H2O2 production must be measured always in functional mitochondria obtained from fresh tissue, and never from frozen tissue samples. The measurement of H2O2 in functional intact mitochondria requires the use of appropriate protocols of isolation. There are methods specifically designed to obtain functional mitochondria from different tissues. Possible choices are those of Mela and Seitz (1979) for heart mitochondria, the method of Lai and Clark (1979) for brain mitochondria (which uses Ficoll gradients specially useful in this tissue), and the protocol described by Lopez-Torres et al. (2002) for liver mitochondria. Using these methods, well-coupled mitochondria can be isolated in a few hours. During all the steps, including homogenization, centrifugation and resuspensions, the mitochondrial preparations must be maintained at 4°C. Between isolation and measurements the tube containing the mitochondria must be kept over ice, and the concentration of mitochondria must be high. This helps avoid loss of mitochondrial functional properties during a few hours.
Just after this isolation, the mitochondrial protein concentration must be measured. Immediately after isolation is finished, oxygen consumption and H2O2 production are assayed at the same temperature (37° C), using the same buffer and with the same concentrations of substrates and inhibitors. This allows calculating the percent free radical leak (see below).
Oxygen consumption is measured with a Clark-type electrode calculating the respiratory control index (RCI), which is the ratio state 3/state 4 respiration. State 3 is the mitochondrial oxygen consumption in the presence of saturating ADP (phosphorylating state), and state 4 is the mitochondrial oxygen consumption without ADP (nonphosphorylating state). The RCI indicates the degree of coupling and metabolic activity of the mitochondrial preparations. A RCI of 1 or less indicates damaged nonfunctional mitochondria. The appropriate RCI must be substantially higher than 1, with values varying depending on the tissue and the substrate used.
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