Ursodeoxycholic acid (UDCA) has beneficial

Transcription

1 Effect of High-Dose Ursodeoxycholic Acid on its Biliary Enrichment in Primary Sclerosing Cholangitis Daniel Rost, Gerda Rudolph, Petra Kloeters-Plachky, and Adolf Stiehl Ursodeoxycholic acid (UDCA) has beneficial effects in cholestatic liver diseases. In primary sclerosing cholangitis (PSC), there is evidence that high doses ( 20 mg/kg) of UDCA may be more effective than average doses. Biliary enrichment of UDCA at such high doses may represent the decisive factor for its beneficial effect. Up to now it is not clear how high-dose UDCA correlates with its biliary enrichment and whether bacterial degradation of large amounts of UDCA may lead to an increased bacterial formation of more toxic hydrophobic bile acids. We determined the biliary bile acid composition in 56 patients with PSC including 30 patients with repeat bile samples treated with various doses of UDCA. At a UDCA dose of mg/kg/d (n 18) biliary UDCA represented 43.1% 0.3% (mean SD) of total bile acids; at a UDCA dose of mg/kg (n 14), its biliary content increased to 46.9% 0.3%, at mg/kg (n 34) to 55.9% 0.2%, at mg/kg (n 12) to 58.6% 2.3%, and at mg/kg (n 8) to 57.7% 0.4%. During UDCA treatment, the biliary content of all other bile acids was unchanged or decreased. In conclusion, biliary enrichment of UDCA increases with increasing dose and reaches a plateau at mg/kg. There was no increase of toxic hydrophobic bile acids. If biliary enrichment of UDCA represents the decisive factor for its clinical effect, it seems likely that UDCA doses of up to mg/kg may be more effective than lower doses. (HEPATOLOGY 2004;40: ) Ursodeoxycholic acid (UDCA) has beneficial effects in cholestatic liver diseases. In primary biliary cirrhosis, UDCA treatment slows progression of the disease and improves survival. 1 6 In primary sclerosing cholangitis (PSC), UDCA improves laboratory parameters, 7 12 and its effect on the outcome is currently being evaluated. There is evidence 11,12 that high doses ( 20 mg/kg/d) of UDCA may be more effective than average doses (15 mg/kg/d). The biliary enrichment of UDCA, which may represent the key factor for its beneficial effect, 13 has not been studied at such high doses. The intestinal absorption of UDCA is incomplete 14,15 and the bacterial degradation of unabsorbed UDCA in the intestine may affect its own biliary enrichment and that of other bile acids. 16,17 The aim of the present study Abbreviations: UDCA, ursodeoxycholic acid; PSC, primary sclerosing cholangitis; ERC, endoscopic retrograde cholangiography. From the Department of Medicine, University of Heidelberg, Heidelberg, Germany. Received March 15, 2004; accepted May 21, Address reprint requests to: Adolf Stiehl, M.D., Medizinische Universitätsklinik, Bergheimer Str., 58 D-69115, Heidelberg, Germany. adolf_stiehl@ med.uni-heidelberg.de; fax: Copyright 2004 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep was to determine the optimal dose of UDCA with respect to its biliary enrichment. Patients and Methods The selection criteria for enrolling patients with PSC in the study included typical endoscopic retrograde cholangiographic findings, serum alkaline phosphatase activity of at least twice the normal range, negative antimitochondrial antibody, and a liver biopsy compatible with the diagnosis of PSC. Criteria for exclusion were decompensation of cirrhosis, patients in whom liver transplantation was foreseen, patients with a history of neoplastic disease and/or coexisting hepatic disease. Table 1 shows the clinical data of the patients. Of 56 patients, 29 had ulcerative colitis, and 2 had Crohn s disease. Patients with ulcerative colitis and previous colectomy with ileoanal pouch were excluded. Twenty-six patients with ulcerative colitis and 1 with Crohn s disease received 5-aminosalicylic acid (3 g/d), and 7 with ulcerative colitis and the 2 with Crohn s disease received intermittently corticosteroids (methylprednisolone, mg/d) for periods of up to 3 months. No differences between patients treated with 5-aminosalicylic acid and/or corticosteroids and those who needed no treatment of their inflammatory bowel disease were noted. The biliary system was visualized by endoscopic 693

2 694 ROST ET AL. HEPATOLOGY, September 2004 Table 1. Patient Data at Entry Into Study (n 56) Age, yr Weight, kg Ulcerative colitis, n (%) 29 (52%) Crohn s disease, n (%) 2 (4%) Alkaline phosphatase, IU Gamma-glutamyltransferase, IU Alanine aminotransferase, IU Serum bilirubin, mg/dl Serum albumin, g/l International normalized ratio retrograde cholangiography (ERC) in each patient. In all patients with narrowing of the common bile duct, ERC was repeated at yearly intervals. 19 In patients without narrowing of the common duct at the first visit, ERC was repeated whenever alkaline phosphatase and/or gammaglutamyltransferase and/or serum aminotransferases and/or serum bilirubin increased by more than 20%. For prevention of bacterial cholangitis as a consequence of ERC, antibiotics were administered in each of the patients. All patients received 3 2 g of mezlocillin (Baypen) intravenously, both on the day before and after the ERC. In addition, 50 mg of netilmycin (Certomycin) were added to each 20-mL portion of the contrast medium iopamidol (Solutrast). In patients with a total or subtotal stenosis of a major duct (stenosis of common duct and/or of the hepatic ducts within 2 cm of the bifurcation) and biochemical evidence of cholestasis, a dilatation of the stenoses was performed. Balloon dilatation was repeated at 4-week intervals up to success, as assessed by opening of the stenosis at repeat cholangiography. Details of the treatment regimen have previously been published. 19 When the original stenosis was open at ERC, the next control ERC was performed after 3 months and afterward after doubling of the time interval (6 and 12 months). In general, patients with dominant stenosis needed 1 repeat ERC with dilatation per year to prevent stenosis. During ERC, the bile duct was selectively intubated, and bile was collected via the intubation catheter and immediately frozen at 20 C. Since cholestasis may lead to reduced absorption of UDCA 20, and increasing evidence suggests that higher doses may be more effective, 11,12 in our patients the UDCA dose has been increased to mg/kg per day. 12 To study the effect of even higher UDCA doses, 11 in 20 arbitrarily selected patients the dose has been increased to mg/kg (in three divided doses). The 22 patients without dominant stenosis in whom one single bile sample was obtained belong to the group of patients in whom the ERC was performed due to an increase of alkaline phosphatase, gamma-glutamyltransferase, or serum bilirubin, and who did not have a dominant stenosis. The 30 patients with repeat bile samples belong to the group of patients with dominant stenoses who needed repeat ERC for therapeutic purposes. In patients with biliary obstruction, bile samples were not collected before at least 1 week after relief of the obstruction. In 11 out of 30 patients, baseline bile samples before treatment with UDCA were obtained. In 12 out of 30 patients, repeat bile samples were obtained at 4-week intervals at the same UDCA dose (8 with 2 bile samples, and 4 with 4 bile samples, each). In all 30 patients the UDCA dose was increased to mg/kg, and repeat bile samples were obtained in 3- to 12-month intervals. In 13 of these patients, the dose was increased from mg/kg to mg/kg, in 7 patients from mg/kg to mg/kg, in 7 patients from mg/kg to mg/kg, and in another 3 patients from to mg/kg. Analytical Methods The methods for analysis of biliary bile acids have been described earlier. 21,22 After solvolysis of bile acid sulfates and hydrolysis of bile acid glucuronides by glucuronidase, the bile acids were separated on DEAP-LH 20 into nonamidated bile acids, and glycine and taurine conjugates. Gas-liquid chromatography of methylated and trimethylsilyl-substituted bile acid derivatives was performed on 4.5 m 0.5% HiEff 8BP (Applied Science Laboratories Inc., State College, PA) conventional glass columns and in addition on 50 m OV 101 (Machery and Nagel, Dueren, Germany) glass capillary columns. An HP 5710A gas chromatograph (Hewlett Packard, Bad Homburg, Germany) equipped with a flame ionization detector was used. Statistical Analysis. Continuous data were compared with the nonparametric Wilcoxon rank-sum test. Results are given as mean SD. Informed consent was obtained from each patient, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval by the institution s human research review committee. Results Intraindividual variation of UDCA enrichment was studied in 12 patients (Fig. 1). In 8 patients with 2 bile samples obtained at an average UDCA dose of (range, ) mg/kg/d, intraindividual UDCA enrichment varied up to 19.84% (mean, 11.91%; range, 4.14% 19.84%). In 4 patients with 4 values at an average UDCA dose of (range, ) mg/kg, maximal intraindividual variation of UDCA in bile was 2.07%, 5.70%, 16.46%, and 18.19% (Fig. 1). On the

3 HEPATOLOGY, Vol. 40, No. 3, 2004 ROST ET AL. 695 Fig. 1. Variation of UDCA enrichment in individuals treated with a given dose of UDCA. In 8 patients, 2 bile samples and in 4 patients 4 bile samples were obtained while the UDCA dose remained unchanged. At a given dose, UDCA enrichment in bile samples obtained at different time points in 1 patient varied up to 20%. UDCA, ursodeoxycholic acid. basis of the dose, 5 dose groups were evaluated (Table 2): mg/kg, mg/kg, mg/kg, mg/ kg, and mg/kg. With increasing dose, UDCA enrichment in bile increased and reached a plateau at mg/kg (Fig. 2). An almost identical curve was obtained when all the lower values of patients with multiple samples were excluded. At a UDCA dose of mg/kg (n 18) biliary UDCA ranged from 20.2% to 62.0% of total bile acids, at mg/kg (n 14) from to 26.3% to 66.8%, at mg/kg (n 34) from 31.5% to 68.8%, at mg/kg (n 12) from 42.0% to 71.9%, and at mg/kg (n 8) from 42.5% to 73.4%. No differences were observed between patients with and without colitis. In the 6 study groups, serum parameters of cholestasis (alkaline phosphatase, gamma-glutamyltransferase, bilirubin) were not significantly different. Up to a UDCA dose of 22 mg/kg, in all patients with repeat bile samples a further increase of the dose led to an increased biliary enrichment of UDCA, whereas this was Fig. 2. Biliary enrichment of UDCA at various UDCA doses. The data of 86 bile samples obtained in 54 patients were included. The enrichment of UDCA in bile reaches a plateau at mg/kg/d UDCA. UDCA, ursodeoxycholic acid. not the case at doses 22 mg/kg (Fig. 3). With increasing UDCA dose, the sum of all other bile acids in bile decreased with the same percentage in which UDCA increased (Table 2). The individual bile acids cholic acid and chenodeoxycholic acid and their bacterial degradation products deoxycholic acid and lithocholic acid decreased slightly (not significant; Table 2). With increasing dose, the conjugation of UDCA and all other bile acids with glycine changed little (not significant; Table 3). Discussion UDCA has been shown to improve liver function tests, liver histology, and survival in primary biliary cirrhosis, 1 6 and similar effects have been observed in other cholestatic diseases. 7 12,23 25 The mechanism of action of UDCA in hepatobiliary diseases is still not completely understood Decreased intestinal absorption of hydrophobic, hepatotoxic bile acids, 39,40 and biliary enrichment with hydrophilic, nontoxic UDCA changes the balance of Table 2. Biliary Bile Acid Composition Bile Acid Before Treatment (n 11) (n 18) (n 14) UDCA Treatment mg/kg/d (n 34) (n 12) (n 8) CA, mmol/l (%) ( ) ( )* ( )* ( )* ( )* ( )* CDCA, mmol/l (%) ( ) ( )* ( )* ( )* ( )* ( )* DCA, mmol/l (%) ( ) ( ) ( ) ( ) ( ) ( ) UDCA, mmol/l (%) ( ) ( )* ( )* ( )* ( )* ( )* LCA, mmol/l (%) ( ) ( ) ( ) ( ) ( ) ( ) NOTE. Values are means SD. Abbreviations: CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid. *Significantly different from values before treatment (P.05). Significantly different from the value at lower dose.

4 696 ROST ET AL. HEPATOLOGY, September 2004 Fig. 3. Biliary enrichment of UDCA in patients who were treated with 2 different UDCA doses. In all patients treated with doses 22 mg/kg/d, an increase of the dose led to an increased biliary enrichment of UDCA. UDCA, ursodeoxycholic acid. biliary bile acids in favor of nontoxic hydrophilic bile acids, and this change may represent one of the mechanisms of action of UDCA. 13 In patients, studies on biliary enrichment are difficult to perform. Ideally, biliary enrichment of UDCA should be studied at regular intervals coinciding with scheduled dosage changes. To do such a study, however, obviously will be clinically difficult. All bile samples have been collected after selective intubation of the bile duct during clinically indicated endoscopic measures. After oral administration of UDCA, there is a considerable interindividual variation in the biliary enrichment of this bile acid, which makes it difficult to determine the optimal dose of UDCA in the individual patient. At low UDCA doses, its biliary enrichment ranges from very low values to values that in other patients are achieved only at high-dose treatment. In the low-dose group (10-13 mg/kg), the difference between the highest and lowest biliary enrichment rate of UDCA was 40%, and with increasing dose this value decreased (Fig. 2), indicating that the biliary enrichment is especially unpredictable at low UDCA doses. The minimum percentage of UDCA in bile needed for a therapeutic effect is unknown. It is obvious that if UDCA enrichment represents the key for its clinical effect, in some patients a low dose may be sufficient, and in others it may be insufficient. Our data indicate that by increasing the UDCA dose, the number of patients with presumably sufficient biliary UDCA enrichment increases. The wide range of biliary enrichment of UDCA makes it difficult to determine the dose above which no further benefit in enrichment can be expected. The analysis of 86 bile samples obtained with different UDCA doses indicates that at mg/kg UDCA, the biliary enrichment curve reaches a plateau. This means that above this dose the majority of patients will not respond to a further increase of the dose with a further increase of UDCA enrichment in bile (Fig. 2). However, more data at doses above 26 mg/kg are needed to confirm this assumption. The extent to which biliary enrichment with UDCA varies from day to day in a given patient up to now was unknown. 41 In the present study the variation of enrichment at a given dose in the same patient was approximately half of that in different subjects (Figs. 1 and 2). Therefore, paired data obtained in the same patient allow more precise evaluation of the effect of an increased dose. In general, patients with low UDCA percentage at a low dose also had a relatively low percentage at higher doses (Fig. 3). A UDCA dose of up to 22 mg/kg in all 27 individuals tested a further increase of the dose was followed by an increase of UDCA in bile (Fig. 3). In 3 patients in whom UDCA doses of 22 mg/kg were further increased this increase in dose led to in part to an increase and in part to a decrease of UDCA. These data obtained in patients with paired bile samples are in Table 3. Conjugation Profile of Biliary Bile Acids (%) Bile Acid Conjugate Before Treatment (n 11) (n 18) (n 14) UDCA Treatment mg/kg/d (n 34) (n 12) (n 8) CA Glycine Taurine CDCA Glycine Taurine UDCA Glycine Taurine DCA Glycine Taurine LCA Glycine Taurine Total Glycine Taurine NOTE. Values are means SD. During treatment, glycine conjugation of all bile acids was significantly increased (P.05). Abbreviations: CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid.

5 HEPATOLOGY, Vol. 40, No. 3, 2004 ROST ET AL. 697 agreement with those of the whole study group (Fig. 2) and support the concept that for the majority of patients the optimal dose is between 22 and 25 mg/kg. This finding does not exclude the possibility that in individual patients even higher doses of UDCA may have an additional effect on its biliary enrichment. In a recent study in patients with primary biliary cirrhosis, after 750 mg/d tauroursodeoxycholic acid biliary enrichment was 32%, and after 750 mg/d UDCA it was 29%. 42 The present data in PSC patients indicate that by increasing the UDCA dose, much higher biliary enrichment values for UDCA can be obtained. Absorption of orally administered UDCA is slow and incomplete. 14,15 Therefore it can be expected that at high doses of UDCA relatively large amounts of unabsorbed UDCA are subject to bacterial degradation. This may lead to an increased bacterial formation of 7ketolithocholic acid and lithocholic acid and by the hydroxylation of 7ketolithocholic acid in the liver, also to an increase of chenodeoxycholic acid. 17,18,43,44 In the patients studied, biliary chenodeoxycholic acid and lithocholic acid decreased. In agreement with previous recommendations, 45 all our patients received antibiotics during endoscopic procedures and therefore the bacterial formation of secondary bile acids may be somewhat reduced. Since bile acids are transported into the bile by specific transporters not used by antibiotics, a direct effect of antibiotics on bile composition seems unlikely. The data obtained in this study indicate that under the given conditions there is no increase of other bile acids, even at very high UDCA doses (Table 2). When administered orally, unconjugated UDCA on the first pass through the liver is rapidly conjugated with glycine or taurine. 46 After UDCA, bile was mainly enriched with the glycine conjugate of UDCA, and at very high UDCA doses this percentage increased only a little (Table 3). Several studies have suggested that the taurine conjugates may have more pronounced hepatoprotective effects than UDCA. 28,29,47 Studies with glycine-conjugated UDCA, however, indicate a similar hepatoprotective effect for taurine and glycine conjugates of UDCA. 48 The little differences in conjugation between the average and high doses of UDCA make it unlikely that this factor is of relevance when patients with PSC are treated with high doses of UDCA. After high-dose UDCA treatment of patients with PSC, its biliary content increased on average to approximately 60%, which is much more than what may be achieved with conventional doses. These findings may represent a rationale for the improved efficacy of such high UDCA doses. References 1. Leuschner U, Fischer H, Kurtz W, Guldutuna S, Hubner K, Hellstern A, et al. Ursodeoxycholic acid in primary biliary cirrhosis: results of a controlled double-blind trial. Gastroenterology 1989;97: Poupon RE, Balkau B, Eschwege E, Poupon R. A multicenter controlled trial of ursodiol for the treatment of primary biliary cirrhosis. N Engl J Med 1991;324: Combes B, Carithers RL, Maddrey WC, Lin D, McDonald MF, Wheeler DE, et al. A randomized, double-blind, placebo-controlled trial of ursodeoxycholic acid in primary biliary cirrhosis. HEPATOLOGY 1995;22: Lindor KDL, Dickson ER, Baldus WP, Jorgensen RA, Ludwig J, Murtaugh PA, et al. Ursodeoxycholic acid in the treatment of primary biliary cirrhosis. Gastroenterology 1994;106: Heathcote EJL, Cauch-Dudek K, Walker V, Bailey RJ, Blendis LM, Ghent CN, et al. The Canadian multi-center double blind randomized controlled trial of ursodeoxycholic acid in primary biliary cirrhosis. HEPATOLOGY 1994;19: Poupon RE, Lindor KD, Cauch-Dudek K, Dickson ER, Poupon R, Heathcote EJ. Combined analysis of randomized controlled trials of ursodeoxycholic acid in primary biliary cirrhosis. Gastroenterology 1997; 113: Beuers U, Spengler U, Kruis W, Aydemir U, Wiebecke B, Heldwein W, et al. Ursodeoxycholic acid for treatment of primary sclerosing cholangitis: a placebo controlled trial. HEPATOLOGY 1992;16: Stiehl A, Walker S, Stiehl L, Rudolph G, Hofmann WJ, Theilmann L. Effects of ursodeoxycholic acid on liver and bile duct disease in primary sclerosing cholangitis. A 3 year pilot study with a placebo-controlled study period. J Hepatol 1994;20: Lindor KD and the Mayo PSC/UDCA study group. Ursodiol for the treatment of primary sclerosing cholangitis. N Engl J Med 1997;336: Stiehl A, Rudolph G, Sauer P, Benz C, Stremmel W, Walker S, Theilmann L. Efficacy of ursodeoxycholic acid and endoscopic dilation of major duct stenoses in primary sclerosing cholangitis. An 8-year prospective study. J Hepatol 1997;26: Harnois DM, Angulo P, Jorgensen RA, LaRusso NF, Lindor KD. Highdose ursodeoxycholic acid as a therapy for patients with primary sclerosing cholangitis. Am J Gastroenterol 2001;96: Mitchell SA, Bansi DS, Hunt N, von Bergmann K, Fleming KA, Chapman RW. A preliminary trial of high dose ursodeoxycholic acid in primary sclerosing cholangitis: results of a randomized double-blind placebo-controlled study. Gastroenterology 2001;121: Hofmann AF. Bile acid hepatotoxicity and the rationale of UDCA therapy in chronic cholestatic liver disease: some hypotheses. In: Paumgartner G, Stiehl A, Barbara L, Roda E, eds. Strategies for the treatment of hepatobiliary diseases, Dordrecht, The Netherlands: Kluwer Academic, 1990; Stiehl A, Raedsch R, Rudolph G. Ileal excretion of bile acids: comparison with biliary bile acid composition and effect of ursodeoxycholic acid treatment. Gastroenterology 1988;94: Walker S, Rudolph G, Raedsch R, Stiehl A. Intestinal absorption of ursodeoxycholic acid in patients with extrahepatic biliary obstruction and bile drainage. Gastroenterology 1992;102: Fedorowski T, Salen G, Colallilo A, Tint GS, Mosbach EH, Hall JC. Metabolism of ursodeoxycholic acid in man. Gastroenterology 1977;73: Fedorowski T, Salen G, Tint GS, Mosbach E. Transformation of chenodeoxycholic acid and ursodeoxycholic acid by human intestinal bacteria. Gastroenterology 1979;77: Fromm H, Carlson GL, Hofmann AF, Farivar S, Amin P. Metabolism in man of 7ketolithocholic acid: precursor of cheno- and ursodeoxycholic acid. Am J Physiol 1980;239:G Stiehl A, Rudolph G, Kloeters-Plachky P, Sauer P, Walker S. Development of dominant bile duct stenoses in patients with primary sclerosing cholan-

6 698 ROST ET AL. HEPATOLOGY, September 2004 gitis treated with ursodeoxycholic acid: outcome after endoscopic treatment. J Hepatol 2002;36: Sauer P, Benz C, Rudolph G, Kloeters-Plachky P, Stremmel W, Stiehl A. Influence of cholestasis on absorption of ursodeoxycholic acid. Dig Dis Sci 1999;44: Stiehl A, Raedsch R, Rudolph G, Czygan P, Walker S. Analysis of bile acid glucuronides in urine: group separation on a lipophilic anion exchanger. Clin Chim Acta 1982;123: Alme B, Bremmelgaard A, Sjövall J, Thomassen P. Analysis of metabolic profiles of bile acid in urine using lipophilic anion exchanger and computerized gas liquid chromatography mass spectrometry. J Lipid Res 1977;18: Palma J, Reyes H, Ribalta J, Iglesias J, Gonzalez MC, Hernandez I, et al. Effects of ursodeoxycholic acid in patients with intrahepatic cholestasis of pregnancy. HEPATOLOGY 1992;15: Cotting J, Lentze MJ, Reichen J. Effects of ursodeoxycholic acid therapy on nutrition and liver function in patients with cystic fibrosis and longstanding cholestasis. Gut 1990;31: Colombo C, Setchell KDR, Podda M, Crosignani A, Roda A, Curcio L, et al. Effects of ursodeoxycholic acid therapy for liver disease associated with cystic fibrosis. J Pediatr 1990;117: Kitani K, Kanai S. Tauroursodeoxycholate prevents taurocholate-induced cholestasis. Life Sci 1982;30: Galle PR, Theilmann L, Raedsch R, Otto G, Stiehl A. Ursodeoxycholate reduces hepatotoxicity of bile salts in primary human hepatocytes. HEPA- TOLOGY 1990;12: Heuman DM, Mills AS, McCall J, Hylemon PB, Pandak M, Vlahcevic ZR. Conjugates of ursodeoxycholate protect against cholestasis and hepatocellular necrosis caused by more hydrophobic bile salts: in vivo studies in rat. Gastroenterology 1991;100: Tsukahara K, Kanai S, Ohta M, Kitani K. Taurine conjugate of ursodeoxycholate plays a major role in the hepatoprotective effect against cholestasis induced by taurochenodeoxycholate in rats. Liver 1993;13: Heuman DM, Dajaj R. Ursodeoxycholate conjugates protect against disruption of cholesterol-rich membranes by bile salts. Gastroenterology 1994;106: Benz C, Angermueller S, Töx U, Kloeters-Plachky P, Riedel HP, Sauer P, et al. Effect of tauroursodeoxycholic acid on bile acid induced apoptosis and cytolysis in rat hepatocytes. J Hepatology 1998;28: Rodrigues CM, Ma X, Linehan-Stieers C, Fan G, Kren BT, Steer J. Ursodeoxycholic acid prevents cytochrome C release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation. Cell Death Differ 1999;6: Que FG, Phan VA, Phan VH, LaRusso NF, Gores GJ. GUDC inhibits cytochrome C release from human cholangiocyte mitochondria. J Surg Res 1999;83: Benz C, Angermüller S, Otto G, Stremmel W, Stiehl A. Effect of tauroursodeoxycholic acid on bile acid-induced apoptosis in primary human hepatocytes. Eur J Clin Invest 2000;30: Rodrigues CMP, Kren BT, Steer CJ, Setchell KD. Tauroursodeoxycholate increases rat liver ursodeoxycholate levels and limits lithocholate formation better than ursodeoxycholate. Gastroenterology 1995;109: Beuers U, Throckmorton DC, Anderson MS, Isales CM, Thasler W, Kullak-Ublick GA, et al. Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes. Gastroenterology 1996;110: Kurz AK, Graf D, Schmitt M, Vom Dahl S, Haussinger D. Tauroursodeoxycholate induced choleresis involves p38 (MAPK) activation and translocation of the bile salt export pump in rats. Gastroenterology 2001;121: Rost D, Herrmann T, Sauer P, Schmidts HL, Stieger B, Meier PJ, et al. Regulation of rat organic anion transporters in bile salt-induced cholestatic hepatitis: effect of ursodeoxycholate. HEPATOLOGY 2003;38: Stiehl A, Raedsch R, Rudolph G. Acute effects of ursodeoxycholic acid and chenodeoxycholic acid on the small intestinal absorption of bile acids. Gastroenterology 1990;98: Marteau P, Chazouilleres O, Myara A, Jian R, Rambaud JC, Poupon R. Effect of chronic administration of ursodeoxycholic acid on the ileal absorption of endogenous bile acids in man. HEPATOLOGY 1990;12: Combes B, Carithers RL, Maddrey WC, Munoz S, Garcia-Tsao G, Bonner GF, et al. Biliary bile acids in primary biliary cirrhosis: effect of ursodeoxycholic acid. HEPATOLOGY 1999;29: Invernizzi P, Setchell KDR, Crosignani A, Battezzati PM, Larghi A, O Conell N, et al. Differences in the metabolism and disposition of ursodeoxycholic acid and of its taurine-conjugated species in patients with primary biliary cirrhosis. HEPATOLOGY 1999;29: Batta AK, Salen G, Arora R, Shefer S, Tint GS, Abroon J, et al. Effect ofursodeoxycholic acid on bile acid metabolism in primary biliary cirrhosis. HEPATOLOGY 1989;10: Crosignani A, Battezzati PM, Bertolini M, Zuin M, Watson D, Setchell KDR. Changes in bile acid composition in patients with primary biliary cirrhosis induced by ursodeoxycholic acid administration. HEPATOLOGY 1991;14: Olsson R, Björnsson E, Bäckman L, Friman S, Hockerstedt K, Kaijser B, et al. Bile duct bacterial isolates in primary sclerosing cholangitis: a study of explanted livers. J Hepatol 1998;28: Rudolph G, Kloeters-Plachky P, Sauer P, Stiehl A. Intestinal absorption and biliary secretion of ursodeoxycholic acid and its taurine conjugate. Eur J Clin Invest 2002;32: Crosignani A, Battezzati PM, Setchell KDR, Invernizzi P, Covini G, Zuin M, et al. Taurursodeoxycholic acid for the treatment of primary biliary cirrhosis. A dose-response study. Dig Dis Sci 1996;41: Kanai S, Kitani K. Glycoursodeoxycholate is as effective as tauroursodeoxycholate in preventing the taurocholate-induced cholestasis in the rat. Res Commun Chem Pathol Pharmacol 1983;42: