Metabolism of [14C]monocrotaline by isolated perfused rat liver.

The metabolism of the pyrrolizidine alkaloid [14C]monocrotaline [( 14C]MCT) was examined using the in situ isolated perfused rat liver. Hepatic tissue was perfused in a recirculatory fashion for 90 min and the distribution of metabolites between the bile and perfusate was analyzed. Monocrotalic acid (MCA) was found to be the major acidic metabolite of [14C]MCT, with trace amounts of 1-formyl-7-hydroxy-6,7-dihydro-5H-pyrrolizine, 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), and 1-hydroxymethyl-7-oxo-6,7-dihydro-5H-pyrrolizine (tentative assignment) being identified in the perfusates using GC/MS. MCT N-oxide was also identified but represented less than 4% of the perfusate 14C. The simple necine base retronecine was not present at detectable levels in the perfusion medium. A large portion of the 14C recovered from both the bile and perfusate was not extractable, under acidic or basic conditions, into organic solvents. Using fast atom bombardment MS/MS, a portion of this material was identified as a glutathione conjugate of DHP. In addition, this nonextractable material retained a portion of the radioactivity that was equivalent to the acidic fraction. Given these findings and the absence of retronecine, the major pathway for the metabolism of MCT could potentially involve the production of MCT pyrrole, which subsequently reacts with cellular nucleophiles producing MCA in addition to highly water-soluble conjugated pyrroles and possibly macromolecular adducts.     

The effect of pregnancy and estradiol-17 beta treatment on the biliary transport maximum of dibromosulfophthalein, and the glucuronide conjugates of 5-phenyl-5-p-hydroxyphenyl[14C]hydantoin and [14C]morphine in the isolated perfused rat liver

The biliary transport maximum (Tm) of three organic axions was determined in the isolated perfused livers of untreated female (control), estradiol-17 beta (E2)-treated female (1 mg/kg/day, s.c. for 14 days), and pregnant (19-21 days of gestation) rats. Dibromosulfophthalein (DBSP), 5-phenyl-5-p-hydroxyphenyl[14C]hydantoin (HPPH) and [14C]morphine were infused continuously into the perfusate for a total dose of 41.2, 18, or 40.5 mumol, respectively. The concentration of [14C]HPPH and [14C]morphine declined in the perfusate, whereas the concentrations of [14C]HPPH glucuronide and [14C]morphine glucuronide increased during the 90-min experiment, indicating that the rate of formation of the glucuronide exceeded its rate of excretion in bile. E2 treatment decreased the Tm (nmol/min/g liver) for [14C]HPPH glucuronide and [14C]morphine glucuronide but not for DBSP, whereas pregnancy decreased the Tm for all three organic anions. Pregnancy, and to a lesser extent E2 treatment, increased liver weight. When expressed per whole liver, the Tm was not altered by pregnancy for any of three organic anions. E2 treatment increased the Tm for DBSP, had no effect on the Tm for HPPH glucuronide and decreased the Tm for [14C]morphine glucuronide. These data suggest the presence of multiple carriers for organic anions which are differentially affected by estrogen treatment and pregnancy.     

Hepatic extraction, metabolism, and biliary excretion of irinotecan in the isolated perfused rat liver

Irinotecan (CPT-11) is a semisynthetic derivative of camptothecine that has proved activity in the treatment of colorectal carcinoma. The metabolites identified in humans include SN-38, SN-38 glucuronide, and several CYP3A-derived metabolites. We have studied the hepatic extraction, metabolism, and biliary excretion of irinotecan in the isolated perfused rat liver. After injection of a bolus dose of 5 micromol in the reservoir, irinotecan lactone disappeared from the perfusate following a two-exponential decay with half-lives of 3.5 and 120 min and a total clearance of 1.54 +/- 0.07 mL/min per gram of liver. The area under the curve (AUC) ratio lactone/total drug was 0.212 +/- 0.098 and the half-life of interconversion was 5.02 +/- 0.10 min. Bolus administrations of 2.5, 5, and 25 micromol of irinotecan gave AUCs proportional to the doses administered, indicating that no saturation occurred during dose increase. However, the relative formation of SN-38 and SN-38 glucuronide decreased at the high dose. This result was not the case for the CYP3A metabolites, which had identical metabolic ratios at all three doses. Infusions of 30 and 90 min of a dose of 5 micromol led to the same AUCs and metabolic ratios as a bolus of the same dose. Biliary elimination of irinotecan and metabolites represented 18-22% of the dose administered at 2.5 and 5 micromol but only 7-9% at 25 micromol, suggesting a saturation of this process. These data indicate that the hepatic disposition of irinotecan may vary at high dose, both at the level of biliary excretion and of activation to SN-38.     

Metabolism of [14C]phenol in the isolated perfused mouse liver.

A previous report from this laboratory focused on the metabolism of [14C]benzene (BZ) in the isolated, perfused, mouse liver (C. C. Hedli, et al., 1997, Toxicol. Appl. Pharmacol. 146, 60-68). Whereas administration of BZ to mice results in bone marrow depression (R. Snyder et al., 1993, Res. Commun. Chem. Pathol. Pharmacol. 20, 191-194), administration of phenol (P), the major metabolite of BZ, does not. It was, therefore, of interest to determine whether the metabolic fate of P produced during BZ metabolism differed from that of P metabolized in the absence of BZ. Mouse livers were perfused with a solution of [14C]P in both the orthograde (portal vein to central vein) and retrograde (central vein to portal vein) direction to investigate the metabolic zonation of enzymes involved in P hydroxylation and conjugation. Perfusate samples were collected, separated by HPLC, and tested for radioactivity. Unconjugated metabolites were identified by comparing their retention times with nonradiolabeled standards, which were detected by UV absorption. Conjugated metabolites were identified and collected on the basis of radiochromatogram results, hydrolyzed enzymatically, and identified by co-chromatography with unlabeled BZ metabolites. The objective was to compare and quantify the metabolites formed during the perfusion of P in the orthograde and retrograde directions and to compare the orthograde P-perfusion results with the orthograde BZ results reported previously. Regardless of the direction of P perfusion, the major compounds released from the liver were P. phenylgucuronide, phenylsulfate, hydroquinone (HQ), and HQ glucuronide. A comparison of the results of perfusing P in the orthograde versus the retrograde direction showed that more P was recovered unchanged and more HQ was formed during retrograde perfusion. The results suggest that enzymes involved in P hydroxylation are generally closer to the central vein than those involved in conjugation, and that during retrograde perfusion, P metabolism may be limited by the sub-optimal conditions of perfusion. Comparison of the orthograde perfusion studies of P and BZ revealed that a larger percentage of the radioactivity released from the liver was identified as unconjugated HQ after BZ perfusion than after P perfusion. In addition, the amount of radioactivity covalently bound to liver macromolecules was measured after each perfusion and determined to be proportional to the amount of HQ and HQG detected in the perfusate samples.     

Malaria infection impairs glucuronidation and biliary excretion by the isolated perfused rat liver

1. The effect of the erythrocytic stage of malaria infection on hepatic glucuronidation, biliary excretion and oxidation processes was investigated using harmol, salbutamol, taurocholate and propranolol. Livers from rats infected with the rodent malaria parasite P. berghei were isolated and perfused in a single-pass (harmol, taurocholate, propranolol) or recirculating (harmol, salbutamol) design. The degree of erythrocytic parasitaemia ranged from 16% to 63%.

2. The hepatic clearance (Cl) of harmol decreased from 7˙8±0˙4 ml/min in controls to 5˙7±1˙1 ml/min in the malaria-infected group in single-pass studies. This corresponded to a 40–60% reduction in hepatic intrinsic clearance (Cl_int). Similar changes were observed using the recirculating design when glucuronidation accounted for >90% of harmol metabolism.

3. The Cl of salbutamol, metabolized exclusively by glucuronidation under the conditions used, also decreased significantly from 8˙5±0˙8 in controls to 6˙6±1˙4 ml/min in the malaria-infected group. This corresponded to a 40–70% reduction in Cl_int.

4. The Cl of taurocholate, excreted unchanged in bile, decreased slightly but significantly from 9˙6±0˙3 ml/min in controls to 8˙3±0˙9 ml/min in the malaria-infected group. In the same livers, there was also a slight but significant decrease in propranolol Cl (10˙0±0˙1 ml/min and 9˙9±0˙1 ml/min, respectively). Both these compounds undergo flow-limited hepatic clearance; the decreases in Cl_int of taurocholate and propranolol were 87% and 35%, respectively.

5. Cl and Cl_int of each of the compounds studied were found to correlate significantly with the degree of erythrocytic parasitaemia. This study shows that glucuronidation, biliary excretion and oxidation by liver are impaired in malaria infection in rats, with biliary excretion being the most affected. The data indicate that there is a general decrease in hepatic elimination processes during the erythrocytic phase of malaria infection.     

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.     

The excretion of [14C] butylated hydroxytoluene in the rat

The excretion of small doses of [¹⁴C]labelled butylated hydroxytoluene has been examined in the rat. Parenteral doses are excreted slowly in urine and faeces over 4 days. Biliary excretion is rapid. The biliary radioactivity is absorbed readily from the gut and a rapid enterohepatic cycle has been found to operate over at least 96 hr.     

Malaria infection impairs glucuronidation and biliary excretion by the isolated perfused rat liver.

1. The effect of the erythrocyte stage of malaria infection on hepatic glucuronidation, biliary excretion and oxidation processes was investigated using harmol, salbutamol, taurocholate and propranolol. Livers from rats infected with the rodent malaria parasite P. berghei were isolated and perfused in a single-pass (harmol, taurocholate, propranolol) or recirculating (harmol, salbutamol) design. The degree of erythrocytic parasitaemia ranged from 16% to 63%. 2. The hepatic clearance (Cl) of harmol decreased from 7.8 +/- 0.4 ml/min in controls to 5.7 +/- 1.1 ml/min in the malaria-infected group in single-pass studies. This corresponded to a 40-60% reduction in hepatic intrinsic clearance (Clint). Similar changes were observed using the recirculating design when glucuronidation accounted for greater than 90% of harmol metabolism. 3. The Cl of salbutamol, metabolized exclusively by glucuronidation under the conditions used, also decreased significantly from 8.5 +/- 0.8 in controls to 6.6 +/- 1.4 ml/min in the malaria-infected group. This corresponded to a 40-70% reduction in Clint. 4. The Cl of taurocholate, excreted unchanged in bile, decreased slightly but significantly from 9.6 +/- 0.3 ml/min in controls to 8.3 +/- 0.9 ml/min in the malaria-infected group. In the same livers, there was also a slight but significant decrease in propranolol Cl (10.0 +/- 0.1 ml/min and 9.9 +/- 0.1 ml/min, respectively). Both these compounds undergo flow-limited hepatic clearance; the decreases in Clint of taurocholate and propranolol were 87% and 35%, respectively. 5. Cl and Clint of each of the compounds studied were found to correlate significantly with the degree of erythrocytic parasitaemia. This study shows that glucuronidation, biliary excretion and oxidation by liver are impaired in malaria infection in rats, with biliary excretion being the most affected. The data indicate that there is a general decrease in hepatic elimination processes during the erythrocytic phase of malaria infection.     

Prednisolone metabolism and excretion in the isolated perfused rat kidney

The isolated perfused rat kidney was used to identify factors responsible for the renal elimination of prednisolone (Pn). Pn was recirculated at initial concentrations varying from 100 to 1000 ng/ml for 90 min. Perfusate and urine samples were assayed for Pn and prednisone by HPLC. Protein binding of Pn was measured by using 3H-Pn and equilibrium dialysis at 37 degrees C. There were no significant differences in perfusate flow, glomerular filtration rate, urine flow, or sodium excretion between control and steroid experiments. Partial metabolism of Pn to prednisone occurred in all studies. The total kidney clearance (CIT) of Pn ranged from 0.39 to 1.24 ml/min/100 g of rat body weight with approximately half of the Pn dose unaccountable for as either Pn or prednisone. The apparent percentage of the Pn dose excreted unchanged in the urine ranged from 1.9 to 6.4% and was not related to Pn dose. The apparent urinary clearances of Pn and its metabolite, prednisone, normalized for inulin clearance (fractional excretion) were variable with means of 0.068 and 0.095, respectively. The fractional excretions of Pn and prednisone were related to the fraction of filtered water excreted but not to perfusate concentration. Thus, the extent of urinary clearance of these corticosteroids is related to glomerular filtration and passive tubular reabsorption. The perfused rat kidney reflects the urinary and renal metabolic clearance of Pn without the complication of dose-dependent disposition.     

The in-vitro metabolism of [14C]pentobarbitone and [14C]phenobarbitone by hamster liver microsomes

The metabolism of [14C]pentobarbitone and [14C]phenobarbitone has been reinvestigated using an in-vitro hepatic microsomal system (Syrian hamsters, Aroclor 1254 induction). The incubation system was routinely supplemented with EDTA (1 mM) and a substrate concentration study revealed the metabolism of [14C]pentobarbitone to be concentration-dependent, with the greatest overall metabolism (greater than 50%) occurring at 0.054 mumol per 3.5 mL. With [14C]phenobarbitone as substrate, overall metabolism was extremely low (3%) and independent of substrate concentration. Addition of further cofactors to the incubation mixture at 20 min intervals over an extended period resulted in almost complete metabolism of [14C]pentobarbitone (100 min), 3'-hydroxypentobarbitone and 3'-oxopentobarbitone being identified as metabolites together with many minor, unidentified products. With [14C]phenobarbitone as the substrate, cofactor addition up to 120 min resulted in 8% overall metabolism; p-hydroxyphenobarbitone was identified as a product of metabolism; other minor products were unidentified. The metabolism studies failed to produce a metabolite having the properties of the N-hydroxylated product of either [14C]pentobarbitone or [14C]phenobarbitone within the detection limits available (0.02% of 0.5 mumol per incubate).     

The fate of saccharin impurities. The excretion and metabolism of [14C]toluene-4-sulphonamide and 4-sulphamoyl[14C]benzoic acid in the rat

1. 4-Sulphamoyl[carboxy-14C]benzoic acid was rapidly eliminateda after oral administration to rats (94% dose in 24 h). After 6 days most of the 14C (73-83% dose) was recovered in the urine with significant amounts (18-32% dose) in the faeces due to incomplete absorption. 2. The 14C in the urine and faeces was unchanged 4-sulphamoylbenzoic acid. No 14CO2 was detected in the expired air. 3. After oral administration of [methyl-14C]toluene-4-sulphonamide to rats the label was rapidly eliminated largely in the urine (66-89% dose) with little in the faeces (2-8% dose). The 14C in the faeces was 4-sulphamoylbenzoic acid, which probably originated in the tissues since the gut flora was unable to effect this biotransformation. 4. The urine of rats given [14C]toluene-4-sulphonamide contained 4-sulphamoylbenzoic acid as the major metabolite (93% of the urinary 14C) together with small amounts of unchanged compound (1.5-2.3% of urinary 14C), 4-sulphamoylbenzyl alcohol (2.0-3.9%), 4-sulphamoylbenzaldehyde (0-1.5%) and at higher doses N-acetyltoluene-4-sulphonamide (2.1-2.3%).     

The metabolism of lysergic acid di[14C]ethylamide ([14C]LSD) in the isolated perfused rat liver

Isolated female rat livers were perfused with a medium containing [¹⁴C]LSD (1 mg and 0.13 μCi/g liver) and (+)-tartaric acid (15 mg). After 4.5 hr, the bile collected contained some 44% of the added radioactivity, the perfusate, 20% and the liver itself, 20%.The radioactive compounds in the bile were identified as 14-hydroxy-LSD glucuronide (21% of the added ¹⁴C), 13-hydroxy-LSD glucuronide (8%), 2-oxo-LSD (7%) and unchanged LSD (1%). Those in the pooled perfusate and homogenised liver were unchanged LSD (18%), 2-oxo-LSD (5%), a naphthostyril derivative of LSD (4%; probably derived from 2-oxo-LSD), nor-LSD (4%), hydroxy-LSD glucuronides (3%) and de-ethyl-LSD (2%).     

地尔硫对大鼠离体肝脏灌流中安替比林代谢的影响

以安替比林（AP）为模型药,采用大鼠离体肝脏灌流模型（IPRL）,研究钙离子拮抗剂地尔硫本（DZ）对AP代谢的影响。方法：15只雄性Sprague-Dawley大鼠随机分为3组,A、B组灌胃（iP）生理盐水3d,第4天分别用5mgAP及加用2mgDZ循环灌流3h,C组ipDZ（100·kg-1）3d,第4天同B组灌流。HPLC法测定灌流液中AP及其代谢物的浓度。结果：B、C组中,AP的T1/2从（127．9±9.4）min分别延长至（2742±33.6）和（303.8±80．2）min（P＜0．01）;Cls从（0．67±0.08）ml·min－1分别减少至（0．36±0.06）和（0．34±0．09）ml·min－1（P＜0．01）;4-羟基AP的AUC0-3h分别降低73．95％和68.1％（P＜0．01）;3-羟甲基AP和N-去甲基AP和没有明显变化（P＞0．05）。结论：DZ明显抑制TAP在IPRL中的消除,且选择性地抑制AP的4-羟基化代谢途径。     

Loperamide inhibits the biliary excretion of irinotecan (CPT-11) in the rat isolated perfused liver

Patients treated with irinotecan (CPT-11) occasionally suffer from severe diarrhoea and aggressive treatment with loperamide at the first signs of loose stools is recommended. We have examined the effect of loperamide on the hepatic metabolism and biliary excretion of CPT-11 in the isolated perfused rat liver (IPRL). CPT-11 (0.5 mumol) was injected as a bolus into the IPRL reservoir, and perfusate and bile samples were collected over 3 h. Experiments were conducted using loperamide-free perfusate (n = 5) or perfusate containing 10 muM loperamide (n = 6). Perfusate and bile concentrations of total CPT-11 and the major metabolites SN-38 (7-ethyl-10-hydroxy-camptothecin), SN-38G (7-ethyl-10-hydroxy-camptothecin glucuronide) and APC (7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidine] carbonyloxycamptothecin) were determined by HPLC. The unchanged parent drug was the predominant species in bile, with approximately 4% of the dose recovered over 180 min as compared with only 1% for the metabolites. Loperamide significantly reduced the biliary excretion of CPT-11 by approximately 50% (2.0 +/- 0.9% dose compared with 3.8 +/- 1.0% in the control group, P = 0.019) over the same period. In contrast, the biliary excretion of SN-38, SN-38G and APC was not significantly affected by loperamide (P > 0.05). Furthermore, bile flow rate was not affected by loperamide. Loperamide appeared to selectively inhibit the biliary excretion of CPT-11, although the extent to which loperamide altered the disposition of CPT-11 in the clinical setting remains to be determined.     

The metabolism of [2-14C]methimazole in the rat

1 The metabolism of [2-¹⁴C]methimazole was studied in Sprague-Dawley rats after a daily intraperitoneal dose of 17±6?mg/kg for three days.

2. 78±4% of the administered ¹⁴C was excreted in the urine; only 6±7% dose as methimazole. After extraction of the urine with chloroform and n-butanol, 47±5% of administered ¹⁴C remained in the urine.

3. Six solvent-extractable metabolites were isolated and characterized by t.l.c. and high-resolution mass spectrometry, as N-methylimidazole, S-methylmethimazole, 3-methyl-2-thiohydantoin, 1-methyl-2-thiohydantoic acid, N-methylthiourea and a methylhydantoin.     

The effect of salicylate on the biliary excretion of 14C-bishydroxycoumarin in rat

1. The effect of intravenous sodium salicylate on the biliary excretion of 14C-bishydroxycoumarin ((14)C-BHC) was studied in rats.2. Salicylate (88.9 mg/kg) increased the biliary excretion of (14)C-radioactivity from a control value of 12.3 +/- 2.7% to 29.3 +/- 2.5% of the administered dose in 6 h after injection.3. During the 6 h period, 11% of the dose of radioactivity underwent biliary recycling in the salicylate-treated rats compared to only 6% in the absence of salicylate.4. About 15.3% of the radioactivity excreted in the bile of rats given BHC alone was detected as unchanged BHC, 8.6% was present as BHC-conjugate, conjugates of BHC metabolites accounted for 30.9%, and the remainder consisted of unidentified metabolites.5. Salicylate treatment did not significantly alter the excretory pattern of unchanged BHC and its metabolites.     

Taurocholate excretion and bile formation in the isolated perfused rat liver

Summary The excretory transport maximum (Tm) for taurocholate was determined in the perfused rat liver and compared to that obtained in the intact rat. An in situ liver perfusion system employing a semisynthetic perfusion medium containing Krebs-Ringer-bicarbonate solution, bovine erythrocytes and bovine albumin was used.In contrast to the bromosulfophthalein (BSP)-Tm reported previously, the taurocholate-Tm was 45% smaller in vitro (196±17 nmol/min per g liver) than in vivo (357±10 nmol/min per g liver), indicating that bile salt transport is more susceptible to alterations induced by the conditions of the perfusion than BSP transport. These findings add to the differences observed previously between the hepatic handling of anionic dyes and bile salts.At a low taurocholate infusion rat (57 nmol/min per g liver) the normal relationship between bile salt excretion and bile flow observed in vivo was maintained in the perfused liver. At higher taurocholate infusion rates, however, bile flow, for a given bile salt excretion rate, was smaller than in vivo.These findings should be taken into account when the isolated perfused rat liver is employed for studies of bile formation.