Assay of mitochondrial respiratory chain complex I in human lymphocytes and cultured skin fibroblasts.
Respiratory chain complex I (NADH:ubiquinone oxidoreductase) deficiency is one of the most frequent causes of mitochondrial disease in humans. The activity of this complex can be confidently measured in most tissue samples, but not in cultured skin fibroblasts or circulating lymphocytes. Highly contaminating non-mitochondrial NADH-quinone oxidoreductase activity in fibroblasts and the limited access of substrates to complex I in lymphocytes hinder its measurement in permeabilized cells. Complex I assay in these cells requires the isolation of mitochondria, which in turn necessitates large quantities of cells and is not feasible when studying circulating lymphocytes. Here we report a simple method to measure complex I activity in a minute amount of either cell type. The procedure strongly reduces contaminating NADH:quinone oxidoreductase activity and permits measuring high rates of rotenone-sensitive complex I activity thanks to effective cell permeabilization.
Chretien
Dominique
D
Unité de Recherches sur les Handicaps Génétiques de l'Enfant (INSERM U393), 2éme Etage, Hôpital Necker-Enfants Malades, 149, rue de Sèvres, 75743 Paris cedex 15, France.
Bénit
Paule
P
Chol
Marie
M
Lebon
Sophie
S
Rötig
Agnès
A
Munnich
Arnold
A
Rustin
Pierre
P
D002478:Cells, Cultured; D042967:Electron Transport Complex I; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology*; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D007888:Leigh Disease / Q000175:diagnosis / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology / Q000201:enzymology*; D008293:Malates / Q000378:metabolism; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011048:Polarography; D012402:Rotenone / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000378:metabolism; D014475:Uncoupling Agents / Q000378:metabolism
D008293:Malates; D014475:Uncoupling Agents; D012402:Rotenone; D018698:Glutamic Acid; C030298:malic acid; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; D010100:Oxygen