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

ObjectiveTo elucidate the pathophysiology of SCAD deficient patients who have aunique neurological phenotype, among fatty acid oxidation disorders, withearly developmental delay, CNS malformations, intractable seizures, myopathyand clinical signs suggesting oxidative stress. MethodsWe studied skin fibroblast cultures from patients homozygous for ACADScommon variant c.625G>A (n = 10), compound heterozygousfor c.625G>A/c.319C>T (n = 3) or homozygous forpathogenic c.319C>T (n = 2) and c.1138C>T (n = 2)mutations compared to fibroblasts from patients with carnitine palmitoyltransferase2 (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 µMmenadione at 37°C. Additonal conditions included exposure to 39°Cand/or hypoglycemia. Time to 100% cell death was confirmed with trypanblue dye exclusion. Experiments were repeated with antioxidants (VitaminsC and E or N-acetylcysteine), Bezafibrate or glucose and temperature rescue. ResultsThe most significant risk factor for vulnerability to menadione-inducedoxidative stress was the presence of a FAO defect. SCADD fibroblasts werethe most vulnerable compared to other FAO disorders and controls, and weresimilarly affected, independent of genotype. Cell death was exacerbated byhyperthermia and/or hypoglycemia. Hyperthermia was a more significant independentrisk factor than hypoglycemia. Rescue significantly prolonged survival. Incubationwith antioxidants and Bezafibrate significantly increased viability of SCADDfibroblasts. InterpretationVulnerability to oxidative stress likely contributes to neurotoxicity ofSCADD regardless of ACADS genotype and is significantly exacerbatedby hyperthermia. We recommend rigorous temperature control in SCADD patientsduring acute illness. Antioxidants and Bezafibrate may also prove instrumentalin their management.

目的：本研究旨在阐明脂肪酸氧化代谢障碍（fatty acid oxidation disorders）中，表现出独特神经表型的短链酰基辅酶A脱氢酶（SCAD）缺陷患者的病理生理学机制；此类患者存在早期发育迟缓、中枢神经系统（Central Nervous System, CNS）畸形、难治性癫痫、肌病以及提示氧化应激的临床体征。
方法：本研究收集了携带不同ACADS基因突变的患者皮肤成纤维细胞：包括c.625G>A纯合突变（n=10）、c.625G>A/c.319C>T复合杂合突变（n=3）、致病性c.319C>T纯合突变（n=2）以及c.1138C>T纯合突变（n=2）的患者；同时设置对照组，纳入肉碱棕榈酰转移酶2（carnitine palmitoyltransferase 2, CPT2）缺陷患者（n=5）、线粒体三功能蛋白（mitochondrial trifunctional protein, MTP）/长链L-3-羟酰基辅酶A脱氢酶（long-chain L-3-hydroxyacyl-CoA dehydrogenase, LCHAD）缺陷患者（n=7）、中链酰基辅酶A脱氢酶（medium-chain acyl-CoA dehydrogenase, MCAD）缺陷患者（n=4）的成纤维细胞，以及9名健康对照者的成纤维细胞。所有细胞均在37℃条件下于含50μM甲萘醌的培养液中孵育。额外实验设置包括39℃孵育及/或低糖培养环境。采用台盼蓝排斥试验确认细胞100%死亡的时间。此外，我们使用抗氧化剂（维生素C、维生素E或N-乙酰半胱氨酸）、苯扎贝特、葡萄糖以及温度恢复条件重复了全部实验。
结果：甲萘醌诱导氧化应激的易感性最显著的危险因素为存在脂肪酸氧化代谢缺陷（FAO）。与其他脂肪酸氧化代谢障碍患者及健康对照相比，短链酰基辅酶A脱氢酶缺陷症（SCADD）成纤维细胞的易感性最高，且其受损伤程度与基因型无关。高热及/或低糖环境会加重细胞死亡；相较于低糖环境，高热是更显著的独立危险因素。温度恢复干预可显著延长细胞存活时间；抗氧化剂与苯扎贝特孵育可显著提升SCADD成纤维细胞的存活率。
解读：无论ACADS基因的基因型如何，氧化应激易感性均可能参与短链酰基辅酶A脱氢酶缺陷症的神经毒性过程，且高热会显著加重该损伤。我们建议在SCADD患者急性发病期间严格控制体温。抗氧化剂与苯扎贝特或可在其临床管理中发挥重要作用。