Alexandre Umpierrez Amaral

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.

Mitochondrial dysfunction in fatty acid oxidation disorders: insights from human and animal studies.

Patients affected by FAOD commonly present with hepatopathy, cardiomyopathy, skeletal myopathy and encephalopathy. Human and animal evidences indicate that mitochondrial functions are disrupted by fatty acids and derivatives accumulating in these disorders, suggesting that lipotoxicity may contribute to their pathogenesis.

Neurochemical evidence that phytanic acid induces oxidative damage and reduces the antioxidant defenses in cerebellum and cerebral cortex of rats

AIMS In the present work we investigated the in vitro effects of phytanic acid (Phyt), that accumulates in Refsum disease and other peroxisomal diseases, on important parameters of oxidative stress in cerebellum and cerebral cortex from young rats. MAIN METHODS The parameters thiobarbituric acid-reactive substances levels (TBA-RS; lipid peroxidation), carbonyl formation and sulfhydryl oxidation (protein oxidative damage) and the concentrations of the most important nonenzymatic antioxidant defense reduced glutathione (GSH) were determined. KEY FINDINGS It was observed that Phyt significantly increased TBA-RS levels in both cerebral structures. This effect was prevented by the antioxidants alpha-tocopherol and melatonin, suggesting the involvement of free radicals. Phyt also provoked protein oxidative damage in both cerebellum and cerebral cortex, as determined by increased carbonyl content and sulfhydryl oxidation. Furthermore, Phyt significantly diminished the concentrations of GSH, while melatonin and alpha-tocopherol treatment totally blocked this effect. We also verified that Phyt does not behave as a direct acting oxidant, since Phyt did not oxidize commercial solutions of GSH and reduced cytochrome c to Phyt in a free cell medium. SIGNIFICANCE Our data indicate that oxidative stress is elicited in vitro by Phyt, a mechanism that may contribute at least in part to the pathophysiology of Refsum disease and other peroxisomal disorders where Phyt is accumulated.

Evidence that 3-hydroxy-3-methylglutaric acid promotes lipid and protein oxidative damage and reduces the nonenzymatic antioxidant defenses in rat cerebral cortex.

In the present work we investigated the in vitro effect of 3‐hydroxy‐3‐methylglutarate (HMG) that accumulates in 3‐hydroxy‐3‐methylglutaryl‐CoA lyase deficiency (HMGLD) on important parameters of oxidative stress in rat cerebral cortex. It was observed that HMG induced lipid peroxidation by significantly increasing chemiluminescence and levels of thiobarbituric acid‐reactive substances (TBA‐RS). This effect was prevented by the antioxidants α‐tocopherol, melatonin, N‐acetylcysteine, and superoxide dismutase plus catalase, suggesting that free radicals were involved in the lipid oxidative damage. On the other hand, HMG did not change TBA‐RS levels in intact or disrupted mitochondrial preparations, indicating that generation of oxidants by this organic acid was dependent on cytosolic mechanisms. HMG also induced protein oxidative damage in cortical supernatants, which was reflected by increased carbonyl content and sulfhydryl oxidation. Furthermore, HMG significantly reduced the nonenzymatic antioxidant defenses total‐radical trapping antioxidant potential, total antioxidant reactivity, and reduced glutathione (GSH) levels in rat cerebral cortex. HMG‐induced GSH reduction was totally blocked by melatonin pretreatment. We also verified that the decrease of GSH levels provoked by HMG in cortical supernatants was not due to a direct oxidative effect of this organic acid, because exposition of commercial GSH and purified membrane protein‐bound thiol groups to HMG in the absence of cortical supernatants did not decrease the reduced sulfhydryl groups. Finally, the activities of the main antioxidant enzymes were not altered by HMG exposure. Our data indicate that oxidative stress elicited in vitro by HMG may possibly contribute at least in part to the pathophysiology of the brain injury in HMGLD.

Induction of oxidative stress by the metabolites accumulating in 3-methylglutaconic aciduria in cerebral cortex of young rats

3-methylglutaconic (MGT), 3-methylglutaric (MGA) and occasionally 3-hydroxyisovaleric (OHIVA) acids accumulate in a group of diseases known as 3-methylglutaconic aciduria (MGTA). Although the clinical presentation of MGTA is mainly characterized by neurological symptoms, the mechanisms of brain damage in this disease are poorly known. In the present study we investigated the in vitro effect of MGT, MGA and OHIVA on various parameters of oxidative stress in cerebral cortex from young rats. Thiobarbituric acid-reactive substances (TBA-RS) and chemiluminescence were significantly increased by MGT, MGA and OHIVA, indicating that these metabolites induce lipid oxidative damage. Furthermore, the addition of melatonin, alpha-tocopherol and superoxide dismutase plus catalase fully prevented MGT-induced increase on TBA-RS, suggesting that free radicals were involved in this effect. These metabolites also provoked protein oxidative damage determined by increased carbonyl formation and sulfhydryl oxidation, but did not induce superoxide generation in submitochondrial particles. It was also verified that MGA and MGT significantly decreased the non-enzymatic antioxidant defenses in cerebral cortex supernatants and that melatonin and alpha-tocopherol totally blocked MGA-induced GSH reduction. The data indicate that the metabolites accumulating in MGTA elicit oxidative stress in vitro in the cerebral cortex. It is therefore presumed that this pathomechanism may be involved in the brain damage observed in patients affected by MGTA.

Long-chain 3-hydroxy fatty acids accumulating in long-chain 3-hydroxyacyl-CoA dehydrogenase and mitochondrial trifunctional protein deficiencies uncouple oxidative phosphorylation in heart mitochondria

Cardiomyopathy is a common clinical feature of some inherited disorders of mitochondrial fatty acid β-oxidation including mitochondrial trifunctional protein (MTP) and isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies. Since individuals affected by these disorders present tissue accumulation of various fatty acids, including long-chain 3-hydroxy fatty acids, in the present study we investigated the effect of 3-hydroxydecanoic (3 HDCA), 3-hydroxydodecanoic (3 HDDA), 3-hydroxytetradecanoic (3 HTA) and 3-hydroxypalmitic (3 HPA) acids on mitochondrial oxidative metabolism, estimated by oximetry, NAD(P)H content, hydrogen peroxide production, membrane potential (ΔΨ) and swelling in rat heart mitochondrial preparations. We observed that 3 HTA and 3 HPA increased resting respiration and diminished the respiratory control and ADP/O ratios using glutamate/malate or succinate as substrates. Furthermore, 3 HDDA, 3 HTA and 3 HPA decreased ΔΨ, the matrix NAD(P)H pool and hydrogen peroxide production. These data indicate that these fatty acids behave as uncouplers of oxidative phosphorylation. We also verified that 3 HTA-induced uncoupling-effect was not mediated by the adenine nucleotide translocator and that this fatty acid induced the mitochondrial permeability transition pore opening in calcium-loaded organelles since cyclosporin A prevented the reduction of mitochondrial ΔΨ and swelling provoked by 3 HTA. The present data indicate that major 3-hydroxylated fatty acids accumulating in MTP and LCHAD deficiencies behave as strong uncouplers of oxidative phosphorylation potentially impairing heart energy homeostasis.

Lysine induces lipid and protein damage and decreases reduced glutathione concentrations in brain of young rats.

The present work investigated the in vitro effects of lysine on important parameters of oxidative stress in cerebral cortex of young rats. Our results show that lysine significantly induced lipid peroxidation, as determined by increase of thiobarbituric acid‐reactive substances and chemiluminescence levels, as well as protein oxidative damage since carbonyl formation and sulfhydryl oxidation were enhanced by this amino acid. Furthermore, the addition of free radical scavengers significantly prevented lysine‐induced lipid oxidative damage, suggesting that free radicals were involved in this effect. Lysine also significantly diminished glutathione levels in cortical supernatants, decreasing, therefore, the major brain antioxidant defense. Finally, lysine markedly oxidized a glutathione commercial solution in a medium devoid of brain supernatants, indicating that it behaved as a direct acting oxidant. The present data indicate that lysine induces oxidative stress in cerebral cortex of young rats. Therefore, it is presumed that this pathomechanism may be involved at least in part in the neurological damage found in patients affected by disorders with hyperlysinemia.

Disturbance of mitochondrial energy homeostasis caused by the metabolites accumulating in LCHAD and MTP deficiencies in rat brain

AIMS We investigated the in vitro effects of 3-hydroxydodecanoic (3HDA), 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, which accumulate in tissues of patients affected by mitochondrial trifunctional protein (MTP) and isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies, on various parameters of energy homeostasis in mitochondrial preparations from brain of young rats. MAIN METHODS We measured the respiratory parameters state 4, state 3, respiratory control ratio (RCR) and ADP/O ratio by the rate of oxygen consumption, as well as the mitochondrial membrane potential and the matrix NAD(P)H levels in the presence of the fatty acids. KEY FINDINGS We found that 3HDA, 3HTA and 3HPA markedly increased state 4 respiration and diminished the RCR using glutamate plus malate or succinate as substrates. 3HTA and 3HPA also diminished the mitochondrial membrane potential and the matrix NAD(P)H levels. In addition, 3HTA decreased state 3 respiration using glutamate/malate, but not pyruvate/malate or succinate as substrates. Our data indicate that the long-chain 3-hydroxy fatty acids that accumulate in LCHAD/MTP deficiencies act as uncouplers of oxidative phosphorylation, while 3HTA also behaves as a metabolic inhibitor. SIGNIFICANCE It is presumed that impairment of brain energy homeostasis caused by these endogenous accumulating compounds may contribute at least in part to the neuropathology of LCHAD/MTP deficiencies.

Induction of oxidative stress in brain of glutaryl-CoA dehydrogenase deficient mice by acute lysine administration

In the present work we evaluated a variety of indicators of oxidative stress in distinct brain regions (striatum, cerebral cortex and hippocampus), the liver, and heart of 30-day-old glutaryl-CoA dehydrogenase deficient (Gcdh(-/-)) mice. The parameters evaluated included thiobarbituric acid-reactive substances (TBA-RS), 2-7-dihydrodichlorofluorescein (DCFH) oxidation, sulfhydryl content, and reduced glutathione (GSH) concentrations. We also measured the activities of the antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD) and glucose-6-phosphate dehydrogenase (G6PD). Under basal conditions glutaric (GA) and 3-OH-glutaric (3OHGA) acids were elevated in all tissues of the Gcdh(-/-) mice, but were essentially absent in WT animals. In contrast there were no differences between WT and Gcdh(-/-) mice in any of the indicators or oxidative stress under basal conditions. Following a single intra-peritoneal (IP) injection of lysine (Lys) there was a moderate increase of brain GA concentration in Gcdh(-/-) mice, but no change in WT. Lys injection had no effect on brain 3OHGA in either WT or Gcdh(-/-) mice. The levels of GA and 3OHGA were approximately 40% higher in striatum compared to cerebral cortex in Lys-treated mice. In the striatum, Lys administration provoked a marked increase of lipid peroxidation, DCFH oxidation, SOD and GR activities, as well as significant reductions of GSH levels and GPx activity, with no alteration of sulfhydryl content, CAT and G6PD activities. There was also evidence of increased lipid peroxidation and SOD activity in the cerebral cortex, along with a decrease of GSH levels, but to a lesser extent than in the striatum. In the hippocampus only mild increases of SOD activity and DCFH oxidation were observed. In contrast, Lys injection had no effect on any of the parameters of oxidative stress in the liver or heart of Gcdh(-/-) or WT animals. These results indicate that in Gcdh(-/-) mice cerebral tissue, particularly the striatum, is at greater risk for oxidative stress than peripheral tissues following Lys administration.

Marked reduction of Na(+), K(+)-ATPase and creatine kinase activities induced by acute lysine administration in glutaryl-CoA dehydrogenase deficient mice.

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by a severe deficiency of the mitochondrial glutaryl-CoA dehydrogenase activity leading to accumulation of predominantly glutaric (GA) and 3-hydroxyglutaric (3HGA) acids in the brain and other tissues. Affected patients usually present with hypotonia and brain damage and acute encephalopathic episodes whose pathophysiology is not yet fully established. In this study we investigated important parameters of cellular bioenergetics in brain, heart and skeletal muscle from 15-day-old glutaryl-CoA dehydrogenase deficient mice (Gcdh(-/-)) submitted to a single intra-peritoneal injection of saline (Sal) or lysine (Lys - 8 μmol/g) as compared to wild type (WT) mice. We evaluated the activities of the respiratory chain complexes II, II-III and IV, α-ketoglutarate dehydrogenase (α-KGDH), creatine kinase (CK) and synaptic Na(+), K(+)-ATPase. No differences of all evaluated parameters were detected in the Gcdh(-/-) relatively to the WT mice injected at baseline (Sal). Furthermore, mild increases of the activities of some respiratory chain complexes (II-III and IV) were observed in heart and skeletal muscle of Gcdh(-/-) and WT mice after Lys administration. However, the most marked effects provoked by Lys administration were marked decreases of the activities of Na(+), K(+)-ATPase in brain and CK in brain and skeletal muscle of Gcdh(-/-) mice. In contrast, brain α-KGDH activity was not altered in WT and Gcdh(-/-) injected with Sal or Lys. Our results demonstrate that reduction of Na(+), K(+)-ATPase and CK activities may play an important role in the pathogenesis of the neurodegenerative changes in GA I.