Estimation of the hydrogen peroxide producing capacities of liver and cardiac mitochondria isolated from C57BL/6N and C57BL/6J mice.

Here, we examined the hydrogen peroxide (H2O2) producing capacities of pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGDH), proline dehydrogenase (PRODH), glycerol-3-phosphate dehydrogenase (G3PDH), succinate dehydrogenase (SDH; complex II), and branched-chain keto acid dehydrogenase (BCKDH), in cardiac and liver mitochondria isolated from C57BL/6N (6N) and C57BL/6J (6J) mice. Various inhibitor combinations were used to suppress ROS production by complexes I, II, and III and estimate the native rates of H2O2 production for these enzymes. Overall, liver mitochondria from 6N mice produced ∼2-fold more ROS than samples enriched from 6J mice. This was attributed, in part, to the higher levels of glutathione peroxidase-1 (GPX1) and catalase (CAT) in 6J mitochondria. Intriguingly, PDH, KGDH, and SDH comprised up to ∼95% of the ROS generating capacity of permeabilized 6N liver mitochondria, with PRODH, G3PDH, and BCKDH making minor contributions. By contrast, BCKDH accounted for ∼34% of the production in permeabilized 6J mitochondria with KGDH and PRODH accounting for ∼23% and ∼19%. G3PDH produced high amounts of ROS, accounting for ∼52% and ∼39% of the total H2O2 generating capacity in 6N and 6J heart mitochondria. PRODH was also an important ROS source in 6J mitochondria, accounting for ∼43% of the total H2O2 formed. In addition, 6J cardiac mitochondria produced significantly more ROS than 6N mitochondria. Taken together, our findings demonstrate that these other generators can also serve as important sources of H2O2. Additionally, we found that mouse strain influences the rate of production from the individual sites that were studied.