UW Division of Gastroenterology - Gut Course Syllabus (HuBio 551)

	Updated: 10/25/07 11:31 AM

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Authors: W. Volwiler, R.A. Willson, A.M. Larson, and J.D. Ostrow
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N. Formation of Gallstones "I got stones in my passway And my road seems dark at night I have pains in my heart They have taken my appetitie" Robert Johnson, 1937
1. Incidence and definition of gallstones
Gallstone disease is very common		Approximately 20% of North American men and 30% of women develop gallstones over a lifetime. The incidence is much higher (80%) in Native Americans. Gallstones are most common after age 40.
Gallstones consist of a 3-dimensional matrix of mucin and small polypeptides on which crystals form and grow		Gallstones (biliary calculi) consist of poorly-soluble components of bile precipitated on a three-dimensional matrix of mucins and proteins. The major precipitates are cholesterol, calcium bilirubinates, and calcium salts of phosphate, carbonate and palmitate. The matrix consists of a network of large, polymeric mucin glycoproteins (the scaffolding) to which are bound small amphipathic, anionic polypeptides (the mortar). Cholesterol is deposited directly on the hydrophobic domains of mucins, whereas the calcium salts are bound to the anionic amino acid sidechains of the polypeptides. Bacteria are readily cultured from the core of almost all brown pigment stones and their RNA can be detected in cholesterol, and black pigment stones.
2. Regulation of biliary lipid secretion
Bile salt secretion mediates the passage of lecithin and cholesterol into bile		The secretion of cholesterol and phospholipids (>90% lecithin) into bile is regulated by the independent secretion of bile salts. Lecithin is translocated to the external leaflet of the canalicular membrane by a phosphatidylcholine flippase (MDR3). Bile salt micelles within the canalicular lumen leach lecithin-cholesterol vesicles from the external leaflet of the canalicular membrane. As bile flows down the biliary tree, these vesicles are gradually converted to mixed micelles, whose composition is more conducive to solubilizing cholesterol.
3. Modification of bile by the gallbladder (Table 4)
Between meals, bile is diverted into the gallbladder.		Over a 24-hour period, a normal adult human produces from 400 - 1100 ml of hepatic bile. The sphincter of Oddi holds the common bile duct relatively closed at the ampulla of Vater during the interdigestive period, diverting most of the hepatic bile into the relaxed gallbladder. Within the gallbladder, the hepatic bile is converted into concentrated gallbladder bile, which is stored until ejected into the duodenum upon eating.
Bile is concentrated and acidified in the gallbladder Cholesterol, lecithin and unconjugated bilirubin are partly reabsorbed in the gallbladder		In the gallbladder, the bile is concentrated up to 10-fold by active mucosal absorption of water and electrolytes (mainly NaC1). Isotonicity with plasma is maintained, however, because the bile salt anions and associated cations (mainly Na⁺ and Ca⁺⁺) aggregate into larger micelles, decreasing their osmotic activity. The bile is also acidified by absorption of sodium ions (Na⁺) in exchange for hydrogen ions (H⁺), while K⁺ and Ca⁺⁺ equilibrate passively with unbound K⁺ and Ca⁺⁺ in plasma. Most of the HCO₃- is lost due to neutralization by H⁺ and diffusion of the CO₂ formed. Some of the phospholipid and an even greater proportion of biliary cholesterol is absorbed, so that the concentration of cholesterol relative to bile salts and phospholipid is lower in gallbladder bile than hepatic bile. A small proportion of the bilirubin glucuronides is hydrolyzed, and some of the unconjugated bilirubin thus formed is absorbed as well.
Table 4 [Click for Larger Image] Comparison of Normal Hepatic and Gallbladder Bile
4. Emptying of gallbladder bile into the duodenum
During eating, CCK release mediates emptying of gallbladder bile into the duodenum		Gallbladder bile is emptied into the duodenum during eating by a co-ordinated contraction of the gallbladder and relaxation of the sphincter of Oddi. This is mediated by the release of CCK (cholecystokinin) from the mucosa of the proximal small intestine, stimulated by free fatty acids and aromatic amino acids liberated during intraluminal digestion. The action of CCK on the gallbladder is enhanced by increased vagal activity during eating.
Enterohepatic cycling (EHC) of bile salts enhanced during meals		In the duodenum, the concentrated mixed bile salt-lipid micelles aid in fat digestion and inhibit the further release of CCK. The gallbladder then relaxes, sphincter tone returns and hepatic bile again flows into the gallbladder. CCK also promotes intestinal motility and thus the passage of bile salts to their major site of reabsorption in the distal ileum. CCK thus enhances the entero-hepatic circulation of bile salts during eating.
5. Requirements and stages of gallstone formation
		Gallstone formation requires a) supersaturation, b) an excess of promotors over inhibitors of crystallization, c) a matrix template on which crystallization can occur (biomineralization), and d) bile stasis (retention in the gallbladder to allow time to grow).
		a. Supersaturation
Supersaturation necessary but not sufficient for gallstones to form		Gallstones can form only when bile becomes supersaturated with one or more of the components mentioned above because i) the bile secreted by the hepatocyte is already supersaturated with one of its components, and/or ii) extrahepatic bile ducts and gallbladder alter the composition and/or pH of the bile.
		b. Precipitation
Inhibitors versus promotors of precipitation Sludge		Supersaturated bile is necessary, but not sufficient for precipitation and/or gallstone formation to occur. Bile contains proteins and glycoproteins, some of which stabilize, and some of which promote precipitation of the supersaturated component(s). The balance between these inhibitors and promotors, as well as the degree of supersaturation, determines whether the supersaturated component(s) will precipitate. The excess of the supersaturated component may precipitate as crystals, which may aggregate loosely with mucins to form sludge; sludge particles are small and soft and normally are easily emptied from the gallbladder when it contracts.
		c. Growth
Growth on a matrix Bile stasis, and the kinetics of stone formation		If suitable matrix is present, the minerals crystallize on the matrix template in an orderly fashion, forming a miniature calculus or nidus. If the nidus is retained in the gallbladder, progressive accretion of matrix and the insoluble mineral can occur, with growth of the nidus into a gallstone. Stasis occurs when emptying of the gallbladder is impaired, or bile flow in the ducts is impeded by partially obstructed.
6. Types of Gallstones
There are three major types of gallstones		There are three major types of gallstones (Table 5): cholesterol stones, black pigment stones, and brown pigment stones, that differ in composition, structure, and pathogenesis. Cholesterol stones with a significant proportion of pigment are often called "mixed" stones. About 80% of human gallstones in the Western countries are of the cholesterol or mixed type. Although the majority of stones in Asian countries are pigment gallstones, cholesterol stones have become predominant among Asians who have switched to a more Western-style diet.
Table 5 [Click for Larger Image] Three Major Types of Gallstones
7. Cholesterol Gallstones
		a. Bile supersaturation with cholesterol
Cholesterol in bile is solubilized by lecithin ± bile salts (vesicles and mixed micelles) Normal bile is supersaturated with cholesterol, but more markedely so in patients with cholesterol gallstones		Cholesterol monomers are essentially insoluble in an aqueous solution. In normal bile, cholesterol is transported either complexed with lecithins in vesicles, or aggregated with phospholipids and bile acids as mixed micelles. Small, unilamellar vesicles with a relatively low proportion of cholesterol are thermodynamically stable. As more cholesterol is added, the small unilamellar vesicles aggregate and fuse to yield less stable, large, multilamellar vesicles, from which cholesterol is more prone to precipitate. The total concentrations and relative proportions of cholesterol to bile salts + lecithin determine if cholesterol can be kept in solution. Preferential transfer of lecithin from vesicles to mixed micelles increases vesicular supersaturation as the bile flows to the duodenum. Thus, bile is normally mildly supersaturated with cholesterol.
		b. Causes of increased supersaturation of bile with cholesterol.
		One or more factors may play a role in an individual patient.
Increased hepatic cholesterol synthesis and secretion is most common mechanism		i. Hepatic Factors: The liver can produce more supersaturated bile in two ways: a) by a relative decrease in synthesis and secretion of bile salts and/or phospholipids; and b) by a relative increase in synthesis and secretion of cholesterol. Impaired bile salt secretion is important in subjects with a history or family history of recurrent cholestasis of pregnancy. The second mechanism is more common, however, especially among Native Americans, diabetics and obese subjects.
Genetic factors Female preponderance		The composition of hepatic bile is strongly influenced by genetic factors, which may explain: a) the extremely high incidence of cholesterol gallstones (more than 50%) in certain ethnic groups (Native Americans) and absence in others (Masai of Africa); b) the well-established familial tendency to cholesterol gallstone formation; and c) the strong predominance of cholesterol gallstones in females. Genes have been identified in inbred mice that influence the lipid composition of bile, often by disturbing the feedback mechanisms that control hepatic bile acid and cholesterol synthesis.
Decreased EHC of bile salts sometimes plays a role but more often leads to formation of black pigment gallstones		ii. Intestinal factors: When the enterohepatic recycling of bile salts is decreased, there is limited capacity for an increase in hepatic synthesis to compensate for the increased loss of bile salts. Therefore total hepatic secretion of bile salts declines and cholesterol synthesis is enhanced due to diminished inhibition by recycled bile salts. However, the enhanced cholesterol synthesis is overbalanced by the increased conversion of hepatic cholesterol to bile salts, so cholesterol saturation of bile does not increase. Thus, this scenario, which occurs with a) resections or mucosal disease of the distal ileum, or b) intraluminal sequestration of bile salts, usually (for uncertain reasons) results in formation of black pigment gallstones, but rarely cholesterol gallstones.
Stasis, and altered absorptive function of the gallbladder		iii. Cholecystic factors: Stasis of bile in the gallbladder, occurs in pregnancy and with prolonged fasting (e.g. when patients are fed parenterally). This favors chronic infection of gallbladder bile, with enhanced deconjugation of bile salts plus increased mucosal permeability, selectively increasing absorption of bile salts from gallbladder bile, increasing cholesterol supersaturation. On the other hand, chronic cholecystitis impairs mucosal absorption of water and electrolytes, resulting in decreases in biliary lipid concentrations and cholesterol supersaturation. Thus, the main effect of gallbladder stasis is not via an increase in cholesterol saturation, but retention of small niduses for biomineralization, allowing time for their growth into gallstones.
		c. Therapy of cholesterol gallstones
Laparoscopic cholecystectomy is the preferred treatment Oral ursodeoxy-cholic acid can desaturate bile and dissolve gallstones		Surgical removal of the gallbladder by laparoscopy or laparotomy is the definitive therapy for cholelithiasis. For patients at high risk for surgery, non-surgical alternatives exist. Oral administration of ursodeoxycholic acid enhances hepatic secretion of bile salts, reduces hepatic synthesis and intestinal absorption of cholesterol, and diminishes supersaturation of bile with cholesterol. In the 40% of patients in whom this treatment renders the bile undersaturated with cholesterol, the cholesterol gallstones will gradually dissolve. If the gallbladder is not surgically removed, however, the gallstones recur in over 50% of the patients within 5 years, especially those who initially had multiple gallstones. Long-term prophylaxis with ursodeoxycholic acid, to keep bile saturation low, and with aspirin, to limit mucin secretion, may help prevent such recurrences.
8. Pigment Gallstones
		a. Solubility of bilirubins and their calcium salts
Bilirubin conjugates and their calcium salts are soluble UCB and its calcium salts are poorly soluble		Normally, over 98% of the bilirubin in hepatic and gallbladder bile is in the conjugated form (>80% diglucuronide), which is water-soluble; its calcium salts are soluble also. The <2% of unconjugated bilirubin (UCB), formed by hydrolysis of the secreted conjugates by biliary β-glucuronidases, is, by contrast very poorly soluble in water. The calcium salts of UCB are also extremely insoluble, and may precipitate if the product of the concentrations of unbound calcium and UCB anions exceeds the solubility product of Ca-UCB. Normally the unbound concentrations of calcium and UCB in bile are kept low by binding to bile salts, albumin, mucins and anionic polypeptides.
		b. Factors leading to precipitation of calcium bilirubinates in bile
Increase in calcium and/or bilirubinate ions can lead to pigment stone formation		Precipitation of pigments in bile and formation of pigment gallstones, can occur if there are increases in concentrations of unbound calcium and/or bilirubinate ions in bile. Increased ionized calcium in bile occurs mainly due to equilibration with increased plasma calcium, as in hyperparathyroidism. An increase in unbound bilirubinate anions in bile may result from an increase in pH, an increase in total UCB in bile, and/or decreased concentrations of bile salts available to solubilize UCB.
↑ plasma Ca²⁺ leads to ↑ bile Ca²⁺		i) Increased ionized calcium in bile can occur only through an increase in unbound, ionized calcium in plasma (e.g. hyperparathyroidism) (free Ca²⁺ ions diffuse passively across the gallbladder and biliary tree)
		ii) Increased Total UCB in bile may be casued by:
Hemolysis increases bile bilirubin Increased hydrolysis of bilirubin conjugates due to increased activity of β-glucuronidase		Increased secretion of bilirubin conjugates into bile with normal rates of hydrolysis to UCB, as in patients with chronic hemolysis (e.g., in sickle-cell disease and cirrhosis). Increased β-glucuronidase activity in bile, either released by damaged hepatocytes or cholangiocytes, or by bacteria (e.g., E. coli) in infected bile. In infected bile, hydrolysis of lecithins by bacterial phospholipases also occurs and the released fatty acids (mainly palmitate) precipitate as calcium soaps, giving brown stones their characteristic soft, greasy consistency. The increased proportion of bilirubin monoglucuronide in the bile in Gilbert's syndrome enhances the hydrolytic formation of UCB, as one less glucuronide group is removed than for diglucuronide.
Polymerization of pigment		Finally, by mechanisms likely initiated by oxidation of precipitated calcium bilirubinates, a 3-dimensional network polymer of pigments and matrix may be formed. The mixture of varicolored oxidative derivatives of UCB in the polymer gives the polymer its black or brown color.
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