The effect of pyridoxine deficiency on the metabolism of the aromatic amino acids by isolated rat liver cells

The total activity of three key enzymes and the flux through eight steps of aromatic amino acid metabolism have been determined in liver cells isolated from rats fed either control or pyridoxine-free diet for 5-6 weeks. The pyridoxine-free diet caused a decrease in the catabolism of tyrosine and phenylalanine because of a drop in the flux through tyrosine aminotransferase. This decrease of expressed cellular tyrosine aminotransferase activity can be fully explained in terms of loss of cofactor. Larger decreases in the catabolism of tryptophan were seen after pyridoxine deprivation. The decreased extent of tryptophan catabolism can be solely attributed to loss of cofactor or increased degradation of kynureninase. Inhibition of tryptophan 2,3-dioxygenase was seen in pyridoxine deficiency, probably because of the buildup of the kynurenine metabolites. The control strength of kynureninase, for flux through kynureninase, was calculated to be less than or equal to 0.004, but 0.41 after pyridoxine deprivation. The sensitivity of the three pathways to pyridoxine deprivation is interpreted and discussed in terms of the different affinities for pyridoxal phosphate and the control strengths of the pyridoxal phosphate-dependent enzymes, tyrosine aminotransferase and kynureninase.     

Role of coenzyme in aminotransferase turnover.

The role of coenzyme in determining intracellular contnet of pyridoxal enzymes was assessed by analyzing effects of pyridoxine deficiency on the rapidly degraded, readily dissociable tyrosine aminotransferase (EC 2.6.1.5) and the slowly degraded, nondissociable alanine aminotransferase (EC 2.6.1.2) of rat liver. Synthesis of the tyrosine enzyme was reduced, leading to a decreased amount of this enzyme, much of which was present as active apoenzyme. Synthesis of alanine aminotransferase was unchanged but much of this enzyme was present as an inactive apoenzyme which retained immunological reactivity. Degradation rates of both enzymes (t1/2 about 1.5 h, tyrosine aminotransferase; about 3 days, alanine aminotransferase) were not changed in pyridoxine deficiency. Hence, interaction with coenzyme is not a significant determinant in intracellular degradation of these aminotransferases. Coenzymes dissociation and intracellular stability probably reflect structural features of the proteins which determine both properties.     

Pyridoxine improves hippocampal cognitive function via increases of serotonin turnover and tyrosine hydroxylase,and its association with CB1 cannabinoid receptor-interacting protein and the CB1 cannabinoid receptor pathway

BackgroundIn the present study, we investigated the effects of pyridoxine on hippocampal functions and changes in protein profiles based on the proteomic approach.MethodsEight-week-old mice received intraperitoneal injections of physiological saline (vehicle) or 350 mg/kg pyridoxine twice a day for 21 days.ResultsPhosphoglycerate mutase 1 was up-regulated, while CB1 cannabinoid receptor-interacting protein 1 (CRIP1) was down-regulated, in the pyridoxine-treated group. Additionally, the serotonin and tyrosine hydroxylase was increased in the hippocampus of the pyridoxine-treated group than in that of the vehicle-treated group. Furthermore, discrimination indices based on the novel object recognition test were significantly higher in the pyridoxine-treated group than in the vehicle-treated group. Administration of CRIP1a siRNA significantly increases the discrimination index as well as cell proliferation and neuroblast differentiation in the dentate gyrus. In addition, the administration of rimonabant, a CB1 cannabinoid receptor antagonist, for 3 weeks significantly decreased the novel object recognition memory, the tyrosine hydroxylase level, the amount of cell proliferation, and neuroblast differentiation in the dentate gyrus. Treatment with pyridoxine significantly increased novel object recognition memory, but slightly ameliorated rimonabant-induced reduction in serotonin, the tyrosine hydroxylase level, the amount of cell proliferation, and neuroblast differentiation in the dentate gyrus.ConclusionThese results suggest that pyridoxine promotes hippocampal functions by increasing serotonin and tyrosine hydroylase immunoreactivity in the hippocampus. This positive effect may be associated with CRIP1a and CB1 cannabinoid receptor function.General significanceVitamin-B₆ enhances hippocampal functions and this is closely associated with CRIP1a and CB1 cannabinoid receptors.     

Induction of tyrosine aminotransferase by pyridoxine in Drosophila hydei

Salivary glands incubated in various concentrations of pyridoxine (Vitamin B₆) show increasing tyrosine aminotransferase (TAT) activity at concentrations up to 10^–5 M and then decreasing activity up to 10^–2 M but in all cases the activity is greater than that of the controls. This increase in activity is demonstrable for up to 6 h, the longest period tested, and is dependent on the synthesis of new mRNA. A similar increase in TAT activity is observed in salivary glands subjected to heat shock. Antibodies prepared against purified tyrosine aminotransferase precipitate a peptide of the same molecular weight (40 KD) as that induced by pyridoxine.     

The effects of salicylate on the activity of rat liver tyrosine–2-oxoglutarate aminotransferase in vitro and in vivo

1. Salicylate, in concentrations of 0.25mm and above, enhances the basal activity of tyrosine–2-oxoglutarate aminotransferase in homogenates of rat liver incubated in the absence of added pyridoxal 5′-phosphate (endogenous activity). The effect is decreased by increasing the concentration of the cofactor. 2. The intraperitoneal administration of sodium salicylate enhances the activity of rat liver tyrosine aminotransferase; the major effect during the first hour being on the enzyme in the absence of added pyridoxal phosphate. Actinomycin D prevents the induction of the enzyme by cortisol and tryptophan. Induction by pyridoxine or salicylate is 50% inhibited by actinomycin D. The effects of the injections of various combinations of cortisol, pyridoxine and salicylate were also studied in the absence or presence of actinomycin D. 3. It is suggested that salicylate induces rat liver tyrosine aminotransferase by displacing its protein-bound cofactor and that a cofactor-type induction of the hepatic enzyme occurs in pyridoxine-treated rats.     

Isoleucine and Threonine Can Prolong Protein and Ribonucleic Acid Synthesis in Pyridoxine-Starved Mutants of Escherichia coli B

Pyridoxineless mutants of Escherichia coli B stopped incorporation of nucleosides into trichloroacetic acid-insoluble material about 40 to 60 min after pyridoxine starvation was initiated, whereas incorporation of amino acids (measured the same way) slowed but did not stop for several hours. Both these incorporations and cell density were increased most effectively by the presence of either threonine or isoleucine. Arginine, glutamate, histidine, methionine, tryptophan, and tyrosine also caused significant but less dramatic increases. Inducibility of beta-galactosidase continued beyond the point where nucleic acids appeared to stop their synthesis, suggesting that messenger ribonucleic acid synthesis continued beyond ribosomal ribonucleic acid synthesis. This inducibility was also increased by isoleucine and threonine. The overall results suggest that the threonine-isoleucine biosynthetic pathway is the most sensitive to starvation for pyridoxine.     

Effect of cofactor depletion on liver tyrosine aminotransferase expression.

Hepatic tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) was partially purified from pyridoxine depleted and control rats and subsequently resolved by electrophoresis on polyacrylamide gels. Enzyme activity was detected histochemically in situ on the gel. Six enzymatically active forms were detected. Cofactor depletion effected further resolution of the enzyme into seven active forms as revealed by the bifurcation of the major active peak.     

Light-dependent generation of reactive oxygen species in cell culture media

Cell culture media (RPMI 1640, Dulbecco’s Minimal Essential Medium and yeast extract-peptone-glucose medium) were found to oxidize dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, and to generate spin adduct of 5,5′-dimethyl-1-pyrroline N-oxide, which indicates formation of reactive oxygen species (ROS). The production of ROS was light dependent. The main component of the media responsible for the generation of ROS was riboflavin, but tryptophan, tyrosine, pyridoxine, and folic acid enhanced the effect of riboflavin. These observations point to exposure of cells to ROS under in vitro culture conditions.     

Formation of β-glucosylpyridoxines in soybean and rice callus

5′-O-(β-Glucosyl)pyridoxine accumulated in soybean calluses and cultured cells grown on a sucrose medium in the presence of pyridoxine. In rice calluses, 5′-O-(β-glucosyl)pyridoxine as the main metabolite was accompanied by small amounts of 4′-O-(β-glucosyl)pyridoxine.     

Vitamin B-6 requirement for irreversible inactivation of rat liver tyrosine aminotransferase.

In vitro inactivation of tyrosine aminotransferase at pH 7.0 did not occur in liver homogenates prepared from vitamin B-6-deficient rats, although it was previously demonstrated that the enzyme was inactivated in liver homogenates from vitamin B-6-adequate rats (R. D. Reynolds and S. D. Thompson, 1974, Arch. Biochem. Biophys.164, 43–51). Addition of 2 mm pyridoxine or pyridoxal-P to the incubated homogenate did not restore the inactivation, but injection of 1 mg of pyridoxine to deficient rats restored full inactivating activity by 12 h. All forms of vitamin B-6 injected restored inactivating activity in vitro. This effect appears to be specific for vitamin B-6, since no restoration of in vitro inactivation of tyrosine aminotransferase was observed following injection of riboflavin, thiamin, niacin, or folic acid. The restoration of inactivating activity in vitro following injection of pyridoxine was not inhibited by repeated injections of puromycin or cycloheximide. Apparently, in vivo protein synthesis is not required for the restoration of the in vitro inactivating activity. However, in vivo inactivation was similar in the vitamin B-6-adequate and -deficient rats. Inactivating activity is present in homogenates of liver and kidney, but not of abdominal muscle, small intestine, heart, testes, whole blood, or erythrocyte ghosts, and is found only in the plasma membrane fraction of liver. Similar to liver, the activity in the kidney homogenate requires the presence of l-cysteine and depends upon the vitamin B-6 status of the animal. Rapid inactivation in the liver occurs between pH 6.75 and 7.75 (final pH), with minimal inactivation above or below this range. No inhibition of inactivation was observed with homogenates incubated in the presence of several protease inhibitors.     

团头鲂幼鱼吡哆醇适宜需求量的研究

试验采用单因素试验设计, 以饲料中吡哆醇浓度为影响因素, 研究了团头鲂幼鱼的适宜吡哆醇需求量。试验共配置了7组等氮等能的半纯化饲料, 其吡哆醇的实际含量分别为0、1.04、1.99、4.07、5.91、7.96和9.22 mg/kg。选用840尾均重:(6.810.17) g团头鲂幼鱼, 随机分为7组, 每组4重复, 每重复30尾鱼, 日投饵3次, 养殖期为8周。结果表明, 当饲料中吡哆醇含量由0升高至5.91 mg/kg时, 团头鲂的增重率、特定生长率、饲料利用率、成活率、蛋白效率比和氮保留率均得到显著改善(P0.05); 当吡哆醇含量进一步升高至9.22 mg/kg时, 蛋白效率比和氮保留率均显著下降(P0.05), 而其他指标则无显著变化(P0.05)。饲料中的吡哆醇含量显著影响团头鲂的肝体比(P0.05)且以5.91 mg/kg组为最低, 但对肥满度和胴体率均无显著影响(P0.05)。当饲料吡哆醇含量由0升高至5.91 mg/kg时, 肝脏谷草转氨酶和谷丙转氨酶活性以及吡哆醇含量均显著升高(P0.05); 当吡哆醇含量进一步升高至9.22 mg/kg时, 三者均无显著变化(P0.05)。以肝脏中的谷丙和谷草转氨酶活性以及吡哆醇含量为评价指标, 拟合折线模型得到团头鲂幼鱼的适宜吡哆醇的需求量为4.175.02 mg/kg。       

Transport and metabolism of pyridoxine in rat liver

Evidence, obtained with in situ perfused rat liver, indicated that pyridoxine is taken up from the perfusate by a non-concentrative process, followed by metabolic trapping. These conclusions were reached on the basis of the fact that at low concentrations (0.125 μM), the ³H of [³H]pyridoxine accumulated against a concentration gradient, but high concentrations (333 μM) of pyridoxine or 4-deoxypyridoxine prevented this apparent concentrative uptake. Under no conditions did the tissue water : perfusate concentration ratio of [³H]pyridoxine exceed unity.The perfused liver rapidly converted the labeled pyridoxine to pyridoxine phosphate, pyridoxal phosphate and pyridoxamine phosphate and released a substantial amount of pyridoxal and some pyridoxal phosphate into the perfusate. Since muscle and erythrocytes failed to oxidize pyridoxine phosphate to pyridoxal phosphate, it is suggested that the liver plays a major role in oxidizing dietary pyridoxine and pyridoxamine as their phosphate esters to supply pyridoxal phosphate which then reaches to other organs chiefly as circulating pyridoxal.     

High-yield recombinant xylanase production by Aspergillus nidulans under pyridoxine limitation

The present study investigated the limitation of pyridoxine on an Aspergillus nidulans culture that produces xylanase B (XynB) as a client enzyme and was unable to synthesize pyridoxine. This technique was used to limit cell growth and divert substrate to product formation for a surface grown culture that could be used in trickle bed reactors. It was observed that growth was limited when pyridoxine was absent, while enzyme production was unaffected. Enzyme production was 1,026 U after 480 h of continuous fermentation, which was similar to a culture that grew on medium with pyridoxine. Furthermore, the present study investigated the growth rate of A. nidulans with pyridoxine in the medium and determined the productivity of XynB production with and without pyridoxine. A maximum growth rate of 0.311/h was observed. The maximum XynB productivity of 21.14 U/g h was achieved when pyridoxine was not added to the medium.     

Possible influence of gonadotrophins on formation in vivo of pyridoxal phosphate

FSH administered to normal rats increased the activity of pyridoxine phosphate oxidase of both liver and kidney and, consequently, pyridoxal phosphate levels in these tissues were elevated. LH administration, on the other hand, decreased the activity of pyridoxine phosphate oxidase, resulting in diminished pyridoxal phosphate level in the tissues. The stimulatory effect of FSH on the activity of liver and kidney pyridoxine phosphate oxidase was not observed in castrated-adrenalectomised rats unless supplemented with cortisone and testosterone, respectively. Puromycin treatment prevented the FSH-induced rise in the activity of liver and kidney pyridoxine phosphate oxidases. It is suggested that FSH stimulates the activity of liver and kidney pyridoxine phosphate oxidase by increasing the synthesis of apoproteins of the enzyme, and the effect of FSH on liver is dependent on the presence of adrenal corticoids while the presence of testosterone is a prerequisite for the FSH to have its effect on kidney pyridoxine phosphate oxidase.     

Physical and kinetic properties of a pyridoxal reductase purified from bakers' yeast

Pyridoxine dehydrogenase (1.1.1.65) (pyridoxal reductase), purified to homogeneity from baker's yeast, is a monomer of Mr approximately 33,000. It catalyzes the reversible oxidation of pyridoxine by NADP to yield pyridoxal and NADPH; equilibrium lies far in the direction of pyridoxine formation (Keq approximately 1.4 X 10(11) l/mol at 25 degrees C). Reduction of pyridoxal occurs most rapidly at pH 6.0-7.0; oxidation of pyridoxine is optimal at pH 8.6. NAD and NADH do not replace NADP and NADPH as substrates; pyridoxine, pyridoxal and pyridoxal 5'-phosphate are the only naturally occurring cosubstrates found. Several other aromatic aldehydes also are reduced, but substrate specificity and other properties of the enzyme distinguish it clearly from other alcohol dehydrogenases or aldehyde reductases. Between pH 6.3 and 7.1 (the intracellular pH of yeast), V/Km with pyridoxal and NADPH as substrates is greater than 600 times that observed with pyridoxine and NADPH as substrates is greater than 600 times that observed with pyridoxine and NADP as substrates. These and other considerations strongly indicate that the dehydrogenase functions in vivo to reduce pyridoxal to pyridoxine, which is the preferred substrate for pyridoxal (pyridoxine) kinase in yeast.     

A Combination of Zinc and Pyridoxine Supplementation to the Diet of Laying Hens Improves Performance and Egg Quality

The objective of this study was to investigate whether zinc, along with pyridoxine, is effective in improving performance and egg quality of laying hens. One hundred and twenty, 28-week-old Hy-Line laying hens were assigned to four treatment groups, 30 hens each. The birds were fed a basal diet or the basal diet supplemented with either 30 mg of zinc/kg of diet, 8 mg of pyridoxine/kg of diet, or 30 mg of zinc plus 8 mg of pyridoxine/kg of diet. Feed conversion (P < 0.01) and egg production (P < 0.01) improved most when both zinc and pyridoxine were supplemented to the diet. Eggshell weights were also greatest (P < 0.01) when the diet was supplemented with both pyridoxine and zinc. Egg-shape index was, however, greatest with zinc-supplemented diet (P < 0.004). Haugh unit was greatest in eggs of hens fed a diet supplemented with both zinc and pyridoxine (P < 0.01). Dietary zinc and pyridoxine supplementations together increased plasma calcium and phosphorous concentrations (P < 0.002). The results of the present study suggested that zinc (30 ppm) and pyridoxine (8 ppm) supplements, when used together, are recommended in terms of a better performance and egg quality in laying hens.     

Isolation of PDX2, a second novel gene in the pyridoxine biosynthesis pathway of eukaryotes, archaebacteria, and a subset of eubacteria

In this paper we describe the isolation of a second gene in the newly identified pyridoxine biosynthesis pathway of archaebacteria, some eubacteria, fungi, and plants. Although pyridoxine biosynthesis has been thoroughly examined in Escherichia coli, recent characterization of the Cercospora nicotianae biosynthesis gene PDX1 led to the discovery that most organisms contain a pyridoxine synthesis gene not found in E. coli. PDX2 was isolated by a degenerate primer strategy based on conserved sequences of a gene specific to PDX1-containing organisms. The role of PDX2 in pyridoxine biosynthesis was confirmed by complementation of two C. nicotianae pyridoxine auxotrophs not mutant in PDX1. Also, targeted gene replacement of PDX2 in C. nicotianae results in pyridoxine auxotrophy. Comparable to PDX1, PDX2 homologues are not found in any of the organisms with homologues to the E. coli pyridoxine genes, but are found in the same archaebacteria, eubacteria, fungi, and plants that contain PDX1 homologues. PDX2 proteins are less well conserved than their PDX1 counterparts but contain several protein motifs that are conserved throughout all PDX2 proteins.     

High-yield production of aryl alcohol oxidase under limited growth conditions in small-scale systems using a mutant <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> strain

Aryl alcohol oxidase (MtGloA) is an enzyme that belongs to the ligninolytic consortium and can play an important role in the bioenergy industry. This study investigated production of an MtGloA client enzyme by a mutant strain of Aspergillus nidulans unable to synthesize its own pyridoxine. Pyridoxine limitation can be used to control cell growth, diverting substrate to protein production. In agitated culture, enzyme production was similar when using media with 1 mg/L and without pyridoxine (26.64 ± 6.14 U/mg mycelia and 26.14 ± 8.39 U/mg mycelia using media with and without pyridoxine, respectively). However, the treatment lacking pyridoxine had to be supplemented with pyridoxine after 156 h of fermentation to sustain continued enzyme production. Use of extremely diluted pyridoxine levels allowed reduced fungal growth while maintaining steady enzyme production. Concentrations of 9 and 13.5 µg/L pyridoxine allowed MtGloA production with a growth rate of only 5% of that observed when using the standard 1 mg/L pyridoxine media.     

Metabolism of tritium-labelled pyridoxine and pyridoxine 5'-phosphate in the central nervous system

Abstract— [³H]Pyridoxine and [³H]pyridoxine 5′-phosphate have been injected into rats and mice. The uptake in brain tissue has been studied by comparing the concentrations of labelled compounds in serum, cerebrospinal fluid and brain tissue. Labelled pyridoxine passes rapidly into brain tissue, whereas the uptake of pyridoxine 5′-phosphate occurs at a much slower rate. Perchloric acid extracts of brain have been fractionated by ion-exchange chromatography and the distribution of isotope between the different forms of the vitamin has been determined at different times after the administration. The time sequence of the metabolic transformation is: pyridoxine+→ pyridoxine 5′-phosphate → pyridoxal 5′-phosphate → pyridoxamine 5′-phosphate. After the initial transformation period about 40 per cent of the isotope is recovered in each of the pyridoxal 5′-phosphate and pyridoxamine 5′-phosphate fractions.     

Vitamin B6 suppresses growth of the feline mammary tumor cell line FRM

Growth of FRM cells was inhibited by the addition of pyridoxine in a dose-dependent manner. Use of 5 mM pyridoxine caused an almost complete arrest of cell growth. Pyridoxal was as effective as pyridoxine, but pyridoxamine showed weak inhibitory action. Electron-microscopic examination of control cells revealed large nuclei and cellular membranes with villi, but, in pyridoxine-treated cells, condensed or degraded nuclei were observed. Many vacuoles and cholesterol crystals were widely distributed inside the cellular membrane of pyridoxine-treated cells. One of the vacuoles was identified as a lipid droplet. The DNA ladder was observed in the pyridoxine-treated cells. It is suggested that pyridoxine treatment of FRM cells causes cytolysis of cells by apoptosis.