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Abstract
To examine the effects of methylcobalamin on glutamate-induced neurotoxicity in the cultured retinal neurons.
Primary cultures obtained from the fetal rat retina (gestation days 16 to 19) were used for the experiment. The neurotoxicity was assessed quantitatively using the trypan blue exclusion method.
Glutamate neurotoxicity was prevented by chronic exposure to methylcobalamin and S-adenosylmethionine (SAM), which is formed in the metabolic pathway of methylcobalamin. Chronic exposure to methylcobalamin and SAM also inhibited the neurotoxicity induced by sodium nitroprusside that release nitric oxide. By contrast, acute exposure to methylcobalamin did not protect retinal neurons against glutamate neurotoxicity.
Chronic administration of methylcobalamin protects cultured retinal neurons against N-methyl-D-aspartate-receptor-mediated glutamate neurotoxicity, probably by altering the membrane properties through SAM-mediated methylation.
... In this study, the majority of retinal cells cultured for 5-7 days were positive for anti-neurofilament antibody, manifested as brownish-yellow particles in the cell body and processes, with darkly stained cell body and lightly stained processes. This indicated that neurofilament mainly accumulated in the cell body and processes, demonstrating that these cells have properties of retinal neurons [30] , consistent with Nissl staining. Glial fibrillary acidic protein is specific to glial cells. ...
... For immunocytochemical staining, retinal neuronal and glial cells were observed using anti-neurofilament and anti-glial fibrillary acidic protein immunocytochemical staining methods [2] . Briefly, cells were harvested from the coverslips, fixed with 4% paraformaldehyde at room temperature for 30 Glial cells were identified as follows: after nuclei were hematoxylin counterstained for 25 seconds, cells were washed with tap water, dehydrated with gradient alcohol, cleared with xylene, and mounted with neutral gum. Cells stained brownish-yellow were neuron-positive staining, cells with brownish-yellow or brown stained particles in the cytoplasm and membrane were glial cell-positive staining. ...
The retina of Wistar rats within 1-3 days of birth were dissociated into a retinal cell suspension using 0.05% trypsin digestion. The cell suspension was incubated in Dulbecco's modified Eagle's medium for 24 hours, followed by neurobasal medium for 5-7 days. Nissl staining showed that 79.86% of primary cultured retinal cells were positive and immunocytochemical staining showed that the purity of anti-neurofilament heavy chain antibody-positive cells was 71.53%, indicating that the primary culture system of rat retinal neurons was a reliable and stable cell system with neurons as the predominant cell type. The primary cultured retinal neurons were further treated with 0, 5.5, 15, 25, and 35 mM glucose for 24, 48, and 72 hours. The thiazolyl blue tetrazolium bromide test and flow cytometry showed that with increasing glucose concentration and treatment duration, the viability of retinal neurons was reduced, and apoptosis increased. In particular, 35 mM glucose exhibited the most significant effect at 72 hours. Thus, rat retinal neurons treated with 35 mM glucose for 72 hours can be used to simulate a neuronal model of diabetic retinopathy.
... L-methylfolate, MeCbl, and N-acetylcysteine improved memory, emotional functions, and communication with other people among Alzheimer's patients [7,8]. MeCbl also has neuronal protection including promoting injured nerve and axonal regeneration [9,10] and confronting against glutamate-induced neurotoxicity [9,11]. In addition, MeCbl improved nerve conduction in either patients of diabetic neuropathy [12][13][14] or streptozotocin-diabetic rats [15] and experimental acrylamide neuropathy [16]. ...
... L-methylfolate, MeCbl, and N-acetylcysteine improved memory, emotional functions, and communication with other people among Alzheimer's patients [7,8]. MeCbl also has neuronal protection including promoting injured nerve and axonal regeneration [9,10] and confronting against glutamate-induced neurotoxicity [9,11]. In addition, MeCbl improved nerve conduction in either patients of diabetic neuropathy [12][13][14] or streptozotocin-diabetic rats [15] and experimental acrylamide neuropathy [16]. ...
Methylcobalamin (MeCbl), the activated form of vitamin B12, has been used to treat some nutritional diseases and other diseases in clinic, such as Alzheimer's disease and rheumatoid arthritis. As an auxiliary agent, it exerts neuronal protection by promoting regeneration of injured nerves and antagonizing glutamate-induced neurotoxicity. Recently several lines of evidence demonstrated that MeCbl may have potential analgesic effects in experimental and clinical studies. For example, MeCbl alleviated pain behaviors in diabetic neuropathy, low back pain and neuralgia. MeCbl improved nerve conduction, promoted the regeneration of injured nerves, and inhibited ectopic spontaneous discharges of injured primary sensory neurons. This review aims to summarize the analgesic effect and mechanisms of MeCbl at the present.
... Agents that are able to inhibit NOS could prevent induction of opioid tolerance [ 17 ] . It was also shown that Vit B12 could inhibit NO synthesis through inhibition of NMDA receptors [ 19 ] . Kikuchi et al. had shown that methylcobalamin and compound that are formed in its metabolic pathway, S-adenosylmethionine (SAM), could protect retinal cells against neurotoxicity of nitroprusside, a potent NO releaser, when they were chronically administered. ...
... Kikuchi et al. had shown that methylcobalamin and compound that are formed in its metabolic pathway, S-adenosylmethionine (SAM), could protect retinal cells against neurotoxicity of nitroprusside, a potent NO releaser, when they were chronically administered. However, the acute administration of these compounds did not show a protective eff ect against glutamate neurotoxicity [ 19 ] . Several mechanisms are suggested for morphine tolerance and dependence, some of these mechanisms include: alter in neurotransmitters (such as catecholamines, γ-amino-butyric acid (GABA), serotonin and acetylcholine) concentrations and activities [ 23 ] , cAMP [ 24 ] , c-fos [ 25 ] and infl ammatory cytokines like TNFα [ 26 ] . ...
The antinociceptive effect of cyanocobalamin (Vit B12) has been reported in animal models and human studies. Our previous study showed the effect of Vit B12 on morphine tolerance. The dependence and tolerance were induced in male mice using subcutaneous morphine injections, 3 times a day (50, 50 and 75 mg/kg/day) for 3 days. Mice also received Vit B12 (100, 250 and 500 µg/kg), clonidine, memantine and saline intraperitoneally before morphine administration. On fourth day mice received only 7 mg /kg morphine just before tail-flick test. To determine the expression of morphine dependence and tolerance, all compounds were injected once intraperitoneally on the day of experiment. The tolerance was evaluated by the tail-flick test. The effect of Vit B12 and other agents on dependence were evaluated by counting the number of jumps (induced by naloxone 5 mg/kg). Co-administration of Vit B12 (100-500 µg/kg) and morphine in 3 days reduced the development of tolerance to morphine analgesic effect (8.2±0.5 and 7.83±0.5 s. vs. normal saline, 3.57±0.3 s). Repeated administration of Vit B12, also, diminished the reduced naloxane withdrawal signs of naloxone withdrawal test (100-500 µg/kg: 5±1.9 and 1.2±0.8 jumps vs. normal saline 72.6±12.2). However, Vit B12 had no effect on the expression of morphine tolerance and physical dependence. It is concluded that co-administration of Vit B12 and morphine could reduce tolerance to analgesic effect of morphine chronic administration and also reduce its withdrawal symptoms.
... The procedures have been described previously. 15,[21][22][23][24] In brief, retinal tissues were mechanically dissociated, and single-cell suspensions were plated on plastic coverslips (1.0 ϫ 10 6 cells/ml). Ten coverslips were placed in a 60-mm dish (Falcon Labware, Oxnard, CA). ...
... The neurotoxic effects of glutamate and the protective effects of drugs on the retinal cultures were quantitatively assessed by the trypan blue exclusion method, as described previously. 15,[21][22][23][24]27 At each session of the experiment, we randomly chose five coverslips from different dishes, which constituted the number of samples (n ϭ 5) for measurement of neurotoxicity. All experiments were performed in Eagle's solution at 37°C. ...
Purpose. To examine the effects of nicotine on glutamate-induced neurotoxicity using cultured rat retinal neurons. Methods. Primary cultures were obtained from the retina of fetal rats (gestation day 17-19). The neurotoxic effects on the retinal cultures were quantitatively assessed by the trypan blue exclusion method. The protective effects of nicotine were assessed by application for 1 to 24 hr prior to glutamate exposure. Results. Cell viability was markedly reduced by 10 min-exposure to 500 uM glutamate followed by 1 hr-incubation in glutamate-free medium. Incubating the cultures with 1 uM nicotine for 1 to 24 hr prior to glutamate exposure reduced its neurotoxicity. The maximal protection was observed in the culture pretreated with nicotine (1 uM) 12 hr prior to glutamate exposure. Twelve-hr pretreatment of nicotine at concentrations of 0.001 to 1 uM demonstrated dose dependent protection against glutamate neurotoxicity. Conclusions. These results suggest that pretreatment of nicotine had protective action against glutamate neurotoxicity in cultured retinal neurons.
... The synthesis of methionine from cysteine depends on both folate and methylcobalamin [23]. Cultured neurons are protected from glutamateinduced neurotoxicity by administration of methylcobalamin, likely through membrane alteration by SAMe [24]. ...
... For e.g:-apart from chelation therapy, diets rich in vitamins and minerals are prescribed for people who are at risk of cadmium and lead toxicity [16]. There are similar reports available on the positive effects of vitamin B 12 and MeB 12 on the recovery of metal induced toxicity [17][18][19]. Reports show that MeB 12 helps our body to eliminate the toxic heavy metal arsenic by methylating it along with selenium and methionine [20,21]. ...
Methylcobalamin (MeB12) a vitamin B12 analogue is used in the treatment of several neurological disorders was used in this study to check for its role in cadmium toxicity. Cadmium is a highly toxic heavy metal can cause considerable damage to airway epithelium which can lead to many pulmonary disorders and even lung cancer. Human A549 cells were grown in medium supplemented with MeB12 for 48 hours, followed by 24 hours exposure to increasing concentrations of CdCl2 . MeB12 enhanced cadmium induced cells death. Isobolographic analysis revealed a synergistic effect of these two compounds. Studies on the revealed while MT1A promoter is already heavily methylated in A549 cell line, MT2A appeared to be methylated only in the presence of CdCl2 and MeB12. Our results suggest that the epigenetic modification of MT2A gene may be one of the contributing factors towards the enhanced cadmium sensitivity in A549 cells in the presence of MeB12.
... 126 Preclinical studies indicated that this compound has antiglutamatergic properties. 127 Animal studies (wobbler mouse) indicate an effect on survival. 128 Clinical studies in ALS have been promising regarding ventilation-free survival in ALS participants. ...
Numerous potential amyotrophic lateral sclerosis (ALS)-relevant pathways have been hypothesized and studied preclinically, with subsequent translation to clinical trial. However, few successes have been observed with only modest effects. Along with an improved but incomplete understanding of ALS as a neurodegenerative disease is the evolution of more sophisticated and diverse in vitro and in vivo preclinical modeling platforms, as well as clinical trial designs. We highlight proposed pathological pathways that have been major therapeutic targets for investigational compounds. It is likely that the failures of so many of these therapeutic compounds may not have occurred because of lack of efficacy but rather because of a lack of preclinical modeling that would help define an appropriate disease pathway, as well as a failure to establish target engagement. These challenges are compounded by shortcomings in clinical trial design, including lack of biomarkers that could predict clinical success and studies that are underpowered. Although research investments have provided abundant insights into new ALS-relevant pathways, most have not yet been developed more fully to result in clinical study. In this review, we detail some of the important, well-established pathways, the therapeutics targeting them, and the subsequent clinical design. With an understanding of some of the shortcomings in translational efforts over the last three decades of ALS investigation, we propose that scientists and clinicians may choose to revisit some of these therapeutic pathways reviewed here with an eye toward improving preclinical modeling, biomarker development, and the investment in more sophisticated clinical trial designs.
... It acts as a coenzyme by donating methyl in homocysteine remethylation reaction and prevents neuronal degeneration by reducing the levels of homocysteine, the deposit which relates to neuronal injury in ALS patients [181,182]. In addition, experimental studies described that methylcobalamin protects neural cells against glutamate neurotoxicity [183], and induces nerve regeneration [184]. Kaji et al. [185] showed no significant differences between Methylcobalamin and placebo. ...
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease with high mortality and morbidity rate affecting both upper and lower motor neurons (MN). Muscle force reduction, behavioral change, pseudobulbar affect, and cognitive impairments are the most common clinical manifestations of ALS. The main physiopathology of ALS is still unclear, though several studies have identified that oxidative stress, proteinopathies,
glutamate-related excitotoxicity, microglial activation, and neuroinflammation may be involved in the pathogenesis of ALS. From 1995 until October 2022, only Riluzole, Dextromethorphan Hydrobromide (DH) with Quinidine sulfate (Q), Edaravone, and Sodium phenylbutyrate with Taurursodiol (PB/TUDCO) have achieved FDA approval for ALS treatment. Despite the use of these four approved agents, the survival rate and quality of life of ALS patients are still low. Thus, finding novel treatments for ALS patients is an urgent requirement. Masitinib, a tyrosine kinase inhibitor, emphasizes the neuro-inflammatory activity of ALS by targeting macrophages, mast cells, and microglia cells. Masitinib downregulates the proinflammatory cytokines, indirectly re
duces inflammation, and induces neuroprotection. Also, it was effective in phase 2/3 and 3 clinical trials (CTs) by increasing overall survival and delaying motor, bulbar, and respiratory function deterioration. This review describes the pathophysiology of ALS, focusing on Masitinib’s mechanism of action and explaining why Masitinib could be a promising actor in the treatment of ALS patients. In addition, Masitinib CTs and other competitor drugs in phase 3 CTs have been discussed.
... 5 Methylcobalamin, a type of vitamin B12 defends against neurological issues and maturing. 6 As compared to other water soluble vitamins, vitamin B12 is not passed out rapidly in urine. It collects and gets stored in the liver, kidney etc. ...
Background
The objective of this study was to estimate serum vitamin B12 levels and its correlation with severity of clinical presentation in patients with trigeminal neuralgia (TN).
Methods
A total of 80 participants were included and rendered into study group: 40 TN patients and control group: 40 healthy individuals. The serum vitamin B12 estimation of each participant was carried out by using serum vitamin B12 ELISA kit and pain characteristics of TN patients were recorded in details.
Results
The mean serum vitamin B12 level was noticed to be significantly lower (p = 0.042) in study group (296.87 ± 248.75pg/mol) as compared to control group (423.5 ± 296.41pg/mol). There was a statistically significant difference in serum vitamin B12 level between vegetarian TN patients and those who were on mixed diet (p = 0.001). The pain associated with TN predominantly reported unilaterally involving the right side (55%), female gender (55%) and mandibular division (50%). Although pain intensity showed no significant relation (p = 0.024), duration of pain reported a strong negative association with mean serum vitamin B12 levels in TN patients (p = 0.001).
Conclusions
Vitamin B12 supplements can be added to the established treatment protocol for the holistic management of TN patients particularly those who are on vegetarian diet.
... Preclinical studies have shown that cobalamin inhibits glutamate exocytosis, as an excitatory pain neurotransmitter, and when infused intracerebroventricularly, increases GABA cell contents, as an inhibitory pain neurotransmitter [6,9]. The inhibitory effects of chronic exposure of vitamin B12 on glutamateinduced neurotoxicity were determined in cell culture, probably by altering the membrane properties through S-adenosylmethionine (SAM)-mediated methylation [26,27]. SAM is one of the major methyl donors for methylation reactions throughout the body, including the methylation of myelin basic protein. ...
Background
Fibromyalgia (FM) as a prototypical nociplastic pain condition displays a difficult therapeutic situation in many cases. Given the promising data on the effect of vitamin B12 in improving pain and cognitive functions in various nociplastic pain conditions, we aimed to determine the efficacy of 1000 mcg daily dose of oral vitamin B12 on the symptom severity and psychological profile of FM patients.
Methods
This open-label, pre-post study was performed on FM patients whose diagnoses were confirmed by a rheumatologist based on the 2016 American College of Rheumatology (ACR). Patients were instructed to take a daily dose of 1000mcg vitamin B12 for fifty days. Outcome measures including the Revised Fibromyalgia Impact Questionnaire (FIQR), Hospital Anxiety and Depression Scale (HADS), 12-item Short-Form health survey (SF-12), and pain Visual Analog Scale (pain-VAS) were fulfilled by patients before and after the treatment.
Results
Of 30 eligible patients, 28 patients completed the study protocol. Patients were female with a mean age of 47.50 ± 8.47 years. FIQR scores in all domains improved significantly after treatment (total FIQR: 49.8 ± 21.86 vs 40.00 ± 18.36, p value < 0.01; function: 13.17 ± 7.33 vs 10.30 ± 5.84, p value: 0.01; overall: 10.32 ± 6.22 vs 8.25 ± 6.22, p value: 0.03; symptoms: 26.30 ± 10.39 vs 21.44 ± 8.58, p value < 0.01). Vitamin B12 also improved anxiety scores from 9.33 ± 4.30 to 7.70 ± 3.60, p value: 0.01. Depression, pain-VAS, and SF-12 didn’t improve following the treatment. The Generalized estimating equations (GEE) analysis showed the improvement in total FIQR score is not cofounded by the improvement of anxiety and patients’ baseline characteristics.
Conclusions
This study showed a short course of sublingual vitamin B12, 1000 mcg daily, significantly improves the severity of FM and anxiety score. We postulate that vitamin B12 has a strong potential to consider, at least, as adjunctive therapy of FM.
... The primary form that was prescribed to patients was methylcobalamin. In patients diagnosed with FM, vitamin B12 may play a role in modulating pain through neuronal protection, [25][26][27][28][29][30][31][32] and improving fatigue in patients who have a B12 deficiency. [33][34][35] Higher-quality studies with larger FM patient populations are needed. ...
Background: Fibromyalgia (FM) is characterized by chronic pain, with allodynia and hyperalgesia being the most common signs. Many patients with FM explore, express interest, and use complementary and alternative medicine to help manage symptoms and improve quality of life. However, little is known about the clinical recommendations provided by naturopathic doctors (NDs). Objective: To describe trends in assessment and treatment of patients with FM by NDs. Methods: Retrospectively, medical records of 200 patients with the FM ICD-10 code were reviewed from the Robert Schad Naturopathic Clinic. Of these records, 70 met inclusion criteria and were further analyzed. Comorbid conditions, health concerns, physical and psychological examinations, and treatment were recorded. Patients were excluded if informed consent for research was not signed. The project was approved by the Research Ethics Board of the Canadian College of Naturopathic Medicine. Results: Seventy patients met criteria and were included in the current analysis. Most patients identified as female (96%). Vitamin D (57%), magnesium (54%), omega-3 fish oil (53%), acupuncture by an acupuncturist (53%) or an ND (40%), B12 orally or by injection (40%), and probiotics (40%) were highly utilized treatments. A past/current medical history of digestive complaints (64%) and depression/mental illness (63%) were common comorbidities, alongside a history of arthritic conditions (53%) and anxiety (43%). A family history of arthritic conditions (47%) was also prevalent. The Widespread Pain Index and Symptom Severity tool (43%) was used to assess pain and other symptoms. No adverse effects of treatment were readily identifiable. Conclusion: Findings from this study reveal elements of both consistency and variability in the treatment recommendations from NDs in a teaching clinic environment. Future research that assesses or compares treatment recommendations for FM in other settings may be informative to better understand health services, the nature of individualized care, and patient experiences.
... Methylcobalamin is considered to be used in the cyanide detoxication process via cyanocobalamin synthesis. Based on an in-vitro study, it is suggested that the altered membrane properties through SAM-mediated methylation are the primary mechanism of the neuroprotective effect of methylcobalamin in multiple types of CNS injuries [19]. ...
... Methylcobalamin Methylcobalamin is an active form of vitamin B 12 which has demonstrated neuroprotective effects in vitro and in vivo (23,24). The importance of vitamin B 12 for neural function is clear, and deficiency can manifest as central or peripheral nervous system pathology. ...
Background: Amyotrophic lateral sclerosis (ALS) is a terminal condition, which is increasing in incidence. Therapeutic interventions have enjoyed limited success. However, research is progressing, with some promising drug candidates emerging. Patients cannot always wait for the results of large trials. The general practitioner has a central role in management of ALS. Objective: To review and summarise available evidence evaluating the disease-modifying and life-extending potential of approved medications available off-label or over the counter. To inform doctors and patients of updates in the field and assist their decision-making. Discussion: Three experimental candidates were deemed to have a reasonable likelihood of efficacy, and doses/routes of administration were clarified. Possibilities for novel therapies were outlined, and the importance of research support was highlighted.
... MeCbl, an analog of vitamin B12, promotes nerve regeneration and is effective for neuronal cell survival; however, a high concentration of MeCbl is required to maximize its effectiveness. [6][7][8][9] Therefore, we developed a novel electrospun nanofiber sheet incorporating MeCbl to locally deliver a high-concentrated compound to the peripheral nerve injury site, and showed its effectiveness both in vitro and in vivo in the axonal outgrowth of neurons and the differentiation of Schwann cells. 10 In this study, the local administration of MeCbl promoted nerve regeneration and functional recovery in a rat sciatic nerve crush injury model, assuming the case of nerve injury was in continuity such as entrapment neuropathy. ...
Outcomes of peripheral nerve repair after injury are often suboptimal. Therefore, developing biological approaches to augment nerve regeneration is important. In this in vivo study, we tested the hypothesis that augmentation with an electrospun nanofiber sheet incorporating methylcobalamin (MeCbl) would be effective for regeneration after peripheral nerve transection and repair.
Methods:
Rats were divided into 3 groups that either underwent sciatic nerve repair with or without the MeCbl sheet, or a sham operation. At 4 and/or 8 weeks after the operation, sensory and motor functional recovery, along with histological findings, were compared among the groups using the toe-spreading test, mechanical and thermal algesimetry tests, tibialis anterior muscle weight measurements, electrophysiological analyses, which included nerve conduction velocity (NCV), compound muscle action potential (CMAP), and terminal latency (TL), and histological analyses involving the myelinated axon ratio, axon diameter, and total axon number.
Results:
Compared with the repair group without the MeCbl sheet, the repair group with the MeCbl sheet showed significant recovery in terms of tibialis anterior muscle weight, NCV and CMAP, and also tended to improve in the toe-spreading test, mechanical and thermal algesimetry tests, and TL. Histological analyses also demonstrated that the myelinated axon ratios and axon diameters were significantly higher. Among these findings, the repair group with the MeCbl sheet demonstrated the same recovery in NCV as the sham group.
Conclusion:
This study demonstrated that electrospun nanofiber MeCbl sheets promoted nerve regeneration and functional recovery, indicating that this treatment strategy may be viable for human peripheral nerve injuries.
... Because of the large number of medications (both prescription and over-the-counter), medical conditions and lifestyle choices that can precipitate vitamin B12 deficiency, combined with the ease, cost, and effectiveness of replacement therapy, widespread screening is recommended within the chronic pain population. In addition to replenishing vitamin B12 stores, treatment with various forms of cobalamin has been shown to provide pain relief, alleviate pain behaviors, improve nerve conduction, and exert neuronal protection by promoting regeneration of injured nerves and antagonizing glutamate-induced neurotoxicity (28)(29)(30)(31)(32)(33). ...
Background:
Because of the subjective nature of current pain assessments, limited efficacy of treatment options and risks associated with opioid abuse and diversion, the need for objective data to assist with chronic pain management has never been greater. Successful identification of mechanistic biomarkers would not only improve our understanding and ability to accurately diagnose pain disorders but would also facilitate the development of disease-modifying pain drugs.
Objectives:
The objective of this study was to determine and evaluate the prevalence of abnormal biomarker findings in a population of patients with chronic pain.
Study design:
Retrospective, observational study.
Setting:
Data analysis of biomarker test results was performed at a single industry site (Ethos Research & Development, Newport, KY) from clinical samples collected and analyzed from July to December 2018.
Methods:
A novel, pain-specific biomarker test panel that evaluates biomarkers of systemic inflammation, oxidative stress, neurotransmitter turnover, and micronutrient status was employed to determine the prevalence of abnormal findings in 17,834 unique patient samples analyzed at a national reference laboratory (Ethos Laboratories, Newport, KY). Patient biomarker results were considered abnormal if they were outside of the 95% confidence interval reference ranges established using a healthy population of donors who had no history of chronic pain or opioid use.
Results:
A total of 77% of patients with chronic pain exhibited at least one abnormal biomarker result (n = 13,765). The most common abnormal biomarker finding was elevated quinolinic acid, which was observed in 29% of patients (n = 5,107). Elevated pyroglutamate, indicative of glutathione depletion, was observed in 19% of patients (n = 3,314). Elevated xanthurenic acid, indicative of vitamin B6 insufficiency, was observed in 17% of patients (3,025). Elevated levels of the acrolein metabolite 3-hydroxypropyl mercapturic acid were observed in 21% of patients (n = 3,667). Elevated methylmalonic acid, indicative of a vitamin B12 deficiency, was observed in 10% of patients (n = 1,827), whereas abnormally low levels of neurotransmitter metabolites were observed in 8% of patients (n = 1,456).
Limitations:
Medications and/or conditions other than those associated with chronic pain were not evaluated as potential causes of abnormal biomarker findings.
Conclusions:
A novel biomarker assay that measures objective correlates to the neurobiological processes underlying chronic pain reveals a high prevalence of atypical biochemistry in a population of patients with pain. Abnormal biomarker findings presented here provide objective support for the role of cytokine-mediated inflammation, oxidative stress, abnormally low production of neurotransmitters, and micronutrient deficiencies in the development or worsening of chronic pain. This unique panel of functional pain biomarkers provides practitioners with novel, objective insight into the underlying causes of pain, which will pave the way for truly personalized pain medicine. Correcting abnormal biomarker findings with targeted, nonopioid therapies to improve patient function and alleviate pain potentially could lessen the opioid burden and drastically reduce health care costs.
Key words:
Biomarker, pain, inflamation, oxidative stress, neurotransmitter, micronutrient deficiency, Kynurenine Pathway.
... The main risk factors for Vitamin B12 deficiency are: low intakes of animal products; malabsorption from food due to gastric atrophy induced by Helicobacter pylori, or bacterial overgrowth; genetic disorder of vitamin B12 metabolism. Deficiency linked to improper absorption rather than low consumption is the main reason of vitamin B12 in developed countries [21,22]. However, in developing or in transition countries, However, in developing or in transition countries, the primary cause of vitamin B12 deficiency might be insufficient consumption of animal products. ...
... Hence, the development of a drug that extends the survival time or ameliorates clinical symptoms of ALS is widely anticipated. High-dose methylcobalamin (an active form of vitamin B12) has been suggested to have a protective action against neurodegeneration in vitro and in vivo [4][5][6][7], and clinical research reports have suggested that administration of high-dose methylcobalamin to patients with ALS has yielded clinically beneficial results [8][9]. ...
Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects the upper and lower motor neurons. Currently, only riluzole and edaravone are approved as drugs for ALS in the world, and new agents with larger effect size are warranted. Our previous study (E0302-J081-761) has suggested that high-dose methylcobalamin (E0302) prolonged the overall survival and suppressed progression in ALS patients with a disease duration less than 12 months in exploratory analyses.
Objective: This clinical trial aims to evaluate the efficacy and safety of E0302 for treatment of ALS patients within 1 year from onset.
Methods: The Japan Early-stage Trial of high dose methylcobalamin for ALS (JETALS) is a prospective, multicenter, placebo-controlled, double-blind, randomized phase III study conducted at 24 tertiary neurology centers and is funded by the Japan Agency for Medical Research and Development.
A total of 128 ALS patients within 1 year from onset were randomized at a 1:1 ratio to receive intramuscular injection with E0302 50 mg or placebo twice a week for 16 weeks. The primary endpoint is changes in the ALS Functional Rating Scale (ALSFRS-R) total score at 16 weeks.
If the patients expect to receive E0302 50 mg after double-blind administration period, E0302 will be provided to those who wish to receive by March 2020 in the continuous administration period.
Results: This study was started in October 2017 and is being implemented by 24 participating institutions in Japan. This study is currently in progress. The patient enrollment period is scheduled to end on August 2019 and the follow-up is scheduled to end on March 2020.
Conclusions: This study is being performed to revalidate the efficacy and safety of E0302 in patients with early-stage ALS in the 1st year of symptom onset. If positive results are obtained, the aim is to apply for E0302 approval as a new drug for treating ALS.
... [39,40] It also can stimulate phosphatidylcholine synthesis to increase myelinogenesis, and then speed up the motor and sensory NCVs. [41,42] Additionally, M accelerates NCVs directly by improving blocked nerve impulse conduction and decreased neurotransmitter levels. [43] Many studies suggested that M monotherapy or polytherapy with other drugs is an effective and safe therapy for patients with DPN. ...
Background:
Prostaglandin E1 (P) or methylcobalamin (M) treatment has been suggested as a therapeutic approach for diabetic peripheral neuropathy (DPN) in many clinical trial reports. However, the combined effects of 2 drugs still remain dubious.
Objective:
The aim of this report was to evaluate the efficacy of M plus P (M + P) for the treatment of DPN compared with that of P monotherapy, in order to provide a reference resource for rational drug use.
Methods:
Randomized controlled trials (RCTs) of M + P for DPN published up to September 2017 were searched. Risk ratio (RR), mean difference (MD), and 95% confidence interval (CI) were calculated and heterogeneity was assessed with the I test. Subgroup and sensitivity analyses were also performed. The outcomes measured were as follows: the clinical efficacy, median motor nerve conduction velocities (MNCV), median sensory nerve conduction velocity (SNCV), peroneal MNCV, peroneal SNCV, and adverse effects.
Results:
Sixteen RCTs with 1136 participants were included. Clinical efficacy of M + P combination therapy was significantly better than P monotherapy (fifteen trials; RR 1.25, 95% CI 1.18-1.32, P < .00001, I = 27%). Compared with P monotherapy, the pooled effects of M + P combination therapy on nerve conduction velocity were (MD 6.29, 95% CI 4.63-7.94, P < .00001, I = 90%) for median MNCV, (MD 5.68, 95% CI 3.53-7.83, P < .00001, I = 94%) for median SNCV, (MD 5.36, 95% CI 3.86-6.87, P < .00001, I = 92%) for peroneal MNCV, (MD 4.62, 95% CI 3.48-5.75, P < .00001, I = 86%) for peroneal SNCV. There were no serious adverse events associated with drug intervention.
Conclusions:
M + P combination therapy was superior to P monotherapy for improvement of neuropathic symptoms and NCVs in DPN patients. Moreover, no serious adverse events occur in combination therapy.
... Vit. B 12 also exhibits anti-inflammatory and anti-apoptotic effects (Kikuchi et al. 1997;Masuda et al. 1998;Zhang et al. 2008 andDas 2008). These mechanisms could explain the observed beneficial effects of Vit. ...
Metabolites of proper fatty acids modulate the inflammatory response and are essential for normal brain development; equally, abnormal fatty acid metabolism plays a critical role in the pathology of autism. Currently, dietary supplements are often used to improve the core symptoms of Autism spectrum disorder (ASD). The present study analyzed the effects of orally supplemented omega-3 (ω-3) and vitamin B12 on ameliorating oxidative stress and impaired lipid metabolism in a propionic acid (PPA)-induced rodent model of autism, together with their effect on the gut microbial composition, where great fluctuations in the bacterial number and strains were observed; interestingly, polyunsaturated fatty acids such as omega-3 induced higher growth of the gram-positive bacterium Staphylococcus aureus and decreased the survival rates of Clostridia sp. as well as other enteric bacterial strains. Thirty-five young male western albino rats were divided into five equal groups. The first group served as the control; the second group was given an oral neurotoxic dose of PPA (250 mg/kg body weight/day) for 3 days. The third group received an oral dose of ω-3 (200 mg/kg body weight/day) for 30 days after the 3-day PPA treatment. Group four was given an oral dose of vitamin B12 (16.7 mg/kg/day) for 30 days after PPA treatment. Finally, group five was given a combination of both ω-3 and vitamin B12 at the same dose for the same duration after PPA treatment. Biochemical parameters related to oxidative stress and impaired fatty acid metabolism were investigated in the brain homogenates of each group. The effects of the dietary supplements on the gut microbiota were also observed. The PPA-treated autistic model expressed significantly higher levels of lipid peroxides and 5-lipoxygenase (5-LOX) and significantly less glutathione (GSH), glutathione S-transferase (GST), and cyclooxygenase 2 (COX2) than the control group. However, a remarkable amelioration of most of the impaired markers was observed with oral supplementation with ω-3 and vitamin B12, either alone or in combination. Our results concluded that impairment at various steps of the lipid metabolic pathways may contribute to the development of autism; however, supplementation with ω-3 and vitamin B12 can result in a positive therapeutic effect.
... Hence, the development of a drug that extends the survival time or ameliorates clinical symptoms of ALS is widely anticipated. High-dose methylcobalamin (an active form of vitamin B12) has been suggested to have a protective action against neurodegeneration in vitro and in vivo [4][5][6][7], and clinical research reports have suggested that administration of high-dose methylcobalamin to patients with ALS has yielded clinically beneficial results [8][9]. ...
BACKGROUND
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects the upper and lower motor neurons. Currently, only riluzole and edaravone are approved as drugs for ALS in the world, and new agents with larger effect size are warranted. Our previous study (E0302-J081-761) has suggested that high-dose methylcobalamin (E0302) prolonged the overall survival and suppressed progression in ALS patients with a disease duration less than 12 months in exploratory analyses.
OBJECTIVE
This clinical trial aims to evaluate the efficacy and safety of E0302 for treatment of ALS patients within 1 year from onset.
METHODS
The Japan Early-stage Trial of high dose methylcobalamin for ALS (JETALS) is a prospective, multicenter, placebo-controlled, double-blind, randomized phase III study conducted at 24 tertiary neurology centers and is funded by the Japan Agency for Medical Research and Development. A total of 128 ALS patients within 1 year from onset were randomized at a 1:1 ratio to receive intramuscular injection with E0302 50 mg or placebo twice a week for 16 weeks. The primary endpoint is changes in the ALS Functional Rating Scale (ALSFRS-R) total score at 16 weeks. If the patients expect to receive E0302 50 mg after double-blind administration period, E0302 will be provided to those who wish to receive by March 2020 in the continuous administration period.
RESULTS
This study was started in October 2017 and is being implemented by 24 participating institutions in Japan. This study is currently in progress. The patient enrollment period is scheduled to end on August 2019 and the follow-up is scheduled to end on March 2020.
CONCLUSIONS
This study is being performed to revalidate the efficacy and safety of E0302 in patients with early-stage ALS in the 1st year of symptom onset. If positive results are obtained, the aim is to apply for E0302 approval as a new drug for treating ALS.
CLINICALTRIAL
NCT 03548311; Pre-results
... 6,7 Clinically, the administration of vitamin B 12 through oral, subcutaneous, intramuscular, intravenous, intra-nasal, and eye-drop routes are now used for the restoration of neurological function. In the retina, a study with the primarycultured rat retinal neurons revealed the neuroprotective effect of vitamin B 12 , 8 and an in vivo study with vitamin B 12 -difficient monkeys showed a vision disorder and loss of retinal ganglion cells, 9 supporting a reduction in the thickness of the peripapillary retinal nerve fiber layer in patients with vitamin B 12 deficiency. 10 The intramuscular administration of vitamin B 12 has been reported to suppress the retinal exudate production in patients with diabetic retinopathy, 11 and the intramuscular administration of hydroxocobalamin has been reported to contribute to the recovery of vision in tobacco amblyopia. ...
The blood-to-retina supply of cyanocobalamin (vitamin B12) across the blood-retinal barrier (BRB) was investigated by synthesizing a fluorescence-labeled cyanocobalamin (Cy5-cyanocobalamin). In the in vivo analysis following internal jugular injection of Cy5-cyanocobalamin, confocal microscopy showed the distribution of Cy5-cyanocobalamin in the inner plexiform layer (IPL), the outer plexiform layer (OPL) and the retinal pigment epithelium (RPE). In the in vitro analysis with TR-iBRB2 cells, an in vitro model cell line of the inner BRB, Cy5-cyanocobalamin uptake by TR-iBRB2 cells exhibited a time-dependent increase after preincubation with transcobalamin II (TCII) protein, during its residual uptake without preincubation with TCII protein. The Cy5-cyanocobalamin uptake by TR-iBRB2 cells was significantly reduced in the presence of unlabeled cyanocobalamin, chlorpromazine and chloroquine, and was also significantly reduced under Ca2+-free conditions. Confocal microscopy of the TR-iBRB2 cells showed fluorescence signals of Cy5-cyanocobalamin and GFP-TCII protein, and these signals merged with each other. The RT-PCR, Western blot and immunohistochemistry clearly suggested the expression of TCII receptor (TCII-R) in the inner and outer BRB. These results suggested the involvement of receptor-mediated endocytosis in the blood-to-retina transport of cyanocobalamin at the inner BRB with implying its possible involvement at the outer BRB.
... Vit B12 possesses the antioxidant activity and is involved in the synthesis of phospholipids and myelin. It also shows anti-inflammatory, anti-apoptotic, and anti-necrotic effects (32)(33)(34)(35). These mechanisms could explain the observed beneficial effects of Vit B12 in the present study. ...
Lead belongs to the heavy metal group and is considered as an environmental contaminant. Acute or chronic contact to lead can change the physiological function of human organs. One of the most important disorders following the lead exposure is neurotoxicity. Lead neurotoxicity consists of the neurobehavioral disturbances like cognitive impairment. The aim of the current study is to evaluate the possible protective effect of vitamin C (Vit C), vitamin B12 (Vit B12), omega 3 (ω-3), or their combination on the lead-induced memory disorder. Adult wistar rats were orally administered Vit C (120 mg/kg/day) or Vit B12 (1 mg/kg/day) or ω-3 (1000 mg/kg/day) or their combination for 3 weeks in groups of 7 animals each. Then lead acetate (15 mg/kg/day) was injected intraperitoneally for one week to all pretreated animals. The control group received normal saline as a vehicle while the positive control for cognitive impairment received just lead acetate. At the end of treatments animal memories were evaluated in Object Recognition Task. The results showed, although 15 mg/kg lead acetate significantly declines the memory-evaluating parameters, pretreatment with Vit C, Vit B12, ω-3, or their combination considerably inverted the lead induced reduction in discrimination (d2) index (P < 0.001) and recognition (R) index (P < 0.001, P < 0.05, P < 0.05, and P < 0.001, respectively). Our findings indicate while lead acetate impairs spatial memory in rat, administration of Vit C, Vit B12, ω-3, or their combination prior to the lead exposure inhibits the lead induced cognitive loss. There was no remarkable difference in this effect between the used supplements.
... 30 It has also been reported to promote nerve regeneration and myelination 48 and to protect against glutamate-induced neurotoxicity. 49,50 In the present study, we showed that intraperitoneal injection of MeCbl, one active form of vitamin B12 that can directly participate in homocysteine metabolism, dose-dependently inhibited the mechanical allodynia and thermal hyperalgesia induced by vincristine. The results are consistent with previous studies showing that continuous administration of MeCbl of high dose could ameliorate neuropathic pain associated with diabetic neuropathy 51 or with CCD. 31 Besides used alone, MeCbl is usually combined with other drugs, such as vitamin E. 52 MeCbl, alpha lipoic acid, and pregabalin combination provides additional pain relief compared to pregabalin alone in type 2 diabetes mellitus associated peripheral neuropathy. ...
Background:
Vincristine, a widely used chemotherapeutic agent, often induces painful peripheral neuropathy and there are currently no effective drugs to prevent or treat this side effect. Previous studies have shown that methylcobalamin has potential analgesic effect in diabetic and chronic compression of dorsal root ganglion model; however, whether methylcobalamin has effect on vincristine-induced painful peripheral neuropathy is still unknown.
Results:
We found that vincristine-induced mechanical allodynia and thermal hyperalgesia, accompanied by a significant loss of intraepidermal nerve fibers in the plantar hind paw skin and an increase in the incidence of atypical mitochondria in the sciatic nerve. Moreover, in the spinal dorsal horn, the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the protein expression of p-p65 as well as tumor necrosis factor a was increased, whereas the protein expression of IL-10 was decreased following vincristine treatment. Furthermore, intraperitoneal injection of methylcobalamin could dose dependently attenuate vincristine-induced mechanical allodynia and thermal hyperalgesia, which was associated with intraepidermal nerve fibers rescue, and atypical mitochondria prevalence decrease in the sciatic nerve. Moreover, methylcobalamin inhibited the activation of NADPH oxidase and the downstream NF-kB pathway. Production of tumor necrosis factor a was also decreased and production of IL-10 was increased in the spinal dorsal horn following methylcobalamin treatment. Intrathecal injection of Phorbol-12-Myristate-13-Acetate, a NADPH oxidase activator, could completely block the analgesic effect of methylcobalamin.
Conclusions:
Methylcobalamin attenuated vincrinstine-induced neuropathic pain, which was accompanied by inhibition of intraepidermal nerve fibers loss and mitochondria impairment. Inhibiting the activation of NADPH oxidase and the downstream NF-kB pathway, resulting in the rebalancing of proinflammatory and anti-inflammatory cytokines in the spinal dorsal horn might also be involved. These findings might provide potential target for preventing vincristine-induced neuropathic pain.
... Some studies suggest that MeCbl and SAM have a protective role in retinal cell culture and that elevated homocysteine levels may affect eye development. 21 Treatment Treatment of Cbl-C deficiency is based on supplementation of vitamin B 12 , betaine, and folic acid (►Table 2). Pharmacological doses of vitamin B 12 , preferably in the form of hydroxocobalamin (OHCbl), are given to maximize the enzyme activity. ...
Cobalamin C (Cbl-C) defects are inherited autosomal recessive disorders of vitamin B12 (or cyanocobalamin [CNCbl]) metabolism. These defects are caused by mutations in the methylmalonic aciduria and homocystinuria Cbl-C type (MMACHC; MIM # 609831) gene located on chromosome 1p34.1, which catalyzes the reductive decyanation of CNCbl, thus impairing the biosynthesis of 5′-deoxyadenosylcobalamin, adenosylcobalamin, and methylcobalamin. This impairment results in methylmalonic acidemia [MMA; MIM # 277400] combined with hyperhomocysteinemia and hypomethioninemia. Clinically, Cbl-C defects are characterized by a constellation of systemic signs and symptoms, including neurological, cognitive, psychiatric, and thromboembolic events. Retinal phenotypes, including maculopathy, pigmentary retinopathy, and optic atrophy, are common in the early-onset form of the disease, but are rare in its adult-onset counterpart. Administration of hydroxocobalamin (OHCbl), betaine, and folinic acid represents main therapeutic approaches. No proven efficacy has been demonstrated for carnitine and dietary protein restrictions. Although early introduction of OHCbl is crucial, no standardized protocols regarding dose adjustment exist. Despite these measures, the long-term outcome is unsatisfactory especially in patients with early onset, who experience frequent progression of their neurological and ocular impairment. The unfavorable outcome suggests that a better understanding of the pathophysiology of the disease is needed to improve treatment protocols and to develop new therapeutic approaches.
... There are several ways that MeCbl could be useful in slowing ALS progression. In cell culture studies, MeCbl has been shown to ameliorate oxidative stress (4), glutamate toxicity (5,6) and apoptosis (5,7). Oxidative stress (8), glutamate toxicity (9) and apoptosis (10) are believed to play important roles in ALS progression and have been targeted in previous clinical trials; riluzole, the only medication reproducibly shown to prolong survival in PALS, is believed to act at least in part through ameliorating glutamate toxicity (11). ...
... Some studies suggest that methylcobalamin and Sadenosylmethionine (SAM), both predicted to be deficient in untreated cblC, have protective roles in retinal cell culture [66] and that hyperhomocysteinemia may cause vascular endothelial injury, hypercoagulability, occlusion and ischemia, and may be disruptive to normal ocular development [67,68]. Plasma homocysteine has been positively correlated with retinopathy of prematurity (ROP) in premature infants [69], as well as with progression of glaucoma, age-related macular degeneration (AMD), and proliferative diabetic retinopathy [70][71][72]. ...
... In addition, Metformin-related B12 deficiency with resultant increased severity of symptomatic diabetic neuropathy has been described [30]. The ability of methylcobalamin to protect against glutamate induced neurotoxicity in vitro [31], enhance myelin node genesis and nerve regeneration [32], and to reduce symptoms of diabetic neuropathy [7] has been demonstrated in previous studies. The property of folate to effectively metabolize elevated homocysteine levels has also similarly been described [33]. ...
Objective: To study the effect of addition of Metanx on burning parasthesias in patients with symptomatic diabetic neuropathy who had obtained only partial symptom resolution with pregabalin. Research design and methods: This was an open-label pilot study. There were 16 patients (7 males, 9 females) in the study group and 8 patients (3 males, 5 females) in the control group. A patient numeric rating scale (0 - 10) of neuropathy associated pain was obtained at baseline and at 20 weeks. The control group continued on a fixed dosage of pregabalin without additional medication, while the study group was supplemented with Metanx (a proprietary blend of bioactive B-vitamins), in addition to being continued on a fixed dosage of pregabalin. Results: After 20 weeks, patients in the study group experienced greater pain relief compared to the control group, (87.5% vs. 25% respectively, p = 0.005). The average pain score reduction after 20 weeks in the study group was 3 compared to 0.25 in the control group (p < 0.001). Conclusion: Our study suggested that addition of Metanx may be used for the relief of pain in patients that have obtained only partial resolution of symptomatology from pregabalin.
... ALA was well tolerated over 4 years. [17] Methylcobalamin has been shown be useful in Bell's palsy, [18] protect against glutamate-induced neurotoxicity in the cultured retinal neurons [19] and also promote regeneration of motor nerve terminals in mouse and thus, could benefit diabetic neuropathy. [20] A systemic review has reported some promise for methylcobalamin in treatment of diabetic neuropathy. ...
To assess whether methylcobalamin and alpha lipoic acid (ALA) added to pregabalin provide additional benefit compared to pregabalin alone in type 2 diabetes mellitus associated peripheral neuropathy.
An open label, randomized, controlled parallel-group pilot study.
Thirty adult patients with type 2 diabetes mellitus with symptoms of peripheral neuropathy for ≥6 months were randomized to receive pregabalin 75 mg, methylcobalamin 750 μg, and ALA 100 mg (PMA, n = 15); or pregabalin 75 mg (PG, n = 15) for 12 weeks. Assessment variables were numeric rating scale (NRS), sleep interference scores (SIS), response rate to pain, and global assessment for the usefulness of therapy. The nerve conduction velocity was assessed for sensory and motor nerves. Safety assessment included adverse events reported by the patients, clinical laboratory, and general medical, neurological examinations.
Efficacy analyses were done on per-protocol (PP) population, whereas safety analyses were done on intent-to-treat (ITT) population.
Significant improvement was seen in pain and sleep interference in both groups. Mean nerve conduction velocity of left common peroneal nerve (CPN) showed significant improvement in PMA group at week 12 compared to baseline (P = 0.018). For right CPN both groups showed significant improvement. (PMA, P = 0.002, PG, P = 0.007). For sensory testing, at week 12, right superficial peroneal nerve showed reduction in nerve conduction velocity in PG group compared to baseline (P = 0.043).
Methylcobalamine, ALA and pregabalin combination provides pain relief and improves sleep interference. Addition of methylcobalamin and ALA to pregabalin improves the nerve function. Due to small sample size, most of the efficacy parameters could not reach significant difference between groups; hence benefit of the 3-drug-combimation should be interpreted with reservation.
... Our study group considered it unethical in Japan to use a placebo for this disease, and mecobalamin was thus used as the control drug because it can be administered either orally or intravenously and promotes recovery of experimentally induced neuronal degeneration. 12,13 Differences in visual function test results for the two treatment groups became evident as early as 1-4 weeks and then less apparent with time. Recovery of visual acuity, HFA mean deviation, correct scores on the Ishihara pseudochromatic plates, and contrast sensitivity all showed essentially the same rate of recovery with statistically significant differences at 1, 3, or 4 weeks. ...
Background: A randomized, controlled clinical trial was conducted in 1991 to compare an intravenous megadose of methylprednisolone with a control drug (mecobalamin) for treating acute idiopathic optic neuritis.Cases: Sixty-six cases from 22 clinical centers throughout Japan were examined to evaluate the treatment on visual function parameters, such as visual acuity, visual field, color vision, contrast sensitivity, and critical flicker frequency.Observations: The methylprednisolone pulse treatment group showed faster recovery of visual function, particularly the visual acuity at 1 week (P < .05), Humphrey field analyzer mean deviation at 3 weeks (P < .05), and color vision at 1 week (P < .05). Recovery of contrast sensitivity at several different spatial frequencies was significant in the pulse treatment group at 1 (P < .01), 2 (P < .05), and 4 weeks (P < .05) after the start of treatment. Visual function test results at 12 weeks and 1 year were essentially the same in the two treatment groups. Side effects appeared more frequently in the pulse treatment group than in the control (P < .05).Conclusions: Pulse treatment does not appear effective for idiopathic optic neuritis even though visual function in the pulse treatment group of this trial recovered more quickly during the initial phase compared to the controls. More effective and specific treatment should be established for optic neuritis.
... As demonstrated inTable 3, in a cellular model designed to better understand vitamin B12 deficiency in the brain, the growth and differentiation of neuronal cells were affected [89] . Additionally, supplementation with certain cobalamin compounds protected the cells from neurotoxicity and increased cell growth [170, 171]. Unfortunately, in vitro research demonstrating a direct link between vitamin B12 deficiency or overload and genomic stability in human cells has not yet been published. ...
Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5-10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed.
... It is possible that the MMACHC protein may play a specific role in eye tissues. Some studies suggest that MeCbl and SAM have a protective role in retinal cell culture (Kikuchi et al. 1997) and that elevated homocysteine levels may affect eye development (Maestro de las Casas et al. 2003). ...
Combined methylmalonic acidemia and homocystinuria, cblC type, is stated to be the most common inborn error of intracellular cobalamin metabolism. The disorder can display a wide spectrum of clinical manifestations, spanning the prenatal period through late adulthood. While increased homocysteine concentrations and impaired methyl group metabolism may contribute to disease-related complications, the characteristic macular and retinal degeneration seen in many affected patients appears to be unique to cblC disease. The early detection of cblC disease by newborn screening mandates a careful assessment of therapeutic approaches and provides a new opportunity to improve the outcome of affected patients. The following article reviews the current knowledge on the complications, pathophysiology, and outcome of cblC disease in an effort to better guide clinical practice and future therapeutic trials.
Blue light induced eye damage (BLED) belongs to modern diseases. It is an ophthalmic disease caused by prolonged exposure to electronic devices or screens containing a large amount of high-energy short waves (blue light). Specific symptoms include dryness and discomfort in the eyes, blurred vision, headache, insomnia, and in severe cases, it may also cause various eye diseases such as cataracts and glaucoma. At present, the development of health products and drugs for eye blue light injury faces many difficulties. Therefore, further exploration and research are needed on the pathogenesis, pathophysiology, and pharmacological mechanisms of blue light injury. Natural medicine ingredients and preparations have unique advantages in targeting eye blue light injury fatigue products due to their multi-component synergistic effects, overall regulation, and mild and safe characteristics. Starting from the disease-related mechanisms and pathophysiological characteristics of eye blue light injury, this article elucidates the pharmacological mechanisms of various drugs for treating eye blue light injury. At the same time, it reviews the research on in vitro cultured cell and animal model conditions for blue light injury eyes, in order to provide reference for subsequent blue light injury modeling experiments. And explore future research directions to provide new ideas and methods for the prevention and treatment of BLED.
Background: Prolonged elevation of extracellular glutamate levels triggers intracellular events, increases glutamate excitotoxicity, and activates apoptotic pathways, causing Alzheimer's disease (AD). The literature has reported that vitamin B12 exhibits anti-inflammatory and anti-apoptotic activities in various diseases. Hydrocortisone (HC) therapy also substantially inhibits microglia and astrocyte hyperactivation, minimizing pro-inflammatory cytokines and reducing neuroinflammation. That is why our study aimed to evaluate the therapeutic effects of HC and B12 combination on oxidative stress and VIP and GAL levels in an in vitro Alzheimer's model. Method: To create the Alzheimer's model, the neuroblastoma cell line (SH-SY5Y) was cultured. Then, all cells except the control group were treated with different doses of HC and B12 combination for 24 hours by applying Glutamate (10-5 mM) to create excitotoxicity. The results were evaluated using MTT and ELISA tests. Results: When the results were examined, it was determined that exceptionally high-dose combination groups showed protective activity against glutamate excitotoxicity. HC+B12 25 µg/ml groups observed the most statistically significant results. According to our results, oxidative stress decreased in the HC+B12 25 µg/ml group, and cell viability increased. Significant changes were also observed in Vasoactive Peptide (VIP) and Galanin (GAL) levels in correlation with other analyses obtained. Conclusion: This study is the first to report the potential of vitamin B12 combined with hydrocortisone to prevent oxidative stress and glutamate excitotoxicity in primary neurons. It provides a basis for further investigating its clinical application in neurodegenerative diseases.
Purpose of review
Recent development in understanding the pathophysiology of amyotrophic lateral sclerosis (ALS) has led to increasing number of promising test drugs in the pipeline along with the existing ones. We will review these agents focusing on ultra-high dose methylcobalamin, which is pending approval in Japan. Clinical trial design best suited for ALS will also be discussed.
Recent findings
The most recent phase 3 trial (JETALS) of ultra-high dose methylcobalamin demonstrated significant slowing of ALSFRSR changes (0.5/month), with marked reduction of serum homocysteine levels in the initial double-blind period. The post hoc analysis of the previous phase 2/3 study (E761 trial; Eisai) showed that it prolonged survival of ALS patients, if started within 1 year of onset, but the previous studies suggested its efficacy even in later stages, depending upon the rate of progression. Phase 3 trial of AMX0035 or Relyvrio on the other hand showed negative results despite the promising phase 2 data. The latter did not adjust the disease progression rate before entry.
Summary
Ultra-high dose methylcobalamin is not a vitamin supplement but a novel disease-modifying therapy for ALS, and it emphasizes homocysteine as a key factor in the disease process. Clinical trial design must include entering patients early and with similar rates of progression using pretrial observation periods for meaningful results, since ALS is a chronologically heterogenous condition with similar phenotypes.
Autism is a neurodevelopmental disorder, the prevalence of which has increased dramatically in the United States over the past two decades. It is characterized by stereotyped behaviors and impairments in social interaction and communication. In this paper, we present evidence that autism can be viewed as a PIN1 deficiency syndrome. Peptidyl‐prolyl cis/trans isomerase, NIMA‐Interacting 1 (PIN1) is a peptidyl‐prolyl cis/trans isomerase, and it has widespread influences in biological organisms. Broadly speaking, PIN1 deficiency is linked to many neurodegenerative diseases, whereas PIN1 over‐expression is linked to cancer. Death‐associated protein kinase 1 (DAPK1) strongly inhibits PIN1, and the hormone melatonin inhibits DAPK1. Melatonin deficiency is strongly linked to autism. It has recently been shown that glyphosate exposure to rats inhibits melatonin synthesis as a result of increased glutamate release from glial cells and increased expression of metabotropic glutamate receptors. Glyphosate's inhibition of melatonin leads to a reduction in PIN1 availability in neurons. In this paper, we show that PIN1 deficiency can explain many of the unique morphological features of autism, including increased dendritic spine density, missing or thin corpus callosum, and reduced bone density. We show how PIN1 deficiency disrupts the functioning of powerful high‐level signaling molecules, such as nuclear factor erythroid 2‐related factor 2 (NRF2) and p53. Dysregulation of both of these proteins has been linked to autism. Severe depletion of glutathione in the brain resulting from chronic exposure to oxidative stressors and extracellular glutamate leads to oxidation of the cysteine residue in PIN1, inactivating the protein and further contributing to PIN1 deficiency. Impaired autophagy leads to increased sensitivity of neurons to ferroptosis. It is imperative that further research be conducted to experimentally validate whether the mechanisms described here take place in response to chronic glyphosate exposure and whether this ultimately leads to autism. image
Background
The primary objective of the present study was to evaluate the effects of a Nerve Support Formula NeuropAWAY® on diabetic neuropathic pain.
Methods
This double-blind, placebo-controlled, randomized trial was conducted between August 2020 and February 2021. Patients aged ≥40 and ≤65 years with a history of type 2 diabetes (T2D) with a confirmed diagnosis of diabetic neuropathic pain were included in the study. The primary efficacy endpoint was to assess the effect of the 42 days administration of the Nerve Support Formula on the neuropathic pain as assessed by the 11 point Pain Intensity Numeric Rating Scale (PI-NRS). The secondary objectives were to assess the effect on plasma vitamin B12 levels, nerve conduction velocity, blood flow velocity, Brief Pain Inventory, Neuropathy Total Symptom Score, and Insomnia Severity Index.
Results
The enrolled study population (n=59) was randomized in two study groups; the Investigational Product (IP) group - Nerve Support Formula (n=27) and placebo group (n=32). The mean age of these participants was 52.63 and 53.72 for IP and placebo group, respectively. The mean (SD) HbA1c levels for IP and placebo group were 8.37 (0.85) and 8.16 (0.86), respectively. By the end of the study (Day 42) the decrease in PI-NRS scores for the IP group was maximal (↓61.32%) and highly significant (p<0.001) in comparison to the placebo group (↑2.47%). Significant improvements (p<0.05) were also noted in the secondary efficacy variables after 42 days of IP intake.
Conclusion
The formula was found to be significantly effective as compared to placebo in reducing pain and other sensory symptoms related to the diabetic peripheral neuropathy.
Background
Regulation of glutamate release is crucial for maintaining normal brain function, but excess glutamate release is implicated in many neuropathological conditions. Therefore, the minimum glutamate release from presynaptic nerve terminals is an important neuroprotective mechanism.
Objective
In this mini-review, we analyze the three B vitamins, namely vitamin B2 (riboflavin), vitamin B6 (pyridoxine), and vitamin B12 (cyanocobalamin), that affect the 4-aminopyridine (4-AP)-evoked glutamate release from presynaptic nerve terminal in rat and discuss their neuroprotective role.
Methods
In this study, the measurements include glutamate release, DiSC3(5), and Fura-2.
Results
The riboflavin, pyridoxine, and cyanocobalamin produced significant inhibitory effects on 4-aminopyridine-evoked glutamate release from rat cerebrocortical nerve terminals (synaptosomes) in a dose-dependent relationship. These presynaptic inhibitory actions of glutamate release are attributed to inhibition of physiologic Ca2+-dependent vesicular exocytosis but not Ca2+-independent nonvesicular release. These effects also did not affect membrane excitability, while diminished cytosolic [Ca2+]c through a reduction of direct Ca2+ influx via Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels, rather than through indirect Ca2+ induced Ca2+ release from ryanodine-sensitive intracellular stores. Furthermore, their effects were attenuated by GF109203X and Ro318220, two protein kinase C (PKC) inhibitors, suggesting suppression of PKC activity. Taken together, these results suggest that riboflavin, pyridoxine, and cyanocobalamin inhibit presynaptic vesicular glutamate release from rat cerebrocortical synaptosomes, through the depression Ca2+ influx via voltage-dependent Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels, and PKC signaling cascade.
Conclusion
Therefore, these B vitamins may reduce the strength of glutamatergic synaptic transmission and is of considerable importance as potential targets for therapeutic agents in glutamate-induced excitation-related diseases.
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
To evaluate the efficacy and safety of intramuscular ultra-high-dose methylcobalamin in patients with amyotrophic lateral sclerosis (ALS).
Methods
373 patients with ALS (El Escorial definite or probable; laboratory-supported probable; duration ≤36 months) were randomly assigned to placebo, 25 mg or 50 mg of methylcobalamin groups. The primary endpoints were the time interval to primary events (death or full ventilation support) and changes in the Revised ALS Functional Rating Scale (ALSFRS-R) score from baseline to week 182. Efficacy was also evaluated using post-hoc analyses in patients diagnosed early (entered ≤12 months after symptom onset).
Results
No significant differences were detected in either primary endpoint (minimal p value=0.087). However, post-hoc analyses of methylcobalamin-treated patients diagnosed and entered early (≤12 months’ duration) showed longer time intervals to the primary event (p<0.025) and less decreases in the ALSFRS-R score (p<0.025) than the placebo group. The incidence of treatment-related adverse events was similar and low in all groups.
Conclusion
Although ultra-high-dose methylcobalamin did not show significant efficacy in the whole cohort, this treatment may prolong survival and retard symptomatic progression without major side effects if started early.
Trial registration number
NCT00444613 .
Background and Objectives: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized clinically
by progressive memory loss and subsequent dementia. The aim of this study was to evaluate the synergist effect
of folic acid and vitamin B12 on spatial memory in adult male rat Alzheimer's disease.
Material and methods: In this experimental study, 49 male Wistar rats were randomly divided into 7 groups: (7
rats in each group): control(intact), NBM lesion group, which received electrically- induced lesion (0.5 m A, 3s)
in NBM, sham group ( the electrode was impaled in to the NBM with no lesion), NBM lesion + folic acid
5mg/kg, NBM lesion+ vitamin B12 5mg/kg, NBM lesion+ folic acid- vitamin B12, NBM lesion + saline.
Acquisition and retention testing was done by using an eight-radial arm maze, in which the patterns of arm
entries were recorded in each group for calculating working memory errors, reference memory error ,and
latency. Data were analyzed using one-way ANOVA and Tukey's post hoc test.
Results: The results showed that there was a significant difference between the control and lesion groups in the
parameters of the reference memory error, working memory error ,and elapsed time (P˂0.05). The
administration of folic acid and vitamin B12alone did not have any significant effect on spatial memory
parameters compared with the lesion group. Co-administration folic acid- vitamin B12 resulted in a reduction in
the reference and working memory errors and the time spent in the eight-arm radial laser maze.
Conclusion: According to the results, the NMB lesion resulted in a reduction in spatial memory and coadministration
of folic acid and vitamin B12 resulted in improved spatial memory.
Key words: Alzheimer disease, Nucleus Basalis Magnocellularis, Folic acid, Vitamin B12, Radial maze
Funding: This research was funded by Shahid Chamran University.
Conflict of interest: None declared.
Ethical approval: The Ethical Committee of Shahid Chamran University approved the study.
How to cite this article: Eskandary A, Moazedi A.A, Mamani N. The Synergist Effects of Folic Acid and
Vitamin B12 on Spatial Memory in Adult Male Rat Model of Alzheimer's Disease. J Rafsanjan Univ Med Sci
2018; 16(10): 925-38. [Farsi]
Objective: To use one of such mechanism like bacteriocins, produced by Lactobacilli activities against pathogens. Study Design: Analytical/observational study Place and Duration of Study: This study was carried out at Gulab Devi Chest Hospital, Lahore from November 2012 to January 2013. Materials and Methods: This study included 203 clinical samples. Multidrug resistant clinical isolates were selected on the basis of their MAR (Multiple antibiotic resistances) index, antibiotic susceptibility testing, methicillin resistant Staphylococcus aureus (MRSA) with oxacillin disc, double disc synergism and combination disc test. Plasmid isolation, conjugation was performed. Well-Diffusion assay was used for screening of putative bacteriocins produced by Lactobacillus strains against MDRs. Physiological characterization of antimicrobial compounds and protein estimation was analyzed. Results: Twenty five strains were selected based on MAR index (>0.2). In which 6 MRSA and 19 extended spectrum beta-lacatmases (ESBL) producers were further proceeded for antimicrobial activity with putative bactericins. Plasmid was easily transferred their resistance by the process of conjugation. Five bacteriocins were obtained from Lactobacillus strains isolated from commercial products. These bacteriocins showed a strong antibacterial activity against selected MDRs. Decrease in zone sizes was observed when putative bacteriocins were treated with heat, SDS (Sodium dodecyl sulfate) and Protinase k. Putative bacteriocins produced by Lactobacilli exhibit significant antibacterial activity against MDRs. SA1 has high antibacterial activity with high protein content of 13mg/ml. Conclusion: Putative bacteriocins produced by Lactobacilli exhibit significant antibacterial activity against selected MDRs. MDRs have ability to transfer their resistance to other bacteria. The peptidal component of these bacteriocins can be used as an alternative therapy. Proper hospital policies require minimizing the horizontal spread of MDRs. Hence, it is necessary to purify the antibacterial molecule out of putative bacteriocin for further analysis.
Objective: To determine the frequency of non-alcoholic fatty liver disease in patients with type 2 diabetes and to compare the diabetic control and dyslipidemia in diabetic patients with and without non-alcoholic fatty liver disease. Study Design: Cross sectional study. Place and Duration of Study: This study was conducted in Outpatient Department of Medicine, Jinnah Hospital, Lahore from 31st August 2013 to 28th February 2014. Materials and Methods: Both male and female with age ranging from 30-60 years having type 2 diabetes for more than 5 years duration were included. Two hundred and sixty diagnosed diabetes patients presenting to outpatient department were enrolled. Screening was done for NAFLD on the basis of ultrasonography. Sampling for HbA1C and lipid profile was done. Data was analyzed using SPSS 17. Results: Proportion of NAFLD was quite high i.e. 70%. Seventy five patients (28.8%) had abnormal triglyceride level and 72 patients (27.7%) had raised serum cholesterol. Low density lipoprotein was abnormally high in all individuals and high density lipoprotein was low in all individuals. One hundred and six (40.8%) diabetics patients had good control while rest has poor control and 135 patients (51.1%) were obese. Conclusion: Proportion of NAFLD was quite high in diabetic patients. We should screen every patient for NAFLD as it may reduce the co morbidity.
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.
Amyotrophic lateral sclerosis (ALS) is an incurable progressive neurodegenerative disorder that causes motor dysfunction. Treatments and drugs that slow progression of ALS have garnered great interest. In the present study, we show that the vitamin B12 analog methylcobalamin (MBL) effectively and dose dependently prevented embryonic stem cell-derived motor neuron death induced by cocultivation with astrocytes expressing mutant human superoxide dismutase-1 (G93A). Moreover, cotreatment of MBL with a conventional ALS drug, riluzole, further enhanced survival of motor neurons in this in-vitro ALS model. Our results show the potential use of MBL as a treatment for ALS and suggest a possible combination therapy strategy with other types of approved ALS drugs.
If the entire retina of an experimental animal is subjected to ischemia for a certain period of time and is then reperfused, subsequent cell death occurs particularly in the inner part of the retina, manifesting in a delayed fashion (Fig. 1). This delayed neuronal death due to retinal ischemia is now believed to reflect impaired cellular ion homeostasis especially concerning Ca2+.
Abstract Methylcobalamin is a vitamin B12 analog that is necessary for nervous system maintenance. Although methylcobalamin has some positive effects on peripheral nervous system disorders, the mechanism through which it affects neurons are not entirely known. Recent studies have revealed its intracellular signaling pathway and some of its molecular actions on neurons. In this article, I review interactions between methylcobalamin and neurons that have been revealed through in vitro studies, in vivo studies, and clinical use. Methylcobalamin participates in nervous system maintenance through several mechanisms. Methylcobalamin is an active form of vitamin B12, and a coenzyme of methionine synthase, which is required for DNA and protein methylation. In addition, methylcobalamin facilitates neurite outgrowth and inhibits neural apoptosis through the Erk1/2 and Akt signaling pathways. Treatment with high doses of methylcobalamin ameliorates symptoms and negative electrophysiological findings in animal models of peripheral nerve neuropathy and in patients with carpal tunnel syndrome and amyotrophic lateral sclerosis. Thus, high-dose methylcobalamin has great potential for treating nervous system disorders. Further investigations with methylcobalamin may help elucidate its mechanisms of action, which may further enable us to treat many nervous system disorders.
Glaucoma often exists and progresses at normal or even low intraocular pressure (IOP) levels, on the basis of IOP-independent
risk factors. Pressure-independent risk factors have only begun to be explored. Decreased perfusion of the optic nerve head
may result from orthostatic hypotension, nocturnal hypotension, atrial fibrillation, migraine, Raynaud’s phenomenon, abnormally
low intracranial pressure, autoimmune phenomena, and sleep apnea. Hemorheologic abnormalities, such as increased erythrocyte
agglutinability, decreased erythrocyte deformability, increased serum viscosity, or increased platelet aggregability, may
also play a role. Recent evidence has implicated oxidative stress as playing a significant role in retinal ganglion cell (RGC)
damage in glaucoma. Gamma-aminobutyric acid (GABAA) receptors are expressed on RGCs and may play a role in apoptosis induced by oxidative stress.
Objectives: The objectives of this prospective study were to examine the efficacy of methylcobalamin in preventing the deterioration of glaucomatous visual field defects, and to evaluate the relationship between the progression of visual field defects and other clinical characteristics using the Cox proportional hazards model. Methods: Patients with normal-tension glaucoma (NTG) who met the selection criteria were divided into a treatment group and an untreated control group. The treatment group consisted of 14 patients and 14 eyes; they were administered 1500 μg/d of methylcobalamin orally for 4 years beginning in April 1994. The control group consisted of 22 patients and 22 eyes. Both groups had similar characteristics in terms of age, systemic complications, intraocular pressure (IOP), refraction, and baseline visual field (mean deviation and corrected pattern standard deviation). Results: After 4 years of peroral methylcobalamin treatment, 2 eyes of 2 patients (14%) in the treatment group and 13 eyes of 13 patients (59%) in the control group showed deterioration of visual field defect. The deterioration in the control group was significant compared with that in the treatment group (P = 0.008). According to the Cox proportional hazards model, peroral methylcobalamin treatment and mean IOP during the follow-up period showed significant correlation to the progression of visual field defect. Conclusions: These findings suggest that peroral methylcobalamin might be useful in the treatment of visual field defects in patients with NTG.
The pathomechanism of sporadic amyotropic lateral sclerosis is not clearly understood, although a proportion of familial amyotropic lateral sclerosis is caused by superoxide dismutase 1 mutations. Theories based on studies of human post-mortem tissue, research on animal models and in vitro work have been proposed for the pathogenesis of amyotropic lateral sclerosis, but the pathogenesis is not the same between sporadic and familial amyotropic lateral sclerosis.
Drug candidates were tested using superoxide dismutase 1 mutant mice. Although the candidates were shown to be effective in mice, clinical trials in humans have failed to identify any truly effective pharmacotherapies in sporadic amyotropic lateral sclerosis, with only riluzole providing a modest improvement in survival. Ongoing or planned trials are exploring the value of antiglutamatergic drugs, antioxidants, neurotrophic factors, anti-inflammatory drugs and anti-aggregation drugs.
A combination of drugs acting on different mechanisms is needed for effective therapy. Moreover, gene expression profiling and genome-wide association studies, together with inhibitory RNA techniques, are helpful for developing new pharmacotherapeutic strategies including gene therapy. It is also likely that the recently advanced generation of induced pluripotent stem cells will lead to the development of cell therapy for amyotropic lateral sclerosis. In addition to finding effective therapies, research is also needed in order to detect early disease markers since pharmacotherapy is most beneficial when given early in the course of sporadic amyotropic lateral sclerosis.
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.
Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite.
The central neurotoxicity of the excitatory amino acid neurotransmitter glutamate has been postulated to participate in the pathogenesis of the neuronal cell loss associated with several neurological disease states, but the complexity of the intact nervous system has impeded detailed analysis of the phenomenon. In the present study, glutamate neurotoxicity was studied with novel precision in dissociated cell cultures prepared from the fetal mouse neocortex. Brief exposure to glutamate was found to produce morphological changes in mature cortical neurons beginning as quickly as 90 sec after exposure, followed by widespread neuronal degeneration over the next hours. Quantitative dose-toxicity study suggested an ED50 of 50-100 microM for a 5 min exposure to glutamate. Immature cortical neurons and glia were not injured by such exposures to glutamate. Uptake processes probably do not limit GNT in culture, as the uptake inhibitor dihydrokainate did not potentiate GNT. Possibly reflecting the lack of uptake limitation, glutamate was found to be actually more potent than kainate as a neurotoxin in these cultures, a dramatic reversal of the in vivo potency rank order. Some neurons regularly survived brief glutamate exposure; these possibly glutamate-resistant neurons had electrophysiologic properties, including chemosensitivity to glutamate, that were grossly similar to those of the original population.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
The electrophysiologic study using patch-clamp techniques demonstrated that NMDA-induced currents had properties similar to those recorded in the brain.
Primary cultures obtained from the fetal rat retina (gestation days 16 to 19) were used for the experiment. Immunocytochemical and electrophysiologic studies were done to identify the cultured cells. The neurotoxic effects of glutamate or N-methyl-D-aspartate (NMDA) on the retinal cultures were quantitatively assessed using the trypan blue exclusion method.
The immunocytochemical study revealed that the major component of the rat retinal cultures was neurons including amacrine cells. The electrophysiologic study using patch-clamp techniques demonstrated that exposure to NMDA-induced currents with properties characteristic of those recorded in the brain. Brief exposure of these neurons to glutamate or NMDA induced delayed cell death. Glutamate neurotoxicity was prevented by the application of dopamine and forskolin. The protective action of dopamine was antagonized by a D1 receptor antagonist (SCH 23390) but not by D2 receptor antagonists (domperidone and sulpiride). A D1 receptor agonist (SKF 38393) protected glutamate-induced neurotoxicity in a concentration-dependent manner, whereas a D2 receptor agonist (quinpirole) did not affect it.
These findings demonstrate that dopamine protects retinal neuronal cells against NMDA receptor-mediated glutamate neurotoxicity via D1 receptors.
There is an increasing amount of experimental evidence that oxidative stress is a causal, or at least an ancillary, factor in the neuropathology of several adult neurodegenerative disorders, as well as in stroke, trauma, and seizures. At the same time, excessive or persistent activation of glutamate-gated ion channels may cause neuronal degeneration in these same conditions. Glutamate and related acidic amino acids are thought to be the major excitatory neurotransmitters in brain and may be utilized by 40 percent of the synapses. Thus, two broad mechanisms--oxidative stress and excessive activation of glutamate receptors--are converging and represent sequential as well as interacting processes that provide a final common pathway for cell vulnerability in the brain. The broad distribution in brain of the processes regulating oxidative stress and mediating glutamatergic neurotransmission may explain the wide range of disorders in which both have been implicated. Yet differential expression of components of the processes in particular neuronal systems may account for selective neurodegeneration in certain disorders.
A 17-year-old boy with vitamin B12 deficiency that occurred after a small bowel resection developed bilateral centrocecal scotomas during folic acid therapy and improved on therapy including vitamin B complex.
A 19-year-old man presented with blurring of vision for 2 weeks. He also complained of anorexia with weight loss during the past 4 months. Eight years ago, his small bowel from midportion of the jejunum, ileum, ascending colon and transverse colon were resected because of gangrene. He gave no history of exposure to tobacco, alcohol or other toxins. The bone marrow aspiration showed hypocellular with panhypoplasia. Serum vitamin B12 level was low while serum and red cell folate were within normal limits. His visual acuity was 5/200 in both eyes with centrocecal scotomas in both eyes. Other neurologic and ophthalmic examinations were found to be normal. The patient was given intramuscular injections of 1,000 micrograms of cyanocobalamin. Four months later, his visual acuity improved, serum vitamin B12 level and the bone marrow returned to be normal. This is a frank case of optic neuropathy in a patient with vitamin B12 deficiency due to a massive small bowel resection.
The number of muscarinic receptors in the striatum and hippocampus of aged rats is significantly lower than the number measured in young animals. The treatment of aged rats for 30 days with S-adenosyl-L-methionine (SAM) restored the number of muscarinic receptors to levels found in the striatum and hippocampus from young animals. We did not observe a clear-cut difference between the dissociation constants of untreated young and untreated or SAM-treated aged rats, whereas the binding capacity varied. Moreover, in vitro addition of SAM to hippocampal membranes from aged rats resulted in a significant increase in the number of binding sites. This in vitro effect was antagonized by S-adenosyl-L-homocysteine, a specific in vitro inhibitor of methyltransferase activity. The reduction in the muscarinic receptor density could be related to a decrease in neuronal membrane fluidity induced by aging, while its increase after SAM treatment might be ascribed to the ability of this methyl donor to increase the fluidity of cell membranes by stimulating phospholipid synthesis.
Here we describe a new model for inducing ischemic neuronal degeneration in the adult rat retina. Rose bengal dye was injected intravenously; then the retina was exposed to intense light which caused vascular photothrombosis resulting in acute degeneration of retinal neurons. By either light or electron microscopical criteria, the acute neurodegernative reaction was judged identical in pattern, cytopathological appearance, and time course to the excitotoxic type of reaction typically seen in the retina following exposure to exogenous glutamate. These results reinforce other accumulating evidence suggesting that ischemic CNS damage is mediated by glutamate or related excitotoxins. The advantages of this model of CNS ischemia include its noninvasiveness, ease of application, authentic simulation of vascular thrombosis, and accessibility for application of neuroprotective drugs.
Chick embryo retinas were exposed for 40 min to anoxic conditions and the resulting histological changes were studied by light microscopy. A strong edematous response with severe swelling of the inner nuclear, inner plexiform and ganglion cell layers and numerous cells with pyknotic nuclei but very few dead cells were observed. These changes were qualitatively similar to those observed on retinas exposed to high potassium concentration or to glutamic acid. The deleterious effects of anoxia and high potassium concentration were not affected by the removal of calcium ions from the retina incubation medium but were prevented by gamma-D-glutamylglycine, an antagonist of excitatory amino acid receptors. These results suggest that the damage observed in retinas maintained under anoxia for 40 min is likely to be due to the release, from non-synaptic pools, of excitatory neurotransmitters, possibly glutamic and/or aspartic acids, evoked by the dissipation of the membrane ionic gradients. These events would be the first in a chain of toxic damage leading ultimately to cell death.
Retinal ischemia was induced in rabbits by increasing intraocular pressure above systolic blood pressure for 60 or 75 minutes, and retinal function was monitored by electroretinography. Pretreatment with intravenous dextromethorphan, a nonprescription antitussive and selective antagonist of N-methyl-D-aspartate receptors, enhanced greatly the post-ischemic recovery of b-wave amplitude. Dextromethorphan may prove to be useful clinically in the management of retinal ischemic disease.
The nervous system has been involved in the majority (at least 75%), of cases of acquired immune deficiency syndrome (AIDS) examined postmortem, but the pathogenetic mechanisms involved are not well understood. The predominant pathological process is opportunistic infection secondary to the decrease of T-helper (T4) cells and includes toxoplasmosis, encephalitis due to cytomegalovirus and progressive multifocal leucoencephalopathy. On the other hand, mycoses (mainly cryptococcosis) are relatively uncommon. Primary lymphomas are three times more common than secondary lymphoma spreading from other sites. Cerebral involvement by Kaposi sarcoma is metastatic. probably from primary foci in the lungs.
Lesions due to the direct involvement of the nervous system by the human immune deficiency virus (HIV) include subacute encephalitis and vacuolar myelopathy. The former is reported with increasing frequency and is localized predominantly to the white matter in which multinucleated giant cells can be found. These are considered typical of AIDS and have been shown to contain HIV particles in their cytoplasm.
AIDS lesions due to infectious agents do not always conform to the typical pattern of the uncomplicated disease and not uncommonly there is evidence of more than one infectious agent in the same area.
Peripheral nervous system lesions in I-IIV infections, responsible for a variety of clinical symptoms, usually appear, in biopsy material, as nonspecific inflammatory in type. CMV inclusions and lymphomatous infiltrations of peripheral nerve have been reported in autopsy cases.
The effect of S-adenosyl-L-methionine (SAM) on neuronal degeneration induced by transient forebrain ischemia was studied in rats. Bilateral occlusion of the common carotid arteries for 30 min in a 4-vessel occlusion model caused degeneration of CA1 neurons of the hippocampus. When SAM-HCl or SAM sulphate tosylate (SAM-ST, 100 mg/kg as the free form of SAM, i.p.) was administered just after recirculation and every hour for 5 h after recirculation, the degeneration and loss of pyramidal cells were prevented. However, adenosine, a metabolite of SAM, and glycerol, which has the same osmotic pressure as the solution of SAM-ST, did not show any effects on the neuronal damage. The results showed that SAM has a beneficial effect on neuronal damage induced by ischemia.
Twenty of 89 consecutive patients with the acquired immunodeficiency syndrome (AIDS) in whom autopsies were performed over a 3 1/2-year period had a vacuolar myelopathy that was most severe in the lateral and posterior columns of the thoracic cord. Light and electron microscopy showed that vacuoles were surrounded by a thin myelin sheath and appeared to arise from swelling within myelin sheaths. Signs and symptoms referable to the spinal-cord lesions, including paraparesis, often accompanied by spasticity or ataxia (or both), were present in all five patients with marked pathological changes, in five of seven patients with moderate changes, and in two of eight patients with mild changes. Fourteen patients were demented. The clinical presentation was sufficiently distinctive to provide a guide for antemortem diagnosis. Possible causes of the vacuolar changes include uncharacterized viral infection or a metabolic derangement related to selective nutritional deficiency.
Brain ischemia was produced in gerbils (Meriones unguiculatus) by the bilateral ligation of the carotid arteries with reported procedures. Changes in the energy status of brain demonstrated that carotid ligation was effective. At different time intervals from ligation, groups of gerbils were given either saline of S-Adenosyl-L-methionine (SAMe) by the intraventricular (i.v.) route (1.6 mg/Kg body wt. twice, at each 10 min interval), or by the intraperitoneal (i.p.) administration (200 mg/Kg body wt.) or subcutaneously (s.c.) with 40 mg/Kg body wt, daily, for two weeks. Control animals, with and without SAMe, together with the ischemic groups, were decapitated directly into liquid nitrogen, 10 min after ligation. Brain neutral and polar lipid, together with free fatty acids, which were all labeled in vivo by the intraventricular injection of [1-14C]arachidonic acid 2 hr prior to ligation, were extracted, purified and separated by conventional procedures. SAMe when injected i.v. or i.p. noticeably corrected the changes in polar lipid by reversing the decrease of brain phosphatidylcholine and choline plasmalogen, as well as of their labeling, which was due to ischemia. Concurrently with this action, SAMe treatment (i.v. and i.p.) also provided to some extent to re-establish the normal level of labeling of ethanolamine lipids. When SAMe was given s.c., no effect was present. SAMe had no effect on the increase of free fatty acid and diglyceride due to ischemia. The prevention by SAMe of the changes of choline lipids suggests that a stimulation of the methyltransferase reaction may occur in the ischemic brain, due to increased substrate (SAMe) availability. This effect may be important for cell survival, since membrane phospholipid derangements alter the properties of the membrane.
This review focuses on the biochemical and clinical aspects of methylation in neuropsychiatric disorders and the clinical potential of their treatment with ademetionine (S-adenosylmethionine; SAMe). SAMe is required in numerous transmethylation reactions involving nucleic acids, proteins, phospholipids, amines and other neurotransmitters. The synthesis of SAMe is intimately linked with folate and vitamin B12 (cyanocobalamin) metabolism, and deficiencies of both these vitamins have been found to reduce CNS SAMe concentrations. Both folate and vitamin B12 deficiency may cause similar neurological and psychiatric disturbances including depression, dementia, myelopathy and peripheral neuropathy. SAMe has a variety of pharmacological effects in the CNS, especially on monoamine neurotransmitter metabolism and receptor systems. SAMe has antidepressant properties, and preliminary studies indicate that it may improve cognitive function in patients with dementia. Treatment with methyl donors (betaine, methionine and SAMe) is associated with remyelination in patients with inborn errors of folate and C-1 (one-carbon) metabolism. These studies support a current theory that impaired methylation may occur by different mechanisms in several neurological and psychiatric disorders.
To examine the effects of Zn2+ on glutamate-induced neurotoxicity in cultured retinal neurons.
Primary cultures obtained from fetal rat retinas (16 to 19 days gestation) were used. The neurotoxic effects of excitatory amino acids were quantitatively assessed using the trypan blue exclusion method.
A brief exposure of retinal cultures to glutamate or N-methyl-D-aspartate (NMDA) induced delayed cell death. Zn2+ at concentrations of 3 to 30 microM ameliorated glutamate- and NMDA-induced neurotoxicity in a dose-dependent manner. By contrast, neurotoxicity induced by a 1-hour exposure to kainate was not affected by Zn2+.
These findings demonstrate that Zn2+ protects retinal neurons from NMDA receptor-mediated glutamate neurotoxicity.
1. The ratio of the methyl donor, S-adenosylmethionine, to the co-product, S-adenosylhomocysteine (the methylation ratio) is known to control the activity of methyltransferases in tissues. Inactivation of the vitamin B12-dependent enzyme, methionine synthase, reduces the methylation ratio in rats and pigs in vivo.
2. We have determined the effect that such alterations have on neural protein ‘O’ and ‘N’ methyltransferases using an in vitro assay in rats, pigs and humans in the presence of the normal methylation ratio and the abnormal methylation ratios found experimentally in vivo in rats and pigs.
3. The methylation ratio found in the neural tissues of vitamin B12-inactivated pigs significantly inhibits the protein methyltransferases of pigs and humans.
4. By contrast, the altered methylation ratio found in vitamin B12-inactivated rats only marginally inhibits the equivalent rat methyltransferases.
5. This is consistent with the induction of a myelopathy by such treatment in pigs and humans, but not in the rat.
6. Dietary supplements of methionine given to vitamin B12-inactivated pigs have been shown to prevent the myelopathy in vivo by both elevating the neural S-adenosylmethionine level and resetting the methylation ratio. We find in our in vitro assay that these events reinstate the methyltransferase activity to near normal levels, thus explaining its protective effect in vivo.
The effects of methylcobalamin, a vitamin B12 analog, on glutamate-induced neurotoxicity were examined using cultured rat cortical neurons. Cell viability was markedly reduced by a brief exposure to glutamate followed by incubation with glutamate-free medium for 1 h. Glutamate cytotoxicity was prevented when the cultures were maintained in methylcobalamin-containing medium. Glutamate cytotoxicity was also prevented by chronic exposure to S-adenosylmethionine, which is formed in the metabolic pathway of methylcobalamin. Chronic exposure to methylcobalamin and S-adenosylmethionine also inhibited the cytotoxicity induced by N-methyl-D-aspartate or sodium nitroprusside that releases nitric oxide. In cultures maintained in a standard medium, glutamate cytotoxicity was not affected by adding methylcobalamin to the glutamate-containing medium. In contrast, acute exposure to MK-801, a NMDA receptor antagonist, prevented glutamate cytotoxicity. These results indicate that chronic exposure to methylcobalamin protects cortical neurons against NMDA receptor-mediated glutamate cytotoxicity.
There is an increasing amount of experimental evidence that oxidative stress is a causal, or at least an ancillary, factor
in the neuropathology of several adult neurodegenerative disorders, as well as in stroke, trauma, and seizures. At the same
time, excessive or persistent activation of glutamate-gated ion channels may cause neuronal degeneration in these same conditions.
Glutamate and related acidic amino acids are thought to be the major excitatory neurotransmitters in brain and may be utilized
by 40 percent of the synapses. Thus, two broad mechanisms--oxidative stress and excessive activation of glutamate receptors--are
converging and represent sequential as well as interacting processes that provide a final common pathway for cell vulnerability
in the brain. The broad distribution in brain of the processes regulating oxidative stress and mediating glutamatergic neurotransmission
may explain the wide range of disorders in which both have been implicated. Yet differential expression of components of the
processes in particular neuronal systems may account for selective neurodegeneration in certain disorders.
Enzymatic step-wise methylation of membrane phosphatidylethanolamine (PE) to phosphatidyl-N-methylethanolamine (PME) and then phosphatidyl-choline (PC) has been known to alter membrane properties and responsiveness of cells for activation of receptors by chemical transmitters. Conversion of PE to PME and PME to PC in the presence of S-adenosyl-L-methionine (SAM) are catalyzed by two phospholipid N-methyltransferases, PMT I and PMT II, of which PMT I is the rate limiting enzyme. Retina is a good neuronal model for chemical transmission. However, retina was not studied for PMT activity. Therefore, we studied the rat retina for PMT I activity. Methylation of PE in the rat retinal sonicates was assayed using 3H-SAM (2 microM) at 37 degrees C in Tris-glycylglycine buffer (50 mM, pH 8.0) and methylated phospholipids were extracted with chloroform/methanol/HCl (2/1/0.02, v/v) and separated by thin layer chromatography on Silica Gel G plates. Chromatograms were developed in a solvent system of propionic acid/n-propyl alcohol/chloroform/water (2/2/1/1, v/v). This study gave the following results: (a) the total methylated phospholipids were (M +/- SE, N = 5) 19.90 +/- 4.03 fmol/mg protein/min; (b) the major methylated phospholipid was PME (4.21 +/- 0.68 fmol/mg protein/min; (c) the fatty acid methylesters formed by fatty acid carboxymethylase (FACM) which accumulated in the solvent front amounted to 18.82 +/- 2.84 fmol/mg protein/min. Both PMT I and FACM were inhibited by S-adenosyl-L-homocysteine (I50, 1.2-5 microM). These observations indicate that rat retina contains both PMTs and FACM.
The effects of methylcobalamin, a vitamin B12 analogue, on the hypoxia/hypoglycemia- or glutamate-induced reduction in hippocampal CA1 presynaptic fiber spikes elicited by Schaffer collateral stimulation in rat brain slices were evaluated. Hippocampal slices were exposed to 15 min of hypoxia/hypoglycemia, and then these slices were returned to oxygenated and glucose-containing buffer for 3 h. Hypoxia/hypoglycemia reduced CA1 presynaptic potentials in vitro. Treatment with 10 microM methylcobalamin attenuated the impairment of CA1 presynaptic potentials induced by hypoxia/hypoglycemia or glutamate application (10 mM). Daily injection of methylcobalamin (0.5 mg/kg i.p./day) for 3 days in vivo also attenuated the hypoxia/hypoglycemia- or glutamate-induced reduction in presynaptic potentials in hippocampal slices. Pretreatment with cyanocobalamin at 10 microM failed to attenuate the impairment of CA1 presynaptic potentials. However, daily injection of cyanocobalamin (0.5 mg/kg i.p./day) for 3 days caused a protective action against the hypoxia/hypoglycemia- or glutamate-induced functional deficit. Furthermore, co-treatment of L-arginine (100 microM), a substrate for nitric oxide synthase, with methylcobalamin in vitro reversed the methylcobalamin-induced functional recovery. The present results demonstrate that methylcobalamin application in vivo or in vitro leads to functional recovery from hypoxia/hypoglycemia- or glutamate-induced impairment of CA1 presynaptic potentials. Neuroprotection was obtained by in vivo application of cyanocobalamin, but not by its in vitro application. It is reported that in vivo injected cyanocobalamin converted to methylcobalamin in the hepatic cells. Therefore, the results suggest that a transmethylation reaction in the hippocampal regions may be involved in the methylcobalamin-induced functional recovery from ischemic impairment.
This study was performed to elucidate the role of nitric oxide (NO) in N-methyl-D-aspartate (NMDA) receptor-mediated glutamate neurotoxicity in the retina. The experiments were done with primary retinal cultures obtained from 17- to 19-day-old rat fetuses. The NOS activity measured by monitoring the conversion of [3H]arginine to [3H]citrulline was approximately 5 pmol/min/mg protein. A 10-min exposure of the cultured cells to glutamate (1 mM) or NMDA (1 mM) followed by a 1-h incubation in a normal medium consistently resulted in 60% cell death. The concomitant addition of an inhibitor of NOS, Nomega-nitro-L-arginine (300 microM), with glutamate or NMDA reduced cell death by 70%. A brief exposure of the cells to sodium nitroprusside (SNP, 500 microM) or S-nitrosocysteine (SNOC, 500 microM), NO-generating agents, caused 60% cell death. Depletion of NO by reduced hemoglobin prevented the cell death induced by either glutamate, NMDA, or NO generating agents. Fifty microM SNOC alone had no effect on the cell viability. However, pretreatment with 50 microM SNOC as well as simultaneous application of 50 microM SNOC with NMDA inhibited cell death induced by NMDA. These findings indicate that a low concentration of NO plays a protective role in glutamate neurotoxicity via closing the NMDA receptor gated ion channel. However, elevated concentrations of NO, interacting with oxygen radicals, become toxic and mediate glutamate-induced neurotoxicity in the cultured retinal neurons.
The clinical potential of ademethionine (S-adenosylmethionine) in neurological disorders Baldessarini RJ. Neuropharmacology of S-adenosyl-Lmethionine
- T Bottigllieri
- K Hyland
- Reynolds
Bottigllieri T, Hyland K, Reynolds EH. The clinical potential of ademethionine (S-adenosylmethionine) in neurological disorders. Drugs. 1994;48:137-152. 5. Baldessarini RJ. Neuropharmacology of S-adenosyl-Lmethionine. Am]Med. 1987;83:95-103.
- G Trovarelli
- Ge De Medio
- S Porcellati
Trovarelli G, de Medio GE, Porcellati S, et al. NeurochemRes. 1983;8:1597-1609.
- Rj Baldessarini
- Neuropharmacology
Baldessarini RJ. Neuropharmacology of S-adenosyl-Lmethionine. Am]Med. 1987;83:95-103.