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Background Vitamin B-12 deficiency is widespread in many parts of the world, affecting all age groups and increasing with age. It is primarily due to a low intake of animal source foods or malabsorption. The measurement of bioavailability of vitamin B-12 is etiologically important in deficiency but is limited due to the use of radioactive isotopes like [57Co]- or [14C]-cyanocobalamin. Objectives The aim of this study was to measure the bioavailability of [13C]-cyanocobalamin in humans and to assess the effect of parenteral replenishment of vitamin B-12 on the bioavailability. Methods We synthesized a stable isotope-labeled vitamin B-12, [13C]-cyanocobalamin, using Salmonella enterica by providing [13C2]-ethanolamine as a sole carbon source. After purification and mass spectrometry-based characterization, its oral bioavailability was measured in the fasted state with high and low oral doses, before and after parenteral replenishment of vitamin B-12 stores, from the kinetics of its plasma appearance in a 2-compartment model. Results [13C]-cyanocobalamin was completely decyanated to [13C]-methylcobalamin describing metabolic utilization, and its plasma appearance showed early and late absorption phases. At a low dose of 2.3 µg, the mean bioavailability was 46.2 ± 12.8 (%, mean ± SD, n = 11). At a higher dose of 18.3 µg, the mean bioavailability was 7.6 ± 1.7 (%, mean ± SD, n = 4). Parenteral replenishment of the vitamin B-12 store in deficient individuals prior to the measurement resulted in a 1.9-fold increase in bioavailability. Conclusions Vitamin B-12 bioavailability is dose dependent and at a low dose that approximates the normal daily requirement (46%). The stable isotope method described here could be used to define the etiology of deficiency and to inform the dietary requirement in different physiologic states as well as the dose required for supplementation and food fortification. This trial was registered at the Clinical Trials Registry of India as CTRI/2018/04/012957.

Vitamin B12 is a vital water-soluble micronutrient required for maintaining the human health. However, one of the major encountered problems related to human health is a deficiency of vitamin B12 due to a strict vegetarian diet, malabsorption, pernicious anemia, surgical resection, etc. The deficiency of vitamin B12 can be overcome by taking vitamin supplements like methylcobalamin. Oral delivery of vitamin supplements is the most widely preferred route for administration as well as it gives good patient compliance. Particle size reduction of active ingredient enhances the dissolution rate which is very important in oral delivery. In the present study, methylcobalamin was re-crystallized using antisolvent crystallization technique and effect of various parameters like time (5–15 min), volume of solvent (0.5–1.5 mL), rate of addition (0.056–0.16 mL/min) and temperature (15–31 °C) on particle size was investigated. The aciculate shape of methylcobalamin was observed when re-crystallized using water as a solvent and acetone as an antisolvent. Increase in time from 5 min to 15 min increased the particle size (4.85 μm to 8.18 μm) while the increase in the volume of solvent from 0.5 mL to 1.5 mL has decreased the particle size (8.18 μm to 5.32 μm) and less effect was observed on particle size with a decrease in temperature (31 °C to 15 °C). The maximum impact on particle size (8.18 μm to 3.21 μm) was observed with the rate of addition from 0.16 mL/min to 0.056 mL/min. For water-acetone system, the particle size of methylcobalamin was found to decrease from 40.5 µm to 2.37 µm at 31 °C, 10 mL antisolvent, 0.5 mL solvent, 0.067 mL/min and 15 min. Hence, ∼18 times reduction in particle size was observed using antisolvent crystallization.

Background This study aimed to report the case of a patient who presented with depression, cognitive impairment, ataxic gait, and urinary incontinence associated with vitamin B12 deficiency. Case Description Serum vitamin B12 level was low in this patient, and anti-intrinsic factor antibody was positive. Neuroimaging revealed abnormal hyperintense signals in the cerebellum and dorsal and lateral columns of the spinal cord, and obstructive hydrocephalus. A biopsy of the stomach revealed chronic gastritis, intestinal metaplasia, and atrophy. After 3 months of initiating methylcobalamin therapy, significant improvement was noticed clinically, and brain magnetic resonance imaging was near to normal. Conclusions This study was novel in reporting subacute combined degeneration of the spinal cord and hydrocephalus associated with vitamin B12 deficiency in adults.

Chub mackerel (Scomber colias), an underutilised fish species, has been identified as an excellent source of selenium and vitamin B12, which may prevent and delay cognitive decline and dementia. Given these nutrients’ contents seasonal variability, a complete monthly sampling, involving a specifically adjusted methodology for the determination of vitamin B12 (LC-MS/MS) and its main vitamers and a sensitive analytical technique (ICPMS) for Se, was carried out. Regarding total vitamin B12 in the chub mackerel muscle, its content varied from 10.0 ± 1.0 μg/100 g in February to 18.9 ± 1.9 μg/100 g in August. It was also shown that methylcobalamin content ranged from 1.12 ± 0.43 (March) to 6.77 ± 0.80 μg/100 g (August), a sixfold increase. Hydroxocobalamin content varied between 3.49 ± 0.12 (February) and 9.62 ± 0.41 μg/100 g (August). The content of Se also had seasonal variability, ranging from 451 ± 14 μg/kg (December) to 606 ± 11 μg/kg (July). In the worst-case scenario, a weekly 160 g meal of this fish would almost guarantee B12 requirements. Three weekly 160 g meals of S. colias would cover, at least, 56% of the Se requirements. Therefore, as first explored here, it is highly relevant a complete, year-round assessment of the chub mackerel’s nutritional value, in order to elaborate finely tuned dietary recommendations. Highlights: Methylcobalamin rose with temperature from 1.12 ± 0.43 μg/100 g to 6.77 ± 0.80 μg/100 g; Hydroxocobalamin rose with temperature from 3.49 ± 0.12 μg/100 g to 9.62 ± 0.41 μg/100 g; A weekly meal of at least 160 g chub mackerel would cover vitamin B12 requirement; 3 weekly meals of at least 160 g chub mackerel may cover 56% of the Se requirement. This work was supported by national funds through FCT - Fundação para a Ciência e a Tecnologia, I.P., within the scope of projects New- Food4Thought PTDC/SAU-NUT/30455/2017 (Exploitation of the bioactive nutrients’ potential for the prevention of Alzheimer’s Disease and cognitive ageing through in vitro assays, in vivo verification, and in silico quantification of the health benefits) and CIISA (Project UIDB/ 00276/2020). Researchers were supported by the following Post- Doctoral Grants: Ref.: SFRH/BPD/102689/2014 (“Fundação para a Ciência e a Tecnologia”, FCT) for the author Carlos Cardoso and DIVERSIAQUA (MAR2020, Ref.: 16-02-01-FEAM-66) for the author Cláudia Afonso. The author Ana Gomes-Bispo also acknowledges FCT and IPMA for the DL57/2016 Junior Researcher Contract. info:eu-repo/semantics/publishedVersion

In this study, it was found that methylcobalamin-C-10-Br (MeCbl-C-10-Br) from stinky tofu exerts an inhibitory effect on Listeria monocytogenes, and the minimum inhibitory concentration was 0.125 ng/mL. Here, we studied the effect of MeCbl-C-10-Br on the adenosylcobalamin (AdoCbl)-dependent enzyme glycerol dehydratase, the methylcobalamin (MeCbl)-dependent enzyme methionine synthetase and three types of vitamin B12 riboswitches from L. monocytogenes. The results show that MeCbl-C-10-Br inhibits about 100% of glycerin dehydrase activity and about 37% methionine synthase activity. While the expression of the downstream genes of the vitamin B12 riboswitch is suppressed, the ratios of inhibition of transport, synthetic, and functional vitamin B12 riboswitches from 1.00 μg/mL MeCbl-C-10-Br are approximately 60%, 30%, and 40%, respectively, which suggests that MeCbl-C-10-Br effectively controls the growth of L. monocytogenes. According to the results of the simulated fermentation, MeCbl-C-10-Br did not destroy the balance of intestinal microbes after ingestion, which indicates that MeCbl-C-10-Br is a safe antibacterial agent and can be applied to food in the future.

Folate metabolism in the brain is critically important and serves a number of vital roles in nucleotide synthesis, single carbon metabolism/methylation, amino acid metabolism, and mitochondrial translation. Genetic defects in almost every enzyme of folate metabolism have been reported to date, and most have neurological sequelae. We report 2 patients presenting with a neurometabolic disorder associated with biallelic variants in the MTHFS gene, encoding 5,10-methenyltetrahydrofolate synthetase. Both patients presented with microcephaly, short stature, severe global developmental delay, progressive spasticity, epilepsy, and cerebral hypomyelination. Baseline CSF 5-methyltetrahydrolate (5-MTHF) levels were in the low-normal range. The first patient was treated with folinic acid, which resulted in worsening cerebral folate deficiency. Treatment in this patient with a combination of oral L-5-methyltetrahydrofolate and intramuscular methylcobalamin was able to increase CSF 5-MTHF levels, was well tolerated over a 4 month period, and resulted in subjective mild improvements in functioning. Measurement of MTHFS enzyme activity in fibroblasts confirmed reduced activity. The direct substrate of the MTHFS reaction, 5-formyl-THF, was elevated 30-fold in patient fibroblasts compared to control, supporting the hypothesis that the pathophysiology of this disorder is a manifestation of toxicity from this metabolite.

Epidemiological studies have indicated importance of folate and vitamin (B12) during pregnancy. Also available evidence on efficacy of B12 forms viz. Cyanocobalamin (Cbl), Methylcobalamin (MeCbl), Adenosylcobalamin (AdCbl) and Hydroxycobalamin (HCbl) in preventing or treating cobalamin deficiency is limited. The present study examines the effect of various forms of B12 in combination with folate during pregnancy and their effect on gestational outcomes.In the present study, we examined the effect of various vitamin B12 forms in presence of recommended folate (RFol: 400μg/day) and high folate (HFol: 5mg/day) on gestational outcomes in female Wistar rats.Dams dosed with excessive folate (HFol group) delivered low birth weight (LBW) offsprings (p0.01) as compared to RFol dams. Plasma homocysteine levels were found to be significantly higher (p0.05) in dams of HFol group and were reduced after vitamin B12 supplementation. Excessive folate supplementation and homocysteine levels showed inverse association with placental weight (p0.01) and placental efficiency (p0.05). B12 supplementation significantly up-regulated placental miR-16 and miR-21, associated with fetal growth which in turn reflected in improved birthweights. Supplementation with vitamin B12 forms, especially combination of active forms of cobalamins: MeCbl+AdCbl significantly increased birth weights (p0.05) and modulated gestational outcomes in RFol as well as HFol supplemented dams.Our results indicated supplementing vitamin B12 along with folate during pregnancy had positive impact on the gestational outcomes. We have shown for the first time that combination of active forms of vitamin B12: MeCbl+AdCbl has better efficacy as compared to Cbl, MeCbl, AdCbl and HCbl alone.

We investigated extracellular vitamin B12-producing Lactobacillus strains and their characteristics in tolerance to environmental stresses, gastric acid and bile salts. Two isolates, Lactobacillus plantarum LZ95 and CY2, showed high extracellular B12 production, 98±15μg/L and 60±9μg/L respectively. Extracellular B12 from LZ95 were identified as adenosylcobalamin and methylcobalamin using a combination of solid phase extraction and reverse-phase HPLC, while that from CY2 was adenosylcobalamin. Both strains grew under environmental stresses, and LZ95 exhibited better tolerance to low temperature and high ethanol concentration. LZ95 also showed good viability when exposed to gastric acid (pH 2.0 and 3.0) and bile salts (0.3%) as well as good adhesion to Caco-2 cells. The viability of CY2 was significantly reduced under low pH and exposure to bile salt. Together, extracellular B12 producer LZ95 with good probiotic properties might be a candidate for in situ B12 fortification in the food industry.

Despite advances in the understanding of the pathophysiology of ischemic stroke, therapeutic options remain limited. Methylcobalamin is an endogenous vitamin B12 that exhibits anti-inflammatory and antiapoptotic activities in a variety of diseases. In this study, we aimed to explore the neuroprotective effects and mechanism of action of methylcobalamin on cerebral ischemic injury in vitro and in vivo. The oxygen and glucose deprivation/reperfusion model and middle cerebral artery occlusion model were used to simulate cerebral ischemic injury in vitro and in vivo. Cell viability, inflammatory factors, cell apoptosis, and protein expression levels were determined. Further, autophagy flux and the cerebral infarction volume were measured. The modified neurological severity score, Longa score, Rotarod assay, and foot-fault test were used to evaluate behavioral changes and neurological deficits in rats. In vitro, methylcobalamin significantly increased cell viability, decreased lactate dehydrogenase release, attenuated inflammatory cytokine expression, reduced the apoptotic proportion, and enhanced autophagy flux after OGD treatment. In addition, Bcl-2 and Beclin1 expression levels and the LC3 II/I ratio were increased, whereas levels of Bax and cleaved caspase-3 were decreased. In vivo, methylcobalamin significantly reduced the cerebral infarction volume and neurological deficits in the rats. Furthermore, methylcobalamin activated the ERK1/2 pathway, whereas ERK1/2 inhibitors diminished its effects in the in vitro and in vivo models. In conclusion, methylcobalamin may exert a neuroprotective effect on cerebral ischemia and is a promising drug candidate for developing novel neuroprotective therapies.

Objective This article describes two patients with renal lithiasis who received a megadose of 25-hydroxy vitamin D (25[OH]D) and had a good outcome. Methods The first case reports a 74-year-old man with a long-term history of renal lithiasis and about four episodes of renal crisis. He was treated once with extracorporeal shock wave lithotripsy. He also had a history of dyslipidemia, myocardial infarction, and stroke. Laboratory tests demonstrated 25(OH)D of 28 ng/mL (normal range (nr): >30 ng/mL), normal lipid levels, creatinine of 1.1 mg/dL, and homocysteine of 26.6 mcmol/L (nr: 5–15 mcmol/L); parathyroid hormone (PTH) was high at 67.3 pg/mL (nr: 10–65 pg/mL), serum total calcium was 8.6 mg/dL, 24-h urinary calcium was 139 mg/d (normal range 100–300 mg/d), and urinary sediment was normal. He received 50 000 IU per week of vitamin D for 3 mo, and 25(OH)D increased to 36.6 ng/mL. Urinary calcium was 142 mg/d, PTH was 46.7 pg/mL, and serum calcium was 9.6 mg/dL. No renal crisis was perceived. He asked for an alternative form of medication since he usually would forget to take drugs. Vitamin D in a single dose of 600 000 IU intramuscular was prescribed. He was asked to increase water intake to 2 to 3 L/d. After 3 mo his 25(OH)D was 75.0 ng/mL, serum calcium was 9.2 mg/dL, urinary calcium was 148 mg/d, and PTH was 38.7 pg/mL. He had no episodes of lithiasis renal crisis. Folic acid and methylcobalamin were added, and homocysteine normalized. At follow-up 3 y later, the patient was asymptomatic, cardiologic evaluation was stable without any other renal lithiasis crises, 25(OH)D continued to be normal at 62 ng/mL, and he received a megadose of vitamin D every 6 mo. Renal ultrasound revealed only microlithiasis. The second case reports a 52-year-old man with a long-term history of renal lithiasis experienced since he was 30 y old, with three renal crisis episodes. He was treated with an extracorporeal shock wave three times. Laboratory tests demonstrated 25(OH)D 18 ng/mL, normal biochemistry, total serum calcium of 10.2 mg/dL, 24-h urinary calcium of 154 mg/d, and normal urinary sediment. He received 50 000 IU per week of 25(OH)D for 3 mo, and 25(OH)D increased to 40.3 ng/mL. Urinary calcium was 167 mg/d, PTH was 35.3 pg/mL, and serum calcium was 10.1 mg/dL. No renal crisis was perceived. He asked for an alternative form of medication, and vitamin D in a single dose of 600 000 IU intramuscular was prescribed. He was asked to increase water intake to 2 to 3 L/d. After 3 mo, his 25(OH)D was 82.0 ng/mL, serum calcium was 9.6 mg/dL, urinary calcium was 175 mg/d, and PTH was 35.3 pg/mL. The renal ultrasound was unchanged. He had no episodes of lithiasis renal crisis. At follow-up 4 y later, the patient was asymptomatic without any other renal lithiasis crises, a renal ultrasound revealed a reduction of calculi size to microlithiasis, 25(OH)D continues normal, and he received a megadose of this vitamin every 4 mo. Conclusion To the best of our knowledge, this is the first description of a megadose of vitamin D used in patients with nephrolithiasis. Furthermore, this shows the safety of this strategy in patients without hypercalciuria.

Summary B vitamin therapy lowers plasma total homocysteine concentrations, and might be a beneficial intervention for stroke prevention; however, cyanocobalamin (a form of vitamin B12) can accelerate decline in renal function and increase the risk of cardiovascular events in patients with impaired renal function. Although early trials did not show benefit in reduction of stroke, these results might have been due to harm in participants with impaired renal function. In patients with diabetic nephropathy, cyanocobalamin is harmful, whereas B vitamins appear to reduce cardiovascular events in study participants with normal renal function. Our meta-analysis of individual patient data from two large trials of B vitamin therapy (VISP and VITATOPS) indicates that patients with impaired renal function who are exposed to high-dose cyanocobalamin do not benefit from therapy with B vitamins for the prevention of stroke (risk ratio 1·04, 95% CI 0·84–1·27), however, patients with normal renal function who are not exposed to high-dose cyanocobalamin benefit significantly from this treatment (0.78, 0·67–0·90; interaction p=0·03). The potential benefits of B vitamin therapy with folic acid and methylcobalamin or hydroxycobalamin, instead of cyanocobalamin, to lower homocysteine concentrations in people at high risk of stroke warrant further investigation.

Peripheral nerve injury is one of common traumas. Although injured peripheral nerves have the capacity to regenerate, axon regeneration proceeds slowly and functional outcomes are often poor. Pharmacological enhancement of regeneration can play an important role in increasing functional recovery. In this study, we developed a novel electrospun nanofiber sheet incorporating methylcobalamin (MeCbl), one of the active forms of vitamin B12 homologues, to deliver it enough locally to the peripheral nerve injury site. We evaluated whether local administration of MeCbl at the nerve injury site was effective in promoting nerve regeneration. Electrospun nanofiber sheets gradually released MeCbl for at least 8 weeks when tested in vitro. There was no adverse effect of nanofiber sheets on function in vivo of the peripheral nervous system. Local implantation of nanofiber sheets incorporating MeCbl contributed to the recovery of the motor and sensory function, the recovery of nerve conduction velocity, and the promotion of myelination after sciatic nerve injury, without affecting plasma concentration of MeCbl. Statement of Significance Methylcobalamin (MeCbl) is a vitamin B12 analog and we previously reported its effectiveness in axonal outgrowth of neurons and differentiation of Schwann cells both in vitro and in vivo. Here we estimated the effect of local administered MeCbl with an electrospun nanofiber sheet on peripheral nerve injury. Local administration of MeCbl promoted functional recovery in a rat sciatic nerve crush injury model. These sheets are useful for nerve injury in continuity differently from artificial nerve conduits, which are useful only for nerve defects. We believe that the findings of this study are relevant to the scope of your journal and will be of interest to its readership.

Background Chronic postthoracotomy pain (CPTP) consists of different types of pain. Some characteristics of CPTP are the same as those of recognized neuropathic pain syndromes. We aimed to determine the safety and efficacy of pregabalin and methylcobalamin combination (PG-B12) in comparison with diclofenac potassium (DP) in patients with CPTP. Methods One hundred consecutive patients with CPTP after posterolateral/lateral thoracotomy were prospectively randomly assigned and evaluated. Fifty patients were given PG-B12 and another 50 patients were given DP treatment. Visual Analogue Scale (VAS) and the Leeds Assessment of Neuropathic Symptoms and Signs (LANSS) scorings were performed previous to the treatment (day 0) and on the 15th, 30th, 60th, and 90th days. Adverse events were questioned. Results The mean ages were 58.7 ± 12.2 and 54.6 ± 14.5 years, and the mean durations of pain were 4.01 ± 1.04 and 3.8 ± 1.02 months, respectively. The number of patients with a VAS score less than 5 at the latest follow-up (VAS90 Conclusions PB-B12 is safe and effective in the treatment of CPTP with minimal side effects and a high patient compliance. These results should be supported by multidisciplinary studies with larger sample sizes and longer follow-ups.

April 2017 issue of Current Opinion in Chemical Biology is a special issue: "Biocatalysis & biotransformation * Bioinorganic Chemistry", The ability of cobalamin to coordinate different upper axial ligands gives rise to a diversity of reactivity. Traditionally, adenosylcobalamin is associated with radical-based rearrangements, and methylcobalamin with methyl cation transfers. Recently, however, a new role for adenosylcobalamin has been discovered as a light sensor, and a methylcobalamin-dependent enzyme has been identified that is suggested to transfer a methyl anion. Additionally, recent studies have provided a wealth of new information about a third class of cobalamin-dependent enzymes that do not appear to use an upper ligand. They function in reductive dehalogenations and epoxide reduction reactions. Finally, mechanistic details are beginning to emerge about the cobalamin-dependent S-adenosylmethionine radical enzyme superfamily for which the role of cobalamin has been largely enigmatic. ©2017 Elsevier Ltd, National Institute of Health (Grant no. R01-GM69857), National Institute of Health Grant (F32-GM108189)

Folic acid (FA) and iron are essential supplements during pregnancy. Similarly effects of vitamin B12 (B12) inadequacy and high folate and low B12 status, on pregnancy outcome are available. However there are no mandatory recommendations for B12. There are many forms of B12 viz. Cyanocobalamin (Cbl), Methylcobalamin (MeCbl), Adenosylcobalamin (AdCbl), and Hydroxycobalamin (HCbl) though there is limited consensus on which form has better efficacy. In the present study we have determined effect of various forms of B12 in the presence of two FA concentrations namely normal physiological (20ng/mL; NPFA) and supra-physiological (2000ng/mL; SPFA) concentration to mimic real time situation where FA is in excess due to supplementation. We assessed trophoblastic proliferation, viability, TNFα and EGFr mRNA expression, homocysteine, β-hCG and MDA levels. Trophoblastic viability was significantly suppressed at SPFA concentration and was restored by B12 treatment with Cbl, AdCbl and combination of MeCbl+AdCbl. The mRNA expressions of TNFα were up-regulated, while EGFr were down-regulated at SPFA concentrations, as validated by RT-PCR. Treatment with MeCbl+AdCbl significantly decreased homocysteine and MDA levels at SPFA concentrations. High levels of FA alone had a detrimental effect on placental health and functions as reflected by decreased viability, EGFr expression and increased TNFα expression, homocysteine and MDA levels. Combination of B12 active forms i.e. MeCbl+AdCbl was found to be most effective in neutralising excess folate effect in-vitro.

The purpose of this narrative review is to highlight insights into the importance and frequency of metabolic vitamin B12 (B12) deficiency, reasons why it is commonly missed, and reasons for the widespread but mistaken belief that treatment of B12 deficiency does not prevent stroke or improve cognitive function. Metabolic B12 deficiency is common, being present in 10%-40% of the population; is frequently missed; is easily treated; and contributes importantly to cognitive decline and stroke in older people. Measuring serum B12 alone is not sufficient for diagnosis; it is necessary to measure holotranscobalamin or functional markers of B12 adequacy such as methylmalonic acid or plasma total homocysteine. B-vitamin therapy with cyanocobalamin reduces the risk of stroke in patients with normal renal function but is harmful (perhaps because of thiocyanate accumulation from cyanide in cyanocobalamin) in patients with renal impairment. Methylcobalamin may be preferable in renal impairment. B12 therapy slowed gray matter atrophy and cognitive decline in the Homocysteine and B Vitamins in Cognitive Impairment Trial. Undiagnosed metabolic B12 deficiency may be an important missed opportunity for prevention of dementia and stroke; in patients with metabolic B12 deficiency, it would be prudent to offer inexpensive and nontoxic supplements of oral B12, preferably methylcobalamin or hydroxycobalamin. Future research is needed to distinguish the effects of thiocyanate from cyanocobalamin on hydrogen sulfide, and effects of treatment with methylcobalamin on cognitive function and stroke, particularly in patients with renal failure.

The purpose of this study was to compare the intestinal permeation of vitamin B12 and various derivatives thereof. Permeation behavior and cytotoxicity of four derivatives (coenzyme B12, hydroxocobalamin, methylcobalamin and 4-ethylphenylcobalamin) in comparison to vitamin B12 were evaluated in two different in vitro models, Caco-2 cells and freshly excised rat intestinal mucosa. Resazurin assay was used to evaluate cytotoxicity of the test substances. All test compounds were used at a concentration of 200 μg/ml. Permeation experiments were carried out for 3h and test compounds were quantified via reversed phase high performance liquid chromatography (HPLC). Cytotoxicity studies showed all test compounds are not toxic to cells. HPLC analyses of test compounds revealed the following rank order of increasing hydrophobicity: hydroxocobalamin

Objective This study was conducted to investigate and compare the effects of add-on folic acid and vitamin B12 supplementation on glycaemic control, insulin resistance and serum lipid profile in subjects with type 2 diabetes mellitus. Study design & Intervention This study was a randomized, multi-arm, open-label clinical trial. 80 patients with type 2 diabetes and on stable oral antidiabetics were enrolled and 20 patients each were randomly allocated to one of the four groups – Group A: add-on Folic acid (5 mg/day); Group B: add-on Methylcobalamin (500 mcg/day); Group C: add-on Folic acid (5 mg/day) + Methylcobalamin (500 mcg/day) and Group D: Standard oral anti-diabetic drugs. The patients were followed up after 8 weeks. Results HbA1c improved significantly in Groups B and C [median changes from baseline – 1.2 % (– 13 mmol/mol) and – 1.5 % (– 16 mmol/mol) respectively, p values 0.04 and 0.02 respectively] compared to Group D. Groups B and C also showed significant improvements in plasma insulin, insulin resistance and serum adiponectin compared to Group D. Serum homocysteine declined significantly in all three groups with add-on supplementation compared to standard treatment. No improvement in the lipid profile was noted in any of the groups. Conclusions Add-on supplementation with vitamin B12 improved glycaemic control and insulin resistance in patients with type 2 diabetes mellitus.

OBJECTIVES: The dietary supplement industry has grown substantially, and advances in technology have played important roles in the development of new forms of dietary supplements and delivery systems. While it is recommended to use compendia methods for testing dietary supplements, the analytical methods in place are often developed for single ingredients or simple matrices and are not always fit to be used for solving modern matrix challenges. Oil- and beadlet-filled, gastric acid-resistant capsule (OBGRC) is designed to improve nutrient delivery and absorption. However, it is challenging to accurately analyze labile nutrients in OBGRCs because nutrients within different physical locations are difficult to separate, and lengthy sample preparations lead to nutrient degradation. We developed and validated analytical methods to determine methylcobalamin (MeB12), 5-methyltetrahydrofolate (5-MTHF), and trans menaquinone-7 (MK-7) in OBGRCs. METHODS: MeB12 and 5-MTHF were extracted by completely dissolving the OBGRC shells using aqueous buffers and then applying hexane-facilitated liquid-liquid extraction to remove the oil phase and determined by RP-HPLC using superficially porous particles columns. For MK-7 analysis, the whole OBGRC was cut to open and the oil phase was collected to dissolve in a mixture of dimethyl sulfoxide/tetrahydrofuran/ethanol before analyzed by RP-HPLC with fluorescence detection after on-line, post-column zinc reduction. The methods were subjected to single-laboratory validations according to the United States Pharmacopeia (USP) guidelines for linearity, suitability, detection limits, specificity, accuracy, and precision. RESULTS: The methods achieved chromatographic resolution of target analytes without the expensive requirement of ultra-high pressure liquid chromatography and/or mass spectrometry. The methods have wide analytical ranges (from at least 50% to 250% of the input concentrations), high precision (repeatability relative standard deviations ranging from 1.04% to 4.90%), and high accuracy (spike recovery rate ranging from 89.2% to 108%). CONCLUSIONS: All three methods passed USP method validation criteria and have the potential to be widely adopted to analyze MeB12, 5-MTHF, and MK-7 in similar matrices for quality control purposes. FUNDING SOURCES: The research funding was provided by Ritual and Eurofins.

OBJECTIVES: Tempeh is a plant-based fermented food consumed as an alternative source of protein. This study aims to compare the nutritional contents of some cooked tempeh and understand which way tempeh contributes to daily needs regarding its protein composition, B12 (cobalamins) and B9 (folates) vitamin contents. METHODS: Amino acid profile was determined by UPLC-PDA. Protein scores to access protein quality were calculated especially for methionine. Folate vitamers (folic acid, 5- methyltetrahydrofolate, tetrahydrofolate, 5–10-formyltetrahydrofolate, 5-formyltetrahydrofolate and 10- formyltetrahydrofolate) and the different B12 vitamers (hydroxocobalamin, methylcobalamin, and cyanocobalamin) were determined by UPLC-MS/MS. Tempeh was analyzed “raw” and after two different cooking methods (sautéed and stewed). Samples were purchased in commercial surfaces from different brands and production sets. Sampling was performed in accordance with the requirements of the Food Composition Databases in compliance with available Data Quality Evaluation Systems. RESULTS: The sautéed sample is the one which most contribute for the population needs, with 34,7% and 27,3% of total amino acids (to woman with 55 kg and a man with 70 kg, respectively), 7% of folates and 32% of B12 vitamin (particularly, hydroxocobalamin), following the “raw” sample and, finally, the marinated sample. In this way, the culinary method chosen for cooking the tempeh reveals to be an essential aspect in order to obtain higher nutritional value from it. All tempeh samples present limiting amino acids, with methionine being one of them, whose contribution to daily needs, in the sautéed sample, is 26.4% for women and 20.7% for men, whose weight is 55 kg and 70 kg, respectively. CONCLUSIONS: This study reveals that tempeh is a source of B12 vitamin but should be consumed in combination with other foods as buckwheat (methionine rich source) to avoid the confining of the methylation cycle. The production of hydroxocobalamin during the process of fermentation of soy for tempeh manufacturing constitutes ongoing research. FUNDING SOURCES: This work was funded by Instituto Nacional de Saúde Doutor Ricardo Jorge as the funding entity of the DAN Department under the project PortFOODVeg.

Radical S-adenosylmethionine (SAM) (RS) methylases perform methylation reactions at unactivated carbon and phosphorus atoms. RS enzymes typically abstract a hydrogen from their substrates, generating a substrate-centered radical; class B RS methylases catalyze methyl transfer from SAM to cobalamin and then to a substrate-centered carbon or phosphorus radical. Radle et al. now show that Mmp10, an RS enzyme implicated in the methylation of Arg-285 in methyl coenzyme M reductase, binds a methylcobalamin cofactor required for methyl transfer from SAM to a peptide substrate. However, Mmp10 has little sequence homology to known methylases, suggesting this enzyme belongs to a new subclass of B12-dependent RS methylases.

Vitamin B12 is among the most essential biomolecules required for crucial metabolic processes in humans. Vitamin B12 was extracted from Chlorella vulgaris biomass under aqueous conditions, partially purified by passing the extract through amberlite XAD-2, Sep-Pak columns, and further purified by HPLC. The target peak eluent was subjected to characterisation by tandem mass spectrometry (MS/MS), selected ion recording (SIR) and multiple reaction monitoring (MRM) and identified as methylcobalamin (Me-Cbl). Quantification of Me-Cbl was carried out by microbiological and chemiluminescence methods, and found to be 29.87±2 μg/100 g and 26.84±2 μg/100 g dry weight, respectively. The presence of Me-Cbl was further substantiated using gold nanoparticle (AuNPs) based aptamer analysis, and found to be 28.02±2 μg/100 g dry weight. Good similarity was observed among all the methods. Methylcobalamin, a form of vitamin B12 was identified in C. vulgaris and this finding enhances its use as a nutritional supplement.

The aim of this work was to evaluate the vitamin B12 content and profile of cobalamin bioactive forms in raw and cooked beef. The study showed that vitamin B12 distribution is unequal among beef muscles. The content of this nutrient ranges from 0.7 to 1.5 μg/100 g of tissue. In beef, three biologically active forms of vitamin B12 were determined, i.e.: methylcobalamin, adenosylcobalamin and hydroxycobalamin; the dominating form of vitamin B12 was adenosylcobalamin (68% of total cobalamins). The process of roasting and grilling had little effect on the vitamin B12 content in the final product as compared to the raw meat. The fried product was characterised by about a 32% lower content of cobalamins than in raw meat.

Methylcobalamin (MeCbl) is a vitamin B12 analog that has some positive effects on peripheral nervous disorders. Although some previous studies revealed the effects of MeCbl on neurons, its effect on the muscle, which is the final target of motoneuron axons, remains to be elucidated. This study aimed to determine the effect of MeCbl on the muscle. We found that MeCbl promoted the proliferation and migration of C2C12 myoblasts in vitro and that these effects are mediated by the Erk1/2 signaling pathway without affecting the activity of the Akt signaling pathway. We also demonstrated that MeCbl inhibits C2C12 cell apoptosis during differentiation. Our results suggest that MeCbl has beneficial effects on the muscle in vitro. MeCbl administration may provide a novel therapeutic approach for muscle injury or degenerating muscle after denervation.

Aims To compare the efficacy and safety of daily lipoic acid (300–600 mg i.v.) plus methylcobalamin (500–1000 mg i.v. or im.) (LA–MC) with that of methylcobalamin alone (MC) on diabetic peripheral neuropathy (DPN). Methods Electronic database were searched for studies published up to November 1, 2012 and study quality was assessed in duplicate. A random or a fixed effect model was used to analyse outcomes which were expressed as risk ratios (RRs) or mean difference (MD). I 2 statistic was used to assess heterogeneity. Results Seventeen studies were included. Combined data from all studies showed that the LA–MC combination therapy was significantly superior to MC monotherapy (RR = 1.47; 95% CI: 1.37–1.58). Superiority of the LA–MC combination was shown in nerve conduction velocity (NCV) with WMDs of 6.89 (95% CI: 4.24–9.73) for median motor nerve conduction velocity (MNCV), 5.24 (4.14–6.34) for median sensory nerve conduction velocity (SNCV), 4.34 (3.03–5.64) for peroneal MNCV, and 4.53 (3.2–5.85) for peroneal SNCV. There were no serious adverse events associated with treatment. Conclusions The results of the meta-analysis show that treatment with LA–MC for 2–4 weeks is associated with better outcomes in NCV and neuropathic symptoms relative to MC treatment. However larger well-designed studies are required to confirm this conclusion.

Aim The purpose of this study is to develop simple, economic, specific, rapid, reliable, and reproducible method for simultaneous estimation of the Gabapentin, Methylcobalamin and Alpha lipoic acid. Method In this method the overlapping spectra of Gabapentin, Methylcobalamin and Alpha lipoic acid were well resolved by making use of the first-derivative of the ratios of their direct absorption spectra. The derivative ratio absorbance of Gabapentin, Methylcobalamin and Alpha lipoic acid were measured at 731.10 nm, 768.53 nm and 242.21 nm for their quantification. The method was validated for accuracy, precision, linearity, robustness and sensitivity. Result & discussion Gabapentin, Methylcobalamin and Alpha lipoic acid were shown linearity in the concentration range of 100–500 μg/ml, 0.5–2.5 μg/ml and 100–500 μg/ml respectively. The LOD & LOQ were found to be 3.09 μg/ml and 9.37 μg/ml; 0.03 μg/ml and 0.10 μg/ml; and 4.79 μg/ml and 14.52 μg/ml respectively. The % labelled claim for Gabapentin, Methylcobalamin and Alpha lipoic acid were found to be 98.71, 98.94 and 98.44 respectively. Conclusion Thus, the described method is suitable for routine analysis and quality control of pharmaceutical preparations containing these drugs in combination.

Vitamin B12 (cobalamin) is required for proper red blood cell formation, neurologic function, and DNA synthesis. Cobalamins in solutions are light sensitive, but no comprehensive study has been performed to compare the photostability of different cobalamins under UVA exposure. Their indirect photodegradation due to their antioxidant properties and their photostability in vivo have also not been studied so far. The photodegradation of four cobalamins (methylcobalamin (MeCbl), adenosylcobalamin (AdCbl), hydroxocobalamin (OHCbl) and cyanocobalamin (CNCbl)) under UVA exposure in aqueous solutions (pH=7.4) have been investigated by absorption spectroscopy. The photodegradation of OHCbl in the absence and presence of the endogenous photosensitizer riboflavin was studied. Serum vitamin B12 concentrations before and after summer were measured in four patients with psoriasis. All studied cobalamins are photolabile. The biologically active forms of cobalamin, AdCbl and MeCbl, are converted to OHCbl within seconds during UVA exposure. OHCbl is the most stable cobalamin. However, reactive oxygen species increases the degradation rate of OHCbl. Our pilot study on humans demonstrates that serum vitamin B12 concentrations are not significantly affected during summertime in Norway. Further work is needed to determine vitamin B12 photostability in humans living at lower latitudes or using sunbeds.

Mutations in cobalamin or B 12 trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cbl D, affects both the mitochondrial and cytoplasmic pathways for B 12 processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5′-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ∼116–296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways.

Background Since 1975 cells lines from patients with suspected inborn errors of vitamin B 12 metabolism have been referred to our laboratory because of elevations of homocysteine, methylmalonic acid, or both. Design Cultured fibroblasts from patients were subjected to a battery of tests: incorporation of labelled propionate and methyltetrahydrofolate into cellular macromolecules, to test the functional integrity of methylmalonyl-CoA mutase and methionine synthase, respectively; uptake of labelled cyanocobalamin and synthesis of adenosylcobalamin and methylcobalamin; and, where applicable, complementation analysis. Results This approach has allowed for the discovery of novel steps in the cellular transport and metabolism of vitamin B 12 , including those involving cellular uptake, the efflux of vitamin B 12 from lysosomes, and the synthesis of adenosylcobalamin and methylcobalamin. For all of these disorders, the responsible genes have been discovered. Conclusion The study of highly selected patients with suspected inborn errors of metabolism has consistently resulted in the discovery of previously unknown metabolic steps and has provided new lessons in biology.

Purpose To determine whether a combination of L-methylfolate, methylcobalamin, and pyridoxal-5′-phosphate (LMF-MC-PLP [Metanx; Pamlab LLC, Covington, La]) improves sensory neuropathy. Research Design and Methods This multicenter, randomized, double-blind, placebo-controlled trial involved 214 patients with type 2 diabetes and neuropathy (baseline vibration perception threshold [VPT]: 25-45 volts), who were randomly assigned to 24 weeks of treatment with either L-methylfolate calcium 3 mg, methylcobalamin 2 mg, and pyridoxal-5′-phosphate 35 mg or placebo. The primary end point was effect on VPT. Secondary end points included Neuropathy Total Symptom Score (NTSS-6) and Short Form 36 (SF-36), as well as plasma levels of folate, vitamins B 6 and B 12 , methylmalonic acid (MMA), and homocysteine. Results There was no significant effect on VPT. However, patients receiving LMF-MC-PLP consistently reported symptomatic relief, with clinically significant improvement in NTSS-6 scores at week 16 ( P =.013 vs placebo) and week 24 ( P =.033). Improvement in NTSS scores was related to baseline MMA and inversely related to baseline PLP and metformin use. Quality-of-life measures also improved. Homocysteine decreased by 2.7±3.0 μmol/L with LMF-MC-PLP versus an increase of 0.5±2.4 μmol/L with placebo ( P =.0001). Adverse events were infrequent, with no single event occurring in ≥2% of subjects. Conclusions LMF-MC-PLP appears to be a safe and effective therapy for alleviation of peripheral neuropathy symptoms, at least in the short term. Additional long-term studies should be conducted, as the trial duration may have been too short to show an effect on VPT. In addition, further research on the effects in patients with cobalamin deficiency would be useful.

An early step in the intracellular processing of vitamin B(12) involves CblC, which exhibits dual reactivity, catalyzing the reductive decyanation of cyanocobalamin (vitamin B(12)), and the dealkylation of alkylcobalamins (e.g. methylcobalamin; MeCbl). Insights into how the CblC scaffold supports this chemical dichotomy have been unavailable despite it being the most common locus of patient mutations associated with inherited cobalamin disorders that manifest in both severe homocystinuria and methylmalonic aciduria. Herein, we report structures of human CblC, with and without bound MeCbl, which provide novel biochemical insights into its mechanism of action. Our results reveal that CblC is the most divergent member of the NADPH-dependent flavin reductase family and can use FMN or FAD as a prosthetic group to catalyze reductive decyanation. Furthermore, CblC is the first example of an enzyme with glutathione transferase activity that has a sequence and structure unrelated to the GST superfamily. CblC thus represents an example of evolutionary adaptation of a common structural platform to perform diverse chemistries. The CblC structure allows us to rationalize the biochemical basis of a number of pathological mutations associated with severe clinical phenotypes.

The identification of eight genes involved in inherited cobalamin (Cbl) disorders has provided insight into the complexity of the vitamin B₁₂ trafficking pathway. Detailed knowledge about the structure, interaction, and physiological functions for many of the gene products, including the MMACHC and MMADHC proteins, is lacking. Having cloned, expressed, and purified MMACHC in Escherichia coli, we demonstrated its monodispersity by dynamic light scattering and measured its hydrodynamic radius, either alone or in complex with each of four vitamin B₁₂ derivatives. Using solution-phase intrinsic fluorescence and label-free, real-time surface plasmon resonance (SPR), MMACHC bound cyanocobalamin and hydroxycobalamin with similar low micromolar affinities (K(D) 6.4 and 9.8 μM, respectively); adenosylcobalamin and methylcobalamin also shared similar binding affinities for MMACHC (K(D) 1.7 and 1.4 μM, respectively). To predict specific regions of interaction between MMACHC and the proposed partner protein MMADHC, MMACHC was subjected to phage display. Five putative MMACHC-binding sites were identified. Finally, MMADHC was confirmed as a binding partner for MMACHC both in vitro (SPR) and in vivo (bacterial two-hybrid system).

Using ulnar nerve as donor and musculocutaneous nerve as recipient we recently demonstrated that end-to-end neurorrhaphy in young adult male Wistar rats resulted in good recovery following protracted survival. Here we explored whether anti-inflammatory drug- methylprednisolone, regeneration/myelination-enhancing agent- methylcobalamin and neurite growth-enhancing and angiogenic factor- pleiotrophin accelerated its recovery. Methylprednisolone suppressed the perineuronal microglial reaction and periaxonal ED-1 expression while pleiotrophin increased the blood vessel density and nerve fiber densities in the reconnected nerve as expected. Neither methylprednisolone nor methylcobalamin altered the expression of growth associated protein 43 in the neurons examined suggesting that they did not interfere with axonal regeneration attempt. Surprisingly methylcobalamin enhanced the recovery of compound muscle action potentials and motor end plate innervation and the performance on sticker removal grooming test and augmented the diameters and myelin thicknesses of regenerated axons dramatically while enhancing S-100 expression in Schwann cells; remarkable recovery was achieved 1 month following neurorrhaphy. Simultaneous methylcobalamin and pleiotrophin treatment resulted in quick and persistent supernumerary reinnervation but failed to enhance the recovery over that of the former alone. Methylprednisolone transiently suppressed the enumeration of regrowing axons. In conclusion, methylcobalamin may be preferred over methylprednisolone to facilitate the recovery of peripheral nerves following end-to-end neurorrhaphy. The long-term effect of this treatment however remains to be clarified.

Patients with the cblC vitamin B(12) (cobalamin, cbl) disorder are defective in the intracellular synthesis of adenosylcobalamin and methylcobalamin and have combined homocystinuria and methylmalonic aciduria. While other vitamin B(12) disorders are treatable with high dose cyanocobalamin (CNCbl) or hydroxocobalamin (OHCbl), cblC patients respond well to OHCbl but not to CNCbl. Patient mutations were introduced into recombinant MMACHC (cblC) protein and the binding of CNCbl and OHCbl was examined. Three mutations were analyzed: G147D, associated with early onset, vitamin B(12) unresponsive disease; R161Q, associated with late onset disease that is highly responsive to OHCbl; and H122A, selected to test the hypothesis that H122 is central to a proposed vitamin B(12) binding motif on MMACHC. We report here that wild-type MMACHC binds both OHCbl and CNCbl with similar, tight affinity (K(d)=5.7 microM). We also report that MMACHC binds CNCbl in the base-off form, with the dimethylbenzimidazole (DMB) base of cobalamin displaced from coordination with the cobalt. In this form, wild-type MMACHC is able to reductively decyanate CNCbl to cob(II)alamin requiring only the presence of NADPH and FAD. We demonstrate that MMACHC with the G147D mutation is unable to bind either CNCbl or OHCbl, providing a straight forward explanation for the absence of response to either vitamin form. However, we show that MMACHC containing the R161Q mutation binds OHCbl with wild-type affinity, but is disturbed in binding CNCbl and has impaired decyanation. Finally, we show that H122A has reduced binding, but like R161Q, it binds OHCbl more tightly than CNCbl, suggesting that this histidine is not absolutely required for binding. These studies suggest that the ability of mutant MMACHC to respond to vitamin therapy depends on its ability to bind the vitamin with significant affinity, and for CNCbl, also on its ability to bind in the base-off form to facilitate reductive decyanation. These studies emphasize the continued use of OHCbl with cblC patients for maximum therapeutic effect.

Pathways for tailoring and processing vitamins into active cofactor forms exist in mammals that are unable to synthesize these cofactors de novo. A prerequisite for intracellular tailoring of alkylcobalamins entering from the circulation is removal of the alkyl group to generate an intermediate that can subsequently be converted into the active cofactor forms. MMACHC, a cytosolic cobalamin trafficking chaperone, has been shown recently to catalyze a reductive decyanation reaction when it encounters cyanocobalamin. In this study, we demonstrate that this versatile protein catalyzes an entirely different chemical reaction with alkylcobalamins using the thiolate of glutathione for nucleophilic displacement to generate cob(I)alamin and the corresponding glutathione thioether. Biologically relevant thiols, e.g. cysteine and homocysteine, cannot substitute for glutathione. The catalytic turnover numbers for the dealkylation of methylcobalamin and 5'-deoxyadenosylcobalamin by MMACHC are 11.7 +/- 0.2 and 0.174 +/- 0.006 h(-1) at 20 degrees C, respectively. This glutathione transferase activity of MMACHC is reminiscent of the methyltransferase chemistry catalyzed by the vitamin B(12)-dependent methionine synthase and is impaired in the cblC group of inborn errors of cobalamin disorders.

The MMACHC gene product of the cblC complementation group, referred to as the cblC protein, catalyzes the in vitro and in vivo decyanation of cyanocobalamin (vitamin B 12 ). We hypothesized that the cblC protein would also catalyze the dealkylation of newly internalized methylcobalamin (MeCbl) and 5′-deoxyadenosylcobalamin (AdoCbl), the naturally occurring alkylcobalamins that are present in the diet. The hypothesis was tested in cultured endothelial cells using [ 57 Co]-AdoCbl and MeCbl analogs consisting of [ 57 Co]-labeled straight-chain alkylcobalamins ranging from C2 (ethylcobalamin) to C6 (hexylcobalamin). [ 57 Co]-AdoCbl was converted to [ 57 Co]-MeCbl by cultured bovine aortic endothelial cells, suggesting that a dealkylation process likely involving the cblC protein removed the 5′-deoxyadenosyl alkyl group. Surprisingly, all of the straight-chain alkylcobalamins served as substrates for the biosynthesis of both AdoCbl and MeCbl. Dealkylation was then assessed in normal skin fibroblasts and fibroblasts derived from three patients with mutations in the MMACHC gene. While normal skin fibroblasts readily converted [ 57 Co]-propylcobalamin to [ 57 Co]-AdoCbl and [ 57 Co]-MeCbl, there was little or no conversion in cblC mutant fibroblasts. These studies suggest that the CblC protein is responsible for early processing of both CNCbl (decyanation) and alkylcobalamins (dealkylation) in mammalian cells.

Objectives To describe 3 patients with the cblD disorder, a rare inborn error of cobalamin metabolism caused by mutations in the MMADHC gene that can result in isolated homocystinuria, isolated methylmalonic aciduria, or combined homocystinuria and methylmalonic aciduria. Study design Patient clinical records were reviewed. Biochemical and somatic cell genetic studies were performed on cultured fibroblasts. Sequence analysis of the MMADHC gene was performed on patient DNA. Results Patient 1 presented with isolated methylmalonic aciduria, patient 3 with isolated homocystinuria, and patient 2 with combined methylmalonic aciduria and homocystinuria. Studies of cultured fibroblasts confirmed decreased synthesis of adenosylcobalamin in patient 1, decreased synthesis of methylcobalamin in patient 3, and decreased synthesis of both cobalamin derivatives in patient 2. The diagnosis of cblD was established in each patient by complementation analysis. Mutations in the MMADHC gene were identified in all patients. Conclusions The results emphasize the heterogeneous clinical, cellular and molecular phenotype of the cblD disorder. The results of molecular analysis of the MMADHC gene are consistent with the hypothesis that mutations affecting the N terminus of the MMADHC protein are associated with methylmalonic aciduria, and mutations affecting the C terminus are associated with homocystinuria.

The methylation reaction of arsenic trioxide conducted at 37 °C and pH 7.0 for 24 h using hydrophobic methylated vitamin B12, (methyl) (aquo) heptamethylcobyrinate perchlorate, CH3B12 ester, as a methyl donor in the presence of reduced glutathione (GSH) yielded monomethylarsonous acid (MMA), dimethylarsinic acid (DMA), and trimethylarsine oxide (TMAO) as products with a methylation rate over 95%. In contrast, when methylcobalamin (CH3B12) was used as the methyl donor, only MMA and DMA were produced and the methylation rate dropped to around 20%. Reductive demethylation of a methyl-corrinoid coordination complex mediated by GSH is suggested as a mechanism of methyl transfer to arsenic trioxide. The differences observed for different corrinoid coordination complexes with respect to the reactivity of methyl transfer to arsenic is ascribable to differences inherent in the base-on (CH3B12) and base-off (CH3B12 ester) natures of the compounds.

Background: Metabolic abnormalities and targeted treatment trials have been reported for several neurobehavioral disorders but are relatively understudied in autism. Objective: The objective of this study was to determine whether or not treatment with the metabolic precursors, methylcobalamin and folinic acid, would improve plasma concentrations of transmethylation/transsulfuration metabolites and glutathione redox status in autistic children. Design: In an open-label trial, 40 autistic children were treated with 75 μg/kg methylcobalamin (2 times/wk) and 400 μg folinic acid (2 times/d) for 3 mo. Metabolites in the transmethylation/transsulfuration pathway were measured before and after treatment and compared with values measured in age-matched control children. Results: The results indicated that pretreatment metabolite concentrations in autistic children were significantly different from values in the control children. The 3-mo intervention resulted in significant increases in cysteine, cysteinylglycine, and glutathione concentrations (P < 0.001). The oxidized disulfide form of glutathione was decreased and the glutathione redox ratio increased after treatment (P < 0.008). Although mean metabolite concentrations were improved significantly after intervention, they remained below those in unaffected control children. Conclusion: The significant improvements observed in transmethylation metabolites and glutathione redox status after treatment suggest that targeted nutritional intervention with methylcobalamin and folinic acid may be of clinical benefit in some children who have autism. This trial was registered at clinicaltrials.gov as NCT00692315.

Resolution of binary mixtures of vitamin B12, methylcobalamin and B12 coenzyme with minimum sample pre-treatment and without analyte separation has been successfully achieved by methods of partial least squares algorithm with one dependent variable (PLS1), orthogonal signal correction/partial least squares (OSC/PLS), principal component regression (PCR) and hybrid linear analysis (HLA). Data of analysis were obtained from UV-vis spectra. The UV-vis spectra of the vitamin B12, methylcobalamin and B12 coenzyme were recorded in the same spectral conditions. The method of central composite design was used in the ranges of 10-80 mg L(-1) for vitamin B12 and methylcobalamin and 20-130 mg L(-1) for B12 coenzyme. The models refinement procedure and validation were performed by cross-validation. The minimum root mean square error of prediction (RMSEP) was 2.26 mg L(-1) for vitamin B12 with PLS1, 1.33 mg L(-1) for methylcobalamin with OSC/PLS and 3.24 mg L(-1) for B12 coenzyme with HLA techniques. Figures of merit such as selectivity, sensitivity, analytical sensitivity and LOD were determined for three compounds. The procedure was successfully applied to simultaneous determination of three compounds in synthetic mixtures and in a pharmaceutical formulation.

The acquisition of resistance by malaria parasites towards existing antimalarials has necessitated the development of new chemotherapeutic agents. The effect of vitamin B(12) derivatives on the formation of beta-haematin (synthetic haemozoin) was determined under conditions similar to those in the parasitic food vacuole (using chloroquine, a known inhibitor of haemozoin formation for comparison). Adenosylcobalamin (Ado-cbl), methylcobalamin (CH(3)-cbl) and aquocobalamin (H(2)O-cbl) were approximately forty times more effective inhibitors of beta-haematin formation than chloroquine, cyanocobalamin (CN-cbl) was slightly more inhibitory than chloroquine, while dicyanocobinamide had no effect. It is proposed that the cobalamins exert their inhibitory effect on beta-haematin formation by pi-interactions of their corrin ring with the Fe(III)-protoporphyrin ring and by hydrogen-bonding using their 5,6-dimethylbenzimidazole/ribose/sugar side-chain. The antimalarial activity for the cobalamins (Ado-cbl>CH(3)-cbl>H(2)O-cbl>CN-cbl) was found to be less than that for chloroquine or quinine. Ado-cbl, CH(3)-cbl and CN-cbl do not accumulate in the parasite food vacuole by pH trapping, but H(2)O-cbl does. Unlike humans, the malaria parasite has only one enzyme that uses cobalamin as a cofactor, namely methionine synthase, which is important for growth and metabolism. Thus cobalamins in very small amounts are necessary for Plasmodium falciparum growth but in larger amounts they display antimalarial properties.

Inborn errors of vitamin B12 (cobalamin, Cbl) metabolism are autosomal recessive disorders and have been classified into nine distinct complementation classes (cblA-cblH and mut). Disorders affecting methylcobalamin metabolism cause megaloblastic anemia, which may be accompanied by leukopenia and thrombocytopenia, and a variety of neurological problems. Disorders affecting adenosylcobalamin cause methylmalonic acidemia and metabolic acidosis. Previous studies have shown that cobalamin binds to two enzymes in humans: methylmalonyl-CoA mutase in mitochondria and methionine synthase in the cytosol. In this study, cobalamin binding patterns were analyzed in crude mitochondrial fractions obtained from both control and patient fibroblasts that had been incubated with [57Co]cyanocobalamin. Crude mitochondrial fractions from control fibroblasts confirmed that the majority of [57Co]Cbl eluted with methylmalonyl-CoA mutase. However, in six of the nine disorders, at least one previously unidentified mitochondrial cobalamin binding protein was observed to bind [57Co]Cbl. The proportion of [57Co]Cbl that binds, is increased compared to controls when a deficiency in either adenosylcobalamin synthesis or utilization prevents binding to methylmalonyl-CoA mutase. Furthermore, unique cobalamin binding profiles emerged demonstrating how known mutations in these patients affect cobalamin binding to as yet unidentified proteins.

The electronic structure of hydroxocobalamin (OHCbl) has been calculated by a density functional method, using the orthogonalized linear combination of the atomic orbitals method (OLCAO). The X-ray crystal structure has been determined from synchrotron X-ray diffraction data and the geometry determined was used in the calculations. Comparison with the recently reported electronic structures of cyanocobalamin (CNCbl), methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) shows that Mulliken charges (Q*) and bond orders (BO) vary only on the axial fragment.

Soft X-ray fluorescence measurements are used to characterize three groups of biomaterials: Vitamin B 12 and derivatives, antioxidants (aspirin and paracetamol), and human teeth. We show that the chemical bonding in Vitamin B 12 is characterized by the strong CoC bond and the relatively weak CoN bond. The CoC bond in cyanocobalamin is found to be stronger than that of methylcobalamin leading to their different biological activity. The chemical bonding of paracetamol and aspirin is characterized by the formation of oxygen lone-pair π-orbitals, which can neutralize free radicals and therefore be related to antioxidant activity of these compounds. Carbon Kα emission spectra of a caries lesion suggest that the CaCO 3 like phase exists in sound enamel and that a selective loss of carbonate occurs during the early stages of a caries attack.

Micro-high-performance liquid chromatography interfaced to inductively coupled plasma mass spectrometry was optimized for the determination and separation of a mixture of cobalt containing species. Four cobalamin species (cyanocobalamin, hydroxocobalamin, methylcobalamin, and 5′-deoxyadenosylcobalamin) representing the various forms of vitamin B12 as well as the harmful corrinoid analogue cobinamide dicyanide were separated using reversed-phase microcapillary chromatography with columns containing C18 packing material with a 2-μm particle size. Selection of organic solvents for the separation took into consideration compatibility with the inductively coupled plasma mass spectrometer being used for element specific detection. Optimized method conditions included use of a methanol gradient and make-up solution for the nebulizer. Some issues associated with dead volume were overcome by the extension of the gradient program. The total analysis time was 52 min. The column-to-column variability was evaluated and was found to be very reasonable (9% RSD on average), confirming that this method is rugged and that the technology should be easily transferred to other laboratories.