Multidimensional mitochondrial energetics: Application to the study of electron leak and hydrogen peroxide metabolism

Comparative studies of mitochondrial reactive oxygen species in animal longevity: Technical pitfalls and possibilities

“…The rate of superoxide/H 2 O 2 formation has been shown to decline with time after isolation of mitochondria from rat skeletal muscle. This effect seems to primarily involve superoxide production at site I Q as it is observed in the presence of succinate as the sole respiratory substrate, but absent when rotenone is added (Treberg, Braun, Zacharias, & Kroeker, ). Preliminary tests for our recent study (Munro, Baldy, Pamenter, & Treberg, ) showed that the time spent (in the absence of substrates) by mice skeletal muscle mitochondria inside the chambers of the Oroboros, before addition of substrates, also leads to a pronounced decline in the capacity for superoxide/H 2 O 2 generation at site I Q (succinate alone, Figure ).…”

“…Finally, the affinity of cytochrome C oxidase for oxygen seems much higher than that of important ROS production sites, particularly for site I Q (Treberg et al, ). This means that the rate of H 2 O 2 formation will diminish before (below 80 µM molecular oxygen) oxygen consumption rates significantly decline (Treberg et al, ).…”

“…Finally, the affinity of cytochrome C oxidase for oxygen seems much higher than that of important ROS production sites, particularly for site I Q (Treberg et al, ). This means that the rate of H 2 O 2 formation will diminish before (below 80 µM molecular oxygen) oxygen consumption rates significantly decline (Treberg et al, ). This whim of mitochondrial metabolism can be an issue when the fluorescence assay is conducted in closed chambers so as to monitor oxygen consumption in parallel, for example, as is commonly done with an oxygraph Oroboros 2K.…”

“…One limitation of using CDNB and auranofin, however, is potential species‐specific off‐target effects (Treberg et al, ). For example, in our recent study (Munro et al, ), the protocols developed for rat skeletal muscle mitochondria required adaptation to work with murine mitochondria, which generally seem more fragile (Treberg et al, ).…”

“…One limitation of using CDNB and auranofin, however, is potential species‐specific off‐target effects (Treberg et al, ). For example, in our recent study (Munro et al, ), the protocols developed for rat skeletal muscle mitochondria required adaptation to work with murine mitochondria, which generally seem more fragile (Treberg et al, ). Specifically, the concentration of auranofin was reduced from 2 to 0.5 µM, and the duration of the CDNB pretreatment was reduced from five to four minutes reaction time at room temperature in order to respect our threshold of a 5% maximal effect on coupled respiration in convergent electron flow (glutamate + malate + succinate + ADP).…”