Krumschnabel Gerhard, Fontana-Ayoub Mona, Sumbalova Zuzana, Heidler Juliana, Gauper Kathrin, Fasching Mario, Gnaiger Erich
OROBOROS INSTRUMENTS, Innsbruck, Austria.
Methods Mol Biol. 2015;1264:245-61. doi: 10.1007/978-1-4939-2257-4_22.
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.
线粒体呼吸与活性氧的形成有关，主要以超氧阴离子（O2(•-)）的形式存在，尤其是过氧化氢（H2O2）。由于H2O2在生理和病理过程中发挥着重要作用，多年来，过氧化氢的测量受到了广泛关注。在这里，我们描述了如何使用成熟的Amplex Red检测法结合高分辨率呼吸测定法同时评估线粒体生物能量学来检测H2O2的产生。对基本的仪器和方法参数进行了优化，以分析各种底物、解偶联剂和抑制剂滴定（SUIT）对呼吸与H2O2产生的影响。H2O2检测的灵敏度受到不同线粒体呼吸培养基中所含化合物的强烈影响，这些化合物也对化学背景荧光变化产生显著影响。荧光信号的近线性仅限于窄范围的试卤灵积累浓度，与线粒体呼吸培养基的性质无关。最后，我们展示了一个应用实例，使用分离的小鼠脑线粒体作为实验模型，在SUIT方案中同时测量线粒体呼吸和H2O2的产生。
