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SARS‐CoV‐2 is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. Like other members of this family, the virus possesses a positive‐sense single‐stranded RNA genome. The genome encodes for the nsp12 protein, which houses the RNA‐dependent‐RNA polymerase (RdRP) activity responsible for the replication of the viral genome. A homology model of nsp12 was prepared using the structure of the SARS nsp12 (6NUR) as a model. The model was used to carry out in silico screening to identify molecules among natural products, or Food and Drug Administration‐approved drugs that can potentially inhibit the activity of nsp12. This exercise showed that vitamin B12 (methylcobalamin) may bind to the active site of the nsp12 protein. A model of the nsp12 in complex with substrate RNA and incoming NTP showed that vitamin B12 binding site overlaps with that of the incoming nucleotide. A comparison of the calculated energies of binding for RNA plus NTP and methylcobalamin suggested that the vitamin may bind to the active site of nsp12 with significant affinity. It is, therefore, possible that methylcobalamin binding may prevent association with RNA and NTP and thus inhibit the RdRP activity of nsp12. Overall, our computational studies suggest that methylcobalamin form of vitamin B12 may serve as an effective inhibitor of the nsp12 protein.

Derivatives of vitamin B12 (cobalamin) are required for activity of two enzymes: methylmalonyl-CoA mutase, which catalyses conversion of methylmalonyl-CoA to succinyl-CoA, and methionine synthase, which converts homocysteine to methionine. Cobalamin deficiency results in the accumulation of methylmalonic acid and homocysteine in blood and urine. Clinically, deficiency results in megaloblastic anaemia and subacute combined degeneration of the spinal cord, loss of sensation, ataxia, dementia and psychosis. Dietary cobalamin deficiency is rare in developed countries, except for individuals consuming a vegan diet. A more common cause of cobalamin deficiency is pernicious anaemia, an autoimmune disease that results in inability to absorb cobalamin from the intestine. A number of rare genetic disorders have been identified that result in either failure to absorb dietary cobalamin, to transport cobalamin from the intestine to cells that require it for metabolism or to convert intracellular cobalamin to one or both of its coenzyme derivatives, adenosylcobalamin and methylcobalamin. Key Concepts Cobalamin (vitamin B12) is required for activity of two enzymes, methylmalonyl-CoA mutase and methionine synthase, in mammalian cells. Cobalamin deficiency results in accumulation of methylmalonic acid and homocysteine in blood and urine. Clinically, deficiency is characterised by megaloblastic anaemia and/or neurological problems. Uptake of dietary cobalamin requires the binding protein intrinsic factor, secreted by parietal cells of the stomach, and the intestinal receptor cubam. Autoimmune destruction of parietal cells results in pernicious anaemia. Mutations in the genes encoding intrinsic factor or the components of cubam (cubilin and amnionless) result in heritable cobalamin malabsorption and signs of cobalamin deficiency. Peripheral cells take up cobalamin bound to the transport protein transcobalamin by carrier-mediated endocytosis. Cells convert cobalamin to adenosylcobalamin, required by methylmalonyl-CoA mutase, and methylcobalamin, required by methionine synthase. Rare mutations affecting genes involved in cobalamin metabolism result in decreased synthesis of either or both cobalamin derivatives. Mutations in certain transcription regulators can affect expression of genes encoding cobalamin metabolising enzymes, resulting in altered cobalamin coenzyme synthesis. Keywords: vitamin B12; cobalamin; homocysteine; methylmalonic acid; anaemia

BACKGROUND: Diabetic peripheral neuropathy (DPN) is a common and severe complication that affects 50% of people with diabetes. Painful DPN is reported to occur in 16% to 24% of people with diabetes. A complete and comprehensive management strategy for the prevention and treatment of DPN, whether painful or not, has not yet been defined. Research into treatment for DPN has been characterised by a series of failed clinical trials, with few noteworthy advances. Strategies that support peripheral nerve regeneration and restore neurological function in people with painful or painless DPN are needed. The amino acid acetyl‐L‐carnitine (ALC) plays a role in the transfer of long‐chain fatty acids into mitochondria for β‐oxidation. ALC supplementation also induces neuroprotective and neurotrophic effects in the peripheral nervous system. Therefore, ALC supplementation targets several mechanisms relevant to potential nerve repair and regeneration, and could have clinical therapeutic potential. There is a need for a systematic review of the evidence from clinical trials. OBJECTIVES: To assess the effects of ALC for the treatment of DPN. SEARCH METHODS: On 2 July 2018, we searched the Cochrane Neuromuscular Specialised Register, CENTRAL, MEDLINE, Embase, LILACS, ClinicalTrials.gov, and the World Health Organization International Clinical Trials Registry Platform. We checked references, searched citations, and contacted study authors to identify additional studies. SELECTION CRITERIA: We included randomised controlled trials (RCTs) and quasi‐RCTs of ALC compared with placebo, other therapy, or no intervention in the treatment of DPN. Participants could be of any sex and age, and have type 1 or type 2 diabetes mellitus, of any severity, with painful or painless DPN. We accepted any definition of minimum criteria for DPN, in accordance with the Toronto Consensus. We imposed no language restriction. Pain was the primary outcome, measured as the proportion of participants with at least 30% (moderate) or 50% (substantial) decrease in pain over baseline, or as the score on a visual analogue scale (VAS) or Likert scale for pain. DATA COLLECTION AND ANALYSIS: We followed standard Cochrane methods. MAIN RESULTS: We included four studies with 907 participants, which were reported in three publications. Three trials studied ALC versus placebo (675 participants); in one trial the dose of ALC was 2000 mg/day, and in the other two trials, it was 1500 mg/day or 3000 mg/day. The fourth trial studied ALC 1500 mg/day versus methylcobalamin 1.5 mg/day (232 participants). The risk of bias was high in both trials of different ALC doses and low in the other two trials. No included trial measured the proportion of participants with at least moderate (30%) or substantial (50%) pain relief. ALC reduced pain more than placebo, measured on a 0‐ to 100‐mm VAS (MD ‐9.16, 95% CI ‐16.76 to ‐1.57; three studies; 540 participants; P = 0.02; I² = 56%; random‐effects; very low‐certainty evidence; a higher score indicating more pain). At doses of 1500 mg/day or less, the VAS score after ALC treatment was little different from placebo (MD ‐0.05, 95% CI ‐10.00 to 9.89; two studies; 159 participants; P = 0.99; I² = 0%), but at doses greater than 1500 mg/day, ALC reduced pain more than placebo (MD ‐14.93, 95% CI ‐19.16 to ‐10.70; three studies; 381 participants; P < 0.00001; I² = 0%). This subgroup analysis should be viewed with caution as the evidence was even less certain than the overall analysis, which was already of very low certainty. Two placebo‐controlled studies reported that vibration perception improved after 12 months. We graded this evidence as very low certainty, due to inconsistency and a high risk of bias, as the trial authors did not provide any numerical data. The placebo‐controlled studies did not measure functional impairment and disability scores. No study used validated symptom scales. One study performed sensory testing, but the evidence was very uncertain. The fourth included study compared ALC with methylcobalamin, but did not report effects on pain. There was a reduction from baseline to 24 weeks in functional impairment and disability, based on the change in mean Neuropathy Disability Score (NDS; scale from zero to 10), but there was no important difference between the ALC group (mean score 1.66 ± 1.90) and the methylcobalamin group (mean score 1.35 ± 1.65) groups (P = 0.23; low‐certainty evidence). One placebo‐controlled study reported that six of 147 participants in the ALC > 1500 mg/day group (4.1%) and two of 147 participants in the placebo group (1.4%) discontinued treatment because of adverse events (headache, facial paraesthesia, and gastrointestinal disorders) (P = 0.17). The other two placebo‐controlled studies reported no dropouts due to adverse events, and more pain, paraesthesia, and hyperaesthesias in the placebo group than the 3000 mg/day ALC group, but provided no numerical data. The overall certainty of adverse event evidence for the comparison of ALC versus placebo was low. The study comparing ALC with methylcobalamin reported that 34/117 participants (29.1%) experienced adverse events in the ALC group versus 33/115 (28.7%) in the methylcobalamin group (P = 0.95). Nine participants discontinued treatment due to adverse events (ALC: 4 participants, methylcobalamin: 5 participants), which were most commonly gastrointestinal symptoms. The certainty of the adverse event evidence for ALC versus methylcobalamin was low. Two studies were funded by the manufacturer of ALC and the other two studies had at least one co‐author who was a consultant for an ALC manufacturer. AUTHORS' CONCLUSIONS: We are very uncertain whether ALC causes a reduction in pain after 6 to 12 months' treatment in people with DPN, when compared with placebo, as the evidence is sparse and of low certainty. Data on functional and sensory impairment and symptoms are lacking, or of very low certainty. The evidence on adverse events is too uncertain to make any judgements on safety.

Aims/Introduction To assess the efficacy and safety of acetyl‐L‐carnitine (ALC) on diabetic peripheral neuropathy compared with methylcobalamin (MC). Materials and methods This was a multicenter, randomized, parallel‐group, double‐blind, double‐dummy, positive‐controlled, non‐inferior phase II clinical trial. Diabetic patients with abnormal nerve conduction test results were randomized in a 1:1 ratio to receive oral ALC 500 mg t.i.d. or MC 0.5 mg t.i.d. for 24 weeks. The neuropathy symptom score, neuropathy disability score and neurophysiological parameters were measured during follow up. Results A total of 232 patients were randomized (ALC n = 117, MC n = 115), 88% of which completed the trial. At week 24, patients from both groups had significant reductions in both neuropathy symptom score and neuropathy disability score with no significant difference between two groups (neuropathy symptom score reduction: ALC vs MC 2.35 ± 2.23, P

The feasible fabrication of nerve guidance conduits (NGCs) with good biological performance is important for translation in clinics. In this study, poly(d,l-lactide-co-caprolactone) (PLCL) films loaded with various amounts (wt; 5%, 15%, 25%) of methylcobalamin (MeCbl) are prepared, and are further rolled and sutured to obtain MeCbl-loaded NGCs. The MeCbl can be released in a sustainable manner up to 21 days. The proliferation and elongation of Schwann cells, and the proliferation of Neuro2a cells are enhanced on these MeCbl-loaded films. The MeCbl-loaded NGCs are implanted into rats to induce the regeneration of 10 mm amputated sciatic nerve defects, showing the ability to facilitate the recovery of motor and sensory function, and to promote myelination in peripheral nerve regeneration. In particular, the 15% MeCbl-loaded PLCL conduit exhibits the most satisfactory recovery of sciatic nerves in rats with the largest diameter and thickest myelinated fibers.

Eur J Clin Invest 2011; 41 (4): 442–450 Abstract Background Methyl-base-attached cobalamin (Methycobalamin) (MC) has a special affinity for nerve tissues to promote myelination and transport of axonal cytoskeleton. It is not known, however, how MC influences on peripheral nerve in experimental diabetic neuropathy. Materials and methods We studied the effects of MC on expressions and activities of protein kinase C (PKC) in peripheral nerve of streptozotocin-induced diabetic rats. Wistar rats, 8 weeks of age, were rendered diabetic by streptozotocin (40 mg kg−1, iv) and followed for 16 weeks. A half of diabetic animals were treated with MC (10 mg kg−1 per every other day, im) after the induction of diabetes. Normal Wistar rats were served as control. Results At the end, untreated diabetic animals developed significant delay of nerve conduction velocity (NCV), and MC treatment normalized the NCV. Nerve PKC activity was significantly suppressed in untreated diabetic rats, while the activity was normalized in treated animals. While PKCα located in Schwann cells, PKCβΙα and βII distributed in axoplasm, vascular walls and macrophages. The decreased PKC activity in diabetic nerve was associated with reduced expression of membrane PKCα and increased membrane expression of PKCβII, and MC treatment corrected these changes. Diabetic nerve contained an increased number of macrophages and 8-hydroxydeoxyguanosine-positive cells in the endoneurium, the latter of which was significantly suppressed by MC treatment. Elevated nerve polyol levels in diabetic nerve were partially corrected by MC treatment. Conclusions This study suggested that correction of impaired neural signalling of PKC and oxidative stress–induced damage may be a major attribute to the beneficial effects of MC on diabetic nerve.

Background: Methionine synthase (MS) is a ubiquitous enzyme that requires vitamin B12 (cobalamin) and 5-methyl-tetrahydrofolate for the methylation of homocysteine to methionine. Previous studies have shown that acute or chronic ethanol (ETOH) administration results in the inhibition of MS and depletion of glutathione (GSH), and it has been proposed that GSH is required for the synthesis of methylcobalamin (MeCbl). Methods: We measured GSH levels and investigated the ability of different cobalamin cofactors [cyano- (CNCbl), glutathionyl- (GSCbl), hydroxo- (OHCbl), and MeCbl] to support MS activity in liver and brain cortex from control and ETOH-treated rats. Results: In control animals, MS activity was higher in liver than in cortex for all cobalamins and MeCbl-based activity was higher than for other cofactors. S-adenosylmethionine (SAM) was required for OHCbl, CNCbl, and GSCbl-based activity, but not for MeCbl. Feeding an ETOH-containing diet for four weeks caused a significant decrease in liver MS activity, in a cobalamin-dependent manner (OHCbl ≥ CNCbl > GSCbl > MeCbl). In brain cortex, OHCbl, CNCbl, and GSCbl-based activity was reduced by ETOH treatment, but MeCbl-based activity was unaffected. GSH levels were reduced by ETOH treatment in both liver and cortex homogenates, and addition of GSH restored OHCbl-based MS activity to control levels. Betaine administration had no significant effect on GSH levels or MS activity in either control or ETOH-fed groups. Conclusions: The ETOH-induced decrease in OHCbl-based MS activity is secondary to decreased GSH levels and a decreased ability to synthesize MeCbl. The ability of MeCbl to completely offset ETOH inhibition in brain cortex, but not liver, suggests tissue-specific differences in the GSH-dependent regulation of MS activity.

Cobalamin C (Cbl-C) defect is the most common inborn cobalamin metabolism error; it causes impaired conversion of dietary vitamin B12 into its two metabolically active forms, methylcobalamin and adenosylcobalamin. Cbl-C defect causes the accumulation of methylmalonic acid and homocysteine and decreased methionine synthesis. The gene responsible for the Cbl-C defect has been recently identified, and more than 40 mutations have been reported. MMACHC gene is located on chromosome 1p and catalyzes the reductive decyanation of CNCbl. Cbl-C patients present with a heterogeneous clinical picture and, based on their age at onset, can be categorized into two distinct clinical forms. Early-onset patients, presenting symptoms within the first year, show a multisystem disease with severe neurological, ocular, haematological, renal, gastrointestinal, cardiac, and pulmonary manifestations. Late-onset patients present a milder clinical phenotype with acute or slowly progressive neurological symptoms and behavioral disturbances. To improve clinical course and metabolic abnormalities, treatment of Cbl-C defect usually consists of a combined approach that utilizes vitamin B12 to increase intracellular cobalamin and to maximize deficient enzyme activities, betaine to provide a substrate for the conversion of homocysteine into methionine through betaine-homocysteine methyltransferase, and folic acid to enhance remethylation pathway. No proven efficacy has been demonstrated for carnitine and dietary protein restriction. Despite these measures, the long-term follow-up is unsatisfactory especially in patients with early onset, with frequent progression of neurological and ocular impairment. The unfavorable outcome suggests that better understanding of the pathophysiology of the disease is needed to improve treatment protocols and to develop new therapeutic approaches.

Plasma and buffy coat specimens of CML patients with untreated disease, in chronic and accelerated phase, and in overt blastic crises were analysed for the cobalamin patterns using non-polar extraction, thin-layer chromato-graphy and bioautography. Splenic tissue specimens from four splenectomized patients in accelerated phase were analysed similarly. Cellular extracts were separated into two compartments by adsorption to haptocorrin antibodies. Plasma concentrations of all cobalamin forms were increased in CML. The proportion of methylcobalamin was significantly lower than in a reference population, and a low plasma proportion of methylcobalamin was associated with a poor prognosis. Buffy coat cells and splenic tissue had a higher proportion of 5′-deoxyadenosylcobalamin and a lower proportion of methylcobalamin than plasma; still, there was relatively more methylcobalamin than in normal tissue. The haptocorrin-bound compartment differed from the non-haptocorrin compartment in untreated or chronic phase patients by binding less methylcobalamin. An estimate of cobalamin analogues in plasma was achieved by comparing two different isotope dilution assays employing a cobalamin-specific binder, intrinsic factor, and a nonspecific binder, hog non-intrinsic factor. Values for total cobalamin and analogues were increased to the same degree in CML plasma.

The methylation of inorganic Hg(II) by methylcobalamin in aquatic systems was studied using high-performance liquid chromatography coupled with UV-digestion cold vapor atomic fluorescence spectrometry (HPLC-UV-CV AFS). Monomethylmercury (MMC) could be positively identified as the reaction product in the aqueous solution. The salinity and pH of the aquatic system have great effects on the formation of MMC, because they could change the species of the reactants in the solution. From an electrophile reaction point of view, salinity and pH alter the electron density of the methyl donor and the electrophilicity of metal ion in the reaction system. This methylation of inorganic Hg(II) is shown to be possible even in highly saline solutions, which indicates its importance in aquatic environments. Kinetic experiments showed that the methylation reaction was fast and first-order for Hg(II). The first-order reaction rate was determined to be 0.00612 and 0.000287 min(-1) for pH 5.0 and 1.5, respectively. It is suggested that this methylation could occur in the absence of enzymes, in which Hg(II) acts as an electrophile to attack methylcobalamin with a subsequent transfer of carbanion methyl group to the higher oxidized state of Hg(II). Copyright (c) 2007 John Wiley & Sons, Ltd.

The preparation of a covalent DNA conjugate of vitamin B12 by means of heterogeneous solid-phase synthesis is reported. The cyano-corrinoid made available, dipotassium Co(beta)-cyanocobalamin-(3''-->2'),(3''-->5')-bis-2''-deoxythymidyl-3''-ate (K(2)-4), was cleanly methylated at the Co center by electrosynthetic means. Aqueous solutions of the resulting organometallic DNA-B12 conjugate K(2)-5 exhibited spectroscopic properties indicative of significant weakening of the axial (Co-N) bond, together with a 25-times higher basicity relative to Co(beta)-methylcobalamin (2). Methyl-transfer equilibria of pH-neutral aqueous solutions of K(2)-5 and cob(I)alamin (K-7) on one side, and of cob(I)alamin-(3''-->2'),(3''-->5')-bis-2''-deoxythymidyl-3''-ate (K(3)-8) and methylcobalamin (2) on the other, were studied at room temperature (Scheme 3). The NMR-derived data provided an equilibrium constant of ca. 0.3. Activation of K(2)-5 for abstraction of its Co-bound Me group by a nucleophile (such as cob(I)alamin) was, thus, indicated.

Glutathione (y-Glu-Cys-Gly, GSH) and methylcobalamin (CH 3 -B 12 ) may play a role in the biomethylation process of antimony. To understand better the transformation of antimony in biological systems, we studied abiotic and biomethylation processes and the influence of GSH in the methylation. CH 3 -B 12 , acting as a possible methylating agent for antimony, was studied with GSH and in the absence of GSH. The most abundant product of this reaction was monomethylantimony, with a small concentration of the dimethylantimony species, as identified by hydride generation cryotrapping gas chromatography inductively coupled plasma mass spectrometry (HG-CT-GC-ICP-MS). In the same experiments we found that tris(y-Glu-Cys-Gly)trithioantimonite [Sb(GS) 3 ] and di(y-Glu-Cys-Gly)methyldithioantimonite [(CH 3 )Sb(GS) 2 ] complexes were present using flow-injection electrospray ionization MS. Both complexes were also identified in a fermented sewage sample, suggesting that these complexes may play a role as intermediates in the biomethylation of antimony. However, CH 3 -B 12 is not the sole methylation agent, since it does not produce any trimethylantimony species as identified in anaerobic sewage sludge cultures inoculated with enriched 123 Sb(V). Species-specific 123/121 Sb isotope ratio measurements of the different methylantimony species suggest a stepwise methylation of antimony according to the Challenger mechanism.

Insolubilized antibody to transcobalamin I was used to separate transcobalamin I and transcobalamin II. By bioautography of the extracted cobalamins it was shown that transcobalamin II bound more deoxyadenosylcobalamin than did transcobalamin I, and that methylcobalamin accounts for most of the cobalamins attached to transcobalamin I. This finding may indicate that transcobalamin I has a function in the metabolism of methylcobalamin in man.

Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of chloromethane to formate. The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA 96, 4615-4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol x min-1 x (mg protein)-1. The apparent kinetic constants for H4folate were: Km = 240 microM, Vmax = 28.5 nmol x min-1 x (mg protein)-1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mM, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.

To develop a symptomatic treatment for amyotrophic lateral sclerosis, we compared the effects of ultrahigh-dose and low-dose (25 and 0.5 mg/day, intramuscularly, for 14 days) methylcobalamin on averaged compound muscle action potential amplitudes (CMAPs) in a double-blind trial. No significant changes in CMAP amplitude were found in 12 patients who had the low-dose treatment at either 2 or 4 weeks after start of treatment. By contrast, 12 patients assigned to the ultrahigh-dose group demonstrated a significant increase at 4 weeks. This method may provide a clinically useful measure to improve or retard muscle wasting, if a larger extended trial fulfills its promise.

Functional methionine synthase deficiency due to abnormal methylcobalamin metabolism causes megaloblastic anemia, moderate to severe developmental delay, lethargy, and anorexia in association with homocystinuria. Patients with this disorder of cobalamin metabolism can be classified into two separate groups, cblE or cblG, primarily on the basis of complementation analysis with cultured skin fibroblasts. We describe two unrelated boys, ages 3 and 5 years, with the cblG defect in methylcobalamin synthesis. Both children presented with severe developmental delay, lethargy, anorexia, and megaloblastic anemia. The diagnosis of homocystinuria was delayed in each case due to difficulties with detection of small amounts of homocystine in physiologic samples. The clinical course of cblG disease is favorably altered by treatment with intramuscular hydroxycobalamin. Megaloblastosis in the presence of adequate supplies of cobalamin and folate in the blood must alert the clinician to the possibility of functional methionine synthase deficiency and should prompt a careful search for associated biochemical hallmarks, including homocystinuria/emia. Am. J. Med. Genet. 71:384–390, 1997. © 1997 Wiley-Liss, Inc.

We compared the metabolic response of a methionine(Met)-dependent (P60) human glioma cell line with that of a Met-independent variant (P60H) when cultured in a homocysteine (Hcy) medium and exposed to N2O. In Hcy medium (without Met), remethylation of Hcy in P60H cells was enhanced and supported growth, whereas remethylation was low in P60 cells, which failed to thrive under these conditions. Both cell types seemed to contain adequate amounts of folates and total cobalamin (Cbl). P60 cells showed increased total and methylcobalamin (CH3Cbl) content after the shift to a Hcy medium, but the high, stable level of CH3Cbl detected in P60H cells was not attained. Further metabolic differences were induced by N2O exposure, which markedly reduced Met-synthase activity in cell-free extracts in both cell lines and completely blocked intact-cell Hcy remethylation in P60, whereas Hcy remethylation was only partly inhibited in P60H cells cultured in Met medium. The residual Hcy remethylation in P60H cells may be related to only a moderate depletion of CH3Cbl. The resulting high CH3Cbl level relative to Met-synthase activity during N2O exposure was even higher in Hcy medium. These findings in P60H cells probably reflect increased provision of Cbl to support Hcy remethylation under metabolic strain. The inability of P60 to furnish CH3Cbl to the enzyme may explain both the Met-dependent phenotype and the increased sensitivity of Hcy remethylation to N2O exposure in these cells.

Summary: Vibration perception thresholds (VPT) were measured using a new machine, the SMV-5 (Technolog Company, Tokyo, Japan) in 25 patients receiving maintenance haemodialysis without overt uraemic neuropathy. Patients who had other medical complications such as diabetes mellitus, dialysis amyloidosis or central nervous system dysfunction or peripheral vascular disease were excluded from this study. Forty per cent of the upper extremity VPT and 68% of the lower extremity VPT of the dialysis patients were abnormal, which was defined as greater than 2s.d. above the mean of the normal subjects. the VPT of smokers (n= 7) were significantly higher than those of non-smokers (n= 18; P

A new approach was used to determine the reaction products of methylcobalamin and ionic mercury: purge-and-trap gas chromatography in line with Fourier transform infrared spectroscopy (PT GCIFTIR). This technique simultaneously and specifically determines the spectrum of dimethylmercury (DMeHg) and methylmercury produced by the reaction. No interference from other known organic mercury species could be detected. The method is different from others because it does not require solvent extraction of the organomercurials from aqueous solution, but relies on immediate volatilization from the reaction vessel by addition of 100 pl of 10 m~ NaBH,,. The sample was purged with nitrogen for 10 min. The volatile species of mercury were trapped in a column at -120 "C, injected into the gas chromatograph and detected by FTIR. The efficiency of DMeHg and MeHg formation depended on different parameters: pH, temperature, reaction time, and the methylcobalamin/ionic mercury ratio. The initial reaction product was MeHg which was further transformed to DMeHg. The first methylation rate was two times faster than the second. MeHg formed first, reaching a maximum at higher temperatures (28 "C and 37 "C) and later decreasing as DMeHg formed. At lower temperatures (2OOC) the rate of MeHg formation was slower, being similar to the formation rate of DMeHg. Different species of inorganic mercury such as

Cobalamin cofactors play critical roles in radical-catalyzed rearrangements and in methyl transfers. This Account focuses on the role of methylcobalamin and its structural homologues, the methylcorrinoids, as intermediaries in methyl transfer reactions, and particularly on the reaction catalyzed by cobalamin-dependent methionine synthase. In these methyl transfer reactions, the cobalt(I) form of the cofactor serves as the methyl acceptor. Biological methyl donors to cobalamin include N5-methyltetrahydrofolate, other methylamines, methanol, aromatic methyl ethers, acetate, and dimethyl sulfide. The challenge for chemists is to determine the enzymatic mechanisms for activation of these unreactive methyl donors and to mimic these amazing biological reactions.

Inorganic germanium(II) in micromolar concentrations was reacted with methyl iodide (CH3I) and methylcobalamin (CH3-CoB12) at various pH values and with different salt matrices. In all experiments monomethylgermanium was the only product. The reaction with CH3-CoB12 at pH 1 yielded approximately 1.3% of the added germanium, whereas no methylation occurred at pH 7. Reaction yields with CH3I were lowest at pH 1 in 0.1 mol dm−3 KCl (1.6%) and highest at pH 7.6 in artificial seawater (6%). For the reaction of CH3−CoB12 with germanium(II) a free-radical mechanism is assumed, whereas methylation by CH3I is most likely an oxidative addition mechanism.

An unprecedented methylation of alkyl radicals is possible with the diamagnetic vitamin B(12) derivative methylcobalamin (see schematic representation). Methylcorrinoids can thus also be considered as methylating cofactors in radical C-methylations.

A simple and fast synthesis of radioactive methylmercury is presented. It is based on the methylation of inorganic 203Hg(II) by methylcobalamin and isolation of resulting CH3203Hg(II) in a single extraction step. Time required is less than 4 hours and the final yield is ⩾90%. © 1997 John Wiley & Sons, Ltd.

Functional methionine synthase deficiency is generally characterized by homocystinuria and hypomethioninemia in the absence of methylmalonic aciduria. Patients are divided into two classes, cblE and cblG, on the basis of complementation analysis. Presentation has usually been in the first 2 years of life, but one patient came to medical attention at age 21 years with symptoms initially diagnosed as multiple sclerosis. Common findings among 11 patients (4 with cblE and 7 with cblG) have included megaloblastic anemia (all patients) and various neurological deficits including developmental retardation (10 patients), cerebral atrophy (8 patients), hypotonia (7 patients), EEG abnormalities (6 patients), and nystagmus (5 patients). Hypertonia, seizures, blindness, and ataxia were less frequent. All patients have responded to therapy with cobalamin with resolution of anemia and biochemical abnormalities; neurological deficits resolved more slowly and in some cases incompletely. Hydroxycobalamin has been more effective than cyanocobalamin. Fibroblasts from patients with cblE (5 patients) and cblG (6 patients) all showed decreased intracellular levels of methylcobalamin (MeCbl) and decreased incorporation of label from 5-methyltetrahydrofolate into macromolecules, suggesting decreased activity of the MeCbl-dependent enzyme methionine synthase. Methionine synthase specific activity in extracts of all cblE fibroblasts was normal or near-normal under standard reducing conditions; synthase specific activity in extracts of 5 cblG patients was low but was high in a 6th patient measured in another laboratory. Thus, there is heterogeneity among patients with functional methionine synthase deficiency both in clinical presentation and in the results of biochemical studies of cultured cells.

Aqueous solutions of methylcobalamin (CH3B12) react with various water-insoluble metal oxides. The decomposition of methylcobalamin follows a kinetic pattern of two parallel first-order reactions. A proposed reaction mechanism involves the attachment of methylcobalamin to the oxide surface, followed by methyl transfer and dissolution of the oxide.

Here we report on a girl who presented with failure to thrive, developmental delay, minor facial anomalies, stomatitis, skin rashes, macrocytosis, mild homocystinemia(uria), and methylmalonic acidemia(uria). Fibroblast studies showed abnormal intracellular cobalamin (vitamin B12) metabolism. Reduced incorporation of 14C from [14C] propionate and [14C] methyltetrahydrofolate into TCA-precipitable macromolecules reflected decreased synthesis of adenosylcobalamin and methylcobalamin respectively. The diagnosis of cb1F mutation was established by demonstrating the accumulation of unmetabolized free cyanocobalamin in fibroblasts and by lack of genetic complementation with fibroblasts from the only other known cb1F patient. The defect is in the lysosomal release of endocytosed cobalamin. Administration of hydroxocobalamin resulted in clinical and biochemical improvement but sudden death occurred at age 5 months. The absence of brain pathological changes suggests that early treatment may prevent the neurological complications in cobalamin cofactor deficiency.

The preactivation of the vitamin B12-dependent methionine synthetase (5-methyl-5,6,7,8-tetrahydrofolate-homocysteine methyltransferase) from Escherichia coli B which is known to lead to a methylcobalamin enzyme has been shown by spectrophotometry to be initiated by a reduction of the cob(III)alamin (B12a) to a cob(II)alamin (B12r) enzyme. This one electron reduction is sufficient to add the methyl kryptocation of the potent methyl donor S-adenosylmethionine to the coenzyme, since B12r, to a small extent, disproportionates to yield B12a and B12s (cob(I)alamin), the latter being trapped from the equilibrium by S-adenosylmethionine. Within the transmethylation cycle, methylcobalamin enzyme donates its methyl group to homocysteine, the resulting B12s enzyme is remethylated by the less potent methyl donor 5-methyltetrahydrofolate thus closing the cycle. The latter reaction can be simulated by model experiments with the free coenzyme. From the experimental conditions, conclusions may be drawn concerning the active site of the holoenzyme.

Summary. The forms of vitamin B12 in guinea-pig ileum, liver and portal plasma were studied using a chromatographic-bioautographic technique. 5’Deoxyadeno-sylcobalamin was the principal cobalamin in each tissue although significant amounts of methylcobalamin were detected in plasma. An unidentified cobalamin was also found in all three tissues. During the absorption of labelled cyanocobalamin a proportion of this analogue was converted to 5’deoxyadenosylcobalamin in the ileum; preliminary experiments suggest that the site of inter-conversion was the mitochondria of the ileal epithelial cells. A significant proportion of the cyanocobalamin was transferred to the portal plasma unchanged however, indicating that the mitochondrial localization of vitamin B12 during its absorption, shown in previous studies, is not solely related to the conversion of vitamin B12 analogues from one form to another.

Die Co-Methyl-o-Bindung in Methylcobalamin wird bereits durch Bestrahlung bei niedriger Energie gespalten.

The demethylation of methylcobalamin with mercuric salts containing ClO4−, AcO−, Cl−, Br−, SCN−, or CN− was kinetically investigated by a conventional spectroscopic technique. The rates of reaction for mercuric chloride were proportional to [CH3–Co] and [HgCl2] while those for acetate depended little on [Hg(AcO)2], but increased in proportion with [AcO−]. The rates of reaction varied depending on the concentration and species - of anion, and the following sequence was obtained: AcO−>>Cl−≥SCN−≥Br−>>CN−. The sequence was reversely correlated with the stability constant of the equilibrium reaction, HgLn−1+L\ightleftarrowsHgLn(n=1 or 2), so that it was considered that HgL+ is an attacking electrophile in the cleavage of cobalt-carbon bond by the mercuric salts. The activity of HgCl42− for the reaction was somewhat lower than that of HgCl2 while that of HgClCN was lower to a greater extent.

Die Untersuchung der Reaktion von Methylcobalamin mit [AuX4]" (X: C1 oder Br) in wasriger Losung zeigt, das unter anaerobeu Bedingungen die Reaktion mit einer 1:2-Stochiometrie unter Bildung von Aquocobalumin mit einem oxidierten Corrinring, Methylhalogenid und metallischem Gold verlauft.