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A high-performance liquid chromatographic method was developed for the separation and determination of 13 vitamin B compounds, including thiamin, riboflavin, riboflavin 5′-monophosphate, pyridoxine, hydroxocobalamin, cyanocobalamin, adenosylcobalamin, methylcobalamin, niacin, niacinamide, pantothenic acid, folic acid, and biotin. The method consisted of an ODS column, a gradient elution using pH 3.0 phosphate buffer–acetonitrile, ion-pairing reagent as mobile phase at 1.0 mL min−1, and UV detection at 210 nm. As a result, the 13 vitamin B compounds were separated in 60 min, and the specificity, linearity, range, limit of detection, limit of quantitation, intra- and inter-day precision, and recovery were satisfactory. The limit of detections ranged from 0.03 to 0.41 µg mL−1. Intra- and inter-day precision of peak area, expressed as RSD, were 0.542–5.144% and 0.216–6.367%, respectively. The method was used to evaluate vitamin B compounds in energy drinks and multivitamin pills. In case of energy drink analysis, the method identified and evaluated not only vitamin B compounds but also major ingredients, such as caffeine, vanillin, and benzoic acid. The vitamin B compounds in energy drinks and multivitamin pills were also quantitated and compared with values listed on each product label. Each quantitative value was close to the label values, suggesting high quantitative ability of the developed method.

Inorganic arsenic (iAs) is an established environmental diabetogen. The link between iAs exposure and diabetes is supported by evidence from adult human cohorts and adult laboratory animals. The contribution of prenatal iAs exposure to the development of diabetes and underlying mechanisms are understudied. The role of factors that modulate iAs metabolism and toxicity in adults and their potential to influence diabetogenic effects of prenatal iAs exposure are also unclear. The goal of this study was to determine if prenatal exposure to iAs impairs glucose metabolism in mice and if maternal supplementation with folate and methylcobalamin (B12) can modify this outcome. C57BL/6J dams were exposed to iAs in drinking water (0, 100, and 1000 μg As/L) and fed a folate/B12 adequate or supplemented diet from before mating to birth of offspring. After birth, dams and offspring drank deionized water and were fed the folate/B12 adequate diet. The metabolic phenotype of offspring was assessed over the course of 14 weeks. Male offspring from iAs-exposed dams fed the folate/B12-adequate diet developed fasting hyperglycemia and insulin resistance. Maternal folate/B12 supplementation rescued this phenotype but had only marginal effects on iAs metabolism in dams. The diabetogenic effects of prenatal iAs exposure in male offspring were not associated with changes in global DNA methylation in the liver. Only minimal effects of prenatal iAs exposure or maternal supplementation were observed in female offspring. These results suggest that prenatal iAs exposure impairs glucose metabolism in a sex-specific manner and that maternal folate/B12 supplementation may improve the metabolic phenotype in offspring. Further studies are needed to identify the mechanisms underlying these effects.

An indirect competitive enzyme-linked immunosorbent assay (Ic-ELISA) method and lateral-flow immunochromatographic (ICA) strip assay method were developed for the detection of vitamin B12 (four major forms, cyanocobalamin, hydroxocobalamin, adenosylcobalamin, and methylcobalamin) in different food products. The limit of detection and 50 % inhibitory concentration for the Ic-ELISA method were 0.065 and 0.43 ng/mL, respectively. The visual limit of detection and cutoff value for the lateral-flow ICA strip assay method were 1 and 4 ng/mL, respectively. For the detection of fortified food and nutritional supplements in vitamin tablets, energy drink, and infant milk powder samples, the recovery rates were in the range of 81 to 122 % for the Ic-ELISA method, and even the lowest content in infant milk powder was identified by the lateral-flow ICA strip assay. Therefore, both of these methods are sensitive, rapid, and effective and are suitable for the on-site detection and rapid screening of mass samples.

The vitamin B12-dependent riboswitch is a crucial factor that regulates gene transcription to mediate the growth of and vitamin B12 synthesis by Propionibacterium freudenreichii. In this study, the effect of various wavelengths of light on the growth rate and vitamin B12 synthesis was studied. Red, green, and blue light-emitting diodes (LEDs) were selected, and a dark condition was used as the control. The microorganism growth rate was measured using a spectrophotometer and plate counting, while the vitamin B12 content was determined using an HPLC-based method. The optical density at 600 nm (OD600) values indicated that P. freudenreichii grew better under the continuous and discontinuous blue light conditions. Moreover, under the blue light condition, P. freudenreichii tended to have a higher growth rate (0.332 h(-1)) and vitamin B12 synthesis (ca. 10 μg/mL) in tofu wastewater than in dark conditions. HPLC analysis also showed that more methylcobalamin was produced under the blue light conditions than in the other conditions. The cbiB gene transcription results showed that blue light induced the synthesis of this vitamin B12 synthesis enzyme. Moreover, the results of inhibiting the expression of green fluorescent protein indicated that blue light removed the inhibition by the vitamin B12-dependent riboswitch. This method can be used to reduce fermentation time and produce more vitamin B12 in tofu wastewater.

Double-blind randomized placebo-controlled trial to compare the effects of gabapentin (900–3600 mg/per day)/pregabalin (150–600 mg per day) in improvement of pain in patients with painful diabetic neuropathy. All diabetic patients were subjected to detailed history and thorough clinical examination, and those with painful diabetic neuropathy were taken into account for study after informed consent. Total of 120 cases were examined and investigated (30 patients in each group, for a total of four groups). Each group was allotted one of the four drugs including placebo, methylcobalamin (1500 microgms) which was common to all groups, according to random number table for 6 weeks. Patients were followed at 0, 2, 4 and 6 weeks for relief of pain intensity and severity according to VAS and Likert scale, respectively. At 2, 4 and 6 weeks, means of both VAS and Likert scale comparison showed that difference was statistically significant (p 0.05) when they are compared with each other. According to our study, all three drugs, i.e. pregabalin, gabapentin and tramadol-acetaminophen combinations, are equally effective in improvement of pain in patients with painful diabetic neuropathy, and also methylcobalamin alone is ineffective in improvement of pain in painful diabetic neuropathy (PDN) at 6 weeks. Researches done on drug treatment of PDN are limited, so large multicentric trials are needed to compare the effect of different drugs on pain in PDN.

To observe the clinical effect of electroacupuncture (EA) plus pricking-cupping bloodletting therapy for herpes zoster. A total of 43 patients with herpes zoster were randomly divided into two groups, 23 cases in the treatment group and 20 cases in the control group. The treatment group was given EA plus pricking-cupping bloodletting therapy. The control group was given oral administration of Aciclovir Tablets, Diclofenac Sodium Dual Release Enteric-coated Capsules, Methylcobalamin Dispersible Tablets, and Vitamin B12. The total effective rate was 87.0% in the treatment group and 70.0% in the control group, with a statistical significance in difference of the total effective rate between the two groups (P

Density functional theory has been applied to investigate the methyl transfer from methylcobalamin (MeCbl) cofactor to homocysteine (Hcy) as catalyzed by methionine synthase (MetH). Specifically, the S(N)2 and the reductive elimination pathways have been probed as the possible mechanistic pathways for the methyl transfer reaction. The calculations indicate that the activation barrier for the reductive elimination reaction (24.4 kcal mol(-1)) is almost four times higher than that for the S(N)2 reaction (7.3 kcal mol(-1)). This high energy demand of the reductive elimination pathway is rooted in the structural distortion of the corrin ring that is induced en route to the formation of the triangular transition state. Furthermore, the reductive elimination reaction demands the syn accommodation of the methyl group and the substrate over the upper face of the corrin ring, which also accounts for the high energy demand of the reaction. Consequently, the reductive elimination pathway for MetH-catalyzed methyl transfer from MeCbl to Hcy cannot be considered as one of the possible mechanistic routes.

Vitamin-B(12) is a generic term for corrinoid compounds that exhibit the biological activity of cyanocobalamin and are collectively referred to as cobalamins. Methylcobalamin and 5-deoxyadenosylcobalamin are the active cobalamins in human metabolism. Cobalamin plays a crucial role in the maintenance of homocysteine and methylmalonyl-CoA homeostasis and is required for erythrocyte formation and DNA synthesis. Data from human and animal studies indicate that cobalamin deficiency impairs neuronal function; a process that is thought to contribute to age-related cognitive decline and dementia. Cobalamin deficiency also results in dysfunction of the peripheral nervous system; among other disorders. Although there is a detailed understanding of the biochemical pathways that are perturbed in cobalamin deficiency, the mechanisms underlying age-related dyshomeostasis in such pathways remain to be addressed. Because cobalamin utilization is dependent on its efficient transit through lysosomes, and mounting evidence indicates that lysosomal function deteriorates in aging long-lived post-mitotic cells such as neurons, in the present article we review published data that supports the proposition that impaired lysosomal processing of cobalamin may play a significant role in age-related (neuro) degenerative diseases.

Objective To study the clinical effect of acupuncture combined with methylcobalamin in the treatment of diabetic peripheral neuropathy.

Homocysteine is a predictor of vascular disease and may have an important role in diabetes. In this study, we examined the effects of folic acid and methylcobalamin supplementation on changes in homocysteine (Hcy) levels and homocysteine thiolactonase/paraoxonase (HTase/PON) activity in a short-term trial.Ninety patients with type 2 diabetes were randomly divided into three groups: Group I received no vitamin supplementation, group II received 5 mg/day folic acid (orally), group III received folic acid (5 mg/day) in combination with methylcobalamin (500 microg/day; intramuscularly, on prescription). All patients were treated for 2 weeks. Plasma Hcy, HTase/PON activity, vitamin B(12), and folic acid were measured before and after supplementation in each group. In addition, forty healthy (nondiabetic) controls were enrolled.Serum HTase/PON activity was significantly higher in diabetics compared with controls, plasma Hcy levels were significantly lower (P0.05). After vitamin supplementation there was a significant reduction in plasma Hcy levels. The mean percentage reduction in Hcy was 2.75% in group I, 14% in group II and 37.3% in group III. There was a significant inverse correlation between the changes in HTase/PON activity and Hcy levels (r=-0.29, P=0.004). A 2.72% increase in HTase/PON activity was seen in group I, an 8.03% increase was detected when folic acid was given in group II (P0.001), and a 17.59% increase in HTase/PON activity was seen in group III (P0.001).Short-term oral folic acid (5 mg/day) supplementation with or without methylcobalamin appeared to be an effective approach to decrease Hcy levels and increase HTase/PON activity in patients with type 2 diabetes. A decrease in plasma Hcy levels may partly account for the elevation of serum HTase/PON activity. This could be a novel mechanism to protect against vascular diabetic complications.

The bond dissociation energies of the Co-C bonds in the cobalamin cofactors methylcobalamin and adenosylcobalamin were calculated using the hybrid quantum mechanics/molecular mechanics method IMOMM (integrated molecular orbital and molecular mechanics). Calculations were performed on models of differing complexities as well as on the full systems. We investigated the origin of the different experimental values for the Co-C bond dissociation energies in methylcobalamin and adenosylcobalamin, and have provided an explanation for the difficulties encountered when we attempt to reproduce this difference in quantum chemistry. Additional calculations have been performed using the Miertus-Scrocco-Tomasi method in order to estimate the influence of solvent effects on the homolytic Co-C bond cleavage. Introduction of these solvation effects is shown to be necessary for the correct reproduction of experimental trends in bond dissociation energies in solution, which consequently have no direct correlation with dissociation processes in the enzyme.

Quantum chemical methods were used to investigate the mechanism of interaction of polyhydrated Hg(II) with methylcobalamin — a derivative of vitamin B12. Migration of the methyl group in the B12·Hg(II) nH2O cluster, initiating the formation of methylmercury, is considered. The reaction of substitution of methylmercury for the cobalt atom in the corrin fragment of B12 has been studied. The thermodynamic and activation characteristics of these processes have been obtained. The mercury analog of vitamin B12 is shown to exist in two radically different conformations.

Isolated functional methionine synthase deficiency occurs in the cblE and cblG defects of methylcobalamin metabolism and is one of a number of causes of severely elevated plasma homocysteine. Clinical features are predominantly of a neurological nature but also include functional restriction of the visual system manifesting as loss of visual acuity and nystagmus. As yet, the origin and pathogenesis of impaired vision have not been explained.We investigated a patient who was proven by complementation analysis in cultured fibroblasts to belong to the cblG complementation group. Ganzfeld electroretinograms (ERG) and flash visual evoked potentials (VEP) were recorded over a period of 4 years.Amplitudes of all International Society for Clinical Electrophysiology of Vision (ISCEV) standard responses were below normal. The greatest reductions were of rod response to 24 microV, of standard combined response (SC) b-wave to 120 microV, of oscillatory potentials (OP) to 5 microV, of cone response b-wave to 35 microV, and of 30 Hz flicker response to 8 microV. Except for SC and cone a-waves at age 2.5 and 3.5 years, as well as cone b-wave at 3.5 years, amplitudes remained at a subnormal level at follow-up examinations. Implicit times were slightly prolonged (SC b-wave 6 ms, OPs 2 ms, cone b-wave 2 ms, 30 Hz flicker 4 ms) or fell within the normal range. Responses of the flash VEP were severely deformed but reproducible.This is the first report of detailed investigations of the visual system in a patient with isolated methionine synthase deficiency. Reduced oscillatory potentials suggest microvascular damage to the retina through homocysteine. Decreased photoreceptor function as well as ganglion cell loss as indicated by pathological flash VEPs may reflect a cytotoxic impact of homocysteine on neurons of the visual pathway.

Whole-cell suspensions of Enterobactercloacae SLD1a-1 produced dimethylselenide(DMSe) from selenate, selenite, elementalselenium, dimethylselenone,seleno-DL-methionine, 6-selenoinosine, and6-selenopurine. Cell-free extracts of thebacterium produced the formation of DMSe fromorganic selenium compounds, includingdimethylselenone, dimethylselenoniopropionate,seleno-DL-methionine, seleno-DL-ethionine, and6-selenoguanosine. The highest rate of DMSeproduction occurred from whole-cell suspensionsand cell-free extracts containingdimethylselenone. DMSe was also produced bycell-free extracts containing selenite orelemental selenium and methylcobalamin. Cell-free extracts did not produce DMSe frominorganic selenium when S-adenosyl-L-methionine was present. Additionally, DL-homocysteine and L-methioninewere found to inhibit selenium volatilization. These findings suggest the formation of DMSefrom inorganic selenium occurs through thetransfer of a methyl group frommethylcobalamin.

An 18-month-old girl presented with macrocytic megaloblastic anaemia followed by haemolytic uraemic syndrome. Metabolic investigations led to the identification of an inborn error of cobalamin metabolism consisting of defective methylcobalamin biosynthesis, probably cobalamin G, since methionine synthase activity was decreased under standard reducing conditions. Despite treatment, pulmonary hypertension progressively developed and responded to oxygen therapy. Renal involvement evolved to terminal failure and haemodialysis, while pulmonary hypertension was controlled by oxygen therapy. Such clinical manifestations have never been reported in association with a defect of methylcobalamin and thus of methionine biosynthesis. A congenital abnormality of cobalamin metabolism was suspected then confirmed in the presence of typical haematological features associated with unusual clinical manifestations such as progressive renal failure and pulmonary hypertension.

Objective: To find out the clinical effects of methylcobalamin on the recovery of active elbow flexion after neurotisation in brachial plexus injury.

Deficient activity of an enzyme can result from a defect in the conversion of the vitamin to a co-enzyme as well from an abnormal apo-enzyme or disturbed binding of coenzyme to enzyme. Conversion of dietary vitamin to intracellular active co-enzyme can be complex and require many physiological and biochemical processes including stomach release of bound vitamin, intestinal uptake, carriers/transport, blood transport, cellular uptake, intracellular release and intracellular compartmentalisation. Disorders of malabsorption (food cobalamin malabsorption, intrinsic factor deficiency and abnormal enterocyte cobalamin processing) and transport proteins (transcobalamin II deficiency, R-binder deficiency) mostly lead to disturbed function of the two cobalamin requiring enzymes, methylmalonyl CoA mutase and methionine synthase. Defects of early steps of intracellular cobalamin (cblF, cbl C/D) result in marked deficiencies of both cobalamin co-enzymes and homocystinuria combined with methylmalonic aciduria. Defective synthesis of adenosyl cobalamin in the cbl A/B defects leads to methylmalonyl CoA mutase. Isolated methionine synthase deficiency is also classified as a cobalamin disorder due to its associated deficient formation of methylcobalamin. Folate disorders include methylene-tetrahydrofolate reductase deficiency and glutamate formimino-transferase deficiency. In addition a hereditary disorder of intestinal folate transport has been described. Less well established are disorders of dihydrofolate reductase, methenyl-tetrahydrofolate cyclohydrolase, and defects of cellular folate uptake.

This preliminary study investigates effects of methyl- and cyanocobalamin on circadian rhythms, well-being, alertness, and concentration in healthy subjects. Six women (mean age 35 years) and 14 men (mean age 37 years) were randomly assigned to treatment for 14 days with 3 mg cyano-(CB12) or methylcobalamin (MB12) after 9 days of pre-treatment observation. Levels in the CB12 group increased rapidly in the first, then slowly in the second treatment week, whereas increase in the MB12 group was linear. Urinary aMT6s excretion was reduced by both forms of vitamin B12 over 24 hours with a significant decrease between 0700-1100 hours, whereas urinary excretion of potassium was significantly increased between 0700-1100 hours. Activity from 2300-0700 hours increased significantly under both forms of vitamin B12. Sleep time was significantly reduced under MB12 intake. In this group the change in the visual analogue scales items "sleep quality," "concentration," and "feeling refreshed" between pretreatment and the first week of treatment showed significant correlations with vitamin B12 plasma levels. Cortisol excretion and temperature were not affected by either medication. We conclude that vitamin B12 exerts a direct influence on melatonin. Only MB12 has a positive psychotropic alerting effect with a distribution of the sleep-wake cycle toward sleep reduction.

The goal of our work was a structural and functional analysis of the state of the fat-carbohydrate (energy) and protein metabolism in rats under conditions of complete alimentary fasting while maintaining the drinking regimen and administering of methylcobalamin. The state of the ultrastructural elements ensuring energy homeostasis was studied. The activity of a variety of compounds in the blood was determined. The results of the studies showed methylcobalamin to increase the adaptation mechanisms maintaining both energy and plastic homeostasis in a fasting organism.

The administration of 2g/kg of di(2-ethylhexyl)-phthalate (DEHP)-induced severe testicular atrophy coincident with the reduction of testicular specific lactate dehydrogenase (LDH-X) activity, zinc, magnesium, and potassium concentrations in rats. Co-administration of DEHP and adenosyl cobalamin (AdoCbl), one of the active vitamin B12s, prevented these testicular specific changes including fluctuations in testicular weight. On the other hand, co-administration of DEHP and methylcobalamin (MeCbl), the other active vitamin B12, did not prevent the testicular atrophy induced by DEHP under the present experimental conditions. In the liver, DEHP administration caused hypertrophy with changes in several metal concentrations and serum biochemical parameters. Co-administration of DEHP and AdoCbl or MeCbl did not prevent these hepatic changes, but aggravated hypolipidemia. The results demonstrated that the preventive effect of AdoCbl was a testicular specific action, and this effect may be stimulated solely by AdoCbl in vitamin B12 groups.

Novel vitamin B12 analogues in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analogue containing imidazole instead of 5,6-dimethylbenzimidazole as well as cobinamide methyl phosphate were tested for biological activities with Escherichia coli 215, a B12- or methionine-auxotroph, and Lactobacillus leichmannii ATCC 7830 as test organisms. A cyano form of 5,6-dimethylbenzimidazolyl tetramethylene, trimethylene and hexamethylene analogues supported the growth of L. leichmannii in this order. 5,6-Dimethylbenzimidazolyl dimethylene and imidazolyl trimethylene analogues did not show B12 activity and behaved as weak B12 antagonists when added together with cyanocobalamin. An adenosyl form of the biologically active analogues served as coenzymes for ribonucleotide reductase of this bacterium, whereas that of the inactive analogues did not. The latter acted as weak competitive inhibitors against adenosylcobalamin. On the contrary, all the analogues did not support the growth of E. coli 215 at all by themselves and inhibited the growth when added with a suboptimum level of cyanocobalamin. A methyl form of the analogues also did not support the growth of E. coli 215, although they served as active coenzymes for methionine synthase of the bacterium. Since unlabeled analogues strongly inhibited the uptake of [3H]cyanocobalamin by this bacterium, it seems likely that the analogues exert their anti-B12 activity toward E. coli 215 by blocking the B12-transport system.

Vitamin B12 (methylcobalamin) was administered orally (3 mg/day) to 9 healthy subjects for 4 weeks. Nocturnal melatonin levels after exposure to bright light (ca. 2500 lx) were determined, as well as the levels of plasma melatonin over 24 h. The timing of sleep was also recorded. Vitamin B12 was given blind to the subjects and crossed over with placebo. We found that the 24-h melatonin rhythm was significantly phase-advanced (1.1 h) in the vitamin B12 trial as compared with that in the placebo trial. In addition, the 24-h mean of plasma melatonin level was much lower in the vitamin B12 trial than with the placebo. Furthermore, the nocturnal melatonin levels during bright light exposure were significantly lower in the vitamin B12 trial than with the placebo. On the other hand, vitamin B12 did not affect the timing of sleep. These findings raise the possibility that vitamin B12 phase-advances the human circadian rhythm by increasing the light sensitivity of the circadian clock.

In this study, the effects of methylcobalamin on testicular damage induced by ethylene oxide (EtO) were studied. When Wistar male rats inhaled EtO at 500 ppm, 6 h a day, 3 days a week, for 6 weeks, testicular damage was observed histopathologically and by some other parameters. Subcutaneous injection of methylcobalamin at 500 micrograms/kg, 5 times/week was found to ameliorate the damage. However, the degree of the methylcobalamin effect differed among the parameters examined in this study. Decrease in testicular weight due to EtO exposure was completely prevented by methylcobalamin, and decrease in testicular mature spermatid count and LDH-X activity was fairly well prevented. The degree of prevention of alteration in the epididymis, such as epididymal weight, epididymal sperm count and sperm abnormality rate, was significant but not complete. EtO caused apparent alterations in glutathione metabolism in the testes, but methylcobalamin did not affect such alterations induced by EtO. From these results, it has been determined that methylcobalamin has definite preventive effects on testicular toxicity of EtO.

Two DNA-methylases from nuclei of rat liver are compared for their ability to methylate thymic DNA in the presence of methyl group donors, radiolabeled3H-S-adenosyl-L-methionine and methylcobalamin. DNA-methylase with an isoelectric point of 5.2 is found, which uses both donors of methyl groups although its activity is more expressed with3H-S-adenosyl-L-methionine.3H-S-adenosyl-L-methionine and methylcobalamin presumably interact with different sites of the enzyme.

Objective To compare the efficacy, safety, and tolerability of Epalrestat with Methylcobalamine in patients with diabetic neuropathy. Materials and methods This prospective, open-labeled, comparative, and multicentric study was conducted on an outpatient basis by qualified investigators at four different centers. A total of 242 patients with diabetic neuropathy were enrolled in the study. The enrollment of patients was as per inclusion/exclusion criteria. After obtaining their informed consent, they were individually interviewed, examined, investigated, and treated as per the study protocol. Each patient was administered with one tablet containing Epalrestat 50 mg thrice daily or Methylcobalamine 500 microg thrice daily for 12 weeks. They were followed up at the end of weeks 4, 8, and 12 for their examination. Therapeutic success was assessed in terms of clinical symptoms, physical examinations, and electrophysiological assessments. Results A significant improvement in all the efficacy parameters was observed with Epalrestat treatment compared to Methylcobalamin treatment. The efficacy parameters assessed were loss of sensation, burning sensation, numbness, muscle cramps, spontaneous pain, weakness, mean score of isometric muscle strength, tendon reflexes, and sensation. Epalrestat treatment is associated with very few adverse events. The tolerability to Epalrestat treatment was also reported to be excellent in the majority of the study population compared to tolerability to Methylcobalamine. Patients as well as physicians reported that the Epalrestat treatment is superior in efficacy and safety parameters compared to Methylcobalamin. Conclusion The present study concludes that Epalrestat has better efficacy and safety profile than Methylcobalamine in the treatment of diabetic neuropathy.

A critical review on corrinoid biochemistry and physiology is presented. This includes: chemical synthesis of biologically important organocorrinoids and the correlation between their structures and coenzymatic activity; forms, distribution and transport of physiologically active corrinoids; methylcobalamin- and adenosylcobalamin-dependent enzymatic reactions and their physiological functions; and steric course of the adenosylcobalamin-dependent enzymatic rearrangements. Special attention is paid to the mechanisms of adenosylcobalamin-dependent enzymatic reactions.

Extract: The intracellular content and proportional distribution of B12 (cobalamin) derivatives in fibroblasts cultured from patients with various forms of methylmalonic aciduria, as well as from normal control subjects, has been determined by a two-dimensional chromatobioautographic technique. Each line of fibroblasts was grown in the presence of four concentrations of cobalamin, ranging from the 0.04–0.07 pmol/ml contained in the basal medium to 74 pmol/ml (100 ng/ml), added in form of hydroxocobalamin (OHCBI). Control cells grown in the basal medium contained substantial proportions of both methylcobalamin (MeCbl) and adenosyl-cobalamin (AdoCbl), with the former predominating. As increasing concentrations of OH-CBI were added to the growth medium, the total cellular cobalamin content increased without marked changes in the relative proportions of MeCbl, AdoCbl, and OHCbl. Three different patterns were discernable in the cobalamin distributions of the cells cultured from patients with methylmalonic aciduria (Table 1 and Fig. 1). Speculation: Information about the cobalamin contents of cultured fibroblasts yields further understanding of cobalamin metabolism. Such information, obtained by the technique of two-dimensional chromatobioautography, permits definition of genetically determined abnormalities in the handling of cobalamins, as well as examination of possible correlations between cellular responses to increased concentrations of these compounds and the effectiveness of cobalamins in the clinical treatment of patients.

In this study, cobalamin deficiency was produced in vitro by the use of nitrous oxide, known to inactivate the vitamin. In 14 sets of experiments, normal human lymphocytes stimulated with phytohemagglutinin on day 0 were exposed to nitrous oxide and oxygen on day 2. MeCbl was delivered later to half of the cells. Untreated cells served as a control. On day 3, the cells were harvested, the lymphocytes were lysed, and the obtained extracts were assayed for thymidylate synthetase. In 16 other experiments the same procedure was performed, and the incorporation of radioactive thymidine or deoxyuridine by the intact cells was measured. In additional experiments, a deoxyuridine suppression test of treated and untreated stimulated lymphocytes was also performed. The results indicate that nitrous oxide significantly reduces the activity of thymidylate synthetase and that this reduction is significantly corrected by MeCbl, suggesting a causative relation between the vitamin and the enzyme. However, there was no statistically significant effect of nitrous oxide demonstrated on the nucleoside incorporation nor on the deoxyuridine suppression test.

Clostridium cochlearium T-2C, a mercury sensitive strain, was isolated by treating its original strain, T-2P, carrying a mercury-resistant plasmid with acridine orange. The T-2C strain obtained, having the ability to synthesize various types of cobalamin including methylcobalamin, can methylate inorganic mercury, probably by the participation of methylcobalamin. Results show that microbial methylation of inorganic mercury in the cells to produce less toxic methylmercury, in terms of the direct inhibitory effect on functional proteins, acts as a detoxification mechanism to build up bacterial tolerance to inorganic mercury.

This paper gives the results of a study of the transplacental action of methylcobalamin (MC) on growth of embryonic tissues during organ culture. An organ culture of embryonic mouse kidney tissue, sensitive to the transplacental action of biologically active substances, including vitamins and hormones, and also of chemical carcinogens, was used as the experimental model. /sup 3/H-thymidine was added to the nutrient medium before fixation of the explants. The stimulating effect of MC on growth of mouse kidney tissue in the prenatal period as revealed in this investigation, is said by the authors to be an important mechanism modifying the transplacental action of chemical carcinogens.

In recent years preparations of the vitamin B12 group, synthesized at the "Vitaminy" Scientific-Industrial Association hydroxycobalamin (OHCbl) and adenosylcobalamin (AdoCbl) have been finding use in medicine [i]. Together with methylcobalamin, these substances are natural forms of vitamin B12, which are present in substantial amounts in the human and animal organism. OHCbl and AdoCbl have a number of advantages in comparison with cyanocobalamin (CNCbl) lower toxicity, the presence of an anabolic effect, lipotropic and hematoprotective properties, etc. [2]. In view of this, there is a basis for believing that the spectrum of their medical use will widen. However, the pharmacokinetics of these domestic preparations have not been studied. Nor are there any data in the literature on the pharmacokinetic parameters for CNCbl, AdoCbl and OHCbl. Therefore we have begun a study of the pharmacokinetics of preparations of the vitamin Bl2 group.

Methylcobalamin (10 microgram/kg bw, intramuscularly, on the 3d and 5th days after tumor transplantation) or methylcobalamin chlorpalladat -- MeCbl.PdCl3 (250 mg/kg bw in 2% starch suspension per os on the 2d and 6th days) were given to C57BI mice bearing adenocarcinoma Ca--755. On the 8th day after transplantation the methionine synthetase activity and proliferative pool of tumor cells were estimated. It has been disclosed that under the effect of MeCbl the activity of methionine synthetase rises concurrently with an increase in pool of the proliferating cells. Inhibition of tumor growth (by 80--90%) after the treatment with MeCbl.PdCl3 correlated with a decrease in the holoenzyme level in tumor cells.

A vitamin B12 requiring strain was isolated from Clostridium cochlearium T-2C which is known to synthesize various types of vitamin B12 including methylcobalamin and has an ability to methylate inorganic mercury. The vitamin B12 auxotroph lacking the mercury-methylating activity showed higher sensitivity to inorganic mercury than its original strain, while the sensitivity of both strains to methylmercury was relative low and essentially the same. These data seem to present affirmative evidence to postulate the physiological role of methylcobalamin-dependent methylation of mercury to be a process of detoxication.

INVESTIGATION of the form of vitamin B12 in human blood plasma by chromatographic-bioautographic analysis revealed as the major component a ‘fourth factor’ besides DMB coenzyme (coenzyme B12) and traces of hydroxo-cobalamin and cyanocobalamin1. This factor was also found in liver, E. coli and Streptomyces rimosus. In order to identify this analogue we have tried to isolate it from a natural source material rich in coenzyme B12 and have found good evidence that this analogue is methyl-cobalamin. Methylcobalamin was prepared by partial synthesis a couple of years ago2 and was shown by Guest et al.3 to be active in the enzymatic synthesis of methionine by extracts of an Escherichia coli mutant. However, methylcobalamin has not previously been shown to occur naturally.

Die Zunahme der Leber- und Serumeiweissmenge bei chronischer Leberstorung von Ratten mit partiel extirpierter Leber wird nach Applikation von Methylcobalamin gefordert. Eine Methylcobalamin induzierte Forderung der Eiweissynthese in der Leber wird angenommen.

We have recently described a child who presented early in life with homocystinuria and megaloblastic anemia. Fibroblasts from this patient had low levels of methylcobalamin (CH3-B12) and a decreased incorporation of (14C) methyltetrahydrofolate (CH3-THF) into methionine which was unresponsive to the addition of vitamin B12 to the tissue culture medium. In contrast to the cultured cells, in the patient the megaloblastic anemia and homocystinuria resolved on treatment with OH-B12. Fibroblasts from the parents of this patient had normal levels of CH3-B12 but intermediate levels of CH3-THF incorporation and a variable response to vitamin B12 in the culture medium. When the pro-band's mother became pregnant, prenatal diagnosis was attempted. The incorporation of total vitamin B12 derivatives which was CH3-B12 in the fetal amniocytes was only 7.4%, as compared to 26.1% and 48.7% in control amniocytes, and 37.1% and 32% in fibroblasts from the obligate heterozygotes. Values for CH3-THF incorporation were variable. Because the proband responded well to vitamin B12 therapy, it was decided to treat the mother prenatally. She received IM hydroxycobalamin (OH-Cbl) 1 mg twice weekly beginning at 25 weeks gestation. Diagnostic studies on fibroblasts obtained at birth confirmed that the baby has CH3-B12 deficiency. The baby has been maintained on 1 mg IM OH-Cbl twice weekly and is clinically well.

Inherited methylmalonicacidemia (MM-emia) due to deficient activity of methylmalonyl-CoA mutase may be caused by an abnormal mutase apoenzyme or by reduced holoenzyme activity secondary to impaired synthesis of the mutase-requiring coenzyme, 5'-deoxyadenosylcobalamin (Ado-Cbl). We studied Ado-Cbl synthesis in 5 fibroblast lines from unrelated patients who have MM-emia due to defective cobalamin (vitamin B12) metabolism. Their whole cells in culture fail to synthesize Ado-Cbl but make normal amounts of the other cobalamin coenzyme, methylcobalamin.