Vitamin B12 Analogues Research Articles

A new antioxidative vitamin B6 analogue modulates pathophysiological cell proliferation and damage

The new large scale synthesis of the yellow colored vitamin B6 analogue 5′-O-phosphono-pyridoxylidenerhodanine (2) (B6PR) leads to oligohydrates of its monosodium salt (4). The light-red hemiheptadecahydrate (812 hydrate) (4a) was crystallized and its three-dimensional structure determined by X-ray crystallography. Special nucleotide and protein interaction properties together with scavenging antioxidative function are combined in this simple water-soluble vitamin B6 analogues B6PR. High (mM) concentrations were untoxic to ‘healthy’ not affected cells and primary tissues. Complexation of ions (e.g. Ca2+, Fe2+, and Zn2+), modulation of nitric oxide synthases (NOS I-III), nitric oxide (NO) metabolism, and reactive oxygen species (ROS) was found. Special cytoprotecting, immunomodulating, stimulating and inhibiting activities were observed in vitro, not in comparison with some natural and synthetic pyridoxines. Low B6PR suppressed proliferation, high induced selective cell death of some cancer cell lines. Low B6PR protected HIV-1-infected CD4+ HUT 78 cells against HIV-1-mediated destruction (complete inhibition of HIV-1-induced syncytia formation and cell death) and reduced p24 level. Autoreactive S100β-specific T cells of Lewis rat, a model of multiple sclerosis, could be influenced. Oxidative damage and age, acquired and inherited disease related pathophysiological disorders can be treated by this new cytopathology-selective versatile acting B6PR. ©

Development of novel 19F NMR pH indicators: synthesis and evaluation of a series of fluorinated vitamin B6 analogues

We have synthesized a series of novel fluorinated vitamin B6 analogues (6-fluoropyridoxol derivatives) as potential 19F NMR pH indicators for use in vivo. Modifications included addition of aldehyde, carboxyl or aminomethyl groups at the 4- or 5-ring position, and examination of a trifluoromethyl moiety as an internal chemical shift standard. The variation in chemical shift with respect to acid–base titration showed pKa values in the range 7.05–9.5 with a chemical shift sensitivity in the range 7.4–12ppm. Several of the molecules readily cross cell membranes providing estimates of both intra- and extra-cellular pH in whole blood. 6-Fluoropyridoxamine (6-FPAM) exhibits a pKa=7.05, which is closer to normal physiological pH than the parent molecule 6-fluoropyridoxol (6-FPOL) (pKa=8.2), and should thus, be useful for precise and accurate measurements of pH in vivo. Enhanced spectral resolution for 6-FPAM over 6-FPOL is demonstrated in whole blood and the perfused rat heart.

Comparison of a Microbiological Assay and a Fully Automated Chemiluminescent System for the Determination of Vitamin B12 in Food

A fully automated chemiluminescence analyzer for the determination of vitamin B12 in serum has been commercialized and clinically used. To determine the applicability of this apparatus in food analysis, vitamin B12 was assayed in foods by the chemiluminescent method, which was compared with a microbiological method. In shellfishes and spirulina, the values determined by the microbiological method were ∼6−8-fold greater than the values determined by the chemiluminescence method, although there was good similarity between the values by the two methods in other foods. Except for the shellfishes and spirulina, which contained substantial amounts of vitamin B12-substitutive compounds or inactive vitamin B12 analogues (or both), the observed correlation coefficient between the methods in the foods tested was excellent (r = 0.99, y = 1.2x − 1.1, n = 9). The chemiluminescence method was suitable for the determination of vitamin B12 in foods as well as in serum and was simpler (fully automated) and more rapid (180...

Comparison of methods for determination of vitamin B12 in microbial material

Three different methods for the measurement of vitamin B12 were compared: two spectrophotometric methods and an HPLC one. When the pure vitamin was used, the results obtained using all three methods were similar, but when samples from microbial material were used, the results were different. The HPLC method could distinguish the true vitamin B12 from the different vitamin B12 analogues whereas the spectrophotometric methods could not.

Central administration of vitamin B12 aggravates cataplexy in canine narcolepsy.

Experimental evidence in canine narcolepsy suggests that central cholinergic systems are critically involved in the regulation of cataplexy, an abnormal manifestation of REM sleep atonia. In the current study, we found that intracerebroventricular perfusion of methyl-B12, (10(-5)-10(-2) M), significantly aggravated cataplexy and enhanced REM sleep in narcoleptic dogs. Choline, a direct precursor of acetylcholine, was also found to aggravate cataplexy, while cyano-B12, a vitamin B12 analog without methyl donating abilities, had no effect on cataplexy. Since both methyl-B12 and choline are reported to enhance acetylcholine synthesis, enhancement of the biosynthesis of acetylcholine may be involved in the effects observed in canine narcolepsy. Our results suggest that central administration of methyl-B12 has the potential to modulate both normal and pathological REM sleep.

Abnormal cyanide metabolism in uraemic patients.

We previously investigated the factors involved in uraemic neuropathy in patients undergoing regular haemodialysis and found a significant relationship between the severity of vibration sensation impairment and the patients' smoking habits. The administration of methylcobalamin markedly improved the severity of uraemic neuropathy in terms of vibration perception thresholds. We presumed that abnormal cyanide metabolism is involved in the development of uraemic neuropathy. Serum levels of thiocyanate (SCN-), the detoxication product of cyanide, were determined in 12 patients with preterminal chronic renal failure (PCRF), 30 patients undergoing regular haemodialysis (HD patients), and 13 healthy volunteers as a control group. Nine of the 30 HD patients were smokers. In addition, in 10 HD patients without smoking habits and 10 non-smoking healthy volunteers, the proportion of each vitamin B12 analogue in total vitamin B12 was estimated. The mean serum SCN- level of the 12 PCRF patients (5.1 +/- 1.5 micrograms/ml) was significantly higher than that of the control (2.8 +/- 0.9 micrograms/ml) (P < 0.01). The mean SCN- level before haemodialysis in the 21 non-smoking HD patients was identical to that in the PCRF group, whereas the level in the nine smoking HD patients (7.2 +/- 1.8 micrograms/ml) significantly higher than that in the non-smoking subgroup (P < 0.01). In 16 HD patients with methylcobalamin treatment, serum SCN- levels were lower than in those without methylcobalamin treatment (4.5 +/- 0.5 micrograms/ml in non-smoking subgroup, P < 0.05). And in the methylcobalamin-treated subgroup (n = 5), the proportion of each vitamin B12 analogue in total vitamin B12 was normal. In the untreated subgroup (n = 5), the proportion of cyanocobalamin fraction (10.5 +/- 2.6%) was as high as the level in Leber's disease patients, while the proportion of methylcobalamin fraction was low. And the serum cyanocobalamin level was higher in the treated subgroup. In uraemic patients, cyanide detoxication capability is impaired because of a reduced SCN- clearance, and increased cyanocobalamin synthesis indicates elevation of cyanide pool, which would be related to the development of uraemic neuropathy. Methylcobalamin was considered to be utilized in cyanide detoxication process via cyanocobalamin synthesis.

Synthesis of 4-aza-5,6-dimethylbenzimidazole and biosynthetic preparation of 4- and 7-aza-5,6-dimethylbenzimidazolylcobamide.

We report on the preparation of 4-aza-5,6-dimethylbenzimidazolylcobamide and 5,6-dimethyl-7-azabenzimidazolylcobamide. These vitamin B12-analogs were required as reference compounds for comparison with a corrinoid previously isolated in small amounts for Eubacterium limosum grown in the presence of 4(5)-aminoimidazole. 4(7)-Aza-5,6-dimethylbenzimidazole was synthesized from N-1-benzyl-4-nitroimidazole which was reduced to N-1-benzyl-4-aminoimidazole and condensed with 1-dimethylamino-2-methylbutan-3-one to yield N-1-benzyl-4-aza-5,6-dimethylbenzimidazole. The benzyl group of this compound was split off by catalytic hydrogenation to form 4(7)-aza-5,6-dimethylbenzimidazole. 4(7)-Aza-5,6-dimethylbenzimidazole was transformed by a growing culture of Propionibacterium shermanii into 4-aza-5,6-dimethylbenzimidazolylcobamide and 5,6-dimethyl-7-azabenzimidazolylcobamide. Both vitamin B12-analogs were almost as active as Vitamin B12 in a growth test with the vitamin B12-dependent Escherichia coli-mutant DSM 4261.

Identification of vitamin B12 analogues by liquid chromatography with electrothermal atomic absorption detection

The coupling of liquid chromatography and electrothermal atomic absorption spectrometry (LC-ETAAS) lowers the detection limit for identification of vitamin B12 analogues. Cobalamins and aqueous cobalt (II) were separated by reversed-phase liquid chromatography using a linear gradient: 26∶74 (v/v) methanol:0.05 M phosphate buffer (pH 4.2) to 50∶50 mixture over 8 min. The vitamins were quantitatively determined in the column effluent by measuring total cobalt by ETAAS. The analysis of meat and liver extracts by LC-ETAAS showed that the matrix did not interfere with the determination of cobalt. Hence, recoveries of cobalt in spiked meat and liver samples were satisfactory.

Circadian time‐dependent modulation of sleep and brain temperature (Tbr) by methylcobalamine and resultant prolongation of Tbr freerunning period in rats

Abstract Vitamin B12 (VB12) is known as a putative modulator for the mammalian circadian clock. In an attempt to analyze the mechanism by which VB12 modulates the clock system, circadian time (CT) dependency of two VB12 analogs, methylcobalamin (methyl‐B12) and cyanocobalamin (cyano‐B12), was investigated by means of recording sleep‐wake and brain temperature (Tbr) rhythms over 3 weeks in rats freerunning under continuous dim illumination (dim LL). A 3‐h intracerebroventricular infusion of methyl‐B12 (30 nmol) was‐done at three different CT phases such as CT 12–15 (starting at activity onset), CT 18–21 (starting mid at active period) and CT 0–3 (starting at activity offset). A significant enhancement of both non‐rapid‐eye‐movement sleep and rapid‐eye‐movement sleep in phase with a significant reduction of Tbr was acutely induced during the late subjective night by the administration of methyl‐B12 at CT 12–15 and at CT 18–21. In contrast, sleep and Tbr were little affected by the infusion at CT 0–3. Subseq...

Nutritional interdependence betweenThermoanaerobacter thermohydrosulfuricus andClostridium thermocellum

Thermoanaerobacter thermohydrosulfuricus strain YM3 andClostridium thermocellum strain YM4, obtained originally as a stable coculture, required yeast extract to grow separately. Cell-free broths ofT. thermohydrosulfuricus strain YM3 andC. thermocellum strain YM4 monocultures replaced yeast extract in supporting the growth of strains YM4 and YM3, respectively.T. thermohydrosulfuricus strain YM3 produced vitamin B6, B12 analog(s),p-aminobenzoic acid and folic acid, which were required byC. thermocellum strain, YM4. Likewise, strain YM4 produced niacin-active compound(s), thiamine, and methionine required by strain YM3.

Biosynthesis of vitamin B12 in anaerobic bacteria. Transformation of 5-hydroxybenzimidazole and 5-hydroxy-6-methylbenzimidazole into 5,6-dimethylbenzimidazole in Eubacterium limosum.

Eubacterium limosum transformed [2-13C]5-hydroxybenzimidazole not only into [2-13C]5-hydroxybenzimidazolylcobamide, but also into [2-13C]5-methoxy-6-methylbenzimidazolylcobamide and into [2-13C]5,6-dimethylbenzimidazolylcobamide (vitamin B12). [2-13C]5-Hydroxy-6-methyl-benzimidazole was used by this bacterium to form [2-13C]5-hydroxy-6-methylbenzimidazolyl-cobamide, [2-13C]5-methoxy-6-methylbenzimidazolylcobamide and [2-13C]5,6-dimethylbenzimidazolylcobamide. The 1H-NMR spectrum of the 5,6-dimethylbenzimidazole isolated from the 13C-labeled vitamin B12 preparations revealed that the externally added bases had been transformed into the vitamin B12 base almost without dilution of the label. This suggests that 5-hydroxybenzimidazole and 5-hydroxy-6-methylbenzimidazole are precursors of 5,6-dimethylbenzimidazole. On the basis of these results, a hypothetical scheme for the biosynthesis of 5,6-dimethylbenzimidazole via 5-hydroxybenzimidazole and 5-hydroxy-6-methylbenzimidazole is discussed. This scheme can also explain the formation of the other benzimidazole bases found in natural vitamin B12 analogs.

Protective effects of a vitamin B12 analog, methylcobalamin, against glutamate cytotoxicity in cultured cortical neurons.

The effects of methylcobalamin, a vitamin B12 analog, on glutamate-induced neurotoxicity were examined using cultured rat cortical neurons. Cell viability was markedly reduced by a brief exposure to glutamate followed by incubation with glutamate-free medium for 1 h. Glutamate cytotoxicity was prevented when the cultures were maintained in methylcobalamin-containing medium. Glutamate cytotoxicity was also prevented by chronic exposure to S-adenosylmethionine, which is formed in the metabolic pathway of methylcobalamin. Chronic exposure to methylcobalamin and S-adenosylmethionine also inhibited the cytotoxicity induced by N-methyl-D-aspartate or sodium nitroprusside that releases nitric oxide. In cultures maintained in a standard medium, glutamate cytotoxicity was not affected by adding methylcobalamin to the glutamate-containing medium. In contrast, acute exposure to MK-801, a NMDA receptor antagonist, prevented glutamate cytotoxicity. These results indicate that chronic exposure to methylcobalamin protects cortical neurons against NMDA receptor-mediated glutamate cytotoxicity.

Existence of vitamin B12 analogs in biological samples: A reality

Due to the fact that the existence of vitamin B12 analogs in biological materials has been much debated, we realized on plasma, bile, and feces series of experiences to demostrate that analogs assayed by radioisotopic method in these biological media using intrinsic factor and haptocorrin as binders could not be extraction products or dosage artifacts. To this purpose, exposure to light was used to transform all corrinoids (true B12 + analogs) to their hydroxo forms. After that all corrinoids were transformed with KCN by cyanilation to their cyano forms. This permitted us to separate corrinoids on high performance liquid chromatography into only two peaks. These peaks will be subsequently identified by radioisotopic dilution assay as cyanocobalamin α and/or β cyanocobinamides. Because the binding ability of an intrinsic factor to corrinoids is limited to its cobalamin forms, our method enabled us to emphasize the existence in biological materials of corrinoids that only recognize haptocorrin (analogs of vitamin B12) and not intrinsic factors.

Assignment of 15N‐NMR resonances of vitamin B12 analogues by 2D‐[15N, 1H] long‐range correlation: Fully [15N]‐labelled Co‐β‐cyano‐5‐hydroxybenzimidazoylcobamide (factor III) and derivatives

AbstractThe 15N‐NMR spectra of vitamin B12 analogues obtained in fully 15N‐labelled form have been measured by direct and inverse (15N, 1H) correlated spectroscopy. All resonances, except those of the NH2 groups, have been assigned to individual N‐atoms. The influences on δ (N) are analyzed and discussed which are caused by changing the α‐face ligand from CN to H2O or CH3 and by switching the β‐face ligand from the base‐on to the base‐off state. An implication of the correct resonance assignment on biosynthetic pathways is demonstrated.

Fluorinated Vitamin B 12 Analogs Are Cofactors of Corrinoid-Dependent Enzymes: a 19 F-Labeled Nuclear Magnetic Resonance Probe for Identifying Corrinoid-Protein Interactions

The homoacetogenic bacterium Sporomusa ovata synthesized the vitamin B(12) analog phenolyl cobamide or 4-fluorophenolyl cobamide when the methanol medium of growing cells was supplemented with 10 mM phenol or 5 mM 4-fluorophenol. Phenol and, presumably, 4-fluorophenol were specifically incorporated into these cobamides, since phenol was not metabolized significantly into amino acids or into acetic acid, the product of the catabolism. The phenol-containing cobamides contributed up to 90% of the protein-bound cobamides of the 1,300 to 1,900 nmol of corrinoid per g of dry cell material formed. Fluorine-19 nuclear magnetic resonance spectroscopy of 4-fluorophenolyl cobamide exhibited a resonance near 30 ppm. An additional signal emerged at 25 ppm when 4-fluorophenolyl cobamide was investigated as the cofactor of a corrinoid-dependent protein. The two resonances indicated distinct cofactor arrangements within the protein's active site. A 5-ppm high-field shift change suggested van der Waal's interactions between the fluorinated nucleotide of the cofactor and adjacent amino acid residues of the enzyme. Similarly, Propionibacterium freudenreichii and Methanobacterium thermoautotrophicum synthesized 5-fluorobenzimidazolyl cobamide. The human corrinoid binders intrinsic factor, transcobalamin, and haptocorrin recognized this corrinoid like vitamin B(12). Hence, it is possible to use F-labeled nuclear magnetic resonance spectroscopy for analyses of protein-bound cobamides.

Effects of Vitamin B12Analogues with Alternations in the Side Chains of the Corrin Ring on Urinary Methylmalonate Excretion in Vitamin B12-Deficient Rats

To study how much the side chains of the corrin ring of vitamin B12 are involved in the physiological roles of the vitamin, five vitamin B12 analogues (cyanocobalamin-b-monocarboxylate, cyanocobalamin-d-monocarboxylate, cyanocobalamin-e-monocarboxylate, cyano-13-epicobalamin, and cyanocobalamin(c-lactam)) with alternations in the side chains were synthesized chemically and then administered orally and intravenously to vitamin B12-deficient rats. Male rats fed a vitamin B12-deficient diet for 11 wk developed a severe vitamin B12 deficiency with a high urinary methylmalonate excretion (223.8 ± 136.2 μmol/d) and ~97% (1.2±0.7ng/g tissue) lower hepatic vitamin B12 content. Oral and intravenous administration of cyanocobalamin-b-,-d-, and -e-monocarboxylates and cyano-13-epicobalamin could not improve the severe vitamin B12-deficient status of the rats, indicating that the b-, d-, and e-propionamide side chains of the corrin ring of vitamin B12 are important in the absorption, transport, and function of the vi...

Development and application of an alpha-face-specific radioimmunoassay for vitamin B12.

The first development of an alpha-face-specific radioimmunoassay for vitamin B12 is described. Sheep, fed a cobalt-deficient diet, and immunized with a conjugate between Co-beta carboxypropyl cobalamin and keyhole limpet hemocyanin, were used to produce antisera. The antisera crossreacted with Co-beta derivatives of vitamin B12, but did not crossreact with the alpha-face vitamin B12 analog cobinamide. The antisera were used to develop a sensitive and reproducible radioimmunoassay that was free from contamination with the nonspecific vitamin B12 binding protein, R-protein. Both the radioimmunoassay and measurements of plasma concentrations of methylmalonic acid were applied to the diagnosis of cobalt/vitamin B12 deficiency in sheep. The assay correlated well with a commercially available radioassay and did not falsely detect normal vitamin B12 concentration in plasma samples containing elevated concentrations of methylmalonic acid.

Irreversible inhibition of thymidylate synthase by pyridoxine (B6) analogues.

Three vitamin B6 analogues have been synthesized and tested as inhibitors of thymidylate synthase. The compounds are: 4',5'-dichloro-, 4',5'-dibromo- and 4',5'-diiodo-pyridoxine. All three analogues inhibited the enzyme irreversibly. The kinetic data for the chloro- and bromo-analogues showed that a limiting rate of inhibition is approached as the inhibitor concentration is increased, which indicates that a reversible enzyme:inhibitor affinity complex is formed prior to the irreversible reaction. 4',5'-Dibromo-pyridoxine exhibited a greater binding affinity (lower Ki) for thymidylate synthase than 4',5'-dichloro-pyridoxine, and it also reacted faster to irreversibly inhibit the enzyme. The presence of the substrate dUMP (10 microM) completely protected thymidylate synthase from inhibition. These data suggest that the halogenated vitamin B6 analogues are active site-directed inhibitors of thymidylate synthase, which first bind reversibly to the catalytic site and then react irreversibly with the enzyme.

Increased hepatic mitochondrial capacity in rats with hydroxy-cobalamin[c-lactam]-induced methylmalonic aciduria.

Treatment of rats with the vitamin B12 analogue hydroxy-cobalamin[c-lactam] (HCCL) impairs methylmalonyl-CoA mutase function and leads to methylmalonic aciduria due to intracellular accumulation of propionyl and methylmalonyl-CoA. Since accumulation of these acyl-CoAs disrupts normal cellular regulation, the present investigation characterized metabolism in hepatocytes and liver mitochondria from rats treated subcutaneously with HCCL or saline (control) by osmotic minipump. Consistent with decreased methylmalonyl-CoA mutase activity, 14CO2 production from 1-14C-propionate (1 mM) was decreased by 76% and 82% after 2-3 wk and 5-6 wk of HCCL treatment, respectively. In contrast, after 5-6 wk of HCCL treatment, 14CO2 production from 1-14C-pyruvate (10 mM) and 1-14C-palmitate (0.8 mM) were increased by 45% and 49%, respectively. In isolated liver mitochondria, state 3 oxidation rates were unchanged or decreased, and activities of the mitochondrial enzymes, citrate synthetase, succinate dehydrogenase, carnitine palmitoyltransferase, and glutamate dehydrogenase (expressed per milligram mitochondrial protein) were unaffected by HCCL treatment. In contrast, activities of the same enzymes were significantly increased in both liver homogenate (expressed per gram liver) and isolated hepatocytes (expressed per 10(6) cells) from HCCL-treated rats. The mitochondrial protein per gram liver, calculated on the basis of the recovery of the mitochondrial enzymes, increased by 39% in 5-6 wk HCCL-treated rats. Activities of lactate dehydrogenase, catalase, cyanide-insensitive palmitoyl-CoA oxidation, and arylsulfatase A in liver were not affected by HCCL treatment. Hepatic levels of mitochondrial mRNAs were elevated up to 10-fold in HCCL-treated animals as assessed by Northern blot analysis. Thus, HCCL treatment is associated with enhanced mitochondrial oxidative capacity and an increased mitochondrial protein content per gram liver. Increased mitochondrial oxidative capacity may be a compensatory mechanism in response to the metabolic insult induced by HCCL administration.

Abnormal fermentations in continuous cultures of rumen micro-organisms given cobalt-deficient hay or barley as the food substrate.

1. Four experiments, each with four concurrent continuous cultures of rumen micro-organisms, were used to investigate the effects of inoculum, food substrate and cobalt supplementation on the course of fermentation and vitamin B12 synthesis. The inocula came from sheep receiving either a Co-rich, complete diet (Ruminant A: Expts 1, 2 and 4) or a Co-deficient hay (Expt 3). In Expt 2, inocula from different donors were used for each culture but for other experiments they were pooled. Co-deficient hay was used as the initial substrate and Co-supplements were given after 8-54 d Co-depletion. 2. In three of four experiments, two using a pooled inoculum, uncharacteristically low acetate (Ac) and high propionate (Pr) outputs were obtained from the hay substrate in some cultures. In all, six cultures gave Ac:Pr values in the effluent less than 2:1 and, in Expt 3, the differences remained evident for at least 6 d after a change to a Co-deficient barley substrate. 3. Abnormal cultures gave lower cobalamin (cbl) and vitamin B12 analogue outputs than normal cultures: when supplemented with Co they showed small responses in cbl and large responses in analogue output with a slow increase in Ac:Pr. 4. We suggest that the use of Co-deficient substrates led to shifts in the microbial populations of some cultures, indicating the cbl-dependence of some species of rumen micro-organisms.