Complex I spectrophotometric assay in cultured cells: detailed analysis of key factors.

Kátia Klug Oliveira, Beatriz Hitomi Kiyomoto, Andresa De Santi Rodrigues, Célia Harumi Tengan

Department of Neurology and Neurosurgery, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP 04039-032, Brazil.

Analytical biochemistry 2013 Apr 1

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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. Copyright © 2012 Elsevier Inc. All rights reserved.

Citation

Kátia Klug Oliveira, Beatriz Hitomi Kiyomoto, Andresa De Santi Rodrigues, Célia Harumi Tengan. Complex I spectrophotometric assay in cultured cells: detailed analysis of key factors. Analytical biochemistry. 2013 Apr 1;435(1):57-9