Development and evaluation of a spectrophotometric assay for complex III in isolated mitochondria, tissues and fibroblasts from rats and humans.

A spectrophotometric method to assay the activity of complex III in isolated mitochondria, tissues and fibroblasts from patients and rats has been developed and validated. Decylubiquinol was shown to be a suitable substrate with a saturating concentration between 100 and 200 mumol/l. The optimal pH was found to range from 7.4 to 8.0. The enzyme reaction was linear for incubations containing up to 15 micrograms/ml mitochondrial protein, 250 micrograms/ml liver tissue, 375 micrograms/ml skeletal muscle or 100 micrograms/ml fibroblast protein. Intraday and interday variability of the assay for different enzyme sources was below 15% and 10%, respectively. Assessment of complex III activity in liver and fibroblasts from patients with signs of mitochondrial dysfunction revealed the usefulness of the newly developed assay in the diagnosis of complex III deficiency.