Optimization of respiratory chain enzymatic assays in muscle for the diagnosis of mitochondrial disorders

The diagnosis of mitochondrial disorders is difficult due to clinical and genetic heterogeneity. Measurements of mitochondrial respiratory chain (RC) enzyme activities are essential for both clinical diagnoses and many basic research questions. Current protocols for RC analysis are not standardized, and so are prone to inter-laboratory variability, and also to biochemical interferences that lead to analytical discrepancies. Moreover, knowledge of the analytical performances of these assays, which is essential to draw meaningful conclusions from the results, is lacking. To understand this variability and to propose possible solutions, we systematically investigated the effect of different homogenization protocols and chemical conditions on RC assays using muscle homogenates. We developed optimized protocols and a novel complex III method with improved sensitivity, precision, and linearity. These methods can be reliably performed on minute muscle samples with a single-wavelength spectrophotometer. Moreover, we measured the variability of the proposed homogenization protocol and we provide a systematic evaluation of each assay's specificity, precision, and linearity. These data will be useful for quality control in both clinical and research laboratories.