Hepatic Microsomal Ethanol Oxidation

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 pmol H,O,/g microsomal protein per minute. The oxygen concentration for half-maximal rates was 50 pM. 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,O,-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 mM); (d) inhibition by catalase inhibitors, e.g. azide (half-maximal effect : 50 pM), 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,O,-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.

Metabolic alterations produced in the liver by chronic ethanol administration. Increased oxidative capacity

“…Dinitrophenol-activated ATPase: controls, 88.8 ±14.6 (5); chronically ethanol-treated, 90.2±9.8 (5)]. Lieber & de Carli (1970a) and Carter & Isselbacher (1971) have reported that the liver microsomal NADPH oxidase, an enzyme system which produces H202 (Gillette, et al, 1957;Thurman et al, 1972), is increased after chronic ethanol treatment. An increased rate of H202 production might be in part responsible through the catalase mechanism for the greater rate of ethanol metabolism after chronic ethanol treatment.…”

“…It appears, therefore, that in vivo the production of H202 is rate-limiting (Aebi, 1960;Portwich & Abei, 1960). Lieber & De Carli (1970a) and Carter & Isselbacher (1971) have reported that a microsomal NADPH oxidase, an enzyme system that can produce H202 (Gillette et al, 1957;Thurman et al, 1972), is increased in rats treated chronically with ethanol. Thus an increased H202 production could conceivably cause an increased metabolism of ethanol by catalase.…”

Lipoteichoic acid-antilipoteichoic acid complexes induce superoxide generation by human neutrophils

“…H202 was determined by the method of Thurman et al (51). Briefly, to stimulated PMNs in HBSS + azide, 200/~1 of TCA (30%) were added.…”

Defence of Rhizobium etli bacteroids against oxidative stress involves a complexly regulated atypical 2‐Cys peroxiredoxin

“…At different time points, samples were taken and the reaction was terminated by adding 12.5% (w/v) TCA. Peroxide concentration was determined with the ferrithiocyanate assay (Thurman et al, 1972).…”