Catalase‐dependent ethanol oxidation in perfused rat liver
Authors: HANDLER Jeffrey A. , THURMAN Ronald G. 
Published: March 2005

The purpose of this study was to measure rates of catalase‐dependent ethanol uptake and rates of H2O2 generation in perfused rat livers in the presence of fatty acids of varying chain length. Rates of ethanol uptake in livers from fasted rats, perfused in a recirculating system, of about 80 μmol g−1 h−1 were decreased to about 10 μmol g−1 h−1 by the addition of an inhibitor of alcohol dehydrogenase (ADH), 4‐methylpyrazole. The medium‐chain‐length fatty acid, laurate (12:0; 1 mM), increased rates of 4‐methylpyrazole‐insensitive ethanol uptake maximally to 80‐85 μmol g−1 h−1. Rates of ethanol uptake diminished as the chain length of fatty acid was decreased [hexanoate (6:0) = 23 μmol g−1 h−1; octanoate (8:0) = 55 μmol g−1 h−1; decanoate (10:0) = 65 μmol g−1 h−1] or increased [myristate (14:0) = 77 μmol g−1 h−1; palmitate (16:0) = 80 μmol g−1 h−1; stearate (18:0) = 29 μmol g−1 h−1; oleate (18:1) = 60 μmol g−1 h−1; erucate (22:3) = 22 μmol g−1 h−1] from 12:0. Oleate did not increase rates of hydroxylation ofp‐nitrophenol, a substrate for the ethanol‐inducible form of cytochrome P‐450, indicating that the stimulation of ethanol uptake by fatty acids was not due to increased mixed‐function oxidation. The increase of ethanol uptake was also not due to displacement of 4‐methylpyrazole from ADH by fatty acids, since oleate stimulated ethanol uptake by about 50% in perfused livers from deermice genetically deficient in ADH. The increase in 4‐methylpyrazole‐insensitive ethanol uptake by fatty acids was blocked by the catalase inhibitor, aminotriazole, indicating the involvement of catalase. Rates of H2O2 generation by livers perfused in a non‐recirculating system with 1.7% albumin were increased from 6:1 to 23:5 μmol g−1 h−1 by oleate (1 mM). Because of the discrepancy between rates of ethanol metabolism and H2O2 production, methods were developed to measure H2O2 production in a recirculating perfusion system. H2O2 generation was determined from the time necessary for steady‐state level of catalase‐H2O2, measured spectrophotometrically (660 640 nm) through a lobe of the liver, to return to basal values after the addition of a known quantity of methanol, which is not metabolized by ADH in the rat. Basal rates of H2O2, generation measued under these conditions were 12:5 μmol g−1 h−1, values similar to basal rates of 4‐methylpyrazole‐insensitive ethanol uptake. Rates of H2O2 generation were stimulated maximally by Iaurate to about 80‐85 μmol g−1 h−1, and decreased as the fatty acid chain length was decreased or increased, analogous to the stimulation of 4‐methylpyrazole‐insensitive ethanol uptake by fatty acids. There was an excellent correlation (r = 0.99) between rates of H2O2 generation, and rates of 4‐methylpyrazole‐insensitive ethanol uptake with all fatty acids studied. Thus, increases in H2O2 generation and ethanol oxidation have similar chain‐length specificities for fatty acids. Rates of H2O2 generation from oleate of about 60 μmol g−1 h−1 were decreased 50% by an inhibitor of peroxisomal β‐oxidation, thioridazine (0.02 mM). Rates were also decreased by 60% by decreasing the albumin concentration in the recirculating system from 4.0% to 1.7%. Rates of ketone body production from oleate were half‐maximal at about 0.13 mM oleate; maximal rates were about 90 μmol g−1 h−1. In contrast, rates of H2O2 production required higher concentrations of fatty acids (e.g., half‐maximal rates were observed with about 0.65 mM oleate; maximal rates were around 60 μmol g−1 h−1). Thus, fatty acyl‐CoA compounds appear to be taken up by peroxisomes at high rates only when uptake into mitochondria is saturated. Hepatic fatty acid oxidation was estimated to be mediated about 25% via peroxisomal β‐oxidation in these studies. Taken together, these data indicate that fatty acids can provide H2O2 for catalase‐dependent peroxidation of ethanol in the perfused liver at high rates.