Measurement of mitochondrial H₂O₂ production under varying O₂ tensions

Mitochondria-derived reactive oxygen species (ROS) play an important role in the development of several pathologies and are also involved in physiological signaling. Molecular oxygen is the direct substrate of complex IV of the respiratory chain, and at the same time, its partial reduction in mitochondria results in the formation of ROS, mainly H₂O₂. The accurate knowledge of the dependence of H₂O₂ production on oxygen concentration is vital for the studies of tissue ischemia/reperfusion, where the relationship between oxygen availability, respiration, and ROS production is critical. In this chapter, we describe a straightforward and reliable protocol for the assessment of H₂O₂ release by mitochondria at varying oxygen concentrations. This method can be used for any ROS-generating system where the effect of oxygen level on H₂O₂ production needs to be assessed.