Magnetic resonance imaging in a patient with nitrous oxide-induced subacute combined degeneration of the spinal cord.

INTRODUCTION: Chronic nitrous oxide use can lead to neurological findings that are clinically and radiographically identical to those found in patients with pernicious anemia, specifically subacute combined degeneration of the spinal cord and peripheral neuropathy. CASE SUMMARY: A 22-year-old man presented with lower extremity weakness and ataxia in the setting of inhaling 250 nitrous oxide cartridges two to three times weekly for two years. IMAGES: Magnetic resonance imaging showed T2 hyperenhancement of the dorsal columns of the cervical spine from the first to the sixth vertebrae, which helped to establish a diagnosis of nitrous oxide-induced subacute combined degeneration of the spinal cord. CONCLUSIONS: Chronic nitrous oxide use should be included in the differential diagnosis of any patient with otherwise unexplained neurological complaints that localize to the dorsal columns and has the changes on magnetic resonance imaging described here.

Nitrous oxide abuse induced subacute combined degeneration despite patient initiated B12 supplementation.

INTRODUCTION: Nitrous oxide (N2O) is a commonly used inhaled anesthetic that is legal to purchase as a food additive and is popular as a recreational euphoric drug. Abuse causes a functional B12 deficiency, leading to clinical features and imaging consistent with subacute combined spinal cord degeneration (SCD). CASES: Poison Center medical records from four patients are reviewed in this series. Four patients presented with lower extremity weakness, paresthesias and gait abnormalities in the setting of chronic N2O abuse. Each reported using 50-150 N2O cartridges ("whippets") almost daily for months to years, and reported supplementing with oral B12 at the recommendation of other users and online forums. None reported prior B12 deficiency or dietary restrictions, and none exhibited hematologic abnormalities. RESULTS: All patients had clinical signs of neurotoxicity including weakness and ataxia. Additionally, all had elevated methylmalonic acid and homocysteine concentrations with normal B12 indicating a functional B12 deficiency. Three had imaging consistent with SCD despite home supplementation The MRI in the fourth case was inconclusive due to movement artifact. CONCLUSION: We report four cases of subacute combined degeneration induced by recreational nitrous oxide abuse despite self-administered vitamin B12 supplementation.

Toxicity induced by multiple high doses of vitamin B12 during pernicious anemia treatment: a case report.

Context: The clinical consequences of excess vitamin B12 induced by multiple oral doses of cyanocobalamin are not well-known.Case details: A young woman was treated with multiple daily doses of 1 mg of cyanocobalamin for severe pernicious anemia. After a total dose of 12 mg, she developed acne, palpitations, anxiety, akathisia, facial ruddiness, headache, and insomnia. She improved two weeks after stopping the drug. There were no sequelae nor complications.Discussion: Although these symptoms of cobalamin toxicity were unexpected and unusual, the case reminds us that the administration of any drug is not entirely safe.

Chemical Pathology of Homocysteine VII. Cholesterol, Thioretinaco Ozonide, Mitochondrial Dysfunction, and Prevention of Mortality.

The purpose of this review is to elucidate how low blood cholesterol promotes mitochondrial dysfunction and mortality by the loss of thioretinaco ozonide from opening of the mitochondrial permeability transition pore (mPTP). Mortality from infections and cancer are both inversely associated with blood cholesterol, as determined by multiple cohort studies from 10 to 30 years earlier. Moreover, low-density lipoprotein (LDL) is inversely related to all-cause and/or cardiovascular mortality, as determined by followup study of elderly cohorts. LDL adheres to and inactivates most microorganisms and their toxins, causing aggregation of LDL and homocysteinylated autoantibodies which obstruct vasa vasorum and produce intimal microabscesses, the vulnerable atherosclerotic plaques. The active site of mitochondrial oxidative phosphorylation and adenosine triphosphate (ATP) biosynthesis is proposed to consist of thioretinaco, a complex of two molecules of thioretinamide with cobalamin, oxidized to the disulfonium thioretinaco ozonide and complexed with oxygen, nicotinamide adenine dinucleotide (NAD+), phosphate, and ATP. Loss of the active site complex from mitochondria results from the opening of the mPTP and from decomposition of the disulfonium active site by electrophilic carcinogens, oncogenic viruses, microbes, and by reactive oxygen radicals from ionizing and non-ionizing radiation. Suppression of innate immunity is caused by the depletion of adenosyl methionine because of increased polyamine biosynthesis, resulting in inhibition of nitric oxide and peroxynitrite biosynthesis. Opening of the mPTP produces a loss of thioretinaco ozonide from mitochondria. This loss impairs ATP biosynthesis and causes the mitochondrial dysfunction observed in carcinogenesis, atherosclerosis, aging and dementia. Cholesterol inhibits the opening of the mPTP by preventing integration of the pro-apoptotic Bcl-2-associated X protein (BAX) in the outer mitochondrial membrane. This inhibition explains how elevated LDL reduces mitochondrial dysfunction by preventing loss of the active site of oxidative phosphorylation from mitochondria.

High methionine, low folate and low vitamin B6/B12 (HM-LF-LV) diet causes neurodegeneration and subsequent short-term memory loss.

Methionine is an essential amino acid found in rich quantities in average American diet such as meats, fish and eggs. Excessive consumption of such food often exceeds the normal requirement of the methionine in our body; which found to be related to the development of neurodegenerative disorders. However, the mechanistic pathways of methionine's influence on the brain are unclear. The present study is focus on the effects of high methionine, low folate and low vitamin B6/B12 (HM-LF-LV) diet on the dysfunction of neuronal and vascular specific markers in the brain. C57BL6/J male mice (8-10 week old) were fed with HM-LF-LV diet for a 6 week period. Cognitive function of mice was determine by measuring short-term memory using a Novel Object Recognition test (NORT). Neuronal dysfunction were evaluate by measuring the levels of Neuronal nuclear antigen (NeuN), Neuron-specific-enolase (NSE) and Fluoro-jade C(FJC) fluorescence; while cerebrovascular disruption were evaluate by assessing levels of endothelial junction proteins Vascular Endothelial-Cadherin (VE-Cadherin) and Claudin-5 in harvested brain tissue. Cerebrovascular permeability was assess by evaluating microvascular leakage of fluorescently labeled albumin in vivo. Endothelial and Neuronal Nitric Oxide Synthase (eNOS, nNOS) regulation and vascular inflammation (ICAM: intercellular adhesion molecules) were also evaluate in brain tissue. All assessments were conduct at weekly intervals throughout the study duration. NORT showed a significant temporal decrease in short-term memory of mice fed on HM-LF-LV diet for 6 weeks compared to the wild-type control group. Our experimental data showed that neuronal dysfunction (decreased NeuN levels and increased FJC positive neurons in brain) was more prominent in HM-LF-LV diet fed mice compared to normal diet fed control mice. In experimental mice, cerebrovascular disruption was found to be elevated as evident from increased pial venular permeability (microvascular leakage) and decreased in VE-Cadherin expression compared to control. Slight decrease in nNOS and increase in eNOS in experimental mice suggest a trend towards the decrease in potential for neuronal development due to the long-term HM-LF-LV diet fed. Collectively, our results suggest that a diet containing high methionine, low folate and low vitamin B6/B12 results in increased neuronal degeneration and vascular dysfunction, leading to short-term memory loss. Interestingly, significant neuronal damage precedes vascular dysfunction.

Effect of methotrexate/vitamin B12 on DNA methylation as a potential factor in leukemia treatment-related neurotoxicity.

Methotrexate (MTX) is administered to treat childhood acute lymphoblastic leukemia (ALL). It acts by inhibiting dihydrofolate reductase which reduces methyltetrahydrofolate, a key component in one carbon metabolism, thus reducing cell proliferation. Further perturbations to one carbon metabolism, such as reduced vitamin B12 levels via the use of nitrous oxide for sedation during childhood ALL treatment, may increase neurotoxicity risk. With B12 as an enzymatic cofactor, methyltetrahydrofolate is essential to produce methionine, which is critical for DNA methylation. We investigated global and gene specific DNA methylation in neuronal cell lines in response to MTX treatment and vitamin B12 concentration individually, and in combination. RESULTS: MTX treatment alone significantly increased LINE-1 methylation in SH-SY5Y (p = 0.040) and DAOY (p < 0.001), and increased FKBP5 methylation in MO3.13 cells (p = 0.009). CONCLUSION: We conclude that altered DNA methylation of brain/central nervous system cells could be one mechanism involved in MTX treatment-related neurotoxicities and neurocognitive late effects in ALL survivors.

Impaired mitochondrial function in HepG2 cells treated with hydroxy-cobalamin[c-lactam]: A cell model for idiosyncratic toxicity.

The vitamin B12 analog hydroxy-cobalamin[c-lactam] (HCCL) impairs mitochondrial protein synthesis and the function of the electron transport chain. Our goal was to establish an in vitro model for mitochondrial dysfunction in human hepatoma cells (HepG2), which can be used to investigate hepatotoxicity of idiosyncratic mitochondrial toxicants. For that, HepG2 cells were treated with HCCL, which inhibits the function of methylmalonyl-CoA mutase and impairs mitochondrial protein synthesis. Secondary, cells were incubated with propionate that served as source of propionyl-CoA, a percursor of methylmalonyl-CoA. Dose-finding experiments were conducted to evaluate the optimal dose and treatment time of HCCL and propionate for experiments on mitochondrial function. 50 µM HCCL was cytotoxic after exposure of HepG2 cells for 2d and 10 and 50 µM HCCL enhanced the cytotoxicity of 100 or 1000 µM propionate. Co-treatment with HCCL (10 µM) and propionate (1000 µM) dissipated the mitochondrial membrane potential and impaired the activity of enzyme complex IV of the electron transport chain. Treatment with HCCL decreased the mRNA content of mitochondrially encoded proteins, whereas the mtDNA content remained unchanged. We observed mitochondrial ROS accumulation and decreased mitochondrial SOD2 expression. Moreover, electron microscopy showed mitochondrial swelling. Finally, HepG2 cells pretreated with a non-cytotoxic combination of HCCL (10 µM) and propionate (100 µM) were more sensitive to the mitochondrial toxicants dronedarone, benzbromarone, and ketoconazole than untreated cells. In conclusion, we established and characterized a cell model, which could be used for testing drugs with idiosyncratic mitochondrial toxicity.

4-ethylphenyl-cobalamin impairs tissue uptake of vitamin B12 and causes vitamin B12 deficiency in mice.

Coß-4-ethylphenyl-cob(III) alamin (EtPhCbl) is an organometallic analogue of vitamin B12 (CNCbl) which binds to transcobalamin (TC), a plasma protein that facilitates the cellular uptake of cobalamin (Cbl). In vitro assays with key enzymes do not convert EtPhCbl to the active coenzyme forms of Cbl suggesting that administration of EtPhCbl may cause cellular Cbl deficiency. Here, we investigate the in vivo effect of EtPhCbl in mice and its ability, if any, to induce Cbl deficiency. We show that EtPhCbl binds to mouse TC and we examined mice that received 3.5 nmol/24h EtPhCbl (n=6), 3.5 nmol/24h CNCbl (n=7) or NaCl (control group) (n=5) through osmotic mini-pumps for four weeks. We analyzed plasma, urine, liver, spleen, submaxillary glands and spinal cord for Cbl and markers of Cbl deficiency including methylmalonic acid (MMA) and homocysteine (tHcy). Plasma MMA (mean±SEM) was elevated in animals treated with EtPhCbl (1.01±0.12 µmol/L) compared to controls (0.30±0.02 µmol/L) and CNCbl (0.29±0.01 µmol/L) treated animals. The same pattern was observed for tHcy. Plasma total Cbl concentration was higher in animals treated with EtPhCbl (128.82±1.87 nmol/L) than in CNCbl treated animals (87.64±0.93 nmol/L). However, the organ levels of total Cbl were significantly lower in animals treated with EtPhCbl compared to CNCbl treated animals or controls, notably in the liver (157.07±8.56 pmol/g vs. 603.85±20.02 pmol/g, and 443.09±12.32 pmol/g, respectively). Differences between the three groups was analysed using one-way ANOVA and, Bonferroni post-hoc test. EtPhCbl was present in all tissues, except the spinal cord, accounting for 35-90% of total Cbl. In conclusion, treatment with EtPhCbl induces biochemical evidence of Cbl deficiency. This may in part be caused by a compromised tissue accumulation of Cbl.

17 e- rhenium dicarbonyl CO-releasing molecules on a cobalamin scaffold for biological application.

Cyanocobalamin (B(12)) offers a biocompatible scaffold for CO-releasing 17-electron dicarbonyl complexes based on the cis-trans-[Re(II)(CO)(2)Br(2)](0) core. A Co-C≡N-Re conjugate is produced in a short time and high yield from the reaction of [Et(4)N](2)[Re(II)Br(4)(CO)(2)] (ReCORM-1) with B(12). The B(12)-Re(II)(CO)(2) derivatives show a number of features which make them pharmaceutically acceptable CO-releasing molecules (CORMs). These cobalamin conjugates are characterized by an improved stability in aqueous aerobic media over the metal complex alone, and afford effective therapeutic protection against ischemia-reperfusion injury in cultured cardiomyocytes. The non-toxicity (at µM concentrations) of the resulting metal fragment after CO release is attributed to the oxidation of the metal and formation in solution of the ReO(4)(-) anion, which is among the least toxic of all of the rare inorganic compounds. Theoretical and experimental studies aimed at elucidating the aqueous chemistry of ReCORM-1 are also described.

Antioxidant activities of Ganoderma lucidum polysaccharides and their role on DNA damage in mice induced by cobalt-60 gamma-irradiation.

In this study, the radio-protective effects of Ganoderma lucidum polysaccharides (GLP) were investigated in a mouse animal model exposed to (60)Co gamma-irradiation. Each of three batches of mice were divided into five groups (negative control, positive gamma irradiated control, and low, middle and high dosage GLP groups). Different batches of animals were used to evaluate the impact of GLP on peripheral white blood cell count, immune organ index; DNA damage, lipid peroxidation; micronuclei formation, and nucleated cell count in bone marrow induced by (60)Co gamma-irradiation. DNA strand-break and micronuclei frequency were significantly reduced and glutathione peroxidase activity and nucleated cell count in bone marrow were significantly increased by GLP treatment in a dose-dependent manner. GLP intervention also increased the activity of superoxide dismutase and decreased the level of malondialdehyde in middle and high GLP treatment groups. No adverse effects were observed on peripheral white blood cells and immune organ or body weight in either the control groups or GLP treated gamma exposed mice. These findings suggest that GLP possesses marked antioxidant capacity which plays an important role in the prevention of radiation damage in mice induced by (60)Co gamma-irradiation.

The octapeptide repeat PrP(C) region and cobalamin-deficient polyneuropathy of the rat.

INTRODUCTION: Cobalamin (Cbl) deficiency affects the peripheral nervous system (PNS) morphologically and functionally. We investigated whether the octapeptide repeat (OR) region of prion protein (PrP(C)) (which is claimed to have myelinotrophic properties) is involved in the pathogenesis of rat Cbl-deficient (Cbl-D) polyneuropathy. METHODS: We intracerebroventricularly administered antibodies (Abs) against the OR region (OR-Abs) to Cbl-D rats to prevent myelin damage and maximum nerve conduction velocity (MNCV) abnormalities, and PrP(C)s to normal rats to reproduce PNS Cbl-D-like lesions. We measured nerve PrP(C) levels and MNCV. RESULTS: The OR-Abs normalized myelin ultrastructure, MNCV values, and tumor necrosis factor (TNF)-α levels in the sciatic and tibial nerves of Cbl-D rats. PrP(C) levels increased in Cbl-D nerves. The nerves of the PrP(C)-treated rats showed typical Cbl-D lesions, significantly decreased MNCV values, and significantly increased TNF-α levels. CONCLUSIONS: OR-Abs prevent the myelin damage caused by increased OR regions, and excess TNF-α is involved in the pathogenesis of Cbl-D polyneuropathy.

Vitamin B12 as a carrier for targeted platinum delivery: in vitro cytotoxicity and mechanistic studies.

It is attractive to use vitamin B12 as a carrier for targeted delivery of cytotoxic agents such as platinum complexes owing to the high demand for vitamin B12 by fast proliferating cells. The basic {B12-CN-Pt(II)} conjugates are recognized by intracellular enzymes and converted to coenzyme B12 in an enzymatic adenosylation assay. The reductive adenosylation of {B12-CN-Pt(II)} conjugates leads to the release of the Pt(II) complexes; thus, {B12-CN-Pt(II)} conjugates can be considered as prodrugs. It is important not only to elucidate the activity of the cisplatin-B12 conjugates, but also to understand the mode of action on a molecular level. Chemical reduction of {B12-CN-Pt(II)} conjugates with cobaltocene yielded cob(II)alamin and induced release of the corresponding Pt(II) species. Kurnakov tests and coordination of 2'-deoxyguanosine or GMP to the released Pt(II) complexes allowed isolation and characterization of Pt(II) complexes as released during enzymatic adenosylation. The biological activity of these Pt(II) complexes was evaluated. Since the cleaved Pt(II) complexes show cytotoxicity, the {B12-CN-Pt(II)} conjugates can be used for specific targeting of cancer cells and therapeutic drug delivery. Preliminary in vitro cytotoxicity studies indicated lower activity (IC(50) between 8 and 88 µM) than found for pure cisplatin. Since active transport and receptor-mediated uptake limits the intracellular {B12-CN-Pt(II)} concentration, comparison with pure cisplatin is of limited use. We could show that the Pt(II) complexes cleaved from B12 exerted a cytotoxicity comparable to that of cisplatin itself. Cytotoxicity studies in vitamin B12 free media showed a dependence on the addition of transcobalamin II for B12-Pt(II) conjugates.

Vitamin B2 as a tracer for intraoperative pulmonary sentinel node navigation surgery.

BACKGROUND: We investigated whether fluorescent agents, especially vitamin B2, can act as tracers for intraoperative pulmonary sentinel node mapping. MATERIALS AND METHODS: Vitamin B2, fluorescent beads, and green fluorescent protein (GFP) were each injected into pulmonary parenchyma in 4 pigs (experiment 1). The safety of each tracer was also verified in 12 rats (experiment 2). RESULTS: Experiment 1: In all groups, the sentinel lymph node was identified in 3 out of the 4 pigs (75%). Speed of agent dispersion: vitamin B2>GFP >fluorescent beads. Level of fluorescence judged as: vitamin B2>GFP=fluorescent beads. Experiment 2: In all groups, all rats survived until sacrifice without complications. In the fluorescent beads group, the fluorescent beads remained in the blood vessels. CONCLUSION: Vitamin B2 is inexpensive, safe and easy to apply. It is anticipated that clinical application of vitamin B2 for intraoperative pulmonary sentinel node mapping will become possible.

Micronutrients and women of reproductive potential: required dietary intake and consequences of dietary deficiency or excess. Part I--Folate, Vitamin B12, Vitamin B6.

This two-part review highlights micronutrients for which either public health policy has been established or for which new evidence provides guidance as to recommended intakes during pregnancy. One pivotal micronutrient is folate, the generic name for different forms of a water-soluble vitamin essential for the synthesis of thymidylate and purines and, hence, DNA. For non-pregnant adult women the recommended intake is 400 µg/day dietary folate equivalent. For women capable of becoming pregnant an additional 400 µg/day of synthetic folic acid from supplements or fortified foods is recommended to reduce the risk of neural tube defects (NTD). The average amount of folic acid received through food fortification (grains) in the US is only 128 µg/day, emphasising the need for the supplemental vitamin for women of reproductive age. Vitamin B12 (cobalamin) is a cofactor required for enzyme reactions, including generation of methionine and tetrahydrofolate. B12 is found almost exclusively in foods of animal origin (meats, dairy products); therefore, vegetarians are at greatest risk for dietary vitamin B12 deficiency and should be supplemented. Vitamin B6 is required for many reactions, primarily in amino acid metabolism. Meat, fish and poultry are good dietary sources. Supplementation beyond routine prenatal vitamins is not recommended.

Vitamin B12b enhances the cytotoxicity of dithiothreitol.

It has been found previously that vitamin B12b amplifies significantly the cytotoxic effects of ascorbic acid by catalyzing the formation of reactive oxygen species, and the antioxidant dithiothreitol (DTT), in contrast to catalase, does not prevent the cytotoxicity. Therefore, in this study we examined whether B12b is able to enhance the cytotoxicity of DTT. It was revealed that B12b strongly increases the cytotoxic effect of DTT. Vitamin B12b added to DTT catalyzed the generation and drastic accumulation of hydrogen peroxide in culture medium to a concentration of 260 microM within 7 min. The extracellular oxidative burst induced by the combination of B12b and DTT (DTT + B12b) was accompanied by intracellular oxidative stress, the destabilization of lysosomes, and damage to DNA. The accumulation of DNA lesions led to the initiation of apoptotic cell death, including the activation of caspase-3 and the release of cytochrome c. The antioxidants pyruvate and catalase completely prevented the DTT + B12b-induced oxidative stress and cell death. The iron chelators desferrioxamine and phenanthroline prevented the geno- and cytotoxic action of the combination although they did not reduce the exogenous oxidative burst, indicating a key role for intracellular iron in the cytotoxicity of the combination. Thus, vitamin B12b dramatically enhances the cytotoxicity of DTT, catalyzing the generation of hydrogen peroxide and inducing extra- and intracellular oxidative stress, early destabilization of lysosomes, and iron-dependent DNA damage.

Effect of folate deficiency and folate and B12 excess on memory functioning in young chicks.

The results of this series of experiments with chicks trained on a single trial, passive avoidance task, demonstrate that methotrexate-induced folate deficiency, and excess levels of folate and B12 lead to amnesia in these subjects. The amnesia appears only after 50 min following learning, leaving the earlier processing stages of memory formation unaffected. The application of methotrexate resulted in disruption of righting reflex in a dose dependent manner, however the ataxia did not appear to be the cause of the memory deficit. The deficit in memory induced by methotrexate-induced folate deficiency could be ameliorated with methionine. These studies suggest that cellular processes involving folate metabolism may play an important role in the memory formation of the young chick and that the observed disruption of memory may well occur due to its affect on protein synthesis mediated by alterations in methionine metabolism.

Influence of cobalamin on the survival of mice bearing ascites tumor.

The effect of cobalamin (vitamin B12) on the survival time of mice bearing P388 leukemia has been examined. Among the three cobalamins studied, the enzymatically active derivatives, methylcobalamin and 5'-deoxyadenosylcobalamin, were able to significantly increase the survival time of mice implanted intraperitoneally with the tumor cells. The pharmaceutical form, cyanocobalamin, was not active. The antitumor activity of these cobalamins may be associated with their functions in metabolism.

Antinociceptive properties of thiamine, pyridoxine and cyanocobalamin following repeated oral administration to mice.

Following repeated oral administration for 7 days thiamine (thiamine nitrate, CAS 532-43-4), pyridoxine (pyridoxol hydrochloride, CAS 58-56-0), and cyanocobalamin (CAS 68-19-9) exhibited at high dose levels alone or in combination dose-related antinociceptive properties in the writhing test in the mouse. Cyanocobalamin exerted a potentiating effect in the combination of the 3 vitamins.

Mutagenic activity of pyrolysates of cyanocobalamin and some other water-soluble vitamins in the model system with the Salmonella/mammalian microsomes.

Pyrolysates of cyanocobalamin, thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, and ascorbic acid were tested for mutagenicity in the histidine-requiring mutants Salmonella typhimurium TA98 and TA100. Each vitamin was sealed in a glass tube and heated at 100-600 degrees C in a muffle furnace. Methanol-chloroform extracts of the pyrolysate of each vitamin tested did not show any mutagenicity in either TA98 or TA100 without rat liver 9000 x g supernatant fraction (S9) added. In the presence of S9, the B-group vitamins (cyanocobalamin, thiamine hydrochloride, riboflavin, and pyridoxine hydrochloride) were all mutagenic in TA98 and TA100, with the highest activity among the vitamins tested found in the pyrolysate of cyanocobalamin. The pyrolysate of 0.25 mumole cyanocobalamin produced 3200 revertants, while the pyrolysates of 0.25 mumole thiamine hydrochloride and riboflavin produced only 910 revertants, and the pyrolysate of pyridoxine hydrochloride did not show any mutagenicity at that amount. The mutagenicity was generally more active to TA98 than to TA100, indicating that frameshift-type mutagens were contained in the pyrolysates. The pyrolysate of ascorbic acid did not show any mutagenic activity in either TA98 or TA100 under the present experimental conditions.