Zarazuela Zolkipli

Vulnerability to Oxidative Stress In Vitro in Pathophysiology of Mitochondrial Short-Chain Acyl-CoA Dehydrogenase Deficiency: Response to Antioxidants

Objective To elucidate the pathophysiology of SCAD deficient patients who have a unique neurological phenotype, among fatty acid oxidation disorders, with early developmental delay, CNS malformations, intractable seizures, myopathy and clinical signs suggesting oxidative stress. Methods We studied skin fibroblast cultures from patients homozygous for ACADS common variant c.625G>A (n = 10), compound heterozygous for c.625G>A/c.319C>T (n = 3) or homozygous for pathogenic c.319C>T (n = 2) and c.1138C>T (n = 2) mutations compared to fibroblasts from patients with carnitine palmitoyltransferase 2 (CPT2) (n = 5), mitochondrial trifunctional protein (MTP)/long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD) (n = 7), and medium-chain acyl-CoA dehydrogenase (MCAD) deficiencies (n = 4) and normal controls (n = 9). All were exposed to 50 µM menadione at 37°C. Additonal conditions included exposure to 39°C and/or hypoglycemia. Time to 100% cell death was confirmed with trypan blue dye exclusion. Experiments were repeated with antioxidants (Vitamins C and E or N-acetylcysteine), Bezafibrate or glucose and temperature rescue. Results The most significant risk factor for vulnerability to menadione-induced oxidative stress was the presence of a FAO defect. SCADD fibroblasts were the most vulnerable compared to other FAO disorders and controls, and were similarly affected, independent of genotype. Cell death was exacerbated by hyperthermia and/or hypoglycemia. Hyperthermia was a more significant independent risk factor than hypoglycemia. Rescue significantly prolonged survival. Incubation with antioxidants and Bezafibrate significantly increased viability of SCADD fibroblasts. Interpretation Vulnerability to oxidative stress likely contributes to neurotoxicity of SCADD regardless of ACADS genotype and is significantly exacerbated by hyperthermia. We recommend rigorous temperature control in SCADD patients during acute illness. Antioxidants and Bezafibrate may also prove instrumental in their management.

Abnormal fatty acid metabolism in spinal muscular atrophy may predispose to perioperative risks

A 15 year old boy with SMA type II underwent spinal fusion and suffered a mitochondrial Reye-like catabolic crisis 4 days postop with hypoketotic hypoglycemia, lactic acidaemia, hyperammonemia and liver failure, with 90% coagulative necrosis and diffuse macro- and microvesicular steatosis, requiring orthotopic liver transplantation. This crisis responded in part to mitochondrial therapy and anabolic rescue. He made a dramatic sustained neurological recovery, though his post-transplant liver biopsies revealed micro- and macrosteatosis. We hypothesize that a combination of surgical stress-catecholamine induced lipolysis, prolonged general anaesthesia with propofol and sevoflurane, and perioperative fasting on a background of decreased β-oxidation were potential risk factors for the mitochondrial decompensation.

The mdx mouse as a model for carnitine deficiency in the pathogenesis of Duchenne muscular dystrophy.

Introduction: Muscle and cardiac metabolism are dependent on the oxidation of fats and glucose for adenosine triphosphate production, for which L‐carnitine is an essential cofactor. Methods: We measured muscle carnitine concentrations in skeletal muscles, diaphragm, and ventricles of C57BL/10ScSn‐DMDmdx/J mice (n = 10) and compared them with wild‐type C57BL/6J (n = 3), C57BL/10 (n = 10), and C3H (n = 12) mice. Citrate synthase (CS) activity was measured in quadriceps/gluteals and ventricles of mdx and wild‐type mice. Results: We found significantly lower tissue carnitine in quadriceps/gluteus (P < 0.05) and ventricle (P < 0.05), but not diaphragm of mdx mice, when compared with controls. CS activity was increased in mdx quadriceps/gluteus (P < 0.03) and ventricle (P < 0.02). This suggests compensatory mitochondrial biogenesis. Conclusions: Decreased tissue carnitine has implications for reduced fatty acid and glucose oxidation in mdx quadriceps/gluteus and ventricle. The mdx mouse may be a useful model for studying the role of muscle carnitine deficiency in DMD bioenergetic insufficiency and providing a targeted and timed rationale for L‐carnitine therapy. Muscle Nerve 46: 767–772, 2012

O7-5 Muscle carnitine deficiency in pathogenesis of myopathy and cardiomyopathy of Duchenne muscular dystrophy (DMD)

Objective: To determine tissue Carnitine (Cn) levels in the C57BL/10ScSn-Dmdmdx male mouse, murine model of DMD. Background:Muscle and cardiacmetabolism and function are dependent on the oxidation of fatty acids and glucose for ATP production, for which Cn serves as an essential cofactor. Skeletal muscle contains >90% of total body Cn stores and uptake across the muscle plasma membrane occurs against a 50 to 70 fold concentration gradient, mediated by the plasmalemmal Cn transporter, OCTN2. Muscle Cn deficiency and reduced long-chain fatty acid oxidation (FAO) have been reported in DMD muscle. We speculate that dystrophin deficiency in DMD, which causes disruption of the plasma membrane, likely disrupts OCTN2, leading to significant secondary tissue Cn depletion. Methods:We determined tissue Cn concentrations according to Cederblad and Lindstedt (1972) in the skeletal muscle (gluteus/quadriceps), diaphragm and ventricles of mdx mice (n = 10) compared to normal male controls: C57BL/6 (n=6), C3H (n=12) and C57BL/10 (n=10) mice. Results: Comparison of the mean Cn concentrations (± standard error mean) in mmol/g dry weight of muscle of the controls vs mdx mice is shown in the table.