Choleresis and hepatic transport mechanisms

Summary To investigate, whether binding to micelles has a function in hepatic transport, biliary excretion of three organic anions, phenolphthalein-\-D-glucuronide (PG), dibromosulphthalein (DBSP) and indocyanine green (ICG) was studied in rats during saline, taurocholate or dehydrocholate administration. Taurocholate causes a weak choleresis with formation of biliary micelles, dehydrocholate a strong choleresis with little micelle formation. The two bile salts did not uniformly influence biliary excretion of the organic anions: biliary excretion of ICG (12.9 moles/kg) and DBSP (75.0 moles/kg) was stimulated by both bile salts: ICG excretion most pronounced by taurocholate and DBSP excretion most strongly by dehydrocholate. Biliary output of PG (25.8 and 200 moles/kg) was not stimulated by bile salt administration. Binding of PG, DBSP and ICG to biliary micelles was studied in sedimentation experiments by ultracentrifugation. PG, DBSP and ICG in bile showed a similar sedimentation pattern as ³H-taurocholate in bile, which indicates an association of all three anions with biliary micelles.Thus, the influence of bile salts on biliary transport of organic anions varies with the compound studied and the bile salt used, effects which cannot be explained by differences in binding to biliary micelles.The previously published paper of Vonk et al. (1974) is regarded as paper I in this series.     

Effect of Bile Acids on Hepatobiliary Transport of Cisplatin by Perfused Rat Liver

Abstract: The liver and kidney collaborate in the excretion of the cytostatic drug, cis-diamminedichloroplatinum(II) (cisplatin) from the body. Enhancement of this procces is envisaged as a way of reducing cisplatin toxicity, thus allowing increases in the doses administered. In this sense, using different compounds, several attempts have been made to enhance cisplatin biliary excretion. In this study, the ability of endogenous compounds belonging to the bile acid family to improve cisplatin excretion by the isolated perfused rat liver was investigated. A highly choleretic bile acid (ursodeoxycholic acid) and two others bile acids with marked micelle-forming properties (glycocholic acid and chenodeoxycholic acid) were chosen for study. When these drugs were given at concentrations (1 μM) that did not affect the viability of liver preparations, a correlation between the biliary excretion of platinum and bile acid output was found. This was not due to the incorporation of cisplatin into mixed micelles because no correlation between the biliary output of lecithin or cholesterol and platinum was observed. Moreover, a wash-out effect of bile acids was probably not the cause of bile acid-induced platinum output into bile because no correlation between this and bile flow was found. An enhancement in cisplatin transport processes by the hepatocyte or by direct binding of cisplatin to bile acid monomers or aggregates cannot be ruled out. In spite of the biliary induction of cisplatin output, the net excretion of platinum was reduced under bile acid administration. This was related to lower platinum contents in the liver tissue, probably due to an inhibition of the ability of the hepatocyte to take up and/or retain cisplatin while subject to bile acid infusion. In summary, our results indicate that bile acids reduce the net excretion of cisplatin by the liver even though they induce an enhancement in the transport of this compound from the hepatocyte into bile.     

3alpha-6alpha-Dihydroxy-7alpha-fluoro-5beta-cholanoate (UPF-680), physicochemical and physiological properties of a new fluorinated bile acid that prevents 17alpha-ethynyl-estradiol-induced cholestasis in rats

3alpha-6alpha-Dihydroxy-7alpha-fluoro-5beta-cholanoate (UPF-680), the 7alpha-fluorine analog of hyodeoxycholic acid (HDCA), was synthesized to improve bioavailability and stability of ursodeoxycholic acid (UDCA). Acute rat biliary fistula and chronic cholestasis induced by 17alpha-ethynyl-estradiol (17EE) models were used to study and compare the effects of UPF-680 (dose range 0.6-6.0 micromol/kg min) with UDCA on bile flow, biliary bicarbonate (HCO(3)(-)), lipid output, biliary bile acid composition, hepatic enzymes and organic anion pumps. In acute infusion, UPF-680 increased bile flow in a dose-related manner, by up to 40.9%. Biliary HCO(3)(-) output was similarly increased. Changes were observed in phospholipid secretion only at the highest doses. Treatment with UDCA and UPF-680 reversed chronic cholestasis induced by 17EE; in this model, UDCA had no effect on bile flow in contrast to UPF-680, which significantly increased bile flow. With acute administration of UPF-680, the biliary bile acid pool became enriched with unconjugated and conjugated UPF-680 (71.7%) at the expense of endogenous cholic acid and muricholic isomers. With chronic administration of UPF-680 or UDCA, the main biliary bile acids were tauro conjugates, but modification of biliary bile acid pool was greater with UPF-680. UPF-680 increased the mRNA for cytochrome P450 7A1 (CYP7A1) and cytochrome P450 8B (CYP8B). Both UDCA and UPF-680 increased the mRNA for Na(+) taurocholate co-transporting polypeptide (NCTP). In conclusion, UPF-680 prevented 17EE-induced cholestasis and enriched the biliary bile acid pool with less detergent and cytotoxic bile acids. This novel fluorinated bile acid may have potential in the treatment of cholestatic liver disease.     

Targeting the ABCB4 gene to control cholesterol homeostasis

Introduction: Multidrug resistance 3 (MDR3) P-glycoprotein is a lipid floppase that is encoded by the ATP-binding cassette sub-family B member 4 (ABCB4) gene and plays a crucial role in proper bile formation by transporting phosphatidylcholine across the canalicular plasma membrane of the hepatocyte into bile. The relevance of this function is underscored by the severe pathology that develops in patients with ABCB4 deficiency. This deficiency leads to the destruction of hepatocytes and cholangiocytes by bile salts, because their cytolytic action is not reduced by formation of mixed micelles with phospholipid.

Areas covered: Evidence that phospholipid secretion into bile is also essential for biliary cholesterol secretion as cholesterol dissolves much better in mixed micelles of bile salts and phospholipid than in pure bile salt micelles. As a consequence, net biliary cholesterol secretion depends on the amount of phospholipid secreted and hence, the expression of ABCB4 indirectly determines biliary cholesterol output.

Expert opinion: It can be argued that upregulation of the ABCB4 gene expression may not only be beneficial for liver pathology in patients with partial ABCB4 deficiency, but also for the prevention of gallstone formation and optimal cholesterol disposition in a much larger population.     

Bile formation and biliary lipid composition under the influence of clofibrate and phenobarbital pretreatment in the rat

Summary The effect of the two enzyme inducing agents, clofibrate and phenobarbital, on bile formation and biliary lipid composition was compared in male rats. Clofibrate (100 mg per kg body weight per day for 14 days) and phenobarbital (at first 60 mg per kg body weight per day for 3 days, then 100 mg per kg body weight per day for 11 days) increase the spontanous bile flow, the ¹⁴C-erythritol clearance but do not alter the bile salt excretion, indicating a stimulation of the bile acid independent fraction of bile. The bile of rats pretreated with clofibrate contains less cholesterol than the bile of saline treated control animals, whereas the concentrations of bile acids, phospholipids and cholesterol are reduced in the bile of the rats of the phenobarbital group. Both drugs diminish the cholesterol saturation of bile. If the biliary bile acid concentration and excretion are augmented by an infusion of sodium taurocholate (1000 nmol per min per 100 g body weight), the biliary concentration of cholesterol remains unchanged in the clofibrate group but increases in the phenobarbital group as compared with the saline control animals. The biliary phospholipid concentration is enhanced after clofibrate as well as after phenobarbital pretreatment. These studies indicate that the biliary excretion of cholesterol and phospholipids is at least to some extent regulated by the bile acid excretion. The importance of the synthesis of cholesterol and phospholipids for their biliary excretion, however, seems to be limited: a reduced cholesterol synthesis by clofibrate results in a reduced biliary cholesterol elimination. By contrast, an increased synthesis of cholesterol by phenobarbital and of the phospholipids by both drugs, however, may enlarge the intrahepatic lipid pools and may place more lipids available for biliary secretion.     

Improvement of absolute bioavailability of normally poorly absorbed drugs: inducement of the intestinal absorption of streptomycin and gentamycin by lipid-bile salt mixed micelles in rat and rabbit

The effect of lipid—bile salt mixed micelles on the intestinal absorption of streptomycin and gentamycin was investigated in the loop of small and large intestine of rat. While bile salts micellar solutions did not enhance the absorption of aminoglycosides, their mixed micellar solutions including monoolein, oleic or lauric acid markedly enhanced their absorption. However, lecithin—bile salt mixed micellar solution did not affect the absorption. Pretreatment with mixed micellar solutions showed neither a direct action on the mucosal membrane nor a transient increase in permeability to aminoglycosides.Improvement of bioavailability in the rabbit was evaluated using various formulations and different routes of administration. In the rectal administration, not only mixed micellar solutions but also lyophilized mixed micelles powder improved bioavailability. The duodenal administration of mixed micellar solution, however, was not effective, indicating that enhanced absorption of drugs by mixed micelles may be more pronounced with rectal administration.     

Biliary and urinary excretion of five cardiac glycosides and its correlation with their physical and chemical properties

Summary Biliary and urinary excretion of five tritiumlabelled cardiac glycosides, i.e. Ouabain, K-strophanthoside, Digoxin, Digitoxin and Deslanatoside C, were investigated in anaesthetized guinea-pigs 5 h after i.v. or enteral administration. Urinary excretion in the main route of elimination in the case of Ouabain and Deslanatoside C. Conversely, biliary excretion is predominant in the case of Digoxin and Digitoxin. K-strophanthoside is excreted both via bile and urine. In conscious guinea-pigs treated i.v. with the same cardiac glycosides the highest levels were observed in urine, bile, kidneys and liver. The relative values of those levels were in agreement with the excretion pattern observed in anaesthetized animals. An inverse linear relation (P<0.05) was encountered between biliary excretion rate and polarity of glycoside molecula. This correlation has been previously observed by ohter authors in other species, but not in the rabbit. This suggests that the correlation may not be considered generally applicable at present.     

Targeting the ABCB4 gene to control cholesterol homeostasis

INTRODUCTION: Multidrug resistance 3 (MDR3) P-glycoprotein is a lipid floppase that is encoded by the ATP-binding cassette sub-family B member 4 (ABCB4) gene and plays a crucial role in proper bile formation by transporting phosphatidylcholine across the canalicular plasma membrane of the hepatocyte into bile. The relevance of this function is underscored by the severe pathology that develops in patients with ABCB4 deficiency. This deficiency leads to the destruction of hepatocytes and cholangiocytes by bile salts, because their cytolytic action is not reduced by formation of mixed micelles with phospholipid. AREAS COVERED: Evidence that phospholipid secretion into bile is also essential for biliary cholesterol secretion as cholesterol dissolves much better in mixed micelles of bile salts and phospholipid than in pure bile salt micelles. As a consequence, net biliary cholesterol secretion depends on the amount of phospholipid secreted and hence, the expression of ABCB4 indirectly determines biliary cholesterol output. EXPERT OPINION: It can be argued that upregulation of the ABCB4 gene expression may not only be beneficial for liver pathology in patients with partial ABCB4 deficiency, but also for the prevention of gallstone formation and optimal cholesterol disposition in a much larger population.     

Metabolism of Digoxigenin,digoxigeninmonodigitoxoside and digoxigeninbisdigitoxoside in rats

Summary The biliary and renal excretion products after i.d. administration of ³H-digoxigenin, ³H-digoxigenin-mono-digitoxoside and ³H-digoxigenin-bis-digitoxoside were studied in biliary fistula rats 65% and 58% of the given dose were excreted in bile and 10% and 5% in urine within 12 h after administration of mono-and-bis-glycoside respectively. Within 8 h, 45% and 4% of the given dose was eliminated in bile and urine after administration of digoxigenin.93–95% of the excreted radioactivity consisted of CHCl₃-soluble substances after administration of the bisglycoside, whereas the CHCl₃-soluble fraction accounted for only 30–45% of the biliary eliminated radioactivity after administration of the monoglycoside and the genin. The main excretion product after administration of bis-digitoxoside was the unchanged bisglycoside. After administration of monodigitoxoside, digitoxoside, the CHCl₃-soluble fraction in bile and urine was mainly represented by the monoglycoside and the 3-epigenin. Only traces of 3-ketogenin and no digoxigenin at all were detectable. The only CHCl₃-soluble excretion product in bile after administration of digoxigenin was its epimer, while in urine a few per cent of unchanged digoxigenin and 3-ketogenin could be identified.The CHCl₃-insoluble fraction after administration of bis- and-monodigitoxoside consisted of conjugation products with glucuronic and sulfuric acid. The monoglycoside was identified as the main conjugation partner after encymatic splitting of the polar fraction with -glucuronidase and sulfatase. Therefore the hydroxyl in C₃ of the steroid moiety cannot be conjugated preferentially with glucuronic or sulfuric acid during the metabolic decomposition of glycosides. This finding led to a degradation scheme of digoxin already discussed. Due to rapid metabolic inactivation of the monoglycoside to polar metabolites a therapeutic significance of this substance is very unlikely.     

Choleresis and hepatic transport mechanisms

Summary The influence of the bile salts taurocholate and dehydrocholate on the hepatic transport of two quaternary ammonium compounds,d-tubocurarine (dTc) andN ⁴-acetylprocainamide ethobromide (APAEB) was investigated in rats. The biliary excretion of APAEB and dTc in vivo was not enhanced by 106 moles/h of taurocholate or dehydrocholate. Infusion of 268 moles/h dehydrocholate caused an inhibition of the plasma disappearance and hepatic transport of dTc. This inhibition, which presumably occurred at the hepatic uptake level, was also observed in isolated perfused rat liver experiments. In animals with an intact renal function, the high dose of dehydrocholate caused a decreased biliary excretion and an increased renal excretion of dTc.The observed concentration gradients, plasma/liver cytosol and bile/liver cytosol 20 min after injection of both drugs were 1.6 and 23 for APAEB and 2.2 and 190 for dTc. These concentration ratios were based on free drug concentrations; corrections were made for plasma protein binding, intracellular binding and biliary micelle binding. No substantial binding of both compounds to ligandin and Z proteins was found. From the amount in the liver 20 min after injection of both drugs 70% of APAEB and 90% of dTc was bound to cellular particles.The rate limiting step in hepatic transport of APAEB from plasma into bile was concluded to be the hepatic uptake, which may explain the lack of effect of bile salt induced choleresis on its biliary excretion.     

INFLUENCE OF BILE ACIDS ON THE BILIARY TRANSPORT MAXIMUM OF PHENOLSULPHONPHTHALEIN IN THE RAT

1. The effect of changes in bile acid secretion induced by cholestyramine treatment or taurocholate infusion on the biliary transport maximum (Tm) of phenolsulfonphthalein (PSP) was studied in Wistar rats. 2. Five hours after oral administration of cholestyramine (1.5 g/kg bodyweight) the biliary output of bile acids decreased to 51% and bile flow to 76% of control values. The percentage of conjugated and unconjugated PSP excreted into bile and the Tm of the dye were not significantly modified by cholestyramine pretreatment. 3. Administration of sodium taurocholate at increasing rates (60-480 nmol/100 g bodyweight per min) enhanced bile flow and the biliary output of bile acids in a linear dose-related fashion. The Tm of PSP increased progressively until a maximum of 29% above the control values was reached at a taurocholate dose of 240 nmol/100 g bodyweight per min). The enhancement corresponded mainly to the unconjugated dye, the excretion of conjugated PSP not being significantly modified by the infusion of the bile acid. 4. The results indicate that bile acids can influence to some extent biliary excretion of PSP in the rat, although this component is of minor importance at low bile acid secretory rates.     

Biliary and renal excretion,hepatic metabolism,and hepatic subcellular distribution of metronidazole in the rat

We studied the biliary and renal excretion, hepatic metabolism, and hepatic subcellular distribution of [¹⁴C]metronidazole in bile fistula rats. An average of 71.1 per cent of an intraduodenal or intravenous dose of [¹⁴C]metronidazole was excreted in 24 hr, 23.9 per cent in bile and 47.6 per cent in urine. Renal pedicle ligation caused a 150 per cent increase in biliary excretion of label, whereas phenobarbital pretreatment had no effect. The majority of label in bile and urine was associated with a polar derivative, tentatively identified by thin-layer chromatography and enzymatic hydrolysis as the monoglucuronide conjugate of metronidazole. After intraduodenal administration of purified conjugated [¹⁴C]metronidazole to rats with ligated renal pedicles, only a small amount of label (12.6 per cent of dose in 24 hr) appeared in bile. Growth inhibition studies showed the glucuronide conjugate to be devoid of antimicrobial activity against a metronidazole-sensitive organism, Tritrichomonas foetus. Uptake studies indicated that these organisms were incapable of concentrating conjugated metronidazole. Fractionation of rat liver homogenates by differential centrifugation after intravenous [¹⁴C]metronidazole showed that 90 per cent of label present in liver was in the non-particulate fraction. Our results in rats indicate that metronidazole undergoes an enterohepatic circulation and that the liver plays a major role in the metabolism and excretion of this compound.     

Choleretic Effect of Curcuma comosa Rhizome Extracts in Rats

Choleretic properties of various extracts of Curcuma comosa Roxb. (Zingiberaceae) were investigated in rats. Butanol and ethyl acetate extracts of the rhizome were the most effective in producing a choleretic effect. Intraduodenal administration of the ethyl acetate extract caused a dose-dependent stimulation of bile secretion. A high dose of the extract increased bile flow rate with decreased bile salt concentration whereas low doses increased the bile flow rate without altering bile salt concentration. With regard to the total output of biliary constituents, the high dose of extract did not alter secretory bile salt output, but markedly increased biliary cholesterol output. Concurrent with increased biliary cholesterol secretion, C. comosa caused a decrease in plasma cholesterol level. This result suggests potential development of this plant as a source of choleretic and hypocholesterolemic drugs.     

Altered hepatobiliary permeability induced by Amanita phalloides in the rat and the protective role of bile duct ligation

The effect of an extract of the toxic mushroom Amanita phalloides (Fr. Secr.) on the transfer of metabolically inert, water-soluble solutes across the biliary tree epithelium of male Sprague-Dawley rats was studied by the segmented retrograde intrabiliary injection (SRII) technique. The purpose was to see if indications for a change in the permeability of the biliary tree could be obtained in vivo. This technique involved the introduction into the biliary system, through a bile duct cannula, of an initial “segment” of solution containing a radioactive marker compound followed by a volume of 0.9% saline given in excess of the biliary tree capacity. Bile flow was immediately reestablished and bile drops were serially collected. The radioactive content was determined and expressed as a percentage of the total administered dose of radioactivity. Evaluated 24 hr following the ip administration of an extract of Amanita phalloides, the biliary recoveries of [³H]mannitol, [³H]sucrose, [³H]inulin, and [³H]dextran were all found to be significantly decreased when compared to controls. These decreased recoveries were consistent with an increase in hepatobiliary permeability. This increase in permeability was not accompanied by any change in liver weight or bile flow. Bile duct ligation, 24 hr before administration of a lethal dose of the extract, protected rats against the alteration in permeability as well as the lethality. Bile duct ligation itself increased bile flow and increased the distended biliary tree capacity.     

Biliary excretion of 7Be and its distribution after intravenous administration of 7BeCl2 in rats

Summary Wistar female rats were given two i.v. doses of ⁷BeCl₂ (dose A = 0.025 mg Be²⁺/kg b.w.; dose B=0.25 mg Be²⁺/kg b.w.). The rats were decapitated at 5, 24, and 48 hrs after administration. The kinetics of ⁷Be bile excretion during the 5 hrs after administration, as well as ⁷ Be retention in selected organs and the urine and stool excretion of beryllium were investigated. Significant differences between the effect of both doses were found particularly in the shape of biliary excretion curves of ⁷Be. Unproportionally higher ⁷Be blood levels after a higher dose persisted for a longer period of time. The decrease of ⁷Be in blood after a higher dose between the 5th and 24th hr after the administration was accompanied by an increased content of ⁷Be in the liver and spleen as well as by an increased urine excretion. The results obtained tend to prove that the reticuloendothelial system mainly participated in beryllium retention. Urine represents the main excretion route of beryllium after a parenteral administration.     

Estimation of cholesterol solubilization by a mixed micelle binding model in aqueous tauroursodeoxycholate:lecithin:cholesterol solutions

In order to interpret the clinical efficacy of conjugated ursodeoxycholate (UDC) in cholesterol (Ch) gallstone patients, the Ch solubilization in mixed micelles in 40:40:32 mM tauroursodeoxycholate (TUDC):taurochenodeoxycholate (TCDC):lecithin (L) and 80:32 mM TUDC:L systems was estimated by using a model of Ch binding to mixed micelles. The Ch solubilization limit in mixed TUDC:L micelles was found to be higher than that in mixed TUDC:TCDC:L micelles. In the 80:32 mM TUDC:L system, the dissolution of the Ch pellet decreased after vesicles (liposomes) formed on the surface of the Ch pellet whereas the dissolution of microcrystalline Ch was rapid before and after vesicle formation in the solution, indicating that the total surface area of solid Ch exposed to the solution may be another important factor in inducing the dissolution of Ch gallstones. These phenomena suggest that although vesicles, occasionally formed in the bile of patients under the therapy of conjugated UDC, make a contribution to the solubilization of Ch gallstones, the model of Ch binding to mixed TUDC:L micelles can be used to estimate Ch solubility in TUDC:L system.     

Ketoconazole impairs biliary excretory function in the isolated perfused rat liver

Summary The effects of ketoconazole (KT) on the hepatic excretory function was investigated in the isolated perfused rat liver. KT, at the concentrations of 5 × 10^–5 M or 10^–4 M caused dose-dependent decreases of the biliary bile acid concentration and excretion rate, with no significant effect on bile flow rates. Neither dose altered perfusate flow through the liver. Furthermore, at the same two concentrations, KT impaired the sulfobromophthalein transport in a dose-dependent manner. In contrast, the drug did not alter ¹⁴C-sucrose bile to perfusate ratio and did not cause enzyme release from the liver into the perfusate. The study demonstrates that KT possesses an intrinsic toxicity in the isolated perfused rat liver and suggests caution in the use of this drug in hepatopathic patients. Send offprint requests to G. B. Gaeta at the above address     

The bilary excretion of flavanones in the rat

1. The major biliary metabolites of flavanones in the rat have been identified by chromatographic and spectral methods. 2. Evidence is presented that flavanones and flavanone glycosides, following oral or parenteral administration, undergo glucuronylation and are selectively excreted via the bile. 3. Flavanone glycosides but not unconjugated aglycones may be excreted to a significant extent in bile. 4. The percentage of parenterally administered flavanones excreted in bile varies inversely with the amount administered.     

Biliary excretion and enterohepatic circulation of two beta-adrenergic blocking drugs,exaprolol and propranolol in rats

Plasma levels and excretion of two beta-adrenoceptor blocking drugs ³H-exaprolol and ³H-propranolol wereobserved up to 96 h after a single i.v. administration to rats. Terminal half-lives of 26·8 ± 9·1 h and 51·3 ± 7·5 h were found for exaprolol and propranolol, respectively. The recovery of ³H radioactivity in feces following i.v. administration of the drus (34·2 ± 0·8 per cent and 12·0 ± 1·3 per cent ³H of exaprolol and propranolol, respectively) is of biliary origin, as 30·7 ± 3.5 per cent and 13·4 ± 3.6 per cent ³H of exaprolol and propranolol, respectively, was excreted in the bile after i.v. administration. Enterohepatic circulation of the drugs was studied using the donor-recipient rat method. After intraduodenal administration of donor bile to the recipient rat approximately 50 per cent and 40 per cent of the biliary ³H activity of exaprolol and propranolol, respectively, was re-excreted following absorption. A formula for calculating the amount of the substance together with its metabolites excreted in the bile, urine or feces as a result of enterohepatic circulation has been proposed.     

Incidence of experimental ochratoxicosis on hepatic disposition of [3h]tetracycline and chloramphenicol in rats

[³H]Tetracycline or chloramphenicol were injected i.v. into anaesthetized controls and 12-day ochratoxin A-administered rats. The biliary excretion and hepatic levels of [³H]tetracycline were decreased whereas bile flow did not vary and plasma bioavailability of radioactivity increased in comparison with control rats. Ochratoxicosis induced also lower biliary excretion of glucuronide conjugated form of chloramphenicol without any change in its plasma or liver concentration.