Lester A. Reinke

Co-regulation of the mixed-function oxidation of p-nitroanisole and glucuronidation of p-nitrophenol in the perfused rat liver by carbohydrate reserves.

Maximal rates of mixed-function oxidation of p-nitroanisole and the glucuronidation of p-nitrophenol in perfused livers from phenobarbital-treated rats varied directly with the nutritional state of the rat (i.e., fasted < fed < fasted-refed). Rates correlated with intracellular concentrations of NADPH, UDP-glucuronic acid, and glycogen but not with amounts of cytochrome P-450 or glucuronyltransferase activity. These data support the hypothesis that mixed-function oxidation and glucuronidation are coregulated in intact cells by carbohydrate-dependent cofactor synthesis.

Stimulation of p-nitroanisole O-demethylation in perfused livers by xylitol and sorbitol.

Xylitol and sorbitol, two sugar alcohols which readily enter into pathways of hepatic carbohydrate metabolism, stimulated p-nitroanisole O-demethylation in perfused livers from fasted, but not fed, phenobarbital-treated rats. The increase in mixed-function oxidation correlated well with the production of NADH from the metabolism of xylitol and sorbitol (half-maximal stimulation for both processes was observed with concentrations between 0.1 and 0.2 mM). p-Nitroanisole metabolism by isolated hepatic microsomes was unaffected by the addition of xylitol and sorbitol; however, when NADH was added to microsomes, or was generated from sorbitol, sorbitol dehydrogenase and NAD(+), a synergistic increase in p-nitroanisole metabolism occurred. Ethanol (0.2 mM), which does not enter into pathways of carbohydrate metabolism, also caused an increase in the pyridine nucleotide redox state and stimululated p-nitroanisole O-demethylation in livers from fasted rats. In addition, sorbitol and xylitol stimulated p-nitrophenol conjugation in livers from fasted, phenobarbital-treated animals, probably by supplying substrate for increased UDP-glucuronic acid synthesis. The data indicate that sugars which influence the pyridine nucleotide redox state alter rates of mixed-function oxidation and conjugation in whole cells.

Regulation of NADPH-dependent mixed-function oxidation in perfused livers: Comparative studies with sorbitol and ethanol☆

Sorbitol and ethanol were shown to have opposite effects on p-nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats. Sorbitol (2 mM) stimulated drug metabolism by 50% while ethanol (20 mM) caused 80% inhibition. Both sorbitol and ethanol infusion decreased the NAD+/NADH ratio and increased fluorescence of pyridine nucleotides monitored from the liver surface; however, the NADP+/NADPH ratio was decreased by sorbitol but tended to be increased by ethanol. Stimulation of drug metabolism by sorbitol was abolished by pretreatment of fasted rats with 6-aminonicotinamide, an inhibitor of the pentose phosphate shunt, but was not affected by aminooxyacetate, a transaminase inhibitor. These results indicate that sorbitol stimulated p-nitroanisole metabolism by providing NADPH via the pentose phosphate shunt. Ethanol and sorbitol caused changes in intracellular concentrations of NADPH in livers from fasted rats which correlated directly with changes in hepatic levels of citrate and aspartate. Furthermore, aspartate infusion reduced the inhibition of p-nitroanisole O-demethylation by ethanol. This inhibition was also reversed partially by sorbitol in livers from 6-aminonicotinamide-treated rats. It is concluded that ethanol inhibits mixed-function oxidation primarily by decreasing the concentrations of citric acid cycle intermediates which leads to depletion of cytosolic NADPH.

Inhibition of mixed-function oxidation of p-nitroanisole in perfused rat liver by 2,4-dinitrophenol☆

Abstract The effect of dinitrophenol (52 μ m ), an uncoupler of oxidative phosphorylation, on p-nitroanisole O-demethylation in the perfused rat liver was examined. Dinitrophenol inhibited p-nitroanisole metabolism 70% in perfused livers from fasted, phenobarbital-treated rats, and 30% in livers from normal rats, but had no effect on this reaction in isolated microsomes. Rates of p-nitroanisole O-demethylation in livers from fed, phenobarbitaltreated rats were not inhibited by dinitrophenol unless the pentose phosphate shunt was first inhibited by 6-aminonicotinamide pretreatment. Dinitrophenol diminished cellular concentrations of ATP and NADPH 30 and 50%, respectively. Since mixed-function oxidation requires NADPH, these data are consistent with the hypothesis that dinitrophenol interrupts the synthesis and/or transfer of reducing equivalents from the mitochondria into the extramitochondrial space by interfering with energy-dependent NADPH synthesis and substrate shuttle mechanisms. In addition, dinitrophenol diminished conjugation reactions 57 and 89% in all metabolic states studied, most likely because it decreased UDP-glucose levels considerably (40 to 60%).

Effects of 3-methylcholanthrene on oxidized nicotinamide-adenine dinucleotide phosphatedependent dehydrogenases and selected metabolites in perfused rat liver.

Treatment of adult rats with 3-methylcholanthrene over a 3-day period produced significant decreases in hepatic ATP concentrations and elevated the activities of 6-phosphogluconate dehydrogenase and malic enzyme. The decline in ATP was accompanied by a decrease in total adenine nucleotides; however, ATP/ADP ratios were essentially the same in livers of normal and 3-methylcholanthrene-treated rats, and no significant changes were noted in glycolytic and citric acid cycle intermediates. Thus, hepatic energy metabolism does not appear to be altered grossly after 3-methylcholanthrene treatment. NADP(+)/NADPH ratios calculated from substrates assumed to be in near equilibrium with malic enzyme, isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase increased after 3-methylcholanthrene treatment; these changes were accompanied by significant increases in the activity of malic enzyme and 6-phosphogluconate dehydrogenase. Oxidation of hepatic NADPH and elevation of activities of several NADPH-generating enzymes by 3-methylcholanthrene indicates that this agent which induces components of the mixed-function oxidase system also elevates the capacity of the liver to form NADPH.

Inhibition of p-nitroanisole O-demethylation in perfused rat liver by oleate

p-Nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats was rapidly and reversibly inhibited by sodium oleate (0.3 to 0.6 mM). Xylitol partially reversed this inhibitory effect. The inhibition was not mediated by a direct effect of oleate on microsomal components since concentrations of oleate ranging up to 1.0 mM did not affect p-nitroanisole O-demethylation by isolated microsomes. Infusion of 0.6 mM oleate did not alter the measured intracellular NAD+/NADH ratio but did cause a significant increase in the intracellular NADP+/NADPH ratio. A significant decrease in the ATP/ADP ratio was also observed. Oleoyl CoA inhibited p-nitroanisole O-demethylation in microsomes (Ki about 30 microM), and both oleoyl CoA and palmitoyl CoA inhibited the energy-linked nicotinamide nucleotide transhydrogenase in submitochondrial particles (Ki about 1 microM). Thus, inhibition of mixed-function oxidation in the intact liver by oleate is most likely mediated by oleoyl CoA. Oleoyl CoA inhibits mixed-function oxidation in the intact liver by acting directly on cytochrome P-450 and by decreasing generation of NADPH via inhibition of key enzymes of the citric acid cycle and the energy-linked transhydrogenase.

Oxidation-reduction state of free NADP+ during mixed-function oxidation in perfused rat livers—Evaluation of the assumptions of near equilibrium by comparisons of surface fluorescence changes and calculated NADP+:NADPH ratios

Abstract Changes in cellular pyridine nucleotide and flavoprotein oxidation-reduction states associated with mixed-function oxidation of p -nitroanisole, hexobarbital and aminopyrine by perfused rat livers were studied. Surface fluorescence techniques were compared with NAD(P) + /NAD(P)H ratios calculated from substrates assumed to be in near equilibrium with various dehydrogenases in freeze-clamped liver samples. p -Nitroanisole and p -nitrophenol caused a large decrease in pyridine nucleotide (366 → 450 nm) fluorescence as a result of fluorescence quenching. This decrease, therefore, did not reflect oxidation of pyridine nucleotides. Moreover, p -nitroanisole infusion decreased the free NADP + :NADPH ratios calculated from malic enzyme and isocitrate dehydrogenase. Hexobarbital, which did not produce fluorescence quenching, caused an oxidation in pyridine nucleotides as indicated by both a decrease in surface fluorescence and an increase in the calculated NADP + /NADPH ratio. These data indicate that free NADP + /NADPH ratios calculated from substrates, which are assumed to be in near equilibrium with NADPH-generating enzymes, indeed reflect the NADPH redox state in intact liver cells.

Maintenance of nicotinamide dinucleotide phosphate content and oxidation-reduction state during mixed-function oxidation of p-nitroanisole in isolated perfused livers of various species

The influence of p-nitroanisole, a substrate for mixed-function oxidation, on total NADP+ and NADPH and NADP+/NADPH ratios was examined in perfused livers from three different species. Studies were performed using livers from Sprague-Dawley rats, Syrian golden hamsters and C57BL/6J mice. Although rates of p-nitroanisole O-demethylation varied more than 16-fold in perfused livers from these species, NADP+/NADPH ratios calculated from measured concentrations of NADP+ and NADPH and from ratios calculated from substrate pairs assumed to be in near equilibrium with NADP+-dependent dehydrogenases remained remarkably constant under most conditions. Thus, rates of NADPH utilization and generation must be tightly coupled in perfused livers during high rates of mixed-function oxidation. Exceptions to the general pattern noted above occurred in livers of fasted, phenobarbital-treated rats where carbohydrate reserves were depleted and in livers from 3-methyl-cholanthrene-treated mice where rates of p-nitroanisole O-demethylation were exceptionally high. Livers from fed phenobarbital-treated rats displayed a paradoxical decrease in NADP+/NADPH ratios reflecting reduction calculated from substrates assumed to be in near equilibrium with 6-phosphogluconate dehydrogenase during mixed-function oxidation, suggesting that NADPH generation exceeded NADPH utilization in the rat in the fed state. In contrast, the NADP+/NADPH ratio calculated from measured pyridine nucleotides increased in livers of 3-methylcholanthrene-treated mice perfused with p-nitroanisole, reflecting oxidation. Moreover, the NADP+/NADPH ratio calculated from substrates assumed to be near equilibrium with 6-phosphogluconate dehydrogenase increased in livers of fasted rats, suggesting that utilization of NADPH exceeded generation. Thus, adequate carbohydrate reserves appear essential for maintenance of NADPH during high rates of mixed-function oxidation.

THE INFLUENCE OF THE NUTRITIONAL STATE ON RATES OF p-NITROANISOLE O-DEMETHYLATION AND p-NITROPHENOL CONJUGATION IN PERFUSED RAT LIVERS

p-Nitroanisole (PNA) O-demethylation and the subsequent conjugation of p-nitrophenol (PNP) were studied in perfused livers from fed, fasted, and fasted-refed control and phenobarbital (PB) -treated rats. In livers from phenobarbitaltreated rats, rates of mixed-function oxidation varied with the glycogen content of the liver (fasted<fed<fasted-refed) but not with changes in microsomal metabolism. A direct correlation was found between rates of mixed-function oxidation and NADPH/NADP ratios, as well as with intracellular concentrations of isocitrate and malate. Conjugation of p-nitrophenol also varied with the glycogen content of the liver but not with glucuronyl transferase activity or ATP/ADP ratios.

Mechanism of inhibition of mixed-function oxidation by ethanol.

p-Nitroanisole (PNA) O-demethylation in perfused rat livers was inhibited by low concentrations of ethanol (Ki = 1 mM) and acetaldehyde (Ki = 0.5 mM). Ethanol and acetaldehyde also inhibited microsomal PNA metabolism, but only at much higher concentrations (Ki = 130 mM and 5 mM, respectively). Moreover, the dose-dependent inhibition of PNA metabolism in the perfused liver correlated strongly with changes in surface fluorescence of pyridine nucleotides and flavoproteins resulting from the metabolism of ethanol and acetaldehyde. t-Butanol (20 mM) did not cause pyridine nucleotide or flavoprotein reduction, nor did it inhibit PNA metabolism. When ethanol metabolism was blocked with 4-methylpyrazole, the inhibition of PNA metabolism by ethanol was diminished.