Bile acids, the water-soluble, amphipathic end products of cholesterol metabolism, are

Transcription

1 The Continuing Importance of Bile Acids in Liver and Intestinal Disease Alan F. Hofmann, MD REVIEW ARTICLE Bile acids, the water-soluble, amphipathic end products of cholesterol metabolism, are involved in liver, biliary, and intestinal disease. Formed in the liver, bile acids are absorbed actively from the small intestine, with each molecule undergoing multiple enterohepatic circulations before being excreted. After their synthesis from cholesterol, bile acids are conjugated with glycine or taurine, a process that makes them impermeable to cell membranes and permits high concentrations to persist in bile and intestinal content. The relation between the chemical structure and the multiple physiological functions of bile acids is reviewed. Bile acids induce biliary lipid secretion and solubilize cholesterol in bile, promoting its elimination. In the small intestine, bile acids solubilize dietary lipids promoting their absorption. Bile acids are cytotoxic when present in abnormally high concentrations. This may occur intracellularly, as occurs in the hepatocyte in cholestasis, or extracellulary, as occurs in the colon in patients with bile acid malabsorption. Disturbances in bile acid metabolism can be caused by (1) defective biosynthesis from cholesterol or defective conjugation, (2) defective membrane transport in the hepatocyte or ileal enterocyte, (3) defective transport between organs or biliary diversion, and (4) increased bacterial degradation during enterohepatic cycling. Bile acid therapy involves bile acid replacement in deficiency states or bile acid displacement by ursodeoxycholic acid, a noncytotoxic bile acid. In cholestatic liver disease, administration of ursodeoxycholic acid decreases hepatocyte injury by retained bile acids, improving liver tests, and slowing disease progression. Bile acid malabsorption may lead to high concentrations of bile acids in the colon and impaired colonic mucosal function; bile acid sequestrants provide symptomatic benefit for diarrhea. A knowledge of bile acid physiology and the perturbations of bile acid metabolism in liver and digestive disease should be useful for the internist. Arch Intern Med. 1999;159: Bile acids are among the first natural products isolated in pure form. They are readily isolated from gallbladder bile, where they are present in high concentrations. Understanding the functions of these amphipathic molecules has occupied physiologists and physicians for the past century. Currently, the physiological functions of bile acids in the liver and intestine have been at least partly clarified. The key role that the enterohepatic circulation of bile acids plays in cholesterol metabolism has been appreciated, which is leading to new treatment approaches for hypercholesterolemia. At the same time, it has become From the Division of Gastroenterology, Department of Medicine, University of California, San Diego, La Jolla. apparent that bile acids are also important in hepatic, biliary, and intestinal diseases. The 3 bile acids present in human bile are highly toxic to cells when present in abnormally high concentrations. Ursodeoxycholic acid, a natural bile acid that occurs in bears, has been shown to be a safe and rather effective medication to treat cholestatic liver disease, and it has recently been approved for marketing in the United States. This article summarizes the most recent information on the functions of bile acids in the liver and small intestine, including their role in liver, biliary, and intestinal disease and the use of ursodiol in the treatment of cholestatic liver disease. More detailed reviews of these topics are available elsewhere. 1-3 It is beyond the scope of this ar- 2647

2 ticle to review the emerging area of bile acid transport inhibitors as a new therapeutic approach for hypercholesterolemia. 4 OVERVIEW OF BILE ACID FORMATION AND FUNCTION Bile Acid Formation Bile acids are formed in the liver from cholesterol in the pericentral hepatocytes. Bile acid synthesis is a complex, multienzyme process in which a cholesterol molecule an insoluble, uncharged, membrane constituent is converted to a bile acid molecule that, when ionized, is an amphipathic, membranedissolving, water-soluble detergent. After biosynthesis from cholesterol and before excretion from the hepatocyte, bile acid molecules are conjugated with glycine or taurine, which converts a weak acid to a strong acid. As a result, conjugated bile acids are fully ionized at the range of ph values present in the small intestine. Therefore, when considering conjugated bile acids, one is referring to the negatively charged bile acid molecule, ie, the bile acid anion. Physiological Functions of Bile Acids Five major physiological functions of bile acids are now well established (Table 1). First and most important is the elimination of cholesterol. Bile acids eliminate cholesterol from the body by converting it to bile acid and by micellar solubilization of cholesterol in bile, enabling cholesterol to move from the hepatocyte to the intestinal lumen, ultimately leading to elimination via the fecal route. Inborn defects in the conversion of cholesterol to bile acids can cause severe hepatic or systemic disease. Second is lipid transport in the form of mixed micelles. In the small intestine, bile acids promote dietary lipid absorption by solubilizing dietary lipids and their digestion products as mixed micelles. Such mixed micelle formation accelerates diffusion through the unstirred layer, greatly accelerating lipid absorption. Unless bile acids are present in micellar form, fat-soluble vitamins (A, D, E, and K 1 ) will not be absorbed, and a deficiency will occur. In the biliary canaliculus, bile acids solubilize biliary phospholipids and cholesterol in mixed micelles. Such micelle formation promotes cholesterol elimination. The third and fourth functions of bile acids are stimulation of bile flow and stimulation of biliary phospholipid secretion. Bile acids are actively transported into the biliary canaliculi between hepatocytes and induce bile flow by their osmotic properties. Bile acids promote the transfer of phospholipids from the canalicular membrane into bile. The presence of phospholipids in bile results in a greater fraction of bile acids existing in the form of mixed micelles and a lower monomer concentration of bile acids, thereby preventing bile acids from damaging the bile duct epithelium. Fifth is negative feedback regulation of bile acid and cholesterol biosynthesis. The concentration of bile acids in the hepatocyte seems to act as a signal: when high, bile acid synthesis is low; when low, bile acid synthesis increases up to 15-fold. Because bile acids are synthesized from cholesterol, cholesterol synthesis undergoes a parallel increase. 5 There may well be additional functions of bile acids in the intestine. Bile acids solubilize polyvalent metals such as iron and calcium in the duodenum, promoting their absorption. Bile acids also seem to stimulate the release of motilin, which coordinates the interdigestive migrating motility complex. Bile acids have bacteriostatic effects and stimulate mucin secretion; these actions are likely to affect intestinal flora of the small intestine. Bile acids, because of their surface activity, should inhibit bacterial adhesion, and they might bind enterotoxin in the intestinal lumen. Bile acids also affect the absorption of water and electrolytes by the colonic Table 1. Functions of Bile Acids* Process Site Mechanism Whole Organism Cholesterol elimination Hepatocyte Conversion to bile acids Stimulation of biliary cholesterol secretion Biliary tract Solubilization of cholesterol in mixed micelles Intestine Fecal excretion of cholesterol and bile acids Hepatobiliary Stimulation of bile flow Hepatocyte Canalicular secretion of osmotically active membrane-impermeable molecules Cholangiocyte Potentiation of secretin-stimulated bicarbonate secretion Stimulation of biliary PC secretion Hepatocyte Detachment of PC from canalicular membrane Biliary tract Solubilization of PC in mixed micelles Feedback inhibition of bile acid and cholesterol biosynthesis Hepatocyte Repression of cholesterol 7 -hydroxylase and HMG-CoA reductase Intestinal Enhancement of lipid absorption Small intestine Formation of mixed micelles that promote diffusion through the unstirred layer Cleaning of absorptive surface Small intestine Surface activity of the bile acid anions Stimulation of intestinal motility Large intestine Neural arc *PC indicates phoshatidylcholine; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A. Postulated, not an established function. 2648

3 mucosa, and affect colonic motility. There is much to be learned. Enterohepatic Circulation of Bile Acids Among the digestive secretions, which include saliva, gastric juice, and pancreatic juice, bile is unique in that bile acids, its major constituent, are absorbed from the distal intestine, returned to the liver, and secreted into bile. This continuous cycle of secretion, absorption, and resecretion is termed the enterohepatic circulation. 3,6 Absorption of bile acids from the distal intestine leads to the accumulation of a large mass of bile acid molecules, termed the bile acid pool, which is about 2 to 3 g in size in adults. Because the pool cycles several times with each meal, bile acid secretion is 4 to 6gper meal, or 12 to 18 g/d. Bile acid synthesis, which averages about 0.3 g/d, is amplified by the enterohepatic cycling of bile acids. In other words, we digest our meals using recycled bile acids. BILE ACID COMPOSITION OF HUMAN BILE Primary Bile Acid Formation When bile acid chemistry is discussed, it is usual to distinguish the chemical nature of the side chain from that of the steroid nucleus. For the side chain, the key attribute is whether bile acids are conjugated (with glycine or taurine) or unconjugated. For the nucleus, the key attribute is the number, position, and configuration of the hydroxy groups. In bile, bile acids are present almost completely in conjugated form because the conjugation of bile acids in the hepatocyte is a highly efficient process. In humans, most bile acids are conjugated with glycine, the minority with taurine. The situation with respect to the steroid nucleus is more complex. Two bile acids are made in the hepatocyte from cholesterol: chenodeoxycholic acid (CDCA) and cholic acid. Chenodeoxycholic acid, a dihydroxy bile acid with -hydroxy groups at the third and seventh carbon atoms (C-3 and C-7), can be considered the building block of other bile acids because every bile acid must have a hydroxy group at C-3 and C-7 of the steroid nucleus; a hydroxy group at C-3 is present in cholesterol, and one is always present at C-7 because hydroxylation at this site is in the biosynthetic pathway of all bile acids. In most vertebrates, an additional hydroxy group is added to CDCA to form a trihydroxy bile acid. In humans, this hydroxy group is added at C-12 to form cholic acid (which then contains hydroxy groups at C-3, C-7, and C-12). Chenodeoxy cholic acid and cholic acid are termed primary bile acids because they are formed in the hepatocyte. Secondary Bile Acid Formation During the enterohepatic cycling of bile acids, their structure is altered by bacterial enzymes in the distal intestine. Bacterial biotransformations of bile acids occur on the side chain and the nucleus. On the side chain, bile acids undergo deconjugation to form an unconjugated bile acid and glycine or taurine. Some of these unconjugated bile acids are absorbed, returned to the liver, and reconjugated during transit through the hepatocyte: a hidden process of deconjugation and reconjugation that could be thought of as damage and repair. 7 The hydroxy group at C-7 is also attacked by anaerobic bacteria in the colon. Bacterial dehydratases remove the hydroxy group to form 7-deoxy bile acids (the term deoxy means that an oxygen-containing group has been lost). By this process, cholic acid (with hydroxy groups at C-3, C-7, and C-12) is converted to deoxycholic acid (DCA; a dihydroxy bile acid with hydroxy groups at C-3 and C-12). Similarly, 7-dehydroxylation of CDCA results in the formation of a monohydroxy bile acid with a hydroxy group at only C-3. This bile acid is called lithocholic acid because it was first isolated from a gallstone taken from a calf. Deoxycholic and lithocholic acids are called secondary bile acids because they are formed from primary bile acids. Both are absorbed to some extent from the colon and returned to the hepatocyte. Fate of Secondary Bile Acids in the Hepatocyte In the hepatocyte, the secondary bile acids undergo differing fates. Deoxycholic acid is conjugated with glycine or taurine and circulates with the primary bile acids. In most adults, DCA constitutes about 20% of the biliary bile acids, the others being cholic acid and CDCA in roughly equal proportions. Lithocholic acid is conjugated with glycine or taurine and sulfated at the C-3 position. These double conjugates (sulfolithocholylglycine and sulfolithocholyltaurine) are excreted into bile but, in contrast to the conjugates of primary bile acids and of DCA, are not efficiently absorbed from the small intestine. As a result, they are promptly eliminated from the body and never constitute more than 5% of biliary bile acids. Their prompt elimination from the body is fortunate because lithocholic acid is a highly hepatotoxic bile acid in experimental animals. Steady State Composition of Bile Biliary bile acids thus consist mostly ( 90%) of the conjugates of cholic acid and CDCA (primary bile acids) and of DCA (a secondary bile acid). Only trace amounts of the conjugates of lithocholic acid are present. Another bile acid, ursodeoxycholic acid (UDCA, identical to ursodiol), is also present in trace amounts, and is identical in structure to CDCA, except that the hydroxy group at C-7 is in a rather than an configuration. It is termed ursodeoxycholic acid because it was first isolated from the bile of the polar bear. Ursodeoxycholic acid, as mentioned at the beginning of this article, is now used as a therapeutic agent in cholestatic liver disease. Chemical structures of the major bile acids present in human bile are shown in Figure 1. The young infant has only primary bile acids in the bile. The anaerobic flora of the colon develop sometime during the first year, signaled by the presence of DCA in bile. The proportion of DCA increases with age, and it is the dominant biliary bile acid in some adults. Bile ac- 2649

4 Primary (From Cholesterol) Secondary (From Primary Bile Acids) HO HO HO HO OH OH OH OH COOH Cholic COOH Chenodeoxycholic COOH Deoxycholic COOH Lithocholic NH 3 + CH 2 COO + Glycine NH 3 + (CH 2 ) 2 SO Taurine O II R-C-N-CH 2 COO I H 0 II R-C-N-(CH2) 2 SO 2 0 I H Cholylglycine Chenodeoxycholylglycine Deoxycholylglycine Lithocholylglycine Ursodeoxycholylglycine Sulfolithocholylglycine Sulfolithocholyltaurine +SO 4 Cholyltaurine Chenodeoxycholyltaurine Deoxycholyltaurine Lithocholyltaurine Ursodeoxycholyltaurine ids are identical in men and women. A schematic depiction of the enterohepatic circulation of bile acids in humans is shown in Figure 2. Bacterial enzymes undo the work of hepatic enzymes. Bile acid formation involves hydroxylation and conjugation; bacterial modification in the colon involves deconjugation and dehydroxylation. Hydroxylation and conjugation render bile acids soluble; conversely, deconjugation and dehydroxylation in the distal intestine make bile acids virtually insoluble and thereby lower the aqueous concentration and bacteriostatic effect of bile acids. This process should be useful in herbivorous animals, which depend on bacterial production of short-chain fatty acids in the colon as a key energy source. 7 STRUCTURE-ACTIVITY RELATIONS OF BILE ACIDS HO OH COOH Ursodeoxycholic Figure 1. Chemical structures of major bile acids in humans. Primary bile acids formed in the liver; deoxycholic, lithocholic, and secondary bile acids formed in the large intestine by bacterial 7-dehydroxylation of their primary bile acid precursor; ursodeoxycholic acid formed by bacterial epimerization of the hydroxy group at the seventh carbon atom in the distal small intestine or large intestine. Bile acids conjugate with glycine or taurine in the hepatocyte. Before 7-dehydroxylation, bile acids are deconjugated by bacterial enzymes. Sulfation of lithocholic acid conjugates prevents ileal conservation and results in rapid excretion from the body. Figure 1 has been published previously. 1,2 Input of New (Unconjugated) 7-Deoxy BAs (Damaged) Unconjugated BAs (Damaged) Colon Input of New BAs Biosynthesized From Cholesterol (Pericentral Hepatocytes) Ileum Conjugated BAs (Undamaged) Jejunum Transport and Repair of Old BAs (Periportal Hepatocytes) Biliary Ductules Duodenum Cholehepatic Cycling (Unconjugated Lipophilic BAs) Conjugated BAs (Undamaged and Repaired) Figure 2. Metabolism and enterohepatic cycling of bile acids (BAs) in humans. Light gray area indicates the liver, where input of newly biosynthesized BAs, and transport and repair of previously secreted BAs occurs. A small fraction of conjugated BAs can be absorbed from the biliary ductules (cholehepatic shunting). If dihydroxy BAs are secreted into bile in unconjugated form, they will also be absorbed passively by cholangiocytes. Deconjugation in the distal small intestine results in input of unconjugated BAs. Dehydroxylation of primary BAs in the colon forms 7-deoxy BAs, which are partly absorbed. In humans, 7-deoxy BAs do not undergo 7-rehydroxylation in the liver. Data from Hofmann and Hagey. 7 Side Chain Structure and Function Because conjugated bile acids are fully ionized at a physiological ph level, they are membrane impermeable. In addition, the bile acid molecule is also too large to pass through the junctional complexes between cells. The impermeability of the conjugated bile acid anion to the epithelium of the biliary tract and small intestine permits high intraluminal concentrations to be maintained. Conjugation with glycine or taurine also affects the physicochemical properties of bile acids. Conjugated bile acids are more soluble at acidic ph levels than are their corresponding unconjugated derivatives. They are also more resistant to precipitation in the presence of high concentrations of calcium ions. The resistance to acidity prevents bile acids from precipitating from solution as the insoluble protonated acid in the duodenum, which is occasionally quite acidic. The resistance to high calcium concentrations prevents bile salts from precipitating from solution as the calcium salt during concentration in the gallbladder. 8 Structure and Function of the Steroid Nucleus The addition of hydroxy groups to the cholesterol molecule during bile 2650

5 acid biosynthesis is restricted to 1 face of the bile acid molecule. The bile acid molecule has a hydrophobic face (without hydroxy substituents) and a hydrophilic face (with hydroxy substituents). Bile acids are planar, surface-active amphipathic molecules. Above a certain concentration the critical micellization concentration bile acids self-associate to form small polymolecular aggregates called micelles. Micelles composed solely of bile acid molecules do not occur in the body. Rather, when present in sufficient concentration, conjugated bile acid molecules solubilize other lipids to form mixed micelles. Not all types of lipids are solubilized by bile acids. The only lipids that readily form mixed micelles with bile acid anions are lipids that, by themselves, form bilayers in water: molecules such as phosphatidylcholine (PC), fatty acid fatty acid anion (soap) mixtures, and monoglycerides, for which bile acids are the most potent solubilizing agents known; conventional detergents are much weaker. The arrangement of molecules in the bile acid PC mixed micelle has been the subject of intense investigation. Recent work using the complex physical technique of small-angle neutron scattering indicates that the micelle is cylindrically shaped. At high lipid and bile acid proportions, the micelle may become worm shaped, like a piece of spaghetti with the PC molecules arranged radially. The broad bile acid molecules are forced down between the heads of the PC molecules. 9 The micelle is not a static entity but rather, a flickering cluster. There is a continuous exchange of molecules between mixed micelles, mostly by micellemicelle collision. There is also a small exchange through the aqueous phase surrounding the mixed micelles. The mixed micelle that is present in small intestinal content contains fatty acids and monoglycerides that have been solubilized by bile acids. These are formed by the action of pancreatic lipase on dietary triglyceride. The molecular arrangement of the bile acid fatty acid monoglyceride mixed micelle Vesicles of PC and Cholesterol Bilayers of Fatty Acid and Monoglyceride is identical to that of the bile acid PC mixed micelle. However, because fatty acids have a greater aqueous solubility than does PC, the concentration of fatty acids in monomeric form in the aqueous phase between micelles is higher than that of PC between bile acid PC mixed micelles. The conversion of PCcholesterol vesicles and bilayers of fatty acid and monoglyceride to mixed micelles is illustrated in Figure 3. DETERMINANTS OF THE ENTEROHEPATIC CIRCULATION OF BILE ACIDS Events in the Liver + Bile Acid The movement of bile acid molecules in enterohepatic circulation can be considered to begin with the canaliculus. Here, adenosine triphosphate stimulated transporters (the major one is termed the canalicular bile salt export pump) pump bile acids into the canaliculus. 11 The canaliculus is surrounded by a spiral ribbon of actin filaments. The osmotic activity of the bile acids pulls water and filterable solutes into the canaliculus, which is then squeezed Mixed Micelles (and Monomers) Figure 3. Conversion in the biliary canaliculus of bilayer vesicles containing phosphatidylcholine (PC) and cholesterol (upper left) to mixed micelles (right). Conversion in the intestinal lumen during fat digestion of bilayer lamellae-containing fatty acid and 2-monoglyceride (lower left) to mixed micelles (right). Mixed micelle is believed to be cylindrically shaped, with solubilized lipids arranged radially; bile acid molecule rests between polar heads of the lipids, with its hydrophobic side inward and its hydrophilic surface facing the aqueous phase. Fatty acid monomers are shown; the concentrations of PC and cholesterol monomers are extremely low. Published in part previously. 10 by the actin spiral. 12 At the same time, bile acids adsorb to PC molecules on the luminal face of the canalicular membrane and detach the PC molecules. Initially vesicles are formed, but in the presence of a supramicellar concentration of bile acids, the vesicles are transformed into mixed micelles. 13 Processes occurring at the canalicular membrane are shown schematically in Figure 4. The canaliculus is a network of intercellular spaces with a blind end at the pericentral zone, where the canalicular channels empty via the canals of Hering into the finest radicals of the biliary tract. The osmotic forces generated by bile acid secretion, together with the contraction of the pericanalicular filaments, generate a pressure of about 30 cm of water. Bile flows down the biliary tract. If pressure in the common bile duct is greater than in the cystic duct, bile enters the gallbladder. During overnight fasting, the sphincter of Oddi contracts and relaxes. Results of the limited studies that are available suggest that about half of the bile secreted by the liver is stored in the gallbladder. Because some bile acids are absorbed 2651

6 ATP ATP bsep mdr2 Bile Salt (Micelles) Mixed Micelle mal small intestine that transports dihydroxy conjugates preferentially. The importance is this carrier in humans is not known. Not all bile acid absorption is carrier mediated; passive absorption from the distal intestine involves only unconjugated bile acids that are formed by bacterial deconjugation of conjugated bile acids. Unconjugated dihydroxy bile acids, being less hydrophilic than cholic acid, are absorbed much more rapidly. Absorption occurs by passive flip-flop of the nonionized bile acid molecule across the lipid bilayer. Return of Bile Acids to the Liver PC-TP Cytosol ATP Cholesterol mdr2 mdr2 Canaliculus Bile Salt Figure 4. Events at the canalicular membrane. Bile acids are transported into the canalicular lumen by the adenosine triphosphate (ATP) stimulated bile salt export pump (bsep). Phosphatidylcholine (PC) molecules (shown with 2 parallel tails) are transported to the canalicular membrane by the PC transport protein (PC-TP) and across the canalicular membrane by the PC flippase (mdr2). When the PC molecules achieve a sufficient enrichment in the luminal face of the canalicular membrane, they bud out, forming bilayer vesicles that adsorb bile acid molecules. When the proportion of bile acid molecules is sufficiently high, a mixed micelle is formed. Modified from Elferink et al. 14 from the proximal small intestine, bile acids gradually accumulate in the gallbladder. The volume of water removed by the concentration of gallbladder bile is replaced by entering bile. Thus, during overnight fasting, the gallbladder can remain constant in size yet store a progressively increasing fraction of the circulating bile acid pool. When a meal is eaten, cholecystokinin is released from the endocrine cells of the intestinal mucosa. The hormone acts on vagal afferents or the nerve ganglia innervating the gallbladder and induces gallbladder contraction. At the same time, it acts on the nerves innervating the sphincter of Oddi, causing it to relax. The end result is that gallbladder bile is delivered to the duodenum. Events in the Small Intestine Conjugated bile acids move along the small intestine via intestinal propulsive activity. Their absorption is predominantly carrier mediated, the most important of which is in enterocytes present in the terminal ileum. This consists of an apical sodium-dependent cotransporter called the apical bile salt transporter and a basolateral transporter that is an anion exchanger. 15 Uptake is likely to be the ratedetermining step. Based on results of animal studies, there is likely to be a second transporter in the proxi- Bile acids return to the liver in portal blood. About three fourths of the trihydroxy bile acids are protein bound, mostly to albumin. Virtually all ( 99%) dihydroxy bile acids are bound to albumin. Hepatic uptake of conjugated bile acids is remarkably efficient, with the firstpass extraction fraction ranging from 50% to 90%, depending on the bile acid structure. 2 Despite the concentration of unbound dihydroxy conjugated bile acids being only 1 25 that of cholyl conjugates, uptake of dihydroxy bile acids is nearly as efficient as that of trihydroxy bile acids, indicating that the bile acid carriers present in the sinusoidal membrane of the hepatocyte transport dihydroxy conjugate preferentially. A sodium-dependent cotransporter and a sodium-independent transporter are present in the sinusoidal membrane, but their relative importance in conjugated bile acid uptake is not yet clarified. The concentration of bile acids at any place in enterohepatic circulation depends on the relation of the rates of input of bile acids and surrounding aqueous fluid. Concentrations in the canaliculus and biliary ductules are high: 20 to 50 mmol/l. With gallbladder concentration, they increase to as high as 300 mmol/l in some species. Bile delivery is slow, and dilution by gastrointestinal tract secretions lowers bile acid concentration to 10 mmol/l (in humans). All of these concentrations are sufficiently high that bile acids are present in micellar form. In the cecum, the concentration of bile acids in so- 2652

7 lution falls markedly, to less than 1 mmol/l, because of deconjugation and dehydroxylation that permits passive absorption, as well as precipitation from solution and adsorption to bacteria. The concentration of bile acids in plasma is always low. Portal venous plasma has a concentration of 20 to 50 µmol/l in humans. Because of efficient first-pass extraction, the concentration of bile acids in systemic venous plasma is less than 5 µmol/l during the fasting state. Because the first-pass extraction of bile acids is constant, when more bile acids are absorbed during digestion, the concentration of bile acids in systemic plasma rises severalfold. Cholesterol and conjugated bile acid molecules that enter the small intestine during the secretion of bile have quite different fates. Only one fourth to one half of cholesterol is absorbed, whereas bile acids are efficiently absorbed. For a meal in which the bile acid pool might circulate twice, nine tenths of the bile acids are absorbed. Bile Acid Excretion Bile acids that are not absorbed from the colon are eliminated in the stool. Fecal elimination is balanced by biosynthesis from cholesterol. Because the concentration of bile acids in systemic venous plasma is low, and because bile acids are bound to albumin, the amount of bile acids entering the glomerular filtrate is small. The ileal bile acid transport system is also located in the proximal renal tubule, where it efficiently reabsorbs most bile acids present in tubular fluid. As a consequence, in the healthy person, virtually no bile acids are eliminated in urine. Thus, in humans, more than 99% of cholesterol is eliminated by the fecal route. In the adult, about two thirds of cholesterol is eliminated as cholesterol and about one third as bile acids. CYTOTOXICITY OF BILE ACIDS Structural Determinants of Cytotoxicity Bile acids are cytotoxic when their concentrations increase to abnormally high levels, either intracellularly or extracellularly. Bile acid cytotoxicity is strongly affected by its structure: the greater the hydrophobicity, the greater the cytotoxicity. Hydrophobicity is defined operationally by the extent to which bile acids bind to hydrophobic surfaces, and this can be determined by measuring the retention time during liquid chromatography using a hydrophobic adsorbent. 16 The natural dihydroxy bile acids CDCA and DCA bind tightly to the adsorbent, have a long retention time, are hydrophobic by this definition, and are highly cytotoxic. Ursodeoxycholic acid, although a dihydroxy bile acid, does not bind to the adsorbent, has a short retention time, is hydrophilic, and devoid of cytotoxic properties in most model systems. Cholic acid is intermediate, being noncytotoxic at low concentrations, but cytotoxic at very high concentrations. 17,18 Intracellular Toxicity Intracellular toxicity caused by conjugated bile acids occurs in the intact cell only when a transporter is present in the cell membrane that permits conjugated bile acids to enter the cell. To date, intracellular toxicity attributable to conjugated bile acids has been clearly established only for the hepatocyte. In the hepatocyte of the healthy person, uptake is followed by rapid elimination, and cytosolic proteins that bind bile acids are likely to be present. As a result, the concentration of bile acids in cytosolic water is low probably less than 1 µmol/l. When elimination is impaired, bile acids accumulate intracellularly. When their concentration exceeds the binding capacity of the cytosolic proteins, bile acids enter other organelles, possibly interfering with their activity, and damage the canalicular membrane. In the hepatocyte, the accumulation of bile acids leads to mitochondrial damage and ultimately to apoptosis or necrosis. 19,20 Details of the pathways involved in bile acid induced apoptosis or necrosis are being actively investigated. Unconjugated bile acids, being membrane permeable, are highly cytotoxic to isolated cells in vitro because unconjugated bile acids can readily accumulate to pathological levels. However, cytotoxicity attributable to unconjugated bile acids in vivo has not been clearly shown. Extracellular Toxicity For cells that lack a bile acid transporter, conjugated bile acids are not usually cytotoxic until their concentration is sufficiently high to attack the membrane of the cell. This concentration is close to the critical micellization concentration of the bile acid. Because CDCA and DCA have a lower critical micellization concentration than does cholic acid, they are more cytotoxic for a given concentration. In the presence of other lipids, such as PC or fatty acids, the monomeric concentration of bile acids depends on their association with these lipids to form mixed micelles. Such mixed micelle formation occurs at a concentration well below the cytotoxic concentration, explaining the lack of cytotoxicity of bile acids in the biliary tract and small intestine in healthy people. In patients and knockout mice lacking the canalicular PC transporter, PC is absent from bile, the monomeric concentration of bile acids is increased, and damage to the biliary epithelial cells occurs. 14 DISTURBANCES OF ENTEROHEPATIC CIRCULATION Overview Disturbances of the enterohepatic circulation may be classified into 4 groups: (1) disturbances of circulation (ie, movement between organs), (2) disturbances of bile acid formation (synthesis and conjugation), (3) disturbances in membrane transport of bile acids, and (4) disturbances involving bacterial deconjugation and dehydroxylation. 1 Disturbances in Bile Acid Circulation Biliary Obstruction. Biliary obstruction, eg, a stone obstructing the common duct, causes bile acid retention in the hepatocyte, leading to hepatocyte necrosis or apoptosis. When bile acids accumulate in the 2653

8 hepatocyte, conjugates of CDCA undergo sulfation at C-3. Sulfated and unsulfated bile acids regurgitate from the hepatocyte and are eliminated in urine. Plasma concentrations of bile acids rise 10- to 20-fold. In time, with total obstruction, bile acid biosynthesis decreases and is balanced by urinary loss. Biliary lipids such as phospholipids and cholesterol regurgitate from bile into plasma, causing increased plasma levels of phospholipids and cholesterol. With complete obstruction, bile acids are not present in the small intestine, fat-soluble vitamins are not absorbed, and dietary triglyceride is inefficiently absorbed (see below). Because bile acids do not enter the intestine, secondary bile acids are not formed. When biliary obstruction is incomplete, secretion of bile acids into the intestine decreases. Despite this, ileal absorption continues, and the return of cytotoxic bile acids to the liver promotes liver damage. Such continuing ileal absorption in cholestatic disease may be considered inappropriate and has been termed organ warfare. Bile acid sequestrants are administered to decrease the efficiency of ileal absorption. If ileal absorption is sufficiently efficient, less bile acids may enter the colon. Secondary bile acids are formed in decreased amounts, and fecal bile acid output also decreases. Biliary Fistula. In a patient with a biliary fistula, bile acids are diverted to the outside instead of entering the small intestine. Because bile acid biosynthesis is controlled by negative feedback, bile acid synthesis increases markedly up to 20-fold. Because bile acids are made from cholesterol, cholesterol biosynthesis must have a parellel increase. Hepatic function is not impaired, although the actual flux of bile acids through the hepatocyte diminishes because maximal bile acid biosynthesis at 3000 to 6000 mg/d is less than the usual flux of conjugated bile acids through the liver when the enterohepatic circulation is intact ( mg/ d). When no bile acids are in the small intestine, fat-soluble vitamins and lipid-soluble drugs such as cyclosporine have little or no absorption. Lipolysis of dietary triglyceride is nonetheless complete, but the uptake of fatty acids is slowed because micelles are not present, and the site of absorption extends throughout the small intestine. Because unsaturated fatty acids have a greater aqueous solubility than saturated fatty acids, their absorption is less disturbed by an absence of bile acids. For infants with cholestatic liver disease, formula feedings usually contain triglycerides rich in medium-chain fatty acids, which are water soluble and can be absorbed efficiently in the absence of conjugated bile acids. A common clinical occurrence of a biliary fistula is after orthotopic liver transplantation. The transplanted liver increases its bile acid biosynthesis slowly, presumably because of its ischemic damage during storage before transplantation. This greatly decreases the flux of bile acids through the transplanted liver; also, there is an increased load of bilirubin for excretion in the cholestatic recipient. It is unclear whether bile acid supplementation with UDCA to increase the flux of bile acids through the recovering hepatocytes would be of therapeutic value. Some groups 21 have reported benefit; others 22 have not. In the patient with a T tube, the dose of UDCA that has been given (10-15 mg/kg per day) is far below that required to restore bile acid secretion to normal levels. Cholecystectomy. In patients in whom the gallbladder has been removed, the bile acid pool is stored in the small intestine during the fasting state. When a meal is ingested, the pool moves to the terminal ileum, where it is actively absorbed. Bile acids return to the liver and are immediately secreted into bile. The overall effect of cholecystectomy on biliary secretion is small, and daily bile acid secretion after cholecystectomy is not very different than in the healthy person. 23 In a few patients, the movement of the bile acid pool to the distal intestine seems to overwhelm the ileal transport system, and bile acid malabsorption occurs. This is clinically manifest as diarrhea and generally responds to administration of a bile acid sequestrant. Ileal Resection. Resection of the terminal ileum causes bile acid malabsorption. If the resection is small, the effect on bile acid metabolism is minimal. Increased biosynthesis occurs to compensate for increased loss. With larger resections, bile acid synthesis increases even further up to 20 times the usual rate. In this new steady state, unabsorbed bile acids, water, and electrolytes enter the colon in greatly increased amounts. In some patients, bile acids act on the colonic epithelium to inhibit water absorption or induce frank secretion. The result is a mild, watery diarrhea. Symptomatic response is obtained by cholestyramine resin administration. 24 When the length of ileum resected is greater than 100 cm, bile acid secretion decreases because the increased bile acid synthesis ( mg/d) is still well below the normal bile acid secretion rate. The bile acid pool becomes progressively depleted during the day. Because of the lack of micelles, fat malabsorption occurs. Increased fatty acids passing into the colon inhibit water absorption. The loss of water and electrolyte conservation by the distal small intestine, together with the inhibition of colonic water absorption, result in severe diarrhea and steatorrhea. If the diarrhea is sufficiently large, and if there is malabsorption of other nutrients, the patient may be diagnosed as having short-bowel syndrome. Therapy is complex and has only limited success. In some patients, fecal weight and frequency is reduced by the elimination of fat from the diet. Conjugated bile acid replacement therapy is being explored to increase fat absorption (see below). Other therapeutic approaches include proton pump inhibitors, low osmolar diets, growth factors, and glutamine. Portal Systemic Venous Shunting. When patients have portal systemic venous shunting because of portal hypertension or a surgical portal caval anastomosis, bile acids enter the systemic circulation. To our knowledge, if liver function is good, the effect on bile acid metabolism is small. Hepatic arterial blood flow is sufficient that systemic plasma concentrations increase only about 2654

9 4-fold. 25 If hepatic function is impaired, it is conceivable that the markedly elevated bile acid concentration could have cytotoxic effects, such as in the lung. Disturbances in Bile Acid Biosynthesis Overview. Disturbances in bile acid biosynthesis are extremely rare yet important to recognize because they may be palliated and even cured by replacement therapy with exogenous bile acids. Because multiple enzymes are involved in the formation of bile acids from cholesterol, there are multiple possibilities for enzyme defects. Because there are at least 2 pathways of bile acid biosynthesis, a defect in 1 enzyme may be compensated for by the increased activity of another bile acid; a deficiency may be avoided. Defects in Side-Chain Biosynthesis. In the conversion of cholesterol to bile acids, the 8-carbon side chain of cholesterol undergoes oxidative cleavage to form the 5-carbon side chain of bile acids. The process involves initial hydroxylation at C-27, followed by modified oxidation. One defect in side-chain biosynthesis involves a key mitochondrial enzyme, sterol 27-hydroxylase. A defect in this enzyme presents as the rare neurologic disease cerebrotendinous xanthomatosis. In this condition, because hydroxylation at C-27 is blocked, hydroxylation occurs at other sites on the side chain such as C-25 and C-24 to form novel bile alcohols. These are formed in greatly increased amounts and excreted in urine. In addition, cholestanol (a reduction product of cholesterol) is formed in the liver and deposited in tissues. Treatment with CDCA slows disease progression. 26 Later steps in side-chain cleavage involve peroxisomal enzymes. Syndromes of peroxisomal dysfunction or absence (Zellweger syndrome) are associated with the formation of C 27 bileacids,ie,bileacidshavingac 8 side chain rather than a C 5 side chain. Defects in Maturation of the Steroid Nucleus. Defects in biotransformation steps involving the steroid nucleus are rare, with fewer than 50 cases having been identified. In the formation of bile acids, the -hydroxy group at C-3 is oxidized to a 3-oxo group, then reduced to a 3- -hydroxy group. An infant lacking the 3-dehydrogenase enzyme has been described elsewhere. 27 Bile contained 3 hydroxy bile acids, and the liver had a giant cell hepatitis. Several cases of inborn deficiency in 3-reductase have been reported. Infants with this defect present with cholestatic disease because the 3-oxo bile acids accumulate in the hepatocyte, causing its death. 28 Treatment with exogenous primary bile acids of both enzyme defects is lifesaving. A defect in hydroxylation on the B ring involving oxysterol 7 -hydroxylase has recently been reported. 29 To our knowledge a defect in C-12 hydroxylation has not yet been unequivocally identified. Defects in Bile Acid Conjugation. Only a single case of absent bile acid conjugation has been identified, to our knowledge. 30 Because unconjugated bile acids are membrane permeable, they are passively absorbed from the small intestine, decreasing the intraluminal bile acid concentration below that required for micelle formation. The patient with this defect presented clinically with a malabsorption syndrome, with an inability to absorb fat-soluble vitamins and severe fat malabsorption. 30 It is remarkable that a hepatocyte defect presents clinically as an intestinal disease. Disturbances in Membrane Transport of Bile Acids Most of the proteins involved in mediating the transport of bile acids across apical and basolateral membranes of epithelial cells have been cloned, but only in the past few years; few clinical examples of transporter defects have been described. One case of an ileal transport defect in the apical transporter has been studied 31 in detail, and the genetic abnormality was clarified as a point mutation causing nonfunction of the transporter. More such defects are likely to be described. A syndrome suggestive of a defect in the hepatocyte basolateral transporter responsible for bile acid uptake has been reported, 32 but no clear defect in the sodium-dependent transporter could be found. A defect in the hepatocyte canalicular adenosine triphosphate stimulated bile acid transporter has been described elsewhere. 33 Identification of patients with defective bile acid transporters is likely to increase greatly in the coming decades. Disturbances in Bile Acid Deconjugation and Dehydroxylation Increased Bile Acid Deconjugation. In healthy people, bile acid deconjugation begins in the distal small intestine, presumably mediated by bacteria spilling across the ileocecal valve. In patients with intestinal stasis or other pathologic conditions promoting the growth of bacteria, bile acid deconjugation increases in the proximal intestine. The unconjugated bile acids that are formed are absorbed passively from the small intestine, causing a decreased intraluminal concentration and impaired micelle formation. Although in the past it was thought that the absorption of unconjugated bile acids caused damage to the intestinal mucosa, this now seems unlikely, and other bacterial products are likely to cause impaired absorptive function. Increased bile acid deconjugation can be detected by aspirating small intestinal content and showing the presence of unconjugated bile acids (by chromatography) in the presence of an increased density of bacteria. Increased deconjugation can be detected indirectly by finding an increased concentration of unconjugated bile acids in systemic venous plasma or by using a breath test. 34,35 In this test, the patient is given a radioactive conjugated bile acid containing glycine with an isotopically tagged carbon atom ( 14 Cor 13 C) in its carboxyl moiety. Deconjugation liberates the glycine, which is metabolized to 14 CO 2 or 13 CO 2, which can be measured in breath. The technique is investigational at present. IncreasedBileAcidDehydroxylation. In the large intestine, bile acids are first deconjugated, then 7-dehydrox- 2655

10 ylated. In the healthy person, all bile acids undergo nearly complete 7-dehydroxylation. Nonetheless, in a subset of patients with cholesterol gallstones, cholic acid undergoes more rapid 7-dehydroxylation to form DCA for unknown reasons. Such patients have an increased proportion of DCA in their biliary bile acids. If such patients are given ampicillin, their increased rate of 7-dehydroxylation is abolished and, at the same time, their bile becomes less saturated in cholesterol. 36 Identification of these patients suggests a group of patients with autointoxication, in whom increased DCA absorption from the large intestine contributes by an unknown mechanism to hypersecretion of cholesterol in bile. In the healthy person, the longer the transit time in the colon, the greater the input of DCA. 37 When acromegalic patients are given octreotide, intestinal transit is slowed, DCA increases in biliary bile acids, and cholesterol gallstones occur. 38 Nonetheless, it remains unclear whether the increased input of DCA in patients with prolonged colonic transit time who are otherwise healthy has any health consequences. PRINCIPLES OF BILE ACID THERAPY Overview Considering the multiple functions of bile acids, many of which have been known for decades, the length of time to introduce bile acid therapy into clinical medicine is remarkable. It seems useful to divide bile acid therapy into 2 types: displacement and replacement. In displacement therapy, the composition of the circulating bile acids is changed, either to decrease the cytotoxicity of endogenous bile acids or to modulate cholesterol metabolism to decrease biliary cholesterol secretion. Bile acid replacement aims to correct a bile acid deficiency. A summary of the therapeutic uses of bile acids is given in Table 2. Displacement Therapy The modern era of displacement therapy began with the finding from a Mayo Clinic group that administration of the primary bile acid CDCA caused a decrease in biliary cholesterol secretion and gradual dissolution of gallstones. Although the efficacy and safety of CDCA was well established in the National Cooperative Gallstone Study, 40 CDCA was gradually replaced by UDCA because it was completely devoid of any hepatotoxicity. 41 Chenodeoxycholic acid is slightly hepatotoxic in humans, but in certain animals, it is highly hepatotoxic. Despite the efficacy and safety of UDCA administration for cholesterol gallstone dissolution, it is not frequently used today because of the success of laparoscopic cholecystectomy, which provides a rapid cure for symptomatic disease. Medical therapy, in contrast, requires months of therapy, does not always dissolve stones, and is followed by gradual recurrence in some patients. 42 A second use of UDCA was proposed by Leuschner and colleagues, who showed that UDCA caused a remarkable improvement in liver test results in patients with primary biliary cirrhosis. This work was confirmed by studies throughout the world, and evidence 43 indicates that administration of UDCA to patients with primary biliary cirrhosis prolongs the interval between diagnosis and liver transplantation. The therapeutic effect of Table 2. Therapeutic Uses of Bile Acids* Bile Acid Disease Comment Ursodeoxycholic Acid (UDCA) UDCA (ursodiol) Cholestatic liver disease Primary biliary cirrhosis FDA approved Primary sclerosing cholangitis Autoimmune hepatitis with cholestatic features Autoimmune cholangitis TPN-associated cholestasis Cholestasis of pregnancy Pediatric cholestatic syndromes ; symptomatic benefit Drug-induced cholestasis Hepatic complications of allogeneic bone marrow transplantation Cystic fibrosis (for hepatic complications) Prevention of rejection after liver transplantation ; efficacy disputed Cholesterol cholecystolithiasis Treatment of existing stones (radiolucent in functioning gallbladders) FDA approved, but surgery is usually used Prevention of cholecystolithiasis during rapid weight loss FDA approved Choledocholithiasis ; seldom used because invasive methods are better Choledocholithiasis with polycystic liver disease (Caroli disease) Other Bile Acids CDCA (chenodiol) Cerebrotendinous xanthomatosis ; retards disease progression Cholesterol cholecystolithiasis FDA approved, but UDCA preferred for medical therapy CDCA-CA-UDCA Inborn errors of bile acid biosynthesis involving the A ring Lifesaving and curative mixtures Cholylsarcosine Short-bowel syndrome (to enhance lipid absorption) Investigational *FDA indicates Food and Drug Administration; TPN, total parenteral nutrition; CDCA, chenodeoxycholic acid; and CA, cholic acid. References for the use of UDCA in these disease conditions are available in Beuers et al