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

doi:10.1016/j.ab.2012.12.002

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

Anal Biochem. 2013 Apr 1;435(1):57-9. doi: 10.1016/j.ab.2012.12.002. Epub 2012 Dec 12.

Authors

Kátia Klug Oliveira¹, Beatriz Hitomi Kiyomoto, Andresa De Santi Rodrigues, Célia Harumi Tengan

Affiliation

¹ Department of Neurology and Neurosurgery, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP 04039-032, Brazil.

PMID: 23246343
DOI: 10.1016/j.ab.2012.12.002

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cells, Cultured
Electron Transport Complex I / metabolism*
Enzyme Assays / methods
Fibroblasts / metabolism
Humans
Mitochondria / metabolism
Spectrophotometry / methods*
Ubiquinone / analogs & derivatives
Ubiquinone / metabolism

Substances

Ubiquinone
Electron Transport Complex I