RESUMO
Long-chain 3-hydroxylated fatty acids (LCHFA) accumulate in long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies. Affected patients usually present severe neonatal symptoms involving cardiac and hepatic functions, although long-term neurological abnormalities are also commonly observed. Since the underlying mechanisms of brain damage are practically unknown and have not been properly investigated, we studied the effects of LCHFA on important parameters of mitochondrial homeostasis in isolated mitochondria from cerebral cortex of developing rats. 3-Hydroxytetradecanoic acid (3 HTA) reduced mitochondrial membrane potential, NAD(P)H levels, Ca(2+) retention capacity and ATP content, besides inducing swelling, cytochrome c release and H2O2 production in Ca(2+)-loaded mitochondrial preparations. We also found that cyclosporine A plus ADP, as well as ruthenium red, a Ca(2+) uptake blocker, prevented these effects, suggesting the involvement of the mitochondrial permeability transition pore (mPTP) and an important role for Ca(2+), respectively. 3-Hydroxydodecanoic and 3-hydroxypalmitic acids, that also accumulate in LCHAD and MTP deficiencies, similarly induced mitochondrial swelling and decreased ATP content, but to a variable degree pending on the size of their carbon chain. It is proposed that mPTP opening induced by LCHFA disrupts brain bioenergetics and may contribute at least partly to explain the neurologic dysfunction observed in patients affected by LCHAD and MTP deficiencies.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/deficiência , Cardiomiopatias/metabolismo , Córtex Cerebral/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Ácidos Láuricos/farmacologia , Erros Inatos do Metabolismo Lipídico/metabolismo , Mitocôndrias/efeitos dos fármacos , Miopatias Mitocondriais/metabolismo , Proteína Mitocondrial Trifuncional/metabolismo , Ácidos Mirísticos/farmacologia , Doenças do Sistema Nervoso/metabolismo , Ácidos Palmíticos/farmacologia , Rabdomiólise/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Cardiomiopatias/patologia , Córtex Cerebral/metabolismo , Citocromos c/metabolismo , Homeostase , Peróxido de Hidrogênio/metabolismo , Erros Inatos do Metabolismo Lipídico/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Miopatias Mitocondriais/patologia , Poro de Transição de Permeabilidade Mitocondrial , Dilatação Mitocondrial/efeitos dos fármacos , NADP/metabolismo , Doenças do Sistema Nervoso/patologia , Oxidantes/metabolismo , Ratos , Ratos Wistar , Rabdomiólise/patologiaRESUMO
Zellweger syndrome (ZS) and some peroxisomal diseases are severe inherited disorders mainly characterized by neurological symptoms and cerebellum abnormalities, whose pathogenesis is poorly understood. Biochemically, these diseases are mainly characterized by accumulation of pristanic acid (Prist) and other fatty acids in the brain and other tissues. In this work, we evaluated the in vitro influence of Prist on redox homeostasis by measuring lipid, protein, and DNA damage, as well as the antioxidant defenses and the activities of aconitase and α-ketoglutarate dehydrogenase in cerebellum of 30-day-old rats. The effect of Prist on DNA damage was also evaluated in blood of these animals. Some parameters were also evaluated in cerebellum from neonatal rats and in cerebellum neuronal cultures. Prist significantly increased malondialdehyde (MDA) levels and carbonyl formation and reduced sulfhydryl content and glutathione (GSH) concentrations in cerebellum of young rats. It also caused DNA strand damage in cerebellum and induced a high micronuclei frequency in blood. On the other hand, this fatty acid significantly reduced α-ketoglutarate dehydrogenase and aconitase activities in rat cerebellum. We also verified that Prist-induced increase of MDA levels was totally prevented by melatonin and attenuated by α-tocopherol but not by the nitric oxide synthase inhibitor N(ω)-nitro-L-arginine methyl ester, indicating the involvement of reactive oxygen species in this effect. Cerebellum from neonate rats also showed marked alterations of redox homeostasis, including an increase of MDA levels and a decrease of sulfhydryl content and GSH concentrations elicited by Prist. Finally, Prist provoked an increase of dichlorofluorescein (DCFH) oxidation in cerebellum-cultivated neurons. Our present data indicate that Prist compromises redox homeostasis in rat cerebellum and blood and inhibits critical enzymes of the citric acid cycle that are susceptible to free radical attack. The present findings may contribute to clarify the pathogenesis of the cerebellar alterations observed in patients affected by ZS and some peroxisomal disorders in which Prist is accumulated.
Assuntos
Antioxidantes/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Ácidos Graxos/toxicidade , Oxirredução/efeitos dos fármacos , Aconitato Hidratase/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Fluoresceínas/metabolismo , Glutationa/metabolismo , Homeostase/efeitos dos fármacos , Complexo Cetoglutarato Desidrogenase/metabolismo , Malondialdeído/metabolismo , Melatonina/administração & dosagem , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Ratos Wistar , Compostos de Sulfidrila/metabolismo , alfa-Tocoferol/farmacologiaRESUMO
Refsum disease is an autosomal recessive disorder of peroxisomal metabolism biochemically characterized by highly elevated concentrations of phytanic acid (Phyt) in a variety of tissues including the cerebellum. Reduction of plasma Phyt levels by dietary restriction intake ameliorates ataxia, a common clinical manifestation of this disorder, suggesting a neurotoxic role for this branched-chain fatty acid. Therefore, considering that the underlying mechanisms of cerebellum damage in Refsum disease are poorly known, in the present study we tested the effects of Phyt on important parameters of bioenergetics, such as the activities of the respiratory chain complexes I to IV, creatine kinase and Na(+), K(+)- ATPase in cerebellum preparations from young rats. The activities of complexes I, II, I-III and II-III and Na(+), K(+)- ATPase were markedly inhibited (65-85%) in a dose-dependent manner by Phyt. In contrast, creatine kinase and complex IV activities were not altered by this fatty acid. Therefore, it is presumed that impairment of the electron flow through the respiratory chain and inhibition of Na(+), K(+)- ATPase that is crucial for synaptic function may be involved in the pathophysiology of the cerebellar abnormalities manifested as ataxia in Refsum disease and in other peroxisomal disorders in which brain Phyt accumulates.
Assuntos
Cerebelo/enzimologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Proteínas do Tecido Nervoso/antagonistas & inibidores , Ácido Fitânico/farmacologia , Doença de Refsum/enzimologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Sinapses/enzimologia , Animais , Cerebelo/patologia , Cerebelo/fisiopatologia , Transporte de Elétrons/efeitos dos fármacos , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Wistar , Doença de Refsum/patologia , Doença de Refsum/fisiopatologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Sinapses/patologiaRESUMO
Isolated 3-methylcrotonyl-CoA carboxylase deficiency (3MCCD) is an autosomal recessive disorder of leucine metabolism biochemically characterized by accumulation of 3-methylcrotonylglycine (3MCG), 3-methylcrotonic acid (3MCA) and 3-hydroxyisovaleric acid. A considerable number of affected individuals present neurological symptoms with or without precedent crises of metabolic decompensation and brain abnormalities whose pathogenesis is poorly known. We investigated the in vitro effects of 3MCG and 3MCA on important parameters of oxidative stress in cerebral cortex of young rats. 3MCG and 3MCA significantly increased TBA-RS and carbonyl formation, indicating that these compounds provoke lipid and protein oxidation, respectively. In contrast, nitric oxide production was not affected by 3MCG and 3MCA. Furthermore, 3MCG- and 3MCA-induced elevation of TBA-RS values was fully prevented by melatonin, trolox and reduced glutathione, but not by the nitric oxide inhibitor N(ω)-nitro-L-arginine methyl ester or the combination of catalase plus superoxide dismutase, indicating that reactive oxygen species were involved in the oxidative damage caused by these compounds. We also found that the activity of the antioxidant enzymes glutathione peroxidase, catalase, superoxide dismutase and glutathione reductase were not altered in vitro by 3MCG and 3MCA. It is therefore presumed that alterations of the cellular redox homeostasis caused by the major metabolites accumulating in 3MCCD may potentially be involved in the pathophysiology of the neurological dysfunction and structural brain alterations found in patients affected by this disorder.
Assuntos
Química Encefálica/fisiologia , Carbono-Carbono Ligases/deficiência , Córtex Cerebral/metabolismo , Estresse Oxidativo/fisiologia , Fatores Etários , Animais , Córtex Cerebral/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Distúrbios Congênitos do Ciclo da Ureia/enzimologia , Distúrbios Congênitos do Ciclo da Ureia/fisiopatologiaRESUMO
Phytanic acid (Phyt) brain concentrations are highly increased in Refsum disease, a peroxisomal disorder clinically characterized by neurological features, cardiac abnormalities, and retinitis pigmentosa. Considering that the pathogenesis of cerebellar ataxia, a common finding in this disease, is still unknown, in the present work we investigated the in vitro effects of Phyt at concentrations similar to those found in affected patients on important parameters of mitochondrial homeostasis in cerebellum from young rats. The respiratory parameters states 3 and 4 and respiratory control ratio (RCR) determined by oxygen consumption, membrane potential (∆Ψm), NAD(P)H pool content, and swelling were evaluated in mitochondrial preparations from this cerebral structure. Phyt markedly increased state 4 respiration, whereas state 3 respiration, the RCR, the mitochondrial matrix NAD(P)H content, and ∆Ψm were decreased by this fatty acid, being the latter effect partially prevented by N-acetylcysteine. These data indicate that Phyt behaves as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor disrupting mitochondrial homeostasis in cerebellum. It is proposed that these pathomechanisms may contribute at least in part to the cerebellar alterations found in Refsum disease.
Assuntos
Cerebelo/ultraestrutura , Homeostase/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Ácido Fitânico/farmacologia , Difosfato de Adenosina/farmacologia , Animais , Relação Dose-Resposta a Droga , Ácido Glutâmico/farmacologia , Ácidos Cetoglutáricos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/patologia , NADP/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Wistar , Estatísticas não ParamétricasRESUMO
Patients affected by peroxisomal disorders commonly present neurologic dysfunction and brain abnormalities, whose neuropathology is poorly understood. Given that high sustained concentrations of pristanic acid (Prist) are found in the brain of these patients, it is conceivable that this complex branched-chain fatty acid is neurotoxic. Therefore, the present work investigated the in vitro effects of Prist at similar concentrations found in plasma of affected patients with some peroxisomal disorders on important parameters of energy homeostasis, including respiratory parameters determined by oxygen consumption, membrane potential (ΔΨm), NAD(P)H content, and swelling in mitochondrial preparations obtained from brain of young rats using glutamate plus malate or succinate as respiratory substrates. Prist markedly increased state 4 respiration and decreased state 3 respiration, the respiratory control ratio (RCR), and the ADP/O ratio with both substrates. The mitochondrial ΔΨm and the matrix NAD(P)H content were also decreased by Prist, which was also able to provoke mitochondrial swelling. Furthermore, Prist-induced mitochondrial swelling was dependent on oxidative damage to the permeability transition pore (PTP), because cyclosporine A and the thiol-reducing agent N-acetylcysteine totally prevented mitochondrial swelling. These data suggest that Prist impairs mitochondrial homeostasis, acting as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor, besides causing mitochondrial swelling probably mediated by the permeability transition pore. It is proposed that these pathomechanisms may potentially be involved in the neurological abnormalities characteristic of the peroxisomal diseases in which Prist accumulates.
Assuntos
Encéfalo/efeitos dos fármacos , Ácidos Graxos/farmacologia , Homeostase/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Deficiency of 3-methylcrotonyl-CoA carboxylase activity is an inherited metabolic disease biochemically characterized by accumulation and high urinary excretion of 3-methylcrotonylglycine (3MCG), and also of 3-hydroisovalerate in lesser amounts. Affected patients usually have neurologic dysfunction, brain abnormalities and cardiomyopathy, whose pathogenesis is still unknown. The present study investigated the in vitro effects of 3MCG on important parameters of energy metabolism, including CO(2) production from labeled acetate, enzyme activities of the citric acid cycle, as well as of the respiratory chain complexes I-IV (oxidative phosphorylation), creatine kinase (intracellular ATP transfer), and synaptic Na(+),K(+)-ATPase (neurotransmission) in brain cortex of young rats. 3MCG significantly reduced CO(2) production, implying that this compound compromises citric acid cycle activity. Furthermore, 3MCG diminished the activities of complex II-III of the respiratory chain, mitochondrial creatine kinase and synaptic membrane Na(+),K(+)-ATPase. Furthermore, antioxidants were able to attenuate or fully prevent the inhibitory effect of 3MCG on creatine kinase and synaptic membrane Na(+),K(+)-ATPase activities. We also observed that lipid peroxidation was elicited by 3MCG, suggesting the involvement of free radicals on 3MCG-induced effects. Considering the importance of the citric acid cycle and the electron flow through the respiratory chain for brain energy production, creatine kinase for intracellular energy transfer, and Na(+),K(+)-ATPase for the maintenance of the cell membrane potential, the present data indicate that 3MCG potentially impairs mitochondrial brain energy homeostasis and neurotransmission. It is presumed that these pathomechanisms may be involved in the neurological damage found in patients affected by 3-methylcrotonyl-CoA carboxylase deficiency.
Assuntos
Encéfalo/enzimologia , Metabolismo Energético/efeitos dos fármacos , Glicina/análogos & derivados , Homeostase/efeitos dos fármacos , Mitocôndrias/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Sinapses/enzimologia , Animais , Antioxidantes/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Dióxido de Carbono/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Creatina Quinase/metabolismo , Citosol/efeitos dos fármacos , Citosol/enzimologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Glicina/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/metabolismo , Sinapses/efeitos dos fármacos , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
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
Ornithine, ammonia and homocitrulline are the major metabolites accumulating in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome, a genetic disorder characterized by neurological regression whose pathogenesis is still not understood. The present work investigated the in vivo effects of intracerebroventricular administration of ornithine and homocitrulline in the presence or absence of hyperammonemia induced by intraperitoneal urease treatment on a large spectrum of oxidative stress parameters in cerebral cortex from young rats in order to better understand the role of these metabolites on brain damage. Ornithine increased thiobarbituric acid-reactive substances (TBA-RS) levels and carbonyl formation and decreased total antioxidant status (TAS) levels. We also observed that the combination of hyperammonemia with ornithine resulted in significant decreases of sulfhydryl levels, reduced glutathione (GSH) concentrations and the activities of catalase (CAT) and glutathione peroxidase (GPx), highlighting a synergistic effect of ornithine and ammonia. Furthermore, homocitrulline caused increases of TBA-RS values and carbonyl formation, as well as decreases of GSH concentrations and GPx activity. Hcit with hyperammonemia (urease treatment) decreased TAS and CAT activity. We also showed that urease treatment per se was able to enhance TBA-RS levels. Finally, nitric oxide production was not altered by Orn and Hcit alone or in combination with hyperammonemia. Our data indicate that the major metabolites accumulating in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome provoke lipid and protein oxidative damage and a reduction of the antioxidant defenses in the brain. Therefore, it is presumed that oxidative stress may represent a relevant pathomechanism involved in the brain damage found in patients affected by this disease.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Amônia/metabolismo , Encéfalo/metabolismo , Citrulina/análogos & derivados , Homeostase/fisiologia , Ornitina/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/sangue , Erros Inatos do Metabolismo dos Aminoácidos/induzido quimicamente , Amônia/sangue , Animais , Antioxidantes/metabolismo , Catalase/metabolismo , Citrulina/metabolismo , Citrulina/urina , Glutationa Peroxidase/metabolismo , Injeções Intraperitoneais , Injeções Intraventriculares , Masculino , Óxido Nítrico/metabolismo , Ornitina/sangue , Ornitina/toxicidade , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Carbonilação Proteica/efeitos dos fármacos , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , UreaseRESUMO
Hyperornithinemia is the biochemical hallmark of hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, an inherited metabolic disease clinically characterized by mental retardation whose pathogenesis is still poorly known. In the present work, we produced a chemical animal model of hyperornithinemia induced by a subcutaneous injection of saline-buffered Orn (2-5 µmol/g body weight) to rats. High brain Orn concentrations were achieved, indicating that Orn is permeable to the blood brain barrier. We then investigated the effect of early chronic postnatal administration of Orn on physical development and on the performance of adult rats in the open field, the Morris water maze and in the step down inhibitory avoidance tasks. Chronic Orn treatment had no effect on the appearance of coat, eye opening or upper incisor eruption, nor on the free-fall righting reflex and on the adult rat performance in the Morris water maze and in the inhibitory avoidance tasks, suggesting that physical development, aversive and spatial localization were not changed by Orn. However, Orn-treated rats did not habituate to the open field apparatus, implying a deficit of learning/memory. Motor activity was the same for Orn- and saline- injected animals. We also verified that Orn subcutaneous injections provoked lipid peroxidation in the brain, as determined by a significant increase of thiobarbituric acid-reactive substances levels. Our results indicate that chronic early postnatal hyperornithinemia may impair the central nervous system, causing minor disabilities which result in specific learning deficiencies.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/induzido quimicamente , Deficiências da Aprendizagem/induzido quimicamente , Deficiências da Aprendizagem/psicologia , Ornitina/toxicidade , Erros Inatos do Metabolismo dos Aminoácidos/psicologia , Amônia/sangue , Animais , Animais Recém-Nascidos , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Citrulina/análogos & derivados , Citrulina/sangue , Cognição/efeitos dos fármacos , Cognição/fisiologia , Deficiências do Desenvolvimento/induzido quimicamente , Modelos Animais de Doenças , Meia-Vida , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Memória de Longo Prazo/efeitos dos fármacos , Ornitina/farmacocinética , Equilíbrio Postural/efeitos dos fármacos , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Pristanic acid (Prist) accumulates in some peroxisomal disorders characterized by neurologic dysfunction and brain abnormalities. The present work investigated the in vitro effects of Prist on important parameters of energy metabolism in brain cortex of young rats. CO(2) production from labeled acetate and the activities of the respiratory chain complexes I-IV, creatine kinase and synaptic Na(+), K(+)-ATPase were measured. Prist decreased CO(2) production and the activities of complexes I, II and II-III. Prist also reduced Na(+), K(+)-ATPase activity, but did not affect the activity of creatine kinase. Considering the importance of the citric acid cycle and the electron flow through the respiratory chain for brain energy production and of Na(+), K(+)-ATPase for the maintenance of membrane potential, the present data indicate that Prist compromises brain bioenergetics and neurotransmission. It is presumed that these pathomechanisms may be involved in the neurological damage found in patients affected by disorders in which Prist accumulates.
Assuntos
Encéfalo/efeitos dos fármacos , Ácidos Graxos/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Sinapses/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Metabolismo Energético , Ratos , Ratos WistarRESUMO
High concentrations of ethylmalonic acid are found in tissues and biological fluids of patients affected by ethylmalonic encephalopathy, deficiency of short-chain acyl-CoA dehydrogenase activity and other illnesses characterized by developmental delay and neuromuscular symptoms. The pathophysiological mechanisms responsible for the brain damage in these patients are virtually unknown. Therefore, in the present work we investigated the in vitro effect of EMA on oxidative stress parameters in rat cerebral cortex. EMA significantly increased chemiluminescence and thiobarbituric acid-reactive species levels (lipoperoxidation), as well as carbonyl content and oxidation of sulfhydryl groups (protein oxidative damage) and DCFH. EMA also significantly decreased the levels of reduced glutathione (non-enzymatic antioxidant defenses). In contrast, nitrate and nitrite levels were not affected by this short organic acid. It is therefore presumed that oxidative stress may represent a pathomechanism involved in the pathophysiology of the neurologic symptoms manifested by patients affected by disorders in which EMA accumulates.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Malonatos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Butiril-CoA Desidrogenase/deficiência , Córtex Cerebral/metabolismo , Cromanos/farmacologia , Fluoresceínas/metabolismo , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Oxirredução , Carbonilação Proteica/efeitos dos fármacos , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Accumulation of lysine (Lys) in tissues and biochemical fluids is the biochemical hallmark of patients affected by familial hyperlysinemia (FH) and also by other inherited neurometabolic disorders. In the present study, we investigated the in vitro effect of Lys on various parameters of energy metabolism in cerebral cortex of 30-day-old Wistar rats. We verified that total (tCK) and cytosolic creatine kinase activities were significantly inhibited by Lys, in contrast to the mitochondrial isoform which was not affected by this amino acid. Furthermore, the inhibitory effect of Lys on tCK activity was totally prevented by reduced glutathione, suggesting a possible role of reactive species oxidizing critical thiol groups of the enzyme. In contrast, Lys did not affect (14)CO(2) production from [U-(14)C] glucose (aerobic glycolytic pathway) and [1-(14)C] acetic acid (citric acid cycle activity) neither the various activities of the electron transfer chain and synaptic Na(+)K(+)-ATPase at concentrations as high as 5.0 mM. Considering the importance of creatine kinase (CK) activity for brain energy metabolism homeostasis and especially ATP transfer and buffering, our results suggest that inhibition of this enzyme by Lys may contribute to the neurological signs presented by symptomatic patients affected by FH and other neurodegenerative disorders in which Lys accumulates.
Assuntos
Córtex Cerebral/enzimologia , Creatina Quinase/metabolismo , Metabolismo Energético/fisiologia , Hiperlisinemias/enzimologia , Lisina/metabolismo , Análise de Variância , Animais , Modelos Animais de Doenças , Transporte de Elétrons/fisiologia , Glutationa/fisiologia , Isoenzimas , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
3-Hydroxyisobutyric aciduria is an inherited metabolic disease caused by 3-hydroxyisobutyryl-CoA dehydrogenase deficiency. Tissue accumulation and high urinary excretion of 3-hydroxyisobutyric acid is the biochemical hallmark of this disorder. Clinical phenotype is heterogeneous and generally includes dysmorphic features, delayed motor development, profound mental impairment, and acute encephalopathy. Lactic acidemia is also found in the affected patients, indicating that mitochondrial dysfunction may be involved in the pathophysiology of this disorder. Therefore, the aim of the present work was to investigate the in vitro effect of 3-hydroxyisobutyric acid (0.1, 0.5 and 1mM) on essential enzymes of energy metabolism, namely the activities of the respiratory chain complexes I-V, total, cytosolic and mitochondrial creatine kinase and Na(+), K(+)-ATPase in cerebral cortex homogenates of 30-day-old rats. We also measured the rate of oxygen consumption in brain mitochondrial preparations in the presence of 3-hydroxyisobutyric acid. 3-Hydroxyisobutyric acid significantly reduced complex I-III (20%), without affecting the other activities of the electron transport chain. Furthermore, 3-hydroxyisobutyric acid did not change state III, state IV and the respiratory control ratio in the presence of glutamate/malate or succinate, suggesting that its effect on cellular respiration was weak. On the other hand, the activities of total and mitochondrial creatine kinase, but not cytosolic creatine kinase, were inhibited (30%) by 3-hydroxyisobutyric acid. We also observed that 3-hydroxyisobutyric acid-induced inhibition of mitochondrial creatine kinase activity was fully prevented by pre-incubation of the homogenates with reduced glutathione, alpha-tocopherol or the combination of superoxide dismutase plus catalase, suggesting that this inhibition was mediated by oxidation of essential thiol groups of the enzyme probably by superoxide, hydrogen peroxide and/or peroxyl radicals. It was also demonstrated that Na(+), K(+)-ATPase activity from synaptic plasma membranes was markedly suppressed (37%) by 3-hydroxyisobutyric acid and that this effect was prevented by alpha-tocopherol co-incubation implying that peroxyl radicals were probably involved in this action. Considering the importance of the affected enzyme activities for brain metabolism homeostasis and neurotransmision, it is suggested that increased tissue levels of 3-hydroxyisobutyric acid may contribute to the neurodegeneration of patients affected by 3-hydroxyisobutyric aciduria and possibly explain previous reports describing elevated production and excretion of lactate.
Assuntos
Ácido 3-Hidroxibutírico/metabolismo , Encefalopatias Metabólicas Congênitas/enzimologia , Córtex Cerebral/enzimologia , Metabolismo Energético/fisiologia , Ácido 3-Hidroxibutírico/farmacologia , Envelhecimento/metabolismo , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Química Encefálica/efeitos dos fármacos , Encefalopatias Metabólicas Congênitas/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Córtex Cerebral/efeitos dos fármacos , Creatina Quinase/efeitos dos fármacos , Creatina Quinase/metabolismo , Complexo I de Transporte de Elétrons/efeitos dos fármacos , Complexo I de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Frações Subcelulares , Membranas Sinápticas/efeitos dos fármacos , Membranas Sinápticas/enzimologiaRESUMO
The present work investigated the in vitro effects of lysine on important parameters of oxidative stress in cerebral cortex of young rats. Our results show that lysine significantly induced lipid peroxidation, as determined by increase of thiobarbituric acid-reactive substances and chemiluminescence levels, as well as protein oxidative damage since carbonyl formation and sulfhydryl oxidation were enhanced by this amino acid. Furthermore, the addition of free radical scavengers significantly prevented lysine-induced lipid oxidative damage, suggesting that free radicals were involved in this effect. Lysine also significantly diminished glutathione levels in cortical supernatants, decreasing, therefore, the major brain antioxidant defense. Finally, lysine markedly oxidized a glutathione commercial solution in a medium devoid of brain supernatants, indicating that it behaved as a direct acting oxidant. The present data indicate that lysine induces oxidative stress in cerebral cortex of young rats. Therefore, it is presumed that this pathomechanism may be involved at least in part in the neurological damage found in patients affected by disorders with hyperlysinemia.
Assuntos
Encefalopatias Metabólicas/metabolismo , Córtex Cerebral/metabolismo , Glutationa/metabolismo , Peroxidação de Lipídeos/fisiologia , Lisina/metabolismo , Estresse Oxidativo/fisiologia , Animais , Animais Recém-Nascidos , Antioxidantes/metabolismo , Encefalopatias Metabólicas/fisiopatologia , Córtex Cerebral/fisiopatologia , Regulação para Baixo/fisiologia , Sequestradores de Radicais Livres/metabolismo , Sequestradores de Radicais Livres/farmacologia , Compostos Carbonílicos de Ferro/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lisina/toxicidade , Degeneração Neural/induzido quimicamente , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Oxidantes/metabolismo , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismoRESUMO
AIMS: Cerebellar ataxia is commonly observed in hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, an inherited metabolic disorder biochemically characterized by ornithine (Orn), homocitrulline (Hcit) and ammonia accumulation. Since the pathophysiology of cerebellum damage in this disorder is still unknown, we investigated the effects of Hcit and Orn on important parameters of redox and energy homeostasis in cerebellum of young rats. MATERIAL AND METHODS: We determined thiobarbituric acid-reactive substance (TBA-RS) levels, carbonyl content, nitrate and nitrite production, hydrogen peroxide production, GSH concentrations, sulfhydryl content, as well as activities of respiratory chain complexes I-IV, creatine kinase, Na(+),K(+)-ATPase, aconitase and α-ketoglutarate dehydrogenase. KEY FINDINGS: Orn and Hcit significantly increased TBA-RS levels (lipid oxidation), that was totally prevented by melatonin and reduced glutathione (GSH). We also found that nitrate and nitrite production was not altered by any of the metabolites, in contrast to hydrogen peroxide production which was significantly enhanced by Hcit. Furthermore, GSH concentrations were significantly reduced by Orn and Hcit and sulfhydryl content by Orn, implying an impairment of antioxidant defenses. As regards energy metabolism, Orn and Hcit provoked a significant reduction of aconitase activity, without altering the other parameters. Furthermore, Orn-elicited reduction of aconitase activity was totally prevented by GSH, indicating that the critical groups of this enzyme were susceptible to oxidation caused by this amino acid. SIGNIFICANCE: Taken together, our data indicate that redox homeostasis is disturbed by the major metabolites accumulating in HHH syndrome and that this mechanism may be implicated in the ataxia and cerebellar abnormalities observed in this disorder.
Assuntos
Cerebelo/metabolismo , Citrulina/análogos & derivados , Homeostase/efeitos dos fármacos , Hiperamonemia/metabolismo , Ornitina/farmacologia , Distúrbios Congênitos do Ciclo da Ureia/metabolismo , Aconitato Hidratase/metabolismo , Animais , Cerebelo/patologia , Citrulina/farmacologia , Creatina Quinase/metabolismo , Transporte de Elétrons , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Hiperamonemia/patologia , Complexo Cetoglutarato Desidrogenase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Ornitina/deficiência , Ornitina/metabolismo , Oxirredução/efeitos dos fármacos , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Distúrbios Congênitos do Ciclo da Ureia/patologiaRESUMO
High tissue levels of glycine (GLY) are the biochemical hallmark of nonketotic hyperglycinemia (NKH), an inherited metabolic disease clinically characterized by severe neurological symptoms and brain abnormalities. Considering that the mechanisms underlying the neuropathology of this disease are not fully established, the present work investigated the in vivo effects of intracerebroventricular administration of GLY on important parameters of energy metabolism in cerebral cortex and striatum from young rats. Our results show that GLY reduced CO2 production using glucose as substrate and inhibited the activities of citrate synthase and isocitrate dehydrogenase in striatum, whereas no alterations of these parameters were verified in cerebral cortex 30 min after GLY injection. We also observed that GLY diminished the activities of complex IV in cerebral cortex and complex I-III in striatum at 30 min and inhibited complex I-III activity in striatum at 24 h after its injection. Furthermore, GLY reduced the activity of total and mitochondrial creatine kinase in both brain structures 30 min and 24 h after its administration. In contrast, the activity of Naâº, Kâº-ATPase was not altered by GLY. Finally, the antioxidants N-acetylcysteine and creatine, and the NMDA receptor antagonist MK-801 attenuated or fully prevented the inhibitory effects of GLY on creatine kinase and respiratory complexes in cerebral cortex and striatum. Our data indicate that crucial pathways for energy production and intracellular energy transfer are severely compromised by GLY. It is proposed that bioenergetic impairment induced by GLY in vivo may contribute to the neurological dysfunction found in patients affected by NKH.
Assuntos
Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Metabolismo Energético/efeitos dos fármacos , Glicina/farmacologia , Mitocôndrias/metabolismo , Animais , Dióxido de Carbono/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/enzimologia , Glicina/administração & dosagem , Homeostase/efeitos dos fármacos , Infusões Intraventriculares , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Ratos , Ratos WistarRESUMO
Short/branched chain acyl-CoA dehydrogenase (SBCAD) deficiency is an autosomal recessive disorder of isoleucine metabolism biochemically characterized by accumulation of 2-methylbutyrylglycine (2MBG) and 2-methylbutyric acid (2MB). Affected patients present predominantly neurological symptoms, whose pathophysiology is not yet established. In the present study, we investigated the in vitro effects of 2MBG and 2MB on important parameters of oxidative stress in cerebral cortex of young rats and C6 glioma cells. 2MBG increased thiobarbituric acid-reactive species (TBA-RS), indicating an increase of lipid oxidation. 2MBG induced sulfhydryl oxidation in cortical supernatants and decreased glutathione (GSH) in these brain preparations, as well as in C6 cells, indicating a reduction of nonenzymatic brain antioxidant defenses. In contrast, 2MB did not alter any of these parameters and 2MBG and 2MB did not affect carbonyl formation (protein damage). In addition, 2MBG-induced increase of TBA-RS levels and decrease of GSH were prevented by free radical scavengers, implying that reactive species were involved in these effects. Furthermore, the decrease of GSH levels caused by 2MBG was not due to a direct oxidative action since this metabolite did not alter sulfhydryl content from a commercial solution of GSH. Nitric oxide production was not altered by 2MBG and 2MB, suggesting that reactive oxygen species possibly underlie 2MBG effects. Finally, we verified that 2MBG did not induce cell death in C6 cells. The present data show that 2MBG induces lipid oxidative damage and reduces the antioxidant defenses in rat brain. Therefore, it may be postulated that oxidative stress induced by 2MBG is involved, at least in part, in the pathophysiology of the brain damage found in SBCAD deficiency.
Assuntos
Antioxidantes/metabolismo , Córtex Cerebral/metabolismo , Glicina/análogos & derivados , Estresse Oxidativo/fisiologia , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Animais , Butiratos/metabolismo , Glicina/metabolismo , Peroxidação de Lipídeos/fisiologia , Masculino , Ratos , Ratos Wistar , Substâncias Reativas com Ácido TiobarbitúricoRESUMO
Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive disorder caused by a defect in the mitochondrial ornithine transporter, leading to accumulation of ornithine (Orn), homocitrulline (Hcit) and ammonia. Progressive neurological regression whose pathogenesis is not well established is common in this disease. The present work investigated the in vivo effects of intracerebroventricular administration of Orn and Hcit on important parameters of oxidative stress and energy metabolism in cerebral cortex from young rats. Orn and Hcit significantly increased thiobarbituric acid-reactive substances values and carbonyl formation, indicators of lipid and protein oxidative damage, respectively. Furthermore, N-acetylcysteine and the combination of the free radical scavengers ascorbic acid plus α-tocopherol attenuated the lipid oxidation and totally prevented the protein oxidative damage provoked by Orn and Hcit, suggesting that reactive species were involved in these effects. Hcit, but not Orn administration, also decreased glutathione concentrations, as well as the activity of catalase and glutathione peroxidase, indicating that Hcit provokes a reduction of brain antioxidant defenses. As regards to the parameters of energy metabolism, we verified that Orn and Hcit significantly inhibited the citric acid cycle function (inhibition of CO(2) synthesis from [1-(14)C] acetate), the aerobic glycolytic pathway (reduced CO(2) production from [U-(14)C] glucose) and complex I-III activity of the respiratory chain. Hcit also inhibited the activity of aconitase, an enzyme very susceptible to free radical attack. Taken together, our data indicate that mitochondrial homeostasis is disturbed by Orn and especially by Hcit. It is presumed that the impairment of brain bioenergetics and the oxidative damage induced by these metabolites may possibly contribute to the brain deterioration and neurological symptoms affecting patients with HHH syndrome.
Assuntos
Córtex Cerebral/metabolismo , Citrulina/análogos & derivados , Ornitina/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiopatologia , Citrulina/administração & dosagem , Citrulina/metabolismo , Citrulina/toxicidade , Hiperamonemia/metabolismo , Hiperamonemia/fisiopatologia , Injeções Intraventriculares , Peroxidação de Lipídeos/efeitos dos fármacos , Ornitina/administração & dosagem , Ornitina/deficiência , Ornitina/metabolismo , Ratos , Ratos Wistar , Distúrbios Congênitos do Ciclo da Ureia/metabolismo , Distúrbios Congênitos do Ciclo da Ureia/fisiopatologiaRESUMO
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.