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

Abstract

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.