Adaptation of a Mitochondrial Complex III Assay for Automation: Examination of Reproducibility and Precision (Technical Briefs‪)‬

Complex III (ubiquinol:ferricytochrome c reductase; EC 1.10.2.2) catalyzes the reduction of cytochrome c by ubiquinol (1, 2). The assay measures the reduction of cytochrome c as catalyzed by complex III at 550 nm (3) in the presence of reduced decylubiquinone ([DBH.sub.2]) (2). Despite numerous in vivo methods to screen for disorders of ATP production, biochemical assays for the respiratory chain (RC) remain the best way to identify and characterize this group of disorders (4). Although investigators have documented various methodologies to measure complex III in a variety of tissue types (3, 5-7), these are based on manual methods that are unsuitable for screening large numbers of samples. We recently automated mitochondrial and RC-specific enzyme (8) and protein (9) assays on a random access analyzer (Roche Mira S). The benefits of automation include enhanced speed and simplicity; savings in costs, including labor and reagents, and in the amount of sample needed; and improvements in precision. We adapted and automated the complex III assay (2, 6) on the Roche Mira S random access automated analyzer and compared methods of isolation, performed stability studies, and examined the precision of this new automated method.