High-Resolution Respirometry for Simultaneous Measurement of Oxygen and Hydrogen Peroxide Fluxes in Permeabilized Cells, Tissue Homogenate and Isolated Mitochondria
1
Laboratory of Pharmaceutical Pharmacology, Latvian Institute of Organic Synthesis, Riga LV1006, Latvia
2
OROBOROS INSTRUMENTS, Innsbruck 6020, Austria
3
Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck 6020, Austria
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Academic Editors: Michael Breitenbach and Peter Eckl
Biomolecules 2015, 5(3), 1319-1338; https://doi.org/10.3390/biom5031319
Received: 7 April 2015 / Revised: 8 June 2015 / Accepted: 8 June 2015 / Published: 29 June 2015
(This article belongs to the Special Issue Oxidative Stress and Oxygen Radicals)
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&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.
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Keywords:
high-resolution respirometry; H2O2 flux; Amplex Red; HEK 293T; mouse brain homogenate; mouse cardiac mitochondria
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MDPI and ACS Style
Makrecka-Kuka, M.; Krumschnabel, G.; Gnaiger, E. High-Resolution Respirometry for Simultaneous Measurement of Oxygen and Hydrogen Peroxide Fluxes in Permeabilized Cells, Tissue Homogenate and Isolated Mitochondria. Biomolecules 2015, 5, 1319-1338.
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