Estimation of the Rate of Production of Oxygen Radicals by Mitochondria

Alberto Sanz and Gustavo Barja

Oxygen radical production by mitochondria seems to be implicated in various degenerative diseases and aging. In order to estimate the rate of generation of reactive oxygen species, it is necessary to use adequate methods. In this article we describe a fluorometric method appropriate to measure the rate of generation of H2O2 in isolated functional mitochondria. The method is specific for H2O2 and is sensitive enough to assay mitochondrial H2O2 generation in the presence of respiratory substrates without the presence of inhibitors of the respiratory chain. Just after isolating functional mitochondria from fresh tissues, rates of generation of H2O2 are measured by fluorometry in the presence of homo-vanillic acid and horseradish peroxidase. Simultaneous measurement of mitochondrial oxygen consumption allows calculation of the percent of electrons out of sequence, which reduce oxygen to oxygen radicals along the mitochondrial respiratory chain instead of reducing oxygen to water at the terminal cytochrome oxidase. This is known as the free radical leak. This method is also appropriate to study the effect ofdifferent inhibitors and modulators on the rate of mitochondrial oxygen radical production, allowing the localization of the sites of generation in the respiratory chain where reactive oxygen species are specifically formed.

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