RESUMO
Cobalamin is an essential nutrient required for the normal functioning of cells. Its deficiency can lead to various pathological states. Hydroxocobalamin (HOCbl) and cyanocobalamin (CNCbl) are the forms of vitamin B12 that are most commonly used for supplementation. There is substantial evidence indicating that cobalamins can both suppress and promote oxidative stress; however, the mechanisms underlying these effects are poorly understood. Here, it was shown that the oxidation of thiols catalyzed by HOCbl and CNCbl is accompanied by reactive oxygen species (ROS) production and induces, under certain conditions, oxidative stress and cell death. The form of vitamin B12 and the structure of thiol play a decisive role in these processes. It was found that the mechanisms and kinetics of thiol oxidation catalyzed by HOCbl and CNCbl differ substantially. HOCbl increased the rate of oxidation of thiols to a greater extent than CNCbl, but quenched ROS in combination with certain thiols. Oxidation catalyzed by CNCbl was generally slower. Yet, the absence of ROS quenching resulted in their higher accumulation. The aforementioned results might explain a more pronounced cytotoxicity induced by combinations of thiols with CNCbl. On the whole, the data obtained provide a new insight into the redox processes in which cobalamins are involved. Our results might also be helpful in developing new approaches to the treatment of some cobalamin-responsive disorders in which oxidative stress is an important component.
Assuntos
Hidroxocobalamina , Vitamina B 12 , Hidroxocobalamina/química , Hidroxocobalamina/metabolismo , Hidroxocobalamina/farmacologia , Oxirredução , Espécies Reativas de Oxigênio , Compostos de Sulfidrila , Vitamina B 12/metabolismoRESUMO
Vitamin B12 (cobalamin, Cbl, B12) is a water-soluble micronutrient synthesized exclusively by a group of microorganisms. Human beings are unable to make B12 and thus obtain the vitamin via intake of animal products, fermented plant-based foods or supplements. Vitamin B12 obtained from the diet comprises three major chemical forms, namely hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl). The most common form of B12 present in supplements is cyanocobalamin (CNCbl). Yet, these chemical forms cannot be utilized directly as they come, but instead, they undergo chemical processing by the MMACHC protein, also known as CblC. Processing of dietary B12 by CblC involves removal of the upper-axial ligand (beta-ligand) yielding the one-electron reduced intermediate cob(II)alamin. Newly formed cob(II)alamin undergoes trafficking and delivery to the two B12-dependent enzymes, cytosolic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MUT). The catalytic cycles of MS and MUT incorporate cob(II)alamin as a precursor to regenerate the coenzyme forms MeCbl and AdoCbl, respectively. Mutations and epimutations in the MMACHC gene result in cblC disease, the most common inborn error of B12 metabolism, which manifests with combined homocystinuria and methylmalonic aciduria. Elevation of metabolites homocysteine and methylmalonic acid occurs because the lack of an active CblC blocks formation of the indispensable precursor cob(II)alamin that is necessary to activate MS and MUT. Thus, in patients with cblC disease, vitamin B12 is absorbed and present in circulation in normal to high concentrations, yet, cells are unable to make use of it. Mutations in seemingly unrelated genes that modify MMACHC gene expression also result in clinical phenotypes that resemble cblC disease. We review current knowledge on structural and functional aspects of intracellular processing of vitamin B12 by the versatile protein CblC, its partners and possible regulators.
Assuntos
Homocistinúria , Vitamina B 12 , Animais , Homocistinúria/genética , Humanos , Hidroxocobalamina/metabolismo , Ligantes , Oxirredutases , Vitamina B 12/metabolismo , VitaminasRESUMO
Nitric oxide (NO) is a powerful vasodilator in different vascular beds and NO-donors are widely used in clinical practice. Early data suggested that NO and NO-donors activate vascular smooth muscle high-conductance, calcium-activated potassium channels (BK channels). There exist two hypotheses explaining the effect of NO and NO-donors on BK channels-one stating that protein kinase G (PKG) mediates the effect of NO, and the other one stating that NO acts directly on the channel. Thus, the degree of contribution of PKG to the NO-induced activation of the BK channel is still not completely clear. This study tested the hypothesis that the sodium nitroprusside (SNP)-induced activation of vascular smooth muscle BK channels is fully mediated by PKG. This hypothesis was investigated using the patch-clamp technique and freshly isolated smooth muscle cells from rat tail artery. In whole-cell experiments, SNP considerably increased the outward current compared with the addition of the bath solution. SNP did not alter the current in the presence of iberiotoxin, the specific blocker of BK channels, during co-application with hydroxocobalamin, an NO-scavenger, and in the presence of Rp-8-Br-PET-cGMPS, the specific PKG-inhibitor. In inside-out patches, the activity of BK channels was increased by SNP, SNAP, and DEA-NO. However, these effects did not differ from the effect of the application of drug-free bath solution. Furthermore, a similar increase in single BK channel activity was induced by Rp-8-Br-PET-cGMPS, Rp-8-Br-PET-cGMPS together with SNP, hydroxocobalamin, and hydroxocobalamin together with SNP or DEA-NO. Finally, the activity of excised BK channels did not change in the absence of any application but was considerably increased by PKG compared with the addition of drug-free bath solution. These results suggest that NO released from NO-donors stimulates the BK current only through activation of PKG.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico , Canais de Potássio Ativados por Cálcio de Condutância Alta , Animais , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Hidroxocobalamina/metabolismo , Hidroxocobalamina/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , RatosRESUMO
We have shown that hydroxycobalamin (vitamin Ð12b) increases the toxicity of diethyldithiocarbamate (DDC) to tumor cells by catalyzing the formation of disulfiram (DSF) oxi-derivatives. The purpose of this study was to elucidate the mechanism of tumor cell death induced by the combination DDC + Ð12b. It was found that cell death induced by DDC + B12b differed from apoptosis, autophagy, and necrosis. During the initiation of cell death, numerous vacuoles formed from ER cisterns in the cytoplasm, and cell death was partially suppressed by the inhibitors of protein synthesis and folding, the IP3 receptor inhibitor as well as by thiols. At this time, a short-term rise in the expression of ER-stress markers BiP and PERK with a steady increase in the expression of CHOP were detected. After the vacuolization of the cytoplasm, functional disorders of mitochondria and an increase in the generation of superoxide anion in them occurred. Taken together, the results obtained indicate that DDC and B12b used in combination exert a synergistic toxic effect on tumor cells by causing severe ER stress, extensive ER vacuolization, and inhibition of apoptosis, which ultimately leads to the induction of paraptosis-like cell death.
Assuntos
Ditiocarb/farmacologia , Hidroxocobalamina/farmacologia , Neoplasias Laríngeas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ditiocarb/metabolismo , Sinergismo Farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Hidroxocobalamina/metabolismo , Neoplasias Laríngeas/metabolismo , Laringe/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Vacúolos/efeitos dos fármacos , Vitamina B 12/metabolismo , Vitamina B 12/farmacologia , Vitaminas/metabolismo , Vitaminas/farmacologiaRESUMO
The stability of B12 vitamers is affected by interaction with other water-soluble vitamins, UV light, heat, and pH. This study compared the degradation losses in cyanocobalamin, hydroxocobalamin and methylcobalamin due to the physicochemical exposure before and after the addition of sorbitol. The degradation losses of cyanocobalamin in the presence of increasing concentrations of thiamin and niacin ranged between 6%-13% and added sorbitol significantly prevented the loss of cyanocobalamin (p<0.05). Hydroxocobalamin and methylcobalamin exhibited degradation losses ranging from 24%-26% and 48%-76%, respectively; added sorbitol significantly minimised the loss to 10% and 20%, respectively (p < 0.05). Methylcobalamin was the most susceptible to degradation when co-existing with ascorbic acid, followed by hydroxocobalamin and cyanocobalamin. The presence of ascorbic acid caused the greatest degradation loss in methylcobalamin (70%-76%), which was minimised to 16% with added sorbitol (p < 0.05). Heat exposure (100 °C, 60 minutes) caused a greater loss of cyanocobalamin (38%) than UV exposure (4%). However, degradation losses in hydroxocobalamin and methylcobalamin due to UV and heat exposures were comparable (>30%). At pH 3, methylcobalamin was the most unstable showing 79% degradation loss, which was down to 12% after sorbitol was added (p < 0.05). The losses of cyanocobalamin at pH 3 and pH 9 (~15%) were prevented by adding sorbitol. Addition of sorbitol to hydroxocobalamin at pH 3 and pH 9 reduced the loss by only 6%. The results showed that cyanocobalamin was the most stable, followed by hydroxocobalamin and methylcobalamin. Added sorbitol was sufficient to significantly enhance the stability of cobalamins against degradative agents and conditions.
Assuntos
Sorbitol , Vitamina B 12 , Ácido Ascórbico/química , Hidroxocobalamina/metabolismoRESUMO
The cobalamin-dependent radical S-adenosylmethionine (SAM) enzyme TsrM catalyzes the methylation of C2 of L-tryptophan to form 2-methyltryptophan during the biosynthesis of thiostrepton A. Although TsrM is a member of the radical SAM superfamily, unlike all other annotated members, it does not catalyze a reductive cleavage of SAM to a 5'-deoxyadenosyl 5'-radical intermediate. In fact, it has been proposed that TsrM catalyzes its reaction through two polar nucleophilic displacements, with its cobalamin cofactor cycling directly between methylcobalamin (MeCbl) and cob(I)alamin. Nevertheless, the enzyme has been stated to require the action of a reductant, which can be satisfied by dithiothreitol. By contrast, all other annotated RS enzymes require a reductant that exhibits a much lower reduction potential, which is necessary for the reductive cleavage of SAM. Herein, we show that TsrM can catalyze multiple turnovers in the absence of any reducing agent, but only when it is pre-loaded with MeCbl. When hydroxocobalamin (OHCbl) or cob(II)alamin is bound to TsrM, a reductant is required to convert it to cob(I)alamin, which can acquire a methyl group directly from SAM. Our studies suggest that TsrM uses an external reductant to prime its reaction by converting bound OHCbl or cob(II)alamin to MeCbl, and to regenerate the MeCbl form of the cofactor upon adventitious oxidation of the cob(I)alamin intermediate to cob(II)alamin.
Assuntos
Proteínas Ferro-Enxofre/metabolismo , S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Ditiotreitol/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Hidroxocobalamina/metabolismo , Proteínas Ferro-Enxofre/química , Metilação , Oxirredução , S-Adenosilmetionina/química , Triptofano/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/químicaRESUMO
It is known that some metals (Cu, Zn, Cd, Au) markedly increase the toxic effect of thiocarbamates. It was shown in the present study that hydroxycobalamin (a form of vitamin B12, HOCbl), which incorporates cobalt, significantly enhances the cytotoxicity of diethyldithiocarbamate (DDC), decreasing its IC50 value in tumor cells three to five times. The addition of HOCbl to aqueous DDC solutions accelerated the reduction of oxygen. No hydrogen peroxide accumulation was observed in DDCâ¯+â¯HOCbl solutions; however, catalase slowed down the oxygen reduction rate. Catalase as well as the antioxidants N-acetylcysteine (NAC) and glutathione (GSH) partially inhibited the cytotoxic effect of DDCâ¯+â¯HOCbl, whereas ascorbate, pyruvate, and tiron, a scavenger of superoxide anion, had no cytoprotective effect. The administration of HOCbl into DDC solutions (>â¯1â¯mM) resulted in the formation of a crystalline precipitate, which was inhibited in the presence of GSH. The data of UV and NMR spectroscopy and HPLC and Mass Spectrometry (LC/MS) indicated that the main products of the reaction of DDC with HOCbl are disulfiram (DSF) and its oxidized forms, sulfones and sulfoxides. The increase in the cytotoxicity of DDC combined with HOCbl occurred both in the presence of Cu2+ in culture medium and in nominally Cu-free solutions, as well as in growth medium containing the copper chelator bathocuproine disulfonate (BCS). The results indicate that HOCbl accelerates the oxidation of DDC with the formation of DSF and its oxidized forms. Presumably, the main cause of the synergistic increase in the toxic effect of DDC +â¯HOCbl is the formation of sulfones and sulfoxides of DSF.
Assuntos
Cobre/metabolismo , Ditiocarb/metabolismo , Hidroxocobalamina/metabolismo , Íons/metabolismo , Oxirredução , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ditiocarb/química , Ditiocarb/farmacologia , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Hidroxocobalamina/química , Hidroxocobalamina/farmacologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Análise EspectralRESUMO
Foods contain natural vitamin B12 forms, such as hydroxoâ»B12 (HOâ»B12), whereas vitamin pills contain the synthetic cyanoâ»B12 (CNâ»B12). Recent studies in rats showed different tissue distributions of CNâ»B12 and HOâ»B12 24 h after oral administration. Here, we investigate whether these differences are sustained or leveled out with time in both B12-deplete and -replete rats, thereby assessing if the two forms are equally good at maintaining a normal B12 status. Male Wistar rats were fed diets with low (n = 16) or high (n = 12) B12 content for 17 days. At day 10, the rats received a single oral dose of [57Co]-labeled CNâ»B12 or HOâ»B12 (n = 6 and n = 8, respectively, in each diet group). The rats were sacrificed on day 17 and endogenous B12 and [57Co]â»B12 were measured in liver, kidney, and plasma. We found that the low-B12 diet introduced a B12-deplete state as judged from medians of endogenous B12 compared to rats on a (high-B12 diet): Plasma (565 (1410) pmol/L), liver (28.2 (33.2) pmol/g), and kidneys (123 (1300) pmol/g). One week after oral administration, the labeled B12 was distributed as follows: HOâ»B12 > CNâ»B12 (liver) and CNâ»B12 > HOâ»B12 (kidneys, plasma). The tissue/plasma ratios showed different equilibriums for labeled CNâ»B12 and HOâ»B12 in the B12-deplete and -replete groups. The equilibrium of endogenous B12 resembled [57Co]CNâ»B12 in replete rats but differed from both [57Co]CNâ»B12 and [57Co]HOâ»B12 in deplete rats. The data suggest long-term differences in tissue utilization of the two B12 forms and warrant further studies concerning the possible benefits of consuming HOâ»B12 instead of CNâ»B12 in oral B12 replacement.
Assuntos
Rim/metabolismo , Fígado/metabolismo , Plasma/metabolismo , Distribuição Tecidual , Deficiência de Vitamina B 12/metabolismo , Vitamina B 12/farmacocinética , Administração Oral , Animais , Hidroxocobalamina/administração & dosagem , Hidroxocobalamina/metabolismo , Hidroxocobalamina/farmacocinética , Masculino , Ratos Wistar , Vitamina B 12/administração & dosagem , Vitamina B 12/metabolismoRESUMO
INTRODUCTION: Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory condition characterized by fever, cytopenias, hepatosplenomegaly and hemophagocytosis. HLH may be primary or secondary to infection, autoimmune disease or malignancy. Hypertriglyceridemia is a common abnormality in HLH and one of the HLH-2004 diagnostic criteria. CASE OUTLINE: We present an infant with severe hypotonia and hypoproteinemic edema who also had extreme hypertriglyceridemia (21 mmol/l) and was diagnosed with HLH based on six of eight HLH-2004 criteria (fever, hepatosplenomegaly, bicytopenia, hypertriglyceridemia with hypofibrinogenemia, slL-2R > 2400 IU/ml, hemophagocytosis). The presence of IgM antibodies to Epstein-Barr virus and cytomegalovirus indicated a probable infectious trigger. The child was cured by the HLH-2004 protocol for secondary HLH (consisting of dexamethasone and cyclosporine). He was also found to have low serum hydroxycobalamin levels, promptly corrected upon hydroxycobalamin administration. CONCLUSION: The presented case history underlines the need to ascertain the presence or absence of each of the eight HLH-2004 criteria in any patient suspected to suffer from HLH.
Assuntos
Hidroxocobalamina/metabolismo , Hipertrigliceridemia/etiologia , Linfo-Histiocitose Hemofagocítica/diagnóstico , Deficiência de Vitamina B 12/diagnóstico , Humanos , Hipertrigliceridemia/diagnóstico , Lactente , Linfo-Histiocitose Hemofagocítica/complicações , Masculino , Deficiência de Vitamina B 12/complicaçõesRESUMO
Riboswitches represent a family of highly structured regulatory elements found primarily in the leader sequences of bacterial mRNAs. They function as molecular switches capable of altering gene expression; commonly, this occurs via a conformational change in a regulatory element of a riboswitch that results from ligand binding in the aptamer domain. Numerous studies have investigated the ligand binding process, but little is known about the structural changes in the regulatory element. A mechanistic description of both processes is essential for deeply understanding how riboswitches modulate gene expression. This task is greatly facilitated by studying all aspects of riboswitch structure/dynamics/function in the same model system. To this end, single-molecule fluorescence resonance energy transfer (smFRET) techniques have been used to directly observe the conformational dynamics of a hydroxocobalamin (HyCbl) binding riboswitch (env8HyCbl) with a known crystallographic structure.1 The single-molecule RNA construct studied in this work is unique in that it contains all of the structural elements both necessary and sufficient for regulation of gene expression in a biological context. The results of this investigation reveal that the undocking rate constant associated with the disruption of a long-range kissing-loop (KL) interaction is substantially decreased when the ligand is bound to the RNA, resulting in a preferential stabilization of the docked conformation. Notably, the formation of this tertiary KL interaction directly sequesters the Shine-Dalgarno sequence (i.e., the ribosome binding site) via base-pairing, thus preventing translation initiation. These results reveal that the conformational dynamics of this regulatory switch are quantitatively described by a four-state kinetic model, whereby ligand binding promotes formation of the KL interaction. The results of complementary cell-based gene expression experiments conducted in Escherichia coli are highly correlated with the smFRET results, suggesting that KL formation is directly responsible for regulating gene expression.
Assuntos
Hidroxocobalamina/química , Hidroxocobalamina/metabolismo , Riboswitch , Transferência Ressonante de Energia de Fluorescência , Cinética , Ligantes , Modelos Moleculares , Conformação Molecular , TermodinâmicaRESUMO
The anaerobic dehalogenation of organohalides is catalyzed by the reductive dehalogenase (RdhA) enzymes produced in phylogenetically diverse bacteria. These enzymes contain a cobamide cofactor at the active site and two iron-sulfur clusters. In this study, the tetrachloroethene (PCE) reductive dehalogenase (PceA) of the Gram-positive Desulfitobacterium hafniense strain Y51 was produced in a catalytically active form in the nondechlorinating, cobamide-producing bacterium Shimwellia blattae (ATCC 33430), a Gram-negative gammaproteobacterium. The formation of recombinant catalytically active PceA enzyme was significantly enhanced when its dedicated PceT chaperone was coproduced and when 5,6-dimethylbenzimidazole and hydroxocobalamin were added to the S. blattae cultures. The experiments were extended to D. hafniense DCB-2, a reductively dehalogenating bacterium harboring multiple rdhA genes. To elucidate the substrate spectrum of the rdhA3 gene product of this organism, the recombinant enzyme was tested for the conversion of different dichlorophenols (DCP) in crude extracts of an RdhA3-producing S. blattae strain. 3,5-DCP, 2,3-DCP, and 2,4-DCP, but not 2,6-DCP and 3,4-DCP, were reductively dechlorinated by the recombinant RdhA3. In addition, this enzyme dechlorinated PCE to trichloroethene at low rates.
Assuntos
Proteínas de Bactérias/metabolismo , Desulfitobacterium/enzimologia , Halogenação/fisiologia , Oxirredutases/metabolismo , Compostos Alílicos/metabolismo , Proteínas de Bactérias/genética , Benzimidazóis/metabolismo , Catálise , Clorofenóis/metabolismo , Clonagem Molecular , Cobamidas/biossíntese , Meios de Cultura , DNA Bacteriano/genética , Desulfitobacterium/genética , Gammaproteobacteria/enzimologia , Hidrocarbonetos Clorados , Hidroxocobalamina/metabolismo , Oxirredutases/genética , Plasmídeos/genética , Recombinação Genética , Análise de Sequência de DNA , Tricloroetileno/metabolismoRESUMO
Chronic renal failure is a well-known long-term complication of methylmalonic aciduria (MMA-uria), occurring even under apparently optimal metabolic management. The onset of renal dysfunction seems to be dependent on the type of defect and vitamin B12-responsiveness. We report on a patient with a vitamin B12-responsive cobalamin A type (cblA) MMA-uria caused by a homozygous stop mutation (p.R145X) in the cobalamin A gene (MMAA). She was diagnosed with chronic kidney disease (CKD) stage III at the age of 12 years. Following re-evaluation, the patient received vitamin B12 (hydroxocobalamin) treatment, resulting in a significant decrease in the concentration of methylmalonic acid (MMA) in urine and plasma. Until age 29 years glomerular filtration rate remained stable probably due to hydroxocobalamin treatment slowing down progression to end-stage renal failure. Kidney biopsies showed non-specific manifestations of chronic interstitial inflammation. The patient received a renal transplant at age 35 years. Under continuous treatment with hydroxocobalamin there is no evidence of kidney damage due to MMA-uria until the last follow-up 6 years after transplantation. This case report illustrates (i) a long-term follow-up of a patient with MMA-uria due to cblA deficiency, (ii) the involvement of the kidney as a target organ and (iii) the importance of early and adequate vitamin B12 substitution in responsive patients. Further investigation will be necessary to prove the protective effect of hydroxocobalamin in the kidney in vitamin B12-responsive patients.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/patologia , Falência Renal Crônica/patologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Vitamina B 12/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Criança , Feminino , Seguimentos , Taxa de Filtração Glomerular , Humanos , Hidroxocobalamina/metabolismo , Falência Renal Crônica/complicações , Falência Renal Crônica/terapia , Transplante de Rim , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação , Vitamina B 12/genéticaRESUMO
The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalamin (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as 'cblG-variant'. In four 'cblG-variant' cases, we observed a decreased conversion of cyanocobalamin to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full-size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in the proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA, respectively, when compared with control cells. In 3D modelling and docking analysis, both truncated and full-size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of Cbls that is required for Cbl cofactor synthesis.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Anemia Megaloblástica/genética , Proteínas de Transporte/metabolismo , Isoformas de Proteínas/metabolismo , Deficiência de Vitamina B 12/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/química , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Sítios de Ligação/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células Cultivadas , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Hidroxocobalamina/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Oxirredutases , Ligação Proteica/genética , Isoformas de Proteínas/genética , Estrutura Secundária de Proteína , Vitamina B 12/análogos & derivados , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/genéticaRESUMO
We identified a bovine B(12) trafficking chaperone bCblC in Bos taurus that showed 88% amino acid sequence identity with a human homologue. The protein bCblC was purified from E. coli by over-expression of the encoding gene. bCblC bound cyanocobalamin (CNCbl), methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) in the base-off states and eliminated the upper axial ligands forming aquo/hydroxocobalamin (OH(2)/OHCbl) under aerobic conditions. A transition of OH(2)/OHCbl was induced upon binding to bCblC. Interestingly, bCblC-bound OH(2)/OHCbl did not react with reduced glutathione (GSH), while the reaction of free OH(2)/OHCbl with GSH resulted in the formation of glutathionylcobalamin (GSCbl) and glutathione disulfide (GSSG). Furthermore we found that bCblC eliminates the GSH ligand of GSCbl forming OH(2)/ OHCbl. The results demonstrated that bCblC is a B(12) trafficking chaperone that binds cobalamins and protects OH(2)/OHCbl from GSH, which could be oxidized to GSSG by free OH(2)/OHCbl.
Assuntos
Glutationa/metabolismo , Hidroxocobalamina/metabolismo , Chaperonas Moleculares/metabolismo , Vitamina B 12/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Glutationa/química , Humanos , Hidroxocobalamina/química , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Vitamina B 12/química , Complexo Vitamínico B/metabolismoRESUMO
Patients with the cblC vitamin B(12) (cobalamin, cbl) disorder are defective in the intracellular synthesis of adenosylcobalamin and methylcobalamin and have combined homocystinuria and methylmalonic aciduria. While other vitamin B(12) disorders are treatable with high dose cyanocobalamin (CNCbl) or hydroxocobalamin (OHCbl), cblC patients respond well to OHCbl but not to CNCbl. Patient mutations were introduced into recombinant MMACHC (cblC) protein and the binding of CNCbl and OHCbl was examined. Three mutations were analyzed: G147D, associated with early onset, vitamin B(12) unresponsive disease; R161Q, associated with late onset disease that is highly responsive to OHCbl; and H122A, selected to test the hypothesis that H122 is central to a proposed vitamin B(12) binding motif on MMACHC. We report here that wild-type MMACHC binds both OHCbl and CNCbl with similar, tight affinity (K(d)=5.7 microM). We also report that MMACHC binds CNCbl in the base-off form, with the dimethylbenzimidazole (DMB) base of cobalamin displaced from coordination with the cobalt. In this form, wild-type MMACHC is able to reductively decyanate CNCbl to cob(II)alamin requiring only the presence of NADPH and FAD. We demonstrate that MMACHC with the G147D mutation is unable to bind either CNCbl or OHCbl, providing a straight forward explanation for the absence of response to either vitamin form. However, we show that MMACHC containing the R161Q mutation binds OHCbl with wild-type affinity, but is disturbed in binding CNCbl and has impaired decyanation. Finally, we show that H122A has reduced binding, but like R161Q, it binds OHCbl more tightly than CNCbl, suggesting that this histidine is not absolutely required for binding. These studies suggest that the ability of mutant MMACHC to respond to vitamin therapy depends on its ability to bind the vitamin with significant affinity, and for CNCbl, also on its ability to bind in the base-off form to facilitate reductive decyanation. These studies emphasize the continued use of OHCbl with cblC patients for maximum therapeutic effect.
Assuntos
Proteínas de Transporte/metabolismo , Homocistinúria/complicações , Homocistinúria/tratamento farmacológico , Erros Inatos do Metabolismo/complicações , Erros Inatos do Metabolismo/tratamento farmacológico , Ácido Metilmalônico/metabolismo , Vitamina B 12/uso terapêutico , Substituição de Aminoácidos/genética , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Humanos , Hidroxocobalamina/metabolismo , Cinética , Proteínas Mutantes/metabolismo , Oxirredutases , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Espectrofotometria Ultravioleta , Vitamina B 12/metabolismoRESUMO
Cyanide poisoning occurs in many smoke inhalation victims. The newest FDA-approved treatment for acute cyanide intoxication is hydroxocobalamin (Cyanokit). However, hydroxocobalamin exhibits chemical properties that can disrupt several clinical laboratory tests. Knowledge of these effects on laboratory tests can be useful in assisting laboratory technicians and clinicians in managing these patients. This article briefly discusses acute cyanide poisoning and treatment, and summarizes laboratory interferences that have been reported with the use of hydroxocobalamin.
Assuntos
Antídotos/uso terapêutico , Cianetos/intoxicação , Hidroxocobalamina/uso terapêutico , Complexo Vitamínico B/uso terapêutico , Antídotos/química , Antídotos/metabolismo , Técnicas de Laboratório Clínico , Cianetos/antagonistas & inibidores , Interações Medicamentosas , Humanos , Hidroxocobalamina/química , Hidroxocobalamina/metabolismo , Técnicas In Vitro , Kit de Reagentes para Diagnóstico , Complexo Vitamínico B/química , Complexo Vitamínico B/metabolismoRESUMO
The US government considers cyanide to be among the most likely agents of chemical terrorism. Cyanide differs from many other biological or chemical agents for which little or no defense is available because its individual and public health effects are largely remediable through appropriate preparedness and response. Because the toxicity of the cyanide antidote currently available in the United States renders it ill-suited for use in terrorist incidents and other situations requiring rapid out-of-hospital treatment, hydroxocobalamin--an effective and safe cyanide antidote being used in other countries--has been introduced in the United States. Unlike the other available cyanide antidote, hydroxocobalamin can be administered at the scene of a cyanide disaster, and it need not be reserved for cases of confirmed cyanide poisoning but can be administered in cases of suspected poisoning. Both of these attributes facilitate the rapid intervention necessary for saving lives. To realize the potential benefits of hydroxocobalamin, progress also needs to be realized in other aspects of readiness, including but not limited to developing plans for ensuring local and regional availability of antidote, educating emergency responders and health care professionals in the recognition and management of cyanide poisoning, and raising public awareness of the potential for a chemical weapons attack and of how to respond.
Assuntos
Guerra Química , Cianetos/intoxicação , Planejamento em Desastres , Hematínicos/uso terapêutico , Hidroxocobalamina/uso terapêutico , Saúde Pública , Terrorismo , Cianetos/administração & dosagem , Cianetos/química , Hematínicos/metabolismo , Humanos , Hidroxocobalamina/metabolismo , Estados UnidosRESUMO
Carbon tetrachloride (CT) is an important groundwater pollutant which is only subject to biotransformation in the absence of oxygen. The anaerobic biotransformation of CT is influenced by electron shuttling compounds. The purpose of this study was to evaluate the impact of redox active vitamins on CT (100 microM) metabolism in a methanogenic sludge consortium (0.5 g VSS l(-1)) supplied with volatile fatty acids as electron donor (0.2 g COD l(-1)). The redox active vitamins, tested at concentrations ranging from 0.5 to 20 microM, were riboflavin (RF) and two forms of vitamin B12, cyanocobalamin (CNB12) and hydroxycobalamin (HOB12), and these were compared with a redox mediating quinone, anthraquinone-2,6-disulfonate (AQDS). Substoichiometric concentrations of RF, CNB12, HOB12 at molar ratios of vitamin: CT as low as 0.005 significantly increased rates of CT-bioconversion. These are the lowest molar ratios of vitamin B12 reported having an impact on dechlorination. Additionally, this study constitutes the first report of RF having a role in reductive dechlorination. At molar ratios of 0.1 vitamin: CT, RF, CNB12, HOB12 increased the first order rate constant of CT bioconversion by 4.0-, 13.3-and 13.6-fold, respectively. The redox active vitamins also enhanced the rates of abiotic CT conversion in heat killed sludge treatments, but the rates were approximately 4- to 5-fold lower than the corresponding vitamin enhanced rates of biological CT conversion. The addition of CNB12 or HOB12 to the live methanogenic sludge consortium increased the yield of inorganic chloride (Cl-) from CT-converted. Chloroform was a transient intermediate in CNB12 or HOB12 supplemented cultures. In contrast, the addition of RF increased the yield of chloroform from CT-converted. Taken as a whole the results clearly demonstrate that very low concentrations of redox active vitamins could potentially play an important role in accelerating the anaerobic the bioremediation of CT as well as influencing the proportions of biotransformation products formed.
Assuntos
Tetracloreto de Carbono/metabolismo , Esgotos/microbiologia , Anaerobiose , Antraquinonas/metabolismo , Antraquinonas/farmacologia , Biodegradação Ambiental/efeitos dos fármacos , Biotransformação/efeitos dos fármacos , Hidroxocobalamina/metabolismo , Hidroxocobalamina/farmacologia , Cinética , Oxirredução , Riboflavina/metabolismo , Riboflavina/farmacologia , Vitamina B 12/metabolismo , Vitamina B 12/farmacologia , Poluentes Químicos da Água/metabolismoRESUMO
Glycerol dehydrase is an enzyme that catalyzes dehydration of glycerol into beta-propionaldehyde. It requires 5'-deoxyadenosylcobalamin, one of the forms of vitamin B12, as a coenzyme. The enzyme is inactivated in vitro by all forms of vitamin B12 stoichiometrically. The objective of this study was to determine vitamin B12 content by utilizing the inactivation of the enzyme by vitamin B12. After various examinations, an excellent standard curve was obtained up to 1 pmol vitamin B12 using 14 mU of the enzyme per tube. Glycerol dehydrase does not respond to vitamin B12 if it is bound to haptocorrin, a vitamin B12-binding protein. This necessitates a procedure for extraction of vitamin B12 from samples before assay. The enzyme was less inactivated by 5'-deoxyadenosylcobalamin than any other form of vitamin B12. However, this did not matter because all forms of vitamin B12 were converted into cyanocobalamin during the extraction procedure cited above, which was performed in a buffer containing potassium cyanide.
Assuntos
Gliceraldeído/análogos & derivados , Hidroliases/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/análise , Aldeídos , Análise de Variância , Benzotiazóis , Cobamidas/química , Cobamidas/metabolismo , Ativação Enzimática , Escherichia coli/enzimologia , Análise de Alimentos , Gliceraldeído/metabolismo , Glicerol/metabolismo , Hidrazonas , Hidroliases/química , Hidroliases/isolamento & purificação , Hidroxocobalamina/química , Hidroxocobalamina/metabolismo , Klebsiella pneumoniae/enzimologia , Medições Luminescentes , Cianeto de Potássio/química , Propano , Propanodiol Desidratase/isolamento & purificação , Propanodiol Desidratase/metabolismo , Ligação Proteica , Valores de Referência , Análise de Regressão , Reprodutibilidade dos Testes , Espectrofotometria , Estereoisomerismo , Comprimidos/química , Tiazóis/química , Transcobalaminas/metabolismo , Vitamina B 12/química , Vitamina B 12/metabolismoRESUMO
Cyanide poisoning may result from different exposures: residential fires, industrial accidents, drug and plant intoxication. Clinical features include coma, respiratory arrest and cardiovascular collapse. The biological hallmark is lactic acidosis. A plasma lactate concentration > or = 10 mmol/L in fire victims without severe burns and > or = 8 mmol/L in pure cyanide poisoned patients is a sensitive and specific indicator of cyanide intoxication. Many antidotes are available and efficient. However, therapeutic strategies are still debated. Our objective was to compare conventional treatments to hydroxocobalamin. This article reviews the literature on cyanide poisoning treatment. Conventional treatment of cyanide poisoning includes decontamination, supportive and specific treatment. Decontamination should be adapted to the route of poisoning and never postpone supportive treatment. Basic life support includes immediate administration of high flow of oxygen, airway protection and cardiopulmonary resuscitation. Advanced life support includes mechanical ventilation, catecholamine and sodium bicarbonate infusion. Supportive treatment is efficient but does not modify the time course or the body burden of cyanide. Numerous antidotes are available. Oxygen counteracts efficiently cyanide action at the mitochondrial level. Sodium thiosulfate, methemoglobin forming agents and cobalt compounds act efficiently by complexing or transforming cyanide into non-toxic stable derivatives. However, regarding the main clinical condition of cyanide poisoning, i.e. smoke inhalation, we should take into account not only the efficiency of antidotes but also their safety. Sodium thiosulfate is both efficient and safe, but acts with delay. Methemoglobin-forming agents are potent, but due to the transformation of hemoglobin into methemoglobin, they impair tissue delivery of oxygen. Experimental data showed increased mortality in carbon monoxide- and cyanide-poisoned rats treated with these agents. Cobalt EDTA and hydroxocobalamin are efficient and act immediately. Cobalt EDTA is more potent on a molar basis; however, numerous side effects limit its use to evidenced cyanide poisoning. In a prospective study, hydroxocobalamin appeared safe in fire victims with or without cyanide poisoning. The only reported side effect was a red coloration of skin and urine. In conclusion, antidotes are beneficial in cyanide poisoning. In suspected cyanide-poisoned patients, we recommend the use of hydroxocobalamin as first-line antidote, owing to its safety. In massive cyanide poisoning, due to the limited potency of hydroxocobalamin, continuous infusion of sodium thiosulfate should be associated.