Determination of the activities of the enzyme complexes of the electron transport chain in human fibroblasts.

In order to improve the determination of the activities of the enzyme complexes of the electron transport chain (ETC) in fibroblasts, we characterized the isolation of mitochondria and measured enzyme activities in mitochondrial preparations from fibroblasts of control subjects and patients with suspected mitochondrial cytopathy. The isolation procedure yielded 54% of the citrate synthase activity in fibroblasts, with a 6-fold enrichment in this mitochondrial marker enzyme. The activities of the complexes of the ETC were linear with time and with the mitochondrial protein concentration used. The coefficients of variation for the enzyme activities determined were in the range of 10% in mitochondria from identical fibroblast cultures and between 30 and 70% in mitochondria from different fibroblast cultures of the same or of different patients. Decreased activities of one or more enzyme complexes (defined as an activity below the 95% confidence limit of control values) were found in 15 of 22 patients investigated. When compared with activities obtained in liver or skeletal muscle obtained at autopsy, the results were identical in three but different in two patients. The studies show that the activities of the enzyme complexes of the ETC can be determined reliably and reproducibly in mitochondria isolated from fibroblasts and that the results obtained are potentially useful for the diagnosis of mitochondrial cytopathies in patients with suggestive symptoms and signs.