RESUMO
Refsum disease is an inherited peroxisomal disorder caused by severe deficiency of phytanoyl-CoA hydroxylase activity. Affected patients develop severe cardiomyopathy of poorly known pathogenesis that may lead to a fatal outcome. Since phytanic acid (Phyt) concentrations are highly increased in tissues of individuals with this disease, it is conceivable that this branched-chain fatty acid is cardiotoxic. The present study investigated whether Phyt (10-30 µM) could disturb important mitochondrial functions in rat heart mitochondria. We also determined the influence of Phyt (50-100 µM) on cell viability (MTT reduction) in cardiac cells (H9C2). Phyt markedly increased mitochondrial state 4 (resting) and decreased state 3 (ADP-stimulated) and uncoupled (CCCP-stimulated) respirations, besides reducing the respiratory control ratio, ATP synthesis and the activities of the respiratory chain complexes I-III, II, and II-III. This fatty acid also reduced mitochondrial membrane potential and induced swelling in mitochondria supplemented by exogenous Ca2+, which were prevented by cyclosporin A alone or combined with ADP, suggesting the involvement of the mitochondrial permeability transition (MPT) pore opening. Mitochondrial NAD(P)H content and Ca2+ retention capacity were also decreased by Phyt in the presence of Ca2+. Finally, Phyt significantly reduced cellular viability (MTT reduction) in cultured cardiomyocytes. The present data indicate that Phyt, at concentrations found in the plasma of patients with Refsum disease, disrupts by multiple mechanisms mitochondrial bioenergetics and Ca2+ homeostasis, which could presumably be involved in the cardiomyopathy of this disease.
Assuntos
Cardiomiopatias , Doença de Refsum , Ratos , Animais , Doença de Refsum/metabolismo , Ácido Fitânico/farmacologia , Ácido Fitânico/metabolismo , Cálcio/metabolismo , Ratos Wistar , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/metabolismo , Metabolismo Energético , Mitocôndrias Cardíacas/metabolismo , Ácidos Graxos/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , HomeostaseRESUMO
Phytanic acid (Phyt) accumulates in tissues and biological fluids of patients affected by Refsum disease. Although cardiomyopathy is an important clinical manifestation of this disorder, the mechanisms of heart damage are poorly known. In the present study, we investigated the in vitro effects of Phyt on important parameters of oxidative stress in heart of young rats. Phyt significantly increased thiobarbituric acid-reactive substances levels (P < 0.001) and carbonyl formation (P < 0.01), indicating that this fatty acid induces lipid and protein oxidative damage, respectively. In contrast, Phyt did not alter sulfhydryl oxidation. Phyt also decreased glutathione (GSH) concentrations (P < 0.05), an important non-enzymatic antioxidant defense. Moreover, Phyt increased 2',7'-dichlorofluorescin oxidation (DCFH) (P < 0.01), reflecting increased reactive species generation. We also found that the induced lipid and protein oxidative damage, as well as the decreased GSH levels and increased DCFH oxidation provoked by this fatty acid were prevented or attenuated by the reactive oxygen species scavengers melatonin, trolox, and GSH, but not by the nitric oxide inhibitor N: (ω)-nitro-L: -arginine methyl ester, suggesting that reactive oxygen species were involved in these effects. Next, we verified that Phyt strongly inhibited NADH-cytochrome c oxidoreductase (complex I-III) activity (P < 0.001) in heart supernatants, and decreased membrane potential and the NAD(P)H pool in heart mitochondria, indicating that Phyt acts as a metabolic inhibitor and as an uncoupler of the electron transport chain. Therefore, it can be presumed that disturbance of cellular energy and redox homeostasis induced by Phyt may possibly contribute to the cardiomyopathy found in patients affected by Refsum disease.
Assuntos
Cardiomiopatias/metabolismo , Homeostase/efeitos dos fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , Miocárdio/patologia , Ácido Fitânico/farmacologia , Doença de Refsum/metabolismo , Animais , Antioxidantes/farmacologia , Cromanos/farmacologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Glutationa/farmacologia , Técnicas In Vitro , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , NADP/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Oxirredução , Estresse Oxidativo , Carbonilação Proteica , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Phytanic acid (Phyt) tissue concentrations are increased in Refsum disease and other peroxisomal disorders characterized by neurologic damage and brain abnormalities. The present work investigated the in vitro effects of Phyt, at concentrations found in these peroxisomal disorders, on important parameters of energy metabolism in brain cortex of young rats. The parameters analyzed were CO(2) production from labeled acetate and glucose, the activities of the citric acid cycle enzymes citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase, as well as of the respiratory chain complexes I-IV, creatine kinase and Na(+),K(+)-ATPase. Our results show that Phyt did not alter citric acid cycle enzyme activities, or CO(2) production from acetate, reflecting no impairment of the functionality of the citric acid cycle. In contrast, respiratory chain activities were reduced at complexes I, II, I-III, II-III and IV. Membrane synaptical Na(+),K(+)-ATPase activity was also reduced by Phyt, with no alteration of creatine kinase activity. Considering the importance of the electron flow through the respiratory chain for brain energy metabolism (oxidative phosphorylation) and of Na(+),K(+)-ATPase activity for maintaining membrane potential necessary for neurotransmission, the data indicate that Phyt impairs brain bioenergetics at the level of energy formation, as well as neurotransmission. It is presumed that Phyt-induced impairment of these important systems may be involved at least in part in the neurological damage found in patients affected by disorders in which brain Phyt concentrations are increased.
Assuntos
Córtex Cerebral/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Ácido Fitânico/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Acetatos/metabolismo , Animais , Membrana Celular/enzimologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Ratos , Doença de Refsum/tratamento farmacológico , Doença de Refsum/metabolismo , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacosRESUMO
AIMS: In the present work we investigated the in vitro effects of phytanic acid (Phyt), that accumulates in Refsum disease and other peroxisomal diseases, on important parameters of oxidative stress in cerebellum and cerebral cortex from young rats. MAIN METHODS: The parameters thiobarbituric acid-reactive substances levels (TBA-RS; lipid peroxidation), carbonyl formation and sulfhydryl oxidation (protein oxidative damage) and the concentrations of the most important nonenzymatic antioxidant defense reduced glutathione (GSH) were determined. KEY FINDINGS: It was observed that Phyt significantly increased TBA-RS levels in both cerebral structures. This effect was prevented by the antioxidants alpha-tocopherol and melatonin, suggesting the involvement of free radicals. Phyt also provoked protein oxidative damage in both cerebellum and cerebral cortex, as determined by increased carbonyl content and sulfhydryl oxidation. Furthermore, Phyt significantly diminished the concentrations of GSH, while melatonin and alpha-tocopherol treatment totally blocked this effect. We also verified that Phyt does not behave as a direct acting oxidant, since Phyt did not oxidize commercial solutions of GSH and reduced cytochrome c to Phyt in a free cell medium. SIGNIFICANCE: Our data indicate that oxidative stress is elicited in vitro by Phyt, a mechanism that may contribute at least in part to the pathophysiology of Refsum disease and other peroxisomal disorders where Phyt is accumulated.
Assuntos
Antioxidantes/metabolismo , Química Encefálica/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ácido Fitânico/toxicidade , Animais , Cerebelo/química , Cerebelo/metabolismo , Córtex Cerebral/química , Córtex Cerebral/metabolismo , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Ácido Fitânico/sangue , Carbonilação Proteica , Ratos , Ratos Wistar , Doença de Refsum/sangue , Doença de Refsum/metabolismo , Compostos de Sulfidrila/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Biochemical studies with emphasis on peroxisomal functions were conducted in six patients with well-documented rhizomelic chondrodysplasia punctata (RCDP) and compared with findings in patients with Zellweger syndrome and neonatal adrenoleukodystrophy (ALD). Patients with RCDP had three characteristic biochemical abnormalities: (1) profound defect in plasmalogen (ether lipid) synthesis, which is significantly greater than the analogous defect in Zellweger syndrome or neonatal ALD; (2) reduction of phytanic acid oxidation activity to 1% to 5% of control, similar to that observed in Refsum disease, Zellweger syndrome, and neonatal ALD; (3) presence of the unprocessed form of peroxisomal 3-oxoacyl-coenzyme A thiolase in the postmortem liver of two patients. Other peroxisomal functions were normal, including levels of very long chain fatty acids, pipecolic acid, and bile acid intermediates, and immunoblot studies of peroxisomal acyl-CoA oxidase and bifunctional enzyme in postmortem liver. Unlike what is observed in Zellweger syndrome and neonatal ALD, catalase activity in cultured skin fibroblasts was sedimentable, indicating that peroxisome structure is not grossly deficient in RCDP. The biochemical abnormalities in RCDP were consistent and set it apart from all the other known peroxisomal disorders.