High-Resolution Respirometry for Simultaneous Measurement of Oxygen and Hydrogen Peroxide Fluxes in Permeabilized Cells, Tissue Homogenate and Isolated Mitochondria

Whereas mitochondria are well established as the source of ATP in oxidative phosphorylation (OXPHOS), it is debated if they are also the major cellular sources of reactive oxygen species (ROS). Here we describe the novel approach of combining high-resolution respirometry and fluorometric measurement of hydrogen peroxide (H(2)O(2)) production, applied to mitochondrial preparations (permeabilized cells, tissue homogenate, isolated mitochondria). The widely used H(2)O(2) probe Amplex Red inhibited respiration in intact and permeabilized cells and should not be applied at concentrations above 10 µM. H(2)O(2) fluxes were generally less than 1% of oxygen fluxes in physiological substrate and coupling states, specifically in permeabilized cells. H(2)O(2) flux was consistently highest in the Complex II-linked LEAK state, reduced with CI&II-linked convergent electron flow and in mitochondria respiring at OXPHOS capacity, and were further diminished in noncoupled mitochondria respiring at electron transfer system capacity. Simultaneous measurement of mitochondrial respiration and H(2)O(2) flux requires careful optimization of assay conditions and reveals information on mitochondrial function beyond separate analysis of ROS production.