RESUMEN
BACKGROUND: Inborn errors of metabolism (IEM) are diseases which can lead to accumulation of toxic metabolites in the organism. AIM OF THE STUDY: To investigate, by selective screening, mitochondrial fatty acid oxidation defects (FAOD) and organic acidemias in Brazilian individuals with clinical suspicion of IEM. METHODS: A total of 7,268 individuals, from different regions of Brazil, had whole blood samples impregnated on filter paper which were submitted to the acylcarnitines analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS) at the Medical Genetics Service of Hospital de Clínicas de Porto Alegre, Brazil, during July 2008-July 2016. RESULTS: Our results showed that 68 patients (0.93%) were diagnosed with FAOD (19 cases) and organic acidemias (49 cases). The most prevalent FAOD was multiple acyl CoA dehydrogenase deficiency (MADD), whereas glutaric type I and 3-OH-3-methylglutaric acidemias were the most frequent disorders of organic acid metabolism. Neurologic symptoms and metabolic acidosis were the most common clinical and laboratory features, whereas the average age of the patients at diagnosis was 2.3 years. CONCLUSIONS: Results demonstrated a high incidence of glutaric acidemia type I and 3-OH-3- methylglutaric acidemia in Brazil and an unexpectedly low incidence of FAOD, particularly medium-chain acyl-CoA dehydrogenase deficiency (MCADD).
Asunto(s)
Acil-CoA Deshidrogenasa/deficiencia , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Encefalopatías Metabólicas/diagnóstico , Carnitina/análogos & derivados , Ácidos Grasos/metabolismo , Glutaril-CoA Deshidrogenasa/deficiencia , Errores Innatos del Metabolismo Lipídico/diagnóstico , Acil-CoA Deshidrogenasa/sangre , Errores Innatos del Metabolismo de los Aminoácidos/sangre , Encefalopatías Metabólicas/sangre , Brasil , Carnitina/análisis , Preescolar , Cromatografía Liquida , Femenino , Glutaratos/metabolismo , Glutaril-CoA Deshidrogenasa/sangre , Humanos , Recién Nacido , Errores Innatos del Metabolismo Lipídico/sangre , Masculino , Tamizaje Masivo , Oxidación-Reducción , Prevalencia , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
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.
Asunto(s)
Ácidos Acíclicos/metabolismo , Ácidos Acíclicos/toxicidad , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Benzoxazinas/farmacología , Encefalopatías Metabólicas/metabolismo , Encéfalo/metabolismo , Agonistas de Receptores de Cannabinoides/farmacología , Glutaril-CoA Deshidrogenasa/deficiencia , Morfolinas/farmacología , Naftalenos/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Encéfalo/efectos de los fármacos , Glutaratos/metabolismo , Glutaratos/toxicidad , Glutaril-CoA Deshidrogenasa/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ácido Metilmalónico/metabolismo , Ácido Metilmalónico/toxicidad , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Propionatos/metabolismo , Propionatos/toxicidad , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
Astrocytes are crucial for postnatal development of neuronal networks, axon myelination and neurovascular structures. Defects in astrocyte generation or maturation are associated with severe neurological developmental disorders. Glutaric acidemia type I (GAI), an inherited neurometabolic disorder characterized by accumulation of glutaric (GA) and 3-hydroxyglutaric acids, shows a paradigmatic postnatal neuropathology characterized by massive degeneration of neurons in the striatum. While the disorder is caused by genetic mutations on glutaryl-CoA dehydrogenase, the neurological defects usually start months after birth. Pathogenesis of GAI has remained largely unknown, and specifically, it is unclear how accumulation of GAI metabolites may result in neurodegeneration. Recent evidence supports a GAI model involving primary defective astrocyte maturation leading to a co-morbid spectrum of neurologic symptoms similar to those of patients. Astrocytes are vulnerable to GAI metabolites, but instead of dying, they follow long-lasting phenotypic changes leading to striatal neuron degeneration as well as defective myelination and blood brain barrier maturation. Here, we summarized recent findings on the pathogenic role of GA-damaged astrocytes in GAI and discuss if astrocyte dysfunction may be a target of therapeutic interventions.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/patología , Astrocitos/patología , Encefalopatías Metabólicas/patología , Glutaril-CoA Deshidrogenasa/deficiencia , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/terapia , Animales , Astrocitos/metabolismo , Encefalopatías Metabólicas/metabolismo , Encefalopatías Metabólicas/terapia , Glutaratos/metabolismo , Glutaril-CoA Deshidrogenasa/metabolismo , HumanosRESUMEN
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.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Encefalopatías Metabólicas/metabolismo , Encéfalo/metabolismo , Glutaratos/metabolismo , Glutaril-CoA Deshidrogenasa/deficiencia , Ácido Quinolínico/metabolismo , Sinaptosomas/metabolismo , Animales , Encéfalo/efectos de los fármacos , Modelos Animales de Enfermedad , Glutaratos/toxicidad , Glutaril-CoA Deshidrogenasa/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ácido Metilmalónico/metabolismo , Ácido Metilmalónico/toxicidad , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Propionatos/metabolismo , Propionatos/toxicidad , Ácido Quinolínico/toxicidad , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Sinaptosomas/efectos de los fármacosRESUMEN
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.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Glutaratos/toxicidad , Animales , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Creatina/farmacología , Maleato de Dizocilpina/farmacología , Glutaratos/metabolismo , Glutaratos/farmacología , Glutatión/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Malondialdehído/metabolismo , N-Metilaspartato/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
3-Methylglutaconic aciduria (MGTA) comprehends a group of disorders biochemically characterized by accumulation of 3-methylglutaric acid (MGA), 3-methylglutaconic acid (MGT) and occasionally 3-hydroxyisovaleric acid (OHIVA). Although neurological symptoms are common in the affected individuals, the mechanisms of brain damage are poorly known. In the present study we investigated the in vitro effect MGA, MGT and OHIVA, at concentrations ranging from 0.1 to 5.0mM, on bioenergetics and oxidative stress in synaptosomal preparations isolated from cerebral cortex of young rats. MGA significantly reduced mitochondrial redox potential (25%), as determined by resazurin reduction, and inhibited the activity of Na(+),K(+)-ATPase (30%), whereas MGT and OHIVA did not modify these parameters. Moreover, the inhibitory effect elicited by MGA on Na(+),K(+)-ATPase activity was totally prevented by co-incubation with the scavenging antioxidants creatine and melatonin, implying a role for reactive species in this effect. MGA also increased 2',7'-dichlorofluorescein (DCFH) oxidation (30%), reinforcing that this organic acid induces reactive species production. The present data indicate that MGA compromises mitochondrial function, elicits reactive species production and inhibits the activity of a crucial enzyme implicated in neurotransmission. It is therefore presumed that these deleterious effects may play a role in the pathophysiology of the brain damage observed in patients affected by disorders in which MGA accumulates.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Meglutol/análogos & derivados , Estrés Oxidativo/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/enzimología , Animales , Glutaratos/metabolismo , Glutaratos/farmacología , Humanos , Masculino , Meglutol/metabolismo , Meglutol/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción , Ratas , Ratas Wistar , Valeratos/metabolismo , Valeratos/farmacologíaRESUMEN
Glutaryl-CoA dehydrogenase deficiency or glutaric acidemia type I (GA I) is an inherited neurometabolic disorder biochemically characterized by tissue accumulation of predominantly glutaric (GA) and 3-hydroxyglutaric (3OHGA) acids and clinically by severe neurological symptoms and structural brain abnormalities, manifested as progressive cerebral atrophy and acute striatum degeneration following encephalopathic crises, whose pathophysiology is still in debate. Considering that reactive astrogliosis is a common finding in brain of GA I patients, in the present study we investigated the effects of GA and 3OHGA on glial activity determined by S100B release by rat C6-glioma cells. We also evaluated the effects of these organic acids on some parameters of oxidative stress in these astroglial cells. We observed that GA and 3OHGA significantly increased S100B secretion and thiobarbituric acid-reactive substances (lipid peroxidation), whereas GA markedly decreased reduced glutathione levels in these glioma cells. This is the first report demonstrating that the major metabolites accumulating in GA I activate S100B secretion in astroglial cells, indicating activation of these cells. We also showed that GA and 3OHGA induced oxidative stress in C6 lineage cells, confirming previous findings observed in brain fresh tissue. It is therefore presumed that reactive glial cells and oxidative damage may underlie at least in part the neuropathology of GA I.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Astrocitos/metabolismo , Encefalopatías Metabólicas/metabolismo , Glutaratos/metabolismo , Glutaril-CoA Deshidrogenasa/deficiencia , Factores de Crecimiento Nervioso/metabolismo , Proteínas S100/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/patología , Animales , Astrocitos/patología , Atrofia , Encefalopatías Metabólicas/enzimología , Encefalopatías Metabólicas/patología , Línea Celular Tumoral , Cuerpo Estriado/enzimología , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Gliosis/enzimología , Gliosis/metabolismo , Gliosis/patología , Glutaratos/farmacología , Glutatión/antagonistas & inhibidores , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Subunidad beta de la Proteína de Unión al Calcio S100 , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
The present work investigated the in vitro effects of 3-hydroxy-3-methylglutarate, 3-methylglutarate, 3-methylglutaconate and 3-hydroxyisovalerate, which accumulate in 3-hydroxy-3-methylglutaric aciduria, on important parameters of oxidative stress in striatum and liver of young rats, tissues that are injured in this disorder. Our results show that all metabolites induced lipid peroxidation (thiobarbituric acid-reactive substances increase) and decreased glutathione levels in striatum, whereas 3-hydroxy-3-methylglutarate, besides inducing the strongest effect, also altered thiobarbituric acid-reactive substances and glutathione levels in the liver. Furthermore, 3-hydroxy-3-methylglutarate, 3-methylglutarate and 3-methylglutaconate oxidized sulfhydryl groups in the striatum, but not in the liver. Our data indicate that 3-hydroxy-3-methylglutarate behaves as a stronger pro-oxidant agent compared to the other metabolites accumulating in 3-hydroxy-3-methylglutaric aciduria and that the striatum present higher vulnerability to oxidative damage relatively to the liver.
Asunto(s)
Cuerpo Estriado , Hígado , Estrés Oxidativo , Oxo-Ácido-Liasas/deficiencia , Animales , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Glutaratos/metabolismo , Glutatión/metabolismo , Glicina/análogos & derivados , Glicina/metabolismo , Humanos , Hígado/metabolismo , Hígado/patología , Masculino , Meglutol/análogos & derivados , Meglutol/metabolismo , Oxo-Ácido-Liasas/genética , Ratas , Ratas Wistar , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Valeratos/metabolismoRESUMEN
Glutaric acidemia type I (GA-I) is an inherited metabolic disease characterized by accumulation of glutaric acid (GA) and striatal degeneration. Although growing evidence suggests that excitotoxicity and oxidative stress play central role in the neuropathogenesis of this disease, mechanism underlying striatal damage in this disorder is not well established. Thus, we decided to investigate the in vitro effects of GA 10nM (a low concentration that can be present initial development this disorder) on l-[(3)H]glutamate uptake and reactive oxygen species (ROS) generation in synaptosomes from striatum of rats. GA reduced l-[(3)H]glutamate uptake in synaptosomes from 1 up to 30min after its addition. Furthermore, we also provided some evidence that GA competes with the glutamate transporter inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC), suggesting a possible interaction of GA with glutamate transporters on synaptosomes. Moreover, GA produced a significant decrease in the V(MAX) of l-[(3)H]glutamate uptake, but did not affect the K(D) value. Although the GA did not show oxidant activity per se, it increased the ROS generation in striatal synaptosomes. To evaluate the involvement of reactive species generation in the GA-induced l-[(3)H]glutamate uptake inhibition, trolox (0.3, 0.6 and 6muM) was added on the incubation medium. Statistical analysis showed that trolox did not decrease inhibition of GA-induced l-[(3)H]glutamate uptake, but decreased GA-induced reactive species formation in striatal synaptosomes (1, 3, 5, 10, 15 and 30min), suggesting that ROS generation appears to occur secondarily to glutamatergic overstimulation in this model of organic acidemia. Since GA induced DCFH oxidation increase, we evaluate the involvement of glutamate receptor antagonists in oxidative stress, showing that CNQX, but not MK-801, decreased the DCFH oxidation increase in striatal synaptosomes. Furthermore, the results presented in this report suggest that excitotoxicity elicited by low concentration of GA, could be in part by maintaining this excitatory neurotransmitter in the synaptic cleft by non-competitive inhibition of glutamate uptake. Thus the present data may explain, at least partly, initial striatal damage at birth, as evidenced by acute bilateral destruction of caudate and putamen observed in children with GA-I.
Asunto(s)
Cuerpo Estriado/metabolismo , Cuerpo Estriado/fisiopatología , Ácido Glutámico/metabolismo , Glutaratos/metabolismo , Estrés Oxidativo/fisiología , Terminales Presinápticos/metabolismo , Sistema de Transporte de Aminoácidos X-AG/efectos de los fármacos , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Antioxidantes/farmacología , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Cromanos/farmacología , Cuerpo Estriado/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Glutaratos/toxicidad , Masculino , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/metabolismo , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/fisiopatología , Estrés Oxidativo/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , SinaptosomasRESUMEN
Glutaric (GA) and 3-hydroxyglutaric (OHGA) acids accumulate in glutaric acidemia I (GAI), a neurometabolic disease characterized by acute striatal degeneration and chronic progressive cortical atrophy. To explore the hypothesis that astrocytes are involved in GAI pathogenesis and targets of accumulating metabolites, we determined the effects of GA and OHGA on cultured rat cortical astrocytes. Remarkably, both acids induced mitochondria depolarization and stimulated proliferation in confluent cultures without apparent cell toxicity. Newborn rats injected with GA systemically also showed increased cell proliferation in different brain regions. Most of the proliferating cells displayed markers of immature astrocytes. Antioxidant iron porphyrins prevented both mitochondria dysfunction and increased in vitro and in vivo proliferation, suggesting a role of oxidative stress in inducing astrocytosis. Taken together, the data suggest that mitochondrial dysfunction induced by GA metabolites causes astrocytes to adopt a proliferative phenotype, which may underlie neuronal loss, white matter abnormalities and macrocephalia characteristics of GAI.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Astrocitos/fisiología , Encefalopatías Metabólicas Innatas/metabolismo , Glutaratos/metabolismo , Mitocondrias/fisiología , Animales , Animales Recién Nacidos , Antracenos/farmacología , Antioxidantes/farmacología , Astrocitos/citología , Astrocitos/ultraestructura , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiología , Butadienos/farmacología , Recuento de Células , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Glutaratos/farmacología , Inmunohistoquímica , Potencial de la Membrana Mitocondrial , Nitrilos/farmacología , Porfirinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The role of excitotoxicity in the cerebral damage of glutaryl-CoA dehydrogenase deficiency (GDD) is under intense debate. We therefore investigated the in vitro effect of glutaric (GA) and 3-hydroxyglutaric (3-OHGA) acids, which accumulate in GDD, on [(3)H]glutamate uptake by slices and synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Glutamate uptake was significantly decreased by high concentrations of GA in cortical slices of 7-day-old rats, but not in cerebral cortex from 15- and 30-day-old rats and in striatum from all studied ages. Furthermore, this effect was not due to cellular death and was prevented by N-acetylcysteine preadministration, suggesting the involvement of oxidative damage. In contrast, glutamate uptake by brain slices was not affected by 3-OHGA exposure. Immunoblot analysis revealed that GLAST transporters were more abundant in the cerebral cortex compared to the striatum of 7-day-old rats. Moreover, the simultaneous addition of GA and dihydrokainate (DHK), a specific inhibitor of GLT1, resulted in a significantly higher inhibition of [(3)H]glutamate uptake by cortical slices of 7-day-old rats than that induced by the sole presence of DHK. We also observed that both GA and 3-OHGA exposure did not alter the incorporation of glutamate into synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Finally, GA in vivo administration did not alter glutamate uptake into cortical slices from 7-day-old rats. Our findings may explain at least in part why cortical neurons are more vulnerable to damage at birth as evidenced by the frontotemporal cortical atrophy observed in newborns affected by GDD.
Asunto(s)
Animales Recién Nacidos/metabolismo , Corteza Cerebral/metabolismo , Glutamatos/farmacocinética , Glutaratos/administración & dosificación , Glutaratos/metabolismo , Acetilcisteína/administración & dosificación , Acetilcisteína/metabolismo , Animales , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Glutamatos/metabolismo , Glutaril-CoA Deshidrogenasa/deficiencia , Técnicas In Vitro , Ácido Kaínico/análogos & derivados , Ácido Kaínico/metabolismo , Neostriado/metabolismo , Ratas , Ratas Wistar , Sinaptosomas/metabolismoRESUMEN
Glutaric acidemia type I is an inherited metabolic disorder caused by a severe deficiency of the mitochondrial glutaryl-CoA dehydrogenase activity leading to accumulation of predominantly glutaric and 3-hydroxyglutaric acids in the brain tissue of the affected patients. Considering that a toxic role was recently postulated for quinolinic acid in the neuropathology of glutaric acidemia type I, in the present work we investigated whether the combination of quinolinic acid with glutaric or 3-hydroxyglutaric acids or the mixture of glutaric plus 3-hydroxyglutaric acids could alter brain energy metabolism. The parameters evaluated in cerebral cortex from young rats were glucose utilization, lactate formation and (14)CO(2) production from labeled glucose and acetate, as well as the activities of pyruvate dehydrogenase and creatine kinase. We first observed that glutaric (5 mM), 3-hydroxyglutaric (1 mM) and quinolinic acids (0.1 microM) per se did not alter these parameters. Similarly, no change of these parameters occurred when combining glutaric with quinolinic acids or 3-hydroxyglutaric with quinolinic acids. In contrast, co-incubation of glutaric plus 3-hydroxyglutaric acids increased glucose utilization, decreased (14)CO(2) generation from glucose, inhibited pyruvate dehydrogenase activity as well as total and mitochondrial creatine kinase activities. The glutaric plus 3-hydroxyglutaric acids-induced inhibitory effects on creatine kinase were prevented by the antioxidants glutathione and catalase plus superoxide dismutase, indicating the participation of reactive oxygen species. Our data indicate a synergic action of glutaric and 3-hydroxyglutaric acids disturbing energy metabolism in cerebral cortex of young rats.
Asunto(s)
Química Encefálica/fisiología , Encefalopatías Metabólicas/metabolismo , Encéfalo/metabolismo , Metabolismo Energético/fisiología , Glutaratos/metabolismo , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Química Encefálica/efectos de los fármacos , Encefalopatías Metabólicas/fisiopatología , Creatina Quinasa/metabolismo , Sinergismo Farmacológico , Metabolismo Energético/efectos de los fármacos , Glucosa/metabolismo , Glutaratos/toxicidad , Ácido Láctico/metabolismo , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/metabolismo , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/fisiopatología , Técnicas de Cultivo de Órganos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Complejo Piruvato Deshidrogenasa/metabolismo , Ácido Quinolínico/metabolismo , Ácido Quinolínico/toxicidad , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Tissue accumulation of high amounts of D-2-hydroxyglutaric acid (DGA) and l-2-hydroxyglutaric acid (LGA) is the biochemical hallmark of the inherited neurometabolic disorders D-2-hydroxyglutaric aciduria (DHGA) and l-2-hydroxyglutaric aciduria (LHGA), respectively. Patients affected by DHGA predominantly present neurological and cardiomuscular symptoms, while those with LHGA have mainly severe neurological symptoms. Lactic aciduria and/or lactic acidemia may also occur in both disorders, suggesting mitochondrial dysfunction. We have previously reported that cytochrome c oxidase (COX) activity is severely inhibited by DGA in rat cerebral cortex and human skeletal muscle. In the present study, we initially evaluated the role of DGA and LGA on the mitochondrial respiratory chain complex activities, as well as CO2 on production in cardiac and skeletal muscle from 30-day-old Wistar rats. DGA significantly inhibited COX and ATP synthase (F0F1-ATP synthase) activities, in contrast to the other activities of the respiratory chain enzymes which were not affected by DGA in both muscular tissues. In addition, CO2 production was also markedly reduced by DGA in rat skeletal and cardiac muscles. On the other hand, LGA did not interfere with any of the respiratory chain complex activities studied, neither with CO2 generation. We also measured mitochondrial respiratory parameters in rat brain mitochondrial preparations in the presence of DGA and LGA. Both metabolites significantly lowered the respiratory control ratio in the presence of glutamate/malate and succinate. Since the metabolites stimulated oxygen consumption in state IV and compromised ATP formation, it can be presumed that these organic acids might act as endogenous uncouplers of mitochondria respiration. Moreover, COX activity linked to TMPD-ascorbate was significantly reduced by DGA in the brain mitochondrial enriched fractions. Finally, DGA and LGA reduced cell viability of rat cerebral cortex slices, as determined by the MTT assay. In case our in vitro data also occur in vivo, it may be presumed that impairment of energy metabolism may contribute to the understanding of the clinical features mainly of patients affected by DHGA.
Asunto(s)
Glutaratos/metabolismo , Mitocondrias Musculares/metabolismo , Animales , Encéfalo/metabolismo , Dióxido de Carbono/metabolismo , Metabolismo Energético , Masculino , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Ratas , Ratas WistarRESUMEN
The pathophysiology of the striatum degeneration characteristic of patients affected by the inherited neurometabolic disorder glutaryl-CoA dehydrogenase deficiency (GDD), also known as glutaric aciduria type I, is still in debate. We have previously reported that 3-hydroxyglutaric acid (3-OH-GA) considered the main neurotoxin in this disorder, induces oxidative stress in rat cerebral cotex. In the present work, we extended these studies by investigating the in vitro effect of 3-OH-GA, at concentrations ranging from 0.01 to 1.0 mmol/L on the brain antioxidant defences by measuring total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR) and glutathione (GSH) levels, and on the production of hydrogen peroxide (H(2)O(2)), nitric oxide (NO) and malondialdehyde in striatum homogenates from young rats. We observed that TRAP, TAR and GSH levels were markedly reduced (by up to 50%) when striatum homogenates were treated with 3-OH-GA. In contrast, H(2)O(2) (up to 44%), NO (up to 95%) and malondialdehyde levels (up to 28%) were significantly increased by 3-OH-GA. These data indicate that total nonenzymatic antioxidant defences (TRAP) and the tissue capacity to handle an increase of reactive species (TAR) were reduced by 3-OH-GA in the striatum. Furthermore, the results also reflect an increase of lipid peroxidation, probably secondary to 3-OH-GA-induced free radical production. Thus, it may be presumed that oxidative stress is involved in the neuropathology in GDD.
Asunto(s)
Cuerpo Estriado/metabolismo , Glutaratos/metabolismo , Estrés Oxidativo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/deficiencia , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Relación Dosis-Respuesta a Droga , Glutaril-CoA Deshidrogenasa , Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Metabolismo de los Lípidos , Peroxidación de Lípido , Masculino , Malondialdehído/farmacología , Óxido Nítrico/metabolismo , Ratas , Ratas Wistar , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Factores de TiempoRESUMEN
INTRODUCTION: Macrocephaly is a pivotal clinical sign, associated with multiple neurological diseases, particularly neurometabolical ones, such as the glutaric aciduria type I (GA I). This aciduria resulting from the genetical deficiency of the enzyme glutaryl-CoA dehydrogenase (GCDH). Is a relatively common cause of acute metabolic brain damage in early childhood. We report on one case of GA I, with early manifestations since fetal period and a novel mutation. CASE REPORT: Our patient was referred due macrocephaly in utero and occipitofrontal head circumference above the 98 percentile for chronologic age during first few months of life, hypotonia and development delay. The metabolic investigations of organic acids in urine and acylcarnitine profile in blood, the brain magnetic resonance and the molecular analyses of the glutaryl-CoA deshidrogenase gene, confirm the diagnosis. The molecular analysis allowed to identify one previously described mutation A293T and a novel mutation IVS5-2 A>G. CONCLUSION: It is important the recognition of in utero macrocephaly as a sign to early diagnosis of glutaric aciduria type I to initiate specific therapy to prevent the encephalopathic crises and minimize brain damage in patients who are already neurologically impaired.
Asunto(s)
Feto/patología , Glutaratos/metabolismo , Errores Innatos del Metabolismo/complicaciones , Errores Innatos del Metabolismo/genética , Malformaciones del Sistema Nervioso/etiología , Malformaciones del Sistema Nervioso/genética , Análisis Mutacional de ADN , Feto/anatomía & histología , Feto/fisiología , Humanos , Lactante , Recién Nacido , Masculino , Errores Innatos del Metabolismo/diagnóstico , Malformaciones del Sistema Nervioso/metabolismo , Malformaciones del Sistema Nervioso/patologíaRESUMEN
Neurological symptoms are common in patients with glutaric acidemia type I (GA-I). Although the pathophysiology of this disorder is not yet fully established, 3-hydroxyglutaric acid (3-HGA), which accumulates in affected patients, has recently been demonstrated to be excitotoxic to embryonic chick and neonatal rat neurons probably via NMDA glutamate receptors. In the present study, we investigated the in vitro effects of 3-HGA on the [(3)H]glutamate and [(3)H]MK-801 (dizocilpine) binding to rat synaptic plasma membranes from cerebral cortex of young rats in order to elucidate the interactions of 3-HGA with glutamate receptors and its possible contribution to the in vitro excitotoxic properties of 3-HGA. 3-HGA (10-100 microM) significantly decreased Na(+)-dependent (up to 62%) and Na(+)-independent (up to 30%) [(3)H]glutamate binding to synaptic membranes, reflecting a possible competition between glutamate and 3-HGA for the glutamate transporter and receptor sites, respectively. Since a decrease in Na(+)-independent glutamate binding might represent an interaction of 3-HGA with glutamate receptors, we next investigated whether 3-HGA interacts with NMDA receptors by adding NMDA alone or combined with 3-HGA and measuring Na(+)-independent [(3)H]glutamate binding to synaptic membranes (binding to receptors). We verified that 3-HGA and NMDA, at 10 and 100 microM concentrations, decreased glutamate binding by up to 20 and 45%, respectively, and that the simultaneous addition of both substances did not provoke an additive effect, implying that they bind to NMDA receptors at the same site. Furthermore, the binding of the NMDA-channel blocker [(3)H ]MK-801 was significantly increased (approximately 32-40%) by 10 and 100 microM 3-HGA, implying that 3-HGA was able to open the NMDA channel allowing MK-801 binding, which is a characteristic of NMDA agonists. On the other hand, glutamate had a much higher stimulatory effect on this binding (180% increase), reflecting its strong NMDA agonist property. Furthermore, the simultaneous addition of 3-HGA and glutamate provoked an additive stimulatory effect on [(3)H]MK-801 binding to the NMDA receptor. These data indicate that, relatively to glutamate, 3-HGA is a weak agonist of NMDA receptors. Finally, we demonstrated that 3-HGA provoked a significant increase of extracellular calcium uptake by cerebral cortex slices, strengthening therefore, the view that 3-HGA activates NMDA receptors. The present study therefore, demonstrates at the molecular level that 3-HGA modulates glutamatergic neurotransmission and may explain previous findings relating the neurotoxic actions of this organic acid with excitotoxicity.
Asunto(s)
Membrana Celular/metabolismo , Corteza Cerebral/metabolismo , Glutaratos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Animales , Animales Recién Nacidos , Membrana Celular/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/ultraestructura , Relación Dosis-Respuesta a Droga , Glutaratos/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Ratas Wistar , Sinapsis/efectos de los fármacos , Sinapsis/ultraestructuraRESUMEN
Neurological dysfunction is common in patients with D-2-hydroxyglutaric aciduria (DHGA). However, the mechanisms underlying the neuropathology of this disorder are far from understood. In the present study, we investigated the in vitro effects of D-2-hydroxyglutaric acid (DGA) at various concentrations (0.1-1.0 mM) on various parameters of the glutamatergic system, namely the basal and potassium-induced release of L-[3H]glutamate by synaptosomal preparations, Na(+)-dependent L-[3H]glutamate uptake by synaptosomal preparations and Na(+)-independent L-[3H]glutamate uptake by synaptic vesicles, as well as of Na(+)-independent and dependent L-[3H]glutamate binding to synaptic plasma membranes from cerebral cortex of male adult Wistar rats. We observed that DGA significantly increased synaptosomal L-[3H]glutamate uptake, without altering the other parameters. Although these findings do not support a direct excitotoxic action for DGA since the metabolite did not affect important parameters of the main neurotransmission system, they do not exclude a direct action of DGA on NMDA or other glutamate receptors. More comprehensive studies are therefore necessary to evaluate the exact role of DGA on neurotransmission.
Asunto(s)
Encefalopatías Metabólicas Innatas/metabolismo , Corteza Cerebral/metabolismo , Ácido Glutámico/metabolismo , Glutaratos/metabolismo , Terminales Presinápticos/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Encefalopatías Metabólicas Innatas/fisiopatología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiopatología , Modelos Animales de Enfermedad , Glutaratos/farmacología , L-Lactato Deshidrogenasa/metabolismo , Masculino , Neurotoxinas/metabolismo , Neurotoxinas/farmacología , Terminales Presinápticos/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Sinaptosomas/química , Sinaptosomas/metabolismoRESUMEN
This study investigated the effects of glutaric acid, which predominantly accumulates in glutaric acidemia type I, on some in vitro parameters of oxidative stress in brain of young rats. We evaluated chemiluminescence, total radical-antioxidant potential (TRAP) and the activities of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase in brain tissue homogenates in the presence of glutaric acid at concentrations ranging from 0.05 to 2.0 mM. The acid significantly increased chemiluminescence (up to 65%) and reduced total radical-antioxidant potential (up to 28%) and glutathione peroxidase activity (up to 46%), without affecting catalase and superoxide dismutase activities. The results provide evidence that glutaric acid induces oxidative stress in vitro in rat brain. If these findings also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by glutaric acidemia type I.
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Envejecimiento/metabolismo , Encefalopatías Metabólicas/metabolismo , Encéfalo/metabolismo , Glutaratos/metabolismo , Neuronas/metabolismo , Estrés Oxidativo/fisiología , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Encefalopatías Metabólicas/fisiopatología , Catalasa/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Radicales Libres/metabolismo , Glutaratos/farmacología , Glutatión Peroxidasa/metabolismo , Mediciones Luminiscentes , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Wistar , Fracciones Subcelulares , Superóxido Dismutasa/metabolismoRESUMEN
A patient with glutaric aciduria type I had an acute encephalopathic crisis despite early treatment. This report indicates that current therapeutic strategies may be insufficient for some high-risk patients and stresses the demand for new approaches in glutaric aciduria type I.
Asunto(s)
Encefalopatías Metabólicas Innatas/terapia , Glutaratos/metabolismo , Errores Innatos del Metabolismo/terapia , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/deficiencia , Enfermedad Aguda , Encefalopatías Metabólicas Innatas/diagnóstico , Encefalopatías Metabólicas Innatas/dietoterapia , Glutaril-CoA Deshidrogenasa , Homocigoto , Humanos , Lactante , Masculino , Errores Innatos del Metabolismo/diagnóstico , Errores Innatos del Metabolismo/dietoterapia , Resultado del TratamientoRESUMEN
Combined 3-methylglutaconic and 3-methylglutaric aciduria, one of the more common urinary organic acid abnormalities, has been observed in at least three clinical syndromes. We studied an additional seven patients with 3-methylglutaconic aciduria, four of whom were best categorized as having the type II syndrome, two as having an "unspecified" syndrome, and one who may have had a primary urea cycle defect. In cultured cells and autopsy tissues derived from patients with the type II and unspecified syndromes, we were unsuccessful in identifying a defect in the leucine degradative pathway distal to 3-methylcrotonyl-coenzyme A carboxylase and in the cholesterol biosynthetic pathway between 3-hydroxy-3-methylglutaryl-coenzyme A reductase and diphosphomevalonate decarboxylase. Further assessment of the cholesterol biosynthetic pathway in several patients with one of the two types of disease also provided no defined abnormality. The primary metabolic defects in the type II and unspecified syndromes remain undefined.