Biochemical Assays for Mitochondrial Activity: Assays of TCA Cycle Enzymes and PDHc

Publisher Summary This chapter focuses on the biochemical assays for mitochondrial activity, which is assay of tricarboxylic acid (TCA) cycle enzymes and pyruvate dehydrogenase complex (PDHc). Defects in one of the counterparts is likely compatible with life but present clinically in a manner that seems far from a TCA cycle defect, as in mitochondrial DNA (mtDNA) replication defects or tumor formation. With the advancement of linkage analysis techniques, it is expected that molecular defects of most TCA components will be identified. Enzymatic methodologies will, therefore, be indispensable for confirming pathogenicity and the study of disease mechanisms. The assays may be performed on tissue homogenates or in isolated mitochondria. Congenital defects of TCA enzymes were previously thought to be rare, overshadowed by the more common defects in PDHc and the mitochondrial respiratory chain (MRC) disruption of the sample before assay is a crucial step. As opposed to the significant proportion of MRC disorders, which are maternally inherited, all PDHc and TCA defects are inherited as Mendelian traits.The most suitable disruption method depends on the enzyme and the material to be assayed. Several methods are employed, including treatment with various detergents such as Triton X-100, cholic acid, or dodecyl maltoside. A number of assays have been the subject of modifications by many distinguished laboratories. Thus, each laboratory should adapt the preferred method, optimizing sample preparation, disruption, and assay conditions to aim for a linear reaction with increased signal—background ratio.