No laughing matter - Myeloneuropathy due to heavy chronic nitrous oxide abuse.

Chronic nitrous oxide abuse is a known cause of myeloneuropathy. Nitrous oxide irreversibly inactivates vitamin B12 causing demyelination of the dorsal spinal columns, clinically indistinguishable from that which is caused by vitamin B12 deficiency. We report a 37-year-old female who presented with ataxia, loss of lower extremity proprioception, demyelination of her cervical dorsal spinal columns, and other laboratory and physical exam findings consistent with nitrous oxide abuse. The patient reported daily use in excess of 500 nitrous oxide cartridges, also known as "whippits". Nitrous oxide myeloneuropathy should be included in the differential diagnoses for emergency medicine physicians when evaluating a patient with bilateral neurologic deficits and ataxia.

Antivitamins B12 -Some Inaugural Milestones.

The recently delineated structure- and reactivity-based concept of antivitamins B12 has begun to bear fruit by the generation, and study, of a range of such B12 -dummies, either vitamin B12 -derived, or transition metal analogues that also represent potential antivitamins B12 or specific B12 -antimetabolites. As reviewed here, this has opened up new research avenues in organometallic B12 -chemistry and bioinorganic coordination chemistry. Exploratory studies with antivitamins B12 have, furthermore, revealed some of their potential, as pharmacologically interesting compounds, for inducing B12 -deficiency in a range of organisms, from hospital resistant bacteria to laboratory mice. The derived capacity of antivitamins B12 to induce functional B12 -deficiency in mammalian cells and organs also suggest their valuable potential as growth inhibitors of cancerous human and animal cells.

Photodissociation of ethylphenylcobalamin antivitamin B12.

Biologically active forms of cobalamins are crucial cofactors in biochemical reactions and these metabolites can be inhibited by their structurally similar analogues known as antivitamins B12. Phenylethynylcobalamin (PhEtyCbl) or 4-ethylphenylcobalamin (EtPhCbl) exemplify recently synthesized and structurally characterized antivitamins B12. Herein, DFT and TD-DFT studies of EtPhCbl are provided to explore its photochemical behavior, which may lead to design of arylcobalamins that can be used as therapeutic agents in light activated drug applications. To understand the photolability of EtPhCbl, a potential energy surface (PES) for the photodissociation of the Co-C bond was constructed. The S1 PES contains two energy minima, one being metal-to-ligand charge transfer (MLCT) and another the ligand-field (LF) state. There are two possible pathways for photodissociation: the first pathway (path A) involves initially lengthening the Co-C bond from the MLCT minimum and then elongation of Co-NIm while the second pathway (path B) involves the lengthening of the Co-NIm bond through the MLCT region followed by the lengthening of the Co-C bond through the LF region. It is shown that photodissociation involving path A is not energetically favorable whereas preferable photodissociation of the Co-C bond involves path B.

Antivitamin B12 Inhibition of the Human B12 -Processing Enzyme CblC: Crystal Structure of an Inactive Ternary Complex with Glutathione as the Cosubstrate.

B12 antivitamins are important and robust tools for investigating the biological roles of vitamin B12 . Here, the potential antivitamin B12 2,4-difluorophenylethynylcobalamin (F2PhEtyCbl) was prepared, and its 3D structure was studied in solution and in the crystal. Chemically inert F2PhEtyCbl resisted thermolysis of its Co-C bond at 100 °C, was stable in bright daylight, and also remained intact upon prolonged storage in aqueous solution at room temperature. It binds to the human B12 -processing enzyme CblC with high affinity (KD =130 nm) in the presence of the cosubstrate glutathione (GSH). F2PhEtyCbl withstood tailoring by CblC, and it also stabilized the ternary complex with GSH. The crystal structure of this inactivated assembly provides first insight into the binding interactions between an antivitamin B12 and CblC, as well as into the organization of GSH and a base-off cobalamin in the active site of this enzyme.

Antivitamins for Medicinal Applications.

Antivitamins represent a broad class of compounds that counteract the essential effects of vitamins. The symptoms triggered by such antinutritional factors resemble those of vitamin deficiencies, but can be successfully reversed by treating patients with the intact vitamin. Despite being undesirable for healthy organisms, the toxicities of these compounds present considerable interest for biological and medicinal purposes. Indeed, antivitamins played fundamental roles in the development of pioneering antibiotic and antiproliferative drugs, such as prontosil and aminopterin. Their development and optimisation were made possible by the study, throughout the 20th century, of the vitamins' and antivitamins' functions in metabolic processes. However, even with this thorough knowledge, commercialised antivitamin-based drugs are still nowadays limited to antagonists of vitamins B9 and K. The antivitamin field thus still needs to be explored more intensely, in view of the outstanding therapeutic success exhibited by several antivitamin-based medicines. Here we summarise historical achievements and discuss critically recent developments, opportunities and potential limitations of the antivitamin approach, with a special focus on antivitamins K, B9 and B12 .

Antivitamins B12--A Structure- and Reactivity-Based Concept.

B12 -antimetabolites are compounds that counteract the physiological effects of vitamin B12 and related natural cobalamins. Presented here is a structure- and reactivity-based concept of the specific 'antivitamins B12 ': it refers to analogues of vitamin B12 that display high structural similarity to the vitamin and are 'locked chemically' to prevent their metabolic conversion into the crucial organometallic B12 -cofactors. Application of antivitamins B12 to healthy laboratory animals is, thus, expected to induce symptoms of B12 -deficiency. Antivitamins B12 may, hence, be helpful in elucidating still largely puzzling pathophysiological phenomena associated with B12 -deficiency, and also in recognizing physiological roles of B12 that probably still remain to be discovered.

Relationship between metformin use, vitamin B12 deficiency, hyperhomocysteinemia and vascular complications in patients with type 2 diabetes.

Aim of the study was to clarify the relationship between metformin-induced vitamin B12 (B12) deficiency, hyperhomocysteinemia and vascular complications in patients with type 2 diabetes. Serum B12 concentrations, homocysteine plasma levels, the presence of retinopathy and history of macroangiopathy (stroke or coronary heart disease) were analyzed in patients without renal dysfunction (serum creatinine<115 µmol/L). Firstly, B12 status was analyzed in 62 consecutive metformin-treated patients. Secondly, the relationship between B12, homocysteine and vascular complications was analyzed in 46 metformin-treated and 38 age- and sex-matched non-metformin-treated patients. Among the 62 consecutive metformin-treated patients, B12 was deficient (<150 pmol/L) in 8 (13%) and borderline-deficient (150-220 pmol/L) in 18 (29%): the larger the metformin dosage, the lower the B12 (P=0.02, Spearman's ρ=-0.30). There were independent correlations between metformin use and B12 lowering (P=0.02, r = -0.25), and B12 lowering and elevation of homocysteine (P<0.01, r=-0.34). Elevation of homocysteine was a risk for retinopathy (P=0.02, OR 1.26, 95%CI 1.04-1.52). There was no significant relation between homocysteine and macroangiopathy. Correlation between B12 and homocysteine was stronger in metformin-treated (P<0.01, r=-0.48) than non-metformin-treated (P=0.04, r=-0.38) patients. In ten B12 deficient patients, B12 supplementation (1,500 µg/day) for 2.2±1.0 months with continued use of metformin raised B12 levels: 152±42 and 299±97 pmol/L before and after treatment, respectively (P<0.01). Metformin-induced B12 lowering in diabetes was associated with elevation of homocysteine, and hyperhomocysteinemia was independently related to retinopathy. Metformin-induced B12 deficiency was correctable with B12 supplementation.

Nitrous oxide and oxygen sedation: an update.

This course will teach the desirable characteristics of nitrous oxide, indications and contraindications for N2O/O2 use, as well as facts and myths surrounding chronic exposure to nitrous oxide, the biologic effects associated with high levels of the gas, and ways to assess and minimize trace gas contamination in an outpatient setting.

Nitrous oxide and oxygen sedation: an update.

Nitrous oxide/oxygen sedation remains a viable option for managing a patient's pain and anxiety in the dental office. There are several advantages to its use and relatively few contraindications. Knowing how to minimize the operator's exposure to the gas is also an important consideration. N2O/O2 sedation has a long-standing history of safety and success and it is likely that this type of sedation will be used far into the future. It is necessary to educate the entire office team on the biohazard issues of nitrous oxide safety in the dental office and keep abreast of sound scientific literature in this area. Many states are starting to include nitrous oxide administration and monitoring in their state practice acts for dental assistants. Refer to your state practice act for current requirements in your location.

Antivitamins B12 in a Microdrop: The Excited-State Structure of a Precious Sample Using Transient Polarized X-ray Absorption Near-Edge Structure.

Polarized transient X-ray absorption near-edge structure (XANES) was used to probe the excited-state structure of a photostable B12 antivitamin (Coß-2-(2,4-difluorophenyl)-ethynylcobalamin, F2PhEtyCbl). A drop-on-demand delivery system synchronized to the LCLS X-ray free electron laser pulses was implemented and used to measure the XANES difference spectrum 12 ps following excitation, exposing only ∼45 µL of sample. Unlike cyanocobalamin (CNCbl), where the Co-C bond expands 15-20%, the excited state of F2PhEtyCbl is characterized by little change in the Co-C bond, suggesting that the acetylide linkage raises the barrier for expansion of the Co-C bond. In contrast, the lower axial Co-NDMB bond is elongated in the excited state of F2PhEtyCbl by ca. 10% or more, comparable to the 10% elongation observed for Co-NDMB in CNCbl.

Cobalamin Deficiency: Effect on Homeostasis of Cultured Human Astrocytes.

Cobalamin deficiency is an important health problem. The major non-hematological symptoms of hypocobalaminemia are nervous system disorders, but the molecular and cellular mechanisms underlying this phenomenon have not yet been fully explained. Increasing scientific evidence is stressing the pivotal role of astrocyte dysfunction in the pathogenesis of a wide range of neurological disorders. In light of the above, the aim of this study was to develop an in vitro model of cobalamin deficiency by optimizing the conditions of astrocyte culture in the presence of vitamin B12 antagonist, and then the model was used for multidirectional analysis of astrocyte homeostasis using image cytometry, immunoenzymatic and colorimetric assays, and fluorescence spectroscopy. Our results indicated that long-term incubation of normal human astrocytes with hydroxycobalamin(c-lactam) causes an increase of extracellular homocysteine level, a reduction of cell proliferation, and an accumulation of cells in the G2/M cell cycle phase. Moreover, we observed dramatic activation of caspases and an increase of catalase activity. Interestingly, we excluded extensive apoptosis and oxidative stress. The study provided significant evidence for astrocyte homeostasis disturbance under hypocobalaminemia, thus indicating an important element of the molecular mechanism of nervous system diseases related to vitamin B12 deficiency.

Cobalamin inactivation decreases purine and methionine synthesis in cultured lymphoblasts.

The megaloblastic anemia of cobalamin deficiency appears secondary to decreased methionine synthetase activity. Decreased activity of this enzyme should cause 5-methyltetrahydrofolate to accumulate intracellularly, and consequently, decrease purine and DNA synthesis; this is the basis of the "methylfolate trap" hypothesis of cobalamin deficiency. However, only some of the clinical and biochemical manifestations of cobalamin deficiency can be explained by the methylfolate trap. We investigated cobalamin deficiency by treating cultured human lymphoblasts with N2O since N2O inhibits methionine synthetase activity by inactivating cobalamin. We found that 4 h of N2O exposure reduced rates of methionine synthesis by 89%. Rates of purine synthesis were not significantly reduced by N2O when folate and methionine were present at 100 microM in the medium; however, at the physiologic methionine concentration of 10 microM, N2O decreased rates of purine synthesis by 33 and 57% in the presence of 100 microM folate and in the absence of folate, respectively. The dependency of rates of purine synthesis on methionine availability would be expected in cells with restricted methionine synthetic capacity because methionine is the immediate precursor of S-adenosylmethionine, a potent inhibitor of 5-methyltetrahydrofolate synthesis; methionine serves as a source of formate for purine synthesis; and rates of purine synthesis are dependent on the intracellular availability of essential amino acids. We conclude that cobalamin inactivation decreases purine synthesis by both methylfolate trapping and reduction of intracellular methionine synthesis.

Chronic cobalamin inactivation impairs folate polyglutamate synthesis in the rat.

Nitrous oxide, by inactivating cobalamin in vivo, produces a suitable animal model for cobalamin 'deficiency.' The synthesis of folate polyglutamate with tetrahydrofolate as substrate is severely impaired in the N2O-treated rat, but is normal with formyltetrahydrofolate as substrate. Methionine restores the capacity of the N2O-treated rat to utilize tetrahydrofolate the minimum effective dose being 16 mumol. S-Adenosylmethionine was somewhat less effective than methionine but 5'methylthioadenosine, a product of S-adenosylmethionine metabolism, was significantly more effective than methionine in correcting the defect in folate polyglutamate synthesis. 5'Methylthioadenosine is metabolised to yield formate. It is suggested that these compounds have their effect in correcting folate polyglutamate synthesis by supplying formate for the formylation of tetrahydrofolate. Formyltetrahydrofolate, at least in the cobalamin-inactivated animal, is the required substrate for folate polyglutamate synthesis. Cobalamin is concerned with the maintenance of normal levels of methionine and this in turn is a major source of formate through S-adenosylmethionine and 5'methylthioadenosine.

Characterization of ileal vitamin B12 Binding using homogeneous human and hog intrinsic factors.

Elucidation of the mechanism of intrinsic factor (IF)-mediated vitamin B(12) (B(12)) binding to ileal binding sites has been hampered by the use of crude or only partially purified preparations of IF in previous studies. We have used homogeneous human IF and hog IF isolated by affinity chromatography to study [(57)Co]B(12) binding to ileal mucosal homogenates. The following observations were made: (a) Human IF-B(12) and hog IF-B(12) were bound to human, monkey, hog, dog, rabbit, mouse, hamster, and guinea pig ileal, but not jejunal, homogenates in amounts significantly greater than free B(12) or B(12) bound to five other homogeneous B(12)-binding proteins; (b) only IF-mediated B(12) binding was localized to ileal homogenates and was inhibited by EDTA; (c) values for the association constant (K(a)) for the various ileal homogenates mentioned above and human IF-B(12) and hog IF-B(12) ranged from 0.3 x 10(9) M(-1) to 13.0 x 10(9) M(-1). Apparent differences in the K(a) for human IF-B(12) and hog IF-B(12) existed in most species; (d) the number of ileal IF-B(12) binding sites per gram (wet weight) of ileal mucosa ranged from 0.3 x 10(12) to 4.9 x 10(12). The same value was always obtained with human IF-B(12) and hog IF-B(12) for any given homogenate preparation; (c) 100-fold excesses of free B(12) or human IF and hog IF devoid of B(12) did not significantly inhibit human IF-B(12) and hog IF-B(12) binding to human and hog ileal homogenates. THESE EXPERIMENTS PERFORMED WITH HOMOGENEOUS IF INDICATE THAT: (a) gastric factors other than IF are not required for B(12) binding to ileal IF-B(12)-binding sites: (b) the mechanism of ileal IF-B(12) binding is different from that of free B(12) or of B(12) bound to non-IF-B(12)-binding proteins; (c) human IF and hog IF have different structures; (d) human IF-B(12) and hog IF-B(12) bind to the same ileal binding sites; and (c) human and hog ileal IF-B(12) binding sites bind free B(12) and human and hog IF devoid of B(12) poorly, if at all.

Effect of divalent cations and pH on intrinsic factor-mediated attachment of vitamin B 12 to intestinal microvillous membranes.

Calcium, but not other divalent cations, is required for optimal uptake of intrinsic factor-bound (57)Co-labeled cyanocobalamin (IFB(12)) by microvillous membranes isolated from hamster ileal-absorptive cells. Chelation of divalent cations by disodium ethylenediaminetetraacetate (EDTA) promptly removes IFB(12) previously attached to microvillous membranes. High concentrations of CaCl(2) or MgCl(2) also markedly inhibit membrane uptake of IFB(12) and rapidly remove previously attached IFB(12). Similarly, reduction of pH to below 5.4 prevents membrane attachment of IFB(12) and removes virtually all IFB(12) already bound to microvillous membranes. The effects of calcium depletion, increased salt concentrations, and acidification on membrane uptake of IFB(12) were completely reversible. These findings are consistent with the concept that the formation of calcium salt bridges is essential for attachment of IFB(12) to the ileal-absorptive surface.

Impaired formylation and uptake of tetrahydrofolate by rat small gut following cobalamin inactivation.

The effect of inactivation of cobalamin by N2O on the intestinal absorption of folate was studied using rat everted gut sacs. Further, in view of uncertainties about the presence of methionine synthetase in gut [1], this enzyme was measured. Everted gut sacs were incubated with [2-14C]tetrahydrofolate, and the subsequent appearance of labelled formyl- and methyl [14C] tetrahydrofolate in everted segments of small intestine of rats was studied. Considerable methionine synthetase activity was present in washed everted gut sacs but not in gut segments in the absence of such treatment. Methionine synthetase activity declined after exposure to N2O, which oxidizes and inactivates cob(I)alamin. Folate uptake by gut sacs was not affected by 24 h exposure of the animals to N2O but fell significantly after 7 days exposure. There was a significant fall in the amount of formyltetrahydrofolate formed after cobalamin inactivation and this was reversed by supplying either methionine, methylthioadenosine or sodium formate. Serine had no effect. The data support the hypothesis that methionine and methylthioadenosine act by supplying single carbon units at the formate level of oxidation.

Effects of nitrous oxide-induced inactivation of cobalamin on methionine and S-adenosylmethionine metabolism in the rat.

Inhalation of nitrous oxidises cobalamin and, in turn, inactivates methionine synthetase which forms methionine from homocysteine and which requires cob[I]alamin as a co-factor. This study was planned to determine the effect of virtual cessation of methionine synthesis via a cobalamin-dependent pathway, on tissue levels of methionine, S-adenosylmethionine and on related enzymes. The level of methionine in liver fell initially after exposure to N2O but was restored to pre-N2O levels after 6 days despite continuing N2O exposure. Brain methionine fell within 12 h of N2O exposure but the fall was not significant. The restoration of methionine levels is accompanied by an increase in activity of betaine homocysteine methyltransferase in liver but this enzyme was not detected in brain. The activity of methionine synthetase remained very low in both liver and brain as long as N2O inhalation was continued. There was an initial rise in liver S-adenosylmethionine levels followed by a steady fall to 40% of its initial level after 11 days of N2O exposure. However, there was no change in the level of S-adenosylmethionine in brain during this period. The data indicate that either brain meets its requirement by increased methionine uptake from plasma or that there are alternate pathways in brain for methionine synthesis other than those requiring a cobalamin coenzyme.

Cobalamin inactivation induces formyltetrahydrofolate synthetase.

Loss of cobalamin function produces profound changes in the metabolism of formate. There is impaired synthesis of formyltetrahydropteroylglutamate synthetase (CHO-H4PteGlu), accumulation of endogenous formate and impaired utilization of [14C]formate. There are contradictory reports on the effect of cobalamin inactivation on CHO-H4PteGlu synthetase. This study confirms a significant increase in synthetase activity following cobalamin inactivation.

Recovery of tissue folates after inactivation of cobalamin by nitrous oxide. The significance of dietary folate.

The anesthetic gas, nitrous oxide (N2O), oxidizes the cobalt moiety in the vitamin B12 molecule and in this way inactivates methionine synthetase which requires reduced cobalamin. In rats this is followed by a disappearance of folates from the tissues, this loss being most marked in the liver. Returning the animals to a normal atmosphere leads to restoration of most of the pre-N2O folate levels within 5 days. The plasma folate, which rises on exposure to N2O, falls within several hours. The restoration of tissue folates does not take place if the rats are placed on a low folate diet after withdrawal from an N2O environment. Thus the fall in tissue folate levels is due to loss from the body either by excretion or increased catabolism and not to redistribution of folate. Return of normal folate levels requires a dietary source of folate.