RESUMO
Neurological dysfunction is a common finding in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly known. In the present study, we investigated the effect of the in vitro effect of the branched chain alpha-keto acids (BCKA) accumulating in MSUD on some parameters of energy metabolism in cerebral cortex of rats. [14CO(2)] production from [14C] acetate, glucose uptake and lactate release from glucose were evaluated by incubating cortical prisms from 30-day-old rats in Krebs-Ringer bicarbonate buffer, pH 7.4, in the absence (controls) or presence of 1-5 mM of alpha-ketoisocaproic acid (KIC), alpha-keto-beta-methylvaleric acid (KMV) or alpha-ketoisovaleric acid (KIV). All keto acids significantly reduced 14CO(2) production by around 40%, in contrast to lactate release and glucose utilization, which were significantly increased by the metabolites by around 42% in cortical prisms. Furthermore, the activity of the respiratory chain complex I-III was significantly inhibited by 60%, whereas the other activities of the electron transport chain, namely complexes II, II-III, III and IV, as well as succinate dehydrogenase were not affected by the keto acids. The results indicate that the major metabolites accumulating in MSUD compromise brain energy metabolism by blocking the respiratory chain. We presume that these findings may be of relevance to the understanding of the pathophysiology of the neurological dysfunction of MSUD patients.
Assuntos
Encéfalo/metabolismo , Metabolismo Energético/efeitos dos fármacos , Cetoácidos/farmacologia , Doença da Urina de Xarope de Bordo/metabolismo , Animais , Transporte Biológico , Dióxido de Carbono/metabolismo , Modelos Animais de Doenças , Glucose/metabolismo , Hemiterpenos , Humanos , Lactatos/metabolismo , Ratos , Ratos Wistar , Succinato Citocromo c Oxirredutase/metabolismoRESUMO
In 1992-1994, a disorder known as the epidemic neuropathy afflicted more than 50,000 Cubans. Three different forms of the illness were identified: epidemic optic neuropathy, peripheral neuropathy and mixed optic and peripheral neuropathy. The causes are still unknown. Skeletal muscle biopsy samples were analyzed by standard histological techniques and by biochemical assays. Elevated activities of citrate synthase, a non-respiratory-chain mitochondrial matrix enzyme, suggested possible mitochondrial proliferation in 7 of the 8 patients. Nicotinamide adenine dinucleotide phosphate (NADP(+)) levels were higher in the patients than in the controls (p = 0.04). Levels of nicotinamide adenine dinucleotide (NAD) and the reduced compounds NADH and NADPH were comparable in patients and controls. Elevations of succinate dehydrogenase and citrate synthase activities and high NADP(+) levels suggest that alterations of mitochondrial functions may be associated with this disorder.
Assuntos
Doenças Mitocondriais/enzimologia , Doenças do Nervo Óptico/enzimologia , Oxirredutases/metabolismo , Doenças do Sistema Nervoso Periférico/enzimologia , Adulto , Citrato (si)-Sintase/metabolismo , Cuba/epidemiologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Pessoa de Meia-Idade , Doenças Mitocondriais/epidemiologia , Músculo Esquelético/enzimologia , NADP/metabolismo , Doenças do Nervo Óptico/epidemiologia , Doenças do Sistema Nervoso Periférico/epidemiologia , Succinato Citocromo c Oxirredutase/metabolismo , Succinato Desidrogenase/metabolismoRESUMO
This study was aimed at assessing the effects of long-term exposure to NO of respiratory activities in mitochondria from different tissues (with different ubiquinol contents), under conditions that either promote or prevent the formation of peroxynitrite. Mitochondria and submitochondrial particles isolated from rat heart, liver and brain were exposed either to a steady-state concentration or to a bolus addition of NO. NO induced the mitochondrial production of superoxide anions, hydrogen peroxide and peroxynitrite, the latter shown by nitration of mitochondrial proteins. Long-term incubation of mitochondrial membranes with NO resulted in a persistent inhibition of NADH:cytochrome c reductase activity, interpreted as inhibition of NADH:ubiquinone reductase (Complex I) activity, whereas succinate:cytochrome c reductase activity, including Complex II and Complex III electron transfer, remained unaffected. This selective effect of NO and derived species was partially prevented by superoxide dismutase and uric acid. In addition, peroxynitrite mimicked the effect of NO, including tyrosine nitration of some Complex I proteins. These results seem to indicate that the inhibition of NADH:ubiquinone reductase (Complex I) activity depends on the NO-induced generation of superoxide radical and peroxynitrite and that Complex I is selectively sensitive to peroxynitrite. Inhibition of Complex I activity by peroxynitrite may have critical implications for energy supply in tissues such as the brain, whose mitochondrial function depends largely on the channelling of reducing equivalents through Complex I.
Assuntos
Mitocôndrias/enzimologia , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/antagonistas & inibidores , Óxido Nítrico/farmacologia , Oxirredutases/metabolismo , Ácido Peroxinitroso/metabolismo , Succinato Desidrogenase/metabolismo , Superóxidos/metabolismo , Tirosina/análogos & derivados , Animais , Encéfalo/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Complexo I de Transporte de Elétrons , Complexo II de Transporte de Elétrons , Feminino , Coração/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Immunoblotting , Fígado/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , NAD/metabolismo , NADH NADPH Oxirredutases/metabolismo , Ratos , Ratos Sprague-Dawley , Succinato Citocromo c Oxirredutase/metabolismo , Succinatos/metabolismo , Superóxido Dismutase/metabolismo , Tirosina/metabolismoRESUMO
Some protozoa of the Trypanosomatidae family harbor in their cytoplasm bacterial endosymbionts that provide essential nutrients to and induce morphological alterations in the protozoa. In the present study, a close association between endosymbionts and glycosomes, a peroxisome-like organelle where most of the enzymes of the glycolytic pathway are compartmentalized, was identified by conventional transmission electron microscopy in Crithidia deanei. Such an association was further supported by the cytochemical localization of catalase in the glycosome and also confirmed by 3-D reconstruction of the protozoan. The enzymes cytochrome oxidase and succinate dehydrogenase were detected by ultrastructural cytochemistry. A positive reaction was observed in the protozoan mitochondrion but not in the endosymbiont envelope. Enzymatic assays for succinate cytochrome c reductase reinforced these results, as a low enzymatic activity was detected in an endosymbiont-enriched fraction, while high activity was observed in a purified protozoan mitochondrion fraction. We also demonstrated that a purified symbiont fraction was able to hydrolyze ATP. This activity was Mg+2 dependent, since it was highly stimulated by the presence of physiological concentrations of this ion. Taken together, these observations suggest that no electron transporting system is active in the symbionts of Crithidia deanei and that they might obtain energetic molecules derivated from the protozoan glycosomes.
Assuntos
Crithidia/enzimologia , Crithidia/ultraestrutura , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glicólise , Succinato Desidrogenase/metabolismo , Simbiose/fisiologia , Adenosina Trifosfatases/metabolismo , Animais , Mitocôndrias/ultraestrutura , Monoéster Fosfórico Hidrolases/metabolismo , Succinato Citocromo c Oxirredutase/metabolismoRESUMO
Electron transport and production of O2-/H2O2 by the NADH dehydrogenase flavin-semiquinone (FMNH.) and ubisemiquinone (UQH.) were studied in a model of in vivo ischemia-reperfusion in rat kidney. H2O2 production rates were assessed in isolated mitochondria using either succinate, with and without antimycin, or malate-glutamate, with and without rotenone. Respiratory activities of isolated mitochondria and activity of NADH- and succinate-cytochrome c reductase and of NADH- and succinate-dehydrogenase in submitochondrial particles were measured to evaluate the electron flux throughout respiratory carriers. The mitochondrial H2O2 production rate was approximately 1.5- and 4-times increased in ischemic and ischemic-reperfused kidneys, respectively. Ischemia caused a marked decrease in the electron transport throughout the NADH-UQ segment with no significant changes either in the NADH dehydrogenase activity or in the electron flux trough the succinate-cytochrome oxidase segment. Reperfusion did not further affect the NADH-ubiquinone segment but markedly inhibited the succinate-supported oxygen consumption, succinate-cytochrome c reductase and succinate dehydrogenase activity. Our results show a redistribution of the electron flux with an increased rate of superoxide anion/hydrogen peroxide production at NADH dehydrogenase in mitochondria subjected to ischemia only. After 10 min reperfusion an impairment of the electron flow at succinate-cytochrome c segment is established and hydrogen peroxide production by UQH. increases up to maximal values becoming the major source of superoxide anion/hydrogen peroxide.
Assuntos
Peróxido de Hidrogênio/metabolismo , Isquemia/metabolismo , Córtex Renal/irrigação sanguínea , Mitocôndrias/metabolismo , Reperfusão , Animais , Transporte de Elétrons , Córtex Renal/ultraestrutura , Cinética , Masculino , NADH Desidrogenase/metabolismo , Consumo de Oxigênio , Ratos , Ratos Wistar , Partículas Submitocôndricas/metabolismo , Succinato Citocromo c Oxirredutase/metabolismo , Succinato Desidrogenase/metabolismo , Superóxidos/metabolismoRESUMO
We report two unrelated children with onset of chronic diarrhea and villous atrophy in the first years of life. Elevated plasma lactate concentrations and lactate/pyruvate and ketone body molar ratios suggested a genetic defect of oxidative phosphorylation. Analysis of the mitochondrial respiratory chain showed a complex III deficiency in muscle of both patients. Southern blot analysis provided evidence of heteroplasmic mitochondrial DNA rearrangements that involve deletion and deletion-duplication. Directly repeated sequences (10 and 11 base pairs, respectively) were present in the wild type of mitochondrial genome at the boundaries of the deletion. Neither parent of either patient had rearranged molecules in their circulating lymphocytes. It appears that a mitochondrial disorder can have chronic diarrhea and villous atrophy as the initial clinical feature. On the basis of these observations, we suggest that genetic defects of mitochondrial energy supply be considered in elucidating the origin of unexplained chronic diarrheas, especially when other, unrelated symptoms occur in the course of the disease.