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 ß-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.

Toxicity of octanoate and decanoate in rat peripheral tissues: evidence of bioenergetic dysfunction and oxidative damage induction in liver and skeletal muscle.

The accumulation of octanoic (OA) and decanoic (DA) acids in tissue is the common finding in medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD), the most frequent defect of fatty acid oxidation. Affected patients present hypoketotic hypoglycemia, rhabdomyolysis, hepatomegaly, seizures and lethargy, which may progress to coma and death. At present, the pathophysiological mechanisms underlying hepatic and skeletal muscle alterations in affected patients are poorly known. Therefore, in the present work, we investigated the in vitro effects of OA and DA, the accumulating metabolites in MCADD, on various bioenergetics and oxidative stress parameters. It was verified that OA and DA decreased complexes I-III, II-III and IV activities in liver and also inhibit complex IV activity in skeletal muscle. In addition, DA decreased complexes II-III activity in skeletal muscle. We also verified that OA and DA increased TBA-RS levels and carbonyl content in both tissues. Finally, DA, but not OA, significantly decreased GSH levels in rat skeletal muscle. Our present data show that the medium-chain fatty acids that accumulate in MCADD impair electron transfer through respiratory chain and elicit oxidative damage in rat liver and skeletal muscle. It may be therefore presumed that these mechanisms are involved in the pathophysiology of the hepatopathy and rhabdomyolysis presented by MCADD-affected patients.

Neurotoxic effects of trans-glutaconic acid in rats.

trans-Glutaconic acid (tGA) is an unsaturated C5-dicarboxylic acid which may be found accumulated in glutaric aciduria type I, whose pathophysiology is still uncertain. In the present work it was investigated the in vitro effect of increasing tGA concentrations on neurochemical and oxidative stress parameters in rat cerebral cortex. We observed that Na(+), K(+)-ATPase activity was reduced by tGA, but not creatine kinase, respiratory chain complex IV, and ATP synthase activities. On the other hand, tGA significantly increased lipid peroxidation (thiobarbituric acid-reactive species levels and spontaneous chemiluminescence), as well as protein oxidative damage (oxidation of sulfhydryl groups). tGA also significantly decreased nonenzymatic antioxidant defenses (TRAP and reduced glutathione levels). Our data suggest that tGA may be neurotoxic in rat brain.

Disruption of redox homeostasis in cerebral cortex of developing rats by acylcarnitines accumulating in medium-chain acyl-CoA dehydrogenase deficiency.

Medium-chain fatty acids and acylcarnitines accumulate in medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most frequent fatty acid oxidation defect clinically characterized by episodic crises with vomiting, seizures and coma. Considering that the pathophysiology of the neurological symptoms observed in MCADD is poorly known and, to our knowledge, there is no report on the involvement of acylcarnitines in the brain damage presented by the affected patients, the objective of the present study was to investigate the in vitro effects of hexanoylcarnitine (HC), octanoylcarnitine, decanoylcarnitine (DC) and cis-4-decenoylcarnitine (cDC) at concentrations varying from 0.01 to 1.0mM on important oxidative stress parameters in cerebral cortex of young rats. HC, DC and cDC significantly induced lipid peroxidation, as determined by increased thiobarbituric acid-reactive substances (TBA-RS) values. In addition, carbonyl formation was significantly augmented and sulfhydryl content diminished by DC, reflecting induction of protein oxidative damage. HC, DC and cDC also decreased glutathione (GSH) levels, the most important brain antioxidant defense. Furthermore, DC-induced elevation of TBA-RS values and decrease of GSH levels were prevented by the free radical scavengers melatonin and α-tocopherol, indicating the involvement of reactive oxygen species in these effects. We also found that l-carnitine itself did not induce lipid and protein oxidative damage, neither reduced the antioxidant defenses. Our present data show that the major medium-chain acylcarnitines accumulating in MCADD elicit oxidative stress in rat brain. It is therefore presumed that these compounds may be involved to a certain extent in the pathogenesis of the neurologic dysfunction of MCADD.

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.

Long-chain 3-hydroxy fatty acids accumulating in LCHAD and MTP deficiencies induce oxidative stress in rat brain.

Accumulation of long-chain 3-hydroxy fatty acids is the biochemical hallmark of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies. These disorders are clinically characterized by neurological symptoms, such as convulsions and lethargy, as well as by cardiomyopathy and muscle weakness. In the present work we investigated the in vitro effect of 3-hydroxydodecanoic (3HDA), 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, which accumulate in these disorders, on important oxidative stress parameters in cerebral cortex of young rats in the hope to clarify the mechanisms leading to the brain damage found in patients affected by these disorders. It was first verified that these compounds significantly induced lipid peroxidation, as determined by increased thiobarbituric acid-reactive substances levels. In addition, carbonyl formation was significantly increased and sulfhydryl content decreased by 3HTA and 3HPA, which indicates that these fatty acids elicit protein oxidative damage. 3HTA and 3HPA also diminished the reduced glutathione (GSH) levels, without affecting nitrate and nitrite production. Finally, we observed that the addition of the antioxidants and free radical scavengers trolox and deferoxamine (DFO) was able to partially prevent lipid oxidative damage, whereas DFO fully prevented the reduction on GSH levels induced by 3HTA. Our present data showing that 3HDA, 3HTA and 3HPA elicit oxidative stress in rat brain indicate that oxidative damage may represent an important pathomechanism involved in the neurologic symptoms manifested by patients affected by LCHAD and MTP deficiencies.

Medium-chain fatty acids accumulating in MCAD deficiency elicit lipid and protein oxidative damage and decrease non-enzymatic antioxidant defenses in rat brain.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most frequent disorder of fatty acid oxidation with a similar prevalence to that of phenylketonuria. Affected patients present tissue accumulation of the medium-chain fatty acids octanoate (OA), decanoate (DA) and cis-4-decenoate. Clinical presentation is characterized by neurological symptoms, such as convulsions and lethargy that may develop into coma and sudden death. The aim of the present work was to investigate the in vitro effect of OA and DA, the metabolites that predominantly accumulate in MCADD, on oxidative stress parameters in rat cerebral cortex homogenates. It was first verified that both DA and OA significantly increased chemiluminescence and thiobarbituric acid-reactive species levels (lipoperoxidation) and decreased the non-enzymatic antioxidant defenses, measured by the decreased total antioxidant capacity. DA also enhanced carbonyl content and oxidation of sulfhydryl groups (protein damage) and decreased reduced glutathione (GSH) levels. We also verified that DA-induced GSH decrease and sulfhydryl oxidation were not observed when cytosolic preparations (membrane-free supernatants) were used, suggesting a mitochondrial mechanism for these actions. Our present data show that the medium-chain fatty acids DA and OA that most accumulate in MCADD cause oxidative stress in rat brain. It is therefore presumed that this pathomechanism may be involved in the pathophysiology of the neurologic symptoms manifested by patients affected by MCADD.