Ângela M. Sgaravatti

Inhibition of brain energy metabolism by the branched-chain amino acids accumulating in maple syrup urine disease.

In the present work we investigated the in vitro effect of the branched-chain amino acids (BCAA) accumulating in maple syrup urine disease (MSUD) on some parameters of energy metabolism in cerebral cortex of rats. 14CO2 production from [1-14C]acetate, [1-5-14C]citrate and [U-14C]glucose, as well as glucose uptake by the brain were evaluated by incubating cortical prisms from 30-day-old rats in the absence (controls) or presence of leucine (Leu), valine (Val) or isoleucine (Ile). All amino acids significantly reduced 14CO2 production by around 20–55%, in contrast to glucose utilization, which was significantly increased by up to 90%. Furthermore, Leu significantly inhibited the activity of the respiratory chain complex IV, whereas Val and Ile markedly inhibited complexes II–III, III and IV by up to 40%. We also observed that trolox (α-tocopherol) and creatine totally prevented the inhibitory effects provoked by the BCAA on the respiratory chain complex activities, suggesting that free radicals were involved in these effects. The results indicate that the major metabolites accumulating in MSUD disturb brain aerobic metabolism by compromising the citric acid cycle and the electron flow through 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.

Tyrosine promotes oxidative stress in cerebral cortex of young rats.

Tyrosine accumulates in inborn errors of tyrosine catabolism, especially in tyrosinemia type II, where tyrosine levels are highly elevated in tissues and physiological fluids of affected patients. In tyrosinemia type II, high levels of tyrosine are correlated with eyes, skin and central nervous system disturbances. Considering that the mechanisms of brain damage in these disorders are poorly known, in the present study, we investigated whether oxidative stress is elicited by l‐tyrosine in cerebral cortex homogenates of 14‐day‐old Wistar rats. The in vitro effect of 0.1–4.0 mM l‐tyrosine was studied on the following oxidative stress parameters: total radical‐trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), ascorbic acid content, reduced glutathione (GSH) content, spontaneous chemiluminescence, thiobarbituric acid‐reactive substances (TBA‐RS), thiol‐disulfide redox state (SH/SS ratio), protein carbonyl content, formation of DNA‐protein cross‐links, and the activities of the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glucose‐6‐phosphate dehydrogenase (G6PDH). TRAP, TAR, ascorbic acid content, SH/SS ratio and CAT activity were significantly diminished, while formation of DNA‐protein cross‐link was significantly enhanced by l‐tyrosine in vitro. In contrast, l‐tyrosine did not affect the other parameters of oxidative stress evaluated. These results indicate that l‐tyrosine decreases enzymatic and non‐enzymatic antioxidant defenses, changes the redox state and stimulates DNA damage in cerebral cortex of young rats in vitro. This suggests that oxidative stress may represent a pathophysiological mechanism in tyrosinemic patients, in which this amino acid accumulates.

Intracerebroventricular administration of N-acetylaspartic acid impairs antioxidant defenses and promotes protein oxidation in cerebral cortex of rats

N-acetylaspartic acid (NAA) is the biochemical hallmark of Canavan Disease, an inherited metabolic disease caused by deficiency of aspartoacylase activity. NAA is an immediate precursor for the enzyme-mediated biosynthesis of N-acetylaspartylglutamic acid (NAAG), whose concentration is also increased in urine and cerebrospinal fluid of patients affected by CD. This neurodegenerative disorder is clinically characterized by severe mental retardation, hypotonia and macrocephaly, and generalized tonic and clonic type seizures. Considering that the mechanisms of brain damage in this disease remain not fully understood, in the present study we investigated whether intracerebroventricular administration of NAA or NAAG elicits oxidative stress in cerebral cortex of 30-day-old rats. NAA significantly reduced total radical-trapping antioxidant potential, catalase and glucose 6-phosphate dehydrogenase activities, whereas protein carbonyl content and superoxide dismutase activity were significantly enhanced. Lipid peroxidation indices and glutathione peroxidase activity were not affected by NAA. In contrast, NAAG did not alter any of the oxidative stress parameters tested. Our results indicate that intracerebroventricular administration of NAA impairs antioxidant defenses and induces oxidative damage to proteins, which could be involved in the neurotoxicity of NAA accumulation in CD patients.

Acute administration of 5-oxoproline induces oxidative damage to lipids and proteins and impairs antioxidant defenses in cerebral cortex and cerebellum of young rats

Abstract5-Oxoproline accumulates in glutathione synthetase deficiency, an autossomic recessive inherited disorder clinically characterized by hemolytic anemia, metabolic acidosis, and severe neurological symptoms whose mechanisms are poorly known. In the present study we investigated the effects of acute subcutaneous administration of 5-oxoproline to verify whether oxidative stress is elicited by this metabolite in vivo in cerebral cortex and cerebellum of 14-day-old rats. Our results showed that the acute administration of 5-oxoproline is able to promote both lipid and protein oxidation, to impair brain antioxidant defenses, to alter SH/SS ratio and to enhance hydrogen peroxide content, thus promoting oxidative stress in vivo, a mechanism that may be involved in the neuropathology of gluthatione synthetase deficiency.

N-Acetylaspartic acid promotes oxidative stress in cerebral cortex of rats

N‐Acetylaspartic acid accumulates in Canavan Disease, a severe leukodystrophy characterized by swelling and spongy degeneration of the white matter of the brain. This inherited metabolic disease, caused by deficiency of the enzyme aspartoacylase, is clinically characterized by severe mental retardation, hypotonia and macrocephaly, and also generalized tonic and clonic type seizures in about half of the patients. Considering that the mechanisms of brain damage in this disease remain not fully understood, in the present study we investigated whether oxidative stress is elicited by N‐acetylaspartic acid. The in vitro effect of N‐acetylaspartic acid (10–80 mM) was studied on oxidative stress parameters: total radical‐trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), chemiluminescence, thiobarbituric acid‐reactive substances (TBA‐RS), reduced glutathione content, sufhydryl content and carbonyl content in the cerebral cortex of 14‐day‐old rats. The effect of the acute administration of N‐acetylaspartic acid (0.1–0.6 mmol/g body weight) was studied on TRAP, TAR, carbonyl content, chemiluminescence and TBA‐RS. TRAP, TAR, reduced glutathione content and sulfhydryl content were significantly reduced, while chemiluminescence, TBA‐RS and carbonyl content were significantly enhanced by N‐acetylaspartic acid in vitro. The enhancement in TBA‐RS promoted by N‐acetylaspartic acid was completely prevented by ascorbic acid plus Trolox, and partially prevented by glutathione and dithiothreitol. The acute administration of N‐acetylaspartic acid also significantly reduced TRAP and TAR, and significantly enhanced carbonyl content, chemiluminescence and TBA‐RS. Our results indicate that N‐acetylaspartic acid promotes oxidative stress by stimulating lipid peroxidation, protein oxidation and by decreasing non‐enzymatic antioxidant defenses in rat brain. This could be another pathophysiological mechanism involved in Canavan Disease.

N-acetylaspartic acid impairs enzymatic antioxidant defenses and enhances hydrogen peroxide concentration in rat brain.

N-Acetylaspartic acid accumulates in Canavan Disease, a severe inherited neurometabolic disease clinically characterized by severe mental retardation, hypotonia, macrocephaly and generalized tonic and clonic type seizures. Considering that the mechanisms of brain damage in this disease remain poorly understood, in the present study we investigated the in vitro and in vivo effects of N-acetylaspartic acid on the activities of catalase, superoxide dismutase and glutathione peroxidase, as well as on hydrogen peroxide concentration in cerebral cortex of 14-day-old rats. Catalase and glutathione peroxidase activities were significantly inhibited, while hydrogen peroxide concentration was significantly enhanced by N-acetylaspartic acid both in vitro and in vivo. In contrast, superoxide dismutase activity was not altered by N-acetylaspartic acid. Our results clearly show that N-acetylaspartic acid impairs the enzymatic antioxidant defenses in rat brain. This could be involved in the pathophysiological mechanisms responsible for the brain damage observed in patients affected by Canavan Disease.