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 kinetics decreased after 2–3 min, especially at the highest protein concentrations (Fig. 1 ). This result indicates that addition of rotenone 4 min after the start of the reaction and measurement of the changes in absorbance during the next 4 min may lead to serious overestimation of the apparent rotenone sensitivity of complex I. To show the consequences of this finding, we performed the assay exactly according to the method described by Janssen et al.(1) by adding rotenone 4 min after the start of the reaction for the protein concentration range shown in Fig. 1 . Obviously the complex I activity, if expressed as specific activity (in mU per mg protein), must be unrelated to the protein concentration. We found, however, a statistically significant linear relation between the specific complex I activity and the mitochondrial protein concentration (R2 = 0.66, P = 0.004, y = 2.0x + 81.9, where y = mU/mg protein and x = μg protein/mL), indicating that the complex I assay performed with the method of Janssen et al. is not reliable. Only with highly diluted fractions does the above-mentioned equation approach a constant, but in the regular assay reported by Janssen et al. the protein concentration amounts to 17.6 μg/mL and the reaction is then no longer first order at a reaction time beyond 2 min (Fig. 1 ). Mitochondrial protein concentration–dependent kinetics of the NADH:2,6-dichloroindophenol oxidoreductase activity in the mitochondrial-enriched fraction of normal human skin fibroblasts, measured exactly according to the method of Janssen et al. (1) but in the absence of rotenone during the entire assay time of 8 min. To find out if the real complex I activity can be assessed when rotenone has enough time to accumulate at the coenzyme Q10–binding site of the enzyme complex, we modified the method by measuring the rotenone-insensitive enzyme activity in a separate cuvette, to which we added the inhibitor 1 min before the start of the reaction with NADH. The specific complex I activity now became independent of the mitochondrial protein concentration up to at least 42 μg/mL (R2 = 0.032; P = 0.622) and amounted to a mean (SD) of 39.6 (18.6) mU/mg protein. This value is only 21% (6%) of the total (i.e., rotenone sensitive plus insensitive) NADH oxidase activity. The intraassay variation was 27% to 63%, and the interassay variation was 47% (n = 6). The consequence of this improved procedure is that the required amount of protein is doubled, which could be a problem if material is limited. With the use of small cuvettes (5), a concentration of 17.6 μg/mL protein per cuvette volume of 142 μL requires only 2.5 μg, which is equivalent to <1×105 fibroblasts. To establish whether this modification indeed measured the correct amount of enzyme, we used our recently reported method (5) to determine complex I in the mitochondrial-enriched fraction by using coenzyme CoQ1, the water-soluble analog to CoQ10, as the only electron acceptor instead of decylubiquinone plus 2,6-dichloroindophenol. We found that with the use of CoQ1 the assay was less influenced by rotenone-insensitive NADH oxidases, increasing the rotenone-sensitive fraction from mean (SD) of 21% (6%) to 62% (9%) (n = 5; Student 2-tailed t-test, P < 0.005), but there was not a statistically significant difference in the complex I activity of 42.6 (11.4) mU/mg mitochondrial protein compared with that found by the improved method of Janssen et al. A noteworthy observation was that our own method(5) appeared more reliable because the intraassay variation was 18% to 37%, the interassay variation was 27% (n = 5), and the SD of the mean complex I activity was smaller by a factor of 2. We conclude that for the correct determination of complex I, it is imperative to add rotenone in a separate cuvette before the start of the measurement to allow rotenone to accumulate to its binding site. Otherwise the complex I activity in the mitochondrial-enriched fraction of human skin fibroblasts will be overestimated. Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data or analysis, and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures of Potential Conflicts of Interest:No authors declared any potential conflicts of interest. Role of Sponsor: The funding organizations played no role in the design of the study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript. Acknowledgments: We kindly thank Dr Kees Schoonderwoerd of our Department of Clinical Genetics for providing the human skin fibroblast samples.