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MECHANISM OF HEPATOPROTECTIVE ACTION OF BILE SALTS IN LIVER DISEASE
BILE SALTS: METABOLIC, PATHOLOGIC, AND THERAPEUTIC CONSIDERATIONS
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MECHANISM OF HEPATOPROTECTIVE ACTION OF BILE SALTS IN LIVER DISEASE Adolf Stiehl, MD, Christine Benz, MD, and Peter Sauer, MD
Ursodeoxycholic acid (UDCA) improves liver enzymes and in many instances liver histology in cholestatic liver diseases, such as primary 38, 83 and primary sclerosing cholangitis biliary cirrhosis (PBC)24, 65, 71, Iol In PBC, UDCA improves survival free of liver trans(PSC).12, plantation4; and in PSC, UDCA improves survival only if dominant strictures of the bile ducts are recognized early and treated endoscopially.^^ UDCA seems to have a beneficial effect in cholestasis of preg21-23, 25, various other cholestatic nan~y,7~ cholestasis of cystic fib~osis,’~, diseases of childhood3as well as cholestasis of total parenteral n ~ t r i t i o n . ~ Besides classic cholestatic diseases, UDCA also improves liver biochemistry in alcoholic liver disease78and in chronic viral hepatitis C.16,31, 79 In chronic hepatitis, UDCA has no effect on liver histology, and its role is unclear; moreover, it is unclear whether UDCA has an effect when combined with interferon treatment. Of considerable interest is the observation that survival of patients undergoing liver transplantation may be improved by treatment with UDCA.4 The main target of UDCA treatment is cholestasis, and consequently the mechanisms responsible for the beneficial effects in these diseases are of interest. ROLE OF BILE ACIDS IN CHOLESTASIS
In cholestasis, bile flow and biliary secretion of bile salts and cholephilic substances are reduced. In humans, bile flow is mainly bile acid From the Department of Medicine, Gastroenterology Unit, University of Heidelberg, Heidelberg, Germany
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Table 1. MECHANISMS OF ACTION OF URSODEOXYCHOLIC ACID
Primary effects Displacement of natural, toxic, endogenous bile salts from the enterohepatic circulation (competitive inhibition of the ileal absorption of endogenous bile salts) Competition for receptors (at sites of cell organelles where toxic bile acids may lead to cell damage) Primary consequences Improvement of hepatic excretory function Cytoprotective effect with prevention of bile acid-induced cytolysis and apoptosis Membrane-protective effect with prevention if bile acid-induced membrane damage (mitochondria, basolateral and canalicular membrane) Antioxidative effect with prevention of bile acid-induced peroxidation Immunomodulative effect Secondary consequences Decrease of cholestasis Improvement of liver function and morphology
dependent, and a shortage of bile salts may lead to cholestasis. Such a situation, however, occurs only in patients with inborn errors of bile acid synthesis, and in such patients replacement of bile salts leads to improvement of cholestasis and cholestasis-related liver cell damage. The problem in the majority of patients with cholestatic disease is not a shortage but the accumulation of bile 36 In patients with cholestasis who do not have a defect in bile acid synthesis, increasing amounts of bile salts appear in peripheral blood and urine? 23, 98 and increased concentrations are found in liver 36 In the early stage of PBC, biliary secretion of bile salts is within the normal range,70 whereas plasma bile acids are markedly elevated. This means that the hepatic clearance of bile salts is reduced.48The increased concentrations of bile salts in the hepatocyte are probably due to impaired canalicular secretion, and it seems likely that the uptake of bile salts into the hepatocyte becomes impaired sec~ndarily.~~ Because in the early stage of cholestatic disease hepatic synthesis and net hepatocyte transport of bile acids remain relatively unchanged, ileal absorption seems to be responsible for the load of bile acids to the liver with the consequence of bile acid The fact that high bile salt concentrations in the liver may be related to the liver cell damage in cholestasis was first recognized by Greim et al.35,36 In systematic studies in patients with extrahepatic cholestasis, Greim et a1 were able to show that a lesion that they called feathery degeneration correlated with the concentration of toxic dhydroxy bile salts in the liver tissue. Subsequently, it became apparent that not only feathery degeneration, but also cholate stasis (hydropic swelling of hepatocytes around the portal field) are bile salt-related forms of liver cell damage in cholestasis. Important was the recognition that it was not the accumulation of atypical monohydroxy bile salts, which are cholestatic in animals and had been extensively investigated for many years, but the accumulation of normal dihydroxy bile salts that was responsible
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for the cell damage in cholestasis. Cholate stasis and feathery degeneration are probably the consequence of the detergent action of high concentrations of bile salts in the liver and are observed only in severe cholestasis, as in extrahepatic biliary obstruction. As has been shown more recently, much lower concentrations of bile salts are sufficient to induce apoptotic cell death? 76, 77, 94 and it appears likely that this form of bile salt toxicity plays the major role in the less severe forms of cholestasis. EFFECTS OF URSODEOXYCHOLIC ACID Displacement of Natural, Toxic, Endogenous Bile Acids from the Enterohepatic Circulation
UDCA changes the balance between toxic hydrophobic and nontoxic hydrophilic bile salts in the bile salt pool in favor of the nontoxic bile salts.22, 26, 29, 30, 48, 49, 88, 97, 98 This change in balance between toxic and nontoxic bile salts may represent the most important mechanism of action of UDCA (Table l).,,Consequently the UDCA molecules displace more toxic bile acid molecules from cell membranes and cell organelles and thus prevent the damage that can be induced by more toxic bile salts. After administration of UDCA, the bile salt is absorbed passively by diffusion in the small intestine. Absorption is slow and incomplete.95, 96, lo6 After absorption, UDCA is transported via the portal blood to the liver, is conjugated with glycine or taurine, and is secreted into bile. Conjugates of UDCA are absorbed by an active process in the ileum and possibly in addition by facilitated diffusion in the jejunum.47The conjugates of UDCA probably are responsible for the beneficial effect of UDCA in cholestatic diseases. In animal studies, glycine and taurine conjugates of UDCA were equally effective.52Shortly after the start of treatment, the bile acid pool becomes enriched in UDCA, whereas the relative content of toxic bile acids is decreased.26* 29, 70* 88, 98 Approximately half of UDCA administered orally is not and is bacterially degradated in the large bowel to lithocholic acid and 33 7-Keto-lithocholic acid may be absorbed and 7-keto-lithocholic acid.30* transformed in the liver to chenodeoxycholic acid.30,33 Lithocholic acid is poorly water soluble, and only small amounts are absorbed, partially sulfated in the liver and other organs, and excreted in bile and urine. In cholestasis, substantial amounts of UDCA and its metabolites are excreted in urine.6,98 The best way to treat bile acid-induced cell destruction seems to be to prevent the accumulation of toxic bile salts at places where they can destroy cell membranes and cell organelles. This prevention is not easy to achieve for several reasons. First, blockage of bile acid synthesis is not feasible because it leads to the accumulation of bile acid precursors, which cause diseases and cholestasis (i.e., cerebrotendinous xanthomatosis). Second, binding of bile salts in the intestine leads to their decreased
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reabsorption. Consequently, bile acid synthesis is increased, and the accumulation of bile salts is not prevented. Moreover, in cholestasis, the amount of bile acids in the intestine is reduced. Thus, the effect of bile acid binding resins on bile salt accumulation is little. Because bile acid binding resins cannot differentiate between good and bud bile acids, they decrease not only the absorption of toxic bile acids, but also, when given together with UDCA, decrease absorption of UDCA, an effect that is undesired. The only beneficial effect observed after treatment of patients with cholestasis with bile acid binding substances is the improvement of pruritus in some patients. Third, competitive inhibition of bile salt absorption represents another option. After treatment with UDCA, bile acid absorption is decreased by competitive i n h i b i t i ~ nIn .~~ contrast to the situation with bile acid binding resins, bile acid synthesis is essentially unaltered.88Therefore the decreased intestinal absorption of potentially toxic endogenous bile acids may represent a factor contributing to the beneficial effect of UDCA.28,69, 96 Finally, substances blocking the ileal bile acid transporter are being developed. The main goal of the pharmaceutical companies is to develop these substances for the treatment of hypercholesterolemia. It is unclear how such substances would affect the patient with cholestasis. It seems possible that in cholestatic patients, treatment with UDCA, which is absorbed by diffusion in the jejunum, conferring blockage of the active absorption of endogenous bile acid conjugates in the ileum may lead to an enrichment of the bile acid pool with UDCA to an extent that is unachievable at present. After UDCA administration to patients who had gallstones, the biliary enrichment with this bile acid increases only up to a dose of 10 to 12 mg/ kg and then reaches a plateau.97In cholestatic diseases, and especially in cystic fibrosis, higher doses may be necessary to achieve sufficient biliary enrichment of UDCA.= Theoretically the combination of UDCA with a bile acid absorption blocking agent should be superior to the currently used combination of UDCA plus cholestyramine as far as the enrichment of UDCA in bile is concerned, whereas the effect on pruritus is unclear.
Effect of Ursodeoxycholic Acid on Hepatic Excretory Function (Bile Flow and Biliary Secretion of Bile Acids and Lipids)
In patients with cholestatic liver disease treated with UDCA, the hepatic excretory function is improved.21,loo The choleretic effect of UDCA is comparable to that of the naturally occurring dihydroxy and trihydroxy bile salts (chenodeoxycholic and cholic acids). In contrast to the naturally occurring dihydroxy and trihydroxy bile salts, UDCA is atoxic and in animal experiments can prevent the cholestatic effect of cholic and chenodeoxycholic acid conjugates. Moreover, cholic acid is bacterially degradated to deoxycholic acid, which is considered a hydrophobic and toxic bile salt. Therefore UDCA, in contrast to endoge-
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nous primary bile acids, can be considered a clinically useful choleretic agent.48 In healthy humans, the administration of 1 g of bile salts is small in comparison to the 3- to 5-g bile acid pool that circulates six to eight times, yielding a biliary secretion of 18 to 40 g/d of bile salts. In noncholestatic individuals, the administration of unconjugated bile salts induces only a slight increase in bile acid secretion and, consequently, probably little change in bile flow. The situation may be different in patients with cholestatic liver disease in whom the biliary secretion of endogenous bile acids may be considerably reduced and the administration of 1 g of UDCA may lead to a substantial improvement in the biliary secretion of bile salts.'" Taurine conjugates of UDCA induce vesicular exocytosis," which is impaired in cholestasis. In contrast to taurine conjugates of primary bile acids, UDCA conjugates cause a sustained stimulation of horseradish peroxidase excretion into bile in isolated perfused rat liver, indicating an improvement of vesicular exocytosis." The ability of taurine-conjugated UDCA to induce vesicular exocytosis depends on its ability to induce the influx of extracellular Ca2+,which activates protein kinase C.Q l3 Vesicular exocytosis may be essential for targeting and insertion of apical membrane transport proteins into the canalicular membrane and may represent a major pathway for bile acid transport in bile acid-loaded liver cells. The molecular mechanisms underlying the beneficial effect of UDCA, however, are still not completely clear, and controversy exists on the molecular signaling processes involved. Alternatively to the activation via protein kinase C,I3 activation of mitogen-activated protein (MAP) kinases may play a role:s9 In rat hepatocytes, tauro-UDCA (TUDCA) leads to an activation of MAP kinases, which are potential regulators of bile acid secretion into bile. TUDCA leads to rapid, microtubule-dependent increase in canalicular bile acid secretion and stimulation of exocytosis. This activity may reflect a microtubule-dependent apical targeting of vesicles containing bile acid transporters in response to MAP kinase activation. Protein kinase C is not involved in the activation of MAP kinases. Therefore at present, two opposing concepts concerning the molecular mechanisms involved in the effect of UDCA exist-one in which protein kinase C plays a role and one in which it does not play a role-and it is not clear which hypothesis is correct. An alternative way of increasing bile flow represents the direct stimulation of C1- efflux through opening of C1- channels in biliary cells.91Such a direct stimulation of ductular secretion by UDCA may be involved in cystic fibrosis patients. Although it seems likely that the beneficial effect of UDCA is linked to the improvement of bile flow and of the biliary secretion of cholephilic substances, a positive effect of UDCA on liver histology has also been reported in bile duct ligated rat, in which bile flow and biliary secretion do not play a role.32,~oProvided that these observations are correct, the beneficial effects of UDCA on the liver might be independent of its effect on the biliary secretion.
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In patients with cholestatic disease, UDCA treatment leads to a markedly increased biliary secretion of phospholipids and a moderate increase of The increased secretion of phospholipids is of interest because these molecules seem to trap bile acids in the biliary canaliculus to form vesicles and mixed micelles and thus prevent their detrimental effect.5,74, 85, Io5 The increase in the secretion of cholesterol is remarkable because in individuals without cholestasis UDCA reduces biliary cholesterol secretion and dissolves cholesterol gallstones. The increase in biliary cholesterol secretion in severe cholestasis indicates that UDCA improves the hepatic excretory function, although the effect is modest.loO
Effect of Ursodeoxycholic Acid on Bile Acid-Induced Cholestasis in Perfused Rat Liver
Naturally occurring bile salts, as cholic acid and chenodeoxycholic acid, increase bile flow in perfused rat liver when infused in physiologic amounts but induce cholestasis with reduction of bile flow when the infused amount exceeds a certain limit.55Besides cholestasis, hepatocellular damage can also be demonstrated. The hepatocytes close to the portal triad are those that are mainly damaged by bile acid infusions, whereas the hepatocytes around the central vein remain relatively unaltered.90This situation is in good agreement with the early observation that cholestasis mainly affects the periportal hepatocytes. The hepatic injury is associated with an increased excretion of alkaline phosphatase and protein into the bile.55Simultaneous infusion of UDCA prevents the cholestatic effect of toxic bile salts and reduces the morphologic alterations in periportal hepatocytes induced by lipophilic bile In addition, the biliary enzyme and protein excretion induced by toxic bile acids is reduced by coinfusion with UDCA.55These were, the first observations that UDCA conjugates can directly prevent the toxic action of common bile salts.
Ursodeoxycholic Acid in Estrogen-Induced Cholestasis
Estrogens, such as contraceptives or postmenopausal replacement therapy, often cause intrahepatic cholestasis. Moreover, estrogens are implicated in the pathogenesis of recurrent jaundice in the last trimester of pregnancy. Metabolites of natural estrogens (i.e., estradiol-17 p-glucu53 ronide) have been used to study drug-induced intrahepatic cholestasis.2* In rats with bile fistulae, estradiol-17 P-glucuronide infused intravenously induces a marked reduction in bile flow. In ultrastructural studies, the reduction of bile flow was accompanied by fragmentation and
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loss of canalicular microvilli, dilation of canaliculi, and thickening of pericanalicular ectoplasm. Both reduction of bile flow and ultrastructural alterations induced by estrogen metabolites can be prevented by the simultaneous infusion of TUDCA.*,53 TUDCA increased the biliary secretion of estradiol-17 P-glucuronide, and it was suggested that the beneficial effect of UDCA in this model is due to the enhanced biliary secretion of the estrogen metabolite.
Effect of Ursodeoxycholic Acid on Bile Acid-Induced Cell Death in Hepatocytes in Culture At concentrations of greater than 100 pmol/L, hepatocytes are destroyed by the detergent action of bile s a l t ~ .40-43 ~ ~ ,In rat and human hepatocytes in culture, cytolysis is dose and time dependent.34,43 Hepatocytes in culture subjected to detergent bile salts in high concentration (> 100 pM) swell and finally disrupt. The toxicity of 3-, 7-, and 12ahydroxylated bile salts increases with the detergent power of the molecule. In general, the toxicity increases with decreasing number of hydroxyl groups, but the position of the hydroxyl group also plays a role. Of the common bile salts, deoxycholic acid (3a-12a-dihydroxy bile salt) is more toxic than chenodeoxycholic acid (3a7a-dihydroxy bile salt), and both are more toxic than cholic acid (3a-,7a-, 12a-trihydroxy bile Bile salts with a 7P or 601 hydroxyl group (UDCA, hyodeoxycholic acid, muricholic acid) are not toxic and can prevent the toxicity of other bile salts. Thus the cytolysis induced by deoxycholic acid and chenodeoxy43 cholic acid conjugates is markedly reduced by UDCA conjugates.34, It has become evident that not only cytolysis, but also apoptosis, the programmed form of cell death, plays a role in bile acid-related cell toxicity.s,58, 76, 77, 94 Hepatocytes undergoing apoptosis shrink and show nuclear condensation with DNA fragmentation. The finding that cell death by apoptosis is induced by bile acid concentrations far below 94 Because those that induce cytolysis (<50 pM) appears irnp~rtant.~~, bile salt concentrations in the livers of patients with cholestasis rarely reach concentrations greater than 100 pM, it seems likely that apoptosis represents the more important form of cell death in the majority of patients with cholestatic diseases. UDCA conjugates decrease apoptosis induced by toxic bile acids in rat and human hepatocytes in culture.*, This effect does not involve the Fas receptor system (Christine Benz, personal communication, January 1998) and probably is mediated by endonu~leases.~~ The protective effect of UDCA on bile acid-induced apoptosis in hepatocytes may represent one of the more important mechanisms of action of UDCA. The toxic effects of bile salts on cholangiocytes have not been studied in detail, but it appears likely that the effects are similar to those in hepatocytes. UDCA conjugates reduce cytolysis as well as apoptosis
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induced by toxic bile acids. It seems, however, that the effect on apoptosis represents the physiologically more relevant effect.8
Effect of Ursodeoxycholic Acid on Membranes and Mitochondria
Bile salts exert their detergent action not only on hepatocytesIMbut also on red blood leading to cytolysis of the cells. The membranedamaging effect of lipophilic bile acids can also be demonstrated in model membranes.40, In all these models, the toxic effects of lipophilic bile acids are, in part, reversed by UDCA conjugates. Studies with red blood cells and both canalicular and basolateral hepatocyte membranes using electron paramagnetic resonance spectroscopy show that the membrane-damaging effect of toxic bile salts is accompanied by an increased polarity of the apolar domain of the membranes.37This polarity is accompanied by a loss of cholesterol and phospholipids from the membranes. UDCA pretreatment prevents this toxic effect of bile salts by binding to the apolar domain, whereas UDCA conjugates are incorporated into the interface of the membranes and thus protect them from the detergent action of lipophilic bile These findings indicate that UDCA and its taurine conjugate both have protective effects by decreasing the membrane polarity but exert their effects at different sites. UDCA may have an influence on the membrane fluidity, which may be decreased in cholestasis associated with total parenteral nutrition In an animal model using newborn piglets, UDCA infusion reduced cholestasis and prevented the decrease of basolateral membrane fluidity that was observed after total parenteral nutrition.27In this model, there was no effect of UDCA on the fluidity of the canalicular membrane. The effect of UDCA has been attributed to the membrane-Stabilizing effect of UDCA. It seems likely that a membrane-stabilizing effect is also responsible for the beneficial effect of UDCA on the bile acid-induced preservation injury to bile ducts of explanted livers.39 Studies in model membranes composed of egg phosphatidylcholine and cholesterol indicate that such membranes disrupt with increasing bile salt concentration, hydrophobicity, and increasing ionic strength.40 UDCA and its conjugates reduced the disruption of cholesterol-rich model membranes.4oAccording to these data, UDCA can reduce the toxicity of other bile salts via a purely physicochemical mechanism.50 Mitochondria1 dysfunction may contribute to liver failure and 63 Bile acids can disturb the lipid composition of chronic chole~tasis.~~, biologic membranes, leading to decreased function of membrane-bound enzymes. Bile acids are particularly damaging to hepatocyte mitochondria and inhibit the activities of enzyme complexes of the electron 63 UDCA transport chain, including succinate cytochrome C reducta~e.~~, protects oxidative mitochondria1 metabolism from toxicity of lipophilic
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bile acids. The protective effect of UDCA may partially be explained by decreased incorporation of bile acids into mitochondrial membranes.37 Toxic bile salts may induce mitochondrial membrane permeability transition characterized by a rapid permeability of the inner mitochondrial membrane, mitochondrial swelling, and a collapse of the mitochondrial p0tentia1.l~This effect leads to necrosis of the hepatocyte. UDCA inhibits the bile salt-induced mitochondrial membrane permeability transition, and this indicates a protective effect of UDCA on bile acidinduced mitochondrial damage.15 Mitochondria1 membranes appear to represent an important target for the deleterious effects of toxic bile acids, and UDCA and its conjugates, at least in part, may prevent this detergent effect of lipophilic bile acids. Ursodeoxycholic Acid as Antioxidant Hydrophobic bile salts can damage hepatocytes by increasing the 93 Bile acid-induced apoptosis is generation of reactive oxygen species.77, associated with increased lipid peroxidation and can be inhibited by antioxidant^.^, 77, 93 Hydrophobic bile salts can oxidatively activate Kupffer 's cells. These cells, when stimulated, secrete reactive oxygen species, nitric oxide, eicosanosoids, and cytokines. Some of these products are cytotoxic and attack nucleic acids, thiol proteins, or membrane lipids causing lipid peroxidation. UDCA can block the activation of Kupffer 's cells by toxic bile salts and reduce lipid per~xidation.~~ Therefore the beneficial effect of UDCA, at least in part, may be related to its ability to act as an antioxidant. Ursodeoxycholic Acid and lmmunomodulation The human leukocyte antigen (HLA) system codes for genes that are believed to play a role in autoimmune diseases. In the normal liver, class I antigens are expressed by hepatic sinusoidal cells and biliary epithelial cells but not hepatocytes. In contrast, class I1 antigens are expressed by hepatic sinusoidal cells and dendritic cells but not biliary epithelial cells or hepatocytes. In patients with PBC, an increased expression of class I antigens has been observed.', 17, l8 In patients with extrahepatic cholestasis, there was increased class I expression on hepatocytes and bile ducts, whereas class I1 expression was increased only on bile In patients with PBC treated with UDCA, HLA class I expression on hepatocytes was significantly reduced, whereas class I1 expression on biliary epithelial cells was unaltered.18,Io3 It has been suggested that the reduction in HLA class I expression may lead to a decrease in the cytotoxicity of T cells, which are involved in lobular necrosis in PBC. UDCA may interfere either at the levels of antigen presentation via MHC molecules or at the level of T-cell polarization into T-cell subsets. The problem is that it is not known what triggers the inflammatory bile
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duct processes in PBC.‘O Although bile ducts are mainly destroyed by T cells, there is still no convincing evidence that the cellular target is the autoantigen recognized by autoantibodies circulating in the periphery, because after liver transplantation antimitochondrial antibodies remain positive, but the grafted liver is generally not affected. Because the exact immunopathomechanism responsible for the progression of disease in PBC is not completely clear, it is not possible to clarify the immunopathomechanism, if one exists, responsible for the beneficial effect of UDCA in PBC. Bile acids, such as chenodeoxycholic acid, have a profound effect on mononuclear cell cytokine production and inhibit secretion of interleukin-2 and other cytokines.” The immunosuppressive effects of endogenous bile acids could explain, at least in part, the occurrence of infections of various origins during cholestasis. UDCA inhibits cytokine release much less than hydrophobic bile ~a1ts.l~ By displacement of endogenous bile salts, UDCA might reduce the cholestasis-related immunosuppression, and it has been speculated that the beneficial effect of UDCA may, at least in part, be due to the prevention of immunosuppression induced by endogenous bile ~a1ts.l~. 2o This hypothesis, however, is in contrast to reports of a decreased release of interleukin-2, interleukin4, and interferon-y according to which the effect of UDCA is due to its immunosuppressive effect.lo7Depending on the experimental design, UDCA may stimulate56or inhibit the release of cytokine~,’~ and at present the principles of immunomodulation by bile acids are unclear. OTHER PROTECTIVE BILE ACIDS
Studies in perfused rat liver indicate that a-muricholate and tauroP-muricholate are as effective as TUDCA in preventing bile acidinduced liver damage.54Although tauro-a-muricholate does not possess a 7P-hydroxyl group as UDCA and instead has a 6P-hydroxyl group, it was able to reduce the toxic effect of hydrophobic bile acids, indicating that the protective effect is not linked only to a 7P-hydroxyl group. These findings have been confirmed in studies with taurohyodeoxycholic acid (301, 6a, dihydroxy bile acid), in which this 6a-hydroxylated bile acid prevented hepatotoxicity induced by hydrophobic bile acids.86It has been shown that both tauro-a-muricholate and tauro-P-muricholate, similar to UDCA, also protect against the cholestatic effect of estrogen metabolites.lo2At present, bile acid molecules with a 7P-hydroxyl or 6ahydroxyl group are candidates for hepatoprotection. The only bile acid, however, for which a beneficial effect has been shown in extensive clinical studies is UDCA, and it can be expected that similar effects will be obtained with its taurine conjugate TUDCA. References 1. Adams DH, Hubscher SG, Shaw J, et al: Increased expression of intercellular adhesion molecule 1 on bile ducts in primary biliary cirrhosis and primary sclerosing cholangitis. Hepatology 14:426, 1991
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2. Albertani CO, Zimmerman HJ, Ishak KG, et al: Drug-induced cholestasis in the perfused rat liver and its reversal by tauroursodeoxycholate: An ultrastructural study. Proc SOCExp Biol Med 199:54, 1992 3. Balisteri WF: Bile acid therapy in pediatric hepatobiliary disease: The role of ursodeoxycholic acid. J Pediatr Gastroenterol Nutr 24:573, 1997 4. Barnes D, Talenti D, Cammell G, et al: A randomized clinical trial of ursodeoxycholic acid as adjuvant treatment to prevent liver transplant rejection. Hepatology 262353, 1997 5. Bamwell SG, Lowe PJ, Coleman R Effect of taurochenodeoxycholate or tauroursodeoxycholate upon biliary output of phospholipids and plasma-menbrane enzymes, and the extent of cell damage, in isolated perfused rat livers. Biochem J 216:107, 1983 6. Batta AK, Salen G, Arora R, et al: Effect of ursodeoxycholic acid on bile acid metabolism in primary biliary cirrhosis. Hepatology 10:414, 1989 7. Beau P, Labat-Labourdette J, Ingrand P, Beauchant M: Is ursodeoxycholic acid an effective therapy for total parenteral nutrition-related liver disease? J Hepatol 20:240, 1994 8. Benz C, Angermuller S, Tox U, et al: Effect of tauroursodeoxycholic acid on bile acid induced apoptosis and cytolysis in rat hepatocytes. J Hepatol 28:99, 1998 9. Benz C, Sauer P, Kloters-Plachky P, et al: Effect of S-adenosylmethionine versus tauroursodeoxycholic acid on bile acid induced apoptosis and cytolysis in rat hepatocytes. Eur J Clin Invest 28:577, 1998 10. Berg PA, Klein R, Rocken M: Cytokines in primary biliary cirrhosis. Semin Liver Dis 17115, 1997 11. Beuers U, Nathanson MH, Isales CM, et a1 Tauroursodeoxycholic acid stimulates hepatocellular exocytosis and mobilizes extracellular Ca mechanisms defective in cholestasis. J Clin Invest 922984, 1993 12. Beuers U, Spengler U, Kruis W, et al: Ursodeoxycholic acid for treatment of primary sclerosing cholangitis: A placebo controlled trial. Hepatology 16:707, 1992 13. Beuers U, Throckmorton DC, Anderson MS, et al: Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes. Gastroenterology 110:1553, 1996 14. Bittner PO, Posselt HG, Sailer T, et al: The effect of treatment with ursodeoxycholic acid in cystic fibrosis and hepatopathy: Results of a placebo-controlled study. In Paumgartner G, Stiehl A, Gerok W (eds): Bile Acids as Therapeutic Agents. Dordrecht, The Netherlands, Kluwer, 1991, p 345 15. Botla R, Spivey JR, Aguilar H, et al: Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane transition induced by chenodeoxycholate: A mechnism of UDCA protection. J Pharmacol Exp Ther 272930, 1995 16. Buzzelli G, Moscarella S, Focardi G, et al: Long-term treatment with ursodeoxycholic acid in patients with chronic active cirrhosis. Curr Ther Res 50:635, 1991 17. Calmus Y, Arvieux C, Gone P, et al: Cholestasis induces major histocompatibility class I expression in hepatocytes. Gastroenterology 102:1371, 1992 18. Calmus Y, Gane P, Rouger P, et al: Hepatic expression of class I and class I1 major histocompatibility complex molecules in primary biliary cirrhosis: Effect of ursodeoxycholic acid. Hepatology 11:12, 1990 19. Calmus Y, Guechot J, Podevin P, et al: Differential effects of chenodeoxycholic and ursodeoxycholic acids on interleukin 1, interleukin 6 and tumor necrosis factor-alpha production by monocytes. Hepatology 16:719, 1992 20. Calmus Y, Weill B, Ozier Y, et al: Immunosuppressive properties of chenodeoxycholic and ursodeoxycholic acids in the mouse. Gastroenterology 103:617, 1992 21. Colombo C, Castellani MR, Balisteri WF, et al: Scintigraphic documentation of an improvement in hepatobiliary excretory function after treatment with ursodeoxycholic acid in patients with cystic fibrosis and associated liver disease. Hepatology 15:677, 1992 22. Colombo C, Crossignani A, Assaido M, et al: Ursodeoxycholic acid therapy in cystic fibrosis-associated liver disease: A dose-response study. Hepatology 16:924, 1992 23. Colombo C, Setchell KDR, Podda M,et a1 Effects of ursodeoxycholic acid therapy for liver disease associated with cystic fibrosis. J Pediatr 117482, 1990 24. Combes B, Carithers RL, Maddrey WC, et al: A randomized, double-blind, placebo+
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controlled trial of ursodeoxycholic acid in primary biliary cirrhosis. Hepatology 22:759, 1995 Cotting J, Lentze MJ, Reichen J: Effects of ursodeoxycholic acid therapy on nutrition and liver function in patients with cystic fibrosis and long-standing cholestasis. Gut 31:918, 1990 Crosignani A, Battezzati PM, Bertolini M, et al: Changes in bile acid composition in patients with primary biliary cirrhosis induced by ursodeoxycholic acid administration. Hepatology 14:1000, 1991 Duerksen DR, Van Aerde JE, Gramlich L, et al: Intravenous ursodeoxycholic acid reduces cholestasis in parenterally fed newborn piglets. Gastroenterology 111:1111, 1996 Eusufzai S, Ericson S, Cederlund T, et a1 Effect of ursodeoxycholic acid treatment on ileal absorption of bile acids in man as determined by the SEHCAT test. Gut 321044, 1991 Fedorowski T, Salen G, Colallilo A, et al: Metabolism of ursodeoxycholic acid in man. Gastroenterology 731131, 1977 Fedorowski T, Salen G, Tint GS, et al: Transformation of chenodeoxycholic acid and ursodeoxycholic acid by human intestinal bacteria. Gastroenterology 771068, 1979 Floreani A, Chiaramonte M, Fabris P, et al: Poor effect of ursodeoxycholic acid in anti-hepatitis C virus positive chronic liver disease. Curr Ther Res 50:579, 1991 Frezza EE, Gerunda GE, Plebani M, et al: Effect of ursodeoxycholic acid administration on bile duct proliferation and cholestasis in bile duct ligated rat. Dig Dis Sci 38:1291, 1993 Fromm H, Carlson GL, Hofmann AF, et al: Metabolism in man of 7-ketolithocholic acid: Precursor of cheno- and ursodeoxycholic acid. Am J Physiol 239:G161, 1980 Galle PR, Theilmann L, Raedsch R, et al: Ursodeoxycholate reduces hepatotoxicity of bile salts in primary human hepatocytes. Hepatology 12486, 1990 Greim H, Triilzsch D, Czygan P, et al: Mechanisms of cholestasis: 6. Bile acids in human livers with and without biliary obstruction. Gastroenterology 63:846, 1972 Greim H, Triilzsch D, Roboz J, et al: Mechanisms of cholestasis: 5. Bile acids in normal rat livers and those after bile duct ligation. Gastroenterology 63:837, 1972 Giildiituna S, Zimmer G, Imhof M, et al: Molecular aspects of membrane stabilization by ursodeoxycholate. Gastroenterology 104:1736, 1993 Heathcote EJL, Cauch-Dudek K, Walker V, et al: The Canadian multi-center double blind randomized controlled trial of ursodeoxycholic acid in primary biliary cirrhosis. Hepatology 19:1149, 1994 Hertl M, Harvey PR, Swanson PE, et a1 Evidence of preservation injury to bile ducts by bile salts in the pig and its prevention by infusions of hydrophilic bile salts. Hepatology 21:1130, 1995 Heuman DM, Bajaj R Ursodeoxycholate conjugates protect against disruption of cholesterol-rich membranes by bile salts. Gastroenterology 106:1333, 1994 Heuman DM, Bajaj RS, Lin Q: Adsorption mixtures of bile salt taurine conjugates to lecithin-cholesterol membranes: Implications for bile salt toxicity and protection. J Lipid Res 37562, 1996 Heuman DM, Mills AS, McCall JB, et al: Conjugates of ursodeoxycholate protect against cholestasis and hepatocelllular necrosis caused by more hydrophobic bile salts: In vivo studies in the rat. Gastroenterology 100:203, 1991 Heuman DM, Pandak WM, Hylemon PB, et al: Conjugates of ursodeoxycholate protect against cytotoxicity of more hydrophobic bile salts: In vitro studies in rat hepatocytes and human erythrocytes. Hepatology 14:920, 1991 Hillaire S, Boucher E, Calmus Y, et a1 Effects of bile acids and cholestasis on major histocompatibility complex class I in human and rat hepatocytes. Gastroenterology 107781, 1994 Hirano F, Tanaka H, Makino Y, et al: Regulation of the major histocompatibility complex class I mRNA expression by bile salts in cultured human hepatoma cells. Biochim Biophys Res Commun 208:935, 1995 Hirano F, Tanaka H, Makino Y, et al: Effects of ursodeoxycholc acid and chenodeoxy-
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cholic acid on major histocompatibility complex class I gene expression. J Gastroenterol 31:55, 1996 47. Hofmann A F Pharmacolozv of chenodeoxvcholic acid and ursodeoxvcholic acid in man. In Paumgartner G, giehl A, Gerok’W (eds): Bile Acids and ’Cholesterol in Health and Disease. Dordrecht, The Netherlands, Kluwer, 1983, p 301 48. 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, et a1 (eds): Strategies for the Treatment of Hepatobiliary Diseases. Dordrecht, The Netherlands, Kluwer, 1990, p 13 49. Hofmann AF, Popper H Ursodeoxycholic acid treatment for primary biliary cirrhosis. Lancet 2398,1987 50. Igimi H, Carey MC: pH solubility relations of chenodeoxycholic and ursodeoxycholic acids: Physical chemical basis for dissimilar solution and membrane phenomena. J Lipid Res 21:72, 1980 51. Invemizzi P, Setchell KDR, Crossignani A, et al: Comparison between tauroursodeoxycholic and ursodeoxycholic acids in patients with primary biliary cirrhosis: A crossover study [abstr]. J Hepatology 26(suppl 1):166, 1997 52. Kanai S, Kitani K Glycoursodeoxycholate is as effective as tauroursodeoxycholate in preventing the taurocholate-induced cholestasis in the rat. Res Commun Chem Pathol Pharmacol42:423, 1983 53. Kinbara S, Ishizaki K, Sakakura H, et a1 Improvement of estradiol-17beta-D-glucuronide-induced cholestasis by sodium tauroursodeoxycholate therapy in rats. Scand J Gastroenterol 32947, 1997 54. Kitani K, Kanai S, Sat0 Y, et a1 Tauro-alpha-muricholate is as effective as taurobeta-muricholate and tauroursodeoxycholate in preventing taurochenodeoxycholateinduced liver damage in the rat. Hepatology 19:1007, 1994 55. Kitani K, Ohta M, Kanai S: Tauroursodeoxycholate prevents biliary protein excretion induced by other bile salts in the rat. Am J Physiol 248:G407, 1985 56. Klein R, Leuschner M, Leuschner U, et al: Cytokine profiles in primary biliary cirrhosis and primary sclerosing cholangitis. In Berg PA, Leuschner U (eds): Bile Acids and Immunology. Dordrecht, The Netherlands, Kluwer, 1996, p 195 57. Krahenbiihl S, Fischer S, Talos C, et a1 Ursodeoxycholate protects oxidative mitochondrial metabolism from bile acid toxicity: Dose-response study in isolated rat liver mitochondria. Hepatology 20:1595, 1994 58. Kwo P, Bronk SF, Gores GJ: Nuclear serine protease activity contributes to bile acidinduced apoptosis in hepatocytes. Am J Physiol268:G613, 1995 59. Lacaille F, Paradis K The immunosuppressive effect of ursodeoxycholic acid: A comparative in vitro study of human peripheral blood mononuclear cells. Hepatology 18:165. 1993 60. Lepage G, Paradis K, Lacaille F, et al: Ursodeoxycholic acid improves the hepatic metabolism of essential fatty acids and retinol in children with cystic fibrosis. J Pediatr 130:52, 1997 61. Leuschner U, Fischer H, Kurtz W, et al: Ursodeoxycholic acid in primary biliary cirrhosis: Results of a controlled double-blind trial. Gastroenterology 971268, 1989 62. Leuschner U, Leuschner M, Sieratzki J, et al: Gallstone dissolution with ursodeoxycholic acid in patients with chronic active hepatitis and two years’ follow-up. Dig Dis Sci 30642, 1985 63. Lim AG, Ahmed HA, Jazrawi RP, et al: Effects of bile acids on human hepatic mitochondria. Eur J Hepatol Gastroenterol 6:1157, 1994 64. Lindor KDL, Dickson ER, Baldus WP, et al: Ursodeoxycholic acid in the treatment of primary biliary cirrhosis. Gastroenterology 106:1284, 1994 65. Lindor KD, Mayo PSC/UDCA study group: Ursodiol for the treatment of primary sclerosing cholangitis. N Engl J Med 336:691, 1997 66. Ljubuncic P, Fuhrman B, Oiknine J, et al: Effect of deoxycholic acid and ursodeoxycholic acid on lipid peroxidation in cultured macrophages. Gut 3 9 ~ 7 5 1996 , 67. Loria P, Bozzoli M, Concari M, et al: Effect of taurohyodeoxcholic acid on biliary lipid secretion in humans. Hepatology 25:1306, 1997 v
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68. Lotterer E, Stiehl A, Raedsch R, et al: Ursodeoxycholic acid in primary biliary cirrhosis: No evidence of toxicity in the stages I to 111. J Hepatol 10284, 1990 69. Marteau P, Chazouilleres 0, Myara A, et al: Effect of chronic administration of ursodeoxycholic acid on the ileal absorption of endogenous bile acids in man. Hepatology 12I1206, 1990 70. Mazzella G, Parini P, Bazzoli F, et al: Ursodeoxycholic acid administration on bile acid metabolism in patients with early stages of primary biliary cirrhosis. Dig Dis Sci 382396, 1993 71. Mitchell SA, Bansi D, Hunt N, et al: High dose ursodeoxycholic acid (UDCA) in primary sclerosing cholangitis (PSC): Results after two years of a randomized doubleblind, placebo-controlled trial [abstr]. Gut 4O(suppl 1):TH115, 1997 72. Nakagawa M, Colombo C, Setchell KDR: Comprehensive study of the biliary bile acid composition of patients with cystic fibrosis and associated liver disease before and after UDCA administration. Hepatology 12:322, 1990 73. Nittono H, Tokita A, Hayashi M, et al: Ursodeoxycholic acid therapy in the treatment of biliary atresia. Biomed Pharamacother 43:37, 1989 74. Oude Elferink RPJ, Ottenhoff R, van Wijland M, et al: Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse. J Clin Invest 95:31, 1995 75. Palma J, Reyes H, Ribalta J, et al: Effects of ursodeoxycholic acid in patients with intrahepatic cholestasis of pregnancy. Hepatology 15:1043, 1992 76. Patel T, Bronk SF, Gores GJ: Increases of intracellular magnesium promote glycochenodexycholate-induced apoptosis in rat hepatocytes. J Clin Invest 942183, 1994 77. Patel T, Gores GJ: Inhibition of bile salt-induced hepatocyte apoptosis by the antioxidant Lazaroid U83836E. Toxic01 Appl Pharmacol 142:116, 1997 78. Plevris JN, Hayes PC, Bouchier IAD Ursodeoxycholic acid in the treatment of alcoholic liver disease. Eur J Gastroenterol Hepatol 3:653, 1991 79. Podda M, Ghezzi C, Battezzati PM, et al: Effects of ursodeoxycholic acid and taurine on serum liver enzymes and bile acids in chronic hepatitis. Gastroenterology 98:1044, 1990 80. Po0 JL, Feldmann G, Erlinger S, et al: Ursodeoxycholic acid limits liver histologic alterations and portal hypertension induced by bile duct ligation in the rat. Gastroenterology 102:1752, 1992 81. Poupon RE, Balkau B, Eschwege E, et a1 A multicenter controlled trial of ursodiol for the treatment of primary biliary cirrhosis. N Engl J Med 3241548, 1991 82. Poupon R, Chretien Y, Poupon R, et al: Serum bile acids in primary biliary cirrhosis: Effect of ursodeoxycholic acid therapy. Hepatology 17599, 1993 83. Poupon R, Poupon RE, Calmus Y, et al: Is ursodeoxycholic acid an effective treatment for primary biliary cirrhosis. Lancet 1:834, 1987 84. Poupon RE, Lindor KD, Cauch-Dudek K, et al: Combined analysis of tandomized controlled trials of ursodeoxycholic acid in primary biliary cirrhosis. Gastroenterology 113:884, 1997 85. Pualielli L, Amiao L, Arrese M, et al: Protective role of biliarv cholesterol and phgspholipid lamellae against bile acid-induced cell damage. 'Gastroenterology 107244. ~. , 1994 86. Roda A, Piazza F, Baraldini M, et a1 Taurohyodeoxycholic acid protects against hepatotoxicity induced by taurochenodeoxycholic acid in rat. Hepatology 27520,1998 87. Rolandi E, Franceschini R, Cataldi A, et al: Effects of ursodeoxycholic acid on serum liver damage indices in patients with chronic active hepatitis. Eur J Clin Pharmacol 40:473, 1991 88. Rudolph G, Endele R, Senn M, et al: Effect of ursodeoxycholic acid on the kinetics of cholic acid and chenodeoxycholic acid in patients with primary sclerosing cholangitis. J Hepatol 171028, 1993 89. Schliess F, Kurz AK, vom Dahl S, et a1 Mitogen-activated protein kinases mediate the stimulation of bile acid secretion by tauroursodeoxycholate in rat liver. Gastroenterology 1133306, 1997 90. Schmucker DL, Ohta M, Kanai S, et al: Hepatic injury induced by bile salts: Correlation between biochemical and morphological events. Hepatology 12:1216, 1990 91. Shimokura GH, McGill J, Schlenker T, et al: Ursodeoxycholate increases cytosolic v
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calcium concentration and activates CL- currents in a biliary cell line. Gastroenterology 109:965, 1995 92. Smit JJM, Schinkel AH, Oude Elferink RPJ, et a1 Homozygous disruption of the murine mdr 2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 75:451, 1993 93. Sokol RJ, Devereaux M, Khandwala R, et al: Evidence for involvement of oxygen free radicals in bile acid toxicity to isolated rat hepatocytes. Hepatology 17869, 1993 94. Spivey JR, Bronk SF, Gores GJ: Glycochenodeoxycholate-inducedlethal hepatocellular injury in rat hepatocytes. J Clin Invest 92:17, 1993 95. Stiehl A, Raedsch R, Rudolph G: Ileal excretion of bile acids: Comparison with biliary bile acid composition and effect of ursodeoxycholic acid treatment. Gastroenterology 94:1201, 1988 96. Stiehl A, Raedsch R, Rudolph G: Acute effects of ursodeoxycholic acid and chenodeoxycholic acid on the small intestinal absorption of bile acids. Gastroenterology 98:424, 1990 97. Stiehl A, Raedsch R, Rudolph G, et al: Effect of ursodeoxycholic acid on biliary bile acid and bile lipid composition in gallstone patients. Hepatology 4:107, 1984 98. Stiehl A, Rudolph G, Raedsch R, et al: Ursodeoxycholic acid-induced changes of plasma and urinary bile acids in patients with primary biliary cirrhosis. HepaFology 12:492. 1990 99. Stiehl A, Rudolph G, Sauer P, et al: Efficacy of ursodeoxycholic acid and endoscopic dilation of major duct stenoses in primary sclerosing cholangitis: An 8-year prospective study. J Hepatol 26:560, 1997 100. Stiehl A, Rudolph G, Sauer P, et al: Biliary secretion of bile acids and lipids in primary sclerosing cholangitis: Influence of cholestasis and effect of ursodeoxycholic acid treatment. J Hepatol 23:283, 1995 101. Stiehl A, Walker S, Stiehl L, et a1 Effects of ursodeoxycholic acid on liver and bile duct disease in primary sclerosing cholangitis: A 3 year pilot study with a placebocontrolled study period. J Hepatol 20:57, 1994 102. Takikawa H, Sano N, Aiso M, et al: Effect of tauro-alpha-muricholate and tauro-betamuricholate on oestradiol 17beta-glucuronide-induced cholestasis in rats. J Gastroenterol Hepatol 1284, 1997 103. Taresaki S, Nakanuma Y, Ogino H, et a1 Hepatocellular and biliary expression of HLA antigens in primary biliary cirrhosis before and after ursodeoxycholic acid therapy. Am J Gastroenterol 86:1194, 1991 104. Tsukahara K, Kanai S, Ohta M, et al: Taurine conjugate of ursodeoxycholate plays a major role in the hepatoprotective effect against cholestasis induced by taurochenodeoxycholate in rats. Liver 13:262, 1993 105. Van Nieuwkerk CMJ, Oude Elferink RPJ, Groen AK, et al: Effects of ursodeoxycholate and cholate feeding on liver disease in FVB mice with a disrupted mdr2 P-glycoprotein gene. Gastroenterology 111:165, 1996 106. Walker S, Rudolph G, Raedsch R, et a1 Intestinal absorption of ursodeoxycholic acid in patients with extrahepatic biliary obstruction and bile drainage. Gastroenterology 102:810, 1992 107. Yoshikawa M, Tsujii T, Matsumura K, et al: Immunomodulatory effects of ursodeoxycholic acid on immune responses. Hepatology 16:358, 1992 108. Zhao XM, Montet AM, Montet JC: p muricholic acid: A new hepatoprotective agent. J Nutr Biochem 4105, 1993
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MECHANISM OF HEPATOPROTECTIVE ACTION OF BILE SALTS IN LIVER DISEASE Adolf Stiehl, MD, Christine Benz, MD, and Peter Sauer, MD
Ursodeoxycholic acid (UDCA) improves liver enzymes and in many instances liver histology in cholestatic liver diseases, such as primary 38, 83 and primary sclerosing cholangitis biliary cirrhosis (PBC)24, 65, 71, Iol In PBC, UDCA improves survival free of liver trans(PSC).12, plantation4; and in PSC, UDCA improves survival only if dominant strictures of the bile ducts are recognized early and treated endoscopially.^^ UDCA seems to have a beneficial effect in cholestasis of preg21-23, 25, various other cholestatic nan~y,7~ cholestasis of cystic fib~osis,’~, diseases of childhood3as well as cholestasis of total parenteral n ~ t r i t i o n . ~ Besides classic cholestatic diseases, UDCA also improves liver biochemistry in alcoholic liver disease78and in chronic viral hepatitis C.16,31, 79 In chronic hepatitis, UDCA has no effect on liver histology, and its role is unclear; moreover, it is unclear whether UDCA has an effect when combined with interferon treatment. Of considerable interest is the observation that survival of patients undergoing liver transplantation may be improved by treatment with UDCA.4 The main target of UDCA treatment is cholestasis, and consequently the mechanisms responsible for the beneficial effects in these diseases are of interest. ROLE OF BILE ACIDS IN CHOLESTASIS
In cholestasis, bile flow and biliary secretion of bile salts and cholephilic substances are reduced. In humans, bile flow is mainly bile acid From the Department of Medicine, Gastroenterology Unit, University of Heidelberg, Heidelberg, Germany
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Table 1. MECHANISMS OF ACTION OF URSODEOXYCHOLIC ACID
Primary effects Displacement of natural, toxic, endogenous bile salts from the enterohepatic circulation (competitive inhibition of the ileal absorption of endogenous bile salts) Competition for receptors (at sites of cell organelles where toxic bile acids may lead to cell damage) Primary consequences Improvement of hepatic excretory function Cytoprotective effect with prevention of bile acid-induced cytolysis and apoptosis Membrane-protective effect with prevention if bile acid-induced membrane damage (mitochondria, basolateral and canalicular membrane) Antioxidative effect with prevention of bile acid-induced peroxidation Immunomodulative effect Secondary consequences Decrease of cholestasis Improvement of liver function and morphology
dependent, and a shortage of bile salts may lead to cholestasis. Such a situation, however, occurs only in patients with inborn errors of bile acid synthesis, and in such patients replacement of bile salts leads to improvement of cholestasis and cholestasis-related liver cell damage. The problem in the majority of patients with cholestatic disease is not a shortage but the accumulation of bile 36 In patients with cholestasis who do not have a defect in bile acid synthesis, increasing amounts of bile salts appear in peripheral blood and urine? 23, 98 and increased concentrations are found in liver 36 In the early stage of PBC, biliary secretion of bile salts is within the normal range,70 whereas plasma bile acids are markedly elevated. This means that the hepatic clearance of bile salts is reduced.48The increased concentrations of bile salts in the hepatocyte are probably due to impaired canalicular secretion, and it seems likely that the uptake of bile salts into the hepatocyte becomes impaired sec~ndarily.~~ Because in the early stage of cholestatic disease hepatic synthesis and net hepatocyte transport of bile acids remain relatively unchanged, ileal absorption seems to be responsible for the load of bile acids to the liver with the consequence of bile acid The fact that high bile salt concentrations in the liver may be related to the liver cell damage in cholestasis was first recognized by Greim et al.35,36 In systematic studies in patients with extrahepatic cholestasis, Greim et a1 were able to show that a lesion that they called feathery degeneration correlated with the concentration of toxic dhydroxy bile salts in the liver tissue. Subsequently, it became apparent that not only feathery degeneration, but also cholate stasis (hydropic swelling of hepatocytes around the portal field) are bile salt-related forms of liver cell damage in cholestasis. Important was the recognition that it was not the accumulation of atypical monohydroxy bile salts, which are cholestatic in animals and had been extensively investigated for many years, but the accumulation of normal dihydroxy bile salts that was responsible
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for the cell damage in cholestasis. Cholate stasis and feathery degeneration are probably the consequence of the detergent action of high concentrations of bile salts in the liver and are observed only in severe cholestasis, as in extrahepatic biliary obstruction. As has been shown more recently, much lower concentrations of bile salts are sufficient to induce apoptotic cell death? 76, 77, 94 and it appears likely that this form of bile salt toxicity plays the major role in the less severe forms of cholestasis. EFFECTS OF URSODEOXYCHOLIC ACID Displacement of Natural, Toxic, Endogenous Bile Acids from the Enterohepatic Circulation
UDCA changes the balance between toxic hydrophobic and nontoxic hydrophilic bile salts in the bile salt pool in favor of the nontoxic bile salts.22, 26, 29, 30, 48, 49, 88, 97, 98 This change in balance between toxic and nontoxic bile salts may represent the most important mechanism of action of UDCA (Table l).,,Consequently the UDCA molecules displace more toxic bile acid molecules from cell membranes and cell organelles and thus prevent the damage that can be induced by more toxic bile salts. After administration of UDCA, the bile salt is absorbed passively by diffusion in the small intestine. Absorption is slow and incomplete.95, 96, lo6 After absorption, UDCA is transported via the portal blood to the liver, is conjugated with glycine or taurine, and is secreted into bile. Conjugates of UDCA are absorbed by an active process in the ileum and possibly in addition by facilitated diffusion in the jejunum.47The conjugates of UDCA probably are responsible for the beneficial effect of UDCA in cholestatic diseases. In animal studies, glycine and taurine conjugates of UDCA were equally effective.52Shortly after the start of treatment, the bile acid pool becomes enriched in UDCA, whereas the relative content of toxic bile acids is decreased.26* 29, 70* 88, 98 Approximately half of UDCA administered orally is not and is bacterially degradated in the large bowel to lithocholic acid and 33 7-Keto-lithocholic acid may be absorbed and 7-keto-lithocholic acid.30* transformed in the liver to chenodeoxycholic acid.30,33 Lithocholic acid is poorly water soluble, and only small amounts are absorbed, partially sulfated in the liver and other organs, and excreted in bile and urine. In cholestasis, substantial amounts of UDCA and its metabolites are excreted in urine.6,98 The best way to treat bile acid-induced cell destruction seems to be to prevent the accumulation of toxic bile salts at places where they can destroy cell membranes and cell organelles. This prevention is not easy to achieve for several reasons. First, blockage of bile acid synthesis is not feasible because it leads to the accumulation of bile acid precursors, which cause diseases and cholestasis (i.e., cerebrotendinous xanthomatosis). Second, binding of bile salts in the intestine leads to their decreased
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reabsorption. Consequently, bile acid synthesis is increased, and the accumulation of bile salts is not prevented. Moreover, in cholestasis, the amount of bile acids in the intestine is reduced. Thus, the effect of bile acid binding resins on bile salt accumulation is little. Because bile acid binding resins cannot differentiate between good and bud bile acids, they decrease not only the absorption of toxic bile acids, but also, when given together with UDCA, decrease absorption of UDCA, an effect that is undesired. The only beneficial effect observed after treatment of patients with cholestasis with bile acid binding substances is the improvement of pruritus in some patients. Third, competitive inhibition of bile salt absorption represents another option. After treatment with UDCA, bile acid absorption is decreased by competitive i n h i b i t i ~ nIn .~~ contrast to the situation with bile acid binding resins, bile acid synthesis is essentially unaltered.88Therefore the decreased intestinal absorption of potentially toxic endogenous bile acids may represent a factor contributing to the beneficial effect of UDCA.28,69, 96 Finally, substances blocking the ileal bile acid transporter are being developed. The main goal of the pharmaceutical companies is to develop these substances for the treatment of hypercholesterolemia. It is unclear how such substances would affect the patient with cholestasis. It seems possible that in cholestatic patients, treatment with UDCA, which is absorbed by diffusion in the jejunum, conferring blockage of the active absorption of endogenous bile acid conjugates in the ileum may lead to an enrichment of the bile acid pool with UDCA to an extent that is unachievable at present. After UDCA administration to patients who had gallstones, the biliary enrichment with this bile acid increases only up to a dose of 10 to 12 mg/ kg and then reaches a plateau.97In cholestatic diseases, and especially in cystic fibrosis, higher doses may be necessary to achieve sufficient biliary enrichment of UDCA.= Theoretically the combination of UDCA with a bile acid absorption blocking agent should be superior to the currently used combination of UDCA plus cholestyramine as far as the enrichment of UDCA in bile is concerned, whereas the effect on pruritus is unclear.
Effect of Ursodeoxycholic Acid on Hepatic Excretory Function (Bile Flow and Biliary Secretion of Bile Acids and Lipids)
In patients with cholestatic liver disease treated with UDCA, the hepatic excretory function is improved.21,loo The choleretic effect of UDCA is comparable to that of the naturally occurring dihydroxy and trihydroxy bile salts (chenodeoxycholic and cholic acids). In contrast to the naturally occurring dihydroxy and trihydroxy bile salts, UDCA is atoxic and in animal experiments can prevent the cholestatic effect of cholic and chenodeoxycholic acid conjugates. Moreover, cholic acid is bacterially degradated to deoxycholic acid, which is considered a hydrophobic and toxic bile salt. Therefore UDCA, in contrast to endoge-
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nous primary bile acids, can be considered a clinically useful choleretic agent.48 In healthy humans, the administration of 1 g of bile salts is small in comparison to the 3- to 5-g bile acid pool that circulates six to eight times, yielding a biliary secretion of 18 to 40 g/d of bile salts. In noncholestatic individuals, the administration of unconjugated bile salts induces only a slight increase in bile acid secretion and, consequently, probably little change in bile flow. The situation may be different in patients with cholestatic liver disease in whom the biliary secretion of endogenous bile acids may be considerably reduced and the administration of 1 g of UDCA may lead to a substantial improvement in the biliary secretion of bile salts.'" Taurine conjugates of UDCA induce vesicular exocytosis," which is impaired in cholestasis. In contrast to taurine conjugates of primary bile acids, UDCA conjugates cause a sustained stimulation of horseradish peroxidase excretion into bile in isolated perfused rat liver, indicating an improvement of vesicular exocytosis." The ability of taurine-conjugated UDCA to induce vesicular exocytosis depends on its ability to induce the influx of extracellular Ca2+,which activates protein kinase C.Q l3 Vesicular exocytosis may be essential for targeting and insertion of apical membrane transport proteins into the canalicular membrane and may represent a major pathway for bile acid transport in bile acid-loaded liver cells. The molecular mechanisms underlying the beneficial effect of UDCA, however, are still not completely clear, and controversy exists on the molecular signaling processes involved. Alternatively to the activation via protein kinase C,I3 activation of mitogen-activated protein (MAP) kinases may play a role:s9 In rat hepatocytes, tauro-UDCA (TUDCA) leads to an activation of MAP kinases, which are potential regulators of bile acid secretion into bile. TUDCA leads to rapid, microtubule-dependent increase in canalicular bile acid secretion and stimulation of exocytosis. This activity may reflect a microtubule-dependent apical targeting of vesicles containing bile acid transporters in response to MAP kinase activation. Protein kinase C is not involved in the activation of MAP kinases. Therefore at present, two opposing concepts concerning the molecular mechanisms involved in the effect of UDCA exist-one in which protein kinase C plays a role and one in which it does not play a role-and it is not clear which hypothesis is correct. An alternative way of increasing bile flow represents the direct stimulation of C1- efflux through opening of C1- channels in biliary cells.91Such a direct stimulation of ductular secretion by UDCA may be involved in cystic fibrosis patients. Although it seems likely that the beneficial effect of UDCA is linked to the improvement of bile flow and of the biliary secretion of cholephilic substances, a positive effect of UDCA on liver histology has also been reported in bile duct ligated rat, in which bile flow and biliary secretion do not play a role.32,~oProvided that these observations are correct, the beneficial effects of UDCA on the liver might be independent of its effect on the biliary secretion.
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In patients with cholestatic disease, UDCA treatment leads to a markedly increased biliary secretion of phospholipids and a moderate increase of The increased secretion of phospholipids is of interest because these molecules seem to trap bile acids in the biliary canaliculus to form vesicles and mixed micelles and thus prevent their detrimental effect.5,74, 85, Io5 The increase in the secretion of cholesterol is remarkable because in individuals without cholestasis UDCA reduces biliary cholesterol secretion and dissolves cholesterol gallstones. The increase in biliary cholesterol secretion in severe cholestasis indicates that UDCA improves the hepatic excretory function, although the effect is modest.loO
Effect of Ursodeoxycholic Acid on Bile Acid-Induced Cholestasis in Perfused Rat Liver
Naturally occurring bile salts, as cholic acid and chenodeoxycholic acid, increase bile flow in perfused rat liver when infused in physiologic amounts but induce cholestasis with reduction of bile flow when the infused amount exceeds a certain limit.55Besides cholestasis, hepatocellular damage can also be demonstrated. The hepatocytes close to the portal triad are those that are mainly damaged by bile acid infusions, whereas the hepatocytes around the central vein remain relatively unaltered.90This situation is in good agreement with the early observation that cholestasis mainly affects the periportal hepatocytes. The hepatic injury is associated with an increased excretion of alkaline phosphatase and protein into the bile.55Simultaneous infusion of UDCA prevents the cholestatic effect of toxic bile salts and reduces the morphologic alterations in periportal hepatocytes induced by lipophilic bile In addition, the biliary enzyme and protein excretion induced by toxic bile acids is reduced by coinfusion with UDCA.55These were, the first observations that UDCA conjugates can directly prevent the toxic action of common bile salts.
Ursodeoxycholic Acid in Estrogen-Induced Cholestasis
Estrogens, such as contraceptives or postmenopausal replacement therapy, often cause intrahepatic cholestasis. Moreover, estrogens are implicated in the pathogenesis of recurrent jaundice in the last trimester of pregnancy. Metabolites of natural estrogens (i.e., estradiol-17 p-glucu53 ronide) have been used to study drug-induced intrahepatic cholestasis.2* In rats with bile fistulae, estradiol-17 P-glucuronide infused intravenously induces a marked reduction in bile flow. In ultrastructural studies, the reduction of bile flow was accompanied by fragmentation and
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loss of canalicular microvilli, dilation of canaliculi, and thickening of pericanalicular ectoplasm. Both reduction of bile flow and ultrastructural alterations induced by estrogen metabolites can be prevented by the simultaneous infusion of TUDCA.*,53 TUDCA increased the biliary secretion of estradiol-17 P-glucuronide, and it was suggested that the beneficial effect of UDCA in this model is due to the enhanced biliary secretion of the estrogen metabolite.
Effect of Ursodeoxycholic Acid on Bile Acid-Induced Cell Death in Hepatocytes in Culture At concentrations of greater than 100 pmol/L, hepatocytes are destroyed by the detergent action of bile s a l t ~ .40-43 ~ ~ ,In rat and human hepatocytes in culture, cytolysis is dose and time dependent.34,43 Hepatocytes in culture subjected to detergent bile salts in high concentration (> 100 pM) swell and finally disrupt. The toxicity of 3-, 7-, and 12ahydroxylated bile salts increases with the detergent power of the molecule. In general, the toxicity increases with decreasing number of hydroxyl groups, but the position of the hydroxyl group also plays a role. Of the common bile salts, deoxycholic acid (3a-12a-dihydroxy bile salt) is more toxic than chenodeoxycholic acid (3a7a-dihydroxy bile salt), and both are more toxic than cholic acid (3a-,7a-, 12a-trihydroxy bile Bile salts with a 7P or 601 hydroxyl group (UDCA, hyodeoxycholic acid, muricholic acid) are not toxic and can prevent the toxicity of other bile salts. Thus the cytolysis induced by deoxycholic acid and chenodeoxy43 cholic acid conjugates is markedly reduced by UDCA conjugates.34, It has become evident that not only cytolysis, but also apoptosis, the programmed form of cell death, plays a role in bile acid-related cell toxicity.s,58, 76, 77, 94 Hepatocytes undergoing apoptosis shrink and show nuclear condensation with DNA fragmentation. The finding that cell death by apoptosis is induced by bile acid concentrations far below 94 Because those that induce cytolysis (<50 pM) appears irnp~rtant.~~, bile salt concentrations in the livers of patients with cholestasis rarely reach concentrations greater than 100 pM, it seems likely that apoptosis represents the more important form of cell death in the majority of patients with cholestatic diseases. UDCA conjugates decrease apoptosis induced by toxic bile acids in rat and human hepatocytes in culture.*, This effect does not involve the Fas receptor system (Christine Benz, personal communication, January 1998) and probably is mediated by endonu~leases.~~ The protective effect of UDCA on bile acid-induced apoptosis in hepatocytes may represent one of the more important mechanisms of action of UDCA. The toxic effects of bile salts on cholangiocytes have not been studied in detail, but it appears likely that the effects are similar to those in hepatocytes. UDCA conjugates reduce cytolysis as well as apoptosis
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induced by toxic bile acids. It seems, however, that the effect on apoptosis represents the physiologically more relevant effect.8
Effect of Ursodeoxycholic Acid on Membranes and Mitochondria
Bile salts exert their detergent action not only on hepatocytesIMbut also on red blood leading to cytolysis of the cells. The membranedamaging effect of lipophilic bile acids can also be demonstrated in model membranes.40, In all these models, the toxic effects of lipophilic bile acids are, in part, reversed by UDCA conjugates. Studies with red blood cells and both canalicular and basolateral hepatocyte membranes using electron paramagnetic resonance spectroscopy show that the membrane-damaging effect of toxic bile salts is accompanied by an increased polarity of the apolar domain of the membranes.37This polarity is accompanied by a loss of cholesterol and phospholipids from the membranes. UDCA pretreatment prevents this toxic effect of bile salts by binding to the apolar domain, whereas UDCA conjugates are incorporated into the interface of the membranes and thus protect them from the detergent action of lipophilic bile These findings indicate that UDCA and its taurine conjugate both have protective effects by decreasing the membrane polarity but exert their effects at different sites. UDCA may have an influence on the membrane fluidity, which may be decreased in cholestasis associated with total parenteral nutrition In an animal model using newborn piglets, UDCA infusion reduced cholestasis and prevented the decrease of basolateral membrane fluidity that was observed after total parenteral nutrition.27In this model, there was no effect of UDCA on the fluidity of the canalicular membrane. The effect of UDCA has been attributed to the membrane-Stabilizing effect of UDCA. It seems likely that a membrane-stabilizing effect is also responsible for the beneficial effect of UDCA on the bile acid-induced preservation injury to bile ducts of explanted livers.39 Studies in model membranes composed of egg phosphatidylcholine and cholesterol indicate that such membranes disrupt with increasing bile salt concentration, hydrophobicity, and increasing ionic strength.40 UDCA and its conjugates reduced the disruption of cholesterol-rich model membranes.4oAccording to these data, UDCA can reduce the toxicity of other bile salts via a purely physicochemical mechanism.50 Mitochondria1 dysfunction may contribute to liver failure and 63 Bile acids can disturb the lipid composition of chronic chole~tasis.~~, biologic membranes, leading to decreased function of membrane-bound enzymes. Bile acids are particularly damaging to hepatocyte mitochondria and inhibit the activities of enzyme complexes of the electron 63 UDCA transport chain, including succinate cytochrome C reducta~e.~~, protects oxidative mitochondria1 metabolism from toxicity of lipophilic
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bile acids. The protective effect of UDCA may partially be explained by decreased incorporation of bile acids into mitochondrial membranes.37 Toxic bile salts may induce mitochondrial membrane permeability transition characterized by a rapid permeability of the inner mitochondrial membrane, mitochondrial swelling, and a collapse of the mitochondrial p0tentia1.l~This effect leads to necrosis of the hepatocyte. UDCA inhibits the bile salt-induced mitochondrial membrane permeability transition, and this indicates a protective effect of UDCA on bile acidinduced mitochondrial damage.15 Mitochondria1 membranes appear to represent an important target for the deleterious effects of toxic bile acids, and UDCA and its conjugates, at least in part, may prevent this detergent effect of lipophilic bile acids. Ursodeoxycholic Acid as Antioxidant Hydrophobic bile salts can damage hepatocytes by increasing the 93 Bile acid-induced apoptosis is generation of reactive oxygen species.77, associated with increased lipid peroxidation and can be inhibited by antioxidant^.^, 77, 93 Hydrophobic bile salts can oxidatively activate Kupffer 's cells. These cells, when stimulated, secrete reactive oxygen species, nitric oxide, eicosanosoids, and cytokines. Some of these products are cytotoxic and attack nucleic acids, thiol proteins, or membrane lipids causing lipid peroxidation. UDCA can block the activation of Kupffer 's cells by toxic bile salts and reduce lipid per~xidation.~~ Therefore the beneficial effect of UDCA, at least in part, may be related to its ability to act as an antioxidant. Ursodeoxycholic Acid and lmmunomodulation The human leukocyte antigen (HLA) system codes for genes that are believed to play a role in autoimmune diseases. In the normal liver, class I antigens are expressed by hepatic sinusoidal cells and biliary epithelial cells but not hepatocytes. In contrast, class I1 antigens are expressed by hepatic sinusoidal cells and dendritic cells but not biliary epithelial cells or hepatocytes. In patients with PBC, an increased expression of class I antigens has been observed.', 17, l8 In patients with extrahepatic cholestasis, there was increased class I expression on hepatocytes and bile ducts, whereas class I1 expression was increased only on bile In patients with PBC treated with UDCA, HLA class I expression on hepatocytes was significantly reduced, whereas class I1 expression on biliary epithelial cells was unaltered.18,Io3 It has been suggested that the reduction in HLA class I expression may lead to a decrease in the cytotoxicity of T cells, which are involved in lobular necrosis in PBC. UDCA may interfere either at the levels of antigen presentation via MHC molecules or at the level of T-cell polarization into T-cell subsets. The problem is that it is not known what triggers the inflammatory bile
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duct processes in PBC.‘O Although bile ducts are mainly destroyed by T cells, there is still no convincing evidence that the cellular target is the autoantigen recognized by autoantibodies circulating in the periphery, because after liver transplantation antimitochondrial antibodies remain positive, but the grafted liver is generally not affected. Because the exact immunopathomechanism responsible for the progression of disease in PBC is not completely clear, it is not possible to clarify the immunopathomechanism, if one exists, responsible for the beneficial effect of UDCA in PBC. Bile acids, such as chenodeoxycholic acid, have a profound effect on mononuclear cell cytokine production and inhibit secretion of interleukin-2 and other cytokines.” The immunosuppressive effects of endogenous bile acids could explain, at least in part, the occurrence of infections of various origins during cholestasis. UDCA inhibits cytokine release much less than hydrophobic bile ~a1ts.l~ By displacement of endogenous bile salts, UDCA might reduce the cholestasis-related immunosuppression, and it has been speculated that the beneficial effect of UDCA may, at least in part, be due to the prevention of immunosuppression induced by endogenous bile ~a1ts.l~. 2o This hypothesis, however, is in contrast to reports of a decreased release of interleukin-2, interleukin4, and interferon-y according to which the effect of UDCA is due to its immunosuppressive effect.lo7Depending on the experimental design, UDCA may stimulate56or inhibit the release of cytokine~,’~ and at present the principles of immunomodulation by bile acids are unclear. OTHER PROTECTIVE BILE ACIDS
Studies in perfused rat liver indicate that a-muricholate and tauroP-muricholate are as effective as TUDCA in preventing bile acidinduced liver damage.54Although tauro-a-muricholate does not possess a 7P-hydroxyl group as UDCA and instead has a 6P-hydroxyl group, it was able to reduce the toxic effect of hydrophobic bile acids, indicating that the protective effect is not linked only to a 7P-hydroxyl group. These findings have been confirmed in studies with taurohyodeoxycholic acid (301, 6a, dihydroxy bile acid), in which this 6a-hydroxylated bile acid prevented hepatotoxicity induced by hydrophobic bile acids.86It has been shown that both tauro-a-muricholate and tauro-P-muricholate, similar to UDCA, also protect against the cholestatic effect of estrogen metabolites.lo2At present, bile acid molecules with a 7P-hydroxyl or 6ahydroxyl group are candidates for hepatoprotection. The only bile acid, however, for which a beneficial effect has been shown in extensive clinical studies is UDCA, and it can be expected that similar effects will be obtained with its taurine conjugate TUDCA. References 1. Adams DH, Hubscher SG, Shaw J, et al: Increased expression of intercellular adhesion molecule 1 on bile ducts in primary biliary cirrhosis and primary sclerosing cholangitis. Hepatology 14:426, 1991
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2. Albertani CO, Zimmerman HJ, Ishak KG, et al: Drug-induced cholestasis in the perfused rat liver and its reversal by tauroursodeoxycholate: An ultrastructural study. Proc SOCExp Biol Med 199:54, 1992 3. Balisteri WF: Bile acid therapy in pediatric hepatobiliary disease: The role of ursodeoxycholic acid. J Pediatr Gastroenterol Nutr 24:573, 1997 4. Barnes D, Talenti D, Cammell G, et al: A randomized clinical trial of ursodeoxycholic acid as adjuvant treatment to prevent liver transplant rejection. Hepatology 262353, 1997 5. Bamwell SG, Lowe PJ, Coleman R Effect of taurochenodeoxycholate or tauroursodeoxycholate upon biliary output of phospholipids and plasma-menbrane enzymes, and the extent of cell damage, in isolated perfused rat livers. Biochem J 216:107, 1983 6. Batta AK, Salen G, Arora R, et al: Effect of ursodeoxycholic acid on bile acid metabolism in primary biliary cirrhosis. Hepatology 10:414, 1989 7. Beau P, Labat-Labourdette J, Ingrand P, Beauchant M: Is ursodeoxycholic acid an effective therapy for total parenteral nutrition-related liver disease? J Hepatol 20:240, 1994 8. Benz C, Angermuller S, Tox U, et al: Effect of tauroursodeoxycholic acid on bile acid induced apoptosis and cytolysis in rat hepatocytes. J Hepatol 28:99, 1998 9. Benz C, Sauer P, Kloters-Plachky P, et al: Effect of S-adenosylmethionine versus tauroursodeoxycholic acid on bile acid induced apoptosis and cytolysis in rat hepatocytes. Eur J Clin Invest 28:577, 1998 10. Berg PA, Klein R, Rocken M: Cytokines in primary biliary cirrhosis. Semin Liver Dis 17115, 1997 11. Beuers U, Nathanson MH, Isales CM, et a1 Tauroursodeoxycholic acid stimulates hepatocellular exocytosis and mobilizes extracellular Ca mechanisms defective in cholestasis. J Clin Invest 922984, 1993 12. Beuers U, Spengler U, Kruis W, et al: Ursodeoxycholic acid for treatment of primary sclerosing cholangitis: A placebo controlled trial. Hepatology 16:707, 1992 13. Beuers U, Throckmorton DC, Anderson MS, et al: Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes. Gastroenterology 110:1553, 1996 14. Bittner PO, Posselt HG, Sailer T, et al: The effect of treatment with ursodeoxycholic acid in cystic fibrosis and hepatopathy: Results of a placebo-controlled study. In Paumgartner G, Stiehl A, Gerok W (eds): Bile Acids as Therapeutic Agents. Dordrecht, The Netherlands, Kluwer, 1991, p 345 15. Botla R, Spivey JR, Aguilar H, et al: Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane transition induced by chenodeoxycholate: A mechnism of UDCA protection. J Pharmacol Exp Ther 272930, 1995 16. Buzzelli G, Moscarella S, Focardi G, et al: Long-term treatment with ursodeoxycholic acid in patients with chronic active cirrhosis. Curr Ther Res 50:635, 1991 17. Calmus Y, Arvieux C, Gone P, et al: Cholestasis induces major histocompatibility class I expression in hepatocytes. Gastroenterology 102:1371, 1992 18. Calmus Y, Gane P, Rouger P, et al: Hepatic expression of class I and class I1 major histocompatibility complex molecules in primary biliary cirrhosis: Effect of ursodeoxycholic acid. Hepatology 11:12, 1990 19. Calmus Y, Guechot J, Podevin P, et al: Differential effects of chenodeoxycholic and ursodeoxycholic acids on interleukin 1, interleukin 6 and tumor necrosis factor-alpha production by monocytes. Hepatology 16:719, 1992 20. Calmus Y, Weill B, Ozier Y, et al: Immunosuppressive properties of chenodeoxycholic and ursodeoxycholic acids in the mouse. Gastroenterology 103:617, 1992 21. Colombo C, Castellani MR, Balisteri WF, et al: Scintigraphic documentation of an improvement in hepatobiliary excretory function after treatment with ursodeoxycholic acid in patients with cystic fibrosis and associated liver disease. Hepatology 15:677, 1992 22. Colombo C, Crossignani A, Assaido M, et al: Ursodeoxycholic acid therapy in cystic fibrosis-associated liver disease: A dose-response study. Hepatology 16:924, 1992 23. Colombo C, Setchell KDR, Podda M,et a1 Effects of ursodeoxycholic acid therapy for liver disease associated with cystic fibrosis. J Pediatr 117482, 1990 24. Combes B, Carithers RL, Maddrey WC, et al: A randomized, double-blind, placebo+
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controlled trial of ursodeoxycholic acid in primary biliary cirrhosis. Hepatology 22:759, 1995 Cotting J, Lentze MJ, Reichen J: Effects of ursodeoxycholic acid therapy on nutrition and liver function in patients with cystic fibrosis and long-standing cholestasis. Gut 31:918, 1990 Crosignani A, Battezzati PM, Bertolini M, et al: Changes in bile acid composition in patients with primary biliary cirrhosis induced by ursodeoxycholic acid administration. Hepatology 14:1000, 1991 Duerksen DR, Van Aerde JE, Gramlich L, et al: Intravenous ursodeoxycholic acid reduces cholestasis in parenterally fed newborn piglets. Gastroenterology 111:1111, 1996 Eusufzai S, Ericson S, Cederlund T, et a1 Effect of ursodeoxycholic acid treatment on ileal absorption of bile acids in man as determined by the SEHCAT test. Gut 321044, 1991 Fedorowski T, Salen G, Colallilo A, et al: Metabolism of ursodeoxycholic acid in man. Gastroenterology 731131, 1977 Fedorowski T, Salen G, Tint GS, et al: Transformation of chenodeoxycholic acid and ursodeoxycholic acid by human intestinal bacteria. Gastroenterology 771068, 1979 Floreani A, Chiaramonte M, Fabris P, et al: Poor effect of ursodeoxycholic acid in anti-hepatitis C virus positive chronic liver disease. Curr Ther Res 50:579, 1991 Frezza EE, Gerunda GE, Plebani M, et al: Effect of ursodeoxycholic acid administration on bile duct proliferation and cholestasis in bile duct ligated rat. Dig Dis Sci 38:1291, 1993 Fromm H, Carlson GL, Hofmann AF, et al: Metabolism in man of 7-ketolithocholic acid: Precursor of cheno- and ursodeoxycholic acid. Am J Physiol 239:G161, 1980 Galle PR, Theilmann L, Raedsch R, et al: Ursodeoxycholate reduces hepatotoxicity of bile salts in primary human hepatocytes. Hepatology 12486, 1990 Greim H, Triilzsch D, Czygan P, et al: Mechanisms of cholestasis: 6. Bile acids in human livers with and without biliary obstruction. Gastroenterology 63:846, 1972 Greim H, Triilzsch D, Roboz J, et al: Mechanisms of cholestasis: 5. Bile acids in normal rat livers and those after bile duct ligation. Gastroenterology 63:837, 1972 Giildiituna S, Zimmer G, Imhof M, et al: Molecular aspects of membrane stabilization by ursodeoxycholate. Gastroenterology 104:1736, 1993 Heathcote EJL, Cauch-Dudek K, Walker V, et al: The Canadian multi-center double blind randomized controlled trial of ursodeoxycholic acid in primary biliary cirrhosis. Hepatology 19:1149, 1994 Hertl M, Harvey PR, Swanson PE, et a1 Evidence of preservation injury to bile ducts by bile salts in the pig and its prevention by infusions of hydrophilic bile salts. Hepatology 21:1130, 1995 Heuman DM, Bajaj R Ursodeoxycholate conjugates protect against disruption of cholesterol-rich membranes by bile salts. Gastroenterology 106:1333, 1994 Heuman DM, Bajaj RS, Lin Q: Adsorption mixtures of bile salt taurine conjugates to lecithin-cholesterol membranes: Implications for bile salt toxicity and protection. J Lipid Res 37562, 1996 Heuman DM, Mills AS, McCall JB, et al: Conjugates of ursodeoxycholate protect against cholestasis and hepatocelllular necrosis caused by more hydrophobic bile salts: In vivo studies in the rat. Gastroenterology 100:203, 1991 Heuman DM, Pandak WM, Hylemon PB, et al: Conjugates of ursodeoxycholate protect against cytotoxicity of more hydrophobic bile salts: In vitro studies in rat hepatocytes and human erythrocytes. Hepatology 14:920, 1991 Hillaire S, Boucher E, Calmus Y, et a1 Effects of bile acids and cholestasis on major histocompatibility complex class I in human and rat hepatocytes. Gastroenterology 107781, 1994 Hirano F, Tanaka H, Makino Y, et al: Regulation of the major histocompatibility complex class I mRNA expression by bile salts in cultured human hepatoma cells. Biochim Biophys Res Commun 208:935, 1995 Hirano F, Tanaka H, Makino Y, et al: Effects of ursodeoxycholc acid and chenodeoxy-
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cholic acid on major histocompatibility complex class I gene expression. J Gastroenterol 31:55, 1996 47. Hofmann A F Pharmacolozv of chenodeoxvcholic acid and ursodeoxvcholic acid in man. In Paumgartner G, giehl A, Gerok’W (eds): Bile Acids and ’Cholesterol in Health and Disease. Dordrecht, The Netherlands, Kluwer, 1983, p 301 48. 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, et a1 (eds): Strategies for the Treatment of Hepatobiliary Diseases. Dordrecht, The Netherlands, Kluwer, 1990, p 13 49. Hofmann AF, Popper H Ursodeoxycholic acid treatment for primary biliary cirrhosis. Lancet 2398,1987 50. Igimi H, Carey MC: pH solubility relations of chenodeoxycholic and ursodeoxycholic acids: Physical chemical basis for dissimilar solution and membrane phenomena. J Lipid Res 21:72, 1980 51. Invemizzi P, Setchell KDR, Crossignani A, et al: Comparison between tauroursodeoxycholic and ursodeoxycholic acids in patients with primary biliary cirrhosis: A crossover study [abstr]. J Hepatology 26(suppl 1):166, 1997 52. Kanai S, Kitani K Glycoursodeoxycholate is as effective as tauroursodeoxycholate in preventing the taurocholate-induced cholestasis in the rat. Res Commun Chem Pathol Pharmacol42:423, 1983 53. Kinbara S, Ishizaki K, Sakakura H, et a1 Improvement of estradiol-17beta-D-glucuronide-induced cholestasis by sodium tauroursodeoxycholate therapy in rats. Scand J Gastroenterol 32947, 1997 54. Kitani K, Kanai S, Sat0 Y, et a1 Tauro-alpha-muricholate is as effective as taurobeta-muricholate and tauroursodeoxycholate in preventing taurochenodeoxycholateinduced liver damage in the rat. Hepatology 19:1007, 1994 55. Kitani K, Ohta M, Kanai S: Tauroursodeoxycholate prevents biliary protein excretion induced by other bile salts in the rat. Am J Physiol 248:G407, 1985 56. Klein R, Leuschner M, Leuschner U, et al: Cytokine profiles in primary biliary cirrhosis and primary sclerosing cholangitis. In Berg PA, Leuschner U (eds): Bile Acids and Immunology. Dordrecht, The Netherlands, Kluwer, 1996, p 195 57. Krahenbiihl S, Fischer S, Talos C, et a1 Ursodeoxycholate protects oxidative mitochondrial metabolism from bile acid toxicity: Dose-response study in isolated rat liver mitochondria. Hepatology 20:1595, 1994 58. Kwo P, Bronk SF, Gores GJ: Nuclear serine protease activity contributes to bile acidinduced apoptosis in hepatocytes. Am J Physiol268:G613, 1995 59. Lacaille F, Paradis K The immunosuppressive effect of ursodeoxycholic acid: A comparative in vitro study of human peripheral blood mononuclear cells. Hepatology 18:165. 1993 60. Lepage G, Paradis K, Lacaille F, et al: Ursodeoxycholic acid improves the hepatic metabolism of essential fatty acids and retinol in children with cystic fibrosis. J Pediatr 130:52, 1997 61. Leuschner U, Fischer H, Kurtz W, et al: Ursodeoxycholic acid in primary biliary cirrhosis: Results of a controlled double-blind trial. Gastroenterology 971268, 1989 62. Leuschner U, Leuschner M, Sieratzki J, et al: Gallstone dissolution with ursodeoxycholic acid in patients with chronic active hepatitis and two years’ follow-up. Dig Dis Sci 30642, 1985 63. Lim AG, Ahmed HA, Jazrawi RP, et al: Effects of bile acids on human hepatic mitochondria. Eur J Hepatol Gastroenterol 6:1157, 1994 64. Lindor KDL, Dickson ER, Baldus WP, et al: Ursodeoxycholic acid in the treatment of primary biliary cirrhosis. Gastroenterology 106:1284, 1994 65. Lindor KD, Mayo PSC/UDCA study group: Ursodiol for the treatment of primary sclerosing cholangitis. N Engl J Med 336:691, 1997 66. Ljubuncic P, Fuhrman B, Oiknine J, et al: Effect of deoxycholic acid and ursodeoxycholic acid on lipid peroxidation in cultured macrophages. Gut 3 9 ~ 7 5 1996 , 67. Loria P, Bozzoli M, Concari M, et al: Effect of taurohyodeoxcholic acid on biliary lipid secretion in humans. Hepatology 25:1306, 1997 v
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68. Lotterer E, Stiehl A, Raedsch R, et al: Ursodeoxycholic acid in primary biliary cirrhosis: No evidence of toxicity in the stages I to 111. J Hepatol 10284, 1990 69. Marteau P, Chazouilleres 0, Myara A, et al: Effect of chronic administration of ursodeoxycholic acid on the ileal absorption of endogenous bile acids in man. Hepatology 12I1206, 1990 70. Mazzella G, Parini P, Bazzoli F, et al: Ursodeoxycholic acid administration on bile acid metabolism in patients with early stages of primary biliary cirrhosis. Dig Dis Sci 382396, 1993 71. Mitchell SA, Bansi D, Hunt N, et al: High dose ursodeoxycholic acid (UDCA) in primary sclerosing cholangitis (PSC): Results after two years of a randomized doubleblind, placebo-controlled trial [abstr]. Gut 4O(suppl 1):TH115, 1997 72. Nakagawa M, Colombo C, Setchell KDR: Comprehensive study of the biliary bile acid composition of patients with cystic fibrosis and associated liver disease before and after UDCA administration. Hepatology 12:322, 1990 73. Nittono H, Tokita A, Hayashi M, et al: Ursodeoxycholic acid therapy in the treatment of biliary atresia. Biomed Pharamacother 43:37, 1989 74. Oude Elferink RPJ, Ottenhoff R, van Wijland M, et al: Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse. J Clin Invest 95:31, 1995 75. Palma J, Reyes H, Ribalta J, et al: Effects of ursodeoxycholic acid in patients with intrahepatic cholestasis of pregnancy. Hepatology 15:1043, 1992 76. Patel T, Bronk SF, Gores GJ: Increases of intracellular magnesium promote glycochenodexycholate-induced apoptosis in rat hepatocytes. J Clin Invest 942183, 1994 77. Patel T, Gores GJ: Inhibition of bile salt-induced hepatocyte apoptosis by the antioxidant Lazaroid U83836E. Toxic01 Appl Pharmacol 142:116, 1997 78. Plevris JN, Hayes PC, Bouchier IAD Ursodeoxycholic acid in the treatment of alcoholic liver disease. Eur J Gastroenterol Hepatol 3:653, 1991 79. Podda M, Ghezzi C, Battezzati PM, et al: Effects of ursodeoxycholic acid and taurine on serum liver enzymes and bile acids in chronic hepatitis. Gastroenterology 98:1044, 1990 80. Po0 JL, Feldmann G, Erlinger S, et al: Ursodeoxycholic acid limits liver histologic alterations and portal hypertension induced by bile duct ligation in the rat. Gastroenterology 102:1752, 1992 81. Poupon RE, Balkau B, Eschwege E, et a1 A multicenter controlled trial of ursodiol for the treatment of primary biliary cirrhosis. N Engl J Med 3241548, 1991 82. Poupon R, Chretien Y, Poupon R, et al: Serum bile acids in primary biliary cirrhosis: Effect of ursodeoxycholic acid therapy. Hepatology 17599, 1993 83. Poupon R, Poupon RE, Calmus Y, et al: Is ursodeoxycholic acid an effective treatment for primary biliary cirrhosis. Lancet 1:834, 1987 84. Poupon RE, Lindor KD, Cauch-Dudek K, et al: Combined analysis of tandomized controlled trials of ursodeoxycholic acid in primary biliary cirrhosis. Gastroenterology 113:884, 1997 85. Pualielli L, Amiao L, Arrese M, et al: Protective role of biliarv cholesterol and phgspholipid lamellae against bile acid-induced cell damage. 'Gastroenterology 107244. ~. , 1994 86. Roda A, Piazza F, Baraldini M, et a1 Taurohyodeoxycholic acid protects against hepatotoxicity induced by taurochenodeoxycholic acid in rat. Hepatology 27520,1998 87. Rolandi E, Franceschini R, Cataldi A, et al: Effects of ursodeoxycholic acid on serum liver damage indices in patients with chronic active hepatitis. Eur J Clin Pharmacol 40:473, 1991 88. Rudolph G, Endele R, Senn M, et al: Effect of ursodeoxycholic acid on the kinetics of cholic acid and chenodeoxycholic acid in patients with primary sclerosing cholangitis. J Hepatol 171028, 1993 89. Schliess F, Kurz AK, vom Dahl S, et a1 Mitogen-activated protein kinases mediate the stimulation of bile acid secretion by tauroursodeoxycholate in rat liver. Gastroenterology 1133306, 1997 90. Schmucker DL, Ohta M, Kanai S, et al: Hepatic injury induced by bile salts: Correlation between biochemical and morphological events. Hepatology 12:1216, 1990 91. Shimokura GH, McGill J, Schlenker T, et al: Ursodeoxycholate increases cytosolic v
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