Simultaneously monitoring the superoxide in the mitochondrial matrix and extramitochondrial space by micellar electrokinetic chromatography with laser-induced fluorescence

Respiring mitochondria produce superoxide and release it into both sides of the mitochondrial inner membrane: the mitochondrial matrix and the extramitochondrial space. These two pools of superoxide are expected to have very distinctive effects on cellular function. Currently, separate measurements are required to measure superoxide in both pools, which complicates the comparison of superoxide's effects and roles in physiology and pathology. In this study, we describe a highly sensitive strategy to monitor simultaneously these two pools of superoxide in respiring isolated rat skeletal muscle mitochondria using hydroethidine. The oxidation of hydroethidine by superoxide forms the membrane-impermeable 2-hydroxyethidium in these two superoxide pools that can be separated by differential centrifugation. Two technical limitations in using 2-hydroxyethidium as a superoxide reporter are (i) the uncontrolled fluorescence enhancement due to intercalation of 2-hydroxyethidium with variable amounts of mitochondrial DNA and (ii) the spectral interference of ethidium fluorescence. These complications were eliminated by digestion of mitochondrial DNA with DNase and by separation of ethidium and 2-hydroxyethidium using cationic micellar electrokinetic capillary chromatography with laser-induced fluorescence, respectively. Using this method, which has subattomole limits of detection, we compared the levels of 2-hydroxyethidium in normally respiring and antimycin A-treated mitochondria and demonstrated that the strategy can be extended to observe how menadione induces superoxide generation in mitochondria.