Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts

被引:315
作者
Janssen, Antoon J. M. [1 ]
Trijbels, Frans J. M. [1 ]
Sengers, Rob C. A. [1 ]
Smeitink, Jan A. M. [1 ]
Van den Heuvel, Lambert P. [1 ]
Wintjes, Liesbeth T. M. [1 ]
Stoltenborg-Hogenkamp, Berendien J. M. [1 ]
Rodenburg, Richard J. T. [1 ]
机构
[1] UMC St Radboud, Lab Pediat & Neurol, Nijmegen Ctr Mitochondrial Disorders, NL-6525 GA Nijmegen, Netherlands
关键词
D O I
10.1373/clinchem.2006.078873
中图分类号
R446 [实验室诊断]; R-33 [实验医学、医学实验];
学科分类号
1001 ;
摘要
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 deficiencies, the new assay detected the complex I deficiencies in both muscle and fibroblasts. Conclusions: This spectrophotometric assay is reproducible, sensitive, and specific for complex I activity because of its high rotenone sensitivity, and it can be applied successfully to the diagnosis of complex I deficiencies. (c) 2007 American Association for Clinical Chemistry.
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页码:729 / 734
页数:6
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