Since Denham Harman published his Free Radical Theory of Aging in 1956, there has been growing evidence that reactive oxygen species (ROS) of biological origin are implicated in pathological processes. Free radicals damage cellular macromolecules including carbohydrates, lipids, proteins, and most importantly, the nucleic acids of nuclear and mitochondrial DNA. In addition, ROS are continuously generated by aerobic tissues. These two properties make ROS potentially responsible in part for degenerative processes like diabetes, cancer, Parkinson's or Alzheimer's diseases, as well as aging (Barja, 2004; Sohal et al., 2002).

Mitochondria are the main generator of ROS in healthy cells. The mitochondrial electron transport chain (ETC) is responsible for more than 90% of cellular oxygen consumption. During the flow of electrons in the ETC, a small percent of them incompletely reduce oxygen to superoxide and hydrogen peroxide. The first reactive species produced in the ETC is superoxide radical (O2 ), resulting from the univalent reduction of oxygen. O2 dismutates to hydrogen peroxide (H2O2), which can diffuse out of mitochondria to their surrounding medium. In submitochondrial particles O2 is directly secreted outside. For this reason ROS generation is measured as O2 in submitochondrial particles and as H2O2 in intact mitochondria. Although submitochondrial particles are adequate for various particular reasons including the possibility of using NADH directly as a substrate, assays in intact functional mitochondria are closer to the real physiological situation. The ideal situation would be to measure mitochondria ROS production in vivo in the whole animal, but techniques allowing this are still not available.

In the present paper we describe a reliable method for measuring H2O2 production in isolated functional mitochondria. This method was originally designed to measure H2O2 in macrophages and neutrophils (Ruch et al., 1983), and it was afterwards adapted to isolated mitochondria (Barja, 1999; Barja, 2002). We described the method in detail, including the latest improvements.

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