Metabolism of pyridoxine in the liver of vitamin B-6-deficient rats.

The metabolism of [6-3H]pyridoxine - HCl was investigated in the liver of vitamin B-6-deficient rats. Rats were made vitamin B-6 deficient by feeding ad libitum for 42 days a diet lacking pyridoxine but otherwise optimal. Animals were each injected intraperitoneally with 33 muCi of [6-3H] pyridoxine - HCl and killed at different time intervals afterwards up to 7 days. Radioactively labeled hepatic B-6 compounds were extracted with acid and chromatographically separated on Dowex-X8 (H+) columns and the percent radioactivity for each vitamin compound was then calculated. Maximal uptake in control and deficient animals was observed 30 and 60 min, respectively, after administration of label. Radioactivity was not retained by the control animals but decreased steadily in a linear fashion after 30 min, reaching a low level after 3 h. On the other hand, vitamin deficient animals accumulated almost twice as much radioactivity in their liver as the controls and retained it through 7 days. In vitamin B-6 deficient animals 93% of the injected radioactivity was metabolized within 2 min at which time pyridoxine 5'-P and pyridoxal 5'-P reached 36 and 44% levels, respectively. Pyridoxine 5'-P dropped to minimal values (3%) within 15 min and remained unchanged for 7 days while pyridoxal 5'-P reached a peak (79%) level at 15 min and then began to drop linearly reaching a plateau (29%) at 5 days. Further, as the level of pyridoxal 5-P was falling, pyridoxamine 5'-P was linearly synthesized reaching a platuau low level (3%). The specific activity level of pyridoxal kinase decreased 3.2 times and that of pyridoxine 5'-phosphate oxidase increased 1.5 times in the state of deficiency. The results presented show that metabolism of [3H]pyridoxine in deficiency is characterized by (a) a delayed, two-fold increase in label uptake as well as an extended label retention period, (b) a rapid pyridoxal 5'-P synthesis, and (c) a continuous synthesis (and accumulation) of pyridoxamine 5'-P which is not utilized or further metabolized.     

PAF-antagonistic bicyclo[3.2.1]octanoid neolignans from leaves of Ocotea macrophylla Kunth. (Lauraceae)

Di-nor-benzofuran neolignan aldehydes, Δ⁷-3,4-methylenedioxy-3′-methoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal A) 1, Δ⁷-3,4,5,3′-tetramethoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal B) 2, and macrophyllin-type bicyclo[3.2.1]octanoid neolignans (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-5′-methoxy-3,4-methylenedioxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol A) 3, (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-3,4,5′-trimethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol B) 4, (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-3,4,5,5′-tetramethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol C) 5, as well as 2′-epi-guianin 6 and (+)-licarin B 7, were isolated and characterized from leaves of Ocotea macrophylla (Lauraceae). The structures and configuration of these compounds were determined by extensive spectroscopic analyses. Inhibition of platelet activating factor (PAF)-induced aggregation of rabbit platelets were tested with neolignans 1–7. Although compound 6 was the most potent PAF-antagonist, compounds 3–5 showed some activity.     

Pyridoxal 5′-phosphate related changes in retention of 1,25-dihydroxy vitamin D-receptor ligands in rat intestinal mucosa cell nuclei

After feeding rats a vitamin B-6-deficient diet, we observed a decrease in pyridoxal 5′-phosphate concentrations in intestinal mucosa cells to 32 and 48% of control in cytoplasm and cell nuclei, respectively. Correlation analysis suggested that there were two pyridoxal 5′-phosphate pools in the nuclei: a “mobile” pool (equivalent to about 5% the concentration of the cytoplasmic pyridoxal 5′-phosphate), and a “stable” pool, which was independent of cytoplasmic fluctuations of pyridoxal 5′-phosphate (about 9 pmol pyridoxal 5′-phosphate/mg DNA). Reduction in pyridoxal 5′-phosphate content in the cells of vitamin B-6-deficient animals was accompanied by a substantial increase in 1,25-dihydroxyvitamin D-receptor ligand concentration in the cell nuclei (76.6 ± 19.7 vs 762 ± 291 fmol/mg DNA, mean ± SEM). The degree of 1,25-dihydrovitamin D accumulation in the nuclei appeared to be an exponential function of the “mobile” nuclear pyridoxal 5′-phosphate concentration. Semilogarithmic transformation of the data yielded a straight line, representing an inverse correlation between the cytoplasm-related nuclear pool of pyridoxal 5′-phosphate and the logarithm of the 1,25-dihydroxyvitamin D concentration in the nuclei (r=−0.95). These data suggest that pyridoxal 5′-phosphate may be related to 1,25-dihydroxyvitamin D retention in the nuclei, possibly through interaction of the pyridoxal 5′-phosphate with the vitamin D receptor protein in the nuclei.     

Transport and metabolism of vitamin B6 in Salmonella typhimurium LT2.

Salmonella typhimurium LT2 concentrates radioactivity intracellularly from [3H]pyridoxal or [3H]pyridoxine up to 25 times the external concentration. After 1 min of uptake intracellular radioactivity is found as phosphorylated vitamin B6. The process is sensitive to temperature and is maximally active at pH 8.1, but under the conditions tested it is insensitive to monovalent cations or metabolic inhibitors, and does not require an exogenous energy source. The Km values for uptake of pyridoxine and pyridoxal are 2.0 x 10(-7) M and 1.2 x 10(-7) M, respectively; [3H]pyridoxamine is not transported. Evidence is presented for an uptake mechanism involving facilitated diffusion followed by trapping by pyridoxal kinase. S. typhimurium also appears to lack a periplasmic binding protein for vitamin B6.     

Gentisic acid conjugates of Medicago truncatula roots

Three phenolic glycosides 5-O-{[5′′-O-E-(4′′′-O-threo-guaiacylglycerol)-feruloyl]-β-apiofuranosyl-(1→2)-β-xylopyranosyl} gentisic acid, 5-O-[(5′′-O-vanilloyl)-β-apiofuranosyl-(1→2)-β-xylopyranosyl] gentisic acid and 1-O-[E-(4′′′-O-threo-guaiacylglycerol)-feruloyl]-3-O-β-galacturonopyranosyl glycerol were isolated and identified from the roots of Medicago truncatula together with four known 5-O-β-xylopyranosyl gentisic acid, vicenin-2, hovetrichoside C and pterosupin identified for the first time in this species. Structural elucidation was carried out on the basis of UV, mass, ¹H and ¹³C NMR spectral data.     

Studies of nucleosides and nucleotides.: LVIII. Deamination of adenosine analogs with calf intestine adenosine deaminase

Several synthetic adeonosine analogs: 8-fluoro-, 8-azido-, 8-iodo-, 8-methylthioadenosine; 8-bromo-2′-deoxyadenosine, 8-bromoxylofuranosyladenine, 5′-benzoly-8-bromoadenosine; 8,2′-S-, 8,2′-O-, 8,2′-NH-, 8,2′-N-CH₃-, 8,3′,-S-, 8,3′-O-, 8,5′-S- and 8,5′O-cycloadenosine; 1-deaza- and 3-deazaadenosine, as well as tubercidine (7-deazaadenosine), were tested as substrates of calf intestine adenosine deaminase.It was found that the adenine base of adenosine should be in the range φ_rmCN = 0–120° (anti to syn-anti) and 8-fluoroadenosine was hydroylzed very slowly. The purine base should have N¹, N³ or N⁷ atoms for the hydrolysis and only 1-deazaadenosine revealed an inhibitory effect toward the hydrolysis of adenosine.5′-OH group should be in the position of S-configuration and must not be substituted.     

Pyridoxine supplementation corrects vitamin B6 deficiency but does not improve inflammation in patients with rheumatoid arthritis

Patients with rheumatoid arthritis have subnormal vitamin B6 status, both quantitatively and functionally. Abnormal vitamin B6 status in rheumatoid arthritis has been associated with spontaneous tumor necrosis factor (TNF)-α production and markers of inflammation, including C-reactive protein and erythrocyte sedimentation rate. Impaired vitamin B6 status could be a result of inflammation, and these patients may have higher demand for vitamin B6. The aim of this study was to determine if daily supplementation with 50 mg of pyridoxine for 30 days can correct the static and/or the functional abnormalities of vitamin B6 status seen in patients with rheumatoid arthritis, and further investigate if pyridoxine supplementation has any effects on the pro-inflammatory cytokine TNF-α or IL-6 production of arthritis. This was a double-blinded, placebo-controlled study involving patients with rheumatoid arthritis with plasma pyridoxal 5'-phosphate below the 25th percentile of the Framingham Heart Cohort Study. Vitamin B6 status was assessed via plasma and erythrocyte pyridoxal 5'-phosphate concentrations, the erythrocyte aspartate aminotransferase activity coefficient (αEAST), net homocysteine increase in response to a methionine load test (ΔtHcy), and 24 h urinary xanthurenic acid (XA) excretion in response to a tryptophan load test. Urinary 4-pyridoxic acid (4-PA) was measured to examine the impact of pyridoxine treatment on vitamin B6 excretion in these patients. Pro-inflammatory cytokine (TNF-α and IL-6) production, C-reactive protein levels and the erythrocyte sedimentation rate before and after supplementation were also examined. Pyridoxine supplementation significantly improved plasma and erythrocyte pyridoxal 5'-phosphate concentrations, erythrocyte αEAST, urinary 4-PA, and XA excretion. These improvements were apparent regardless of baseline B6 levels. Pyridoxine supplementation also showed a trend (p < 0.09) towards a reduction in post-methionine load ΔtHcy. Supplementation did not affect pro-inflammatory cytokine production. Although pyridoxine supplementation did not suppress pro-inflammatory cytokine production in patients with rheumatoid arthritis, the suboptimal vitamin B6 status seen in rheumatoid arthritis can be corrected by 50 mg pyridoxine supplementation for 30 days. Data from the present study suggest that patients with rheumatoid arthritis may have higher requirements for vitamin B6 than those in a normal healthy population.     

Evidence for the role of vitamin C-6 as a cofactor of lysyl oxidase.

At 24 h after injection of 16-day chick embryos with [C-3H]pyridoxine hydrochloride, some of this label appears in the epiphysial cartilage. Over 35% of this radioactivity appears in the form of [G-3H]pyridoxal and a further 30% as other vitamin B-6 compounds. Partial purification of lysyl oxidase from the labelled epiphysial cartilage reveals a single peak of radioactivity coinciding with a single peak of enzyme activity. On dialysis against phosphate-buffered saline, 75% of this radioactivity is found to be non-diffusible. After incubation with isonicotinic acid hydrazide, a carbonyl reagent that appears to inhibit lysyl oxidase both in vivo and in vitro, a further 70% of the radioactivity is lost, with a roughly corresponding loss of enzyme activity. It is suggested that a form of vitamin B-6 is required as a cofactor of lysyl oxidase, and that this may have important implications in terms of connective-tissue metabolism.     

Simultaneous determination of stable isotopically labelled l-histidine and urocanic acid in human plasma by stable isotope dilution mass spectrometry

A capillary gas chromatographic—mass spectrometric method for the simultaneous determination of stable isotopically labelled l-histidine (l-[3,3-²H₂,1′,3′-¹⁵N₂]histidine, l-His-[M + 4]) and urocanic acid ([3-²H,1′,3′-¹⁵N₂]urocanic acid, UA-[M + 3]) in human plasma was developed using dl-[2,3,3,5′-²H₄,2′-¹³C,1′,3′-¹⁵N₂]histidine (dl-His-[M + 7]) and [2,3,5′-²H₃,2′-¹³C,1′,3′-¹⁵N₂]urocanic acid (UA-[M + 6]) as internal standards. l-Histidine and urocanic acid were derivatized to ^αN-(trifluoroacetyl)-^imN-(ethoxycarbonyl)-l-histidine n-butyl ester and ^imN-(ethoxycarbonyl)urocanic acid n-butyl ester. Quantification was carried out by selected ion monitoring of the molecular ions of the respective derivatives of l-His-[M + 4], dl-His-[M + 7], UA-[M + 3] and UA-[M + 6]. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring plasma concentrations of l-His-[M + 4] and UA-[M + 3] following administration of trace amounts of l-His-[M + 4] to humans.     

P2X<Subscript>7</Subscript> Receptor Stimulation of Membrane Internalization in a Thyrocyte Cell Line

Using fluorescent membrane markers, we have previously shown that extracellular ATP stimulates both exocytosis and membrane internalization in the Fisher rat thyroid cell line FRTL. In this study, we examine the actions of ATP using whole-cell recording conditions that favor stimulation of membrane internalization. ATP stimulation of the P2X₇ receptor activated a reversible, Ca²⁺-permeable, cation conductance that slowly increased in size without changes in ion selectivity. ATP also induced a delayed irreversible decrease in cell capacitance (C_m) that was equivalent to an 8% decrease in membrane surface area. Addition of guanosine 5′-0-2-thiodiphosphate to the pipette solution inhibited the ATP-induced decrease in C_m without affecting channel activation. The effects of ATP on membrane conductance were mimicked by 2′,3′-O-(4-benzoylbenzoyl)-ATP, but not by UTP, adenosine, or 2-methylthio-ATP, and were inhibited by pyridoxal phosphate-6-azophenyl-2′4′-disulfonic acid, adenosine 5′-triphosphate-2′3′-dialdehyde, and Cu²⁺. The capacitance decrease persisted in Na⁺-, Ca²⁺- and Cl⁻-free external saline or with Ca²⁺-free pipette solution. It is concluded that ATP activation of the inotropic P2X₇ receptor stimulates membrane internalization by a mechanism that involves intracellular GTP, but does not require internal Ca²⁺ or influx of Na⁺ or Ca²⁺ through the receptor-gated channel.     

Vitamin A metabolism in rats chronically treated with 3,3′,4,4′,5,′-hexabromobiphenyl

Chronic dietary administration of 3,3′,4,4′,5,5′-hexabromobiphenyl (HBB), 1 mg/kg diet, caused a decrease in retinol (20-fold) and retinyl esters (23-fold) in the livers of female rats, but resulted in a 6.4-fold increase in retinol and 7.4-fold increase in retinyl esters in the kidneys. Liver acyl-CoA: retinol acyltransferase and retinyl palmitate hydrolase activities were reduced while serum concentration of retinol was unaffected by HBB feeding. Metabolism of a physiological dose of [11-³H]retinyl acetate (10 μg), was examined in rats fed either vitamin A-adequate diet, or marginal amounts of vitamin A, or vitamin A-adequate diet containing HBB. A 13-fold greater amount of the administered vitamin A was found in kidneys of HBB-treated rats. In rats fed adequate or low amounts of vitamin A, kidney radioactivity was primarily in the retinol fraction, while in HBB-fed rats the radioactivity was associated mostly with retinyl esters. Fecal and urinary excretion of radioactivity was greatly increased in HBB-treated rats. Chronic HBB feeding results in a loss of ability of liver to store vitamin A, and severely alters the uptake and metabolism of vitamin A in the kidneys. We conclude that HBB causes major disturbances in the regulation of vitamin A metabolism.     

ATP increases chemoattractant induced cyclic GMP accumulation in Dictyostelium discoideum

Changes in guanosine cyclic 3′,5′-monophosphate associated with adenosine cyclic 3′,5′-monophosphate and folic acid addition in the presence of ATP have been examined in Dictyostelium discoideum. Preincubation with 1 mM ATP had no effect on the basal cyclic GMP level but increased the cycli GMP accumulation in response to cylci AMP (5·10⁻⁸ M) or folic acid (5·10⁻⁶ M) 40–50%. ATP could not be replaced by ADP of 5′-adenylyliminodiphosphate. Because ATP has no effect on cyclic AMP receptor binding these results indicate that structural membrane alterations (e.g. membrane phosphorylation) may control the transduction of a chemotactic signal.     

Freezing Resistance and Cryopreservation of Cell Strains of Rhaponticum carthamoides and Thalictrum minus

Effects of long-term (few months) culturing and short pregrowth (up to 7 days prior to deep freezing) in the presence of mannitol (5–6%), ABA (5.0–7.5 × 10^–5 M), or both substances on cryogenic resistance of leusea (Rhaponticum carthamoides, strains Rhs-2 and Rhs-8) and meadow rue (Thalictrum minus L., strain B-233) cell suspension cultures were studied. Cryoprotective capacities of 48 solutions were studied at slow (0.33°C/min) freezing to the temperature of liquid nitrogen with an automatic initiation of crystallization. Cells were stored in liquid nitrogen for several days or months. ABA had a cryoprotective effect, provided that subculturing intervals were 12–14 days. At more frequent subculturing (every 7 days), pregrowth on ABA-containing medium did not increase survival percentage compared to pregrowth with mannitol. Successful cryopreservation of these strains has been achieved due to strict standardization of 7-day subculturing regime, pregrowth in the presence of mannitol prior to freezing, and cryopreservation with dimethyl sulfoxide, sucrose, trehalose, and glycerol. The cell survival rates after thawing were 60% (Rhs-8), 80% (Rhs-2), and 70% (B-233). The cell growth resumed on the third to seventh day. The growth indices and protoberberine synthesizing activity in B-233 strain reached their control values at the ninth subculturing after a post-thaw recovery.     

Preparation and characterization of several new immobilized derivatives of pyridoxal 5′-phosphate

Two types of new Sepharose-bound pyridoxal 5′-phosphate, N-immobilized and 3-0-immobilized pyridoxal 5′-phosphate analogues, were prepared by reacting pyridoxal 5′-phosphate with a bromoacetyl derivative of Sepharose 4B in dimethylformamide (50% v/v) and in potassium phosphate buffer (pH 6.0) for approx. 70 h at room temperature in the dark, respectively. The properties of these immobilized pyridoxal 5′-phosphate derivatives including their catalytic activities in the non-enzymatic cleavage reaction of tryptophan were studied in comparison with those of the 6-immobilized pyridoxal 5′-phosphate analogue reported previously by the present authors. The usefulness of these pyridoxal 5′-phosphate analogues in the preparation of immobilized tryptophanase was demonstrated.     

Pyridoxine induced puffing (II-48 C) and synthesis of a 40 KD protein in Drosophila hydei salivary glands

A specific heat shock puff (II-48 C) has been induced in isolated salivary glands of D. hydei by pyridoxine (vitamin B₆) at concentrations from 10^–7 to 10^–2 M. The puff begins to form within 5 min and continues to increase in size up to 2 h. Under conditions of puff induction the -amanitin sensitive synthesis of a 40 KD protein is induced.     

Effect of tryptophan metabolites on the activities of rat liver pyridoxal kinase and pyridoxamine 5-phosphate oxidase in vitro.

Pyridoxal kinase was purified 4760-fold from rat liver. The Km values for pyridoxine and pyridoxal were 120 and 190 microM respectively, and pyridoxine showed substrate inhibition at above 200 microM. Pyridoxamine 5-phosphate oxidase was also purified 2030-fold from rat liver, and its Km values for pyridoxine 5-phosphate and pyridoxamine 5-phosphate were 0.92 and 1.0 microM respectively. Pyridoxine 5-phosphate gave a maximum velocity that was 5.6-fold greater than with pyridoxamine 5-phosphate and showed strong substrate inhibition at above 6 microM. Among the tryptophan metabolites, picolinate, xanthurenate, quinolinate, tryptamine and 5-hydroxytryptamine inhibited pyridoxal kinase. However, pyridoxamine 5-phosphate oxidase could not be inhibited by tryptophan metabolites, and on the contrary it was activated by 3-hydroxykynurenine and 3-hydroxyanthranilate. Regarding the metabolism of vitamin B-6 in the liver, the effects of tryptophan metabolites that were accumulated in vitamin B-6-deficient rats after tryptophan injection were discussed.     

DNA sequence dependence of guanine-O alkylation by the N-nitroso carcinogens N-methyl- and N-ethyl-N-nitrosourea

After intracellular in vitro exposure to the mutagenic and carcinogenic N-nitroso compounds N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), respectively, the average relative amounts of the premutational lesion O⁶-alkylguanine represent about 6% and 8% of all alkylation products formed in genomic DNA. At the level of individual DNA molecules gunine-O⁶ alkylation does nor occur at random; rather, the probability of a substitution reaction at the nucleophilic O⁶ atom is influenced by nucleotide sequence, DNA conformation, and chromatin structure. In the present study, 5 different double-stranded polydeoxynucleotides and 15 double-stranded oligodeoxynucleotides (24-mers) were reacted with MeNU or EtNU in vitro under standardized conditions. Using a competitive radioimmunoassay in conjunction with an anti-(O⁶-2′-deoxyguanosine) monoclonal antibody, the frequency of guanine-O⁶ alkylation was found to be strongly dependent on the nature of the nucleotides flanking guanine on the 5t́ and 3′ sides. Thus, a 5′ neighboring guanine, followed by 5t́ adenine and 5′ cytosine, provided an up to 10-fold more ‘permissive’ condition for O⁶-alkylation of the central guanine than a 5′ thymine (with a 5-methylcytocine in the 5′ position being only slightly less inhibitory). Thymine and cytosine were more ‘permissive’ when placed 3′ in comparison with their affects in the 5′ flanking position.     

Metabolomic Analysis Reveals Extended Metabolic Consequences of Marginal Vitamin B-6 Deficiency in Healthy Human Subjects

Marginal deficiency of vitamin B-6 is common among segments of the population worldwide. Because pyridoxal 5′-phosphate (PLP) serves as a coenzyme in the metabolism of amino acids, carbohydrates, organic acids, and neurotransmitters, as well as in aspects of one-carbon metabolism, vitamin B-6 deficiency could have many effects. Healthy men and women (age: 20-40 y; n = 23) were fed a 2-day controlled, nutritionally adequate diet followed by a 28-day low-vitamin B-6 diet (<0.5 mg/d) to induce marginal deficiency, as reflected by a decline of plasma PLP from 52.6±14.1 (mean ± SD) to 21.5±4.6 nmol/L (P<0.0001) and increased cystathionine from 131±65 to 199±56 nmol/L (P<0.001). Fasting plasma samples obtained before and after vitamin B6 restriction were analyzed by ¹H-NMR with and without filtration and by targeted quantitative analysis by mass spectrometry (MS). Multilevel partial least squares-discriminant analysis and S-plots of NMR spectra showed that NMR is effective in classifying samples according to vitamin B-6 status and identified discriminating features. NMR spectral features of selected metabolites indicated that vitamin B-6 restriction significantly increased the ratios of glutamine/glutamate and 2-oxoglutarate/glutamate (P<0.001) and tended to increase concentrations of acetate, pyruvate, and trimethylamine-N-oxide (adjusted P<0.05). Tandem MS showed significantly greater plasma proline after vitamin B-6 restriction (adjusted P<0.05), but there were no effects on the profile of 14 other amino acids and 45 acylcarnitines. These findings demonstrate that marginal vitamin B-6 deficiency has widespread metabolic perturbations and illustrate the utility of metabolomics in evaluating complex effects of altered vitamin B-6 intake.