Krumschnabel 2014 Abstract MiP2014
|Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production.|
Mitochondrial respiration is associated with the formation of reactive oxygen species, primarily in the form of superoxide (O2•-) and particularly hydrogen peroxide (H2O2). Since H2O2 plays important roles in physiology and pathology, measurement of hydrogen peroxide has received considerable attention over many years. Here we describe how the well-established Amplex Red assay can be used to detect H2O2 production in combination with the simultaneous assessment of mitochondrial bioenergetics by high-resolution respirometry. Fundamental instrumental and methodological parameters were optimized for analysis of the effects of various substrate, uncoupler and inhibitor titrations (SUIT ) on respiration versus H2O2 production. The sensitivity of the H2O2 assay was strongly influenced by compounds present in different mitochondrial respiration media, which exerted significant effects on chemical background fluorescence changes. Near-linearity of the fluorescence signal was restricted to narrow ranges of accumulating resorufin concentrations independent of the nature of mitochondrial respiration media. Finally, we show an application example using mouse brain mitochondria as an experimental model for the simultaneous measurement of mitochondrial respiration and H2O2 production in SUIT protocols.
Labels: MiParea: Respiration
Stress:Oxidative stress;RONS Organism: Mouse Tissue;cell: Nervous system Preparation: Homogenate, Isolated mitochondria
Coupling state: LEAK, OXPHOS, ET Pathway: N, S, NS HRR: Oxygraph-2k, O2k-Fluorometer Event: B4, Poster MiP2014
1-Oroboros Instruments, Innsbruck, Austria; 2-Present address: Pharmacobioch Lab 3rd Dep Intern Medicine, Comenius Univ, Bratislava, Medical Fac; 3-Daniel Swarovski Research Lab, Mitochondrial Physiol, Dep Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck.
References and acknowledgements
Supported by K-Regio project MitoCom Tyrol.
- Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopsies of human muscle. Methods Mol Biol 810:25-58.