Measurement of mitochondrial ROS production

Abstract

The significance of reactive oxygen species (ROS) as aggravating or primary factors in numerous pathologies is widely recognized, with mitochondria being considered the major intracellular source of ROS. It is not yet possible to routinely measure mitochondrial ROS in animals or cultured cells with a reasonable degree of certainty. However, at the level of isolated mitochondria, one can easily monitor and quantify the rate of ROS production, identify major sites of ROS production, and compare the rates of ROS production in mitochondria isolated from normal and diseased tissue. In this chapter, we describe in detail the most recent and reliable method to measure mitochondrial ROS as the rate of H2O2 emission. This method may be employed with minimal modifications to measure H2O2 production by mitochondria isolated from various tissues and under a wide variety of experimental conditions.

Fig. 1

Calibrating the H₂O₂ assay. See text for the details.

Fig. 2

H₂O₂ emission by mouse brain mitochondria. Incubation medium (37°C) (Subheading 4.2) is supplemented with 4 U/ml horseradish peroxidase, 40 U/ml superoxide dismutase, 0.010 mM Amplex Red, and respiratory substrates as indicated. (a) Mouse brain mitochondria oxidizing succinate. (b) Mouse brain mitochondria oxidizing pyruvate and malate. Additions: Mito, 0.05 mg/ml mouse brain mitochondria; Succinate, 10 mM; Pyruvate:malate, 5:1 mM; Rotenone, 0.5 μM; Antimycin, 1 μg/ml. Numbers near the tracings are the rates of H₂O₂ production expressed in pmol/min/mg of mitochondria protein.