Determination of 3 beta-hydroxy-5-cholenoic acid in serum of hepatobiliary diseases--its glucuronidated and sulfated conjugates

3 beta-Hydroxy-5-cholenoic acid in the serum of control subjects and 62 patients with various hepatobiliary diseases was quantitated by mass fragmentography after separation into nonglucuronidated-nonsulfated, glucuronidated, and sulfated fractions. Deuterium-labeled deoxycholic acid and its glucuronide and sulfate were used as internal standards. Mean concentrations of total 3 beta-hydroxy-5-cholenoic acid in serum (mumole/liter) were as follows: Control subjects (14), 0.184; obstructive jaundice (15), 6.783; liver cirrhosis, compensated (12), 0.433, and decompensated (12), 1.636; chronic hepatitis (12), 0.241; and acute hepatitis (11), 2.364. Most of the 3 beta-hydroxy-5-cholenoic acid was glucuronidated or sulfated. Only in patients with obstructive jaundice did glucuronidation (60 +/- 14%) exceed sulfation (31 +/- 14%), sulfation exceeding glucuronidation in the others. The UDP-glucuronyltransferase might have different substrate specificities for 3 beta-hydroxy-5-cholenoic acid and other common bile acids, especially in the cholestatic state.     

Human cecal bile acids: concentration and spectrum

To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3alpha-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3alpha-hydroxy bile acid concentration (mumol bile acid/ml cecal content) was 0.4 +/- 0.2 mM (mean +/- SD); the total 3-hydroxy bile acid concentration was 0.6 +/- 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 +/- 9%, mean +/- SD); sulfated, nonamidated bile acids were 7 +/- 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 +/- 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 +/- 16%), lithocholic (26 +/- 10%), and ursodeoxycholic (6 +/- 9), as well as their primary bile acid precursors cholic (6 +/- 9%) and chenodeoxycholic acid (7 +/- 8%). In addition, 3beta-hydroxy derivatives of some or all of these bile acids were present and averaged 27 +/- 18% of total bile acids, indicating that 3beta-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.     

Metabolism of intravenously administered 7 alpha-hydroxycholesterol-3 beta-stearate in the hamster.

In order to investigate the metabolic fate of serum esterified 7 alpha-hydroxycholesterol, [4-14C]7 alpha-hydroxycholesterol-3 beta-stearate was synthesized from labeled cholesterol and administered to bile fistula hamsters intravenously. Bile samples were collected at every 20 min for 7 h. Radioactivity was detected in bile 40 min after the beginning of the infusion of the labeled compound and 56.5 +/- 5.7% (48.7-66.0%) of the administered radioactivity was recovered in bile during 7 h. The liver contained appreciable radioactivity (19.5 +/- 7.6% of the administered dose) at the time of sacrifice. Only a trace amount of radioactivity was detected in urine and blood. Cumulative recovery of the radioactivity was 76.3 +/- 8.6% (63.3-90.4%). Major radioactive metabolites in the bile samples were identified to be taurine- and glycine-conjugated cholic acid and chenodeoxycholic acid by radioactive thin-layer chromatographic analysis of the bile samples before and after enzymatic hydrolysis and 3 alpha-hydroxysteroid dehydrogenase treatment. The conversion was nearly complete and we could not detect neutral metabolites, such as the mother compound, free 7 alpha-hydroxycholesterol and bile alcohols, as well as glucuronidated or sulfated bile acids. It is concluded that serum esterified 7 alpha-hydroxycholesterol could be effectively taken up by the liver, hydrolyzed by cholesterol esterase and metabolized via the normal biosynthetic pathway to taurine- or glycine-conjugated primary bile acids to be excreted into bile.     

Formation of three types of glucuronides of 6-hydroxy bile acids by rat liver microsomes

The glucuronidation of 6-hydroxylated bile acids by rat liver microsomes was studied in vitro; for comparison, several major bile acids lacking a hydroxyl group in position 6 were also investigated. The highest reaction rates were found for lithocholic and deoxycholic acid (10.2 +/- 0.2 and 7.3 +/- 1.4 nmol/mg.min, respectively); our results for these substrates agree well with published values. Glucuronidation rates for the 6 beta-hydroxylated bile acids 3 alpha, 6 beta-dihydroxy-5 beta-cholanoate (murideoxycholate) and 3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanoate (beta-muricholate) were only slightly lower (3.7 +/- 0.3 and 3.6 +/- 0.3 nmol/mg.min). 6 alpha-Hydroxylated bile acids were glucuronidated at rates that were lower than those for their 6 beta-hydroxy counterparts. Rigorous product identification by high-field proton NMR of methyl/acetyl derivatives revealed that while bile acids lacking a 6-hydroxyl group gave rise exclusively to the typical 3-O-glucuronide, the presence of a hydroxyl group in position 6 led to the formation, in ratios depending on the substrate, of three types of conjugate: the 3-O-, the 6-O-, and the carboxyl-linked (acyl-) glucurnide. The latter is the first example of an acyl glucuronide of a bile acid of conventional (C24) size.     

Inhibition of carnitine acetyltransferase by bile acids: implications for carnitine analysis

Carnitine acetyltransferase is used in a radioenzymatic assay to measure the concentration of carnitine. While determining the concentration of carnitine in rat bile, we found that the apparent concentration increased as bile was diluted (6.7 +/- 1.0 and 66.6 +/- 9.4 nmol/ml in undiluted and 20-fold diluted bile, respectively). The present study was designed to investigate whether a component of bile inhibited carnitine acetyltransferase. Inhibition was evaluated by measuring carnitine concentration in bile or by determining the recovery of a known amount of carnitine in the presence of bile. Inhibitory activity was extractable in organic solvents, stable to heat and base treatments, resistant to trypsin and lipase digestions, and removable by cholestyramine, a bile acid-binding resin. These results suggested that the inhibitory activity was associated with bile acids. Direct evidence was obtained by showing a reduced detectability of carnitine in the presence of individual bile acids. Chenodeoxycholic acid was the most potent inhibitor. Inhibition was unrelated to the detergent properties of bile acids. Kinetic studies revealed that carnitine acetyltransferase was inhibited competitively by chenodeoxycholic acid with a Ki of 520 microM. Bile acids also interfered in the quantitation of carnitine in cholestatic plasma. Carnitine concentration in such plasma was underestimated (17.5 +/- 2.1 mmol/ml). Reduction of bile acid concentration by a 20-fold dilution of cholestatic plasma resulted in a 3-fold higher carnitine concentration (54.6 +/- 9.0 nmol/ml). Results demonstrate that, because of the inhibition of carnitine acetyltransferase by bile acids, the radioenzymatic assay will underestimate carnitine concentration in bile or in cholestatic plasma. Accurate measurement requires either the removal of bile acids or a marked reduction in their concentration.     

Ion-pair extraction of bile acids with Lipidex gel

A new method for the extraction of bile acids from aqueous solutions, urine, plasma, and bile is described. A buffered solution of decyltrimethylammonium bromide is added to the sample to give a 0.03 m concentration of the counter-ion. The mixture is passed through a bed of Lipidex 1000, which is then washed with the buffered solution of counter-ion followed by water. The decyltrimethylammonium salts of bile acids are sorbed by the Lipidex and are eluted with methanol. Recoveries of unconjugated, taurine- and glycine-conjugated, sulfated, and glucuronidated bile acids are close to 100%. Unconjugated bile acids can also be quantitatively extracted from aqueous solutions and urine after acidification with acetic acid.     

Itch in Liver Disease: Facts and Speculations

Pruritus in hepatobiliary disease is commonly believed to be caused by retention of bile acids with their sequestration in the skin. HOwever, we have recently demonstrated that skin levels of bile acids in patients with cholestasis correlate poorly with pruritus. In this report, we present additional data concerning the relationship of pruritus to bile acid retention: (1) the urinary excretion of sulfated and nonsulfated bile acids was not significantly different in patients with cholestasis who itched compared to those who did not; (2) one patient with itch associated with a liver abscess had normal levels of bile acids in serum, skin, and urine; (3) patients with primary biliary cirrhosis who itched had lower serum bile acid levels than patients with mechanical biliary obstruction who did not itch.These studies support our premise that pruritus in hepatobiliary diseases is not directly related to bile acid retention. They suggest that the type of cholestatic disorder, and not simply the magnitude of the cholestasis, as estimated by the elevation of serum bile acids, is important. We propose that the agent responsible for pruritus is produced in response to cholestasis, possibly through activation of the alternate pathway of bile acid synthesis. Properties of the hypothetical pruritogen are discussed.     

Screening of newborn infants for cholestatic hepatobiliary disease with tandem mass spectrometry

ObjectiveTo assess the feasibility of screening for cholestatic hepatobiliary disease and extrahepatic biliary atresia by using tandem mass spectrometry to measure conjugated bile acids in dried blood spots obtained from newborn infants at 7-10 days of age for the Guthrie test.SettingThree tertiary referral clinics and regional neonatal screening laboratories.DesignUnused blood spots from the Guthrie test were retrieved for infants presenting with cholestatic hepatobiliary disease and from the two cards stored on either side of each card from an index child. Concentrations of conjugated bile acids measured by tandem mass spectrometry in the two groups were compared.Results218 children with cholestatic hepatobiliary disease were eligible for inclusion in the study. Two children without a final diagnosis and five who presented at <14 days of age were excluded. Usable blood spots were obtained from 177 index children and 708 comparison children. Mean concentrations of all four bile acid species were significantly raised in children with cholestatic hepatobiliary disease and extrahepatic biliary atresia compared with the unaffected children (P<0.0001). Of 177 children with cholestatic hepatobiliary disease, 104 (59%) had a total bile acid concentration >33 μmol/l (97.5th centile value for comparison group). Of the 61 with extrahepatic biliary atresia, 47 (77%) had total bile acid concentrations >33 μmol/l. Taurotrihydroxycholanoate and total bile acid concentrations were the best predictors of both conditions. For all cholestatic hepatobiliary disease, a cut off level of total bile acid concentration of 30 μmol/l gave a sensitivity of 62% and a specificity of 96%, while the corresponding values for extrahepatic biliary atresia were 79% and 96%.ConclusionMost children who present with extrahepatic biliary atresia and other forms of cholestatic hepatobiliary disease have significantly raised concentrations of conjugated bile acids as measured by tandem mass spectrometry at the time when samples are taken for the Guthrie test. Unfortunately the separation between the concentrations in these infants and those in the general population is not sufficient to make mass screening for cholestatic hepatobiliary disease a feasible option with this method alone.

Key messages

• The prognosis of cholestatic hepatobiliary disease in infancy, in particular biliary atresia, is improved by early detection
• Infants destined to present with cholestatic jaundice in the first few months of life have raised concentrations of bile acids in the blood spots obtained at 7-10 days for current neonatal screening programmes
• Tandem mass spectrometry can be used to detect this marker of neonatal cholestasis
• Unfortunately there is too much overlap between bile acid concentrations in infants with cholestasis and those in control infants for this to be used as a single screening test for cholestatic hepatobiliary disease in general and biliary atresia
• Tandem mass spectrometry is a powerful tool for neonatal screening but every potential application must be carefully assessed

     

Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice

The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver.     

Plasma levels of ursodeoxycholic acid in black bears, Ursus americanus: seasonal changes

To date, no other studies have examined the seasonal changes in circulating levels of various bile acids in the plasma of wild North American black bears, Ursus americanus. Using gas chromatography, bile acid concentrations were measured in plasma samples obtained during either early or late hibernation, and during summer active periods. Thus, specific compositional changes from individual animals were examined through a given year. Total bile acid concentrations in the plasma of these normal animals were found to range between 0.2 and 3.1 micromol/L (0.9 +/- 0.2 micromol/L, mean +/- SEM). Cholic, ursodeoxycholic and chenodeoxycholic acids were the major bile acid species identified. Ursodeoxycholic acid represented 28.0 +/- 2.6% of the total bile acid pool. Deoxycholic and lithocholic acids were found only in small amounts. In addition, total bile acid concentrations were lower in plasma samples obtained during hibernation compared with those obtained during summer active periods (0.6 +/- 0.1 and 1.2 +/- 0.4 micromol/L, respectively; p < 0.05). However, the relative proportion of ursodeoxycholic acid, was significantly greater in winter than in summer (31.5 +/- 3.2% and 22.2 +/- 4.5%, p < 0.05). Finally, taurine-conjugated bile acids were the predominant species in bear plasma, accounting for >67% of the total bile acids. These data demonstrate that ursodeoxycholic acid is a major bile acid in black bear plasma, mostly conjugated with taurine. Further, the finding of seasonal variation in plasma bile acid composition provides evidence to support the possible role that ursodeoxycholic acid may play in cellular protection in hibernating black bears.     

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.     

Human liver steroid sulphotransferase sulphates bile acids.

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.     

Hydroxylation, conjugation and sulfation of bile acids in primary monolayer cultures of rat hepatocytes

Hydroxylation of lithocholic, chenodeoxycholic, deoxycholic and cholic acids was studied in monolayers of rat hepatocytes cultured for 76 h. The majority of added lithocholic and chenodeoxycholic acids was metabolized to beta-muricholic acid (56-76%). A small part of these bile acids (9%), however, and a considerable amount of deoxycholic and cholic acids (21%) were converted into metabolites more polar than cholic acid in the first culture period. Formation of these compounds decreased during the last day of culture. Bile acids synthesized after addition of [4-14C]-cholesterol were almost entirely (97%) sulfated and/or conjugated, predominantly with taurine (54-66%), during culture. Sulfated bile acids were mainly composed of free bile acids. The ability of hepatocytes to sulfurylate bile acids declined with culture age. Thus, rat hepatocytes in primary monolayer culture are capable to sulfurylate bile acids and to hydroxylate trihydroxylated bile acids, suggesting formation of polyhydroxylated metabolites.     

Bile acid sulfates. II. Formation, metabolism, and excretion of lithocholic acid sulfates in the rat

Sulfate esterification has been shown previously to be a prominent feature of lithocholate metabolism in man. These studies were undertaken to ascertain whether this metabolic pathway is also present in rats, and to investigate the physiological significance of bile acid sulfate formation. Lithocholic acid-24-(14)C was administered to bile fistula rats, and sulfated metabolites were identified in bile by chromatographic and appropriate degradative procedures. They constituted only a small fraction (2-9%) of the total metabolites but a more significant fraction (about 20%) of the secreted monohydroxy bile acids, most of the lithocholate having been hydroxylated by the rat liver. When sulfated glycolithocholate was administered orally, it was absorbed from the intestine without loss of the sulfate, presumably by active transport, and secreted intact into the bile. In comparison with non-sulfated lithocholate, an unusually large fraction (24%) of the sulfated bile acid was excreted in the urine, and fecal excretion took place more rapidly. Both the amino acid and sulfate moieties were extensively removed prior to excretion in the feces. Hydroxylation of bile acid sulfates or sulfation of polyhydroxylated bile acids did not occur to any great extent, if at all.     

Biliary lipid secretion in the rat. The uncoupling of biliary cholesterol and phospholipid secretion from bile acid secretion by sulfated glycolithocholic acid

Glycolithocholic acid and its sulfated derivative are major metabolites of the secondary bile acid lithocholic acid in man. Both compounds are known to induce cholestasis in experimental animals. We compared the effects of these endogenous hepatotoxins on bile production and biliary lipid composition in rats with chronic biliary drainage. The compounds were administered enterally at relatively low rates (5-50% of the rats' endogenous bile acid secretion in these experiments) to simulate enterohepatic circulation. Both compounds were substantially secreted into bile (more than 90% of dose); sulfated glycolithocholic acid unchanged and glycolithocholic acid after hepatic hydroxylation predominantly in the form of glyco-beta-muricholic acid (cf. Kuipers et al. (1986) Am. J. Physiol. 251, G189-G194). Neither glycolithocholic acid nor its sulfated derivative affected the biliary excretion of endogenous bile acids or bile flow in these experiments. In spite of this, phospholipid and cholesterol secretion were significantly reduced by sulfated glycolithocholic acid but were not altered by glycolithocholic acid. Phospholipid and cholesterol secretion rapidly decreased to 25 and 50% of their initial values, respectively, at biliary output rates of sulfated glycolithocholic acid up to 2 mumol/h, and did not further decrease when this output was increased to 6 mumol/h. Small unilamellar liposomes consisting of cholesterol, [Me-14C]choline-labeled phosphatidylcholine, phosphatidylserine and [3H]cholesteryl oleate in a 5:4:1:0.1 molar ratio were employed to label intrahepatic lipid pools. Administration of sulfated glycolithocholic acid slightly reduced bile acid synthesis from [3H]cholesteryl oleate, but significantly reduced the biliary secretion of [14C]phospholipid. Glycolithocholic acid did not affect the hepatic processing of liposomal lipids. It is concluded that sulfated glycolithocholic acid at low doses causes the uncoupling of biliary lipid secretion from that of bile acids, which might represent in initiating event in sulfated glycolithocholic acid hepatotoxicity.     

Effects of chronic glucagon administration on cholesterol and bile acid metabolism

Male adult Wistar rats received daily, at 9 a.m. and 5 p.m., 10 micrograms of Zn-protamine glucagon for 21 days by subcutaneous injections. The blood glucose level was not significantly modified. Cholesterol and triacylglycerol levels were decreased by 40 and 70% in plasma but not in the liver. The rates of cholesterol turnover processes were determined in vivo with an isotope balance method. Internal secretion of cholesterol (13.8 +/- 0.5 mg/day per rat in control rats and 22.4 +/- 0.9 mg/day per rat in glucagon-treated rats) and cholesterol transformation into bile acids were strikingly increased by chronic administration of glucagon. Biliary secretion rates of bile acids measured by a wash-out method were increased by 139%, while the intestinal bile acid pool was not changed. The enterohepatic cycle number was increased from five per day in control rats to nine per day in glucagon-treated rats. An increased turnover rate of the exchangeable cholesterol would explain the hypocholesterolemic effect of glucagon.     

The effect of dietary calcium supplementation on intestinal lipid metabolism.

Population studies in man and experimental animal work support the contention that dietary supplementation with calcium may prevent the development of colorectal cancer. The mechanism of action is postulated to be bile acid chelation in the small-bowed forming non-toxic calcium soap compounds but such substances have yet to be isolated and quantified. In this 2-part study faecal concentrations of acidic lipids and neutral sterols were measured in 93 Sprague-Dawley rats whose calcium intake was modulated by enriching the chow and adding calcium lactate (24 milligrams) to the drinking water. In study-1 (dietary calcium intake doubled from 0.4-0.8%) small bowel resection was used to manipulate colonic lipid concentration for comparison with control rats who had undergone transection with immediate restoration of bowel continuity at an equivalent point. Faecal concentrations of free bile acids were 53-67% less in animals receiving added calcium [1.76 +/- 1.33 vs 0.82 +/- 0.65 mg/g (transection); 2.74 +/- 3.73 vs 1.03 +/- 1.27 mg/g (small bowel resection): P less than 0.001]. In study-2 (dietary calcium intake trebled to 1.21%) faecal bile acid concentration was reduced by 32% (1.86 +/- 0.57 vs 1.27 +/- 0.34 mg/g: NS) whereas long chain fatty acid concentrations were increased by 117% (6.77 +/- 2.39 vs 14.67 +/- 4.82 mg/g: P less than 0.001) in animals receiving added calcium. Serum calcium levels remained unchanged in these animals. Calcium soaps of the bile acids were not detected in faeces and therefore contrary to popular theory these results indicate that conditions within the intestinal lumen favour calcium chelation of long chain fatty acids rather than bile acids.     

Physiological characteristics of allo-cholic acid

The physiological characterstics of allo-cholic acid (ACA), a typically fetal bile acid that reappears during liver regeneration and carcinogenesis were investigated. [(14)C] Tauro-ACA (TACA) uptake by Chinese hamster ovary cells expressing rat organic anion transporter polypeptide (Oatp)1 or sodium-taurocholate cotransporter polypeptide (Ntcp) was lower than that of [(14)C]taurocholic acid (TCA). Although TACA inhibited ATP-dependent TCA transport across plasma membrane vesicles from Sf9 cells expressing rat or mouse bile salt export pump (Bsep), no ATP-dependent TACA transport was found. In rats, TACA was secreted into bile with no major biotransformation and it had lower clearance and longer half-life than TCA. In mice, TACA bile output was lower (-50%) than that of TCA, whereas TACA induced 9-fold higher bile flow than TCA. Even though the intracellular levels were lower for TACA, translocation into the hepatocyte nucleus was higher for TACA than for TCA; however, rate of DNA synthesis, expression levels of alpha-fetoprotein, albumin, Ntcp, and Bsep, cell viability, and apoptosis in rat hepatocytes were similarly affected by both isomers. In conclusion, TACA partly shares hepatocellular uptake system(s) for TCA. Furthermore, in contrast to other "flat" bile acids, TACA is efficiently secreted into bile via transport system(s) other than Bsep and is highly choleretic, hence its appearance during certain situations may prevent accumulation of cholestatic precursors.