Spectral properties of substrate-cytochrome P-450 interaction and catalytic activity of xenobiotic metabolizing enzymes in isolated rainbow trout liver cells

A method for the isolation of intact viable rainbow trout liver cells in high numbers is described. The technique involves perfusion of collagenase through the liver. A major part of the cytochrome P-450 in isolated liver cells was present in the oxidized non-substrate bound form. It was observed that 7-ethoxycoumarin was rapidly taken up by the liver cells and bound to cellular cytochrome P-450. The substrate binding spectrum for isolated trout liver cells was slightly modified compared with that obtained with trout liver microsomes. The microsomal affinity of 7-ethoxycoumarin, calculated as the apparent spectral dissociation constant (ks), was elevated 11-fold after fish were treated with beta-naphthoflavone, indicating a qualitative alteration in the nature of the constitutive cytochrome P-450. The metabolism of 7-ethoxycoumarin in isolated liver cells was found to be of a comparable rate to that obtained in liver microsomes. Pretreatment of fish with Clophen A50 or beta-naphthoflavone significantly increased the content of cytochrome P-450 and elevated the rate of 7-ethoxycoumarin deethylation in isolated liver cells. Furthermore, the rate of conjugation of 7-hydroxycoumarin was significantly elevated in liver cells isolated from beta-naphthoflavone treated fish when compared with the control rate. In isolated liver cells, 90% of the 7-hydroxycoumarin formed from deethylation of 7-ethoxycoumarin was further metabolized to conjugated products. However, in beta-naphthoflavone of Clophen A50 treated fish the fraction of conjugated metabolites was markedly decreased, indicating a changed balance between cytochrome P-450 dependent reactions and conjugation reactions in the cell.     

Hepatic uptake, disposition, and metabolism of rubratoxin B in isolated perfused rat liver

Isolated perfused rat liver preparations were utilized to measure the hepatic uptake, biliary excretion and metabolism of rubratoxin B. Livers were perfused with 30% rat blood perfusate containing 0.24 μmol labeled rubratoxin B, and a series of timed blood and bile samples were analyzed by high-pressure liquid chromatography, and treated enzymatically for the determination of glucuronide and sulfate conjugates. Blood, bile and liver samples were also radioassayed. Rubratoxin B was removed from the perfusate by a first-order process (monophasically) with a half-life of 207.5 ± 23.7 min (mean ± SE). By 3.5 hr of perfusion, 30% of the total rubratoxin B-derived radioactivity was excreted into the bile. More than 8% of the total dose of rubratoxin B was excreted unchanged into the bile by 3.5 hr. The rates of biliary excretion of rubratoxin B- derived radioactivity and parent compound reached a maximum at 30 min, after which time the rates of excretion decreased monophasically with half-lives of 35.5 and 72 min, respectively. Two major metabolites detected in the bile were the glucuronide and sulfate conjugates, together accounting for 22% of the radioactivity excreted into the bile by 3.5 hr. In addition, at least one major unidentified organosoluble metabolite was detected in the bile.     

Metabolism of the human carcinogen, benzidine, in the isolated perfused rat liver

14C-Benzidine (BZ) was added to the recirculating perfusate of the isolated perfused rat liver. The system was monitored at timed intervals for the disappearance of BZ and the appearance of metabolites. BZ was extensively metabolized by this system and after 2 hr of perfusion greater than 95% of the administered radiolabel was in the form of metabolic products. In the perfusate the concentration of BZ declined rapidly whereas the concentration of N-acetyl-BZ (ABZ) increased temporarily and then declined. The concentration of N,N'-diacetyl-BZ (DABZ) increased with time and by 1 hr DABZ had become the major metabolite in the system. In the bile, which contained 22% of the dose after 2 hr, BZ-N-glucuronide and ABZ-glucuronide were the major metabolites initially, but after 1 hr of perfusion N-hydroxy-DABZ-glucuronide had become the major biliary metabolite. Addition of BZ and 35S-Na2SO4 to the perfusate resulted in at least one 35S-containing metabolite. Other major metabolites excreted in bile included 3-hydroxy-DABZ glucuronide, ABZ, and DABZ. DABZ underwent deacetylation to ABZ and N-hydroxy-DABZ underwent rapid reduction to DABZ when added to the isolated liver system. Qualitatively similar biliary metabolite patterns at later times were observed when either BZ, DABZ, or N-hydroxy-DABZ was added to the perfusate.     

Oxidation of barbiturates and the glucuronidation of 1-napthol in perfused rat liver and in microsomes

Summary An attempt has been made to relate quantitatively drug metabolism in perfused liver with that in microsomes. Therefore in both systems microsomal monooxygenase (hexobarbital as substrate) and UDP-glucuronyltransferase (1-naphthol as substrate) have been studied.The rate of hexobarbital oxidation in perfused liver was slightly higher than in microsomes incubated with an NADPH regenerating system, indicating that the generation of reducing equivalents is not rate-limiting in livers of normal rats. The rate of phenobarbital oxidation was only about 3% of the rate of hexobarbital oxidation in perfused liver.Microsomal UDP-glucuronyltransferase can be activated about 10-fold in vitro. The formation of naphthol glucuronide in perfused liver (determined from the appearance of glucuronide in liver tissue, perfusate and bile) corresponded well with UDP-glucuronyltransferase activity in non-activated microsomes when the intracellular UDP-glucuronate level was taken into account. This suggests that UDP-glucuronyltransferase is mostly latent within the cell.     

Metabolism of a benzothiazine compound (SQ 11,579) by the intact rat, isolated perfused rat liver, and rat-liver microsomes.

1. The metabolic dispositions of a benzothiazine compound (SQ 11,579) by the intact rat, isolated perfused rat liver, and rat-liver microsomes have been investigated, and the results compared. 2. The drug was well absorbed after oral administration to rats and was widely distributed in all tissues, which, with the exception of brain, had higher concentrations of the drug, its metabolites, or both, than did plasma. 3. Metabolism by rat-liver microsomes included N-oxidation, N-demethylation, S-oxidation and aryl hydroxylation. Metabolites hydroxylated in the aromatic ring were excreted only in bile, both by the isolated perfused rat livers and by anaesthetized bile-duct-cannulated rats. 4. Liver perfusion of the benzothiazine or its monodesmethyl analogue (V) resulted in temporary cessation of the flow of perfusate through the organ. The benzothiazine sulphoxide (IV) had only a slight effect on the flow of liver perfusate, but IV followed by I caused the flow of perfusate to cease.     

Hepatic glutathione concentrations and the release of pyrrolic metabolites of the pyrrolizidine alkaloid, monocrotaline, from the isolated perfused liver.

We have examined the relationship between the metabolism of the pyrrolizidine alkaloid, monocrotaline, and glutathione concentration in the isolated, perfused rat liver. On perfusion of monocrotaline (300 microM) through the isolated liver, high concentrations (1.1 mM) of its metabolite glutathionyldehydroretronecine are released into bile, while much lower amounts (4.86 microM; 0.05 mumol/g liver) accumulate in the perfusate over a 1 hr perfusion period. Metabolite concentration in both the bile and perfusate increase when the level of monocrotaline perfused is increased to 900 microM. Metabolite release is also elevated in livers pretreated with phenobarbital. Monocrotaline perfusion lowered glutathione concentrations in the liver from 30 min onwards. Livers from animals treated with buthionine sulfoximine or chloroethanol showed much lower glutathione levels after 60 min perfusion. Livers from chloroethanol-treated (but not buthionine sulfoximine-treated) animals showed significantly lower release of pyrroles into the bile on perfusion with monocrotaline, but there is no effect on the rate of build-up of pyrrolic metabolites in the perfusate. We conclude that hepatic glutathione concentrations and the release of pyrrolic metabolites of monocrotaline mutually interact. Exposure of the liver to monocrotaline reduces glutathione concentrations, while marked depletion of liver glutathione concentration leads to a decrease in the release of monocrotaline metabolites.     

Metabolism of 1-[14C]nitropyrene in isolated perfused rat livers

1-Nitropyrene (1-NP), a constituent of diesel exhaust, is carcinogenic to rats and is a bacterial and mammalian mutagen. Biliary and fecal excretion of 1-NP metabolites are the major routes of excretion in rats, suggesting that hepatic metabolism plays a dominant role in determining the biological fate of 1-NP. The purpose of this investigation was to quantitate 1-[14C]NP metabolites formed in isolated perfused rat livers and excreted in bile from rats. Perfused rat livers displayed a capacity for oxidation, reduction, acetylation, and conjugation of 1-NP (or its metabolites). Reduction of 1-NP followed by N-acetylation was the major metabolic pathway observed in the perfused livers. Acetylaminopyrene (AAP) was the major metabolite detected, with total quantities (150 nmol) accounting for about 60% of the total 1-[14C]NP dose (258 nmol) added to the perfusate. Considerably smaller quantities of aminopyrene and hydroxynitropyrenes were also detected. Livers perfused with 1-[14C]NP excreted about 36 nmol equivalents of 1-[14C]NP (12% of the total 1-NP dose) in bile after 60 min. Some of the biliary metabolites were tentatively identified as metabolites of the mercapturic acid pathway. The spectrum of biliary metabolites was qualitatively identical to that seen in bile from intact rats. Quantities of 14C covalently bound to hepatic macromolecules from perfused livers were 0.4 nmol 1-NP eq/g liver. The data from this study indicate that the liver may be an important site for metabolism of 1-NP.     

Species differences in the elimination of a peroxisome proliferator-activated receptor agonist highlighted by oxidative metabolism of its acyl glucuronide.

A species difference was observed in the excretion pathway of 2-[[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy]-2-methylpropanoic acid (MRL-C), an alpha-weighted dual peroxisome proliferator-activated receptor alpha/gamma agonist. After intravenous or oral administration of [14C]MRL-C to rats and dogs, radioactivity was excreted mainly into the bile as the acyl glucuronide metabolite of the parent compound. In contrast, when [14C]MRL-C was administered to monkeys, radioactivity was excreted into both the bile and the urine as the acyl glucuronide metabolite, together with several oxidative metabolites and their ether or acyl glucuronides. Incubations in hepatocytes from rats, dogs, monkeys, and humans showed the formation of the acyl glucuronide of the parent compound as the major metabolite in all species. The acyl glucuronide and several hydroxylated products, some which were glucuronidated at the carboxylic acid moiety, were observed in incubations of MRL-C with NADPH- and uridine 5'-diphosphoglucuronic acid-fortified liver microsomes. However, metabolism was more extensive in the monkey microsomes than in those from the other species. When the acyl glucuronide metabolite of MRL-C was incubated with NADPH-fortified liver microsomes, in the presence of saccharo-1,4-lactone, it underwent extensive oxidative metabolism in the monkey but considerably less in the rat, dog, and human liver microsomes. Collectively, these data suggested that the oxidative metabolism of the acyl glucuronide might have contributed to the observed in vivo species differences in the metabolism and excretion of MRL-C.     

Metabolism and disposition of the antiviral nucleoside analogue AM365 in the isolated perfused rat liver

The present study was designed to investigate the hepatic disposition of the prodrug AM365 and the generated antiviral guanosine analogue, AM188 in the isolated perfused rat liver (IPL). The livers of rats (n=12) were isolated and perfused with Krebs-Henseleit pH 7.4 buffer to which AM365 was added as a bolus to achieve an initial perfusate concentration of 22.4 micromol/L. During the 120-min period after administration of AM365, bile was collected in 10-min intervals and perfusate was collected at the mid-point of these intervals. Concentrations of AM365 and AM188 in perfusate and bile were quantified by HPLC. Following administration of AM365, its concentration in perfusate declined and the concentration of AM188 increased; the sum of the molar concentrations remained constant. The clearance and hepatic extraction ratio of AM365 were 3.3+/-2.4 mL/min and 0.110+/-0.079, respectively. The cumulative amount of AM365 excreted in bile during the 120-min perfusion period was approximately 0.21% of the bolus dose, and 0.36% of the total amount of AM365 cleared by the liver during the period. The cumulative amount of AM188 excreted in bile was about 0.48% of the total amount of AM188 formed during the perfusion period. In conclusion, AM365 was metabolised to AM188, which appeared to be the only metabolite and was not further biotransformed. The biliary excretion of AM365 and AM188 was negligible.     

1H nuclear magnetic resonance study of metronidazole metabolism by perfused rat liver

High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz has been used to monitor the metabolism of metronidazole by the perfused rat liver. Samples of perfusate removed at various times during the perfusion show resonances from metronidazole and its major metabolites, as well as those from organic metabolites released from the liver. The major metabolite of metronidazole is shown to be its glucuronide conjugate. Metronidazole metabolism and glucuronide production are both stimulated in livers from rats pretreated with phenobarbitone.     

Glucuronidation and excretion of nonylphenol in perfused rat liver.

Nonylphenol, an environmental estrogenic chemical, is reported to have adverse effects on the reproductive organs of animals. In this study, the metabolism of nonylphenol and that of other alkylphenols in the rat liver was investigated using liver perfusion. Alkylphenols (nonylphenol, hexylphenol, butylphenol, and ethylphenol) were glucuronidated by rat liver microsomes. Nonylphenol was found to be conjugated with glucuronic acid by an isoform of UDP-glucuronosyltransferase, UGT2B1, expressed in yeast AH22 cells. However, when nonylphenol was perfused into rat liver in situ, it was difficult for free nonylphenol and conjugated metabolite to be excreted into the bile or vein, and most of the perfused nonylphenol remained free and as a glucuronide conjugate in the liver tissue, even after 1 h of perfusion. After 1 h of perfusion of the other alkylphenols, most of them were excreted into the bile as glucuronides. Ethylphenol, which has the shortest alkyl chain, was excreted rapidly into both the bile and vein; however, the excretion rates of alkylphenols having longer alkyl chains tended to be slow. MRP-2-deficient Eisai hyperbilirubinemic rats could not secrete alkylphenol-glucuronides into the bile, indicating that alkylphenol-glucuronides are transported by MRP-2 to the bile in normal Sprague-Dawley rats. The results indicate that the kinetics of excretion of alkylphenol-glucuronides into the bile or vein depends on the length of alkyl chain and suggest that nonylphenol-glucuronide formed in the liver cannot be transported by MRP-2.     

The important role of Bcrp (Abcg2) in the biliary excretion of sulfate and glucuronide metabolites of acetaminophen, 4-methylumbelliferone, and harmol in mice

The role of Mrp2, Bcrp, and P-glycoprotein in the biliary excretion of acetaminophen sulfate (AS) and glucuronide (AG), 4-methylumbelliferyl sulfate (4MUS) and glucuronide (4MUG), and harmol sulfate (HS) and glucuronide (HG) was studied in Abcc2(-/-), Abcg2(-/-), and Abcb1a(-/-)/Abcb1b(-/-) mouse livers perfused with the respective parent compounds using a cassette dosing approach. Biliary clearance of the sulfate conjugates was significantly decreased in Bcrp-deficient mouse livers, resulting in negligible biliary excretion of AS, 4MUS, and HS. It is noteworthy that the most profound decrease in the biliary clearance of the glucuronide conjugates was observed in Bcrp-deficient mouse livers, although the biliary clearance of 4MUG was also approximately 35% lower in Mrp2-deficient mouse livers. As expected, biliary excretion of conjugates was not impaired in P-glycoprotein-deficient livers. An appreciable increase in perfusate recovery due to a shift in the directionality of metabolite excretion, from bile to perfusate, was noted in knockout mice only for conjugates whose biliary clearance constituted an appreciable (> or =37%) fraction of total hepatic excretory clearance (i.e., 4MUS, HG, and HS). Biliary clearance of AG, AS, and 4MUG constituted a small fraction of total hepatic excretory clearance, so an appreciable increase in perfusate recovery of these metabolites was not observed in knockout mice despite markedly decreased biliary excretion. Unlike in rats, where sulfate and glucuronide conjugates were excreted into bile predominantly by Mrp2, mouse Bcrp mediated the biliary excretion of sulfate metabolites and also played a major role in the biliary excretion of the glucuronide metabolites, with some minor contribution from mouse Mrp2.     

Glucuronidation of 7-hydroxycoumarin in periportal and pericentral regions of the lobule in livers from untreated and 3-methylcholanthrene-treated rats

Rates of production of 7-hydroxycoumarin glucuronide were measured in specific zones of the liver lobule using micro-light guides placed on periportal and pericentral regions on the surface of livers from untreated and 3-methylcholanthrene-treated rats. Livers were perfused with sulfate-free buffer under normoxic conditions and fluorescence of free 7-hydroxycoumarin was monitored. The formation of nonfluorescent 7-hydroxycoumarin glucuronide was then inhibited completely by perfusion with N2-saturated perfusate containing 20 mM ethanol. The difference between fluorescence readings under normoxic and hypoxic conditions was used to calculate rates of glucuronidation. Maximal rates of glucuronidation (11.9-13.5 mumol/g/hr) did not differ significantly in periportal and pericentral regions in livers from either 3-methylcholanthrene-treated or untreated rats. In all regions of the liver lobule, glucuronidation was half-maximal with about 20 microM 7-hydroxycoumarin. Glucuronosyltransferase assayed in lyophilized tissue sections with saturating concentrations of UDPGA (9 mM) was 2.3-fold greater in pericentral than in periportal areas in livers from untreated rats. In livers from 3-methylcholanthrene-treated rats, activities were similar in periportal and pericentral regions but were 4- to 7-fold higher than values from untreated rats. In addition, glucuronosyltransferase activity assayed in native microsomes with physiological concentrations of UDP-glucuronic acid (UDPGA) (0.4 mM) with UDP-N-acetylglucosamine (0.3 mM) was 2-fold higher in preparations from 3-methylcholanthrene-treated than untreated rats. Thus, 3-methylcholanthrene treatment increased glucuronosyltransferase activity in vitro but did not alter rates of glucuronide formation in periportal and pericentral regions of the liver lobule of intact liver. Infusion of epinephrine (50 nM) into perfused livers from untreated and 3-methylcholanthrene-treated rats increased rates of glucuronidation by about 35%. Since epinephrine probably acts by increasing the supply of the cofactor UDPGA due to increased breakdown of glycogen, it follows that UDPGA supply limits rates of glucuronidation in perfused livers from both untreated and 3-methylcholanthrene-treated rats.     

A novel biologically active selenooorganic compound--VII. Biotransformation of ebselen in perfused rat liver

Ebselen (PZ 51) is a selenoorganic compound with antioxidant and antiinflammatory properties, and its metabolism was studied in isolated perfused rat liver (hemoglobin-free, open system). 75Se-labelled ebselen was taken up into liver cells and radioactivity was excreted into bile. Biliary excretion of 75Se-compounds reached maximal values of 4 nmol/min per g wet wt. HPLC analysis of bile and effluent perfusate as well as identification of separated metabolites by mass spectrometry were carried out. The biliary metabolites were (a) an interesting novel Se-glucuronide, 2-glucuronylselenobenzanilide, (metabolite IV), as the major metabolite, and (b) an O-glucuronide, N-(4'-glucuronyloxyphenyl)-2-methylselenobenzanilide (metabolite III). The major effluent perfusate metabolites were Se-methylated derivatives (metabolites I and II). There was no evidence for sulfated metabolites. The selenodisulfide with glutathione, S-(2-phenyl-carbamoyl-phenylselenyl)-glutathione, was not detected, probably because of low steady-state concentrations and/or its biochemical lability. The selenium in ebselen is not bioavailable (e.g. for the synthesis of glutathione peroxidase), in contrast to selenite, for example, thus explaining the very low ebselen toxicity. However, the enzymatic steps in Se-methylation could be similar to those in the metabolism of selenite which include hydrogen selenide methylation. Se-glucuronides constitute a novel category of compounds in addition to the O-, N-, C- and S-glucuronide classes known in biology.     

Metabolism of hexachlorobenzene (HCB) in the isolated perfused rat liver

The metabolism of HCB in the isolated perfused rat liver was studied by administration of [14C]HCB diluted with unlabelled HCB at a total dose of 0.1 mg HCB/ml perfusate. Metabolites in bile, perfusate and liver were studied by GLC-mass spectrometry. Histological examination of the livers showed that no hepatic necrosis had developed, although there was a slight increase in ASAT and ALAT in the perfusate and about 50% decrease in hepatic glutathione. About 0.15% of administered radioactivity was recovered in the bile within 2 hr. In the bile, HCB together with the metabolites pentachlorothiophenol and pentachlorophenol, were identified and accounted for about 20% of the radioactivity excreted. In addition, eleven metabolites with 4 or 5 chlorines were isolated. In the perfusate and in the liver, unchanged HCB was responsible for most of the radioactivity. Traces of pentachlorothiophenol and pentachlorophenol were identified in the perfusate and the liver, respectively.     

Metabolism of 14C-2',3'-dideoxyinosine by the in situ perfused rat liver preparation

The metabolism and biliary excretion of 14C-dideoxyinosine (14C-ddI) has been investigated using the in situ perfused rat liver (PRL) preparation. After 2 h of perfusion through the liver, approximately 70-75 per cent of the total 14C-radiolabel was recovered in the perfusion medium, less than 1 per cent was excreted in bile and 15-18 per cent was retained in the liver. Hepatic clearance of ddI was 1.5 +/- 0.1 ml min-1 and half-life for the elimination of ddI from the medium was 22.9 +/- 2.0 min (n = 3). Hepatic extraction was estimated to be 7.5 per cent. HPLC analysis of the effluent perfusate indicated that ddI was metabolized to hypoxanthine, xanthine, uric acid, and to a polar metabolite which was tentatively identified as allantoin. Approximately 60-65 per cent of the ddI dose was converted to allantoin after 2 h of perfusion. Of the other three metabolites, uric acid levels increased to 20-30 per cent of the dose after 45 min and declined to about 5 per cent of the dose by the end of the perfusion period. Levels of hypoxanthine and xanthine were low and both compounds were not detected in the perfusate after 45 min post-infusion. In bile, the major peak, which accounted for about 50 per cent of the 14C-radiolabel co-eluted with the putative metabolite, allantoin (0.4 per cent of the dose). Uric acid (0.06 per cent of the dose) was the only other metabolite detected in bile. These results suggest that biliary excretion is a minor pathway for the elimination of ddI. Furthermore, ddI is rapidly cleared and metabolized by the liver to hypoxanthine, xanthine, uric acid, and to allantoin.     

In vivo intestinal metabolism of 7-ethoxycoumarin in the rat: production and distribution of phase I and II metabolites in the isolated, perfused intestinal loop.

These studies describe the phase I and II metabolism of 7-ethoxycoumarin (7-EC) in the isolated, perfused intestinal loop. Following cytochrome P450-dependent oxidative deethylation of 7-EC by intestinal epithelial cells, the product, 7-hydroxycoumarin (7-HC), undergoes phase II conjugation to form both the glucuronide and sulfate conjugates. The capacity for conjugation of 7-HC within the intestinal epithelium exceeds that of phase I oxidative deethylation, as demonstrated by the absence of increased release of unconjugated 7-HC upon saturation of the conjugation pathways. The formation of both glucuronide (53-62%) and sulfate (41-43%) conjugates contributed to a comparable extent to the overall phase II metabolism of 7-HC within the intestine. This is in contrast to the liver, where sulfate conjugation has been shown to be the predominant phase II metabolic pathway. Furthermore, it was found that unconjugated and sulfate conjugated 7-HC were evenly distributed between the lumenal perfusate and blood compartments, whereas the glucuronide conjugates of 7-HC were preferentially transported at a 4:1 ratio toward the blood. These results indicate that the epithelial cells of the small intestine have the capacity to biotransform orally administered xenobiotics, and the ultimate profile of metabolites generated may influence the biodisposition of these compounds.     

The metabolism of oestrone and some other steroids in isolated perfused rat and guinea pig livers

1. Oestrone is rapidly taken up by isolated perfused rat liver (t 1/2 less than 2 min) to yield at least 10 metabolites excreted in the bile; peak concentration occurs after about 20 min. 2. Sulphated metabolites of oestrone appear in the perfusate, reaching peak concentration at about 10 min, and then slowly disappear. 3. Sulphated metabolites of oestrone accumulate in the liver during the first 10 min. They are partly converted to sulphoglucuronides (steroid 3-sulphates conjugated with glucuronic acid in the D ring) and partly hydrolysed to be reconjugated as glucuronides. 4. The major biliary metabolites of oestrone in isolated perfused rat liver are glucuronides and sulphoglucuronides, but free steroids, sulphates and polar metabolites are also so excreted. 5. The isolated perfused guinea pig liver also rapidly takes up oestrone (t 1/2 less than 2 min) but, in contrast to the rat, a single glucuronide is the only quantitatively important metabolite in the bile: it is also extensively secreted into the perfusate where it reaches peak concentration at about 10 min. 6. In perfused guinea pig liver, oestrone does not form sulphoglucuronides, and sulphates are only minor metabolites; this is not due to lack of the appropriate sulphotransferase because oestradiol 17 beta-(beta-D-glucuronide) is extensively sulphated in this system. 7. Oestradiol 17 beta-(beta-D-glucuronide) is not cholestatic in the isolated perfused guinea pig liver although it is in rat liver. 8. There is a similar species difference in the metabolism of dehydroepiandrosterone in the two species: the rat forms sulphoglucuronides, the guinea pig does not. 9. The perfused rat liver extensively hydroxylates, presumably on the D ring, 17-deoxyoestrone and 17-deoxydehydroepiandrosterone. 10. The inability of perfused guinea pig liver to form sulphoglucuronides from oestrone or dehydroepiandrosterone is probably due to its restricted ability to hydroxylate the D ring of steroids. 11. Both rat and guinea pig biles contain beta-glucuronidase, about 80 and 230 sigma units/ml, respectively.     

The toxicokinetics of (1R, cis)- and (1R, trans)- tetramethrin in the isolated perfused rat liver

1. The toxicokinetics of cis- and trans-tetramethrin isomers were investigated using the isolated perfused rat liver preparation. 2. The concentration of cis- and trans-tetramethrin decreased rapidly in the plasma perfusate and was initially replaced by N-(hydroxymethyl)3,4,5,6-tetrahydrophthalimide (MTI) and then by 3,4,5,6-tetrahydrophthalimide (TPI). Plasma perfusate concentrations of the intact cis-isomer were higher than those of the trans-isomer. Concentrations of MTI and TPI were higher in livers treated with the trans-isomer. 3. Tetramethrin and its metabolites were rapidly excreted in the bile. Bile from livers perfused with trans-isomer contained higher concentrations of parent isomer and metabolites MTI and TPI, than did bile from livers treated with the cis-isomer.     

The Disposition of Morphine and Its Metabolites in the In-situ Rat Isolated Perfused Liver

A specific HPLC method with UV detection was used to investigate the disposition of morphine and its metabolites in the in-situ rat isolated perfused liver preparation. Livers of male Sprague-Dawley rats (n = 4) were perfused under single pass conditions with protein-and erythrocyte-free perfusate, containing 2·66 μm morphine, for up to 90 min. The concentration of morphine, normorphine and morphine-3-glucuronide (M3G) in outflow perfusate, and the biliary excretion of M3G and normorphine glucuronide, all reached steady-state levels within 15–20 min after commencing perfusion. At steady-state, the mean (± s.d.) extraction ratio of morphine was 0·87 ± 0·06 and clearance (26·0 ± 1·7 mL min^?1) approached perfusate flow rate (30 mL min^?1). Although M3G was the main metabolite, accounting for 72·8 ± 12·7% of eliminated morphine, a significant proportion (21·6 ± 13·5%) was N-demethylated to normorphine and was recovered as unchanged normorphine in outflow perfusate and normorphine glucuronide in bile. The biliary extraction ratio of hepatically-formed M3G was 0·61 ± 0·31. Results from an additional six experiments, in which livers were perfused with 1·33 and 2·66 μm of morphine for 30 min each in a balanced cross-over manner, indicated that the disposition of morphine and its metabolites was approximately linear within this concentration range.