Hepatic disposition of the cytochrome P450 2E1 marker chlorzoxazone and its hydroxylated metabolite in isolated perfused rat livers

The steady-state disposition of chlorzoxazone (CZX) and its hydroxylated metabolite 6-hydroxychlorzoxazone (HCZX) was determined in a single-pass isolated perfused rat liver (IPRL) model using constant CZX concentrations of 10-200 microM. The concentrations of CZX, HCZX, and/or HCZX glucuronide in the perfusate, bile, and liver tissues were measured and kinetic parameters calculated. Upon an increase in CZX inlet concentrations from 10 to 200 microM, its extraction ratio sharply declined from 0.681 to 0.087. This was associated with a saturable formation of HCZX, which was rapidly and completely metabolized to its glucuronide conjugate. Whereas the biliary excretion of CZX was negligible, that of HCZX was substantial (up to 40% of the generated metabolite). Overall, 79-93% of the CZX dose (10-200 microM) was recovered in our model as CZX and HCZX. Additionally, HCZX accounted for 56% (200 microM) to 71% (10 microM) of the extracted CZX dose. Further, a preliminary study using the preformed HCZX showed a complete (100%) recovery of the metabolite as its conjugate. Therefore, the unrecovered portion of CZX dose in our study (7-21% of the administered dose or 29-44% of the extracted dose at inlet CZX concentrations of 10-200 microM) is most likely due to parallel metabolism of CZX to other metabolites.     

Effects of normothermic hepatic ischemia-reperfusion injury on the in vivo, isolated perfused liver, and microsomal disposition of chlorzoxazone, a cytochrome P450 2E1 probe, in rats

In vitro studies have shown that the activities of cytochrome P450 (P450) enzymes may be altered after hepatic ischemia-reperfusion (IR) injury. Here, we investigated the effects of 1 h of partial ischemia, followed by 3 (IR3) or 24 (IR24) h of in vivo reperfusion, on the in vivo, isolated perfused rat liver (IPRL), and microsomal disposition of chlorzoxazone (CZX) and its cytochrome P450 2E1 (CYP2E1)-mediated metabolite, 6-hydroxychlorzoxazone (HCZX), in rats. Although IR3 caused a 30% reduction in the in vivo clearance of CZX, the area under the plasma concentration-time curve of HCZX was not affected. IPRL experiments showed that IR3, in addition to a 30% reduction in the clearance of CZX, causes a 70% decrease in the biliary clearance of HCZX. Microsomal data revealed a 50% decline in the intrinsic clearance of HCZX formation due to an IR3-induced significant decline in maximum velocity. Although IR3 did not affect the microsomal CYP2E1 protein, it caused approximately 30% reduction in the cytochrome P450 reductase activity. IR24 did not have any effect on the disposition of CZX or HCZX. In conclusion, metabolism of xenobiotics and endogenous compounds that are substrates for CYP2E1, and possibly other P450 isoenzymes, may be reduced shortly after surgical procedures that require transient interruption of the hepatic blood flow.     

Hepatobiliary disposition of rhodamine 123 in isolated perfused rat livers

The disposition of rhodamine 123 (RH-123), a known marker of P-glycoprotein, and its liver-generated glucuronide metabolite (RH-Glu), a marker of Mrp2, was studied in an isolated perfused rat liver model. Livers were perfused with a buffer containing 0.1 microg ml(-1) RH-123 for 30 or 60 min or for 30 min followed by 90 min of drug-free perfusion, and the concentrations of the drug and its metabolites were determined in the perfusate, bile, and the liver tissue. The outlet perfusate concentrations of RH-123 and RH-Glu reached an apparent plateau during the continuous infusion of the drug, with a very extensive extraction ratio of approximately 96% for the parent drug. However, the biliary excretion rates of both RH-123 and generated RH-Glu continued to rise almost linearly during the entire 60 min of drug infusion. This was associated with a linear increase in the amount of RH-123 recovered in the liver between 30 and 60 min of drug infusion, resulting in a significant (>50% of the administered dose) recovery of the marker in the liver both after 30 and 60 min of perfusion. Additionally, the washout experiments showed that the declines in the biliary excretion rates of RH-123 and RH-Glu were parallel to that of RH-123 concentration in the liver in the absence of drug input. The hepatobiliary disposition of RH-123 in rats is unique because of its substantial and time-dependent accumulation in the liver, resulting in a lack of steady-state in its biliary excretion despite apparent steady-state in the perfusate.     

Hepatobiliary disposition of rhodamine 123 in isolated perfused rat livers

The disposition of rhodamine 123 (RH-123), a known marker of P-glycoprotein, and its liver-generated glucuronide metabolite (RH-Glu), a marker of Mrp2, was studied in an isolated perfused rat liver model. Livers were perfused with a buffer containing 0.1 µg ml^?1 RH-123 for 30 or 60 min or for 30 min followed by 90 min of drug-free perfusion, and the concentrations of the drug and its metabolites were determined in the perfusate, bile, and the liver tissue. The outlet perfusate concentrations of RH-123 and RH-Glu reached an apparent plateau during the continuous infusion of the drug, with a very extensive extraction ratio of approximately 96% for the parent drug. However, the biliary excretion rates of both RH-123 and generated RH-Glu continued to rise almost linearly during the entire 60 min of drug infusion. This was associated with a linear increase in the amount of RH-123 recovered in the liver between 30 and 60 min of drug infusion, resulting in a significant (>50% of the administered dose) recovery of the marker in the liver both after 30 and 60 min of perfusion. Additionally, the washout experiments showed that the declines in the biliary excretion rates of RH-123 and RH-Glu were parallel to that of RH-123 concentration in the liver in the absence of drug input. The hepatobiliary disposition of RH-123 in rats is unique because of its substantial and time-dependent accumulation in the liver, resulting in a lack of steady-state in its biliary excretion despite apparent steady-state in the perfusate.     

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.     

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.     

The effect of diethylstilbestrol treatment on the metabolism and biliary excretion of [14C]phenytoin in the isolated perfused rat liver

The metabolism and biliary excretion of [¹⁴C]phenytoin (DPH) were examined in isolated perfused livers taken from Sprague-Dawley rats pretreated with 0.01, 0.05, 0.1, 0.5, and 1.0 mg/day diethylstilbestrol (DES) sc for 6 days. No difference was seen in the rate of disappearance of DPH from the perfusate or in the perfusate levels of its hydroxylated metabolite, 5-phenyl-5-para-hydroxyphenylhydantoin (HPPH). The biliary excretion of HPPH-glucuronide, however, was significantly depressed in livers from DES-treated rats and resulted in a significant increase in the amount of HPPH-glucuronide appearing in the perfusate. A linear relationship existed between the percentage decrease in biliary excretion of HPPH-glucuronide and the log of the pretreatment dose of DES. Bile flow was significantly depressed at all pretreatment doses of DES such that bile flow was 53.7 and 10.9% of bile flow in controls after 0.01 and 1.0 mg/day DES, respecively. The low bile flow appeared to limit secretion of HPPH-glucuronide in the bile since the maximal concentration of HPPH-glucuronide in bile was greater in livers from DES-treated rats than controls and no significant differences were found in the maximal bile/perfusate concentration ratios of HPPH-glucuronide.     

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.     

Biosynthesis and biliary excretion of S-conjugates of hexachlorobuta-1,3-diene in the perfused rat liver.

The formation and biliary excretion of the glutathione and cysteine S-conjugates of hexachlorobutadiene were studied in the isolated perfused rat liver. Infusion of increasing amounts of hexachlorobutadiene led to an increase in total metabolite excretion. Partitioning of glutathione conjugate release between bile and perfusate depended on the rate of substrate infusion: S-(1,2,3,4,4-pentachlorobutadienyl)glutathione (PCBG) appeared quantitatively in bile at low hexachlorobutadiene infusion rates, and increasing amounts of the glutathione conjugate were found in the perfusate as infusion rates were increased. The cysteine S-conjugate, S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine, was not detected in the perfusate, but the amounts found in the bile were correlated with the concentrations of the glutathione conjugate. Depletion of hepatic glutathione concentrations decreased PCBG formation. Hence, at moderate hexachlorobutadiene infusion rates, PCBG is exclusively excreted into bile, indicating that intestinal absorption of PCBG or its metabolites is required for the induction of kidney damage in vivo.     

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.     

The disposition of primaquine in the isolated perfused rat liver. Effect of dose size

The disposition of 14C-radiolabeled primaquine in the isolated perfused rat liver preparation was investigated after the administration of 0.5-, 1.5-, and 5.0-mg doses of the drug. The pharmacokinetics of primaquine in this experimental model were dependent on dose size. Increasing the dose from 0.5 to 5.0 mg produced a significant reduction in clearance from 11.6 +/- 2.5 to 2.9 +/- 1.0 ml X min-1. This decrease was accompanied by a disproportionate increase in the value of AUC from 25.4 +/- 5.9 to 1128.6 +/- 575.7 micrograms X min X ml-1, elimination half-life from 33.2 +/- 10.7 to 413.0 +/- 239.3 min, and volume of distribution from 547.7 +/- 153.1 to 1489.0 +/- 249.0 ml. Furthermore, primaquine exhibited dose dependency in its pattern of metabolism. While the carboxylic acid derivative of primaquine was not detected in perfusate after the 0.5-mg dose, it was the principal perfusate metabolite after the 5.0-mg dose. Primaquine was subject to extensive biliary excretion at all doses; the total amount of 14C radioactivity excreted in the bile decreased from 60 to 30% as the dose of primaquine was increased from 0.5 to 5.0 mg. The metabolite composition of radioactivity excreted in the bile was also examined. Total recovery of the administered radioactivity from bile, perfusate, and liver was essentially complete at all doses. The perfusate concentrations at the conclusion of each experiment (i.e. 5 hr) did not differ among dosage groups. By contrast, increased dose size produced a reduction in the amount of 14C radioactivity recovered in bile which was associated with increased levels of 14C in the liver.     

Physiological modeling of drug and metabolite: disposition of oxazepam and oxazepam glucuronides in the recirculating perfused mouse liver preparation

The disposition of tracer doses of 3H-oxazepam was studied in the recirculating perfused mouse liver preparation. 3H-Oxazepam was biotransformed primarily to the diastereomeric 3H-oxazepam glucuronides, which either effluxed into the circulation or underwent biliary excretion. Three additional, unknown metabolites constituted a small fraction (5-10%) of the total radioactivity recovered in bile (7% of dose); no other metabolite was detected in perfusate. A physiologically based model, comprising the reservoir, liver blood and tissue, and bile, was fitted to reservoir concentrations of 3H-oxazepam and 3H-oxazepam glucuronides, and the cumulative amount excreted into bile. The model allowed for consideration of elimination pathways other than glucuronidation and the presence of a transport barrier for the oxazepam glucuronides across the hepatocyte membrane. The fitted results suggest a slight barrier existing for the transport of metabolites across the sinusoidal membrane, inasmuch as the transmembrane clearance was comparable to liver blood flow rate. Upon further comparison of estimates of formation, biliary, and transmembrane clearances for the oxazepam glucuronides, the rate-limiting step in the overall (biliary) clearance appears to be a poor capacity for biliary excretion. The influence of the cumulative volume loss that a recirculating perfused organ system incurs upon repeated sampling was discussed, and a compartmental method of correcting the observed concentrations of drug and generated metabolite was presented.     

Effect of chlorpromazine on the function of the perfused isolated liver

The acute effect of chlorpromazine on the function of the perfused rat liver was evaluated by monitoring the removal of sulfobromophthalein (BSP) from the perfusate, the biliary excretion of the dye and the rate of bile and perfusate flow.     

Metabolism of Roxithromycin in the Isolated Perfused Rat Liver

Roxithromycin is a macrolide antibiotic with high clinical potency. N-Demethylation is considered to be one of the main pathways of roxithromycin metabolism in rats. We have studied the hepatic metabolism of roxithromycin in the isolated perfused rat liver. After addition of roxithromycin (30 μM) to the perfusion medium the parent compound and one major metabolite were detected in bile by high-performance liquid chromatography. The metabolite was identified as monodesmethylated roxithromycin by mass spectrometric analysis. Onset of biliary excretion of native roxithromycin was fast, reaching a maximum (130.52 ±43.88 pmol g^?1 min^?1) after only 10 min, whereas excretion of the metabolite was delayed (maximum 75.83 ± 11.92 pmol g^?1 min^?1 at 30 min). The cumulative excretion of roxithromycin and its metabolite into bile during the 60 min of application amounted to only 1.09 ± 0.30 and 0.64 ± 0.22% of the roxithromycin cleared from the perfusate during the same time. The liver content was 0.48 μmol (g liver)^?1, indicating high retention within the organ. No release of the metabolite into the perfusate was detected. In conclusion, this study has demonstrated the importance of phase-I metabolism for the biliary excretion of roxithromycin in rat liver. These findings might be predictive of roxithromycin biotransformation and biliary excretion in man.     

Biotransformation of 7-ethoxycoumarin in isolated perfused rainbow trout liver

A reliable technique for rainbow trout liver perfusion has been developed for studies on xenobiotic biotransformation. Normal function of the perfused liver was indicated throughout the perfusion experiment by 1) a proper oxygen consumption, 2) a low leakage of intracellular enzymes, 3) a stable pH value in the effluent, and 4) a stable release of metabolites into the effluent perfusate and bile for at least 2 hr after maximal rate of metabolism was attained. The main metabolite of 7-ethoxycoumarin in effluent perfusate was identified as 7-hydroxycoumarin glucuronide. Only trace amounts were identified as sulfates. When fish were pretreated with Clophen A50 or beta-naphthoflavone, the amount of metabolites released into the effluent perfusate increased 3.4- and 6.4-fold, respectively, when compared to control livers. Furthermore, in livers from Clophen A50- or beta-naphthoflavone-treated fish, only 80 and 67%, respectively, of excreted products were conjugated. Influence of temperature on 7-ethoxycoumarin metabolism was studied in perfused liver and isolated liver microsomes. Results indicate that the Q10 for the metabolism of 7-ethoxycoumarin in perfused liver deviates from that found in isolated microsomes. The amount of metabolites excreted into the bile consisted of about 25% of the amount found in effluent perfusate. The only metabolite detected in bile from perfused liver from control as well as treated fish was 7-hydroxycoumarin glucuronide.     

The effects of alcohol and diallyl sulphide on CYP2E1 activity in humans: a phenotyping study using chlorzoxazone.

The effects of acute administration of dietary levels of ethanol and the garlic oil extract, diallyl sulphide (DAS), on cytochrome P450 2E1 (CYP2E1) activity in volunteers were studied using the selective probe substrate, chlorzoxazone (CZX). The ratio of the CZX metabolite 6- hydroxychlorzoxazone (6-OHCZX) to CZX was taken to indicate CYP2E1 activity. The mean differences between the baseline and DAS-treated (0.2 mg/kg) CYP2E1 activities were significantly different (two-tailed p value = 0.0242, n = 8). Likewise, the mean differences between the baseline and ethanol-treated (0.8 g/kg) CYP2E1 activities were also significantly different (two-tailed p value = 0.0005, n = 7). The reduction in in vivo CYP2E1 activity by DAS is consistent with reported inhibition observed in vitro. The marked reduction in CYP2E1 activity following acute ingestion of ethanol is consistent with a competitive inhibition mechanism of CZX metabolism. The inhibitory effect of DAS maybe additive with daily consumption of Allium vegetables in particular. This may explain the lower 6-OHCZX/CZX metabolic ratios measured in various European and Mexican cohorts and is consistent with the lower incidence of stomach, liver and colon cancers observed in southern Europeans.     

Biliary excretion of novel pneumotoxic metabolites of the pyrrolizidine alkaloid, monocrotaline

W. M. Lafranconi, S. Ohkuma and R. J. Huxtable. Biliary excretion of novel pneumotoxic metabolites of the pyrrolizidine alkaloid, monocrotaline. Toxicon23, 983–992, 1985. — Perfusion of the pyrrolizidine alkaloid, monocrotaline, through the isolated rat liver resulted in the appearance of Ehrlich-positive metabolites in both the perfusate and bile. Livers from male rats released greater quantities of metabolite into both bile and perfusate. Metabolite release was stimulated by pretreatment with phenobarbital. Livers of phenobarbital-pretreated male rats perfused with 300 μM monocrotaline produced biliary metabolite concentrations in excess of 5 mM. Metabolite release was inhibited by anoxic perfusion; low temperature and pretreatment with SKF-525A. Above 150 μM monocrotaline, bile became the predominant route of excretion. On perfusion through the isolated lung of the rat, bile and perfusate metabolites were equally effective in inhibiting serotonin transport. A single Ehrlich-positive peak was obtained on silica gel column chromatography of bile containing metabolite. Mass spectrometry revealed the major component of this peak to have a molecular weight of 281, indicating a novel pyrrole metabolite in which the esterifying acid present in monocrotaline, monocrotalic acid, had been partially degraded. This compound mimics the pneumotoxic action of monocrotaline giver, in vivo, and its availability should prove a valuable tool in the elucidation of the mechanism of pyrrolizidine toxicity.     

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.     

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.     

Hepatic uptake and biliary secretion of bile acids in the perfused rat liver.

Hepatic uptake and biliary secretion have been evaluated in the isolated perfused rat liver for cholic, chenodeoxycholic, ursodeoxycholic acid, both free and taurine-conjugated; the physicochemical properties of the bile acids have also been calculated and related to these experimental parameters. Cholic acid disappearance rate from the perfusate was the fastest, followed by that of ursodeoxycholic and chenodeoxycholic; it was also faster for taurine-conjugated bile acids than for their respective unconjugated forms. The recovery in bile was higher for conjugated than for unconjugated bile acids, and among each class, was higher for cholic than for chenodeoxycholic and ursodeoxycholic. The hepatic uptake correlated negatively (r = -0.99) with the bile acid lipophilicity, while the biliary secretion correlated with the solubility of the molecules. These results show the effect of the physicochemical properties of BA on their hepatic handling, at the physiological concentration of BA in the portal blood.