Omega-3 prevents behavior response and brain oxidative damage in the ketamine model of schizophrenia.

Supplementation with omega-3 has been identified as an adjunctive alternative for the treatment of psychiatric disorders, in order to minimize symptoms. Considering the lack of understanding concerning the pathophysiology of schizophrenia, the present study hypothesized that omega 3 prevents the onset of symptoms similar to schizophrenia in young Wistar rats submitted to ketamine treatment. Moreover, the role of oxidative stress in this model was assessed. Omega-3 (0.8g/kg) or vehicle was given by orogastric gavage once daily. Both treatments were performed during 21days, starting at the 30th day of life in young rats. After 14days of treatment with omega-3 or vehicle, a concomitant treatment with saline or ketamine (25mg/kg ip daily) was started and maintained until the last day of the experiment. We evaluated the pre-pulse inhibition of the startle reflex, activity of antioxidant systems and damage to proteins and lipids. Our results demonstrate that supplementation of omega-3 prevented: decreased inhibition of startle reflex, damage to lipids in the hippocampus and striatum and damage to proteins in the prefrontal cortex. Furthermore, these changes are associated with decreased GPx in brain tissues evaluated. Together, our results suggest the prophylactic role of omega-3 against the outcome of symptoms associated with schizophrenia.

Acute renal failure potentiates methylmalonate-induced oxidative stress in brain and kidney of rats.

Tissue methylmalonic acid (MMA) accumulation is the biochemical hallmark of methylmalonic acidemia. The disease is clinically characterized by progressive neurological deterioration and kidney failure, whose pathophysiology is still unclear. In the present work we investigated the effects of acute MMA administration on various parameters of oxidative stress in cerebral cortex and kidney of young rats, as well as the influence of acute renal failure on MMA-elicited effects on these parameters. Acute renal failure was induced by gentamicin, an aminoglycoside antibiotic whose utilization over prolonged periods causes nephrotoxicity. The administration of gentamicin alone increased carbonyl content and inhibited superoxide dismutase (SOD) activity in cerebral cortex, as well as increased thiobarbituric acid-reactive substances (TBA-RS) and sulfhydryl levels and diminished glutathione peroxidase activity in kidney. On the other hand, MMA administration increased TBA-RS levels in cerebral cortex and decreased SOD activity in kidney. Furthermore, the simultaneous administration of MMA and gentamicin to the rats provoked an augment in TBA-RS levels and superoxide generation in cerebral cortex and in TBA-RS, carbonyl and sulfhydryl levels in kidney, while diminished SOD activity in both studied tissues. Finally, nitrate/nitrite content, reduced glutathione levels, 2',7'-dihydrodichlorofluorescein oxidation and catalase activity were not affected by this animal treatment in either tissue. In conclusion, our present data are in line with the hypothesis that MMA acts as a toxin in brain and kidney of rats and suggest that renal injury potentiates the toxicity of MMA on oxidative stress parameters in brain and peripheral tissues.

Methylmalonate inhibits succinate-supported oxygen consumption by interfering with mitochondrial succinate uptake.

The effect of methylmalonate (MMA) on mitochondrial succinate oxidation has received great attention since it could present an important role in energy metabolism impairment in methylmalonic acidaemia. In the present work, we show that while millimolar concentrations of MMA inhibit succinate-supported oxygen consumption by isolated rat brain or muscle mitochondria, there is no effect when either a pool of NADH-linked substrates or N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD)/ascorbate were used as electron donors. Interestingly, the inhibitory effect of MMA, but not of malonate, on succinate-supported brain mitochondrial oxygen consumption was minimized when nonselective permeabilization of mitochondrial membranes was induced by alamethicin. In addition, only a slight inhibitory effect of MMA was observed on succinate-supported oxygen consumption by inside-out submitochondrial particles. In agreement with these observations, brain mitochondrial swelling experiments indicate that MMA is an important inhibitor of succinate transport by the dicarboxylate carrier. Under our experimental conditions, there was no evidence of malonate production in MMA-treated mitochondria. We conclude that MMA inhibits succinate-supported mitochondrial oxygen consumption by interfering with the uptake of this substrate. Although succinate generated outside the mitochondria is probably not a sig-nificant contributor to mitochondrial energy generation, the physiopathological implications of MMA-induced inhibition of substrate transport by the mitochondrial dicarboxylate carrier are discussed.

Na+, K+ ATPase activity is markedly reduced by cis-4-decenoic acid in synaptic plasma membranes from cerebral cortex of rats.

We have previously demonstrated that octanoic (OA) and decanoic acids (DA) inhibit Na+, K+ ATPase activity in synaptic plasma membranes from rat brain. The objective of the present study was to investigate the in vitro effects of the other metabolites that accumulate in tissues of medium-chain acyl-CoA dehydrogenase (MCAD)-deficient patients, namely cis-4-decenoic acid (cDA), octanoylcarnitine (OC), hexanoylcarnitine (HC), hexanoylglycine (HG), phenylpropionylglycine (PPG) and suberoylglycine (SG), on Na+, K+ ATPase activity in synaptic plasma membrane from cerebral cortex of 30-day-old rats. cDA, the pathognomonic compound found in this disorder, provoked the strongest inhibition on this enzyme activity at concentrations as low as 0.25 mM, whereas OC inhibited this activity at 1.0 mM and higher concentrations in a dose-dependent manner. In contrast, HC, HG, PPG and SG did not affect Na+, K+ ATPase activity. Furthermore, pre-treatment of cortical homogenates with the antioxidant enzymes catalase plus superoxide dismutase totally prevented cDA-induced Na+, K+ ATPase inhibition. We also provided evidence that cDA, as well as OA and DA, caused lipid peroxidation, which may explain, at least in part, the inhibitory properties of these compounds towards Na+, K+ ATPase. Considering that Na+, K+ ATPase is a critical enzyme for normal brain development and functioning, it is presumed that these findings, especially those regarding to the marked inhibitory effect of cDA, may be involved in the pathophysiology of the neurological dysfunction of MCAD-deficient patients.

Inhibition of energy metabolism by 2-methylacetoacetate and 2-methyl-3-hydroxybutyrate in cerebral cortex of developing rats.

Mitochondrial beta-ketothiolase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiencies are inherited neurometabolic disorders affecting isoleucine catabolism. Biochemically, beta-ketothiolase deficiency is characterized by intermittent ketoacidosis and urinary excretion of 2-methyl-acetoacetate (MAA), 2-methyl-3-hydroxybutyrate (MHB) and tiglylglycine (TG), whereas in MHBD deficiency only MHB and tiglylglycine accumulate. Lactic acid accumulation and excretion are also observed in these patients, being more pronounced in MHBD-deficient individuals, particularly during acute episodes of decompensation. Patients affected by MHBD deficiency usually manifest severe mental retardation and convulsions, whereas beta-ketothiolase-deficient patients present encephalopathic crises characterized by metabolic acidosis, vomiting and coma. Considering that the pathophysiological mechanisms responsible for the neurological alterations of these disorders are unknown and that lactic acidosis suggests an impairment of energy production, the objective of the present work was to investigate the in vitro effect of MAA and MHB, at concentrations varying from 0.01 to 1.0 mmol/L, on several parameters of energy metabolism in cerebral cortex from young rats. We observed that MAA markedly inhibited CO2 production from glucose, acetate and citrate at concentrations as low as 0.01 mmol/L. In addition, the activities of the respiratory chain complex II and succinate dehydrogenase were mildly inhibited by MAA. MHB, at 0.01 mmol/L and higher concentrations, strongly inhibited CO2 production from all tested substrates, as well as the respiratory chain complex IV activity. The other activities of the respiratory chain were not affected by these metabolites. The data indicate a marked blockage in the Krebs cycle and a mild inhibition of the respiratory chain caused by MAA and MHB. Furthermore, MHB inhibited total and mitochondrial creatine kinase activities, which was prevented by the use of the nitric-oxide synthase inhibitor L-NAME and glutathione (GSH). These data indicate that the effect of MHB on creatine kinase was probably mediated by oxidation or other modification of essential thiol groups of the enzyme by nitric oxide and other by-products derived from this organic acid. In contrast, MAA did not affect creatine kinase activity. Taken together, these observations indicate that aerobic energy metabolism is inhibited by MAA and to a greater extent by MHB, a fact that may be related to lactic acidaemia occurring in patients affected by MHBD and beta-ketothiolase deficiencies. If the in vitro effects detected in the present study also occur in vivo, it is tempting to speculate that they may contribute, at least in part, to the neurological dysfunction found in these disorders.

Inhibition of mitochondrial creatine kinase activity from rat cerebral cortex by methylmalonic acid.

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of patients affected by the neurometabolic disorder known as methylmalonic acidemia (MMAemia). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not totally established. In the present study, we investigated the effect of MMA, as well as propionic (PA) and tiglic (TA) acids, whose concentrations are also increased but to a lesser extend in MMAemia, on total (tCK), cytosolic (Cy-CK) and mitochondrial (Mi-CK) creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas Cy-CK and Mi-CK were determined, respectively, in cytosolic and mitochondrial preparations from rat cerebral cortex. We verified that tCK and Mi-CK activities were significantly inhibited by MMA at concentrations as low as 1 mM, in contrast to Cy-CK which was not affected by the presence of the acid in the incubation medium. Furthermore, PA and TA, at concentrations as high as 5 mM, did not alter CK activity. We also observed that the inhibitions provoked by MMA were fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of MMA was possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of MMA may contribute to the neurodegeneration of patients affected by MMAemia and explain previous reports showing an impairment of brain energy metabolism and a reduction of brain phosphocreatine levels caused by MMA.

D-2-hydroxyglutaric acid inhibits creatine kinase activity from cardiac and skeletal muscle of young rats.

BACKGROUND: Tissue accumulation of high amounts of D-2-hydroxyglutaric acid (DGA) is the biochemical hallmark of the inherited neurometabolic disorder D-2-hydroxyglutaric aciduria (DHGA). Patients affected by this disease usually present hypotonia, muscular weakness, hypertrophy and cardiomyopathy, besides severe neurological findings. However, the underlying mechanisms of muscle injury in this disorder are virtually unknown. MATERIALS AND METHODS: In the present study we have evaluated the in vitro role of DGA, at concentrations ranging from 0.25 to 5.0 mM, on total, cytosolic and mitochondrial creatine kinase activities from skeletal and cardiac muscle of 30-day-old Wistar rats. We also tested the effects of various antioxidants on the effects elicited by DGA. RESULTS: We first verified that total creatine kinase (CK) activity from homogenates was significantly inhibited by DGA (22-24% inhibition) in skeletal and cardiac muscle, and that this activity was approximately threefold higher in skeletal muscle than in cardiac muscle. We also observed that CK activities from mitochondrial (Mi-CK) and cytosolic (Cy-CK) preparations from skeletal muscle and cardiac muscle were also inhibited (12-35% inhibition) by DGA at concentrations as low as 0.25 mm, with the effect being more pronounced in cardiac muscle preparations. Finally, we verified that the DGA-inhibitory effect was fully prevented by preincubation of the homogenates with reduced glutathione and cysteine, suggesting that this effect is possibly mediated by modification of essential thiol groups of the enzyme. Furthermore, alpha-tocopherol, melatonin and the inhibitor of nitric oxide synthase L-NAME were unable to prevent this effect, indicating that the most common reactive oxygen and nitrogen species were not involved in the inhibition of CK provoked by DGA. CONCLUSION: Considering the importance of creatine kinase activity for cellular energy homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA might be an important mechanism involved in the myopathy and cardiomyopathy of patients affected by DHGA.

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid.

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32-46%), complex I (61-72%), and complex II+III (15-26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1mM succinate in the assay instead of the usual 16mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11-27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.