Frazier Ann E, Vincent Amy E, Turnbull Doug M, Thorburn David R, Taylor Robert W
Brain and Mitochondrial Research, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia; Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia.
Wellcome Centre for Mitochondrial Research, Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom.
Methods Cell Biol. 2020;155:121-156. doi: 10.1016/bs.mcb.2019.11.007. Epub 2019 Dec 14.
Measurement of the individual enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) forms a key part of diagnostic investigations in patients with suspected mitochondrial disease, and can provide crucial information on mitochondrial OXPHOS function in a variety of cells and tissues that are applicable to many research investigations. In this chapter, we present methods for analysis of mitochondrial respiratory chain enzymes in cells and tissues based on assays performed in two geographically separate diagnostic referral centers, as part of clinical diagnostic investigations. Techniques for sample preparation from cells and tissues, and spectrophotometric assays for measurement of the activities of OXPHOS complexes I-V, the combined activity of complexes II+III, and the mitochondrial matrix enzyme citrate synthase, are provided. The activities of mitochondrial respiratory chain enzymes are often expressed relative to citrate synthase activity, since these ratios may be more robust in accounting for variability that may arise due to tissue quality, handling and storage, cell growth conditions, or any mitochondrial proliferation that may be present in tissues from patients with mitochondrial disease. Considerations for adaption of these techniques to other cells, tissues, and organisms are presented, as well as comparisons to alternate methods for analysis of respiratory chain function. In this context, a quantitative immunofluorescence protocol is also provided that is suitable for measurement of the amount of multiple respiratory chain complexes in small diagnostic tissue samples. The analysis and interpretation of OXPHOS enzyme activities are then placed in the context of mitochondrial disease tissue pathology and diagnosis.
对参与线粒体氧化磷酸化（OXPHOS）的各个酶进行测量，是疑似线粒体疾病患者诊断研究的关键部分，并且可以为多种细胞和组织中线粒体OXPHOS功能提供关键信息，这些信息适用于许多研究调查。在本章中，作为临床诊断研究的一部分，我们介绍了基于在两个地理位置不同的诊断转诊中心所进行的检测，对细胞和组织中线粒体呼吸链酶进行分析的方法。提供了从细胞和组织中制备样品的技术，以及用于测量OXPHOS复合体I-V的活性、复合体II+III的联合活性和线粒体基质酶柠檬酸合酶活性的分光光度测定法。线粒体呼吸链酶的活性通常相对于柠檬酸合酶活性来表示，因为这些比率在解释由于组织质量、处理和储存、细胞生长条件或线粒体疾病患者组织中可能存在的任何线粒体增殖而可能产生的变异性方面可能更可靠。介绍了将这些技术应用于其他细胞、组织和生物体的注意事项，以及与呼吸链功能分析的替代方法的比较。在这种情况下，还提供了一种定量免疫荧光方案，适用于测量小诊断组织样本中多种呼吸链复合体的数量。然后将OXPHOS酶活性的分析和解释置于线粒体疾病组织病理学和诊断的背景下。
