Differential Age-Dependent Mitochondrial Dysfunction, Oxidative Stress, and Apoptosis Induced by Neonatal Hypoxia-Ischemia in the Immature Rat Brain.

Neonatal hypoxia-ischemia (HI) is among the main causes of mortality and morbidity in newborns. Experimental studies show that the immature rat brain is less susceptible to HI injury, suggesting that changes that occur during the first days of life drastically alter its susceptibility. Among the main developmental changes observed is the mitochondrial function, namely, the tricarboxylic acid (TCA) cycle and respiratory complex (RC) activities. Therefore, in the present study, we investigated the influence of neonatal HI on mitochondrial functions, redox homeostasis, and cell damage at different postnatal ages in the hippocampus of neonate rats. For this purpose, animals were divided into four groups: sham postnatal day 3 (ShP3), HIP3, ShP11, and HIP11. We initially observed increased apoptosis in the HIP11 group only, indicating a higher susceptibility of these animals to brain injury. Mitochondrial damage, as determined by flow cytometry showing mitochondrial swelling and loss of mitochondrial membrane potential, was also demonstrated only in the HIP11 group. This was consistent with the decreased mitochondrial oxygen consumption, reduced TCA cycle enzymes, and RC activities and induction of oxidative stress in this group of animals. Considering that HIP3 and the sham animals showed no alteration of mitochondrial functions, redox homeostasis, and showed no apoptosis, our data suggest an age-dependent vulnerability of the hippocampus to hypoxia-ischemia. The present results highlight age-dependent metabolic differences in the brain of neonate rats submitted to HI indicating that different treatments might be needed for HI newborns with different gestational ages.

The effect of WIN 55,212-2 suggests a cannabinoid-sensitive component in the early toxicity induced by organic acids accumulating in glutaric acidemia type I and in related disorders of propionate metabolism in rat brain synaptosomes.

Several physiological processes in the CNS are regulated by the endocannabinoid system (ECS). Cannabinoid receptors (CBr) and CBr agonists have been involved in the modulation of the N-methyl-D-aspartate receptor (NMDAr) activation. Glutaric (GA), 3-hydroxyglutaric (3-OHGA), methylmalonic (MMA) and propionic (PA) acids are endogenous metabolites produced and accumulated in the brain of children affected by severe organic acidemias (OAs) with neurodegeneration. Oxidative stress and excitotoxicity have been involved in the toxic pattern exerted by these organic acids. Studying the early pattern of toxicity exerted by these metabolites is crucial to explain the extent of damage that they can produce in the brain. Herein, we investigated the effects of the synthetic CBr agonist WIN 55,212-2 (WIN) on early markers of GA-, 3-OHGA-, MMA- and PA-induced toxicity in brain synaptosomes from adult (90-day-old) and adolescent (30-day-old) rats. As pre-treatment, WIN exerted protective effects on the GA- and MMA-induced mitochondrial dysfunction, and prevented the reactive oxygen species (ROS) formation and lipid peroxidation induced by all metabolites. Our findings support a protective and modulatory role of cannabinoids in the early toxic events elicited by toxic metabolites involved in OAs.

Toxic synergism between quinolinic acid and organic acids accumulating in glutaric acidemia type I and in disorders of propionate metabolism in rat brain synaptosomes: Relevance for metabolic acidemias.

The brain of children affected by organic acidemias develop acute neurodegeneration linked to accumulation of endogenous toxic metabolites like glutaric (GA), 3-hydroxyglutaric (3-OHGA), methylmalonic (MMA) and propionic (PA) acids. Excitotoxic and oxidative events are involved in the toxic patterns elicited by these organic acids, although their single actions cannot explain the extent of brain damage observed in organic acidemias. The characterization of co-adjuvant factors involved in the magnification of early toxic processes evoked by these metabolites is essential to infer their actions in the human brain. Alterations in the kynurenine pathway (KP) - a metabolic route devoted to degrade tryptophan to form NAD(+) - produce increased levels of the excitotoxic metabolite quinolinic acid (QUIN), which has been involved in neurodegenerative disorders. Herein we investigated the effects of subtoxic concentrations of GA, 3-OHGA, MMA and PA, either alone or in combination with QUIN, on early toxic endpoints in rat brain synaptosomes. To establish specific mechanisms, we pre-incubated synaptosomes with different protective agents, including the endogenous N-methyl-d-aspartate (NMDA) receptor antagonist kynurenic acid (KA), the antioxidant S-allylcysteine (SAC) and the nitric oxide synthase (NOS) inhibitor nitro-l-arginine methyl ester (l-NAME). While the incubation of synaptosomes with toxic metabolites at subtoxic concentrations produced no effects, their co-incubation (QUIN+GA, +3-OHGA, +MMA or +PA) decreased the mitochondrial function and increased reactive oxygen species (ROS) formation and lipid peroxidation. For all cases, this effect was partially prevented by KA and l-NAME, and completely avoided by SAC. These findings suggest that early damaging events elicited by organic acids involved in metabolic acidemias can be magnified by toxic synergism with QUIN, and this process is mostly mediated by oxidative stress, and in a lesser extent by excitotoxicity and nitrosative stress. Therefore, QUIN can be hypothesized to contribute to the pathophysiology of brain degeneration in children with metabolic acidemias.

Reactive nitrogen species mediate oxidative stress and astrogliosis provoked by in vivo administration of phytanic acid in cerebellum of adolescent rats: A potential contributing pathomechanism of cerebellar injury in peroxisomal disorders.

Phytanic acid (Phyt) accumulates in various peroxisomal diseases including Refsum disease (RD) and Zellweger syndrome (ZS). Since the pathogenesis of the neurological symptoms and especially the cerebellar abnormalities in these disorders are poorly known, we investigated the effects of in vivo intracerebral administration of Phyt on a large spectrum of redox homeostasis parameters in the cerebellum of young rats. Malondialdehyde (MDA) levels, sulfhydryl oxidation, carbonyl content, nitrite and nitrate concentrations, 2',7'-dichlorofluorescein (DCFH) oxidation, total (tGS) and reduced glutathione (GSH) levels and the activities of important antioxidant enzymes were determined at different periods after Phyt administration. Immunohistochemical analysis was also carried out in the cerebellum. Phyt significantly increased MDA and nitric oxide (NO) production and decreased GSH levels, without altering tGS, DCFH oxidation, sulfhydryl oxidation, carbonyl content and the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD). Furthermore, immunohistochemical analysis revealed that Phyt caused astrogliosis and protein nitrosative damage in the cerebellum. It was also observed that the NO synthase inhibitor Nω-Nitro-L-arginine methyl ester (L-NAME) prevented the increase of MDA and NO production as well as the decrease of GSH and the immunohistochemical alterations caused by Phyt, strongly suggesting that reactive nitrogen species (RNS) were involved in these effects. The present data provide in vivo solid evidence that Phyt disrupts redox homeostasis and causes astrogliosis in rat cerebellum probably mediated by RNS production. It is therefore presumed that disequilibrium of redox status may contribute at least in part to the cerebellum alterations characteristic of patients affected by RD and other disorders with Phyt accumulation.

Cannabinoid receptor agonists reduce the short-term mitochondrial dysfunction and oxidative stress linked to excitotoxicity in the rat brain.

The endocannabinoid system (ECS) is involved in a considerable number of physiological processes in the Central Nervous System. Recently, a modulatory role of cannabinoid receptors (CBr) and CBr agonists on the reduction of the N-methyl-d-aspartate receptor (NMDAr) activation has been demonstrated. Quinolinic acid (QUIN), an endogenous analog of glutamate and excitotoxic metabolite produced in the kynurenine pathway (KP), selectively activates NMDAr and has been shown to participate in different neurodegenerative disorders. Since the early pattern of toxicity exerted by this metabolite is relevant to explain the extent of damage that it can produce in the brain, in this work we investigated the effects of the synthetic CBr agonist WIN 55,212-2 (WIN) and other agonists (anandamide or AEA, and CP 55,940 or CP) on early markers of QUIN-induced toxicity in rat striatal cultured cells and rat brain synaptosomes. WIN, AEA and CP exerted protective effects on the QUIN-induced loss of cell viability. WIN also preserved the immunofluorescent signals for neurons and CBr labeling that were decreased by QUIN. The QUIN-induced early mitochondrial dysfunction, lipid peroxidation and reactive oxygen species (ROS) formation were also partially or completely prevented by WIN pretreatment, but not when this CBr agonist was added simultaneously with QUIN to brain synaptosomes. These findings support a neuroprotective and modulatory role of cannabinoids in the early toxic events elicited by agents inducing excitotoxic processes.

Disruption of redox homeostasis and histopathological alterations caused by in vivo intrastriatal administration of D-2-hydroxyglutaric acid to young rats.

High accumulation of D-2-hydroxyglutaric acid (D-2-HG) is the biochemical hallmark of patients affected by the inherited neurometabolic disorder D-2-hydroxyglutaric aciduria (D-2-HGA). Clinically, patients present neurological symptoms and basal ganglia injury whose pathophysiology is poorly understood. We investigated the ex vivo effects of intrastriatal administration of D-2-HG on important parameters of redox status in the striatum of weaning rats. D-2-HG in vivo administration increased malondialdehyde (MDA) and carbonyl formation (lipid and protein oxidative damage, respectively), as well as the production of reactive nitrogen species (RNS). D-2-HG also compromised the antioxidant defenses by decreasing reduced glutathione (GSH) concentrations, as well as the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Increased amounts of oxidized glutathione (GSSG) with no significant alteration of total glutathione (tGS) were also found. Furthermore, D-2-HG-induced lipid oxidation and reduction of GSH concentrations and GPx activity were prevented by the N-methyl-d-aspartate (NMDA) receptor antagonist dizocilpine maleate (MK-801) and the nitric oxide synthase (NOS) inhibitor N(ω)-nitro-l-arginine methyl ester (l-NAME), suggesting the participation of NMDA receptors and nitric oxide derivatives in these effects. Creatine also impeded D-2-HG-elicited MDA increase, but did not change the D-2-HG-induced diminution of GSH and of the activities of SOD and GPx. We also found that DCFH oxidation and H2O2 production were not altered by D-2-HG, making unlikely an important role for reactive oxygen species (ROS) and reinforcing the participation of RNS in the oxidative damage and the reduction of antioxidant defenses provoked by this organic acid. Vacuolization, lymphocytic infiltrates and macrophages indicating brain damage were also observed in the striatum of rats injected with D-2-HG. The present data provide in vivo solid evidence that D-2-HG disrupts redox homeostasis and causes histological alterations in the rat striatum probably mediated by NMDA overstimulation and RNS production. It is therefore presumed that disturbance of redox status may contribute at least in part to the basal ganglia alterations characteristic of patients affected by D-2-HGA.

Disruption of redox homeostasis and brain damage caused in vivo by methylmalonic acid and ammonia in cerebral cortex and striatum of developing rats.

Hyperammonemia is a common finding in children with methylmalonic acidemia and propionic acidemia, but its contribution to the development of the neurological symptoms in the affected patients is poorly known. Considering that methylmalonic acid (MMA) and propionic acid (PA) predominantly accumulate in these disorders, we investigated the effects of hyperammonemia induced by urease treatment in 30-day-old rats receiving an intracerebroventricular (ICV) injection of MMA or PA on important parameters of redox homeostasis in cerebral cortex and striatum. We evaluated glutathione (GSH) concentrations, sulfhydryl content, nitrate and nitrite concentrations, 2',7'-dichlorofluorescein (DCFH) oxidation, and the activity of antioxidant enzymes. MMA decreased GSH concentrations and sulfhydryl content and increased nitrate and nitrite concentrations in cerebral cortex and striatum from hyperammonemic rats, whereas MMA or ammonia per se did not alter these parameters. MMA plus hyperammonemia also decreased glutathione reductase activity in rat cerebral cortex, but did not affect catalase, superoxide dismutase and glutathione peroxidase activities, neither DCFH oxidation. Furthermore, ICV PA administration alone or combined with hyperammonemia did not alter any of the evaluated parameters. We also found that pre-treatment with antioxidants prevented GSH reduction and sulfhydryl oxidation, whereas N(ω)-nitro-L-arginine methyl ester (L-NAME) prevented the increased nitrate and nitrite concentrations provoked by MMA plus ammonia treatments. Histological alterations, including vacuolization, ischemic neurons, and pericellular edema, were observed in brain of hyperammonemic rats injected with MMA. The data indicate a synergistic effect of MMA and ammonia disturbing redox homeostasis and causing morphological brain abnormalities in rat brain.

Redox homeostasis is compromised in vivo by the metabolites accumulating in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency in rat cerebral cortex and liver.

3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a disorder biochemically characterized by the predominant accumulation of 3-hydroxy-3-methylglutarate (HMG), 3-methylglutarate (MGA), 3-methylglutaconate and 3-hydroxyisovalerate in tissues and biological fluids of the affected patients. Neurological symptoms and hepatopathy are commonly found in HL deficiency, especially during metabolic crises. Since the mechanisms of tissue damage in this disorder are not well understood, in the present study we evaluated the ex vivo effects of acute administration of HMG and MGA on important parameters of oxidative stress in cerebral cortex and liver from young rats. In vivo administration of HMG and MGA provoked an increase of carbonyl and carboxy-methyl-lysine formation in cerebral cortex, but not in liver, indicating that these metabolites induce protein oxidative damage in the brain. We also verified that HMG and MGA significantly decreased glutathione concentrations in both cerebral cortex and liver, implying a reduction of antioxidant defenses. Furthermore, HMG and MGA increased 2',7'-dichlorofluorescin oxidation, but did not alter nitrate and nitrite content in cerebral cortex and liver, indicating that HMG and MGA effects are mainly mediated by reactive oxygen species. HMG and MGA also increased the activities of superoxide dismutase and catalase in cerebral cortex and liver, whereas MGA decreased glutathione peroxidase activity in cerebral cortex. Our present data showing a disruption of redox homeostasis in cerebral cortex and liver caused by in vivo administration of HMG and MGA suggest that this pathomechanism may possibly contribute to the brain and liver abnormalities observed in HL-deficient patients.

Oxidative stress in phenylketonuric patients.

Phenylketonuria is the most frequent disturbance of amino acid metabolism. Untreated patients present mental retardation whose pathophysiology is not completely established. In this work we discuss the oxidative stress in phenylketonuric patients. Several studies have shown reduction in antioxidant defenses, possibly due to dietary restriction of nutrients with antioxidant properties and increase in oxidative damage to biomolecules, probably secondary to increased formation of reactive species. Therefore, antioxidants could be considered an adjuvant therapy in phenylketonuria.

Evidence that L-carnitine and selenium supplementation reduces oxidative stress in phenylketonuric patients.

It is well established that the involvement of reactive species in the pathophysiology of several neurological diseases, including phenylketonuria (PKU), a metabolic genetic disorder biochemically characterized by elevated levels of phenylalanine (Phe). In previous studies, we verified that PKU patients (treated with a protein-restricted diet supplemented with a special formula not containing L-carnitine and selenium) presented high lipid and protein oxidative damage as well as a reduction of antioxidants when compared to the healthy individuals. Our goal in the present study was to evaluate the effect of Phe-restricted diet supplemented with L-carnitine and selenium, two well-known antioxidant compounds, on oxidative damage in PKU patients. We investigated various oxidative stress parameters in blood of 18 treated PKU patients before and after 6 months of supplementation with a special formula containing L-carnitine and selenium. It was verified that treatment with L-carnitine and selenium was capable of reverting the lipid peroxidation, measured by thiobarbituric acid-reactive species, and the protein oxidative damage, measured by sulfhydryl oxidation, to the levels of controls. Additionally, the reduced activity of glutathione peroxidase was normalized by the antioxidant supplementation. It was also verified a significant inverse correlation between lipid peroxidation and L-carnitine blood levels as well as a significant positive correlation between glutathione peroxidase activity and blood selenium concentration. In conclusion, our results suggest that supplementation of L-carnitine and selenium is important for PKU patients since it could help to correct the oxidative stress process which possibly contributes, at least in part, to the neurological symptoms found in phenylketonuric patients.

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.

Incidence of 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HL) deficiency in Brazil, South America.

3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency (3-hydroxy-3-methylglutaric aciduria, 3-HMG) is a rare autosomal recessive inborn error of metabolism involving the final step of leucine degradation. HL is the key enzyme for the production of glucose-sparing ketone bodies for brain. Positive biochemical findings are metabolic acidosis, hyperammonaemia, and hypoketotic hypoglycaemia in the neonatal period or infancy. In the present study we report 15 Brazilian patients with HL deficiency and present their clinical and biochemical findings. Urine from all patients contained large amounts of 3-hydroxy-3-methylglutaric, 3-methylglutaconic, 3-hydroxyisovaleric and 3-methylglutaric acids, and 3-methylcrotonylglycine was also observed in 13 patients. The main features at clinical presentation were hypoglycaemia (12 patients), seizures (10 patients), metabolic acidosis (9 patients), vomiting (6 patients), and hepatomegaly (5 patients). All but two patients were of Portuguese ancestry. HL deficiency comprised 7.3% of total organic acidurias detected in our laboratory during a 13-year time span, indicating a high incidence of this disorder in Brazil. Limited molecular characterization (4/15 patients only) revealed two mutations common for individuals of Portuguese/Spanish (Iberian Peninsula) ancestry (E37X and V168fs(-2)). Our findings increase the number of HL-deficient patients and reinforce the characteristic phenotypic picture of the disease. Effective dietary interventions based on mild protein restriction and avoidance of fasting and possibly alternative C5 ketone body generating therapy for this disorder may provide further impetus and rationale for expanded newborn screening of HL deficiency.

Evidence for oxidative stress in tissues derived from succinate semialdehyde dehydrogenase-deficient mice.

Animal models of inborn errors of metabolism are useful for investigating the pathogenesis associated with the corresponding human disease. Since the mechanisms involved in the pathophysiology of succinate semialdehyde dehydrogenase (SSADH) deficiency (Aldh5a1; OMIM 271980) are still not established, in the present study we evaluated the tissue antioxidant defences and lipid peroxidation in various cerebral structures (cortex, cerebellum, thalamus and hippocampus) and in the liver of SSADH-deficient mice. The parameters analysed were total radical-trapping antioxidant potential (TRAP) and glutathione (GSH) levels, the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as thiobarbituric acid-reactive substances (TBARS). We first observed that the tissue nonenzymatic antioxidant defences were significantly reduced in the SSADH-deficient animals, particularly in the liver (decreased TRAP and GSH) and in the cerebral cortex (decreased GSH), as compared to the wild-type mice. Furthermore, SOD activity was significantly increased in the liver and cerebellum, whereas the activity of CAT was significantly higher in the thalamus. In contrast, GPx activity was significantly diminished in the hippocampus. Finally, we observed that lipid peroxidation (TBARS levels) was markedly increased in the liver and cerebral cortex, reflecting a high lipid oxidative damage in these tissues. Our data showing an imbalance between tissue antioxidant defences and oxidative attack strongly indicate that oxidative stress is involved in the pathophysiology of SSADH deficiency in mice, and likely the corresponding human disorder.

Cytoskeleton as a potential target in the neuropathology of maple syrup urine disease: insight from animal studies.

In this short review we provide evidence that the branched-chain keto acids accumulating in the neurometabolic disorder maple syrup urine disease disturb rat cerebral cytoskeleton in a developmentally regulated manner. Alterations of protein phosphorylation leading to brain cytoskeletal misregulation and neural cell death caused by these metabolites are associated with energy deprivation, oxidative stress and excitotoxicity that may ultimately disrupt normal cell function and viability.

Investigation of oxidative stress parameters in treated phenylketonuric patients.

Phenylketonuria (PKU) is the most frequent disturbance of amino acid metabolism being caused by severe deficiency of phenylalanine hydroxylase activity. Untreated PKU patients present severe mental retardation whose pathophysiology is not completely estabilished. Despite the low-Phe diet, a considerable number of phenylketonuric patients present a mild to moderate psychomotor delay and decreased cognitive functions. In the present study we evaluated various parameters of oxidative stress namely thiobarbituric acid-reactive species (TBA-RS), total antioxidant reactivity (TAR) and activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in two groups of treated PKU patients, one with well controlled and the other with high Phe blood levels in order to investigate whether blood Phe concentrations could be correlated with the extend of oxidative stress. We initially verified a marked increase of TBA-RS, and a decrease of TAR in plasma, as well as a reduction of erythrocyte GSH-Px activity which were similar in both groups of PKU patients, when compared to controls of similar ages. In contrast, CAT and SOD activities were not altered in PKU patients. These results show that oxidative stress occurs in PKU patients and that this pathogenic process is probably not directly correlated to Phe blood levels.

Isolated 3-methylcrotonyl-coenzyme A carboxylase deficiency in a child with metabolic stroke.

We report a 3-year-old boy with isolated 3-methylcrotonyl-coenzyme A deficiency with unexpectedly severe presentation, seizures and history of cerebral ischae-mic episode.

Creatine prevents behavioral alterations caused by methylmalonic acid administration into the hippocampus of rats in the open field task.

Although a variable degree of psychomotor delay/mental retardation is found in a considerable number of patients affected by methylmalonic acidemia, the mechanisms underlying the neuropathology of this disorder are still poorly defined. The present study investigated the effect of acute intrahippocampal administration of methylmalonic acid (MMA), the biochemical hallmark of this disease, on rat behavior in the open field task. Cannulated 60-day-old male Wistar rats received bilateral intrahippocampal injection of MMA (0.1-1.0 micromol) 10 min before training. Controls received 0.1-1.0 micromol NaCl. Testing session was performed 24 h later. We observed that rats administered with 1.0 micromol MMA, but not with lower doses, did not habituate in the open field task, reflecting a deficit of performance. Motor activity, assessed by the number of crossing responses, was the same at training for the groups infused with MMA or NaCl. The effect of MK-801 (15 nmol) and succinate (1.5 micromol) administered 30 min before MMA injection, and of creatine (50 mg/kg, i.p.) administered twice a day for 3 days on the behavioral alterations provoked by MMA in the open field task revealed that only the energetic substrate creatine prevented these effects, reflecting a possible compromise of brain energy production by MMA. The results indicate that high intrahippocampal concentrations of the major metabolite accumulating in methylmalonic acidemia compromises brain functioning, causing deficit of performance in the open field task that may be related to the psychomotor delay/mental retardation observed in the affected patients.

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.

3-Hydroxyglutaric acid moderately impairs energy metabolism in brain of young rats.

3-Hydroxyglutaric acid (3HGA) accumulates in the inherited neurometabolic disorder known as glutaryl-CoA dehydrogenase deficiency. The disease is clinically characterized by severe neurological symptoms, frontotemporal atrophy and striatum degeneration. Because of the pathophysiology of the brain damage in glutaryl-CoA dehydrogenase deficiency is not completed clear, we investigated the in vitro effect of 3HGA (0.01-5.0mM) on critical enzyme activities of energy metabolism, including the respiratory chain complexes I-V, creatine kinase isoforms and Na(+),K(+)-ATPase in cerebral cortex and striatum from 30-day-old rats. Complex II activity was also studied in rat C6-glioma cells exposed to 3HGA. The effect of 3HGA was further investigated on the rate of oxygen consumption in mitochondria from rat cerebrum. We observed that 1.0mM 3HGA significantly inhibited complex II in cerebral cortex and C6 cells but not the other activities of the respiratory chain complexes. Creatine kinase isoforms and Na(+),K(+)-ATPase were also not affected by the acid. Furthermore, no inhibition of complex II activity occurred when mitochondrial preparations from cerebral cortex or striatum homogenates were used. In addition, 3HGA significantly lowered the respiratory control ratio in the presence of glutamate/malate and succinate under stressful conditions or when mitochondria were permeabilized with digitonin. Since 3HGA stimulated oxygen consumption in state IV and compromised ATP formation, it can be presumed that this organic acid might act as an endogenous uncoupler of mitochondria respiration. Finally, we observed that 3HGA changed C6 cell morphology from a round flat to a spindle-differentiated shape, but did not alter cell viability neither induced apoptosis. The data provide evidence that 3HGA provokes a moderate impairment of brain energy metabolism and do not support the view that 3HGA-induced energy failure would solely explain the characteristic brain degeneration observed in glutaryl-CoA dehydrogenase deficiency patients.