concentrations (P < 0.05). In Experiment 2, 20 pigs of 14 days old (initial BW = 5.0 kg) were subjected to a 4-week vitamin B6 depletion protocol, based on results obtained in Experiment 1. After the depletion period and assessment of baseline status (four pigs), remaining pigs were allocated to one of four dietary vitamin B6 repletion treatments: 0.75, 1.5, 2.25 and 3 mg/kg diet as pyridoxine·HCl (n = 4 per level) for 14 days. Significant dose-dependent increases in plasma PLP and cysteine, and decreases in homocysteine were observed, and these were sensitive to the duration of repletion. In conclusion, data from the current studies support the use of both plasma PLP and homocysteine as sensitive indices of vitamin B6 status in the pig. Additionally, the observed patterns of responses in vitamin B6-sensitive metabolites are supportive of an inclusion level of 2.25 mg/kg diet, as pyridoxine·HCl, in diets for young pigs.     

Production of pyridoxal phosphate by a mutant strain of Schizosaccharomyces pombe

Conditions for extracellular production of vitamin B6 compounds (B6), especially pyridoxal 5'-phosphate (PLP) by Schizosaccharomyces pombe leul strain were examined. The productivity was dependent on concentration of L-leucine in the culture medium: 30 mg/l gave the highest concentrations of total B6 and PLP. The viable cells harvested at different growth phases showed different productivity: middle and late exponential phase cells showed the highest productivity of total B6 and PLP, respectively. D-Glucose (1%, w/v) among other sugars gave the best productivity. Supplementation of air and ammonium sulfate significantly increased extracellular production of PLP. Superoxide anion producers, menadione and plumbagin, and H202 increased the productivity of PLP. Cycloheximide inhibited the increase of PLP by the oxidative stress and, in contrast, increased pyridoxine.     

Vitamin B6 metabolism in microbes and approaches for fermentative production

Vitamin B6 is a designation for the six vitamers pyridoxal, pyridoxine, pyridoxamine, pyridoxal 5′-phosphate (PLP), pyridoxine 5′-phosphate, and pyridoxamine. PLP, being the most important B6 vitamer, serves as a cofactor for many proteins and enzymes. In contrast to other organisms, animals and humans have to ingest vitamin B6 with their food. Several disorders are associated with vitamin B6 deficiency. Moreover, pharmaceuticals interfere with metabolism of the cofactor, which also results in vitamin B6 deficiency. Therefore, vitamin B6 is a valuable compound for the pharmaceutical and the food industry. Although vitamin B6 is currently chemically synthesized, there is considerable interest on the industrial side to shift from chemical processes to sustainable fermentation technologies. Here, we review recent findings regarding biosynthesis and homeostasis of vitamin B6 and describe the approaches that have been made in the past to develop microbial production processes. Moreover, we will describe novel routes for vitamin B6 biosynthesis and discuss their potential for engineering bacteria that overproduce the commercially valuable substance. We also highlight bottlenecks of the vitamin B6 biosynthetic pathways and propose strategies to circumvent these limitations.     

Human brain pyridoxal-5'-phosphate phosphatase: production and characterization of monoclonal antibodies

We cloned and expressed human pyridoxal-5\'-phosphate (PLP) phosphatase, the coenzymatically active form of vitamin B6, in Escherichia coli using pET15b vector. Monoclonal antibodies (mAb) were generated against purified human brain PLP phosphatase in mice, and four antibodies recognizing different epitopes were obtained, one of which inhibited PLP phosphatase. The binding affinities of these four mAbs to PLP phosphatase, as determined using biosensor technology, showed that they had similar binding affinities. Using the anti-PLP phosphatase antibodies as probes, we investigated their cross-reactivities in various mammalian and human tissues and cell lines. The immunoreactive bands obtained on Western blots had molecular masses of ca. 33 kDa. Similarly fractionated extracts of several mammalian cell lines all produced a single band of molecular mass 33 kDa. We believe that these PLP phosphatase mAbs could be used as valuable immunodiagnostic reagents for the detection, identification, and characterization of various neurological diseases related to vitamin B6 abnormalities.     

Pyridoxine supply in human development

Vitamin B(6) has an important role in the function of the human nervous system. Experimental data are not generally available on the role in human development, but significant conclusions may be made from studies of the effect of disorders of B(6) vitamer metabolism. Vitamin B(6) comprises seven compounds - pyridoxal, pyridoxine, pyridoxamine and their respective 5' phosphates. The common active form in human tissue is the 5'-phosphate form of pyridoxal (PLP) most of which is found in muscle bound to phosphorylase. Like many vitamins, B(6) can function both as a co-enzyme and as a chaperone. Pyridoxal-5'-phosphate is the metabolically active form and is involved in 100 enzymatic reactions including carbohydrate, amino acid, and fatty acid metabolism. There is evidence that in some situations B(6) vitamers can function as antioxidants. The fetus is dependent on the placenta for supply of vitamin B(6) and the demand correlates with amino acid metabolism. Few reports are available on the role of B(6) in embryogenesis. Studies of human disorders where B(6) metabolism is blocked show a major role in neurotransmitter function with secondary cerebral and cerebellar hypoplasia. Pyridoxine potentiates vitamin A teratogenicity and an excess leads to peripheral nerve cell degeneration. The key role of vitamin B(6) in the developing human is in metabolism, especially of the neurotransmitters.     

The regulation of alanine and aspartate aminotransferase by different aminothiols and by vitamin B-6 derivatives

We examined the effects on alanine aminotransferase and aspartate aminotransferase of different aminothiols (l-cysteine, d-cysteine, cysteamine, l-cysteine ethyl ester, l-cysteine methyl ester) and several vitamin B-6 derivatives (pyridoxal, pyridoxamine, pyridoxol, pyridoxol 5′-phosphate), before and after treatment with KOCN, which transforms these molecules into the corresponding carbamoyl derivatives. Only GTP, and not GOT, was specifically inhibited by l-cysteine and, to a lesser extent, by d-cysteine. The association reaction: PLP + apo GPT ↔ holo GPT was inhibited by the vitamin B-6 derivatives, and this inhibition was prevented by pretreatment of the vitamin B-6 derivatives with KOCN. All the observed effects occurred at pH 7, 37°C, at mM and even lower concentrations of reagents. Hence, they all potentially play a physiological role, in the regulation of the PLP dependent enzymes and of the vitamin B-6 levels in the cell.     

Improved cognition,mild anxiety-like behavior and decreased motor performance in pyridoxal phosphatase-deficient mice

Pyridoxal 5′-phosphate (PLP) is an essential cofactor in the catalysis of ~140 different enzymatic reactions. A pharmacological elevation of cellular PLP concentrations is of interest in neuropsychiatric diseases, but whole-body consequences of higher intracellular PLP levels are unknown. To address this question, we have generated mice allowing a conditional ablation of the PLP phosphatase PDXP. Ubiquitous PDXP deletion increased PLP levels in brain, skeletal muscle and red blood cells up to 3-fold compared to control mice, demonstrating that PDXP acts as a major regulator of cellular PLP concentrations in vivo. Neurotransmitter analysis revealed that the concentrations of dopamine, serotonin, epinephrine and glutamate were unchanged in the brains of PDXP knockout mice. However, the levels of γ-aminobutyric acid (GABA) increased by ~20%, demonstrating that elevated PLP levels can drive additional GABA production. Behavioral phenotyping of PDXP knockout mice revealed improved spatial learning and memory, and a mild anxiety-like behavior. Consistent with elevated GABA levels in the brain, PDXP loss in neural cells decreased performance in motor tests, whereas PDXP-deficiency in skeletal muscle increased grip strength. Our findings suggest that PDXP is involved in the fine-tuning of GABA biosynthesis. Pharmacological inhibition of PDXP might correct the excitatory/inhibitory imbalance in some neuropsychiatric diseases.     

Nonradioactive enzymatic assay for plasma and serum vitamin B(6)

Pyridoxal-5'-phosphate (PLP) is the biologically active form of vitamin B(6). Clinical studies suggest that low PLP concentrations are an independent risk factor for cardiovascular and other diseases. However, PLP concentrations are not routinely used for diagnosis because of the lack of a homogeneous, nonradioactive assay. We describe a homogeneous, nonradioactive, enzymatic PLP assay that uses the apo form of the recombinant PLP-dependent enzyme, homocysteine-alpha,gamma-lyase (rHCYase). The restoration of enzymatic activity by reconstitution of the holoenzyme is linearly dependant on the amount of PLP bound to the enzyme. Nanomolar concentrations of PLP can then be measured by the conversion (by reconstituted holo-rHCYase) of millimolar concentrations of homocysteine (HCY) to H(2)S. H(2)S combines with DBPDA (N,N-dibutylphenylenediamine) to form 3,7-Bis(dibutylamino)-phenothiazine-5'-ium chloride and the absorbance of this compound is read at 675 nm. The PLP enzymatic assay has a lower limit of detection of 14.8 nmol l(-1) and is linear to 300 nmol l(-1) and requires only 10 microl plasma or serum. This PLP assay is the first homogeneous, nonradioactive method for diagnosing vitamin B(6). The procedure takes approximately 2 h to complete.     

Human brain pyridoxal-5'-phosphate phosphatase (PLPP):protein transduction of PEP-1-PLPP into PC12 cells

Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin B(6) precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin B(6).