Detection and measurement of reactive oxygen intermediates in mitochondria and cells

Reactive oxygen intermediates (ROIs) play a key role in a number of human diseases either by inducing cell death, cellular proliferation, or by acting as mediators in cellular signaling. Therefore, their measurement in vivo and in cell culture is desirable but technically difficult and often troublesome. To address some of the key methodological issues in examining the formation of ROI in cells and mitochondria, this chapter discusses the following: (a) the cellular sources of ROI and their enzymatic removal, (b) common methods used to determine cellular and mitochondrial ROI such as chemiluminescence, electron paramagnetic resonance spectroscopy, fluorescence, and enzymatic techniques, and (c) some common problems associated with these assays and the interpretation of data. We also provide some simple protocols for the estimation of ROI production in cells and mitochondria, and when measuring ROI in cells and mitochondria, we emphasize the need for thorough understanding of results obtained and their interpretation.