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Liver-Cholesterol and Bile Formation
Liver-Cholesterol and Bile Formation Barry Potter Louisiana State University Health Sciences Center, New Orleans, USA ã 2007 Elsevier Inc. All rights reserved.
Cholesterol is partly synthesized de novo by the liver and other tissues, and partly absorbed from the intestine and incorporated into lipoproteins. Absorbed cholesterol is taken up by the chylomicron remnants in the liver and, together with the newly synthesized cholesterol, is redistributed to the extrahepatic tissues in VLD L. A small proportion of this cholesterol, however, is used in the synthesis of bile acids. Bile acids are reabsorbed from the ileum and inhibit 7alpha-hydroxylase, preventing the modification of cholesterol and, thus, regulating bile acid synthesis. Similarly, cholesterol in chylomicron remnants inhibits HMG CoA reductase and therefore regulates hepatic cholesterol synthesis (Fig. 1). Biliary secretion is the only major cholesterol excretory pathway. Approximately 300–350 mg of cholesterol are absorbed from the diet, with another 800 mg synthesized by the liver. This counterbalances losses in the form of bile acids, of which approximately 95% are re-absorbed, and active secretion of cholesterol and cholesterol derivatives into bile Turley and Dietschy (1988). The remainder is used for the production of skin and steroid hormones. As cholesterol is insoluble in water, transportation in the plasma occurs by way of various lipoproteins. Cholesterol is synthesized in the liver from acetate and converted to primary bile acids. The bile acids are excreted in bile into the duodenum. Bile acids, some dietary cholesterol, and bile-secreted cholesterol are absorbed in the ileum. Although bile acids are actively transported into the portal venous blood by secondary active transport, cholesterol diffuses into the enterocytes and is packaged together with triglycerides, phospholipids, and apolipoproteins into very large lipoproteins (chylomicrons) that enter the lymphatic system by way of the terminal intestinal lacteals. Some of the absorbed cholesterol returns to the liver in the chylomicron remnants. The uptake of recycled
Fig. 1. Cholesterol pathways in Humans.
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Liver-Cholesterol and Bile Formation
bile acids or cholesterol by the liver inhibits their de novosynthesis, thus acting as part of the negative feedback system for the production of bile acids and cholesterol (Fig. 2). Unlike bile acids, of which approximately 90–95% are recycled, only 40% of cholesterol is reabsorbed. Fecal excretion eliminates 60%, with almost half being present as neutral sterols and the rest as acidic sterols or bile-acid derivatives. There is an acinar distribution of metabolic activity, with bile acids accumulated primarily in zone 1, and bile-acid synthesis in zone 3 of the acinusGroothuis and Meijer (1992) (see the record on LiverStructure). Both cholesterol and bile acids are synthesized by the liver and secreted into the intestine in bile. As these materials are also absorbed/reabsorbed in the ileum, increased presentation to the liver results in negative feedback inhibition of their hepatic synthesis. However, it should be noted that the enterohepatic pathways are different, relating to their uptake. Bile acids are reabsorbed by a sodium-dependent active transport mechanism and enter the hepatic circulation. Cholesterol is absorbed from the intestine by diffusion and is packaged into chylomicrons. These enter the lymphatic system by way of the intestinal terminal lacteals and enter the general circulation. Thus, the liver normally receives only cholesterol from chylomicrons remnants. Bile Formation and Secretion: One of the most important functions of the liver, and one that relates to its function as a modified exocrine gland, is bile secretion. However, bile differs from most classical exocrine secretions because it is composed of both excretory products and secretory materials. As with most exocrine secretions, the initial product, the primary or canalicular bile, which drives flow Paumgartner (1977), is modified by the duct system (Fig. 3). In the human liver, however, there is additional modification of bile during its storage in the gall bladder (Table 1). Although the active transport mechanisms have been well characterized, the composition of canalicular bile can only be inferred. However, the composition of ductular and gallbladder bile are well known and the changes that occur withstorage and release are well defined. Cholestasis, or stoppage of bile flow, may result from a variety of causes, from intrahepatic caused by xenobiotics to extrahepatic caused by inflammation, tumors, biliary tree atresia, as in primary biliary cirrhosis, or gall stones. Super-saturated gallbladder bile, formed during the storage phase in man, may easily be ‘‘seeded’’ and result in the formation of gallstones. The composition of these gallstones usually reflects associated
Fig. 2. Cholesterol and Bile Acid Homeostasis in Humans.
Liver-Cholesterol and Bile Formation
Fig. 3. Phases of Bile Secretion.
Table 1. Bile Composition*
* Values express as mM, except where noted.
underlying diseases, such as cerebrotendinous xanthomatosis, where there are excessive amounts of cholestanol (dihydrocholesterol) in all tissues and the formation of cholesterol/cholestanol gallstones. Similar gallstones may be seen in those with primary biliary cirrhosis, but not in alcoholics. In cryptogenic liver cirrhosis, the bile salt pool is markedly diminished, as is the synthesis of primary bile acids. In consequence, gallstones tend to be of the pigmented variety. Defective reabsorption of bile acids can also affect bile composition by disruption of the enterohepatic circulation. Ileal diseases, such as Crohn’s disease, lead to defective bile acid reabsorption and subsequent diarrheal complications. The major driving force for bile secretion is the active transport of bile acids into the canaliculus. Bile acids are amphipathic molecules derived from cholesterol that have ionic side chains and, thus, act like detergents on hydrophobic liquid droplets. The basic sterol structure of cholesterol, as well as a simple primary bile acid, cholic acid, are shown in Fig. 4. A variety of substitutions may be made in the basic structure of cholesterol to
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Liver-Cholesterol and Bile Formation
Fig. 4. The structure of cholesterol and a typical primary bile acid, cholic acid.
produce the common primary (C24) bile acids. These alterations includethe addition of hydroxyl groups at C6, C7, or C12 within the sterol nucleus, and at C23 on the carboxylic acid side chain. The term primary bile acid refers to a bile acid synthesized de novofrom cholesterol in the hepatocytes. As is evident from the structure of cholic acid, there is a basic, hydrophobic sterol nucleus and several ionic groups. Stereochemically these are all positioned to one side of the molecule, giving rise to a polar and a nonpolar side to the bile-acid molecule. This generates the amphipathic properties of the molecule as it has the ability to partition into both hydrophilic and lipophilic regions, reducing the surface tension on lipid droplets in aqueous solutions. Bile is generated by active transport mechanisms secreting bile acids and other osmotically active substances into the canaliculus. This results in the paracellular movement of water and small solutes into this space and generates flow Erlinger (1988). The canalicular bile is modified by the ductule cells, which reabsorb secondary solutes such as glucose and amino acids, and add bicarbonate. In the inter-digestive phase, bile is stored in the gallbladder, where concentration by absorption of water and sodium chloride occurs. Acidification also occurs to prevent precipitation. Bile release in the digestive phase is under hormonal control. Cholecystokinin causes contraction of the gallbladder wall smooth muscle and relaxation of the Sphincter of Oddi, with secretin stimulation causing the addition of bicarbonate and water to gallbladder bile. After their synthesis, bile acids are actively transported across the canalicular membrane of the hepatocyte into the canaliculus. As is the case for fatty acids, there are bile acid transport proteins, at least within the cytosol of the hepatocyte. Microtubules do not appear to be involved, and the movement to the canalicular region is thought to occur by diffusion of the protein-bound, and possibly unbound, bile acid monomers. Bile acids are transported across the canalicular plasma membrane by way of a specific 100-kDatransport protein into the canalicular space. While it had been proposed that the active secretion of osmotically active substances, principallybile acids, generates the driving force for bile secretion, it has also been shown more recently that there are contractile elements within the canalicular region of these polarized epithelial cells that generate a rhythmic pumping action that contributes to bile flow. There are several important transport systems involved in the transport of osmotically active substances into the canaliculus (Fig. 5). Many of these substances are initially accumulated by the hepatocyte at the sinusoidal or basolateral plasma membrane by a different transporter. It appears that many hydrophobic substances, which are materials that circulate bound to plasma albumin, have specific transporters across the plasma membrane, and cytosolic transport systems to move them to the canalicular membrane, with transformation reactions occurring to make them more excretable. Uptake
Liver-Cholesterol and Bile Formation
Fig. 5. Transport mechanisms involved in the formation of canalicular bile.
mechanisms at the sinusoidal plasma membrane may be divided into three groups: bile acids, non bile acid cholephils (organic anions), and weak organic acids. Bile acids are cotransported with Na+ that is removed from the cell by the sinusoidal membrane Na+/K+ ATPase. Organic anions, such as bilirubin, are transported into the cell by facilitated diffusion, using a chloride-dependent membrane protein. This protein also transports a variety of molecules, including bromosulphophthalein (BSP) and indocyanine (cardiac) green. Weak organic acids, such as free fatty acids, are thought to be transported across the plasma membrane by way of a 43-kDa-membrane protein. There are also vesicle and endocytic uptake pathways at the plasma membrane for phospholipids and proteins. Formation of canalicular bile requires the active transport of osmotically active materials into this region. The major transported substances are ionized bile acids by way of a specific 100-kDa-transport protein. Bilirubin monoglucuronide (BMG), diglucuronid (BDG), glutathione conjugates, and glutathione (GSH and GSSG) are transported by the multi-organic anion transporter and cations by a 170-kDa p-glycoprotein, both of which require AT P. Bicarbonate, chloride, sulfate, and oxalate ions are also added to the canalicular bile. Uptake of the bile acids, nonbile acid cholephils, and organic acids also occurs by way of an active transport processes (see the record on Hepatic Transport Mechanisms). Once inside the hepatocyte, many of these cholephils are modified during their movement across the hepatocyte to the canalicular region. Bile acids are often hydroxylated or sulfated. Many nonbile-acid cholephils are conjugated to glutathione by one of the glutathione S-transferase family of enzymes. Bilirubin is also bound to glutathione S-transferase B, also known as ligandin, but this molecule is only involved in its cytosolic movement. Bilirubin is, in fact, glucuronidated by UDP-glucuronyl transferase to form either bilirubin monoglucuronide or digluronide for excretion. When plasma bilirubin is measured, the quantity that is conjugated is usually also estimated, giving an indication in pathological conditions as to where the defect occurs (Table 1). Bilirubin glucuronides and glutathione conjugates are transported into the canaliculus by way of a multi-organic anion transporter (MOAT) protein. Both this and the organic cation transport (OCT) proteins require AT P. Other substances that are actively secreted into bile include sulfate, oxalate, and bicarbonate ions. Canalicular bile flow is basically an osmotic response to active solute transport into the area Paumgartner (1977). It is divided into bile acid-dependent flow, where the bile acid output is the major solute generating bile flow, and bile acid-independent flow, where bile is generated at low-bile acid concentrations, or in addition to bile acid-dependent bile
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Table 2. The clinical relevance of altered bilirubin homeostasis in terms of its metabolism and excretion (see the record on Motility and other Disorders of the Biliary Tract).
flow Erlinger (1977). Bile acid-dependent flow occurs as a result of the active secretion of osmotically active bile acids into the canaliculus. Normally, bile acids ‘‘obligate’’ approximately 8–15 ml of water per mmole of bile acid. That is, they cause this volume of solvent to be drawn into the canaliculus, principally by a paracellular route. This is known as choleresis. The most likely source of this liquid is from the plasma by way of the space of Disse. As a result of solvent-drag, small ions and solutes are also moved into the canaliculus. Accordingly, the composition of the solution obligated by the bile acids is very similar that to the kidney glomerulus, being close to iso-osomotic. The canalicular bile generated by bile acids is therefore nearly always hyperosmotic. The quantity of liquid obligated is bile-acid specific and is known as the choleretic activity. A few bile acids, such as ursodeoxycholic acid, may obligate up to 90 ml of water and are termed hypercholeretic. There are also some bile acids that obligate much less than 8ml of water and are termed hypocholeretic or, in the extreme case, cholestatic, resulting in cessation of bile flow, as is seen with the monohydroxybile acids. Bile-acid secretion also induces the canalicular secretion of phospholipid- and cholesterol-rich vesicles. Bile acid-independent flow is thought to play only a minor role in the generation of bile. The mechanism for the generation of bile acid-independent flow is not clear, but is thought to involve a canalicular ATPase. Other transport systems, such as the sinusoidal Na+/H+ and the canalicular Cl-/HCO-3 antiporters, which play a role in bicarbonate secretion into bile, may also be involved in bile acid-independent canalicular bile flow. The addition of bicarbonate to ductular bile is regulated by secretin. Binding of this hormone to its basolateral ductule plasma membrane receptor results in the activation of adenylate cyclase (AC)protein kinase A and increased chloride diffusion at the luminal membrane. The chloride is then transported back into the cell, coupled to bicarbonate counter-transport. Bicarbonate is produced by cellular carbonic anhydrase. Excess protons are secreted across the basolateral membrane into the blood by way of vesicular proton ATPase.
Liver-Cholesterol and Bile Formation
Canalicular bile is modified by the duct cells and during storage in the gallbladder. The bile ducts change the solute composition of the newly secreted bile through the addition of bicarbonate and water, as well as the reabsorption of glucose and other solutes. Bicarbonate is produced by the action of carbonic anhydrase on carbon dioxide and water within the bile-duct cells. Bicarbonate ions are then transported into the bileduct lumen by a chloride/bicarbonate antiporter at a ratio of 2Cl-/3HCO-3. The chloride is then recycled into the lumen through a chloride channel. The hydrogen ions generated by this reaction are secreted into the blood by way of a vesicular proton ATPase. While many hormones, such as gastrin, VIP, and bombesin, influence bile ductule secretions, the major regulator is secretin (Fig. 6). The binding of secretin to its receptor on the basolateral surface of the bile-duct cell results in the phosphorylation of the luminal chloride channels due to activation of adenylate cyclase and the production of cAMPto activate protein kinase A, which, in turn, phosphorylates the chloride channel. This mechanism for bicarbonate secretion is similar to that seen in the salivary gland and the pancreas. Sodium ions and water are generally thought to migrate by way of the paracellular route into the bile to modify its osmolarity, although aquaporins are associated with the bile ductule cells. Mucus is also secreted by associated cells to protect the biliary tract. Carbon dioxide and water are converted to carbonic acid by carbonic anhydrase. This dissociates and bicarbonate is secreted by way of the bicarbonate/chloride luminal antiporter. Excess protons are removed by a basolateral vesicular ATPase. The system is stimulated by secretin binding to a basolateral receptor that generates cAM P. This results in phosphorylation of the chloride channel/pump by protein kinase A, which subsequently increases chloride uptake by the antiporter. In humans, bile is not normally secreted into the small intestine but is stored in an accessory organ, the gallbladder, during the interdigestive phase and during fasting. While the gallbladder is not absolutely essential, being absent in rats and mice, it does serve a useful purpose in that bile is almost immediately available for brief periods of digestion. However, because the gallbladder only has a volume of 50–75 ml, and the liver produces up to 600 ml of bile in a 24-hour period, some form of concentration must occur during storage. The gallbladder is essentially a sac lined with epithelial cells and surrounded by smooth muscle that is under neurohumoral control.
Fig. 6. Regulation of bicarbonate secretion by bile ductule cells.
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Storage of bile in the gallbladder involves two separate processes: concentration and acidification. Concentration is to reduce the volume to make storage feasible and acidification is to lower both bicarbonate concentrations during concentration and the quantity of anions that are likely to precipitate calcium, which could lead to the formation of gallstones. Concentration is achieved by pumping sodium chloride into the intercellular space between the biliary epithelial cells. Approximately two thirds of the sodium transported into the epithelial cells occurs by way of a Na+/H+ exchanger, and the rest by other unspecified mechanisms. Similarly, chloride ions are thought to be transported by an apical Cl-/HCO-3 transporter, although the exact mechanisms have yet to be confirmed. The hydrogen ions transported into the gallbladder lumen react with bicarbonate ions to form carbonic acid, which then decays to carbon dioxide and water. The removal of bicarbonate leads to a transient hypotonicity within the lumen and provides the osmotic driving force that leads to net water movement across the epithelial cells, concentrating the bile (Fig. 7). Sodium in the bile is transported into the biliary epithelial cells, counter-transporting protons into the bile. Chloride diffusion follows sodium. Sodium chloride is then transported into the extracellular fluid, generating an osmotic gradient that causes the movement of water by both transcellular and paracellular pathways, concentration of the remaining gallbladder bile. The low pH also favors the conversion of carbonate and bicarbonate ions to carbon dioxide and water. The solubility of calcium bilirubinate is enhanced by this low pH (pH 5.5–pH 6). As calcium salt and bilirubin gallstones are most frequently encountered in the Western hemisphere, it is clear that maintaining low pH and low-anion concentration tends to inhibit nucleation and their subsequent formation by precipitation/ crystallization. At this lowered pH, the conditions are favorable for micelle formation by bile salts as the bile becomes progressively more concentrated. Mixed in with these micelles are the secreted phospholipids and cholesterol. This micelle formation results in a lower osmotic activity because osmolarity is proportional to the number of particles in solution. As a consequence, bile-salt concentration in the gallbladder may exceed 300 mM without producing hyperosmotic bile. The osmolarity in both the ducts and the gallbladder remains relatively constant at approximately 280–290 mOsm. The acid environment
Fig. 7. Fluid transport by gallbladder epithelium.
Liver-Cholesterol and Bile Formation
does not damage the epithelial cells because, at this pH, the protective mucins secreted by the epithelia assume their maximum hydrodynamic radius, thereby increasing their protective effect. The protective effect is also enhanced by the viscosity and strong anionic charge of the mucins as a result of their sialate residues. Secretion of bile into the duodenum is regulated by two hormones, cholecystokinin (CCK) and secretin. CCK causes relaxation of the sphincter of Oddi and contraction of the musculature around the gallbladder, whereas secretin potentiates bicarbonate and water secretion into the bile duct. Bile flow is dependent on the pressure generated by the gallbladder during contraction and the compliance of the Sphincter of Oddi. The initial pressure, generated at the canalicular level, is created by canalicular secretion of osmotically active materials that drives water movement and, ultimately, if the Sphincter of Oddi is closed, filling of the gallbladder while it is relaxed during the interdigestive phase. Some of the material secreted in bile is reabsorbed in the small intestine, mainly in the ileal region. Approximately 95% of bile acids are reabsorbed in this region and are taken up from the portal blood by zone 1 hepatocytes. Hepatic extraction of bile acids is extremely efficient, with systemic venous blood levels rarely exceeding 20 mM in normal individuals. As a result of the operative homeostatic mechanism for bile acid synthesis, de novo synthesis of bile acids occurs in zone 3 of the acinus. Furthermore, as bile movement in the liver is countercurrent to blood flow, bile acids accumulated at high concentrations in zone 1 are rapidly re-secreted into bile, minimizing potential damage to the organ. As many of these bile acids are also tightly bound to albumin, the filtered load is quite small (about 100–200 mg/day) and these are almost completely reabsorbed by an active transport mechanism in the proximal renal tubules of the kidney. Bile acids in the intestine may be modified by the bacterial flora to form secondary bile acids, such as deoxycholic acid from cholic acid and lithocholic acid from chenodeoxycholic acid. After uptake by the hepatocytes, these bile acids are further modified by conjugation with glycine, taurine, or sulfation before re-secretion into bile. These are known as tertiary bile acids. Other materials are also reabsorbed in the intestine. Thus, cholesterol is reabsorbed unless it is modified, and bilirubin is heavily modified to form compounds such as the uroporphyrinogensfor renal excretion. The absence of bile is deleterious in that, besides lipid solubilization, this material is also required for the solubilzation and uptake of lipid soluble vitamins, in particular vitamin K, which is required by the liver for the synthesis of many of the coagulation cascade proteins. As it is fat soluble, one consequence of cholestasis is a prolonged coagulation time.
Other Information – Web Sites Cholesterol and Bile Metabolism-http://www.indstate.edu/thcme/mwking/cholesterol. html Cholesterol synthesis-http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/ cholesterol.htm Bile metabolism-http://dwb.unl.edu/Teacher/NSF/C11/C11Links/web.indstate.edu/ thcme/mwking/cholesterol.html
Journal Citations Groothuis, G.M.M., Meijer, D.K.F., 1992. Hepatocyte heterogeneity in bile formation and hepatobiliary transport of drugs. Enzyme, 46(1-3), 94–138.
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Book Citations Paumgartner, G., 1977. Physiology I: Bile Acid-Dependent Bile Flow. Bianchi, L., Gerok, W., Sickinger, K. (Ed.), Liver and Bile, pp. 45–53, MTP Press, London. Erlinger, S., 1977. Physiology II: Bile Acid-Independent Secretion. Bianchi, L., Gerok, W., Sickinger, K. (Ed.), Liver and Bile, pp. 55–62, MTP Press, London. Turley, S.D., Dietschy, J.M., 1988. The Metabolism and Excretion of Cholesterol by the Liver. Arias, I.M., Jakoby, W.B., Popper, H., Schachter, D., Shafritz, D.A. (Ed.), The Liver, Biology and Pathobiology, pp. 617–641, Raven Press, New York. Erlinger, S., 1988. Bile flow. Arias, I.M., Jakoby, W.B., Popper, H., Schachter, D., Shafritz, D.A. (Ed.), The Liver, Biology and Pathobiology, pp. 643–661, Raven Press, New York.
Cholesterol is partly synthesized de novo by the liver and other tissues, and partly absorbed from the intestine and incorporated into lipoproteins. Absorbed cholesterol is taken up by the chylomicron remnants in the liver and, together with the newly synthesized cholesterol, is redistributed to the extrahepatic tissues in VLD L. A small proportion of this cholesterol, however, is used in the synthesis of bile acids. Bile acids are reabsorbed from the ileum and inhibit 7alpha-hydroxylase, preventing the modification of cholesterol and, thus, regulating bile acid synthesis. Similarly, cholesterol in chylomicron remnants inhibits HMG CoA reductase and therefore regulates hepatic cholesterol synthesis (Fig. 1). Biliary secretion is the only major cholesterol excretory pathway. Approximately 300–350 mg of cholesterol are absorbed from the diet, with another 800 mg synthesized by the liver. This counterbalances losses in the form of bile acids, of which approximately 95% are re-absorbed, and active secretion of cholesterol and cholesterol derivatives into bile Turley and Dietschy (1988). The remainder is used for the production of skin and steroid hormones. As cholesterol is insoluble in water, transportation in the plasma occurs by way of various lipoproteins. Cholesterol is synthesized in the liver from acetate and converted to primary bile acids. The bile acids are excreted in bile into the duodenum. Bile acids, some dietary cholesterol, and bile-secreted cholesterol are absorbed in the ileum. Although bile acids are actively transported into the portal venous blood by secondary active transport, cholesterol diffuses into the enterocytes and is packaged together with triglycerides, phospholipids, and apolipoproteins into very large lipoproteins (chylomicrons) that enter the lymphatic system by way of the terminal intestinal lacteals. Some of the absorbed cholesterol returns to the liver in the chylomicron remnants. The uptake of recycled
Fig. 1. Cholesterol pathways in Humans.
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Liver-Cholesterol and Bile Formation
bile acids or cholesterol by the liver inhibits their de novosynthesis, thus acting as part of the negative feedback system for the production of bile acids and cholesterol (Fig. 2). Unlike bile acids, of which approximately 90–95% are recycled, only 40% of cholesterol is reabsorbed. Fecal excretion eliminates 60%, with almost half being present as neutral sterols and the rest as acidic sterols or bile-acid derivatives. There is an acinar distribution of metabolic activity, with bile acids accumulated primarily in zone 1, and bile-acid synthesis in zone 3 of the acinusGroothuis and Meijer (1992) (see the record on LiverStructure). Both cholesterol and bile acids are synthesized by the liver and secreted into the intestine in bile. As these materials are also absorbed/reabsorbed in the ileum, increased presentation to the liver results in negative feedback inhibition of their hepatic synthesis. However, it should be noted that the enterohepatic pathways are different, relating to their uptake. Bile acids are reabsorbed by a sodium-dependent active transport mechanism and enter the hepatic circulation. Cholesterol is absorbed from the intestine by diffusion and is packaged into chylomicrons. These enter the lymphatic system by way of the intestinal terminal lacteals and enter the general circulation. Thus, the liver normally receives only cholesterol from chylomicrons remnants. Bile Formation and Secretion: One of the most important functions of the liver, and one that relates to its function as a modified exocrine gland, is bile secretion. However, bile differs from most classical exocrine secretions because it is composed of both excretory products and secretory materials. As with most exocrine secretions, the initial product, the primary or canalicular bile, which drives flow Paumgartner (1977), is modified by the duct system (Fig. 3). In the human liver, however, there is additional modification of bile during its storage in the gall bladder (Table 1). Although the active transport mechanisms have been well characterized, the composition of canalicular bile can only be inferred. However, the composition of ductular and gallbladder bile are well known and the changes that occur withstorage and release are well defined. Cholestasis, or stoppage of bile flow, may result from a variety of causes, from intrahepatic caused by xenobiotics to extrahepatic caused by inflammation, tumors, biliary tree atresia, as in primary biliary cirrhosis, or gall stones. Super-saturated gallbladder bile, formed during the storage phase in man, may easily be ‘‘seeded’’ and result in the formation of gallstones. The composition of these gallstones usually reflects associated
Fig. 2. Cholesterol and Bile Acid Homeostasis in Humans.
Liver-Cholesterol and Bile Formation
Fig. 3. Phases of Bile Secretion.
Table 1. Bile Composition*
* Values express as mM, except where noted.
underlying diseases, such as cerebrotendinous xanthomatosis, where there are excessive amounts of cholestanol (dihydrocholesterol) in all tissues and the formation of cholesterol/cholestanol gallstones. Similar gallstones may be seen in those with primary biliary cirrhosis, but not in alcoholics. In cryptogenic liver cirrhosis, the bile salt pool is markedly diminished, as is the synthesis of primary bile acids. In consequence, gallstones tend to be of the pigmented variety. Defective reabsorption of bile acids can also affect bile composition by disruption of the enterohepatic circulation. Ileal diseases, such as Crohn’s disease, lead to defective bile acid reabsorption and subsequent diarrheal complications. The major driving force for bile secretion is the active transport of bile acids into the canaliculus. Bile acids are amphipathic molecules derived from cholesterol that have ionic side chains and, thus, act like detergents on hydrophobic liquid droplets. The basic sterol structure of cholesterol, as well as a simple primary bile acid, cholic acid, are shown in Fig. 4. A variety of substitutions may be made in the basic structure of cholesterol to
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Liver-Cholesterol and Bile Formation
Fig. 4. The structure of cholesterol and a typical primary bile acid, cholic acid.
produce the common primary (C24) bile acids. These alterations includethe addition of hydroxyl groups at C6, C7, or C12 within the sterol nucleus, and at C23 on the carboxylic acid side chain. The term primary bile acid refers to a bile acid synthesized de novofrom cholesterol in the hepatocytes. As is evident from the structure of cholic acid, there is a basic, hydrophobic sterol nucleus and several ionic groups. Stereochemically these are all positioned to one side of the molecule, giving rise to a polar and a nonpolar side to the bile-acid molecule. This generates the amphipathic properties of the molecule as it has the ability to partition into both hydrophilic and lipophilic regions, reducing the surface tension on lipid droplets in aqueous solutions. Bile is generated by active transport mechanisms secreting bile acids and other osmotically active substances into the canaliculus. This results in the paracellular movement of water and small solutes into this space and generates flow Erlinger (1988). The canalicular bile is modified by the ductule cells, which reabsorb secondary solutes such as glucose and amino acids, and add bicarbonate. In the inter-digestive phase, bile is stored in the gallbladder, where concentration by absorption of water and sodium chloride occurs. Acidification also occurs to prevent precipitation. Bile release in the digestive phase is under hormonal control. Cholecystokinin causes contraction of the gallbladder wall smooth muscle and relaxation of the Sphincter of Oddi, with secretin stimulation causing the addition of bicarbonate and water to gallbladder bile. After their synthesis, bile acids are actively transported across the canalicular membrane of the hepatocyte into the canaliculus. As is the case for fatty acids, there are bile acid transport proteins, at least within the cytosol of the hepatocyte. Microtubules do not appear to be involved, and the movement to the canalicular region is thought to occur by diffusion of the protein-bound, and possibly unbound, bile acid monomers. Bile acids are transported across the canalicular plasma membrane by way of a specific 100-kDatransport protein into the canalicular space. While it had been proposed that the active secretion of osmotically active substances, principallybile acids, generates the driving force for bile secretion, it has also been shown more recently that there are contractile elements within the canalicular region of these polarized epithelial cells that generate a rhythmic pumping action that contributes to bile flow. There are several important transport systems involved in the transport of osmotically active substances into the canaliculus (Fig. 5). Many of these substances are initially accumulated by the hepatocyte at the sinusoidal or basolateral plasma membrane by a different transporter. It appears that many hydrophobic substances, which are materials that circulate bound to plasma albumin, have specific transporters across the plasma membrane, and cytosolic transport systems to move them to the canalicular membrane, with transformation reactions occurring to make them more excretable. Uptake
Liver-Cholesterol and Bile Formation
Fig. 5. Transport mechanisms involved in the formation of canalicular bile.
mechanisms at the sinusoidal plasma membrane may be divided into three groups: bile acids, non bile acid cholephils (organic anions), and weak organic acids. Bile acids are cotransported with Na+ that is removed from the cell by the sinusoidal membrane Na+/K+ ATPase. Organic anions, such as bilirubin, are transported into the cell by facilitated diffusion, using a chloride-dependent membrane protein. This protein also transports a variety of molecules, including bromosulphophthalein (BSP) and indocyanine (cardiac) green. Weak organic acids, such as free fatty acids, are thought to be transported across the plasma membrane by way of a 43-kDa-membrane protein. There are also vesicle and endocytic uptake pathways at the plasma membrane for phospholipids and proteins. Formation of canalicular bile requires the active transport of osmotically active materials into this region. The major transported substances are ionized bile acids by way of a specific 100-kDa-transport protein. Bilirubin monoglucuronide (BMG), diglucuronid (BDG), glutathione conjugates, and glutathione (GSH and GSSG) are transported by the multi-organic anion transporter and cations by a 170-kDa p-glycoprotein, both of which require AT P. Bicarbonate, chloride, sulfate, and oxalate ions are also added to the canalicular bile. Uptake of the bile acids, nonbile acid cholephils, and organic acids also occurs by way of an active transport processes (see the record on Hepatic Transport Mechanisms). Once inside the hepatocyte, many of these cholephils are modified during their movement across the hepatocyte to the canalicular region. Bile acids are often hydroxylated or sulfated. Many nonbile-acid cholephils are conjugated to glutathione by one of the glutathione S-transferase family of enzymes. Bilirubin is also bound to glutathione S-transferase B, also known as ligandin, but this molecule is only involved in its cytosolic movement. Bilirubin is, in fact, glucuronidated by UDP-glucuronyl transferase to form either bilirubin monoglucuronide or digluronide for excretion. When plasma bilirubin is measured, the quantity that is conjugated is usually also estimated, giving an indication in pathological conditions as to where the defect occurs (Table 1). Bilirubin glucuronides and glutathione conjugates are transported into the canaliculus by way of a multi-organic anion transporter (MOAT) protein. Both this and the organic cation transport (OCT) proteins require AT P. Other substances that are actively secreted into bile include sulfate, oxalate, and bicarbonate ions. Canalicular bile flow is basically an osmotic response to active solute transport into the area Paumgartner (1977). It is divided into bile acid-dependent flow, where the bile acid output is the major solute generating bile flow, and bile acid-independent flow, where bile is generated at low-bile acid concentrations, or in addition to bile acid-dependent bile
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Liver-Cholesterol and Bile Formation
Table 2. The clinical relevance of altered bilirubin homeostasis in terms of its metabolism and excretion (see the record on Motility and other Disorders of the Biliary Tract).
flow Erlinger (1977). Bile acid-dependent flow occurs as a result of the active secretion of osmotically active bile acids into the canaliculus. Normally, bile acids ‘‘obligate’’ approximately 8–15 ml of water per mmole of bile acid. That is, they cause this volume of solvent to be drawn into the canaliculus, principally by a paracellular route. This is known as choleresis. The most likely source of this liquid is from the plasma by way of the space of Disse. As a result of solvent-drag, small ions and solutes are also moved into the canaliculus. Accordingly, the composition of the solution obligated by the bile acids is very similar that to the kidney glomerulus, being close to iso-osomotic. The canalicular bile generated by bile acids is therefore nearly always hyperosmotic. The quantity of liquid obligated is bile-acid specific and is known as the choleretic activity. A few bile acids, such as ursodeoxycholic acid, may obligate up to 90 ml of water and are termed hypercholeretic. There are also some bile acids that obligate much less than 8ml of water and are termed hypocholeretic or, in the extreme case, cholestatic, resulting in cessation of bile flow, as is seen with the monohydroxybile acids. Bile-acid secretion also induces the canalicular secretion of phospholipid- and cholesterol-rich vesicles. Bile acid-independent flow is thought to play only a minor role in the generation of bile. The mechanism for the generation of bile acid-independent flow is not clear, but is thought to involve a canalicular ATPase. Other transport systems, such as the sinusoidal Na+/H+ and the canalicular Cl-/HCO-3 antiporters, which play a role in bicarbonate secretion into bile, may also be involved in bile acid-independent canalicular bile flow. The addition of bicarbonate to ductular bile is regulated by secretin. Binding of this hormone to its basolateral ductule plasma membrane receptor results in the activation of adenylate cyclase (AC)protein kinase A and increased chloride diffusion at the luminal membrane. The chloride is then transported back into the cell, coupled to bicarbonate counter-transport. Bicarbonate is produced by cellular carbonic anhydrase. Excess protons are secreted across the basolateral membrane into the blood by way of vesicular proton ATPase.
Liver-Cholesterol and Bile Formation
Canalicular bile is modified by the duct cells and during storage in the gallbladder. The bile ducts change the solute composition of the newly secreted bile through the addition of bicarbonate and water, as well as the reabsorption of glucose and other solutes. Bicarbonate is produced by the action of carbonic anhydrase on carbon dioxide and water within the bile-duct cells. Bicarbonate ions are then transported into the bileduct lumen by a chloride/bicarbonate antiporter at a ratio of 2Cl-/3HCO-3. The chloride is then recycled into the lumen through a chloride channel. The hydrogen ions generated by this reaction are secreted into the blood by way of a vesicular proton ATPase. While many hormones, such as gastrin, VIP, and bombesin, influence bile ductule secretions, the major regulator is secretin (Fig. 6). The binding of secretin to its receptor on the basolateral surface of the bile-duct cell results in the phosphorylation of the luminal chloride channels due to activation of adenylate cyclase and the production of cAMPto activate protein kinase A, which, in turn, phosphorylates the chloride channel. This mechanism for bicarbonate secretion is similar to that seen in the salivary gland and the pancreas. Sodium ions and water are generally thought to migrate by way of the paracellular route into the bile to modify its osmolarity, although aquaporins are associated with the bile ductule cells. Mucus is also secreted by associated cells to protect the biliary tract. Carbon dioxide and water are converted to carbonic acid by carbonic anhydrase. This dissociates and bicarbonate is secreted by way of the bicarbonate/chloride luminal antiporter. Excess protons are removed by a basolateral vesicular ATPase. The system is stimulated by secretin binding to a basolateral receptor that generates cAM P. This results in phosphorylation of the chloride channel/pump by protein kinase A, which subsequently increases chloride uptake by the antiporter. In humans, bile is not normally secreted into the small intestine but is stored in an accessory organ, the gallbladder, during the interdigestive phase and during fasting. While the gallbladder is not absolutely essential, being absent in rats and mice, it does serve a useful purpose in that bile is almost immediately available for brief periods of digestion. However, because the gallbladder only has a volume of 50–75 ml, and the liver produces up to 600 ml of bile in a 24-hour period, some form of concentration must occur during storage. The gallbladder is essentially a sac lined with epithelial cells and surrounded by smooth muscle that is under neurohumoral control.
Fig. 6. Regulation of bicarbonate secretion by bile ductule cells.
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Storage of bile in the gallbladder involves two separate processes: concentration and acidification. Concentration is to reduce the volume to make storage feasible and acidification is to lower both bicarbonate concentrations during concentration and the quantity of anions that are likely to precipitate calcium, which could lead to the formation of gallstones. Concentration is achieved by pumping sodium chloride into the intercellular space between the biliary epithelial cells. Approximately two thirds of the sodium transported into the epithelial cells occurs by way of a Na+/H+ exchanger, and the rest by other unspecified mechanisms. Similarly, chloride ions are thought to be transported by an apical Cl-/HCO-3 transporter, although the exact mechanisms have yet to be confirmed. The hydrogen ions transported into the gallbladder lumen react with bicarbonate ions to form carbonic acid, which then decays to carbon dioxide and water. The removal of bicarbonate leads to a transient hypotonicity within the lumen and provides the osmotic driving force that leads to net water movement across the epithelial cells, concentrating the bile (Fig. 7). Sodium in the bile is transported into the biliary epithelial cells, counter-transporting protons into the bile. Chloride diffusion follows sodium. Sodium chloride is then transported into the extracellular fluid, generating an osmotic gradient that causes the movement of water by both transcellular and paracellular pathways, concentration of the remaining gallbladder bile. The low pH also favors the conversion of carbonate and bicarbonate ions to carbon dioxide and water. The solubility of calcium bilirubinate is enhanced by this low pH (pH 5.5–pH 6). As calcium salt and bilirubin gallstones are most frequently encountered in the Western hemisphere, it is clear that maintaining low pH and low-anion concentration tends to inhibit nucleation and their subsequent formation by precipitation/ crystallization. At this lowered pH, the conditions are favorable for micelle formation by bile salts as the bile becomes progressively more concentrated. Mixed in with these micelles are the secreted phospholipids and cholesterol. This micelle formation results in a lower osmotic activity because osmolarity is proportional to the number of particles in solution. As a consequence, bile-salt concentration in the gallbladder may exceed 300 mM without producing hyperosmotic bile. The osmolarity in both the ducts and the gallbladder remains relatively constant at approximately 280–290 mOsm. The acid environment
Fig. 7. Fluid transport by gallbladder epithelium.
Liver-Cholesterol and Bile Formation
does not damage the epithelial cells because, at this pH, the protective mucins secreted by the epithelia assume their maximum hydrodynamic radius, thereby increasing their protective effect. The protective effect is also enhanced by the viscosity and strong anionic charge of the mucins as a result of their sialate residues. Secretion of bile into the duodenum is regulated by two hormones, cholecystokinin (CCK) and secretin. CCK causes relaxation of the sphincter of Oddi and contraction of the musculature around the gallbladder, whereas secretin potentiates bicarbonate and water secretion into the bile duct. Bile flow is dependent on the pressure generated by the gallbladder during contraction and the compliance of the Sphincter of Oddi. The initial pressure, generated at the canalicular level, is created by canalicular secretion of osmotically active materials that drives water movement and, ultimately, if the Sphincter of Oddi is closed, filling of the gallbladder while it is relaxed during the interdigestive phase. Some of the material secreted in bile is reabsorbed in the small intestine, mainly in the ileal region. Approximately 95% of bile acids are reabsorbed in this region and are taken up from the portal blood by zone 1 hepatocytes. Hepatic extraction of bile acids is extremely efficient, with systemic venous blood levels rarely exceeding 20 mM in normal individuals. As a result of the operative homeostatic mechanism for bile acid synthesis, de novo synthesis of bile acids occurs in zone 3 of the acinus. Furthermore, as bile movement in the liver is countercurrent to blood flow, bile acids accumulated at high concentrations in zone 1 are rapidly re-secreted into bile, minimizing potential damage to the organ. As many of these bile acids are also tightly bound to albumin, the filtered load is quite small (about 100–200 mg/day) and these are almost completely reabsorbed by an active transport mechanism in the proximal renal tubules of the kidney. Bile acids in the intestine may be modified by the bacterial flora to form secondary bile acids, such as deoxycholic acid from cholic acid and lithocholic acid from chenodeoxycholic acid. After uptake by the hepatocytes, these bile acids are further modified by conjugation with glycine, taurine, or sulfation before re-secretion into bile. These are known as tertiary bile acids. Other materials are also reabsorbed in the intestine. Thus, cholesterol is reabsorbed unless it is modified, and bilirubin is heavily modified to form compounds such as the uroporphyrinogensfor renal excretion. The absence of bile is deleterious in that, besides lipid solubilization, this material is also required for the solubilzation and uptake of lipid soluble vitamins, in particular vitamin K, which is required by the liver for the synthesis of many of the coagulation cascade proteins. As it is fat soluble, one consequence of cholestasis is a prolonged coagulation time.
Other Information – Web Sites Cholesterol and Bile Metabolism-http://www.indstate.edu/thcme/mwking/cholesterol. html Cholesterol synthesis-http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/ cholesterol.htm Bile metabolism-http://dwb.unl.edu/Teacher/NSF/C11/C11Links/web.indstate.edu/ thcme/mwking/cholesterol.html
Journal Citations Groothuis, G.M.M., Meijer, D.K.F., 1992. Hepatocyte heterogeneity in bile formation and hepatobiliary transport of drugs. Enzyme, 46(1-3), 94–138.
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Book Citations Paumgartner, G., 1977. Physiology I: Bile Acid-Dependent Bile Flow. Bianchi, L., Gerok, W., Sickinger, K. (Ed.), Liver and Bile, pp. 45–53, MTP Press, London. Erlinger, S., 1977. Physiology II: Bile Acid-Independent Secretion. Bianchi, L., Gerok, W., Sickinger, K. (Ed.), Liver and Bile, pp. 55–62, MTP Press, London. Turley, S.D., Dietschy, J.M., 1988. The Metabolism and Excretion of Cholesterol by the Liver. Arias, I.M., Jakoby, W.B., Popper, H., Schachter, D., Shafritz, D.A. (Ed.), The Liver, Biology and Pathobiology, pp. 617–641, Raven Press, New York. Erlinger, S., 1988. Bile flow. Arias, I.M., Jakoby, W.B., Popper, H., Schachter, D., Shafritz, D.A. (Ed.), The Liver, Biology and Pathobiology, pp. 643–661, Raven Press, New York.