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Postherpetic pain (PHP) is difficult to control. Although Neurotropin® (NTP) and methylcobalamin (MCB) are often prescribed to treat the pain, the efficacy of combined treatment for PHP remains imcompletely understood. In this study, we investigate the combined effects of NTP and MCB on PHP in mice. NTP and MCB were administered from day 10–29 after herpes simplex virus type-1 (HSV-1) infection. The pain-related responses were evaluated using a paint brush. The expression of neuropathy-related factor (ATF3) and nerve repair factors (GAP-43 and SPRR1A) in the dorsal root ganglion (DRG) and neurons in the skin were evaluated by immunohistochemical staining. Nerve growth factor (NGF) and neurotrophin-3 (NT3) mRNA expression levels were evaluated using real-time PCR. Repeated treatment with NTP and MCB after the acute phase inhibited PHP. Combined treatment with these drugs inhibited PHP at an earlier stage than either treatment alone. In the DRG of HSV-1-infected mice, MCB, but not NTP, decreased the number of cells expressing ATF3 and increased the number of cells expressing GAP-43- and SPRR1A. In addition, MCB, but not NTP, also increased and recovered non-myelinated neurons decreased in the lesional skin. NTP increased the mRNA levels of NTF3 in keratinocytes, while MCB increased that of NGF in Schwann cells. These results suggest that combined treatment with NTP and MCB is useful for the treatment of PHP. The combined effect may be attributed to the different analgesic mechanisms of these drugs. • Neurotropin® and methylcobalamin to postherpetic pain (PHP) were evaluated in mice. • Combined treatment with these drugs to PHP showed faster efficacy than either drug. • Methylcobalamin effected the expression of the factors on neuropathy or nerve repair. • These drugs induced the gene expression on neurotropic factors. • Combined treatment with these drugs is more effective to PHP than each drug alone. [ABSTRACT FROM AUTHOR]

Degradation scheme for the photolytic degradation of methylcobalamin (MC) in the presence of ascorbic acid (AH 2). [Display omitted] • Photodegradation of methylcobalamin in the presence of ascorbic acid (AH 2). • Application of linear and non-linear correction of irrelevant absorption. • First-order rate constant for the photodegradation of MC is determined. • Second-order rate constants for photochemical interaction of MC and AH 2. • Fluorescence characteristic of MC in the presence of AH 2 is determined. Photodegradation of drug substances leads to the formation of known and unknown degradation products. These unknown degradation products interfere and give erroneous results because of absorption on analytical wavelengths. This interference could be eliminated using the correction of irrelevant absorbancies. This study is based on the application of linear and non–linear correction of irrelevant absorption for the determination of methylcobalamin (MC) and hydroxocobalamin in the photolytic degradation assisted by ascorbic acid (AH 2). MC follows first–order degradation kinetics and the rate of degradation (k obs) ranges from 1.99–2.34 × 10−2, min−1 at pH 2.0–12.0. The second–order rate constants (k 2) for the photochemical interaction of MC and AH 2 are in the range of 17.9–60.3 × 10−2 M−1, min−1 (acidic region) and 10.3–24.6 × 10−2 M−1, min−1 (alkaline region). The k 2 –pH profile was found to be bell–shaped and the maximum rate of degradation in the presence of AH 2 is at pH 5.0 (60.3 × 10−2 M−1, min−1) due to the protonation of MC. However, in alkaline pH, the rate of photodegradation decreases due to the ionization form of AH 2 which is AH− species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Confirmation of controlling factors affecting polycyclic macromolecular drug solid stability for methylcobalamin. • Stable preparation of three methylcobalamin hydrate powders with isolated site and channel water structures. • Inter-conversion and corresponding structural variation relations of methylcobalamin hydrates were established. • Preferable morphology and particle size distribution control of methylcobalamin crystal product. As one of derivatives of Vitamin B 12 , methylcobalamin (MeCbl) is an indispensable "Life Element" and plays an essential role in maintaining human normal physiology function and clinical medicine application. Because of the intricate molecular structure, strong hygroscopicity and optical instability, maintaining its solid stability is a great challenge in pharmaceutical preparation. Based on the structure features of MeCbl hydrates, this study explored the drug solid stability by designing solid-solid phase transformation (SSPT) experiments. Three hydrate powders of MeCbl that had special structure with isolated site and channel water molecules were discovered. It was found that drying condition and surrounding humidity were controlling factors influencing the final solid form. The inter-conversion relations relevant to heating-induced and humidity-induced structure changes were established among the three hydrate powders. Powder X-ray diffraction, thermogravimetric analysis, differential scanning calorimetry, high performance liquid chromatography and dynamic vapor sorption were used to characterize the differences and related properties of stably prepared MeCbl hydrate powders. The particle size of product could be regulated and controlled by optimizing operating conditions of crystallization process, where ultrasound-assisted and seeding-introduced were applied as promising strategies to enhance solution crystallization process. This study opens up the possibility for the stable preparation and large-scale production of polycyclic macromolecular bulk drugs like methylcobalamin. [ABSTRACT FROM AUTHOR]

The photolysis of methylcobalamin (MC) in aqueous solution (pH 1.0–12.0) under aerobic condition has been studied on irradiation in the visible region. It leads to the formation of aquocobalamin (B 12a ) in acid solution and hydroxocobalamin (B 12b ) in alkaline solution. This is indicated by the changes in the spectra of MC with a shift towards 525 nm in the acidic and alkaline solutions. The reaction follows first-order kinetics and the values of apparent first-order rate constants ( k obs ) using a 125 W lamp range from 2.80 (pH 12.0) to 27.25 × 10 −2  min −1 (pH 5.0). The k-pH profile for the reaction represents a bell-shaped curve with the maximum rate of photolysis around pH 5.0. The rates of the reaction decrease with a decrease in pH in the acid solution due to protonation and with an increase in pH in the alkaline solution due to ionization of the molecule. MC shows highest fluorescence around pH 5.0 with a decrease in pH in the acidic region and with an increase in pH in the alkaline region. MC and B 12a / B 12b in the irradiated solutions have been determined by a two-component spectrometric assay at 461 and 525 nm with a precision of ±2%. A reaction scheme for the photolysis of MC in aqueous solution has been presented. [ABSTRACT FROM AUTHOR]

Vitamin B12 (cobalamin, Cbl) is represented by several molecular variants distinguished by the exchangeable ligand X coordinated to cobalt ion (XCbl). The most typical XCbl-forms are cyanocobalamin (CNCbl), hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and 5′-deoxydeoxyadenosylcobalamin (AdoCbl). Cells convert the "inactive" vitamins CNCbl and HOCbl to the two critically important coenzymes AdoCbl or MeCbl. Surprisingly, little or no MeCbl is usually uncovered in the tissue samples, as compared to AdoCbl and HOCbl. We hypothesized that a low level of MeCbl is an artifact of "harsh" extractions, leading to degradation of MeCbl and/or its conversion to other XCbl-forms. We designed a "mild" extraction protocol, including homogenization of rat liver in ammonium acetate (pH 4.6), dilution with EtOH (final 60%) and heating for 10 min at 70 °C. The XCbls were separated by HPLC and quantified by isotope dilution assays. A "mild" extraction revealed the following composition of Cbls: 37% AdoCbl, 35% MeCbl, 15% HOCbl and 13% CNCbl. The usual "harsh" protocol (pH 7, 20 min at 80 °C) changed this balance to 33%, 5%, 43% and 17%, respectively. A model assay revealed that MeCbl underwent demethylation and conversion to HOCbl at pH 3 and pH > 7, when heated with thiols. Other changes included decyanation of CNCbl and destruction of HOCbl. Our procedure reveals a high content of MeCbl in rat liver. This result challenges previous data and pinpoints the need for new studies to characterize the endogenous Cbl-forms in health and disease. [Display omitted] • Low methylcobalamin (MeCbl) in liver tissues is an artifact of extraction. • The artificial decrease in MeCbl is caused by its conversion to hydroxocobalamin. • A harsh heating of a tissue sample at pH < 4 or pH > 7 accelerates this conversion. • Thiols are possible agents that demethylate MeCbl during its harsh extraction. • Milder extraction methods reveal a high level of MeCbl in rat liver. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (20 ng/mL; NPFA) and supra-physiological (2000 ng/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 . [ABSTRACT FROM AUTHOR]

Aims This study investigated the efficacies of gliclazide (GLZ), methylcobalamin (MCA), and GLZ + MCA combination therapy on DPN by evaluating the treatment-related changes in peripheral nerve function, the polyol pathway, and oxidative stress in the sciatic nerve of streptozotocin-induced diabetic rats. Materials and methods The rat model of streptozotocin-induced diabetes was orally given GLZ (25 mg/kg/day), MCA (175 μg/kg/day), and GLZ + MCA (25 mg/kg/day + 175 μg/kg/day) combination therapy for 8 weeks, in order to observe its effects on the motor nerve conduction velocity (MNCV), on the activities of Na + , K + -ATPase, aldose reductase(AR), AR mRNA expression, on the polyol contents, antioxidative enzyme activities and peroxidation products in the sciatic never tissue. Key findings Most of the indicators of DPN, such as delayed MNCV, altered/damaged nerve structure, inhibited Na + ,K + -ATPase activity, enhanced AR activity and AR mRNA expression, increased polyol contents, altered Cu,Zn-superoxide dismutase, catalase, and glutathione-peroxidase activities, and elevated malondialdehyde level in the sciatic nerves of the diabetic rats, were significantly ameliorated by treatment with either GLZ or MCA. Moreover, the combination of GLZ and MCA was found to enhance the curative effect on DPN in parts of above-mentioned parameters as compared to monotherapy. Significance Monotherapy with GLZ or MCA, and especially the combined application of GLZ and MCA, could be efficient therapeutic strategies for combating experimental DPN in diabetic rats. [ABSTRACT FROM AUTHOR]

The purpose of this study was to compare the intestinal permeation of vitamin B 12 and various derivatives thereof. Permeation behavior and cytotoxicity of four derivatives (coenzyme B 12 , hydroxocobalamin, methylcobalamin and 4-ethylphenylcobalamin) in comparison to vitamin B 12 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 3 h 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 < vitamin B 12 < coenzyme B 12 < methylcobalamin < 4-ethylphenylcobalamin. Apparent permeability coefficients of coenzyme B 12 , hydroxocobalamin, methylcobalamin and 4-ethylphenylcobalamin were 10.2, 0.3, 9.4 and 31.3 fold of vitamin B 12 on Caco-2 cells while 0.2, 0.4, 2.6 and 1.9 fold of vitamin B 12 on rat intestine, respectively. As various vitamin B 12 derivatives showed a significantly higher permeation coefficient than vitamin B 12 , the use of certain derivatives might be a promising strategy for oral vitamin B 12 substitution. [ABSTRACT FROM AUTHOR]

Background High-dose of methylcobalamin promotes nerve regeneration in rats with acrylamide neuropathy. A double-blind controlled trial suggested that high-dose methylcobalamin could increase compound muscle action potentials in patients with amyotrophic lateral sclerosis (ALS). A large-scale extended period human trial is now on-going in ALS ( Clinicaltrial.gov NCT00444613 ). We attempted to study whether high-dose methylcobalamin can improve symptoms or retard progression of motor dysfunction in the wobbler mouse model of ALS. Methods After initial diagnosis of the disease at the postnatal age of 3–4 weeks, wobbler mice received methylcobalamin (3 or 30 mg/kg, n = 10/group) or vehicle (n = 10), daily for 4 weeks by intraperitoneal administration in a blinded fashion. We compared clinical symptoms and pathological changes among all groups. Vitamin B12 concentrations were measured in the serum, the skeletal muscle and the spinal cord of three groups (n = 5/group). Results In comparison with vehicle, mice treated with ultra-high dose (30 mg/kg) of methylcobalamin significantly inhibited muscle weakness and contracture in the forelimb, and increased the weight of the bicep muscles and the number of musculocutaneous nerves. Methylcobalamin-treated mice significantly elevated vitamin B12 concentrations of the serum, the bicep muscle and the spinal cord compared to vehicle. Conclusion Our results suggest that treatment with methylcobalamin could delay progression of motor symptoms and neuropathological changes in wobbler mouse motor neuron disease if very high doses are used. [ABSTRACT FROM AUTHOR]

This report was to evaluate the efficacy of lipoic acid, prostaglandin E1 and methylcobalamin (L + P + M) for the treatment of diabetic peripheral neuropathy (DPN) in comparison with that of prostaglandin E1 plus methylcobalamin (P + M), in order to provide the basis and reference for clinical rational drug use. Randomized controlled trials (RCTs) of L + P + M for DPN published up to 3rd August, 2014 were searched. A random or fixed effect model was used to analyze outcomes which were expressed as risk ratios (RRs) or mean difference (MD) with a 95% confidence interval (CI). Eighteen RCTs with 1410 participants were included. Clinical efficacy of L + P + M therapy was significantly better than P + M therapy (fifteen trials; RR 1.32, 95% CI 1.24–1.41, P < 0.00001, I 2 = 32%). As compared with P + M therapy, the pooled effects of L + P + M therapy on nerve conduction velocities (NCVs) were (fifteen trials; MD 4.70, 95% CI 3.77–5.63, P < 0.00001, I 2 = 79%) for median MNCV, (thirteen trials; MD 4.73, 95% CI 3.69–5.77, P < 0.00001, I 2 = 85%) for median SNCV, (sixteen trials; MD 4.22, 95% CI 3.32–5.12, P < 0.00001, I 2 = 83%) for peroneal MNCV, (fourteen trials; MD 3.09, 95% CI 2.04–4.14, P < 0.00001, I 2 = 82%) for peroneal SNCV. There was no serious adverse events associated with drugs intervention. L + P + M therapy was superior to P + M therapy for improvement of clinical efficacy and NCVs in DPN patients. These findings should be further verified by high-quality RCTs. [ABSTRACT FROM AUTHOR]

Vitamin B 12 is among the most essential biomolecules required for crucial metabolic processes in humans. Vitamin B 12 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 B 12 was identified in C. vulgaris and this finding enhances its use as a nutritional supplement. [ABSTRACT FROM AUTHOR]

Abstract: 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 1.5μg/100g 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. [Copyright &y& Elsevier]

Abstract: Aims: To compare the efficacy and safety of daily lipoic acid (300–600mg i.v.) plus methylcobalamin (500–1000mg 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 2–4weeks 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. [Copyright &y& Elsevier]

Highlights: [•] Adenosylcobalamin- and methylcobalamin are converted to hydroxocobalamin during UVA exposure. [•] Hydroxocobalamin and cyanocobalamin are the most stable cobalamins. [•] Cobalamins may photodegrade indirectly due to their antioxidant properties. [•] Exposure of humans to sun in Norway does not have effect on serum vitamin B12 levels. [ABSTRACT FROM AUTHOR]

Abstract: Mercury (Hg) is assumed to be predominantly methylated by microorganisms in the environment. However, the mechanisms and extent of abiotic methylation are poorly appreciated. The understanding of the mechanisms leading to abiotic methylation and demethylation in the aquatic environment is of special concern since methylmercury (MeHg) biomagnifies in the food web. Bioaccumulating organisms have also been found to preserve specific Hg isotopic signatures that provide direct insight into aquatic Hg transformations. In this study we investigated the influence of chloride on the magnitude of Hg isotope fractionation during abiotic methylation of inorganic Hg (Hg(II)) using methylcobalamin as methyl donor compound. Coupling of gas chromatography with multi-collector inductively coupled plasma mass spectrometry has allowed to determine simultaneously isotopic ratios of inorganic and methyl-Hg species. Kinetic experiments demonstrated that the presence of chloride not only slowed the chemical alkylation of Hg(II) by methylcobalamin, but also decreased the extent of the methylation, which it is especially significant under visible light conditions due to the enhancement of MeHg photodecomposition. Abiotic methylation of Hg(II) by methylcobalamin in the presence of chloride caused significant Hg mass-dependent isotope fractionation (MDF) for both Hg(II) substrate (δ 202Hg(II) from −0.74‰ to 2.48‰) and produced MeHg (δ 202MeHg from −1.44‰ to 0.38‰) both under dark and visible light conditions. The value of this MDF under such saline conditions was higher than that previously reported (δ 202MeHg from −0.73‰ to 0.09‰) in the absence of chloride and appeared mainly related to inorganic Hg speciation in solution, which is predominantly mercuric chloro-complexes (i.e. HgCl4 2−). Different isotopic signatures were observed for the different Hg species at the same time of reaction for either dark or visible light (450–650nm wavelengths) conditions. However, no significant mass-independent fractionation (MIF) was induced under any conditions within the analytical uncertainties (−0.17±0.31<∆201Hg<0.17±0.28‰), suggesting that photo-induced demethylation does not always involve MIF. These results also suggest that methylation by methylcobalamin can be an experimental model to study Hg isotope fractionation extent during elementary reaction of methyl transfer in biotic systems. [Copyright &y& Elsevier]

Abstract: Methylcobalamin (MeCbl), a vitamin B12 analog, promotes neurite outgrowth by activating Akt in neurons. However, Akt is involved in many cellular functions, and the downstream signal of Akt that promotes neurite outgrowth in neurons in the presence of MeCbl remains obscure. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates multiple cellular functions including neurite outgrowth. mTOR is regarded as important for the regeneration of injured nerves. In this study, we examined the relationship between MeCbl and mTOR activity and found that MeCbl increases mTOR activity via the activation of Akt and promotes neurite outgrowth in cerebellar granule neurons via the activation of mTOR. [Copyright &y& Elsevier]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: We have separated the active polypeptides from aqueous extracts of Achyranthes bidentata Blume (ABPP), a commonly prescribed Chinese medicinal plant with a range of pharmaceutical properties. We investigated the effects of ABPP administration on peripheral nerve regeneration in a mouse sciatic nerve crush injury model. After nerve crush, the mice received daily tail vein injections of 1, 4, and 16mg/kg of ABPP, 65μg/kg of methylcobalamin, and vehicle saline, respectively, over a 21-day period. At 1, 3, 6, 9, 12, 15, 18 and 21 days after nerve crush, the animals were subjected to walking track analysis for evaluating the sciatic functional index (SFI) values. At day 21 the animals were anesthetized, and the compound muscle action potential and nerve conduction velocity were respectively recorded. After the animals were killed, the sciatic nerve was examined with immunohistochemistry and electron microscopy, and gastrocnemius muscle was analyzed with Masson trichome staining. The results indicated that treatment with ABPP at a dose range (1–16mg/kg) promoted histological regeneration and functional recovery of the injured sciatic nerve and its target muscle, yielding a desired efficacy greater than that by vehicle treatment and close to that by methylcobalamin (65μg/kg). These findings suggest that plant polypeptides, ABPP, may be a potential agent in ameliorating of neuropathy caused by sciatic nerve crush. [Copyright &y& Elsevier]

• Methylcobalamin protected PC-12 cells against cell apoptosis and inflammatory response. • Methylcobalamin regulated cell autophagy in PC-12 cells. • Methylcobalamin ameliorated cerebral ischemia/reperfusion injury in MCAO rats. • The protective mechanism might be related to the activation of the ERK1/2 signaling pathway. • Methylcobalamin is promising to serve as a neuroprotective agent for the treatment of ischemic stroke. 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. [ABSTRACT FROM AUTHOR]

Abstract: In this manuscript we review our recent work on the structure and dynamics of iron and cobalt complexes with macrocyclic ligands using ab initio molecular dynamics (AIMD). AIMD techniques are used to analyze the short time scale dynamics of these complexes, helping to understand the results obtained by experimental methods with larger time resolution. Firstly, the case of the iron–oxygen bond in myoglobin (Mb) and hemoglobin (Hb), as well as its cobalt analogues, will be discussed. Secondly, we will focus on the B12 coenzyme and its organocobaloxime derivatives. The simulations grasp the role played by temperature on the dynamics of ligands (the FeO2 and CoO2 moieties in heme models and the bridging OH⋯O protons in organocobaloxime crystals), clarifying problems related to average structures and underscoring the need to perform dynamical simulations. [Copyright &y& Elsevier]

The methylation reaction of tin(II) with methylcobalamin (CH3B12) in aquatic systems was modeled in the laboratory. The products were detected by a sensitive gas chromatography-flame photometric detector (GC-FPD), and further identified by gas chromatography-mass spectrometry (GC–MS). Both monomethyltin (MMT) and dimethyltin (DMT) were found as methylation products. Three important effecting factors during the methylation reaction, salinity, pH and aerobic or anaerobic, were studied. The results showed that methyl group can be transferred from CH3B12 to tin in aquatic solutions as a radical or carbonium, as well as a carbanion. Two explanations for the pH-dependency of the methylation reaction between tin(II) and CH3B12 were proposed: pH-dependency of the equilibrium states of CH3B12, and pH-dependency of inorganic tin(II) species whose reactivity vary with the redox potential in the solution. Salinity can influence the activity of the methyl donor, which cause changes in the methylation efficiency. Kinetic experiments showed that the methylation reaction was pseudo-first-order for CH3B12. [Copyright &y& Elsevier]

The reaction between the vitamin B12 coenzyme methylcobalamin and cyanide has been re-investigated by UV–Vis and 1H NMR spectroscopies under the same conditions. On the basis of the results and recent results of other studies on the reaction between other alkylcobalamins and cyanide, the changes in the 1H NMR chemical shifts and linewidths in the aromatic region observed upon addition of cyanide to methylcobalamin can be attributed to the rapid formation of (β-CH3)(α-CN)Cbl−, rather than the formation of MeCbl·CN− as proposed earlier (Inorg. Chem. 36 (1997) 4891). There is excellent agreement between the equilibrium constant for formation of (β-CH3)(α-CN)Cbl− determined by the two methods (0.35±0.03 M−1 and 0.31±0.01 M−1 from the UV–Vis and 1H NMR spectroscopic data, respectively (D2O, pD 12.1, 25.0 °C)). [Copyright &y& Elsevier]

• The methionine synthase process takes place without any energy barriers. The methionine synthase process is regenerating the methionine from cysteine in the S-adenosylmethionine cycle running throughout several interactions and electron transfers between methylcobalamin cofactor and substrates. The methionine synthase process in connection with the methionine synthase reductase process can run, in principle, unlimited number of turnovers without side products or effects. This raises questions about the energy barriers of the methionine synthase process reactions. The CASSCF geometry optimization with 13 electrons and 13 orbitals in the active zone of the methylco(II)balamin cofactor species was used to calculate the full mechanism of the methionine synthase process. We have shown that the anticipated reaction of the methionine synthase process is the cleavage of the Co-N (dimethylbenzimidazole) axial bond under the influence of the homocysteine ion and the histidine substrates. Our results also show that the active species in the methylation of homocysteine is a base-on methylco(II)balamin cofactor with the dimethylbenzimidazole ligand substituted by the histidine trans ligand. This methylation process takes place during the methylco(II)balamin cofactor interaction with the homocysteine negative ion, proving that this reaction is of SN2. We have shown that the triggering factor of this process is the initial electron transfer from a homocysteine negative ion to a base-on methylco(II)balamin-histidine cofactor particle. Another step of the methionine synthase process is the N-C(CH 3) bond cleavage in the protonated 5-methyltetrahydrofolate systems under the influence of the negative total (and the atomic charges of the central part) of the co(I)balamin cofactor. Simultaneously, the cleaved methyl (+1) positive particle is leaving an electron back to tetrahydrofolate. The no energy barrier and the minimum energy of the in-vivo reaction products have been proved for all the methionine synthase process reactions. Finally, the full mechanism of the methionine synthase process has been drawn. [Display omitted] [ABSTRACT FROM AUTHOR]

• This is the first report of mecobalamin (MBL) assay in human plasma by LC–MS/MS. • Instability of MBL under light required minimal light exposure during sample preparation. • MBL was quantifiable from 0.05 ng/mL, which enables detection of endogenous levels. • "Stripped" plasma exposed to light was used for calibration sample preparation. • The developed method was successfully applied to clinical sample assay. Methylcobalamin (mecobalamin) is a vitamin B12 coenzyme and is effective for the treatment of peripheral neuropathies. As mecobalamin is an endogenous substance in human plasma at low levels and is light-labile, a sensitive assay method has been developed by using liquid chromatography with tandem mass spectrometry with particular care taken for light exposure. Stability tests performed under light showed that mecobalamin was stable in plasma in amber tubes only when exposed to <10 lx light, and thus, all the assays used for the method validation and the in-study sample assays should be performed under red light with minimal light exposure. Mecobalamin and its stable isotope, which was used as an internal standard, were extracted from 0.1 mL plasma by protein precipitation with methanol and were quantifiable in a range from 0.05 to 20 ng/mL. The reproducibility assessment showed acceptable accuracy and precision (≤15 %). Mecobalamin was stable in plasma at room temperature for 21 h when exposed to <10 lx light in amber tubes and for 205 days when stored frozen. The validated method was successfully applied to an in-study sample assay by performing a clinical pharmacokinetic study of mecobalamin, in which a successful incurred sample reanalysis was performed. [ABSTRACT FROM AUTHOR]

• Simultaneous determination of four bioactive vitamin B12s from dietary supplements. • The method is simpler, less time-consuming, and more economical than other methods. • The LOQs are less than 0.52 µg/mL. • The method is validated and meets all AOAC SMPR 2016.017 requirements. Vitamin B12 dietary supplement can be critical to the alleviation strategies against micronutrient malnutrition and food insecurity. An HPLC-DAD method has been developed and validated, per AOAC SMPR 2016.017 (Standard Method Performance Requirements), for the quantitation of four bioactive forms of vitamin B12 (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, methylcobalamin) from dietary ingredients and supplements. The method achieves chromatographic baseline resolution of vitamin B12 forms on a modern column platform without the expensive requirement of an ultra-high pressure liquid chromatography and/or mass spectrometry. The method has a wide analytical range (0.0005%w/w–85%w/w), high precision (reproducibility relative standard deviations ranged from 1.43% to 4.67%), and high accuracy (>96% spike recovery rate for 11 out of 12 accuracy testing data points). The method detection and quantification limits are less than 0.16 and 0.52 µg/mL, respectively. To our best knowledge, it is simpler, less time-consuming, and more economical than other published methods for its intended uses. [ABSTRACT FROM AUTHOR]