Correct Assay of Complex I Activity in Human Skin Fibroblasts by Timely Addition of Rotenone

In a recent issue of Clinical Chemistry Janssen et al. (1) reported the development of a new spectrophotometric assay to determine complex I activity in a mitochondrial fraction of human skin fibroblasts, which is based on measuring the reduction of 2,6-dichloroindophenol by electrons accepted from decylubiquinol. This is a potentially important finding because the determination of complex I in fibroblasts is difficult owing to the high activity of contaminating rotenone-insensitive NADH dehydrogenases(2). In the reported method complex I was assayed by measuring the total NADH oxidase activity during a 4-min period, after which rotenone was added to measure the rotenone-insensitive NADH oxidase activity. By subtraction of the reaction rates, the complex I activity was calculated. Because it is well known that the accumulation of rotenone on its binding site is not instantaneous(3)(4), we questioned if the complex I assay might be affected by the delay in the inhibitory effect of rotenone.We investigated the relationship between the amount of mitochondrial protein isolated from normal human skin fibroblasts exactly as described by Janssen et al. (1) and the time course of the NADH oxidase activity in the absence of rotenone. The duration of the first order …