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The bile salt export pump (BSEP), encoded by the abcb11 gene, is the major canalicular transporter of bile acids from the hepatocyte. BSEP malfunction in humans causes bile acid retention and progressive liver injury, ultimately leading to end-stage liver failure. The natural, hydrophilic, bile acid ursodeoxycholic acid (UDCA) is efficacious in the treatment of cholestatic conditions, such as primary biliary cirrhosis and cholestasis of pregnancy. The beneficial effects of UDCA include promoting bile flow, reducing hepatic inflammation, preventing apoptosis, and maintaining mitochondrial integrity in hepatocytes. However, the role of BSEP in mediating UDCA efficacy is not known. Here, we used abcb11 knockout mice (abcb11-/-) to test the effects of acute and chronic UDCA administration on biliary secretion, bile acid composition, liver histology, and liver gene expression. Acutely infused UDCA, or its taurine conjugate (TUDC), was taken up by the liver but retained, with negligible biliary output, in abcb11-/- mice. Feeding UDCA to abcb11-/- mice led to weight loss, retention of bile acids, elevated liver enzymes, and histological damage to the liver. Semiquantitative RT-PCR showed that genes encoding Mdr1a and Mdr1b (canalicular) as well as Mrp4 (basolateral) transporters were upregulated in abcb11-/- mice. We concluded that infusion of UDCA and TUDC failed to induce bile flow in abcb11-/- mice. UDCA fed to abcb11-/- mice caused liver damage and the appearance of biliary tetra- and penta-hydroxy bile acids. Supplementation with UDCA in the absence of Bsep caused adverse effects in abcb11-/- mice.

The transcription factor nuclear factor-E2-related factor 2 (Nrf2) is a key regulator for induction of hepatic detoxification and antioxidant mechanisms, as well as for certain hepatobiliary transporters. To examine the role of Nrf2 in bile acid homeostasis and cholestasis, we assessed the determinants of bile secretion and bile acid synthesis and transport before and after bile duct ligation (BDL) in Nrf2(-/-) mice. Our findings indicate reduced rates of biliary bile acid and GSH excretion, higher levels of intrahepatic bile acids, and decreased expression of regulators of bile acid synthesis, Cyp7a1 and Cyp8b1, in Nrf2(-/-) compared with wild-type control mice. The mRNA expression of the bile acid transporters bile salt export pump (Bsep) and organic solute transporter (Ostα) were increased in the face of impaired expression of the multidrug resistance-associated proteins Mrp3 and Mrp4. Deletion of Nrf2 also decreased ileal apical sodium-dependent bile acid transporter (Asbt) expression, leading to reduced bile acid reabsorption and increased loss of bile acid in feces. Finally, when cholestasis is induced by BDL, liver injury was not different from that in wild-type BDL mice. These Nrf2(-/-) mice also had increased pregnane X receptor (Pxr) and Cyp3a11 mRNA expression in association with enhanced hepatic bile acid hydroxylation. In conclusion, this study finds that Nrf2 plays a major role in the regulation of bile acid homeostasis in the liver and intestine. Deletion of Nrf2 results in a cholestatic phenotype but does not augment liver injury following BDL.

Studies were performed to characterize hepatic and intestinal transport, as well as biotransformation during transport, of a spectrum of fluorescent bile acids containing a fluorophore attached to the side chain. The following two classes of compounds were studied: 1) aminofluorescein (amF) coupled directly to the carboxylic group of a bile acid which was cholic, ursodeoxycholic, or cholylglycine; and 2) nitrobenzoxadiazolyl (NBD) coupled to the epsilon-amino group of a lysine conjugated bile acid, which was cholic or ursodeoxycholic. Fluorescein, a cholephilic organic anion, was studied as a control. Fluorescent bile acids were synthesized and their structures confirmed by nuclear magnetic resonance and mass spectrometry. Using the biliary fistula rat, hepatic transport, biotransformation, and choleretic activity were defined; intestinal absorption was assessed by jejunal or ileal perfusion studies. All fluorescent bile acids had hepatic transport maxima about one-sixth that reported for cholyltaurine, but derivatives of cholylglycine were transported best. Bile acids underwent little (<5%) biotransformation during hepatocyte transport. Only the amF conjugate of cholylglycine had normal choleretic activity; other compounds were hypocholeretic or cholestatic. In contrast, fluorescein was well transported, was partly glucuronidated, and had normal choleretic activity. NBD-tagged, but not amF-tagged, bile acids were actively transported by the intestine (ileum > jejunum), and no fluorescent bile acid had passive intestinal permeability; NBD-tagged bile acids were biotransformed during intestinal transport (jejunum > ileum). We conclude that the structure of the fluorophore as well as that of the bile acid influences transport by the hepatocyte and enterocyte. These fluorescent bile acids differ from fluorescein in being impermeable to cell membranes and undergoing little biotransformation during hepatocyte transport. Of these fluorescent bile acids, cholylglycylamF has hepatocyte transport and choleretic properties most closely resembling those of a natural bile acid.

To assess the effect of side chain length on the metabolism and physiological effects of homologues of chenodeoxycholic acid (CDCA), dinorCDCA, the C22 homologue, was synthesized and its hepatic biotransformation, transport kinetics, and choleretic properties were defined in rat and hamster biliary fistula and in isolated perfused rat liver. Results were compared with those of norCDCA, the C23 homologue, and of CDCA, the natural C24 homologue. In the rat, dinorCDCA was secreted mostly in unconjugated form (the majority as dinor-alpha-muricholic acid); the remainder was glucuronidated. In the hamster, glucuronidation was greater, and the unconjugated fraction contained equal parts of dinorCDCA and 5beta-hydroxy-dinorCDCA. NorCDCA was glucuronidated extensively (70%, rat; 40%, hamster). CDCA, in contrast, was efficiently amidated with taurine or glycine. In the perfused liver, the initial uptake rate of all three homologues was identical; later, regurgitation and/or cholehepatic shunting of dinorCDCA and norCDCA, but not of CDCA, occurred. In rats and hamsters with biliary fistulas, dinorCDCA and norCDCA, but not CDCA, induced a bicarbonate-rich hypercholeresis of canalicular origin. Hypercholeresis was not induced by the taurine conjugate of dinorCDCA. Hepatobiliary retention of both dinorCDCA and norCDCA occurred, consistent with efficient ductular absorption (calculated to be 94%) and cholehepatic cycling of the unmetabolized bile acids. It is concluded that dinorCDCA, as norCDCA, is inefficiently amidated, is metabolized as a xenobiotic, and induces hypercholeresis. DinorCDCA is the first dihydroxy bile acid to be identified that is secreted largely in unconjugated form in bile.

The hepatic biotransformation in the rat and hamster of 23(R)-hydroxychenodeoxycholic acid (23(R)OH-CDCA), the alpha-hydroxy derivative of CDCA, was defined; some physiological and physicochemical properties were also assessed. 23(R)OH-CDCA was isolated from duck bile; [24-14C]23(R)OH-CDCA was synthesized. The compound was administered intravenously to anesthetized biliary fistula rats at doses of 1, 3, or 5 mu mol/kg-min and to hamsters at 3 mu mol/min-kg. Biliary bile acids and radioactivity were analyzed by thin-layer chromatography (TLC), high pressure liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS). Recovery of radioactivity in bile was incomplete (50-70% of infused dose); some was also recovered as breath 14CO2. Radioactivity in bile was present as unchanged compound (25-50%, dose-dependent) and its conjugates (with taurine, with glycine, or with glucuronate). Nor-CDCA (C23) was present in bile (in both unconjugated and conjugated form), indicating that 23(R)OH-CDCA had undergone oxidative decarboxylation (alpha-oxidation) with loss of the C-24 carboxyl group. The alpha-oxidation was 20 +/- 5% (mean +/- SD) of administered dose in the rat and was not dose-dependent; in hamsters, alpha-oxidation was 35 +/- 8%. In rats, the S isomer of 23OH-CDCA also underwent alpha-oxidation (10 +/- 2%). Nor-CDCA also underwent 6beta-hydroxylation to form nor-alpha-muricholic acid, as well as reduction of its C-23 carboxyl group to form the C23 alcohol. The taurine conjugate of 23(R)OH-CDCA [23(R)OH-CDC-tau] was prepared synthetically and characterized by 1H-NMR. By surface tension measurements, it had a critical micellization concentration (CMC) of 3.5 mM (in 0.15 M Na+), as compared to 1.8 mM for CDC-taurine. Aqueous solubility of 23(R)OH-CDCA increased markedly above pH 5, compared to pH 7 for CDCA. When incubated with cholylglycine hydrolase, 23(R)OH-CDC-tau was deconjugated at a rate one-fourth that of CDC-tau. It is concluded that the presence of a 23(R)-hydroxyl group in a 3alpha, 7alpha-dihydroxy bile acid alters its metabolism in the rodent hepatocyte, as evidenced by inefficient conjugation with taurine or glycine, alpha-oxidation to nor (C23) bile acid, and reduction of the nor bile acid to the primary alcohol. The taurine conjugate of 23(R)OH-CDCA, a major biliary bile acid of marine mammals and wading birds, is a biological detergent with properties superior to those of the taurine conjugate of CDCA. Natural C23 nor-bile acids may be formed by alpha-oxidation of alpha-hydroxy C24 bile acids.

The role of bile acids in the regulation of cholesterol 7 alpha-monooxygenase (EC 1.14.13.17) was characterized using primary cultures of rat hepatocytes supplemented with dexamethasone and thyroxine. Taurocholate and taurodeoxycholate (50 microM) repressed cholesterol 7 alpha-hydroxylase mRNA to 44 +/- 9 and 52 +/- 4%, respectively, of control values. Repression by these natural, relatively hydrophobic bile acids was concentration dependent, with an IC50 of about 50 microM, and time dependent with a t1/2 for repression of 22 h. In contrast, two natural hydrophilic bile acids, tauroursodeoxycholate and taurohyodeoxycholate, had no effect on cholesterol 7 alpha-hydroxylase mRNA levels. Taurochenodeoxycholate and taurolithocholate also had no effect, but these hydrophobic bile acids were rapidly hydroxylated to more hydrophilic bile acids. Hydrophilic bile acid analogues (nor (C23) bile acids and beta-hydroxy epimers) repressed cholesterol 7 alpha-hydroxylase mRNA less potently than their corresponding and more hydrophobic C24 or alpha-hydroxy derivatives. Cholesterol 7 alpha-hydroxylase specific activity was decreased by taurocholate or taurodeoxycholate (50 microM) to 26 +/- 9 and 56 +/- 3% of control, respectively; its transcriptional activity was repressed to 52 +/- 5% of control by taurocholate (50 microM). The addition of cholesterol or the induction of cholesterol biosynthesis did not influence repression of cholesterol 7 alpha-hydroxylase mRNA levels by taurocholate. Based on several lines of evidence, cAMP was not involved in bile acid-induced repression. In rat hepatocytes cultured under conditions in which cholesterol 7 alpha-hydroxylase gene expression is maintained at in vivo levels, hydrophobic bile acids repress this enzyme at the level of gene transcription independently of cholesterol availability.

24-Norursodeoxycholic acid (nor-UDCA), when administered into the anesthetized biliary fistula hamster or injected into the perfusate of an isolated liver, was hydroxylated at C-5 to give 5 beta-hydroxynorursodeoxycholic acid 2 (3 alpha,5,7 beta-trihydroxy-24-nor-5 beta-cholan-23-oic acid), which was secreted into bile mainly as such. Similarly, 24-norchenodeoxycholic acid (nor-CDCA) was 5 beta-hydroxylated to give 5 beta-hydroxynor-chenodeoxycholic acid 4 (3 alpha,5,7 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic acid), which was also secreted into bile without appreciable further biotransformation. The site of hydroxylation was assigned by 13C and 1H NMR and mass spectrometry. 5-Hydroxylation was a major biotransformation pathway at physiological bile acid loads. 5-Hydroxylation of UDCA also occurred in the perfused rat liver but to a lesser extent. 5-Hydroxylation of nor-UDCA was not observed in rabbit, dog, or man, indicating that its formation is species-specific. 5-Hydroxylation of nor-CDCA and nor-UDCA is the first reported example of hydroxylation of a tertiary carbon atom of bile acids. Nor-dihydroxy bile acids appear to be useful for the detection of minor hydroxylation pathways, because their prolonged hepatobiliary retention exposes them repeatedly to hydroxylases present in the hepatobiliary system.

The enterohepatic circulation of norursocholic acid (nUC) and its glycine (nUCG) and taurine (nUCT) conjugates was investigated in the rat; cholic acid (C) was studied as control. The biliary recovery of intravenously infused 14C-labeled bile acids was high: nUC, 88%; nUCG, 80%; nUCT, 99%, and C, 90%. Biliary recovery after the same bile acids were infused intraduodenally was similar: nUC, 90%; nUCG, 66%; nUCT, 97%; and C, 99%. The two conjugated bile acids, nUCG and nUCT, were not biotransformed during intestinal or hepatic transport; nUC was also secreted largely unchanged, but approximately 10% was secreted as an unknown conjugate or sulfate; C was completely conjugated with taurine or glycine. To compare the rates of active ileal transport, biliary recovery was measured after an in situ ileal perfusion technique. The rate of absorption of nUC, nUCG, and nUCT was one-fourth to one-half that of cholyltaurine, which served as control. Competition experiments indicated that the same transport system was involved. When infused intravenously, nUC, nUCG, and nUCT induced far less biliary lipid secretion than an identical dose of C; the secretion of both phospholipid and cholesterol was decreased, cholesterol to a greater extent than phospholipid. It is concluded that nUC and its conjugates are well transported by the ileum, are efficiently secreted into bile without undergoing appreciable hepatic biotransformation, and induce bile flow as other hydrophilic bile acids, but in contrast to C induce little phospholipid and cholesterol secretion into bile.

A new bile acid analogue, 3 alpha,7 alpha-dihydroxy-7 beta-methyl-24-nor-5 beta-cholan-23-oic acid (7-Me-norCDCA) was synthesized from the methyl ester of norursodeoxycholic acid, and its hepatic biotransformation was defined in the hamster. To synthesize 7-Me-norCDCA, the 3 alpha-hydroxyl group of methyl norursodeoxycholate was protected as the hemisuccinate, and the 7 beta-hydroxyl group was oxidized with CrO3 to form the 7-ketone. A Grigard reaction with methyl magnesium iodide followed by alkaline hydrolysis gave 7-Me-norCDCA (greater than 70% yield). The structure of the new compound was confirmed by proton magnetic resonance and mass spectrometry. After intraduodenal administration of the 14C-labeled compound into the anesthetized biliary fistula hamster, it was rapidly and efficiently secreted into the bile; 80% of radioactivity was recovered in 2 h. After intravenous infusion, the compound was efficiently extracted by the liver and secreted into the bile (greater than 75% in 3 h). Most (93%) of the biliary radioactivity was present in biotransformation products. The major biotransformation product (48.7 +/- 6.0%) was a new compound, assigned the structure of 3 alpha,5 beta,7 alpha- trihydroxy-7 beta-methyl-24-nor-5 beta-cholan-23-oic acid (5 beta-hydroxy-7- Me-norCDCA). In addition, conjugates of 7-Me-norCDCA with taurine (13.7 +/- 5.0%), sulfate (10.3 +/- 3.0%), or glucuronide (5.1 +/- 1.7%) were formed. 7-Me-norCDCA was strongly choleretic in the hamster; during its intravenous infusion, bile flow increased 2 to 3 times above the basal level, and the calculated choleretic activity of the compound (and its metabolic products) was much greater than that of many natural bile acids, indicating that the compound induced hypercholeresis. It is concluded that the biotransformation and physiological properties of 7-Me-norCDCA closely resemble those of norCDCA. Based on previous studies, the major biological effect of the 7-methyl group in 7-Me-norCDCA is to prevent its bacterial 7-dehydroxylation in the distal intestine.

Ursodeoxycholic acid, the 7 beta-hydroxy epimer of chenodeoxycholic acid, is more hydrophilic and less hepatotoxic than chenodeoxycholic acid. Because "lagodeoxycholic acid," the 12 beta-hydroxy epimer of deoxycholic acid, is also more hydrophilic than deoxycholic acid, it was hypothesized that it should also be less hepatotoxic than deoxycholic acid. To test this, lagodeoxycholic acid was synthesized, and its transport and metabolism were examined in the rat, rabbit, and hamster. The taurine conjugate of lagodeoxycholic acid was moderately well transported by the perfused rat ileum (Tmax = 2 mumol/min.kg). In rats and hamsters with biliary fistulas, the taurine conjugate of lagodeoxycholic acid was well transported by the liver with a Tmax greater than 20 mumol/min.kg; for the taurine conjugate of deoxycholic acid, doses infused at a rate greater than 2.5 mumol/min.kg are known to cause cholestasis and death. Hepatic biotransformation of lagodeoxycholic acid in the rabbit was limited to conjugation with glycine; in the hamster, lagodeoxycholic acid was conjugated with glycine or taurine; in addition, 7-hydroxylation occurred to a slight extent (approximately 10%). When lagodeoxycholic acid was instilled in the rabbit colon, it was absorbed as such although within hours it was progressively epimerized by bacteria to deoxycholic acid. When injected intravenously and allowed to circulate enterohepatically, lagodeoxycholic acid was largely epimerized to deoxycholic acid in 24 hours. Surgical creation of a distal ileostomy abolished epimerization in the rabbit, indicating that exposure to colonic bacterial enzymes was required for the epimerization. Lagodeoxycholic acid was administered for 3 weeks at a dose of 180 mumol/day (0.1% by weight of a chow diet; 2-4 times the endogenous bile acid synthesis rate); other groups received identical doses of deoxycholic acid (hamster) or cholyltaurine, a known precursor of deoxycholic acid (rabbit). After 3 weeks of lagodeoxycholic acid ingestion, liver test results and liver appearance were normal. The total bile acid pool expanded by 37% in the rabbit, lagodeoxycholic acid composing 10% of biliary bile acids. In the hamster, the total bile acid pool was expanded by 95%, lagodeoxycholic acid composing 22% of biliary bile acids; biliary lipid secretion remained unchanged. Tracer studies indicated that the fractional turnover rate of lagodeoxycholic acid was high (157%/day, rabbit; 116%/day, hamster) because of its rapid epimerization to deoxycholic acid in the colon. These studies indicate that lagodeoxycholic acid, the more hydrophilic epimer of deoxycholic acid, is transported and metabolized as other dihydroxy bile acids but is much less toxic than deoxycholic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

To define whether side-chain length influences the physiologic properties of bile acids, nor-ursodeoxycholate (nor-UDC), the C23-nor derivative of ursodeoxycholate (UDC), was synthesized in both nonradioactive and radioactive forms (23-14C). Its hepatic translocation, hepatic biotransformation, and effect on bile flow, biliary bicarbonate, and biliary lipid secretion were compared with that of UDC and those of their respective glycine and taurine conjugates in anesthetized biliary fistula hamsters, rats, and guinea pigs, as well as the isolated perfused hamster liver. Hepatic uptake and biliary output of nor-UDC was slower than that of UDC or cholyltaurine in the isolated perfused hamster liver. In biliary fistula animals, nor-UDC was secreted only in bile. Biliary recovery of nor-UDC as compared to that of UDC was prolonged in the rat and hamster, although not in the guinea pig. Hepatic biotransformation, assessed by chromatography of bile, showed that conjugation of nor-UDC was inefficient, as unconjugated nor-UDC was present in bile; there was little amidation with glycine or taurine in any species, but sulfates and glucuronides, as well as other metabolites, were formed, with the pattern of biotransformation varying among species. When infused over a dosage range of 0.2-30 mumol/kg X min, nor-UDC induced a striking choleresis of canalicular origin. The bile acid-dependent flow was increased threefold in hamsters, ninefold in rats, and nearly twofold in guinea pigs when compared to that induced by UDC. The choleresis was associated with a linear increase in bicarbonate output and concentration in bile, and little phospholipid or cholesterol secretion was induced. A competition experiment in the bile fistula hamster indicated that nor-UDC or its metabolites, or both, appeared to compete for canalicular transport of ursocholyltaurine (a cholyltaurine epimer) when the latter was secreted under its Vmax conditions. Conjugates of nor-UDC and UDC were promptly and almost completely recovered in bile without appreciable hepatic biotransformation; the conjugates did not induce a hypercholeresis or increase biliary bicarbonate concentration. It is proposed that a fraction of nor-UDC is secreted into canalicular bile in the unconjugated form and is protonated by a hydrogen ion derived from carbonic acid that was generated by the hydration of luminal CO2 by carbonic anhydrase present in biliary ductular cells. The protonated bile acid is absorbed, thus generating a bicarbonate anion. The bile acid passes through the cholangiocyte, returns to the sinusoids via the periductular capillary plexus, and is resecreted into bile.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of conjugation and side chain length on dihydroxy bile acid unidirectional hepatic uptake and efflux was studied using the isolated perfused rat liver which was perfused prograde or retrograde in single pass fashion. Deoxycholic acid (DC) and its C23 (nor) derivative nor-DC, as well as the synthetically prepared taurine conjugate of DC, were administered at a constant dose of 1 mumol/min/kg (body weight), upon which a bolus tracer dose of labeled bile acid was superimposed. Analysis of radioactivity recovery in perfusate indicated that unidirectional uptake of all three bile acids was equally rapid, but that only nor-DC showed considerable and continuing efflux into the perfusate; this involved mostly the unchanged acid. Nor-DC was not amidated but was metabolized to mostly ester glucuronides and hydroxylated derivatives; the biotransformation products did not reflux and were secreted into bile; similarly, DC was amidated with taurine; its taurine conjugate did not efflux and was secreted into bile. When nor-DC-taurine was infused, it did not efflux and was secreted rapidly into bile. When the liver was perfused retrograde fashion to increase concentrations of bile acids pericentral cells, only nor-DC showed efflux, which again involved only the unchanged acid. All bile acids were partly 7 alpha-hydroxylated, the magnitude being greater during retrograde perfusion presumably because slower cellular transport exposed bile acid to hydroxylation enzymes for a longer period. It is concluded that bile acid conjugation, whether by esterification with CoA formation adn subsequent amidation or by esterification with glucuronate, restricts the movement of lipophilic dihydroxy bile acids to the hepatocyte and canalicular lumen.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of side chain length on bile acid conjugation by human and rat liver fractions was examined. The rate of conjugation with glucuronic acid, sulfate and coenzyme A of several natural (C24) bile acids was compared with that of their corresponding nor-bile acids. The rate of coenzyme A ester formation by nor-bile acids was much lower than that of the natural bile acids. In human liver microsomes, the rate of coenzyme A formation was less than 8% of the rate for the corresponding C24 bile acid. Rat liver microsomes formed the coenzyme A ester of nor-bile acids less than 20% of the rate of their corresponding C24 homologs. Glucuronidation rates were greater than sulfation rates in both species. With human liver microsomes, nor-bile acids were glucuronidated more rapidly than their corresponding C24 homologs, whereas with rat liver microsomes the reverse was true. Purified 3 alpha-OH androgen UDP-glucuronyltransferase catalyzed the glucuronidation of both nor-bile acids and bile acids. Human liver cytosol sulfated nor-bile acids more slowly than the corresponding bile acids. Rat liver cytosol, however, sulfated nor-bile acids more rapidly than the corresponding bile acids. The highest rate was seen with lithocholylglycine. The results indicate that the novel biotransformation of nor-bile acids seen in vivo--sulfation and glucuronidation rather than amidation--is most likely explained as a consequent of defective amidation, to which the rate of coenzyme A formation contributes. Thus, side chain and nuclear structures as well as species differences in conjugating enzyme activity are determinants of the pattern of bile acid biotransformation by the mammalian liver.

Canalicular transport of bilirubin diglucuronide, dibromosulfophthalein and several glutathione conjugates is deficient in mutant TR- rats. In contrast, transport of cholyltaurine (taurocholate), a conjugated bile acid, is normal. Previous studies using normal rats have shown that C23 nor-dihydroxy bile acids are conjugated with sulfate or glucuronide during hepatic transport in contrast to the natural C24 bile acids, which are amidated with glycine or taurine. Studies were performed to test the hypothesis that (a) in the TR- rat, nordeoxycholate would be conjugated with glucuronate or sulfate just as in the normal rat, and (b) that such conjugates would have defective biliary secretion. [C23-14C]Nordeoxycholate was administered intravenously to bile fistula rats (TR- and normal), and the biliary recovery of metabolites was assessed by chromatography and mass spectrometry. In both groups of rats, the major biotransformation product of nordeoxycholate was the side chain (23-ester) glucuronide. Conjugation on the nucleus with sulfate and glucuronide at the 3-position (ethereal linkage) also occurred, as well as amidation at the C23 carboxylic acid group. In the mutant rats, biliary secretion of the 3-sulfate and 3-glucuronide conjugates was less than 10% and 1%, respectively, of that of normal rats, whereas biliary secretion of the 23-ester glucuronide and the 23-taurine amidate, as well as unchanged nordeoxycholate, was not decreased. Canalicular secretion of nor-bile acid 3-ether glucuronides and 3-sulfates appears to involve the "bilirubin transport system," which is deficient in mutant rats. Canalicular secretion of unconjugated, amidated or esterified nordeoxycholate is mediated via another pathway, probably the "bile acid transport system."(ABSTRACT TRUNCATED AT 250 WORDS)

In the perfused rat liver, ursodeoxycholate in high dose produces an HCO3- -rich hypercholeresis which we have shown previously to be inhibited by replacement of perfusate Na+ with Li+ or by addition of amiloride (or amiloride analogues). In the present studies, we have determined whether such inhibition is associated with altered ursodeoxycholate biotransformation. Under control conditions, ursodeoxycholate infusion produced a 3.7-fold increase in bile flow and a 9.2-fold increase in biliary HCO3- output. By thin-layer chromatography, ursodeoxycholate radioactivity in bile was present in unconjugated form (15%) or as glycine or taurine amidates. Glucuronide conjugates of ursodeoxycholate accounted for less than 1% of biliary bile acids. Li+/Na+ substitution decreased ursodeoxycholate-stimulated bile flow and HCO3- secretion by greater than 90%, but decreased recovery of ursodeoxycholate and metabolites by only 25%. Amiloride or amiloride analogues decreased ursodeoxycholate-stimulated choleresis and HCO3- output by 38%-76%, yet did not cause decreased recovery of ursodeoxycholate and metabolites. Inhibition of the hypercholeresis was associated with a decrease in unconjugated ursodeoxycholate to less than 2% of total biliary bile acids, a striking increase in ursodeoxycholate glucuronides, and a reciprocal decrease in glycine and taurine amidates. With Li+/Na+ substitution, the predominant metabolites were a mixture of the 24-ester and the 3-aketal (ethereal) glucuronide (29%), and amidation with glycine appeared to be selectively inhibited; with amiloride or its analogues, only the 3-ethereal glucuronide was formed (20%-60% of biliary bile acids), and both taurine and glycine amidation were inhibited. Thus, maneuvers that decrease Na+/H+ exchange inhibit ursodeoxycholate hypercholeresis and cause replacement of unconjugated ursodeoxycholate in bile by its glucuronide. The secretion of unconjugated ursodeoxycholate, a lipophilic bile acid, appears to be necessary for hypercholeresis induced by high-dose ursodeoxycholate infusion.

Nor-ursodeoxycholate, the C23 analogue of ursodeoxycholate, is a potent choleretic agent in rodents when given acutely but, to be used in humans, chronic toxicity studies are required. In the rabbit, ingestion of ursodeoxycholate or chenodeoxycholate leads to accumulation of lithocholate, its major bacterial metabolite, in biliary bile acids, which causes inflammation in portal tracts of the liver and bile duct proliferation. To test whether chronic administration of nor-ursodeoxycholate would cause an analogous accumulation of nor-lithocholate and hepatotoxicity, rabbits were fed a Chow diet containing nor-ursodeoxycholate (5 or 50 mg/day): control groups received Chow alone, and "disease control" groups received Chow plus ursodeoxycholate or Chow plus chenodeoxycholate. After 3 wk, animals were killed, liver sections were interpreted by a pathologist, and the steroid moiety of the glycine (and taurine) conjugates of gallbladder bile acids were analyzed by high-pressure liquid chromatography. Ingestion of nor-ursodeoxycholate did not cause hepatotoxicity, and neither it nor its presumed metabolite, nor-lithocholate, accumulated in biliary bile acids. To explain this unexpected finding, the hepatic metabolism of nor-ursodeoxycholate was investigated in biliary fistula rabbits. Nor-ursodeoxycholate was well absorbed from the intestine and secreted in the bile as a glucuronide as well as the unchanged compound, but conjugation with glycine and taurine was not observed. As glucuronides are poorly absorbed from the gut, it is proposed that the hepatic biotransformation of nor-ursodeoxycholate to a glucuronide rather than to a glycine amidate in the liver prevented its accumulation in the bile acid pool. Thus, shortening the side chain of ursodeoxycholate by a single carbon atom resulted in a bile acid with novel metabolism, which when administered chronically, does not accumulate in the enterohepatic circulation and does not cause hepatotoxicity.