Inhibitors of sterol synthesis. Metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one after intravenous administration to bile duct-cannulated rats

The metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one has been studied after intravenous administration to bile duct-cannulated rats. Very rapid and substantial conversion of the 15-ketosterol to polar biliary metabolites was observed in both male and female rats. For example, upon intravenous injection of [4-14C]5 alpha-cholest-8(14)-en-3 beta-ol-15-one to male bile duct-cannulated rats, approximately 86% of the administered 14C was recovered in bile in the first 38 h. Of the total amount of 14C recovered in bile in 38 h, approximately 50% was excreted in bile in the first 70 min and approximately 90% was excreted within 8 h after the injection of the 15-ketosterol. A substantial fraction of the polar biliary metabolites was shown to undergo enterohepatic circulation. Of the radioactivity derived from the labeled 15-ketosterol which was not recovered in bile or other excreta at 48 h after the intravenous administration of the 15-ketosterol, most (approximately 79%) was recovered in the form of cholesterol and cholesteryl esters of blood and the various tissues. The very substantial and rapid biliary excretion of polar metabolites of the 15-ketosterol (or of cholesterol derived from the 15-ketosterol), coupled with inhibition of the intestinal absorption of cholesterol by the 15-ketosterol, may contribute to the overall hypocholesterolemic action of the 15-ketosterol which has been observed in rodents and in nonhuman primates by providing a metabolic pathway(s) wherein a substantial fraction of the absorbed 15-ketosterol is rapidly removed from the body by biliary excretion in the form of polar metabolites.     

Stereoselectivity of biliary excretion of 2-arylpropionates in rats

To examine the stereoselectivity of biliary excretion, the optically pure enantiomers of ketoprofen (KT), ibuprofen (IBU), and flurbiprofen (FLU) were intravenously administered to normal and bile duct-cannulated rats at 10 mg/kg. The recovery of total KT in bile was significantly higher after administration of (S)-KT than after (R)-KT [90.1 ± 3.5% vs 68.8 ± 8.2%, n =3, P < 0.05]. In normal rats the terminal half-life of (R)-KT was significantly shorter than that of (S)-KT after administration of (R)-KT (2.2 ± 0.6 h vs 14.3 ± 4.9 h, n = 3, P < 0.05). The terminal half-life of both enantiomers was significantly shorter in rats with continuous bile drainage as compared to normal rats. No significant differences in pharmacokinetic parameters could be found between both enantiomers in bile duct-cannulated animals. The total amount of IBU in bile was slightly higher after administration of (S)-IBU than after (R)-IBU administration. The percentage of (R)-IBU after (R)-IBU administration, however, was very low [(R)-IBU: 1.5 ± 0.9%, (S)-IBU: 23.4 ± 5.8%]. In normal rats the clearance of (R)-IBU was significantly higher as compared to (S)-IBU. Differences in pharmacokinetic parameters between normal and bile duct-cannulated rats were not statistically significant due to high interindividual variability. The total recovery of FLU, which was excreted in bile to a lower extent than either KT or IBU, also tended to be greater after S-enantiomer administration. Only small amounts of (S)-FLU could be recovered in bile after (R)-FLU administration. The pharmacokinetic parameters did not differ significantly between (R)- and (S)-FLU or between normal and bile duct-cannulated rats due to its low inversion rate and low excretion via bile. © 1993 Wiley-Liss, Inc.     

Comparison of the biliary excretion of the four isomers of bilirubin-IX in Wistar and homozygous Gunn rats.

The biliary excretion of the four isomers of bilirubin-IX was studied in Wistar rats (JJ) and homozygous Gunn rats (jj). Synthetic preparations of 14C-labelled pigments were used. 1. After intravenous administration, the alpha-isomer was rapidly excreted in conjugated form in bile of Wistar rats. In Gunn rats excretion was insignificant. In contrast, both rat species promptly excreted the non-alpha-isomers at rates that were comparable with that found for bilirubin-IXalpha in Wistar rats. 2. In normal rats about 16% of the beta- and delta-isomers and at least 50% of the gamma-isomer were excreted as ester conjugates of the injected parent bile pigments. Conjugation of the beta- and delta-isomers had occurred exclusively at the carboxyl groups of pyrrole ring D and C respectively. For bilirubin-IXgamma no preference for any carboxyl group could be established. 3. In homozygous Gunn rats the non-alpha-isomers were apparently excreted chemically unaltered. This suggests that, as for bilirubin-IXalpha, conjugation of the non-alpha-isomers is also deficient in Gunn rats.     

Extrahpetic distribution of sulfobromophthalein.

Plasma clearance of sulfobromophthalein (BSP) is widely used as a measure of hepatic function. Its validity depends upon its exclusive elimination from the body via bile. For example, in the present study, when BSP was administered intravenously (i.v.) to rats at four different doses (18.75, 37.5, 75, and 150 mg/kg), less than 0.5% of each dose was excreted into the urine and between 70 and 85% was excreted into the bile within 6 h after administration. It has been assumed that the distribution of BSP is limited to the blood and liver witith very little appearing in other tissues. When we measured the amount of BSP in the plasma, liver, and the bile 10 min after the i.v. administration of either a high (150 mg/kg) or a low (18.75 mg/kg) dose of BSP, only 60% of the dose was accounted for. The concentration of BSP and 12-I-labelled albumin (RISA) was measured in various tissue samples 10 min after administration of 17.5 or 150 mg of BSP or RISA per kilogram. More BSP was found in all tissues than was contained in the plasma entrapped therein. Thus, the distribution of BSP is not limited to the liver and plasma. During excretion BSP leaves other tissue (kidney, spleen, lung, etc.) and is ultimately excreted into the bile.     

Oral absorption and excretion of icaritin, an aglycone and also active metabolite of prenylflavonoids from the Chinese medicine Herba Epimedii in rats

Icaritin (ICT) is a main aglycone and also active intestinal metabolite of prenylflavonoids from the Chinese medicine Herba Epimedii. In the present study, the oral absorption and excretion of this compound was investigated using rats for exploring its fate in the body, so as to better understanding its in vivo pharmacological activities. The free (parent) and total (parent plus conjugated metabolites) ICT concentrations in rat plasma, urine and bile, after intravenous (i.v.) and oral administration both at 5mg/kg, were determined before and after enzymatic hydrolysis with β-glucuronidase/sulphatase, respectively, by a HPLC-UV method. The results showed that free ICT plasma concentration after i.v. dose was rapidly decreased with average t(1/2, λ) of 0.43h, while the total ICT concentration was decreased slowly with t(1/2, λ) of 6.86h. The area under the curve of ICT conjugated metabolites was about 11-fold higher than that of free ICT. The majority of ICT in the body was excreted from the bile with 68.05% of dose over 8h after i.v. dosing, in which only 0.15% was in parent form. While very little amount of ICT was excreted from the urine with 3.01% of dose over 24h, in which the parent form was 0.62%. After oral administration, very little amount of parent ICT was detected only in 0.5, 1 or 2h plasma samples with the concentration less than LOQ, however, its total plasma concentration after enzymatic hydrolysis treatment was at relative high level with average maximum concentration of 0.49μg/ml achieved at 1h post dose. The oral bioavailability of ICT was 35% of dose, estimated by its total plasma drug concentrations. It is concluded that ICT can be easily absorbed into the body, and then rapidly conversed to its conjugated metabolites, and finally removed from the body mainly by biliary excretion.     

The influence of (3H)taurocholate on the biliary excretion of (14C)acetylprocaine amide ethobromide.

The biliary excretion rates of [14C]acetylprocaine amide ethobromide (acetyl-PAEB) and [3H]taurocholate, either administered alone or in combination to adult male Wistar rats, were studied. Their renal pedicles were ligated, and the common bile duct and one jugular vein cannulated. Acetyl-PAEB, 20 mg/kg, and sodium taurocholate, 70 mg/kg, were infused over a 5-min period. Blood and bile samples were collected every 10 min for 60 min. Liver samples were taken at 10 and 20 min. Approximately 100% of the administered taurocholate was excreted within 50 min. The simultaneous administration of acetyl-PAEB did not significantly alter the taurocholate excretion. The amount of the acetyl-PAEB dose excreted in 1 h was 9.4%. This was increased significantly to 16.5% when taurocholate was given concomitantly. The concentration of acetyl-PAEB in the bile increased significantly when taurocholate was given, and the ratios of its concentrations in bile-liver and bile-plasma were also increased. Taurocholate did not alter the liver-plasma concentration ratio of acetyl-PAEB. It is suggested that the concomitant administration of taurocholate increased the biliary excretion of acetyl-PAEB by facilitating its secretion by the liver into the bile.     

Absorption, metabolism, and excretion studies of carbon 14- and tritium-labeled derivatives of a ketomethylene containing tripeptide

Tritium and Carbon 14 analogs of the angiotensin converting enzyme inhibitor ketoACE were synthesized and their oral absorption, metabolism and excretion in rats were investigated. KetoACE, a ketomethylene analog of the tripeptide Bz-Phe-Gly-Pro, was slowly absorbed at a 35% level upon oral administration. It is rapidly eliminated from the blood with a half-life of about 10 minutes. Its excretion is primarily via the bile duct and it is excreted as 80% unchanged drug. The only identified metabolite consisting of 5-10% of the excreted radioactivity was determined to be the reduced ketoACE in which the ketone group was reduced to a hydroxyl.     

Metabolism of verruculogen in rats.

Radiolabeled verruculogen was detected in a wide range of body tissues 6 min after intravenous administration, but after a further 20 min it was mainly being excreted via the biliary route. In isolated liver perfusion, [14C]verruculogen was rapidly taken up by the liver and metabolized completely, principally to the related tremorgen TR-2 but also to a desoxy derivative of verruculogen. In addition, a smaller amount of an isomer of TR-2 was detected. These metabolic products were excreted in the bile.     

Biliary excretion of a stretched bilirubin in UGT1A1-deficient (Gunn) and Mrp2-deficient (TR-) rats

The metabolism and biliary excretion of a stretched bilirubin analog with a p-xylyl group replacing the central CH2 hinge were investigated in normal rats, Gunn rats deficient in bilirubin conjugation, and TR- rats deficient in bilirubin glucuronide hepatobiliary transport. Unlike bilirubin, the analog was excreted rapidly in bile unchanged in all three rat strains after intravenous administration. In TR- rats biliary excretion of the analog was diminished, but still substantial, demonstrating that the ATP-binding cassette transporter Mrp2 is not required for its hepatic efflux. These effects are attributable to differences in the preferred conformations of bilirubin and the analog.     

Gastrointestinal absorption of estrone sulfate in germfree and conventional rats

Steroids are extensively excreted in the bile of rats. There was no significant difference in biliary excretion of steroid following administration of [3H]-estrone sulfate into the proximal small intestine (PSI) of conventional (CVL; 17.8 +/- 62%; mean +/- SD) or germfree (GF; 28.2 +/- 5.3) rats. A similar finding resulted from administration into the distal small intestine (DSI)-CVL, 22.3 +/- 11.8%; GF, 11.4 +/- 3.7%. However, when the drug was given into the caecum, excretion in the bile of CVL rats after 5 h was 59.1% whereas in GF rats it was only 1.7%. When estrone was injected into the PSI and DSI of CVL and GF rats, absorption (as judged by excretion in bile) was more rapid than that seen with estrone sulfate. Five hours after injection into the PSI, biliary excretion was, in CVL 88.2% and in GF 81.7% and after injection into the DSI excretion was, in CVL 84.7% and in GF 83.6%. Absorption of estrone from the caeca of GF rats was apparently reduced (49.0% and 25.3% excreted in the bile of CVL and GF rats respectively). There was no significant difference in bile flow rate between CVL and GF rats. These results give unequivocal evidence of intact absorption of estrone sulfate from the small intestine of the rat. The rate of absorption is however very much reduced compared to the non-sulphated steroid. Estrone sulfate is not absorbed intact in the caecum but is hydrolysed by the gut microflora prior to absorption.     

Fecal neutral steroids and bile acids from germfree rats

The amount and composition of fecal neutral sterols and bile acids excreted by adult male germfree and conventional rats have been determined. The amounts of neutral sterols excreted were 12.8 (germfree) and 19.5 (conventional) mg/kg of body wt per day. The germfree rats excreted cholesterol and lathosterol (methostenol was not assayed); the conventional rats excreted coprostanol and coprostanone in addition. The amounts of bile acids excreted were 11.3 (germfree) and 21.4 (conventional) mg/kg of body wt per day. The bile acids excreted by the rats were tentatively identified as tauro--muricholate, tauro-alpha-muricholate, and tauro-cholate, besides an unidentified component. The conventional rats excreted the corresponding unconjugated acids as well as many other unconjugated bile acids. No significant correlation was found between the amount of coprosterols and the total amount of neutral sterols excreted by the conventional rats. This suggests that bacterial reduction of cholesterol is not an important mechanism of increasing neutral sterol excretion of conventional rats as compared to germfree rats. Evidence is presented that suggests that this difference in neutral sterol excretion is due to changes in intestinal secretion and sloughing between the two types of animal. The factors reponsible for the differences in bile acid excretion have not been identified.     

Age dependence of hepatic transport in control and phenobarbital-pretreated rats.

Eosine is excreted in rat bile unchanged, which makes it suitable for the study of age dependent changes in hepatic uptake and excretion. Bile flow was approximately 40 μl/kg/min in 20-day-old rats and twice as high in 30-day-old animals. In 60- and 120-day-old rats the bile volume was decreased, moreover in 220-day-old ones it fell to the level of 20-day-old rats. The biliary excretion of eosine (150 μmol/kg i.v.) was highest in 60-day-old rats, however, the biliary flow reached its peak in 30-day-old rats. After phenobarbital (PB) pretreatment (75 mg/kg i.p. daily for five days) each age group showed enhancement in liver weight and bile volume. On the other hand, the hepatic concentration of eosine did not change after PB pretreatment caused an increase in the biliary excretion of eosine in 30-, 60-, 120- and 220-day-old rats but no significant change in 20-day-old animals. Our results indicate that the hepatic transport in young rats was immature and was not induced by PB. However, PB can increase the low excretion rate in old rats.     

Effect of wheat bran on excretion of radioactively labeled estradiol-17 beta and estrone-glucuronide injected intravenously in male rats.

Urinary and fecal estrogen excretion were studied in male rats fed a non-fiber wheat starch diet (dietary fiber less than 1%; NF group; n = 4), a low-fiber wheat flour diet (dietary fiber 2%; LF group; n = 4) or a high-fiber wheat bran diet (dietary fiber 11.6%; HF group; n = 3). Short-term effects of the experimental diet on estrogen excretion were studied after i.v. injection of 5 microCi (0.185 MBq) of [14C]estradiol-17 beta (E2) into the tail vein of the rats fed the diets for 2 days. After 3 weeks on the experimental diets, the long-term effects were studied after injection of 5 microCi of [14C]E2 and 10 microCi of [3H]estrone-3-glucuronide (E1-gluc). The diet was found to affect estrogen excretion. The short-term effect indicated that rats fed the HF diet excreted a relatively large amount of labeled compounds in the feces during the first day after injection, while rats fed the NF or the LF diets excreted about half that amount over the same period. On the other hand, urinary excretion of labeled compounds was significantly higher in the NF and LF rats. The long-term effect resulted in steeper slopes (P less than 0.05) of the fecal excretion profiles of rats fed the HF diet as compared with rats fed the NF and LF diets, indicating an accelerated fecal excretion of labeled compounds in the HF rats. The kinetic profiles of 14C and 3H radioactivity in blood plasma indicated a fast decrease (t1/2 of less than 2 min) for both [14C]E2 and [3H]E1-gluc. It was concluded that, owing to the short-term effect of wheat bran intake, during the first 24 h after i.v. administration relatively large amounts of radioactively labeled compounds are excreted in feces of rats fed the HF diet. In contrast, excretion is lower in urine of these rats. When the microflora is adapted to the experimental diet the wheat bran diet still results in an accelerated fecal excretion of labeled compounds, which might be attributed to an interruption of the enterohepatic circulation of estrogens. This might result in lowered plasma and/or tissue estrogen levels and hence a decreased exposure of estrogen-sensitive tissue to estrogens, which might decrease risk on mammary (breast) cancer development.     

The biliary excretion of trypsin in rats

The serum half-life of bovine [³H]acetyltrypsin was estimated to be 9 rain following intravenous administration in rats. This was maintained when six successive doses of 200 g each were given at 1-h intervals. The enzyme was removed from the circulation after complexing with 2-macroglobulin (2-M). The amount of³H label appearing in bile increased with each successive dose and this was associated with breakdown products (<10 000 daltons) of the ₂–M/[³H] acetyltrypsin. Intact –M/[³H] acetyltrypsin was recovered from bile but represented only 0.06% of the administered dose of active enzyme.     

Excretion of copper complexed with thiomolybdate into the bile and blood in LEC rats

Copper (Cu) accumulating in a form bound to metallothionein (MT) in the liver of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, was removed with ammonium tetrathiomolybdate (TTM), and the fate of the Cu complexed with TTM and mobilized from the liver was determined. TTM was injected intravenously as a single dose of 2, 10 or 50 mg TTM/kg body weight into LEC and Wistar (normal Cu metabolism) rats, and then the concentrations of Cu and molybdenum (Mo) in the bile and plasma were monitored with time after the injection. In Wistar rats, most of the Mo was excreted into the urine, only a small quantity being excreted into the bile, while Cu excreted into the urine decreased. However, in LEC rats, Cu and Mo were excreted into the bile and blood, and the bile is recognized for the first time as the major route of excretion. The Cu excreted into both the bile and plasma was accompanied by an equimolar amount of Mo. The relative ratio of the amounts of Cu excreted into the bile and plasma was 40/60 for the low and high dose groups, and 70/30 for the medium dose group. The systemic dispositions of the Cu mobilized from the liver and the Mo complexed with the Cu were also determined for the kidneys, spleen and brain together with their urinal excretion. Although Mo in the three organs and Cu in the kidneys and spleen were increased or showed a tendency to increase, Cu in the brain was not increased at all doses of TTM.     

Studies on flavonoid metabolism. Biliary and urinary excretion of metabolites of (+)-[U-14C]catechin

1. The biliary and urinary excretion of (+)-[U-(14)C]catechin was studied in normal male rats after a single injection of the flavonoid. 2. In rats large amounts of radioactivity (33.6-44.3% of the dose in 24h) were excreted in the bile as two glucuronide conjugates [one of which was a (+)-catechin conjugate] and three other unconjugated metabolites. 3. Excretion of radioactivity in the urine when the bile duct was not cannulated amounted to 44.5% of the dose. 4. In both the urine and bile the new metabolites showed maximum excretion in the (1/2)-1(1/2)h after intravenous injection of [(14)C]catechin. 5. The metabolites m-hydroxyphenylpropionic acid, p-hydroxyphenylpropionic acid, delta-(3-hydroxyphenyl)-gamma-valerolactone and delta-(3,4-dihydroxyphenyl)-gamma-valerolactione originate from the action of the intestinal micro-organisms on the biliary-excreted metabolites of (+)-catechin. These phenolic acid and lactone metabolites are then reabsorped and excreted in the urine. 6. It is proposed that, depending on the route of administration of (+)-catechin, there exists an alternative pathway, involving biliary excretion, for the metabolism of (+)-catechin.     

Comparison of biliary excretion and metabolism of lithocholic acid and its sulfate and glucuronide conjugates in rats

Biliary excretion and biotransformation of tracer doses of [14C]lithocholic acid and its sulfate and glucuronide intravenously injected into bile-drainaged rats were compared. Biliary excretion efficiency was in the order of unconjugate sulfate glucuronide and all conjugates were completely excreted into bile within 60 min after injection. Only tracer doses of radioactivity were found in the liver and urine. About 90% of radiolabeled bile acids in bile were conjugated with taurine immediately after injection of lithocholic acid, whereas lithocholic acid-glucuronide was only partly conjugated with taurine all the time (less than 6%) and excreted into bile mainly as native compound. In the first 10 min, 66% of lithocholic acid-sulfate was conjugated with taurine and it gradually proceeded up to 87%. Hydroxylation at C-6 and C-7 positions of lithocholic acid proceeded time-dependently up to 45%. No hydroxylation was observed with lithocholic acid-sulfate or glucuronide. Differences of biliary excretion rate of these conjugates may be one of the reasons for the delayed decrease of sulfated and glucuronidated bile acids in serum after bile drainage to patients with obstructive jaundice of during the recovery of acute hepatitis than non-esterified bile acids.     

Biliary excretion of 110mAg and its kinetics in the isolated perfused liver in rats

The biliary excretion of 110mAg in rats after i.v. administration of an aqueous solution of 110mAgNO3 (4.57 micrograms; 16kBq per rat) was studied for a period of 24 hours. The maximum rate of excretion was reached in 30th minute after the metal administration and over 70% of the silver dosed was excreted during 24 hours. Using the method of isolated perfused liver it was observed that 110mAg is rapidly taken up in the liver. During the five minutes period of the perfusion less than 50% of silver administered was found in the perfusion medium. In following minutes the level of the metal in the medium remained approximately constant. It was suggested that the rate of excretion of silver and its high uptake in the liver tissue is in connection with an unusual binding of it in the bile.     

Tissue distribution and plasma clearance of heparin-binding EGF-like growth factor (HB-EGF) in adult and newborn rats

Heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, can protect intestinal epithelial cells from various forms of injury in vitro and attenuate intestinal ischemia/reperfusion damage in vivo. With the goal of eventual clinical use of HB-EGF to protect the intestines from injury in neonates, children, and adults, the pharmacokinetics and biodistribution of 125I-labeled HB-EGF were investigated. After intravenous bolus, HB-EGF had a distribution half-life of 0.8 min and an elimination half-life of 26.67 min. After gastric administration, the bioavailability was 7.8%, with a 2.38 h half-life in the absorption phase and an 11.13 h half-life in the elimination phase. After intravenous dosing, most radioactivity was found in the plasma, liver, kidneys, bile, and urine, whereas it was mainly distributed in the gastrointestinal tract after intragastric administration. The degradation of 125I-HB-EGF in plasma from newborn rats was lower than that in adult rats after gastric administration. This supports the feasibility of enteral administration of HB-EGF in the treatment of gastrointestinal diseases, including newborns afflicted with necrotizing enterocolitis.     

Influence of microbial bile salt desulfation upon the fecal excretion of bile salts in gnotobiotic rats

The fecal excretion of intraperitoneally injected 24-14C-labeled taurocholate (TCA), taurolithocholate (TLCA) and the respective 3-sulfate esters (TCA-3-S; TLCA-3-S), were compared in germfree (GF) rats, conventional (CV) rats, and in gnotobiotic rats associated with Clostridium Cl-8 or this same strain Cl-8 plus the bile desulfating Clostridium S1, respectively. TCA and TLCA were about two times more rapidly excreted by CV animals than by GF animals; the time required for 50% excretion of total label injected (t 1/2) was 6.6 days vs 14.9 for TCA, and 4.4 vs 8.9 for TLCA. In GF and in CV animals, TCA-3-S and TLCA-3-S were excreted more rapidly than their nonsulfated analogues; the t 1/2 values of TCA-3-S and TCA were 2.7 days vs 14.9 in GF rats, and 3.1 vs 6.6 days in CV animals. The t 1/2 values of TLCA-3-S and TLCA were 2.7 days vs 8.9 in GF rats, and 1.5 vs 4.4 days in CV rats. In gnotobiotic rats associated with Clostridium strains S1 + Cl-8, fecal bile salts were nearly 100% deconjugated and desulfated and the 50% excretion times of TCA-3-S and TLCA-3-S approximated to those of TCA and TLCA in GF animals. T 1/2 of TCA-3-S in gnotobiotic S1 + Cl-8 animals was 12.2 days vs 14.9 for TCA in GF animals. In gnotobiotic S1 + Cl-8 animals the t 1/2 of TLCA and TLCA-3-S was 12.5 and 11.0 days, respectively. These results illustrate clearly the important effect the intestinal microflora has upon the metabolic half-life of bile salts. Moreover, they demonstrate that desulfation of bile salts by the intestinal microflora takes place in intestinal segments from where a certain degree of reabsorption is still possible, and thus point to the fact that microbial desulfation is an important variable in the overall elimination of bile salts.