title: Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production.
abstract: 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 contained in different mitochondrial respiration media, which also 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 isolated mouse brain mitochondria as an experimental model for the simultaneous measurement of mitochondrial respiration and H2O2 production in SUIT protocols.
authors: OROBOROS INSTRUMENTS, Innsbruck, Austria.
authors: Gerhard
authors: G
authors: Krumschnabel
authors: Mona
authors: M
authors: Fontana-Ayoub
authors: Zuzana
authors: Z
authors: Sumbalova
authors: Juliana
authors: J
authors: Heidler
authors: Kathrin
authors: K
authors: Gauper
authors: Mario
authors: M
authors: Fasching
authors: Erich
authors: E
authors: Gnaiger
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D019069:Cell Respiration / Q000187:drug effects; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010101:Oxygen Consumption; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide