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Biliary lipids and cholesterol gallstone disease

2008; Elsevier BV; Volume: 50; Linguagem: Inglês

10.1194/jlr.r800075-jlr200

1539-7262

David Q.‐H. Wang, David E. Cohen, Martin C. Carey,

Pediatric Hepatobiliary Diseases and Treatments

Biliary lipids are a family of four dissimilar molecular species consisting of a mixture of bile salts (substituted cholanoic acids), phospholipids, mostly (>96%) diacylphosphatidylcholines, unesterified cholesterol, and bilirubin conjugates known trivially as lipopigments. The primary pathophysiological defect in cholesterol gallstone disease is hypersecretion of hepatic cholesterol into bile with less frequent hyposecretion of bile salts and/or phospholipids. Several other gallbladder abnormalities contribute and include hypomotility, immune-mediated inflammation, hypersecretion of gelling mucins, and accelerated phase transitions; there is also reduced intestinal motility that augments "secondary" bile salt synthesis by the anaerobic microflora. Cholesterol nucleation is initiated when unilamellar vesicles of cholesterol plus biliary phospholipids fuse to form multilamellar vesicles. From these "plate-like" cholesterol monohydrate crystals, the building blocks of macroscopic stones are nucleated heterogeneously by mucin gel. Multiple Lith gene loci have been identified in inbred mice, paving the way for discovery of an ever-increasing number of LITH genes in humans. Because of the frequency of the metabolic syndrome today, insulin resistance and LITH genes all interact with a number of environmental cholelithogenic factors to cause the gallstone phenotype. This review summarizes current concepts of the physical-chemical state of biliary lipids in health and in lithogenic bile and outlines the molecular, genetic, hepatic, and cholecystic factors that underlie the pathogenesis of cholesterol gallstones. Biliary lipids are a family of four dissimilar molecular species consisting of a mixture of bile salts (substituted cholanoic acids), phospholipids, mostly (>96%) diacylphosphatidylcholines, unesterified cholesterol, and bilirubin conjugates known trivially as lipopigments. The primary pathophysiological defect in cholesterol gallstone disease is hypersecretion of hepatic cholesterol into bile with less frequent hyposecretion of bile salts and/or phospholipids. Several other gallbladder abnormalities contribute and include hypomotility, immune-mediated inflammation, hypersecretion of gelling mucins, and accelerated phase transitions; there is also reduced intestinal motility that augments "secondary" bile salt synthesis by the anaerobic microflora. Cholesterol nucleation is initiated when unilamellar vesicles of cholesterol plus biliary phospholipids fuse to form multilamellar vesicles. From these "plate-like" cholesterol monohydrate crystals, the building blocks of macroscopic stones are nucleated heterogeneously by mucin gel. Multiple Lith gene loci have been identified in inbred mice, paving the way for discovery of an ever-increasing number of LITH genes in humans. Because of the frequency of the metabolic syndrome today, insulin resistance and LITH genes all interact with a number of environmental cholelithogenic factors to cause the gallstone phenotype. This review summarizes current concepts of the physical-chemical state of biliary lipids in health and in lithogenic bile and outlines the molecular, genetic, hepatic, and cholecystic factors that underlie the pathogenesis of cholesterol gallstones. In the United States, cholelithiasis [mostly cholesterol (∼85%) and "black" pigment (∼15%) stones] is one of the most prevalent and costly digestive diseases, with at least 20 million Americans affected (1Liver Disease Subcommittee of the Digestive Disease Interagency Coordinating Committee. 2004. Action Plan for Liver Disease Research. National Institutes of Health, Bethesda, MD.Google Scholar). Prevalence of cholesterol stones is increasing because of the epidemic of obesity, with insulin resistance being a fundamental feature of the metabolic syndrome. Approximately one million new cases of gallstones are discovered each year, 700,000 cholecystectomies are performed, and unavoidable complications result in 3,000 deaths in the United States. Furthermore, medical expenses for the treatment of gallstone disease exceeded $6 billion in the bimillenial year. Recent studies on both humans and mouse models have demonstrated that polygenes underlie the predisposition to develop most cholesterol gallstones (2Wang D.Q-H. Paigen B. Carey M.C. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: physical-chemistry of gallbladder bile.J. Lipid Res. 1997; 38: 1395-1411Abstract Full Text PDF PubMed Google Scholar, 3Katsika D. Grjibovski A. Einarsson C. Lammert F. Lichtenstein P. Marschall H.U. Genetic and environmental influences on symptomatic gallstone disease: a Swedish study of 43,141 twin pairs.Hepatology. 2005; 41: 1138-1143Crossref PubMed Scopus (203) Google Scholar, 4Khanuja B. Cheah Y.C. Hunt M. Nishina P.M. Wang D.Q-H. Chen H.W. Billheimer J.T. Carey M.C. Paigen B. Lith1, a major gene affecting cholesterol gallstone formation among inbred strains of mice.Proc. Natl. Acad. Sci. USA. 1995; 92: 7729-7733Crossref PubMed Scopus (189) Google Scholar). The powerful genetic technique of quantitative trait locus (QTL) analysis has identified multiple cholesterol gallstone (Lith) gene loci in inbred mice (4Khanuja B. Cheah Y.C. Hunt M. Nishina P.M. Wang D.Q-H. Chen H.W. Billheimer J.T. Carey M.C. Paigen B. Lith1, a major gene affecting cholesterol gallstone formation among inbred strains of mice.Proc. Natl. Acad. Sci. USA. 1995; 92: 7729-7733Crossref PubMed Scopus (189) Google Scholar). Dissecting the physical-chemical, pathophysiological, and genetic influences of individual Lith genes has provided a logical framework to elucidate the genetic mechanisms of cholesterol gallstone disease in humans. Here, we summarize biliary lipid physical chemistry and the molecular pathogenesis of cholesterol gallstone formation, focusing principally on recent progress. Cholesterol, phospholipids, and bile salts are the major lipid components of bile (Fig. 1). Biliary cholesterol is nonesterified and accounts for at least 97% of total sterols, the remainder comprising cholesterol precursors and dietary phyto- and conchosterols. The major phospholipids of bile are mixed diacylphosphatidylcholines. These insoluble, swelling amphiphiles are structured with a hydrophilic, zwitterionic phosphocholine headgroup and two hydrophobic fatty acid chains. Because cholesterol and phospholipids are insoluble in an aqueous medium, mixed micelles with bile salts and unilamellar (single-bilayered) vesicles are required to maintain them in solution. The common bile salts are soluble amphiphiles that consist of a hydrophobic steroid nucleus of four fused hydrocarbon rings, subtending polar hydroxyl functions on one face, and a flexible aliphatic side chain amidated with glycine or taurine. Cholate and chenodeoxycholate, the principal bile salts of humans, are "primary" species synthesized directly from cholesterol in the liver. The "secondary" bile salts, deoxycholate and lithocholate, are modifications of the primary bile salts by specialized anaerobic intestinal bacteria (5Hofmann A.F. Biliary secretion and excretion in health and disease: current concepts.Ann. Hepatol. 2007; 6: 15-27Crossref PubMed Google Scholar). "Tertiary" bile salts, ursodeoxycholates, the 7βOH-epimer of chenodeoxycholate, and lithocholate-3-SO4, are the result of modifications of secondary bile salts by intestinal flora and hepatocytes. All of these cycle continuously in humans in a highly efficient enterohepatic circulation. Bile salt monomers self-associate spontaneously to form simple micelles when their concentrations exceed the critical micellar concentration (Fig. 1). These simple micelles are small (∼3 nm in diameter), thermodynamically stable aggregates that solubilize small quantities of biliary cholesterol. Mixed micelles are larger (∼4 to 8 nm in diameter), quasispherical, and thermodynamically stable aggregates that are composed of bile salts, cholesterol, and phospholipids with a high capacity to solubilize cholesterol (Fig. 1). Vesicles are spherical structures (40 to 100 nm in diameter) composed of unilamellar phospholipids and cholesterol bilayers but little bile salt (Fig. 1). Liquid crystals (>500 nm in diameter) are multilamellar vesicles derived from fusion of unilamellar vesicles principally in the gallbladder (6Mazer N.A. Carey M.C. Quasi-elastic light-scattering studies of aqueous biliary lipid systems. Cholesterol solubilization and precipitation in model bile solutions.Biochemistry. 1983; 22: 426-442Crossref PubMed Scopus (150) Google Scholar, 7Crawford J.M. Crawford G.M. Mo¨ckel, A.R. Hagen S.J. Hatch V.C. Barnes S. Godleski J.J. Carey M.C. Imaging biliary lipid secretion in the rat: ultrastructural evidence for vesiculation of the hepatocyte canalicular membrane.J. Lipid Res. 1995; 36: 2147-2163Abstract Full Text PDF PubMed Google Scholar). Unilamellar vesicles are usually stable and constitute an additional transport system for cholesterol molecules, but liquid crystals invariably imply initiation of the cholesterol nucleation sequence. Although conjugated bilirubin molecules in "ridge-tile" conformation (Fig. 1) are water soluble, they are bound to the hydrophilic faces of bile salt molecules in tight π-orbital-OH interactions. As demonstrated by ternary phase diagrams (Fig. 2), cholesterol is solubilized in thermodynamically stable simple and mixed micelles in small one-phase micellar zones. By contrast, "supersaturated" bile implies cholesterol present in concentrations above and beyond what can be solubilized in the micellar zones; however "true" micellar supersaturation is transient, and excess micellar cholesterol becomes dispersed in cholesterol-phospholipid vesicles (8Carey M.C. Small D.M. The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and dissolution in man.J. Clin. Invest. 1978; 61: 998-1026Crossref PubMed Scopus (534) Google Scholar). For a relative lipid composition in a native bile sample plotted on an appropriate triangular phase diagram, the proportional distance along an axis joined to the cholesterol apex (Fig. 2), i.e., ratio of the actual amount of cholesterol present to the maximal micellar solubility, is defined as the cholesterol saturation index (9Carey M.C. Critical tables for calculating the cholesterol saturation of native bile.J. Lipid Res. 1978; 19: 945-955Abstract Full Text PDF PubMed Google Scholar). Physical-chemical and imaging studies demonstrate that bile salts stimulate the biliary secretion of unilamellar vesicles (10Cohen D.E. Angelico M. Carey M.C. Quasielastic light scattering evidence for vesicular secretion of biliary lipids.Am. J. Physiol. 1989; 257: G1-G8Crossref PubMed Google Scholar) from the external hemileaflet of the canalicular membrane (7Crawford J.M. Crawford G.M. Mo¨ckel, A.R. Hagen S.J. Hatch V.C. Barnes S. Godleski J.J. Carey M.C. Imaging biliary lipid secretion in the rat: ultrastructural evidence for vesiculation of the hepatocyte canalicular membrane.J. Lipid Res. 1995; 36: 2147-2163Abstract Full Text PDF PubMed Google Scholar, 11So¨mjen G.J. Marikovsky Y. Lelkes P. Gilat T. Cholesterol-phospholipid vesicles in human bile: an ultrastructural study.Biochim. Biophys. Acta. 1986; 879: 14-21Crossref PubMed Scopus (55) Google Scholar). The radii of vesicles within the canalicular spaces of rat liver by transmission electron microscopy (7Crawford J.M. Crawford G.M. Mo¨ckel, A.R. Hagen S.J. Hatch V.C. Barnes S. Godleski J.J. Carey M.C. Imaging biliary lipid secretion in the rat: ultrastructural evidence for vesiculation of the hepatocyte canalicular membrane.J. Lipid Res. 1995; 36: 2147-2163Abstract Full Text PDF PubMed Google Scholar) and dynamic light-scattering spectroscopy (12Mo¨ckel G.M. Gorti S. Tandon R.K. Tanaka T. Carey M.C. Microscope laser light-scattering spectroscopy of vesicles within canaliculi of rat hepatocytes couplets.Am. J. Physiol. 1995; 269: G73-G84PubMed Google Scholar) vary between 60 and 80 nm. Dynamic light scattering spectroscopy (10Cohen D.E. Angelico M. Carey M.C. Quasielastic light scattering evidence for vesicular secretion of biliary lipids.Am. J. Physiol. 1989; 257: G1-G8Crossref PubMed Google Scholar) has also been applied to quantify the changing proportions of vesicles as they are slowly transformed into mixed micelles in native bile. In contrast with the otherwise insoluble lipids, bile salts enter the canalicular space as monomers and include those that are newly synthesized ( 95%). The principal driving forces for biliary lipid secretion are ATP binding cassette (ABC) transporters located on the canalicular membrane of the hepatocyte. ABCB11 is the dedicated common bile salt export pump (13Gerloff T. Stieger B. Hagenbuch B. Madon J. Landmann L. Roth J. Hofmann A.F. Meier P.J. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver.J. Biol. Chem. 1998; 273: 10046-10050Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar, 14Wang R. Salem M. Yousef I.M. Tuchweber B. Lam P. Childs S.J. Helgason C.D. Ackerley C. Phillips M.J. Ling V. Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis.Proc. Natl. Acad. Sci. USA. 2001; 98: 2011-2016Crossref PubMed Scopus (282) Google Scholar). The relationship between bile salt secretion and both cholesterol and phospholipid secretion is curvilinear. At low bile salt secretion rates (<10 μmol/h/kg in humans), more cholesterol is secreted per molecule of bile salt than at higher rates. At high bile salt secretion rates, for example, during and after eating, biliary saturation is less than during the interprandial periods. This diurnal rhythm persists, but is set to higher cholesterol levels, in the lithogenic state (15Carey M.C. Duane W. Enterohepatic circulation. In The Liver: Biology and Pathobiology. 3rd Edition. I. M. Arias, J. L. Boyer, N. Fausto, W. B. Jakoby, D. Schachter, and D. A. Shafritz. Raven Press, New York1994: 719-767Google Scholar). Hepatic secretion of biliary phosphatidylcholines is dependent on the product of the Abcb4 gene in mice (16Smit J.J. Schinkel A.H. Elferink R.P. Oude Groen A.K. Wagenaar E. Deemter L.van Mol C.A. Ottenhoff R. Lugt N.M. van der Roon M.A. van al et Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.Cell. 1993; 75: 451-462Abstract Full Text PDF PubMed Scopus (1327) Google Scholar). ABCB4 is uniquely responsible for translocation or "flipping" of biliary-destined phosphatidylcholine molecules from the inner to outer bilayer leaflet of the canalicular membrane. As evidenced by transmission electron microscopy, when Abcb4 is knocked out in mice, vesicles disappear from the canalicular space only to reappear with the introduction of the expressed human homolog ABCB4 (17Crawford A.R. Smith A.J. Hatch V.C. Elferink R.P. Oude Borst P. Crawford J.M. Hepatic secretion of phospholipid vesicles in the mouse critically depends on mdr2 or MDR3 P-glycoprotein expression. Visualization by electron microscopy.J. Clin. Invest. 1997; 100: 2562-2567Crossref PubMed Scopus (132) Google Scholar, 18Oude Elferink R.P. Ottenhoff R. Wijland M.van Smit J.J. Schinkel A.H. Groen A.K. Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse.J. Clin. Invest. 1995; 95: 31-38Crossref PubMed Google Scholar). Overexpression of the canalicular heterodimeric ABCG5/ABCG8 transporter increases the cholesterol content of gallbladder bile (19Yu L. Hammer J.Li-Hawkins, R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol.J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (605) Google Scholar). However, ABCG5/ABCG8 requires a functioning ABCB4, indicating the necessity for a hydrophobic "sink" for solubilizing the secreted cholesterol molecules. Gallstone formation is observed in ABCG5 or ABC/G8 knockout mice challenged with a lithogenic diet (20Wang H.H. Patel S.B. Carey M.C. Wang D.Q-H. Quantifying anomalous intestinal sterol uptake, lymphatic transport, and biliary secretion in Abcg8(−/−) mice.Hepatology. 2007; 45: 998-1006Crossref PubMed Scopus (51) Google Scholar), suggesting that an ABCG5/ABCG8-independent pathway for biliary cholesterol secretion exists. The primary pathophysiologic defect in most cholelithogenic humans is hepatic hypersecretion of cholesterol, which may be accompanied by normal, high, or low secretion rates of biliary bile salts and phospholipids. Singly or in combination, this represents the cardinal hepatic pathophysiology inducing cholesterol supersaturation of gallbladder bile (8Carey M.C. Small D.M. The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and dissolution in man.J. Clin. Invest. 1978; 61: 998-1026Crossref PubMed Scopus (534) Google Scholar, 21Admirand W.H. Small D.M. The physicochemical basis of cholesterol gallstone formation in man.J. Clin. Invest. 1968; 47: 1043-1052Crossref PubMed Scopus (792) Google Scholar). Triangular phase diagrams for model systems of cholesterol, egg yolk lecithin, and bile salts in 0.15 M NaCl not only act as templates to determine the physical states of natural biles at equilibrium (8Carey M.C. Small D.M. The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and dissolution in man.J. Clin. Invest. 1978; 61: 998-1026Crossref PubMed Scopus (534) Google Scholar, 21Admirand W.H. Small D.M. The physicochemical basis of cholesterol gallstone formation in man.J. Clin. Invest. 1968; 47: 1043-1052Crossref PubMed Scopus (792) Google Scholar) but also reveal cholesterol crystallization pathways (Fig. 2). Five different crystallization pathways with individual phase transition sequences (A to E) have been identified as functions of bile salt-to-lecithin ratio, common bile salt species (with varying hydrophilic/hydrophobic balance), total lipid concentration, and temperature, as well as cholesterol saturation indices (22Wang D.Q-H. Carey M.C. Complete mapping of crystallization pathways during cholesterol precipitation from model bile: influence of physical-chemical variables of pathophysiologic relevance and identification of a stable liquid crystalline state in cold, dilute and hydrophilic bile salt-containing systems.J. Lipid Res. 1996; 37: 606-630Abstract Full Text PDF PubMed Google Scholar). In region A (Fig. 2) with the lowest phospholipid contents, arc-like crystals, representing a monoclinic polymorph of cholesterol monohydrate of an elongated habit (23Weihs D. Schmidt J. Goldiner I. Danino D. Rubin M. Talmon Y. Konikoff F.M. Biliary cholesterol crystallization characterized by single-crystal cryogenic electron diffraction.J. Lipid Res. 2005; 46: 942-948Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), appear first and evolve via helical and tubular crystalline structures to form classic plate-like triclinic cholesterol monohydrate crystals. In region B (Fig. 2) with higher phospholipid contents, classic cholesterol monohydrate crystals form earlier than polymorphic cholesterol monohydrate crystals. In region C (Fig. 2) with typical physiological phospholipid contents, early multilamellar liquid crystals are followed by classic plate-like cholesterol monohydrate crystals; subsequently, the polymorphic cholesterol monohydrate crystals appear. In region D (Fig. 2) with still higher lecithin contents, liquid crystals are followed by classic plate-like cholesterol monohydrate crystals only. In region E (Fig. 2) at the highest lecithin contents, liquid crystals are stable indefinitely, and solid cholesterol crystals never form. Decreases in temperature (37°C to 4°C), total lipid concentration (7.5–2.5 g/dl), and bile salt hydrophobicity (Fig. 2) progressively shift all crystallization pathways to lower lecithin contents as well as retarding crystallization and reducing micellar cholesterol solubilities (22Wang D.Q-H. Carey M.C. Complete mapping of crystallization pathways during cholesterol precipitation from model bile: influence of physical-chemical variables of pathophysiologic relevance and identification of a stable liquid crystalline state in cold, dilute and hydrophilic bile salt-containing systems.J. Lipid Res. 1996; 37: 606-630Abstract Full Text PDF PubMed Google Scholar). The cholesterol crystallization pathways and sequences observed in human gallbladder bile are identical to those occurring in appropriately matched model bile (24Wang D.Q-H. Carey M.C. Characterization of crystallization pathways during cholesterol precipitation from human gallbladder biles: identical pathways to corresponding model biles with three predominating sequences.J. Lipid Res. 1996; 37: 2539-2549Abstract Full Text PDF PubMed Google Scholar). Nonetheless, the kinetics of these phase transitions are faster in lithogenic human bile compared with either model bile (24Wang D.Q-H. Carey M.C. Characterization of crystallization pathways during cholesterol precipitation from human gallbladder biles: identical pathways to corresponding model biles with three predominating sequences.J. Lipid Res. 1996; 37: 2539-2549Abstract Full Text PDF PubMed Google Scholar) or supersaturated gallbladder bile from control subjects (25Holan K.R. Holzbach R.T. Hermann R.E. Cooperman A.M. Claffey W.J. Nucleation time: a key factor in the pathogenesis of cholesterol gallstone disease.Gastroenterology. 1979; 77: 611-617Abstract Full Text PDF PubMed Scopus (450) Google Scholar). It is believed that the principal heterogenous pronucleating/kinetic agent in gallbladder bile is mucin gel (26Lee S.P. LaMont J.T. Carey M.C. Role of gallbladder mucus hypersecretion in the evolution of cholesterol gallstones.J. Clin. Invest. 1981; 67: 1712-1723Crossref PubMed Scopus (311) Google Scholar). Gallbladder motility is compromised early in cholesterol gallstone formation (27Portincasa P. Ciaula A.Di Vendemiale G. Palmieri V. Moschetta A. Palasciano G.P. Vanberge-Henegouwen, and G. Gallbladder motility and cholesterol crystallization in bile from patients with pigment and cholesterol gallstones.Eur. J. Clin. Invest. 2000; 30: 317-324Crossref PubMed Scopus (58) Google Scholar) in part because of absorption of cholesterol molecules (28Erranz B. Miquel J.F. Argraves W.S. Barth J.L. Pimentel F. Marzolo M.P. Megalin and cubilin expression in gallbladder epithelium and regulation by bile acids.J. Lipid Res. 2004; 45: 2185-2198Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and their incorporation into and stiffening of sarcolemmal membranes (29Yu P. Chen Q. Harnett K.M. Amaral J. Biancani P. Behar J. Direct G protein activation reverses impaired CCK signaling in human gallbladders with cholesterol stones.Am. J. Physiol. 1995; 269: G659-G665PubMed Google Scholar). The result is diversion of more hepatic bile into the intestine and as a consequence elevation in deoxycholate conjugate levels in bile (15Carey M.C. Duane W. Enterohepatic circulation. In The Liver: Biology and Pathobiology. 3rd Edition. I. M. Arias, J. L. Boyer, N. Fausto, W. B. Jakoby, D. Schachter, and D. A. Shafritz. Raven Press, New York1994: 719-767Google Scholar). This in turn further promotes cholesterol hypersecretion as well as more rapid nucleation and crystallization in gallbladder bile (22Wang D.Q-H. Carey M.C. Complete mapping of crystallization pathways during cholesterol precipitation from model bile: influence of physical-chemical variables of pathophysiologic relevance and identification of a stable liquid crystalline state in cold, dilute and hydrophilic bile salt-containing systems.J. Lipid Res. 1996; 37: 606-630Abstract Full Text PDF PubMed Google Scholar). It is still far from settled whether endogenous or infective intestinal bacteria and an activated immune system (30Maurer K.J. Rao V.P. Ge Z. Rogers A.B. Oura T.J. Carey M.C. Fox J.G. T-cell function is critical for murine cholesterol gallstone formation.Gastroenterology. 2007; 133: 1304-1315Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) play facilitating roles in human cholesterol gallstone formation. Studies in many laboratory animals indicate that altering intestinal cholesterol absorption greatly influences cholesterol cholelithogenesis (31Buhman K.K. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese Jr., R.V. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice.Nat. Med. 2000; 6: 1341-1347Crossref PubMed Scopus (296) Google Scholar, 32Wang H.H. Portincasa P. Uribe N.Mendez-Sanchez, M. Wang D.Q-H. Effect of ezetimibe on the prevention and dissolution of cholesterol gallstones.Gastroenterology. 2008; 134: 2101-2110Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), mostly because these diets contain massive amounts of cholesterol and cholic acid to facilitate intestinal absorption. Nonetheless, it appears that the extent of intestinal cholesterol absorption may play only a minor role in human cholesterol gallstone disease (33Kern F., Jr. Effects of dietary cholesterol on cholesterol and bile acid homeostasis in patients with cholesterol gallstones.J. Clin. Invest. 1994; 93: 1186-1194Crossref PubMed Scopus (71) Google Scholar). Evidence for a genetic basis of cholesterol gallstone disease in humans rests on geographic and ethnic differences as well as on family and twin studies (34Paigen B. Carey M.C. Gallstones. In Genetic Basis of Common Diseases. 2nd editon. R. A. King, J. I. Rotter, and A. G. Motulsky. Oxford University Press, London2002: 298-335Google Scholar). A genetic predisposition is present in Pima Indians (age-adjusted prevalence 48%), certain other North and South American Indians, and Chileans (34Paigen B. Carey M.C. Gallstones. In Genetic Basis of Common Diseases. 2nd editon. R. A. King, J. I. Rotter, and A. G. Motulsky. Oxford University Press, London2002: 298-335Google Scholar). By contrast, the overall age-adjusted prevalence in American Caucasians and Europeans is ∼20%, with the lowest prevalence ( A, RFLPBiliary cholesterol secretion ↑ secondary to reverse cholesterol transport ↑Apolipoprotein BAPOB107730GC02M 021135+(+) (China, Poland)c.2488C>T, c.4154G>A+Biliary cholesterol secretion ↑ secondary to hepatic VLDL synthesis ↓ and intestinal cholesterol absorption ↑Apolipoprotein C1APOC1107710GC19P 050109(+) (India)RFLPAPOC1 ↑ remnant-like particle cholesterol ↑Androgen receptorAR313700GC0XP 066680(+) (Greece)c.172(CAG)nCholecystokinin 1 receptorCCK1R118444GC04M 026159+(+) (India)RFLP+Lith13Gallbladder and small intestinal hypomotilityCholesterol ester transfer proteinCETP118470GC16P 055553(+) (Finland)RFLPGene absent in miceHepatic cholesterol uptake ↑ from HDL catabolism ↑Cytochrome P450 7A1CYP7A1Cholesterol 7α-hydroxylase (rate-limiting enzyme of bile salt synthesis)118470GC08M 059565+(+) (China)Promoter SNP −204A>C+Bile salt synthesis ↓Estrogen receptor 2ESR2Estrogen receptor β601663GC14M 063621(+) (Greece)c.1092+3607(CA)n(+)Cholesterol synthesis ↑Low-density lipoprotein receptor-related protein associated protein 1LRPAP1104225GC04M 003551(+) (India)Intron 5 insertion/deletion (rs11267919)+Lith13Hepatic cholesterol uptake ↑ from chylomicron remnants via LRP ↑a Reproduced from Ref. 38Pullinger C.R. Eng C. Salen G. Shefer S. Batta A.K. Erickson S.K. Verhagen A. Rivera C.R. Mulvihill S.J. Malloy M.J. al et Human cholesterol 7α-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype.J. Clin. Invest. 2002; 110: 109-117Crossref PubMed Scopus (450) Google Scholar (slightly modified) by permission of the copyright holders (Springer-Verlag/Falk Foundation) and the authors; individual references are cited therein.b Abbreviations used: OMIM, Online Mendelian inheritance in man; GBD, gallbladder disease; RFLP, restriction fragment length polymorphism; SNP, single nucleotide polymorphism; LRP, low-density lipoprotein receptor-related protein.c http://www.ncbi.nim.nih.gov/entrez.d http://www.genecards.org. Open table in a new tab The table also lists the rare monogenic cholesterol gallstone syndromes. The first evidence that human cholesterol gallstones might be caused by a single gene defect arose from studies showing that homozygous deletion mutation in the rate-limiting cholesterol 7α-hydroxylase (CYP7A1) gene, resulting in loss of enzyme function (38Pullinger C.R. Eng C. Salen G. Shefer S. Batta A.K. Erickson S.K. Verhagen A. Rivera C.R. Mulvihill S.J. Malloy M.J. al et Human cholesterol 7α-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype.J. Clin. Invest. 2002; 110: 109-117Crossref PubMed Scopus (450) Google Scholar). Missense mutations in the ABCB4 gene are the basis for another type of monogenic cholelithiasis (39Rosmorduc O. Hermelin B. Boelle P.Y. Parc R. Taboury J. Poupon R. ABCB4 gene mutation-associated cholelithiasis in adults.Gastroenterology. 2003; 125: 452-459Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar) where cholesterol gallbladder stones and even recurrent cholesterol stones forming in the bile ducts are associated with very low phospholipids-to-bile salt ratios in bile. Moreover, Asian patients with hepatolithiasis also exhibit low levels of ABCB4 plus hepatic phosphatidylcholine transfer protein causing markedly decreased biliary phospholipid secretion rates (40Shoda J. Oda K. Suzuki H. Sugiyama Y. Ito K. Cohen D.E. Feng L. Kamiya J. Nimura Y. Miyazaki H. al et Etiologic significance of defects in cholesterol, phospholipid, and bile acid metabolism in the liver of patients with intrahepatic calculi.Hepatology. 2001; 33: 1194-1205Crossref PubMed Scopus (62) Google Scholar). The genes for cholecystokinin (CCK) and the CCK-1 receptor (CCK-1R), which regulates gallbladder motility, are attractive cholelithogenic candidate genes (Table 1) in the setting of supersaturated bile. An aberrant splicing of CCK-1R, which is predicted to result in a nonfunctional receptor, is found in some obese patients with cholesterol gallstones (34Paigen B. Carey M.C. Gallstones. In Genetic Basis of Common Diseases. 2nd editon. R. A. King, J. I. Rotter, and A. G. Motulsky. Oxford University Press, London2002: 298-335Google Scholar). With respect to the common polygenic gallstone disease (Table 1), associations between gene polymorphisms and cholesterol gallstone formation indicate that the causative genes are highly heterogenous (Table 1). For example, a common variant (D19H) of the canalicular cholesterol transporter ABCG5/G8 gene (Table 1) confers odds ratios of 2 to 3 in heterozygotes and 7 in homozygous carriers based on genome-wide association (41Buch S. Schafmayer C. Volzke H. Becker C. Franke A. Kluck H.von Eller-Eberstein, C. Bassmann I. Brosch M. Lammert F. al et A genome-wide association scan identifies the hepatic cholesterol transporter ABCG8 as a susceptibility factor for human gallstone disease.Nat. Genet. 2007; 39: 995-999Crossref PubMed Scopus (272) Google Scholar) and linkage (42Gru¨nhage F. Acalovschi M. Tirziu S. Walier M. Wienker T.F. Ciocan A. Mosteanu O. Sauerbruch T. Lammert F. Increased gallstone risk in humans conferred by common variant of hepatic ATP-binding cassette transporter for cholesterol.Hepatology. 2007; 46: 793-801Crossref PubMed Scopus (133) Google Scholar) studies from Germany and Chile. Interestingly this common susceptibility gene (Table 1) was predicted by QTL mapping in inbred mice (43Wittenburg H. Lyons M.A. Li R. Churchill G.A. Carey M.C. Paigen B. FXR and ABCG5/ABCG8 as determinants of cholesterol gallstone formation from quantitative trait locus mapping in mice.Gastroenterology. 2003; 125: 868-881Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). By contrast, polymorphisms in the apolipoprotein (APO) AI, B, and C1 genes and the cholesterol ester transfer protein are modifiers, but a cholelithogenic role for APOE polymorphisms has been discounted (36Lammert F. Sauerbruch T. Pathogenesis of gallstone formation: updated inventory of human lithogenic genes. In Future Perspectives in Gastroenterology (Falk Symposium 161). M. C. Carey, P. Dité, A. Gabryelewicz, V. Keim, and J. Mo¨ssner. Springer-Verlang, Dordrecht, The Netherlands2008: 99-107Google Scholar). Hepatocanalicular hypersecretion of cholesterol into bile caused by LITH genes and insulin resistance upregulating ABCG5/G8 via the dysregulation of the FOX01 transcription factor (44Biddinger S.B. Haas J.T. Yu B.B. Bezy O. Jing E. Zhang W. Unterman T.G. 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