Catalase‐dependent ethanol oxidation in perfused rat liver

Abstract

The purpose of this study was to measure rates of catalase-dependent ethanol uptake and rates of HzOz 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 pmol g-' h-' were decreased to about 10 pmol g-' h-' 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][81][82][83][84][85] Oleate did not increase rates of hydroxylation of p-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 H z 0 2 generation by livers perfused in a non-recirculating system with 1.7% albumin were increased from 6 f 1 to 23 f. 5 pmol g-' h-' by oleate (1 mM). Because of the discrepancy between rates of ethanol metabolism and HzOz production, methods were developed to measure H z 0 2 production in a recirculating perfusion system. H 2 0 2 generation was determined from the time necessary for steady-state level of catalase-H20z, 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 H 2 0 2 generation measued under these conditions were 12 f 5 pmol g-' h-', values similar to basal rates of 4-methylpyrazole-insensitive ethanol uptake.Rates of HzOz generation were stimulated maximally by laurate to about 80 -85 pmol g-' h-' , and decreased as the fatty acid chain length was decreased or increased, analogous to the stimulation of 4-methylpyrazoleinsensitive ethanol uptake by fatty acids. There was an excellent correlation (r = 0.99) between rates of HzOz generation and rates of 4-methylpyrazole-insensitive ethanol uptake with all fatty acids studied. Thus, increases in H 2 0 2 generation and ethanol oxidation have similar chain-length specificities for fatty acids. Rates of H 2 0 2 generation from oleate of about 60 pmol g-' h-' were decreased 50% by an inhibitor of peroxisomal p-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 pmol g-' h-'. In contrast, rates of H 2 0 2 production required higher concentrations of fatty acids (e.g., half-...

Response of Hepatic Mitochondrial and Peroxisomal β-Oxidation to Increasing Palmitate Concentrations in Piglets

“…Lower rates of mitochondrial poxidation in piglet liver suggest that entry of palmitate into mitochondria may have been limited by carnitine palmitoyltransferase I (EC 2.3.1.21) activity. However, the progres sively increased CCF production with in crease of palmitate concentration and the greater ratio of CCF production to mitochon drial P-oxidation in piglet liver (33.7%) than in adult pig liver (13.1 %) at 1.0 mM palmitate may indicate an intramitochondrial limita tion of ketogenesis in the neonate, as suggest ed elsewhere [3,4], Our results for rats are in general agree ment with earlier work suggesting that peroxi somal p-oxidation in liver increases more slowly than mitochondrial P-oxidation as the concentration of free fatty acids or fatty acylCoA increases [10,26,27], Others have noted that total oxidation of palmitate by isolated rat hepatocytes increased as the concentration of palmitate in the medium increased [26], We found that 14CO: production and total mitochondrial P-oxidation in rats increased as the palmitate concentration increased to 1.0 mM, but the ratio of 14CC>2 production to mitochondrial P-oxidation was <25% and generally decreased with increase of palmitate concentration. These results suggest that re sponses of mitochondrial p-oxidation to in 290 Biol Neonate 1997;72:284-292…”

“…Previous reports indicated increased ketogenesis by perfused rat liver as the concentration of oleate in the perfusate increased [27], Because we used fed rats, the response of mitochon drial ß-oxidation to increasing palmitate may be confounded to some degree by progressive ly greater inhibition of acetyl-CoA carboxy lase (EC 6.4.1.2) as palmitate or palmitoylCoA concentrations increased, which could lead to decreased concentrations of malonylCoA and thus greater flux through carnitine palmitoyltransferase I [13]. We found that the percentage contribution of peroxisomal ß-oxidation to total ß-oxidation capacity in rats was highly dependent on substrate concentration.…”

Sustained formation of α-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts in mouse liver by peroxisome proliferators is dependent upon peroxisome proliferator-activated receptor-α, but not NADPH oxidase

“…Furthermore, peroxisomal β-oxidation is limited by substrate availability. In fact, it has been shown that treatment with peroxisome proliferators increased H 2 O 2 in vitro, but not in the perfused liver because fatty acid supply is rate-limiting in intact cells [35,36]. Indeed, the timing of the increases in radical production observed in this study did not correlate with that for the induction of ACO protein level and activity.…”

The role of transferrin in the mechanism of cellular iron uptake

“…As to the mechanism by which iron accumulation may interfere with hepatocyte function, most studies focus on the avidity with which iron may accelerate the formation of toxic oxygen species [121]. Iron and ethanol have additive effects on lipid peroxidation [121][122][123]. A possible sequence of reactions is the following: intake of ethanol produces NADH via alcohol dehydrogenase, this in turn mobilizes iron from ferritin, and non-haem iron thus mobilized catalyses the production of toxic oxygen species, of which OH is the most potent.…”