title: Assay of mitochondrial respiratory chain complex I in human lymphocytes and cultured skin fibroblasts.
abstract: 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.
authors.affiliation: 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.
authors.forename: Dominique
authors.initials: D
authors.lastname: Chretien
authors.forename: Paule
authors.initials: P
authors.lastname: Bénit
authors.forename: Marie
authors.initials: M
authors.lastname: Chol
authors.forename: Sophie
authors.initials: S
authors.lastname: Lebon
authors.forename: Agnès
authors.initials: A
authors.lastname: Rötig
authors.forename: Arnold
authors.initials: A
authors.lastname: Munnich
authors.forename: Pierre
authors.initials: P
authors.lastname: Rustin
mesh_terms: 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
chemical_list: D008293:Malates; D014475:Uncoupling Agents; D012402:Rotenone; D018698:Glutamic Acid; C030298:malic acid; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; D010100:Oxygen