Optimized Spectrophotometric Assay for the Completely Activated Pyruvate Dehydrogenase Complex in Fibroblasts

complex (PDHc) activity in human skin fibroblasts is hampered by low enzyme activity in the cells. The most commonly used radiochemical method detects the for-mation of 14CO2, an endproduct of the E1 component of PDHc, from [1-14C]pyruvate. Methods: Here we report a spectrophotometric method for the analysis of PDHc activity in fibroblasts based on detection of NADH formation via a p-iodonitrotetrazo-lium violet (INT)-coupled system. We investigated in detail the specific requirements of this assay, such as cofactor requirements and the effects of suggested stim-ulatory compounds and different cell disruption proce-dures. The reliability of the optimized assay was stud-ied by investigation of patients previously diagnosed with PDHc deficiency and by comparison with result