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PubMed1976

Determination of cyanocobalamin by thermal decomposition of the cyano group using an ion-selective electrode.

A method for the simultaneous determination of cyanocobalamin, cobinamide (Factor B), and hydroxocobalamin in the solid state is described. The method is based on heating at 120 degrees for cobinamide and at 140-145 degrees for cyanocobalamin (15-20 min). The cyano content in the sample is distilled as hydrocyanic acid, trapped in 0.1 M potassium nitrate at pH 12-13, and determined by means of the cyanide ion-selective electrode. The error of this method, statistically established, does not exceed +/-3%.

Electrodes↗

PubMed1994

Antagonism of the lethal effects of cyanide by a synthetic water-soluble cobalt(III) porphyrin compound.

Efficacy of hydroxocobalamin (vitamin B12) as a cyanide antidote is limited by its high molecular weight (1355 g/mol) and by the competitive binding of the cobalamin dimethylbenzimidazole. The present study describes experiments with a lower molecular weight cobalt porphyrin that has a high affinity for cyanide, Co(III)-5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrin (CoTPPS), which was prepared by the method of Herrmann et al. (1978). CoTPPS was synthesized and its efficacy as an antidote to the lethal effects of cyanide either alone or in various combinations with NaNO2 and/or Na2S2O3 was determined. The LD50 value for CoTPPS was found to be 334 mg/kg. These studies were conducted using the CoTPPS LD01, 200 mg/kg. The cyanide antagonists NaNO2 (0.1 g/kg, sc), Na2S2O3 (1.0 g/kg, ip), and CoTPPS (0.2 g/kg, ip) were administered at 45, 15, and 10 min respectively prior to graded doses of KCN (sc). The LD50 values for KCN in male Swiss-Webster mice were calculated by probit analysis at the 95% confidence level and the various treatments were compared by potency ratios. These results indicated that the administration of CoTPPS alone protects against the lethal effects of cyanide. Moreover, CoTPPS adds to the protection provided by Na2S2O3 and/or NaNO2. Efficacy of this antidote is probably related to the binding equilibrium between CoTPPS and cyanide.

Animals↗

PubMed1991

Ether-cleaving enzyme and diol dehydratase involved in anaerobic polyethylene glycol degradation by a new Acetobacterium sp.

A strictly anaerobic, homoacetogenic bacterium was enriched and isolated from anoxic sewage sludge with polyethylene glycol (PEG) 1000 as sole source of carbon and energy, and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The new isolate fermented ethylene glycol and PEG's with molecular masses of 106 to 1000 to acetate and small amounts of ethanol. The PEG-degrading activity was not destroyed by proteinase K treatment of whole cells. In cell-free extracts, a diol dehydratase and a PEG-degrading (ether-cleaving) enzyme activity were detected which both formed acetaldehyde as reaction product. The diol dehydratase enzyme was oxygen-sensitive and was stimulated 10-14 fold by added adenosylcobalamine. This enzyme was found mainly in the cytoplasmic fraction (65%) and to some extent (35%) in the membrane fraction. The ether-cleaving enzyme activity reacted with PEG's of molecular masses of 106 to more than 20000. The enzyme was measurable optimally in buffers of high ionic strength (4.0), was extremely oxygen-sensitive, and was inhibited by various corrinoids (adenosylcobalamine, cyanocobalamine, hydroxocobalamine, methylcobalamine). This enzyme was found exclusively in the cytoplasmic fraction. It is concluded that PEG is degraded by this bacterium inside the cytoplasm by a hydroxyl shift reaction, analogous to a diol dehydratase reaction, to form an unstable hemiacetal intermediate. The name polyethylene glycol acetaldehyde lyase is suggested for the responsible enzyme.

Anaerobiosis↗

PubMed1994

Fermentative degradation of triethanolamine by a homoacetogenic bacterium.

With triethanolamine as sole source of energy and organic carbon, a strictly anaerobic, gram-positive, rod-shaped bacterium, strain LuTria 3, was isolated from sewage sludge and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The G+C content of the DNA was 34.9 +/- 1.0 mol %. The new isolate fermented triethanolamine to acetate and ammonia. In cell-free extracts, a triethanolamine-degrading enzyme activity was detected that formed acetaldehyde as reaction product. Triethanolamine cleavage was stimulated 30-fold by added adenosylcobalamin (co-enzyme B12) and inhibited by cyanocobalamin or hydroxocobalamin. Ethanolamine ammonia lyase, acetaldehyde:acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results establish that triethanolamine is degraded by a corrinoid-dependent shifting of the terminal hydroxyl group to the subterminal carbon atom, analogous to a diol dehydratase reaction, to form an unstable intermediate that releases acetaldehyde. No anaerobic degradation of triethylamine was observed in similar enrichment assays.

Biodegradation, Environmental↗

PubMed1983

A comparison of the effects of vitamin B12 and aspirin in three experimental pain models in rats and mice.

The effects of vitamin B12 are compared to those of aspirin and 0.9% saline in three different experimental pain models in rats and mice; an electric shock test, the hot-plate test, and the phenylbenzoquinone writhing test. Neither cyanocobalamin nor hydroxocobalamin showed any antinociceptive activity in any of the three tests at any of the doses used, whether the drug was administered as a single injection or as a series of daily injections lasting 7 days. These results run counter to those of clinical trials in man, though close scrutiny of the experimental procedures used in these trials often reveals a lack of rigor which casts doubts on their validity. This consideration, together with the negative findings reported here, suggests that the clinical use of vitamin B12 as an analgesic may be inappropriate.

Analgesics↗

PubMed1989

Characterization of cobalamin-dependent methionine synthase purified from the human malarial parasite, Plasmodium falciparum.

Methionine synthase, which catalyzes the reaction, 5-methyltetrahydrofolate (5-CH3-H4PteGlu) + homocysteine----methionine + tetrahydrofolate, was detected and partially purified from the human malarial parasite, Plasmodium falciparum (K1 isolate). Partial purification was achieved using high-performance size-exclusion and anion-exchange chromatography. The apparent relative molecular weight of the enzyme was estimated as 105,000 daltons, and the apparent Km for 5-CH3-H4PteGlu was 24.2 microM. The enzyme was dependent on adenosylcobalamin or methylcobalamin but not on cobalamin, cyanocobalamin, or hydroxocobalamin in either the absence or presence of S-adenosylmethionine. Preincubation with nitrous oxide markedly inhibited the enzyme. Methionine synthase in P. falciparum may play a role in the supply of methionine and in folate salvage using exogenous 5-CH3-H4PteGlu for tetrahydrofolate metabolism.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1996

Atypical vitamin B12-unresponsive methylmalonic aciduria in sibship with severe progressive encephalomyelopathy: a new genetic disease?

UNLABELLED: We report on two siblings, a girl of 7 years and a boy of 2 years, who presented in infancy with hypotonia, athetoid movements, myopathy and severe developmental delay. The progressive clinical course was characterized by ophthalmoplegia, pyramidal tract signs, loss of visual contact and failure to thrive. The older sister died at the age of 7 years. The younger brother followed an almost identical clinical course. MRI of the brain revealed bilateral hypodensities and atrophy of the putamen. Neurophysiological investigations were consistent with peripheral neuropathy. Investigations for neurometabolic disorders in urine, plasma and CSF of both patients revealed a consistent increase of methylmalonic acid in urine, plasma and CSF as well as borderline low free GABA in CSF. Except for an inconstant elevation of lactate in the boy, metabolic acidosis, hypoglycaemia, episodic ketoacidosis, or hyperammonaemia, the usual concomitants of organoacidopathies, were absent in both children. Homocystinuria was excluded. Methylmalonic aciduria did not respond to antibiotic treatment, vitamin B12 therapy nor dietary protein restriction. Incorporation of [14C]propionate into protein in cultured fibroblasts was pathologically but inconsistently decreased. Both patients' cell lines showed only minimal response to hydroxocobalamin and normal methylmalonyl-CoA mutase activity. CONCLUSION: Even though the definitive underlying enzymatic defect in this sibship remains obscure our results suggest a new genetic disorder. This report illustrates that hitherto undescribed metabolic disorders remain to be elucidated even in long investigated areas of intermediary metabolism such as methylmalonic aciduria.

Amino Acid Metabolism, Inborn Errors↗

PubMed1999

Nitric oxide synthase inhibitor decreases NMDA-induced elevations of extracellular glutamate and intracellular Ca2+ levels via a cGMP-independent mechanism in cerebellar granule neurons.

These studies were designed to examine the differential effect of nitric oxide (NO) and cGMP on glutamate neurotransmission. In primary cultures of rat cerebellar granule cells, the glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulates the elevation of intracellular calcium concentration ([Ca2+]i), the release of glutamate, the synthesis of NO and an increase of cGMP. Although NO has been shown to stimulate guanylyl cyclase, it is unclear yet whether NO alters the NMDA-induced glutamate release and [Ca2+]i elevation. We showed that the NO synthase inhibitor, N(G)-monomethyl-L-arginine (NMMA), partially prevented the NMDA-induced release of glutamate and elevation of [Ca2+]i and completely blocked the elevation of cGMP. These effects of NO on glutamate release and [Ca2+]i elevation were unlikely to be secondary to cGMP as the cGMP analogue, dibutyryl cGMP (dBcGMP), did not suppress the effects of NMDA. Rather, dBcGMP slightly augmented the NMDA-induced elevation of [Ca2+]i with no change in the basal level of glutamate or [Ca2+]i. The extracellular NO scavenger hydroxocobalamine prevented the NMDA-induced release of glutamate providing indirect evidence that the effect of NO may act on the NMDA receptor. These results suggest that low concentration of NO has a role in maintaining the NMDA receptor activation in a cGMP-independent manner.

Animals↗

PubMed1983

Determination of cobalamins in biological material. I. Improvement in the unequal recovery of cobalamins by preincubation with cadmium acetate.

Ethanol extraction of 28 specimens of human plasma in the dark showed that the total cobalamin value is increased up to 2 1/2 times by adding 400 micrometers of potassium cyanide. The addition of cyanide significantly changed the substance concentration (fractional content times substance concentration of total cobalamins) for all the cobalamin forms except 5'-deoxyadenosylcobalamin. The ethanol extraction was also studied with 57Co-labeled cobalamin forms. The recovery of [57Co]hydroxo- and [57Co]sulfitocobalamin from both plasma and hemolysate was inadequate. The recovery of [57Co]5'-deoxyadenosylcobalamin was only studied from liver tissue; the extraction recovery of this form was equal to that of 57Co]cyano- and [57Co]methylcobalamin. Preincubation with 0.25 M of cadmium acetate or extraction with 400 micrometers of potassium cyanide almost equalized the recovery of all five cobalamins. It is concluded that (i) the substance concentrations of the individual cobalamins presented hitherto are erroneous, owing to inadequate ethanol extraction of hydroxo- and sulfitocobalamin; (ii) about one-third of the cobalamins in plasma consists of hydroxocobalamin, which is not extractable by the methods used hitherto; (iii) studies of the cobalamin forms in body fluids and tissue cells need to be repeated by methods ensuring equal extraction of all the cobalamins; and (iv) the extraction of the cobalamins must be uniform for determination of the substance concentration of total cobalamins as well as the fractional amounts of all cobalamins.

Acetates↗

PubMed1985

The binding site for the adenosyl group of coenzyme B12 in diol dehydrase.

The binding of cob(II)alamin (CblII) and 5'-deoxyadenosine to diol dehydrase was studied spectroscopically and with [U-14C]5'-deoxyadenosine. CblII was bound to this enzyme forming a tight 1:1 complex which was resistant to oxidation by O2 even in the presence of CN-. An irreversible 1:1:1 ternary complex was formed between enzyme, CblII, and 5'-deoxyadenosine, when the enzyme was incubated first with the nucleoside and then with CblII. When this order of addition of the constituents was reversed, no 5'-deoxyadenosine was bound to the enzyme-CblII complex. Hydroxocobalamin could also bind to the enzyme together with the nucleoside, while other cob(III)alamins bearing a bulkier Co beta ligand displaced the nucleoside upon binding to the enzyme. The binding of [U-14C]5'-deoxyadenosine was strongly inhibited by unlabeled 5'-deoxy-ara-adenosine, 4',5'-anhydroadenosine, adenosine, adenine, and 5',8-cyclic adenosine, in this order, but not by 5'-deoxyuridine. These results constitute direct evidence for the presence of the binding site for the adenosyl group of adenosylcobalamin, which is spatially limited to and highly specific for adenine nucleosides. The binding of 5'-deoxyadenosine to the apoenzyme was reversible.

Binding Sites↗

PubMed1990

Essential histidine residues in coenzyme B12-dependent diol dehydrase: dye-sensitized photooxidation and ethoxycarbonylation.

The apoenzyme of diol dehydrase was inactivated by photoirradiation in the presence of rose bengal or methylene blue, following pseudo-first-order kinetics. The inactivation rates were markedly reduced under a helium atmosphere, suggesting that the inactivation is due to photooxidation of the enzyme under air. The half-maximal rate of methylene blue-sensitized photoinactivation was observed at pH around 7.5. Amino acid analyses indicated that one to two histidine residues decreased upon the dye-sensitized photoinactivation, whereas the numbers of tyrosine, methionine, and lysine did not change. Ethoxyformic anhydride, another histidine-modifying reagent, also inactivated diol dehydrase, with pseudo-first-order kinetics and a half-maximal rate at pH 7.7. It was shown spectrophotometrically that three histidine residues per enzyme molecule were modified by this reagent with loss of enzyme activity. Two tyrosine residues per enzyme molecule were also modified rapidly, irrespective of the activity. The photooxidation or ethoxycarbonylation of the enzyme did not result in dissociation of the enzyme into subunits, but deprived the enzyme of ability to bind cyanocobalamin. The percentage loss of cobalamin-binding ability agreed well with the extent of inactivation. The enzyme-bound hydroxocobalamin showed only partial protecting effect against photoinactivation and resulting loss of the cobalamin-binding ability. These results provide evidence that diol dehydrase possesses essential histidine residues which are required for the coenzyme binding.

Amino Acids↗

PubMed1989

Assay of methylmalonyl CoA mutase with high-performance liquid chromatography.

An assay for methylmalonyl CoA mutase activity is described. Succinyl CoA produced in this method is separated from the substrate, methylmalonyl CoA, by reverse-phase high-performance liquid chromatography and is quantified. This method is useful to differentiate mutase apoenzyme deficiency (mut0, mut-) and the defect in deoxyadenosylcobalamin synthesis using fibroblasts cultured in high concentration of supplementary hydroxocobalamin. In methylmalonic acidemia, measurement of lymphocytes mutase activity offers therapeutical and prognostic informations.

Acyl Coenzyme A↗

PubMed1991

Portal and biliary phases of enterohepatic circulation of corrinoids in humans.

The assimilation of labeled cobalamin and the transport of corrinoids in portal blood, peripheral venous blood, and bile were studied in eight cholecystectomized patients, after ingestion of a dose of cyano[57Co]cobalamin (0.5 microCi). The radioactivity appeared in the portal vein after a delay of 1.5-2 hours and in the peripheral vein 1 hour later. In bile, it reached a maximum at 24-72 hours; the excreted cobalamin corresponded to 1.42% +/- 0.92% of the dose ingested. The output of total corrinoids was 1.85 nmol/day. The high-performance liquid chromatography analysis of bile showed the presence of methylcobalamin, 5'-deoxyadenosylcobalamin, hydroxocobalamin, and an unknown corrinoid. This corrinoid bound to R binder but not to the intrinsic factor, and it had the same retention time as cobinamide. The R binder was the single cobalamin-binding protein found in bile. It was completely saturated in some periods of bile secretion. The corrinoids corresponding to such a period were eluted in Sephacryl S 300 gel filtration (Pharmacia Fine Chemicals, Uppsala, Sweden) in two peaks corresponding to saturated R binder and to free cobalamin. The mean level of total corrinoid was significantly higher in the portal vein (593 +/- 238 pmol/L) than in the peripheral vein (376 +/- 114 pmol/L) (P less than 0.01). This "cobalamin analogue" fraction was hypothetical because it was calculated from the difference between total corrinoid concentration and the so called "true cobalamin" concentration. This difference corresponded to the cobalamin analogue fraction. These data show that bile removes not only cobalamin but also cobalamin analogues and that R binder is the single carrier protein involved in their excretion.

Aged↗

PubMed1980

Peripheral nerve involvement in pernicious anaemia.

Impairment of nervous function and thiamine metabolism were studied in 40 patients suffering from pernicious anaemia, 20 of whom had not been treated and the other 20 were on hydroxocobalamin therapy. Of the untreated patients 13 (65%) showed signs of peripheral nerve dysfunction with reduced conduction velocities, as compared with only 1 (5%) of the treated patients. Reduced blood thiamine levels were found in 16 of the untreated patients (80%), 10 (60%) of whom showed biochemical signs of thiamine deficiency. In the treated group, 7 patients (30%) had reduced blood thiamine levels. Thus, peripheral nerve involvement in pernicious anaemia is much more frequent than would be expected from the literature, and thiamine deficiency was present in the majority of these cases.

Aged↗

PubMed1993

The methylcobalamin metabolism of cultured human fibroblasts.

The effect of supplying exogenous methylcobalamin (MeCbl), a methyl donor to methionine synthase (MS), on the cellular metabolism of MeCbl was tested in cultured fibroblasts from healthy persons and from a subject with an inherited defect in the synthesis of MeCbl. MeCbl bound to transcobalamin II (TCII) was taken up in larger amounts than cyanocobalamin (CN-Cbl), but was equal to the uptake of hydroxocobalamin (OH-Cbl). The form of Cbl in the lysosomes persisted as the same form, bound to TCII, to which the cells were exposed in the medium. Once released from the lysosomes, both MeCbl and OH-Cbl were converted in the same proportions to coenzyme forms, suggesting equivalent entry into common cellular pools of Cbl from which active forms are synthesized. Exogenous MeCbl enjoyed no advantage in binding to MS, in synthesis of MeCbl, and in supporting cell division in the absence of methionine. All evidence supported the concept that in human cells the active MeCbl on MS forms de novo on the enzyme. It appeared unlikely that therapeutic MeCbl would have any advantage over OH-Cbl in the treatment of MeCbl deficiency or Cbl deficiency in general.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1989

Oral epithelial dysplasia in vitamin B12 deficiency.

Widespread soreness and focal ulceration of the oral mucosa developed in a woman with vitamin B12 deficiency shortly after hydroxocobalamin therapy had commenced. An oral mucosal biopsy showed disordered epithelial maturation with marked cytologic atypia. However, during the next month of treatment, the patient's oral mucosa became clinically and histologically normal. Pathologists should be aware that vitamin B12 deficiency may be associated with epithelial changes similar to those associated with premalignancy.

Aged↗

PubMed1988

Cyanide scavengers: kinetics of the reactions of cyanide with a water soluble cobalt(III) porphyrin.

The equilibrium and kinetic aspects of the interaction of cyanide with a model anticyanide drug cobalt(III)-tetrakis(4-sulfonatophenyl)porphyrin [Co-P] were studied at 25 degrees C, I = 0.1 (NaNO3). At the physiologic pH of 7.4, 1.9 +/- 0.1 mol of cyanide were rapidly bound per molecule of Co-P. The dissociation constant of cyanide from Co-P(H2O) (CN) was less than 10(-12), and the formation constant of Co-P(CN)2 from Co-P(H2O) (CN) and CN- was 3.5 X 10(6). From pH 4 to 10.5, the kinetics of mono-cyano Co-P formation were first order in cyanide and porphyrin, with the following specific rate constants (units M-1 s-1): Co-P(H2O)2/CN-, 3.1 X 10(2); Co-P(H2O) (OH)/CN-, 2.4 X 10(3); Co-P(OH)2/CN-, 5.1 X 10(1) and Co-P(H2O)/HCN, 3.1 X 10(-3). At pH 7.4, a second cyanide molecule adds more rapidly than the first: Co-P(H2O) (CN)/CN-, 3 X 10(4) M-1 s-1. It is concluded that low molecular weight water soluble cobalt(III) porphyrins might be used as effectively and at lower dose levels than hydroxocobalamin (B12b), a known in vivo anticyanide agent.

Chemical Phenomena↗

PubMed1991

Vitamin B12 metabolism in a photosynthesizing green alga, Chlamydomonas reinhardtii.

To elucidate vitamin B12 (cobalamin) metabolism in green algae, Chlamydomonas reinhardtii was labeled with radioactive cobalamin for 4 days. The Chlamydomonas cells did not require cobalamin for growth, but nevertheless took up and accumulated exogenous cobalamin. The level of the accumulated cobalamin reached a maximum (about 20 fmol/10(6) cells) at day 1 of the cell growth. The cobalamin accumulated by the 4-day cells was partly converted to the coenzymes (5'-deoxyadenosylcobalamin, 20.7% and methylcobalamin, 4.8%). The cells contained NADH- and NADPH-linked aquacobalamin reductases (35.9 and 31.5 nmol/min per mg protein, respectively) involved in the synthesis of the coenzymes. Most of the accumulated cobalamin was associated with two heat-labile macromolecules; the coenzymes synthesized were recovered in the macromolecular fraction with a molecular weight of 230,000, while non-coenzyme forms (cyanocobalamin and hydroxocobalamin) predominated in that with a molecular weight of 40,000. Evidence for de novo biosynthesis of cobalamin was also obtained in the alga, indicating that it has both ability to synthesize cobalamin and take up the exogenous vitamin.

Chlamydomonas↗