Conjugation Of P-nitrophenol Research Articles

Kinetic characterization of glycosidase activity from disaccharide conjugate to monosaccharide conjugate in Caco-2 cells

Glycosidase activity influences the intestinal absorption of glycosides. Our previous study in rats suggested that disaccharide conjugates might be prototypes for pre-prodrugs aiming at the Na(+)/glucose co-transporter-mediated transport of prodrugs (drug glucoside) as a novel absorption pathway. One of the crucial factors is the formation of a glucoside drug from the disaccharide conjugate. Since there is a large species difference in metabolism, it is necessary to examine the cells and/or enzymes derived from human tissue to confirm this concept. In this paper, we kinetically characterized the glycosidase activity of disaccharide conjugates in Caco-2 cells. Disaccharide conjugates of p-nitrophenol (p-NP) (p-NP beta-cellobioside, p-NP beta-lactoside and p-NP beta-maltoside) were hydrolysed to p-NP beta-glucoside. beta-glucosidase or beta-galactosidase (lactase/phloridzin hydrolase, LPH) and alpha-glucosidase (sucrase-isomaltase) had different pH-dependent activities for disaccharide conjugates. At neutral pH, LPH has low affinity and low capacity, and sucrase-isomaltase has high affinity and high capacity, whereas at acid pH, LPH has high affinity and low capacity, and sucrase-isomaltase has low affinity and high capacity. The hydrolysis clearance calculated with Vmax/Km indicated that sucrase-isomaltase activity is much higher than LPH activity at either neutral or acid pH in Caco-2 cells. Since the hydrolysis rate of the disaccharide conjugate was highly dependent on the pH value and type of glycoside linkage, the appropriate selection of a glycoside form after consideration of these differences is the key to designing a sugar-conjugate prodrug.

Role of Transporter and Metabolic Enzyme in Sequential and Competitive Process of Intestinal Absorption: Study on SGLT1, Disaccharidase and Peptidases.

We have studied intestinal metabolism and transport, which is considered to be sequential (1) or competitive (2) process in absorption (Scheme 1). (1) Disaccharide (maltose, cellobiose, lactose) conjugates of p-nitrophenol were hydrolyzed to p-nitrophenyl β-glucosides (p-NPβglc) on the mucosal side. p-NPβglc was transported by Na+/glucose cotransporter (SGLT1). Transport clearance of p-NPβglc formed from cellobiose and lactose conjugates of p-NP were higher than that from maltose or of p-NPβglc itself. These results suggest that SGLT1 is cooperatively coupled with lactase/phloridzin hydrolase catalyzing hydrolysis of cellobiose and lactose conjugates. There might be cooperative relationship between peptidase and H+/oligopeptide cotransporter or amino acid transporter as well. (2) Kyotorphin (KTP) was too unstable in intestine to be absorbed. KTP appeared on the serosal side in the presence of peptidase inhibitors. Meanwhile, cyclic KTP was stable in intestine to be absorbed. Absorption clearance of cyclic KTP was higher than the overall transport clearance of KTP, which was calculated according to the metabolic inhibition model. Competitive process was observed in intestinal absorption of α-naphthol as well. These results indicate that metabolism degradation and membrane transport are competitive. Unless drug is stabilized against metabolic enzyme, intestinal absorption of drug can not be improved even if membrane transport is increased.

ROLE OF TRANSPORTER AND METAOBLIC ENZYME IN INTESTINAL DRUG ABSORPTION

We have studied intestinal metabolism and transport, which is considered as sequential (1) or competitive (2) process in absorption (Scheme 1). (1) Disaccharide (maltose, cellobiose, lactose) conjugates of p-nitrophenol were hydrolyzed to p-nitrophenyl β-glucosides (p-NPβglc) on the mucosal side. p-NPβglc was transported by Na+/glucose cotransporter (SGLT1). Transport clearance of p-NPβglc formed from cellobiose and lactose conjugates of p-NP were higher than that from maltose or of p-NPβglc itself. These results suggest that SGLT1 may be cooperatively coupled with lactase/phloridzin hydrolase catalizing hydrolysis of cellobiose and lactose conjugates. There might be cooperative relationship between peptidase and H+/oligopeptide cotransporter or amino acid transporter as well. (2) Kyotorphin (KTP) was too unstable in intestine to be absorbed. KTP appeared on the serosal side in the presence of peptidase inhibitors. Meanwhile, cyclic KTP was stable in intestine to be absorbed. Absorption clearance of cyclic KTP was higher than the overall transport clearance of KTP, which was calculated according to the metabolic inhibition model. These results indicate that metabolism degradation and membrane transport are competitive. Unless drug is stabilized against metabolic enzyme, intestinal absorption of drug can not be improved even if membrane transport is increased.

Intestinal Na +/glucose cotransporter-mediated transport of glucose conjugate formed from disaccharide conjugate

Intestinal absorption of β-disaccharide (cellobiose, maltose and lactose) conjugates of p-nitrophenol ( p-nitrophenyl β-disaccharide) were examined in terms of the hydrolysis of disaccharide conjugate to monosaccharide conjugate and the transport of monosaccharide conjugate by Na +/glucose transport carrier (SGLT1). β-Cellobioside, β-maltoside and β-lactoside of p-nitrophenol ( p-NP) were hydrolyzed to p-nitrophenyl β-glucoside ( p-NPβglc) on the mucosal side, and p-NPβglc appeared on the serosal side. Although p-NP β-disaccharide, p-NP and p-NP glucuronide also appeared on the serosal side, their amounts were much lower than that of p-NPβglc. The amount of p-NPβglc transported to the serosal side was decreased in the presence of phloridzin (transport inhibitor of SGLT1) and in the absence of Na + (a cosubstrate of SGLT1), indicating that p-NPβglc was formed from p-NP β-disaccharide on the mucosal side and transported to the serosal side by SGLT1. Furthermore, the absorption clearance of p-NPβglc, which was formed from p-NP β-cellobioside and p-NP β-lactoside by lactase-phloridzin hydrolase (LPH), was much higher than that of p-NPβglc itself, although the absorption clearance of p-NPβglc, which was formed from p-NP β-maltoside by maltase was similar to that of p-NPβglc itself. These results indicated that p-NPβglc was transported by the vectorial cooperation of SGLT1 with LPH from mucosal p-NP β-cellobioside or p-NP β-lactoside.

Effect of aging on mixed-function oxidation and conjugation by isolated perfused rat livers

Aging is known to decrease hepatic cytochrome P450 content in rats. However, limited information is available on the effects of aging on mixed-function oxidation and conjugation in intact liver. The purpose of these studies was to determine the effects of aging on oxidation and conjugation of p-nitrophenol (pNP) in perfused livers from male Sprague-Dawley rats. Livers from senescent (22–24 months) or young adult (3–6 months) rats were perfused in a nonrecirculating hemoglobin-free system and supplemented with pNP (60 μM). Glucuronide and sulfate conjugates of the oxidation product, 4-nitrocatechol, in effluent perfusate were cleaved enzymatically and 4-nitrocatechol was determined colorimetrically. Rates of 4-nitrocatechol production were decreased in senescent compared with young adult rats (0.67 ± 0.14 vs 0.92 ± 0.15 μmol/ g/ hr). However, the rates of oxidation of pNP in microsomes from senescent rats were similar to those in young adult rats. Hepatic malate content was decreased approximately 50% in livers from senescent compared with young adult rats in the presence and absence of pNP, suggesting that movement of reducing equivalents from the mitochondria to the cytosol, and thus cytosolic NADPH supply, may have been diminished by senescence. The rates of conjugation of 60 μM pNP in perfused livers from senescent rats were similar to those in young adult rats, but K m and V max values of microsomal 4-nitrocatechol glucuronyltransferase were about 2.5- and 1.6-fold higher, respectively, in livers from senescent compared with young adult rats. Hepatic glycogen content was about 50% lower in livers from senescent compared with young adult rats, but the contents of UDP-glucose and UDP glucuronic acid were similar between the two groups. Taken together, the data are consistent with the hypothesis that rates of mixed-function oxidation are decreased in intact livers from senescent compared with young adult rats, due possibly to age-related changes in cofactor supplies. Glucuronidation of low, but not high, concentrations of substrates may be affected by age-related, changes in K m and V max values of microsomal glucuronyltransferase.

Food Restriction Stimulates Conjugation of P-Nitrophenol in Perfused Rat Liver

Rates of conjugation of p-nitrophenol were studied in livers from normal and food-restricted rats perfused with either p-nitroanisole or p-nitrophenol. Female Sprague-Dawley rats had ad libitum access to a Purina 5001 nonpurified diet (control) or were given 65% of the intake of controls for 3 weeks. Livers were perfused with oxygenated Krebs-Henseleit buffer using a nonrecirculating system. Maximal rates of conjugation of p-nitrophenol, generated either from the O-demethylation of p-nitroanisole (200 μM) or from the infusion of p-nitrophenol (70 μM), were elevated significantly nearly twofold by food restriction. Thus, food restriction stimulates conjugation in the intact liver cell. Specifically, rates of conjugation were increased from 2.1 ± 0.2 to 3.7 ± 0.4 and from 3.3 ± 0.6 to 5.8 ± 0.5 μmol/g/h when 200 μM p-nitroanisole or 70 μM p-nitrophenol were infused, respectively. On the other hand, rates of conjugation were not affected by food restriction when low concentrations of p-nitroanisole (50 μM) or p-nitrophenol (20 μM) were infused. Further, food restriction did not alter rates of conjugation in isolated microsomes supplemented with excess UDPGA. Interestingly, both UDP-glucose and UDP-glucuronic acid were increased significantly in liver extracts from food-restricted rats when livers were perfused with high but not low concentrations of p-nitrophenol. Under these conditions, the increase in UDP-glucuronic acid was threefold. Moreover, food restriction increased carbohydrate release from the liver about twofold. Glycogen content was also increased significantly in liver extracts from 8.4 ± 1.9 to 60.4 ± 13.8 mmol/kg wet weight by food restriction. Taken together, these data support the hypothesis that food restriction stimulates conjugation of p-nitrophenol concentrations by increasing the supply of the pivotal cofactor UDP-glucuronic acid from carbohydrate reserves (e.g., glycogen).

Endotoxin inhibits glucuronidation in the liver: An effect mediated by intercellular communication

Endotoxin [lipopolysaccharide (LPS) 50 μg/mL] added to the perfusion medium increased glucose production and inhibited the glucuronidation of p-nitrophenol in perfused mouse liver both in recirculating and non-recirculating systems, while sulfation of p-nitrophenol was unchanged. The effects of endotoxin could be prevented by the addition of cyclooxygenase inhibitors, while PGD 2 and PGE 2 also caused a decrease in p-nitrophenol glucuronidation in perfused liver. In isolated hepatocytes endotoxin failed to affect p-nitrophenol conjugation, while PGD 2 and PGE 2 decreased the rate of it. Our results suggest that endotoxin inhibits glucuronidation through an intercellular communication presumably mediated by eicosanoids.

Intraluminal calcium of the liver endoplasmic reticulum stimulates the glucuronidation of p-nitrophenol

The relationship between the intraluminal Ca2+ content of endoplasmic reticulum and the rate of the glucuronidation of p-nitrophenol was investigated in isolated rat hepatocytes. Different agents which decrease the Ca2+ level in the endoplasmic reticulum [calcium ionophores (A23187, ionomycin) or Ca(2+)-ATPase inhibitors(thapsigargin,2,5-di-(t-butyl)-1,4-benzohydroquinone+ ++)] inhibited the conjugation of p-nitrophenol. Depletion of intracellular Ca2+ stores by preincubation of hepatocytes in the absence of free Ca2+ (in the presence of excess EGTA) also decreased the rate of glucuronidation; Ca2+ re-admission to EGTA-treated hepatocytes restored glucuronidation. In intact liver microsomes the p-nitrophenol UDP-glucuronosyl-transferase activity was not modified by varying the external free Ca2+ concentrations within a cytosol-like range. Emptying of the Ca2+ from the lumen of microsomal vesicles by A23187, after MgATP-stimulated Ca2+ sequestration, decreased the glucuronidation of p-nitrophenol. A similar effect was observed in filipin-permeabilized hepatocytes. In native and in detergent-treated microsomes, Ca2+ (1-10 mM) increased the p-nitrophenol UDP-glucuronosyltransferase activity. It is suggested that the physiological concentration of Ca2+ in the lumen of the endoplasmic reticulum is necessary for the optimal activity of p-nitrophenol UDP-glucuronosyltransferase; the depletion of Ca2+ decreases the activity of the enzyme.

Evaluation of Heptane/1-Butanol/Water Systems for Separation of Drug Metabolites by Countercurrent Chromatography

Abstract A small-volume CCC coil (1.07 mm i. d. × 13.5 m, Vc 14.7 ml) has been used to evaluate heptane/ 1-butanol/aqueous buffer systems for potential use in separating drug metabolites. Repeated sample injections can be made and a partition coefficient as high as 40 can be determined in about 4 hrs. Conjugates of p-nitrophenol with glucuronic acid, sulfuric acid and glucose were separated, and the partition coefficients of several N-alkyl anilines, benzylamines and phenylethylamines were measured to emulate chromatographic behavior expected for compounds generated in xenobiotic metabolism. Amines chromatographed well in reverse phase systems above the amine pKa value. But peaks were distorted when the mobile phase pH was slightly below the amine pKa for benzyl and phenylethyl amines and about two units below for anilines. The effect of alcoholic hydroxy! substitution and dealkylation is discussed for anilines and derivatives of the drug chioroquine.

High amounts of glucose and insulin inhibit p-nitrophenol conjugation in mouse hepatocytes

High amounts of glucose and insulin inhibit p-nitrophenol conjugation in mouse hepatocytes

Evidence for glucuronide conjugation of p-nitrophenol in the Caco-2 cell model.

Evidence for glucuronide conjugation of p-nitrophenol in the Caco-2 cell model.

In Situ Structural Analysis of Microsomal UDP-glucuronyltransferases by Radiation Inactivation

The structure of the UDP-glucuronyltransferases in microsomes from guinea pig and rat liver was examined in situ by radiation inactivation analysis. The p-nitrophenol conjugating activity of guinea pig microsomes increased at lower doses of radiation; at higher doses (greater than or equal to 36 megarads), activity showed a first order decline yielding a target size of 71 +/- 9 kDa. Treating microsomes with Triton X-100 eliminated the activation seen at lower doses of radiation and yielded a simple exponential decrease in activity which gave a larger target size (95 +/- 18 kDa). A monoexponential decrease in activity was seen in sonicated microsomes, at greater than or equal to 36 megarads. The same response was obtained when the reaction was assayed in the reverse direction. The estrone conjugating activity of guinea pig microsomes was similarly activated at lower doses of radiation and declined at higher doses (greater than or equal to 36 megarads), with a target size of 57 +/- 11 kDa. Allosteric activation of the enzyme by UDP-N-acetylglucosamine was eliminated by lower doses of radiation. Thus, activation of the enzyme by radiation, detergent, sonication, and UDP-N-acetylglucosamine appear to be interdependent. These activations are postulated to be due to the existence of the enzyme in an oligomeric form which can be dissociated into monomers with higher activity. The same biphasic activation-inactivation curves were obtained for p-nitrophenol conjugation in rat liver microsomes. The target sizes were 54 +/- 8 kDa (p-nitrophenol in the forward direction) and 66 +/- 10 kDa (p-nitrophenol in the reverse direction). Thus, the enzyme appears to be smaller in rat liver as compared with guinea pig liver. Lithocholate glucuronidating activity in rat liver microsomes (at greater than 36 megarads) gave a target size of 74 +/- 1 kDa.

Subcellular Localization of UDP-Glucuronyltransferase by Differential Centrifugation

Subcellular fractionation of liver homogenates from treated rats was carried out in order to study the mechanism of action of the gastrointestinal polypeptides on glucoronidation. Rats were treated for 90 min with an intravenous infusion of secretin (0.4 cU/h/100 g body weight), glucagon (100 micrograms/h/100 g body weight) and vasoactive intestinal polypeptide (VIP) (300 ng/h/100 g body weight); controls were sham-treated rats. For comparison, another group of animals was treated with a daily injection of phenobarbitone (10 mg/kg), a well-established enzyme inducer. Treatment with the different polypeptides produced minor changes in the subcellular localization of the enzyme. The bulk of activity was always recovered in the microsomal fraction, as identified by both differential centrifugation and the enrichment in specific activity of glucose-6-phosphatase, esterase and NADPH-cytochrome c reductase. Secretin produced a specific increase of bilirubin glucuronidation, more evident in all nuclear fractions. Glucagon increased both bilirubin and p-nitrophenol glucuronidation in all subcellular fractions. VIP had a selective action on p-nitrophenol conjugation of similar extent in nuclear and microsomal fractions. The type of changes observed is suggestive of physicochemical modifications occurring into the cell, perhaps at the membrane environment of different organelles, able to modify the overall conjugation of different substrates by the cell.

Glycogenolysis - and not gluconeogenesis - is the source of UDP-glucuronic acid for glucuronidation

Differences in cofactor (NADPH and UDP-glucuronic acid) supply for various processes of biotransformation were studied by investigating the interrelations between glucose production (gluconeogenesis and glycogenolysis) and drug ( p-nitrophenol, aminopyrine, phenolphthalein) biotransformation (hydroxylation and conjugation) in isolated murine hepatocytes. In glycogen-depleted hepatocytes prepared from animals fasted for 48 h (i) p-nitrophenol conjugation was decreased by 80% compared to the fed control, while aminopyrine oxidation was unaltered, (ii) addition of glucose or gluconeogenic substrates failed to increase the rate of p-nitrophenol conjugation, while the rate of p-nitrophenol and also aminopyrine oxidation was increased and (iii) gluconeogenesis was inhibited by 80% by aminopyrine oxidation: it was moderately decreased by p-nitrophenol oxidation and conjugation and remained unchanged by phenolphthalein conjugation. In hepatocytes prepared from fed mice (i) p-nitrophenol conjugation was independent of the extracellular glucose concentration, (ii) it was linked to the consumption of glycogen - addition of fructose inhibited p-nitrophenol glucuronidation only, while sulfation was unaltered and (iii) p-nitrophenol oxidation was not detectable: aminopyrine oxidation was not affected by fructose addition. It is suggested that UDP-glucuronic acid for glucuronidation derives predominantly from glycogen, while the NADPH generation for mixed function oxidation is linked to glucose uptake and / or gluconeogenesis in the liver.

Accumulation of phenols and catechols in isolated mouse hepatocytes in starvation or after pretreatment with acetone

Conditions leading to the accumulation of unconjugated phenols and catechols were investigated in mouse livers. The formation of unconjugated hydroxylated products of added p-nitrophenol and aniline was investigated in isolated hepatocytes prepared from 48 hr fasted or fed mice or from fed mice after acetone pretreatment. 4-Nitrocatechol and p-aminophenol — the hydroxylated products of p-nitrophenol and aniline — were accumulated in cells prepared from fasting animals, while in cells prepared from fed mice these unconjugated derivatives were not detectable. The accumulation of 4-nitrocatechol and p-aminophenol was also shown in isolated hepatocytes prepared from acetone pretreated fed mice. Inhibition of glucuronidation by N 6,O 2-dibutyryl cAMP or by D-galactosamine increased the accumulation of 4-nitrocatechol upon addition of p-nitrophenol in cells prepared from fasted mice. Both 48 hr starvation and acetone pretreatment enhanced the activity of microsomal p-nitrophenol and aniline hydroxylase by 300% and 600%, respectively, whereas p-nitrophenol conjugation in isolated hepatocytes as well as in hepatocyte homogenates was decreased by about 80% after 48 hr starvation. Acetone pretreatment did not alter the rate of p-nitrophenol conjugation measured in liver homogenates. It is suggested that a shift from conjugation toward hydroxylation in starvation gives rise to the formation of hazardous metabolites.

Induction of unscheduled DNA synthesis in primary cultures of rat, mouse, hamster, monkey, and human hepatocytes

Variation in hepatic metabolism between species may be an important factor in the differences observed in chemical carcinogenesis. We examined 6 chemicals representative of 4 chemical classes in the in vitro hepatocyte DNA repair assay using cells isolated from the Fischer-344 rat, B6C3F1 mouse, Syrian golden hamster, cynomolgus monkey and from human liver. Hepatocytes were isolated by in situ or biopsy liver perfusion and incubated with [ 3H]-thymidine and the test chemical. Unscheduled DNA synthesis (UDS) was measured as net grains/nucleus (NG) by quantitative autoradiography. Qualitative and quantitative differences in UDS responses were observed for every chemical. Liver cultures isolated from the rat, mouse, hamster, human, and monkey and treated with aflatoxin B 1 or dimethylnitrosamine all yielded dose-related increases in NG. Human, rat, and hamster hepatocyte cultures yielded positive responses following exposure to the aromatic amines 2-acetylaminofluorene, 4-aminobiphenyl, and benzidine, whereas cultures isolated from the monkey and mouse yielded < 0 NG. Treatment with benzo[ a]pyrene (BAP) produced strong positive responses in monkey and human hepatocyte cultures, weak positive responses in hamster cultures, and equivocal or negative responses in rat and mouse hepatocyte cultures. Hepatocyte function was assessed by measurement of DNA content, glutathione content, BAP hydroxylase activity, p-nitroanisole- O-demethylase activity, p-nitrophenol conjugation, and urea synthesis rates. The functional capabilities of isolated hamster, monkey, and human hepatocyte cultures do not appear to correlate with UDS responses observed for any compound; however, they indicate that the cultures were metabolically competent at the time of chemical exposure. These studies suggest that rat hepatocytes are a suitable model for human hepatocytes, whereas mouse and male monkey hepatocytes may be insensitive to aromatic amines.

Increased retinoic acid metabolism following 3,3',4,4',5,5'-hexabromobiphenyl injection.

Young male Wistar rats received single i.p. injections of 3,3',4,4',5,5'-hexabromobiphenyl. In rats dosed with 40 mg/kg, food consumption and growth as well as liver retinol and retinyl palmitate concentrations decreased, while serum retinol and liver weight increased within 28 days following the injection. In rats receiving a 20-mg/kg dose, food consumption, growth, liver weight, and serum retinol were not affect, although liver retinol and retinyl palmitate concentrations declined to 23 and 21% of their respective control values. Vitamin A metabolism was studied in liver microsomes prepared from rats sacrificed 7 days after the 20-mg/kg injection. The rate of retinoic acid hydroxylation via the cytochrome P-450 system to 4-hydroxyretinoic acid plus the subsequent oxidation to 4-ketoretinoic acid was significantly elevated. Retinoic acid conjugation by UDP-glucuronyl transferase was also significantly increased. These changes corresponded with increased activities of cytochrome P-450-dependent aryl hydrocarbon hydroxylase and UDP-glucuronyltransferase conjugation of p-nitrophenol. These results provide a direct link between enzyme induction due to xenobiotics and specific steps in the vitamin A metabolic pathway.

Cyclic AMP-dependent phosphorylation in the control of biotransformation in the liver

The possibility of a short-term cAMP-dependent regulation of mixed-function oxidation and of glucuronide formation was investigated in isolated mouse hepatocytes and in mouse liver microsomal membranes. N 6, O 2-dibutyryl cAMP (in accordance with its increasing effect on gluconeogenesis) decreased aminopyrine oxidation and p-nitrophenol conjugation in isolated hepatocytes, while the phenolphthalein conjugation remained unaltered. Similar to dibutyryl cAMP the Ca 2+ ionophore A 23187 also decreased aminopyrine oxidation. In cell-free systems the phosphorylation of isolated microsomal membranes by the exogenous cAMP-dependent protein kinase was inhibitory on amino-pyrine oxidation and p-nitrophenol glucuronide formation but aniline oxidation and phenolphthalein glucuronidation were not affected. The correlation between the negative cAMP-dependent control of certain processes of biotransformation and the positive cAMP-dependent regulation of gluconeogenesis is discussed.

Application of molecular-secondary-ion mass spectrometry for drug metabolism studies: I. Direct analysis of conjugates by thin-layer chromatography-secondary-ion mass spectrometry

Molecular-secondary-ion mass spectrometry (SIMS) is a suitable method for the analysis of non-volatile substances such as conjugatedmetabolites of drugs. We have developed a simple method for the direct SIMS measurement of conjugates following thin-layer chromatography without any extraction procedure. After separation with a butanol-acetic acid-ethanol-water (3:1:1:1, v/v) system, the spot was cut out and attached to a SIMS probe. The conjugates of p-nitrophenol and 4-hydroxyantipyrine were measured. The quantitative application of the method is also discussed, using deuterium-labelled internal standards for p-nnitrophenol conjugates.

Stimulation of mixed-function oxidation by NADPH in perfused mouse livers: Studies with saponin-permeabilized tissue

In perfused livers from fed and fasted β-naphthoflavone-treated C57BL/6J mice, maximal rates of p-nitroanisole O-demethylation were 30–40 μmoles/g/hr and 15–20 μmoles/g/hr respectively. The detergent saponin, at concentrations ranging from 0.001 to 0.005%, was infused between 2 and 30 min to establish optimal conditions to permeabilize plasma membranes. Permeabilization was assessed by release of lactate dehydrogenase and stimulation of p-nitroanisole O-demethylation by citrate. Saponin (0.005% for 5 min) alone had little effect on the rates of p-nitroanisole O-demethylation or conjugation of p-nitrophenol by perfused livers. Further, dicarboxylates or NADPH had no effect on rates of monooxygenation by perfused mouse liver in the absence of saponin. In saponin-treated livers from fasted mice, however, rates of monooxygenation were increased rapidly by infusion of dicarboxylates (10 mM malate, citrate, or isocitrate) or an NADPH-generating system (60 and 110% respectively), over a 6–8 min period. During this time period, cellular energetics were not comprised as reflected by normal rates of glucuronidation of p-nitrophenol. Thus, non-permeable metabolites can enter saponin-permeabilized cells in the perfused liver. Rates of monooxygenation were increased 40–60% in livers from fed mice by citrate, NADPH (200 μM) or an NADPH-generating system. In contrast, saponin decreased mixed-function oxidation assayed in isolated microsomes incubated with an NADPH-generating system. Taken together, these data support the hypothesis that maximal rates of monooxygenation in intact hepatocytes from fed as well as fasted mice is limited by the availability of NADPH.