Hepatic Microsomal Ethanol Oxidation: Hydrogen Peroxide Formation and the Role of Catalase

Abstract

Microsomes from rat liver form hydrogen peroxide in the presence of an NADPH‐generating system in proportion to protein concentrations as determined by three independent methods: ferrithiocyanate, cytochrome c peroxidase, and scopoletin fluorescence. Maximal rates observed were about 15 μmol H 2 O 2/g microsomal protein per minute. The oxygen concentration for half‐maximal rates was 50 μM. It is suggested that NADPH‐dependent hydrogen peroxide formation in microsomes is mainly due to NADPH oxidase; however, partial inhibition by carbon monoxide suggests that about one third arises from the autoxidation of cytochrome P‐450. Similarities exist between microsomal acetaldehyde production from ethanol (i.e. the microsomal ethanol‐oxidizing system of Lieber and DeCarli [4]) and hydrogen peroxide formation: viz. requirement for NADPH and oxygen, identical oxygen concentrations for halfmaximal rates, and sensitivity to carbon monoxide. Microsomal acetaldehyde production in the presence of either an NADPH‐ or an H 2 O 2‐generating system exhibits identical characteristics as follows: (a) ethanol concentration for half‐maximal rates (i.e. 12 mM); (b) dependency of maximal rates on rates of hydrogen peroxide formation; (c) competitive inhibition by peroxidatic substrates for catalase, e.g. formate (half‐maximal effect: 150 μM); (d) inhibition by catalase inhibitors, e.g. azide (half‐maximal effect: 50 μM), with identical azide insensitive rates; (e) diminished acetaldehyde production in microsomes from rats pretreated with aminotriazole or pyrazole with identical residual rates. Moreover, NADPH‐dependent acetaldehyde production is suppressed in the presence of an active H 2 O 2‐utilizing system. Thus, it is concluded that the NADPH‐dependent microsomal ethanol‐oxidizing system of Lieber and DeCarli [4] is due to a hydrogen peroxide formation from NADPH and a subsequent peroxidation of ethanol by contaminating catalase. The data indicate that the existence of a unique system in addition to the peroxidatic reaction of catalase as postulated recently [4] is highly doubtful.