Guilhian Leipnitz

Inhibition of cytochrome c oxidase activity in rat cerebral cortex and human skeletal muscle by D-2-hydroxyglutaric acid in vitro

L-2-Hydroxyglutaric (LGA) and D-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as L-2-hydroxyglutaric aciduria and D-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO(2) production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO(2) production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 1.9.3.1) in a dose-dependent manner (35-95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by D-2-hydroxyglutaric aciduria.

Induction of oxidative stress by L-2-hydroxyglutaric acid in rat brain.

L‐2‐hydroxyglutaric acid (LGA) is the biochemical hallmark of L‐2‐hydroxyglutaric aciduria (L‐OHGA), an inherited neurometabolic disorder characterized by progressive neurodegeneration with cerebellar and pyramidal signs, mental deterioration, epilepsy, and subcortical leukoencephalopathy. Because the underlying mechanisms of the neuropathology of this disorder are virtually unknown, in this study we tested the in vitro effect of LGA on various parameters of oxidative stress, namely, chemiluminescence, thiobarbituric acid‐reactive substances (TBA‐RS), protein carbonyl formation (PCF), total radical‐trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), and the activities of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase in cerebellum and cerebral cortex of 30‐day‐old rats. LGA significantly increased chemiluminescence, TBA‐RS, and PCF measurements and markedly decreased TAR values in cerebellum, in contrast to TRAP and the activity of the antioxidant enzymes, which were not altered by the acid. Similar but less pronounced effects were provoked by LGA in cerebral cortex. Moreover, the LGA‐induced increase of TBA‐RS was significantly attenuated by melatonin (N‐acetyl‐5‐methoxytryptamine) and by the combinations of ascorbic acid plus Trolox (soluble α‐tocopherol) and of superoxide dismutase plus catalase but not by the inhibitor of nitric oxide synthase Nω‐nitro‐L‐arginine methyl ester (L‐NAME), creatine, or superoxide dismutase or catalase alone in either cerebral structure. The data indicate that LGA provokes oxidation of lipids and proteins and reduces the brain capacity to modulate efficiently the damage associated with an enhanced production of free radicals, possibly by inducing generation of superoxide and hydroxyl radicals, which are trapped by the scavengers used. Thus, in case these findings can be extrapolated to human L‐OHGA, it may be presumed that oxidative stress is involved in the pathophysiology of the brain damage observed in this disorder.

α-Ketoisocaproic acid and leucine provoke mitochondrial bioenergetic dysfunction in rat brain

Patients affected by maple syrup urine disease (MSUD) present severe neurological symptoms and brain abnormalities, whose pathophysiology is poorly known. In the present study we investigated the in vitro effects of leucine (Leu), alpha-ketoisocaproic acid (KIC) and alpha-hydroxyisovaleric acid (HIV), respectively, the branched-chain amino, keto and hydroxy acids that most accumulate in MSUD, on brain bioenergetic homeostasis, evaluating respiratory parameters obtained by oxygen consumption, membrane potential (Psim), NAD(P)H content, swelling and citric acid cycle enzyme activities in mitochondrial preparations from rat forebrain using glutamate plus malate, succinate or alpha-ketoglutarate as respiratory substrates. KIC increased state 4 and decreased the respiratory control ratio with all substrates, in contrast with Leu and HIV. Furthermore, KIC and Leu, but not HIV, decreased state 3 using alpha-ketoglutarate. A KIC-induced selective inhibition of alpha-ketoglutarate dehydrogenase activity was also verified, with no alteration of the other citric acid cycle activities. The ADP/O ratio and the mitochondrial NAD(P)H levels were also reduced by KIC using glutamate/malate and alpha-ketoglutarate. In addition, KIC caused a reduction in the Psim when alpha-ketoglutarate was the substrate. Finally, KIC was not able to induce mitochondrial swelling. The present data indicate that KIC acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor possibly through its inhibitory effect on alpha-ketoglutarate dehydrogenase activity, while Leu acts as a metabolic inhibitor. It is suggested that impairment of mitochondrial homeostasis caused by the major metabolites accumulating in MSUD may be involved in the neuropathology of this disease.

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.

Promotion of oxidative stress by 3-hydroxyglutaric acid in rat striatum

SummaryThe 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 (H2O2), 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, H2O2 (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.

Inhibition of Creatine Kinase Activity in Vitro by Ethylmalonic Acid in Cerebral Cortex of Young Rats

Short-chain acyl-CoA dehydrogenase deficiency is an inherited metabolic disorder biochemically characterized by tissue accumulation of ethylmalonic (EMA) and methylsuccinic (MSA) acids and clinically by severe neurological symptoms. In the present study we investigated the in vitro effects of EMA and MSA on the activity of creatine kinase (CK) in homogenates from cerebral cortex, skeletal and cardiac muscle of rats. EMA significantly inhibited CK activity from cerebral cortex, but did not affect this activity in skeletal and cardiac muscle. Furthermore, MSA had no effect on this enzyme in all tested tissues. Glutathione (GSH), ascorbic acid and α-tocopherol, and the nitric oxide synthase inhibitor L-NAME, did not affect the enzyme activity per se, but GSH fully prevented the inhibitory effect of EMA when co-incubated with EMA. In contrast, α-tocopherol, ascorbic acid and L-NAME did not influence the inhibitory effect of the acid. The data suggest that inhibition of brain CK activity by EMA is possibly mediated by oxidation of essential groups of the enzyme, which are protected by the potent intracellular, endogenous, naturally occurring antioxidant GSH.

Oxidative stress-mediated inhibition of brain creatine kinase activity by methylmercury

Methylmercury (MeHg), a potent neurotoxicant, easily passes through the blood-brain barrier and accumulates in brain causing severe irreversible damage. However, the underlying neurotoxic mechanisms elicited by MeHg are still not completed defined. In this study, we aimed to investigate the in vitro toxic effects elicited by crescent concentrations (0-1500 microM) of MeHg on creatine kinase (CK) activity, thiol content (NPSH) and protein carbonyl content (PCC) in mouse brain preparations. In addition, CK activity, MTT reduction and DCFH-DA oxidation (reactive oxygen species (ROS) formation) were also measured in C6 glioma cell linage. CK activity was severely reduced by MeHg treatment in mouse brain preparations. This inhibitory effect was positively correlated to the MeHg-induced reduction of NPSH levels and increment in PCC. Moreover, the positive correlation between brain CK activity and NPSH levels was observed at either 15 or 60 min of MeHg pre-incubation. In addition, MeHg-treated C6 cells showed also a significant inhibition of CK activity at MeHg concentrations, as low as, 50 microM in parallel to reduced mitochondrial function and increased ROS production. Taking together, these data demonstrate that MeHg severely affects CK activity, an essential enzyme for brain energy buffering to maintain cellular energy homeostasis. This effect appears to be mediated by oxidation of thiol groups that might cause subsequent oxidative stress.

Inhibition of creatine kinase activity from rat cerebral cortex by D-2-hydroxyglutaric acid in vitro.

D-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as D-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11-34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.

Quinolinic acid reduces the antioxidant defenses in cerebral cortex of young rats

Quinolinic acid (QA), the major metabolite of the kynurenine pathway, is found at increased concentrations in brain of patients affected by various common neurodegenerative diseases, including Huntingtons disease and Alzheimers disease. Recently, a role for QA in the pathophysiology of glutaric acidemia type I (GAI) was postulated. Considering that oxidative stress has been recently involved in the pathophysiology of the brain injury in these neurodegenerative disorders; in the present study, we investigated the in vitro effect of QA on various parameters of oxidative stress, namely total radical‐trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), glutathione (GSH) levels, thiobarbituric acid‐reactive substances (TBA‐RS) measurement and chemiluminescence in cerebral cortex of 30‐day‐old rats. QA diminished the brain non‐enzymatic antioxidant defenses, as determined by the reduced levels of TRAP, TAR and GSH. We also observed that QA significantly increased TBA‐RS and chemiluminescence. Therefore, in vitro QA‐treatment of rat cortical supernatants induced oxidative stress by reducing the tissue antioxidant defenses and increasing lipid oxidative damage, probably as a result of free radical generation. In addition, we examined the effect of QA on TBA‐RS levels in the presence of glutaric acid (GA) and 3‐hydroxyglutaric acid (3HGA), which are accumulated in GAI, as well as in the presence of 3‐hydroxykynurenine (3HK), a tryptophan metabolite of the kynurenine pathway with antioxidant properties. It was verified that the single addition of QA or GA plus 3HGA to the incubation medium significantly stimulated in vitro lipid peroxidation. Furthermore, 3HK completely prevented the TBA‐RS increase caused by the simultaneous addition of QA, GA and 3HGA. Taken together, it may be presumed that QA induces oxidative stress in the brain, which may be associated, at least in part, with the pathophysiology of central nervous system abnormalities of neurodegenerative diseases in which QA accumulates.

Ethylmalonic Acid Inhibits Mitochondrial Creatine Kinase Activity from Cerebral Cortex of Young Rats in Vitro

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an inherited metabolic disorder biochemically characterized by tissue accumulation of predominantly ethylmalonic acid (EMA) and clinically by neurological dysfunction. In the present study we investigated the in vitro effects of EMA on the activity of the mitochondrial (Mi-CK) and cytosolic (Cy-CK) creatine kinase isoforms from cerebral cortex, skeletal muscle, and cardiac muscle of young rats. CK activities were measured in the mitochondrial and cytosolic fractions prepared from whole-tissue homogenates of 30-day-old Wistar rats. The acid was added to the incubation medium at concentrations ranging from 0.5 to 2.5 mM. EMA had no effect on Cy-CK activity, but significantly inhibited the activity of Mi-CK at 1.0 mM and higher concentrations in the brain. In contrast, both Mi-CK and Cy-CK from skeletal muscle and cardiac muscle were not affected by the metabolite. We also evaluated the effect of the antioxidants glutathione (GSH), ascorbic acid, and a-tocopherol and the nitric oxide synthase inhibitor L-NAME on the inhibitory action of EMA on cerebral cortex Mi-CK activity. We observed that the drugs did not modify Mi-CK activity per se, but GSH and ascorbic acid prevented the inhibitory effect of EMA when co-incubated with the acid. In contrast, L-NAME and α-tocopherol could not revert the inhibition provoked by EMA on Mi-CK activity. Considering the importance of CK for brain energy homeostasis, it is proposed that the inhibition of Mi-CK activity may be associated to the neurological symptoms characteristic of SCAD deficiency.