Zheng X X, Shoffner J M, Voljavec A S, Wallace D C
Department of Biochemistry, Emory University, School of Medicine, Atlanta, GA 30322.
Biochim Biophys Acta. 1990 Aug 9;1019(1):1-10. doi: 10.1016/0005-2728(90)90118-n.
The mitochondrial myopathies (MM) are a heterogenous group of neuromuscular diseases associated with abnormal mitochondria and defects in mitochondrial oxidative phosphorylation (OXPHOS). Analysis of a broad spectrum of MM patients has revealed that patients with similar clinical symptoms frequently do not have the same muscle OXPHOS defect. To determine whether some of this variation was due to methodological differences between studies, we have made a detailed survey of OXPHOS enzyme analysis procedures. The coupled OXPHOS assays for Complexes I + III and II + III were found to be variable due to competing reactions and complicated interactions between complexes. These problems were resolved by utilizing specific Complex I and III assays. The muscle mitochondria isolated from surgery patients under general anesthesia and prepared by proteinase digestion were observed to give low and highly variable OXPHOS activities. Mitochondria isolated from muscle biopsies performed under local anesthesia and finely sliced prior to homogenization gave higher and more consistent OXPHOS activities. Assays for Complexes I, III and V required mitochondrial sonication to express maximal activity, but Complex IV was prone to inactivation by excessive mechanical disruption. Mitochondria isolated from frozen muscle or from patients with an OXPHOS disease are more fragile than those isolated from fresh tissue and normal individuals. Hence, Complex IV activity can be preferentially lost from frozen and sonicated myopathy patient samples. These results suggest that variation in muscle OXPHOS analysis techniques may account for some of the discrepancies between clinical manifestations and OXPHOS defects and suggest that no single protocol is sufficient to adequately define the OXPHOS defect in MM patients.
线粒体肌病（MM）是一组异质性神经肌肉疾病，与线粒体异常及线粒体氧化磷酸化（OXPHOS）缺陷相关。对广泛的MM患者进行分析发现，具有相似临床症状的患者其肌肉OXPHOS缺陷往往并不相同。为确定这种差异是否部分归因于不同研究之间的方法学差异，我们对OXPHOS酶分析程序进行了详细调查。发现用于复合体I + III和II + III的偶联OXPHOS检测因复合体之间的竞争反应和复杂相互作用而存在差异。通过使用特定的复合体I和III检测解决了这些问题。观察到从全身麻醉下手术患者分离并经蛋白酶消化制备的肌肉线粒体，其OXPHOS活性较低且变化很大。从局部麻醉下进行的肌肉活检中分离、在匀浆前精细切片的线粒体，其OXPHOS活性更高且更一致。对复合体I、III和V的检测需要线粒体超声处理以表达最大活性，但复合体IV容易因过度机械破坏而失活。从冷冻肌肉或患有OXPHOS疾病的患者中分离的线粒体比从新鲜组织和正常个体中分离的线粒体更脆弱。因此，复合体IV活性可能优先从冷冻和超声处理的肌病患者样本中丧失。这些结果表明，肌肉OXPHOS分析技术的差异可能是临床表现与OXPHOS缺陷之间某些差异的原因，并表明没有单一方案足以充分界定MM患者的OXPHOS缺陷。
