title: High-Resolution Respirometry for Simultaneous Measurement of Oxygen and Hydrogen Peroxide Fluxes in Permeabilized Cells, Tissue Homogenate and Isolated Mitochondria.
abstract: 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 (H2O2) production, applied to mitochondrial preparations (permeabilized cells, tissue homogenate, isolated mitochondria). The widely used H2O2 probe Amplex Red inhibited respiration in intact and permeabilized cells and should not be applied at concentrations above 10 µM. H2O2 fluxes were generally less than 1% of oxygen fluxes in physiological substrate and coupling states, specifically in permeabilized cells. H2O2 flux was consistently highest in the Complex II-linked LEAK state, reduced with CI&amp;II-linked convergent electron flow and in mitochondria respiring at OXPHOS capacity, and were further diminished in uncoupled mitochondria respiring at electron transfer system capacity. Simultaneous measurement of mitochondrial respiration and H2O2 flux requires careful optimization of assay conditions and reveals information on mitochondrial function beyond separate analysis of ROS production.
authors.affiliation: Laboratory of Pharmaceutical Pharmacology, Latvian Institute of Organic Synthesis, Riga LV1006, Latvia. makrecka@biomed.lu.lv.
authors.forename: Marina
authors.initials: M
authors.lastname: Makrecka-Kuka
authors.affiliation: OROBOROS INSTRUMENTS, Innsbruck 6020, Austria. Gerhard.Krumschnabel@oroboros.at.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Krumschnabel
authors.affiliation: OROBOROS INSTRUMENTS, Innsbruck 6020, Austria. erich.gnaiger@i-med.ac.at.|Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck 6020, Austria. erich.gnaiger@i-med.ac.at.
authors.forename: Erich
authors.initials: E
authors.lastname: Gnaiger
mesh_terms: D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000166:cytology*; D019069:Cell Respiration; D005470:Fluorometry / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D051379:Mice; D008929:Mitochondria, Heart / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010539:Permeability
chemical_list: D006861:Hydrogen Peroxide; D010100:Oxygen
keywords: Amplex Red; H2O2 flux; HEK 293T; high-resolution respirometry; mouse brain homogenate; mouse cardiac mitochondria