title: Complex I spectrophotometric assay in cultured cells: detailed analysis of key factors.
abstract: The diagnosis of mitochondrial encephalomyopathies caused by complex I (C-I) deficiency relies mainly on the spectrophotometric C-I assay. Considered difficult, this assay lacks reliability and has high nonspecific activity. We studied the key factors of this assay in cultured cells (cybrid and fibroblast): ubiquinone analogues, rotenone inhibition to determine specific activity, and mode of permeabilization of mitochondrial membranes. We showed that ubiquinone 1 allows a more stable and reliable assay, better results were obtained with rotenone inhibition done in the same assay, and mitochondrial permeability was improved just by freeze/thawing the sample prior to the assay.
authors.affiliation: Department of Neurology and Neurosurgery, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP 04039-032, Brazil.
authors.forename: Kátia Klug
authors.initials: KK
authors.lastname: Oliveira
authors.forename: Beatriz Hitomi
authors.initials: BH
authors.lastname: Kiyomoto
authors.forename: Andresa De Santi
authors.initials: Ade S
authors.lastname: Rodrigues
authors.forename: Célia Harumi
authors.initials: CH
authors.lastname: Tengan
mesh_terms: D002478:Cells, Cultured; D042967:Electron Transport Complex I / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods*; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D014451:Ubiquinone; D042967:Electron Transport Complex I