Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts.
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.
Janssen
Antoon J M
AJ
Department of Pediatrics and Laboratory of Pediatrics and Neurology, The Nijmegen Centre for Mitochondrial Disorders at Radboud University Medical Centre, Nijmegen, The Netherlands.
Trijbels
Frans J M
FJ
Sengers
Rob C A
RC
Smeitink
Jan A M
JA
van den Heuvel
Lambert P
LP
Wintjes
Liesbeth T M
LT
Stoltenborg-Hogenkamp
Berendien J M
BJ
Rodenburg
Richard J T
RJ
D002478:Cells, Cultured; D042967:Electron Transport Complex I / Q000032:analysis* / Q000172:deficiency / Q000235:genetics; D042963:Electron Transport Complex II / Q000032:analysis; D005347:Fibroblasts / Q000737:chemistry*; D006801:Humans; D008928:Mitochondria / Q000737:chemistry*; D028361:Mitochondrial Diseases / Q000175:diagnosis; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000166:cytology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012867:Skin / Q000737:chemistry* / Q000166:cytology; D013053:Spectrophotometry
D042963:Electron Transport Complex II; D042967:Electron Transport Complex I