Spectrophotometric Assay for Complex I of the Respiratory Chain in Tissue Samples and Cultured Fibroblasts

Abstract

Background: A reliable and sensitive complex I assay is an essential tool for the diagnosis of mitochondrial disorders, but current spectrophotometric assays suffer from low sensitivity, low specificity, or both. This deficiency is mainly due to the poor solubility of coenzyme-Q analogs and reaction mixture turbidity caused by the relatively high concentrations of tissue extract that are often required to measure complex I. Methods: We developed a new spectrophotometric assay to measure complex I in mitochondrial fractions and applied it to muscle and cultured fibroblasts. The method is based on measuring 2,6-dichloroindophenol reduction by electrons accepted from decylubiquinol, reduced after oxidation of NADH by complex I. The assay thus is designed to avoid nonspecific NADH oxidation because electrons produced in these reactions are not accepted by decylubiquinone, resulting in high rotenone sensitivity. Results: The assay was linear with time and amount of mitochondria. The K m values for NADH and 2,6-dichloroindophenol in muscle mitochondria were 0.04 and 0.017 mmol/L, respectively. The highest complex I activities were measured with 0.07 mmol/L decylubiquinone and 3.5 g/L bovine serum albumin. The latter was an essential component of the reaction mixture, increasing the solubility of decylubiquinone and rotenone. In patients with previously diagnosed complex I deficien-

Protein Carbonylation and Adipocyte Mitochondrial Function

“…To investigate whether carbonylation affects Complex I activity, the transfer of electrons from NADH to dichlorophenolindophenol was evaluated spectrophotometrically (25) in mitochondria isolated from the GSTA4-silenced and overexpressing cells. As shown in Fig.…”

“…Complex I activity was assessed as described previously but adapted to a microplate format (25). Briefly, Complex I transfers electrons from NADH to decylubiquinone that subsequently delivers them to dichlorophenylindole.…”

Characterization of mtDNA variation in a cohort of South African paediatric patients with mitochondrial disease

“…Informed consent and assent were obtained for all patients before initiation of this study, although a family history was not available for most cases. Muscle enzyme analyses were performed essentially as described elsewhere, [10][11][12] and for 56/71 of the cases, an RC deficiency was confirmed. In addition, mtDNA relative copy number analysis on muscle DNA was performed by real-time polymerase chain reaction (PCR) using the ND1/GAPDH ratio using TaqMan chemistry and commercial probes (Applied Biosystems, Foster City, CA, USA).…”

Effect of antipsychotics on mitochondrial bioenergetics of rat ovarian theca cells

“…Both mitochondrial enriched fraction and cell lysates were prepared following methods described by Spinazzi et al (2012). For complex I assay, Dichlorophenolindophenol (DCIP) was used as a terminal electron acceptor (Janssen et al, 2007).…”