Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent formation and breakdown of hydrogen peroxide during mixed function oxidation reactions in liver microsomes

Abstract

Conditions for the recovery of H 2O 2 from microsomes and for determination of the rate and extent of H 2O 2 formation during oxidation of NADPH by liver microsomes have been investigated. H 2O 2 was determined by two methods that are applicable to conditions existing during microsomal mixed function oxidation reactions, provided that contaminating catalase activity is inhibited by azide and that interference by other mixed function oxidation reactions can be excluded. To estimate the formation of H 2O 2 in absence of azide, H 2O 2 was determined indirectly by the production of HCHO during oxidation of cold and 14C-labeled methanol and an excess of exogenous catalase. As additional catalase-independent decomposition of H 2O 2 also occurs during oxidation of NADPH, the kinetics of H 2O 2 formation in microsomes is influenced by two independent processes. H 2O 2 will be produced under optimal conditions i.e., at V when O 2 and NADPH are in excess. Addition or formation of increasing amounts of H 2O 2 raises the substrate (H 2O 2) concentration and will enhance the rate of breakdown of H 2O 2.