Ursodeoxycholic acid exerts farnesoid X receptor-antagonistic effects on bile acid and lipid metabolism in morbid obesity.

BACKGROUND & AIMS: Bile acids (BAs) are major regulators of hepatic BA and lipid metabolism but their mechanisms of action in non-alcoholic fatty liver disease (NAFLD) are still poorly understood. Here we aimed to explore the molecular and biochemical mechanisms of ursodeoxycholic acid (UDCA) in modulating the cross-talk between liver and visceral white adipose tissue (vWAT) regarding BA and cholesterol metabolism and fatty acid/lipid partitioning in morbidly obese NAFLD patients. METHODS: In this randomized controlled pharmacodynamic study, we analyzed serum, liver and vWAT samples from 40 well-matched morbidly obese patients receiving UDCA (20 mg/kg/day) or no treatment three weeks prior to bariatric surgery. RESULTS: Short term UDCA administration stimulated BA synthesis by reducing circulating fibroblast growth factor 19 and farnesoid X receptor (FXR) activation, resulting in cholesterol 7α-hydroxylase induction mirrored by elevated C4 and 7α-hydroxycholesterol. Enhanced BA formation depleted hepatic and LDL-cholesterol with subsequent activation of the key enzyme of cholesterol synthesis 3-hydroxy-3-methylglutaryl-CoA reductase. Blunted FXR anti-lipogenic effects induced lipogenic stearoyl-CoA desaturase (SCD) in the liver, thereby increasing hepatic triglyceride content. In addition, induced SCD activity in vWAT shifted vWAT lipid metabolism towards generation of less toxic and more lipogenic monounsaturated fatty acids such as oleic acid. CONCLUSION: These data demonstrate that by exerting FXR-antagonistic effects, UDCA treatment in NAFLD patients strongly impacts on cholesterol and BA synthesis and induces neutral lipid accumulation in both liver and vWAT.

Gallstone disease in Swedish twins is associated with the Gilbert variant of UGT1A1.

BACKGROUND & AIMS: The Gilbert syndrome-associated functional TATA box variant UGT1A1*28 (A(TA)7TAA) was found to increase susceptibility to pigment gallstone formation in patients with haemolytic anaemia. Further studies in extensive cohorts demonstrated an increased risk of this variant for cholesterol gallstone disease (GD). We now investigated this polymorphism as a determinant of symptomatic GD in Swedish twins. METHODS: The Swedish Twin Registry was merged with the Hospital Discharge and Causes of Death Registries and searched for GD-related diagnoses among monozygotic (MZ) twins living in the Stockholm area. In addition, we screened the TwinGene database for GD. In total, we found 44 MZ twin pairs with and eight MZ twins without GD to be evaluable. GD-free twins from TwinGene (109 concordantly MZ and 126 independent DZ) served as controls. UGT1A1*28 genotyping was performed using TaqMan assays. RESULTS: Overall, 58 and 8 of 106 twins with GD were hetero- and homozygous UGT1A1 risk allele carriers respectively. The case-control association tests showed a significantly (P < 0.05) increased risk of developing GD (OR = 1.62, 95% CI 1.00-2.63) in heterozygotes carriers and in addition, a trend (P = 0.075) for an increased risk among carriers (OR = 1.52, 95% CI 0.97-2.44) of the risk allele. CONCLUSION: These data from Swedish twins confirm the Gilbert variant as risk factor for GD. Our observation is in line with nucleation in bilirubin supersaturated bile representing an initial step in cholelithogenesis.

The genetic background of gallstone formation: an update.

Gallstone disease is one of the most prevalent gastrointestinal diseases with a substantial burden to health care systems that is expected to increase in ageing populations at risk. This review summarizes recent data on the genetic background of cholesterol gallstones and the role of biliary lipid composition. Three previously unknown non-synonymous mutations in the ABCB4 gene encoding the hepatobiliary phospholipid-flippase MDR3 are presented.

ACAT2 and human hepatic cholesterol metabolism: identification of important gender-related differences in normolipidemic, non-obese Chinese patients.

OBJECTIVE: ACAT2 is a major cholesterol esterification enzyme specifically expressed in hepatocytes and may control the amount of hepatic free (unesterified) cholesterol available for secretion into bile or into HDL. This study aims to further elucidate physiologic roles of ACAT2 in human hepatic cholesterol metabolism. METHODS AND RESULTS: Liver biopsies from 40 normolipidemic, non-obese gallstone patients including some gallstone-free patients (female/male, 18/22) were collected and analyzed for microsomal ACAT2 activity, protein and mRNA expression. Plasma HDL-cholesterol (HDL-C) was significantly higher in females than in males, while triglycerides were significantly lower. ACAT2 activity in females was significantly lower than observed in males, regardless of the presence of gallstone disease. Moreover, the activity of ACAT2 correlated negatively with plasma levels of HDL-C (r=-0.57, P<0.05) and with Apo AI (r=-0.49, P<0.05). CONCLUSION: This is the first description of a gender-related difference in hepatic ACAT2 activity in normolipidemic non-obese Chinese patients suggesting a possible role for ACAT2 in the regulation of cholesterol metabolism in humans. The negative correlation between ACAT2 activity and HDL-C or Apo AI may reflect this regulation. Since ACAT2 activity generally has been found to be pro-atherogenic in animal models, the observed sex-related difference may contribute to female protection from complications of coronary heart disease (CHD).

Increased NPC1L1 and ACAT2 expression in the jejunal mucosa from Chinese gallstone patients.

The incidence of cholesterol gallstones is a very common disease. The aim of this study is to probe for underlying intestinal molecular defects associated with development of gallstones. Twelve Chinese patients with cholesterol gallstone disease (GS) and 31 gallstone-free (GSF) patients were investigated. Quantitation of mRNA levels for individual genes in mucosal biopsies from jejunum was carried out with real-time PCR. The frequency of two SNPs in the ABCG8 gene (Y54C and T400K) was determined by allelic discrimination. The intestinal mRNA expression of NPC1L1 and ACAT2 were significantly higher in GS than GSF (P<0.05). No differences were observed concerning the levels for plasma lipids, plant sterols and 7alpha-hydroxy-4-cholesten-3-one between GS and GSF. No correlations were observed between patients carrying the different genotypes for Y54C or T400K and their mRNA levels for ABCG5 or ABCG8. The increased NPC1L1 and ACAT2 mRNA levels in gallstone patients might indicate an upregulated absorption and esterification of cholesterol in the small intestine.

Cholesterol synthesis inhibition elicits an integrated molecular response in human livers including decreased ACAT2.

OBJECTIVE: The purpose of this study was to identify how different degrees of cholesterol synthesis inhibition affect human hepatic cholesterol metabolism. METHODS AND RESULTS: Thirty-seven normocholesterolemic gallstone patients randomized to treatment with placebo, 20 mg/d fluvastatin, or 80 mg/d atorvastatin for 4 weeks were studied. Based on serum lathosterol determinations, cholesterol synthesis was reduced by 42% and 70% in the 2 groups receiving statins. VLDL cholesterol was reduced by 20% and 55%. During gallstone surgery, a liver biopsy was obtained and hepatic protein and mRNA expression of rate-limiting steps in cholesterol metabolism were assayed and related to serum lipoproteins. A marked induction of LDL receptors and 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase was positively related to the degree of cholesterol synthesis inhibition (ChSI). The activity, protein, and mRNA for ACAT2 were all reduced during ChSI, as was apoE mRNA. The lowering of HDL cholesterol in response to high ChSI could not be explained by altered expression of the HDL receptor CLA-1, ABCA1, or apoA-I. CONCLUSIONS: Statin treatment reduces ACAT2 activity in human liver and this effect, in combination with a reduced Apo E expression, may contribute to the favorable lowering of VLDL cholesterol seen in addition to the LDL lowering during statin treatment.

Hypocholesterolemic effects of fatty acid bile acid conjugates (FABACs) in mice.

Fatty acid bile acid conjugates (FABACs) prevent and dissolve cholesterol gallstones and prevent diet induced fatty liver, in mice. The present studies aimed to test their hypocholesterolemic effects in mice. Gallstone susceptible (C57L/J) mice, on high fat (HFD) or regular diet (RD), were treated with the conjugate of cholic acid with arachidic acid (FABAC; Aramchol). FABAC reduced the elevated plasma cholesterol levels induced by the HFD. In C57L/J mice, FABAC reduced plasma cholesterol by 50% (p<0.001). In mice fed HFD, hepatic cholesterol synthesis was reduced, whereas CYP7A1 activity and expression were increased by FABAC. The ratio of fecal bile acids/neutral sterols was increased, as was the total fecal sterol excretion. In conclusion, FABACs markedly reduce elevated plasma cholesterol in mice by reducing the hepatic synthesis of cholesterol, in conjunction with an increase of its catabolism and excretion from the body.

Increased expression of LXR alpha, ABCG5, ABCG8, and SR-BI in the liver from normolipidemic, nonobese Chinese gallstone patients.

Cholesterol supersaturation of bile is one prerequisite for gallstone formation. In the present study of Chinese patients with gallstones, we investigated whether this phenomenon was correlated with the hepatic expression of genes participating in the metabolism of cholesterol and bile acids. Twenty-two nonobese, normolipidemic patients (female-male, 11:11) with gallstones were investigated with 13 age- and body mass index-matched gallstone-free controls (female-male, 10:3). The bile from the gallstone patients had higher cholesterol saturation than that from the controls. The mRNA levels of ABCG5, ABCG8, and liver X receptor alpha (LXRalpha) in the gallstone patients were increased by 51, 59, and 102%, respectively, and significantly correlated with the molar percentage of biliary cholesterol and cholesterol saturation index (CSI). The mRNA and protein levels of the hepatic scavenger receptor class B type I (SR-BI) were increased, and a significant correlation was found between the protein levels and the CSI. No differences were recorded between the two groups concerning the hepatic synthesis of cholesterol, bile acids, and esterification of cholesterol. Our results suggest that the upregulation of ABCG5/ABCG8 in gallstone patients, possibly mediated by increased LXRalpha, may contribute to the cholesterol supersaturation of bile. Our data are consistent with the possibility that increased amounts of biliary cholesterol may originate from plasma HDL cholesterol by enhanced transfer via SR-BI.

Ethanol stimulates bile acid formation in primary human hepatocytes.

The conversion of cholesterol to bile acids is a key pathway for elimination of cholesterol from the body, thereby reducing the risk of arteriosclerosis. Moderate consumption of ethanol has been shown to have preventive effects on cardiovascular disease and decrease the risk of gallstone formation. In the present study primary human hepatocytes were used to investigate if ethanol affected bile acid synthesis. Hepatocytes were prepared from donor liver (n=11) and treated with ethanol, 7.7 or 50 mM, for 24 h. mRNA levels for enzymes in bile acid synthesis pathways were studied and bile acid synthesis was analyzed. Treatment with 7.7 mM ethanol increased cholic acid synthesis by 20% and treatment with 50 mM ethanol up-regulated cholic acid formation by 60%. The synthesis of cholic acid increased more than that of chenodeoxycholic acid, indicating that the classical pathway for bile acid synthesis was up-regulated. Increased bile acid levels in the cells treated with ethanol were seen after approximately 20 h. mRNA expression of CYP7A1, CYP27A1, and CYP8B1 in the hepatocytes was not affected by alcohol exposure.

Involvement of corepressor complex subunit GPS2 in transcriptional pathways governing human bile acid biosynthesis.

Coordinated regulation of bile acid biosynthesis, the predominant pathway for hepatic cholesterol catabolism, is mediated by few key nuclear receptors including the orphan receptors liver receptor homolog 1 (LRH-1), hepatocyte nuclear factor 4alpha (HNF4alpha), small heterodimer partner (SHP), and the bile acid receptor FXR (farnesoid X receptor). Activation of FXR initiates a feedback regulatory loop via induction of SHP, which suppresses LRH-1- and HNF4alpha-dependent expression of cholesterol 7alpha hydroxylase (CYP7A1) and sterol 12alpha hydroxylase (CYP8B1), the two major pathway enzymes. Here we dissect the transcriptional network governing bile acid biosynthesis in human liver by identifying GPS2, a stoichiometric subunit of a conserved corepressor complex, as a differential coregulator of CYP7A1 and CYP8B1 expression. Direct interactions of GPS2 with SHP, LRH-1, HNF4alpha, and FXR indicate alternative coregulator recruitment strategies to cause differential transcriptional outcomes. In addition, species-specific differences in the regulation of bile acid biosynthesis were uncovered by identifying human CYP8B1 as a direct FXR target gene, which has implications for therapeutic approaches in bile acid-related human disorders.

Differential hepatocellular zonation pattern of cholesterol 7alpha-hydroxylase (Cyp7a1) and sterol 12alpha-hydroxylase (Cyp8b1) in the mouse.

The synthesis of primary bile acids is confined to the hepatocytes. This study aimed to evaluate the expression pattern within the liver architecture of the rate-limiting enzyme of the neutral pathway, cholesterol 7alpha-hydroxylase (Cyp7a1), and sterol 12alpha-hydroxylase (Cyp8b1), the enzyme necessary for the synthesis of cholic acid. Specific Cyp8b1 and Cyp7a1 peptide antiserums were used for immunohistochemical staining of livers from wild type and Cyp8b1 null mice, the latter instead expressing beta-galactosidase (beta-Gal) as a replacement reporter gene. Cyp8b1 was mainly expressed in the hepatocytes in a zonal pattern surrounding the central vein while the areas surrounding the portal zones showed much lower levels. The zonation was maintained in cholic acid-depleted mice using beta-Gal as a reporter protein. Cyp7a1 expression in wild type mice also showed a zonal distribution pattern, although less distinct, with a maximal expression within a 1-2 cell thick layer of hepatocytes surrounding the central vein. In Cyp8b1 null mice, a more intense staining was obtained, in accordance with the higher expression level of Cyp7a1, although the overall expression pattern was maintained. Our results in mice indicate possible differences in the regulation of the cellular zonation of Cyp7a1 and Cyp8b1. Also, cholic acid affects the set-point of Cyp7a1 expression but not its zonal distribution.

Analysis of ileal sodium/bile acid cotransporter and related nuclear receptor genes in a family with multiple cases of idiopathic bile acid malabsorption.

The etiology of most cases of idiopathic bile acid malabsorption (IBAM) is unknown. In this study, a Swedish family with bile acid malabsorption in three consecutive generations was screened for mutations in the ileal apical sodium-bile acid cotransporter gene (ASBT; gene symbol, SLC10A2) and in the genes for several of the nuclear receptors known to be important for ASBT expression: the farnesoid X receptor (FXR) and peroxisome proliferator activated receptor alpha (PPARalpha). The patients presented with a clinical history of idiopathic chronic watery diarrhea, which was responsive to cholestyramine treatment and consistent with IBAM. Bile acid absorption was determined using (75)Se-homocholic acid taurine (SeHCAT); bile acid synthesis was estimated by measuring the plasma levels of 7alpha-hydroxy-4-cholesten-3-one (C4). The ASBT, FXR, and PPARalpha genes in the affected and unaffected family members were analyzed using single stranded conformation polymorphism (SSCP), denaturing HPLC, and direct sequencing. No ASBT mutations were identified and the ASBT gene did not segregate with the bile acid malabsorption phenotype. Similarly, no mutations or polymorphisms were identified in the FXR or PPARalpha genes associated with the bile acid malabsorption phenotype. These studies indicate that the intestinal bile acid malabsorption in these patients cannot be attributed to defects in ASBT. In the absence of apparent ileal disease, alternative explanations such as accelerated transit through the small intestine may be responsible for the IBAM.

Critical role of cholic acid for development of hypercholesterolemia and gallstones in diabetic mice.

We studied bile acid and cholesterol metabolism in insulin-dependent diabetes utilizing genetically modified mice unable to synthesize cholic acid (Cyp8b1-/-). Diabetes was induced in Cyp8b1-/- and wild type animals (Cyp8b1+/+) by alloxan, and the mice were fed normal or cholesterol-enriched diet for 10 weeks. The serum levels of cholesterol were strongly increased in diabetic Cyp8b1+/+ mice fed cholesterol, while diabetic Cyp8b1-/- mice did not show any aberrations regardless of the diet. Diabetic cholesterol-fed Cyp8b1+/+ mice had much higher biliary cholesterol and cholesterol saturation index than all other groups, their bile contained a large number of cholesterol crystals, and their canalicular cholesterol transporter Abcg5/g8 mRNA levels were much higher. Cyp7a1 mRNA levels were similar in all diabetic mice but higher compared to non-diabetic animals. The results indicate a critical role for cholic acid for the development of hypercholesterolemia and gallstones in our animal model.

Gallbladder bile composition in patients with Crohn 's disease.

AIM: To further elucidate the pathogenesis and mechanisms of the high risk of gallstone formation in Crohn's disease. METHODS: Gallbladder bile was obtained from patients with Crohn's disease who were admitted for elective surgery (17 with ileal/ileocolonic disease and 7 with Crohn's colitis). Fourteen gallstone patients served as controls. Duodenal bile was obtained from ten healthy subjects before and after the treatment with ursodeoxycholic acid. Bile was analyzed for biliary lipids, bile acids, bilirubin, crystals, and crystal detection time (CDT). Cholesterol saturation index was calculated. RESULTS: The biliary concentration of bilirubin was about 50% higher in patients with Crohn's disease than in patients with cholesterol gallstones. Ten of the patients with Crohn's disease involving ileum and three of those with Crohn's colitis had cholesterol saturated bile. Four patients with ileal disease and one of those with colonic disease displayed cholesterol crystals in their bile. About 1/3 of the patients with Crohn's disease had a short CDT. Treatment of healthy subjects with ursodeoxycholic acid did not increase the concentration of bilirubin in duodenal bile. Several patients with Crohn's disease, with or without ileal resection/disease had gallbladder bile supersaturated with cholesterol and short CDT and contained cholesterol crystals. The biliary concentration of bilirubin was also increased in patients with Crohn's colitis probably not due to bile acid malabsorption. CONCLUSION: Several factors may be of importance for the high risk of developing gallstones of both cholesterol and pigment types in patients with Crohn's disease.

Successful treatment of severe unconjugated hyperbilirubinemia via induction of UGT1A1 by rifampicin.

We report two patients with uncommon Gilbert's syndrome with severe unconjugated hyperbilirubinemia which was reduced from 200 to 60-90 micromol/L by long-term administration of rifampicin. Hepatic induction of bilirubin-glucuronosyltransferase was suggested by increased relative amounts of conjugated serum bilirubin. This molecular mechanism was confirmed in primary cultures of human hepatocytes.

Complementary stimulation of hepatobiliary transport and detoxification systems by rifampicin and ursodeoxycholic acid in humans.

BACKGROUND & AIMS: Rifampicin (RIFA) and ursodeoxycholic acid (UDCA) improve symptoms and biochemical markers of liver injury in cholestatic liver diseases by largely unknown mechanisms. We aimed to study the molecular mechanisms of action of these drugs in humans. METHODS: Thirty otherwise healthy gallstone patients scheduled for cholestectomy were randomized to RIFA (600 mg/day for 1 week) or UDCA (1 g/day for 3 weeks) or no medication before surgery. Routine biochemistry, lipids, and surrogate markers for P450 activity (4beta-hydroxy cholesterol, 4beta-OH-C) and bile acid synthesis (7alpha-hydroxy-4-cholesten-3-one, C-4) were measured in serum. Bile acids were analyzed in serum, urine, and bile. A wedge liver biopsy specimen was taken to study expression of hepatobiliary ABC transporters as well as detoxification enzymes and regulatory transcription factors. RESULTS: RIFA enhanced bile acid detoxification as well as bilirubin conjugation and excretion as reflected by enhanced expression of CYP3A4, UGT1A1, and MRP2. These molecular effects were paralleled by decreased bilirubin and deoxycholic acid concentrations in serum and decreased lithocholic and deoxycholic acid concentrations in bile. UDCA on the other hand stimulated the expression of BSEP, MDR3, and MRP4. UDCA became the predominant bile acid after UDCA treatment and lowered the biliary cholesterol saturation index. CONCLUSIONS: RIFA enhances bile acid detoxification as well as bilirubin conjugation and export systems, whereas UDCA stimulates the expression of transporters for canalicular and basolateral bile acid export as well as the canalicular phospholipid flippase. These independent but complementary effects may justify a combination of both agents for the treatment of cholestatic liver diseases.

Common polymorphisms in the CYP7A1 gene do not contribute to variation in rates of bile acid synthesis and plasma LDL cholesterol concentration.

Transcriptional regulation of the cholesterol 7alpha-hydroxylase (CYP7AI) gene is of critical importance for bile acid and cholesterol metabolism. We evaluated the physiological significance of two common polymorphisms (-203C/A and -469T/C) in the promoter region of the CYP7AI gene. No evidence was found for physiological differences between either the -203C and -203A alleles or the -469T and -469C alleles in transient transfection studies using native 834bp promoter constructs. Moreover, no association was observed between the CYP7AI promoter polymorphisms and the hepatic cholesterol 7alpha-hydroxylase activity and parameters of bile acid synthesis rates, as analyzed in subjects with gallstone disease. In addition, no relationships were found between the promoter polymorphisms and plasma LDL cholesterol concentration in association studies conducted in three different groups of middle-aged Swedish men. Finally, near complete allelic association was found between the two promoter polymorphisms and the IVS6+363G/A polymorphism at the 3' end of the CYP7AI gene (|D'|=0.98), indicating strong linkage disequilibrium across the whole CYP7AI gene. It is concluded that common polymorphisms of the CYP7A1 gene do not contribute to variation in cholesterol 7alpha-hydroxylase activity, rates of bile acid synthesis and plasma LDL cholesterol concentration in humans.

Human sterol 12a-hydroxylase (CYP8B1) is mainly expressed in hepatocytes in a homogenous pattern.

The liver is the only organ where the complete synthesis of bile acids takes place. The present study was undertaken to investigate whether regional differences exist within the individual human hepatic lobuli regarding the pattern of expression of sterol 12alpha-hydroxylase (CYP8B1), a key enzyme in bile acid synthesis. A specific anti-human CYP8B1 peptide antiserum was developed and used for Western blotting and hepatic immunostaining of livers from various patients. CYP8B1 in human liver was expressed in the cytoplasm of hepatocytes with an even nonzonal distribution within the liver lobulus. Pericentral expression was confirmed for CYP2E1. A weak staining was noted in cholangiocytes and Kupffer cells. Previous studies on hepatic CYP27A1 and CYP7A1 in rats have shown a zonal expression, primarily in the pericentral region. Our studies indicate a different pattern for CYP8B1 expression in human liver, which was even rather than zonal.

Changes in gallbladder bile composition and crystal detection time in morbidly obese subjects after bariatric surgery.

The aim of the present study was to elucidate the mechanisms of development of cholesterol crystals and gallstones during weight reduction in obese subjects. Twenty-five morbidly obese, gallstone-free subjects underwent vertical-banded gastroplasty. Gallbladder bile was collected at the time of the operation via needle aspiration and 1.1-7.3 months after the operation via ultrasound-guided transhepatic puncture of the gallbladder. The mean weight loss was 17 kg. Two patients developed gallstones and 10 patients displayed cholesterol crystals in their bile. In patients with a follow-up time of less than 2 months (n = 13), cholesterol saturation increased from 90% to 114% but tended to decrease in the patients with a follow-up time of more than 2 months. The extraction of the concanavalin-A-binding fraction from gallbladder bile obtained after weight reduction in 7 patients prolonged crystallization detection time from 6 to 10 days. The hexosamine concentration, a marker for mucin, was increased by about 100% in bile obtained in 6 of 7 patients after weight reduction. In conclusion, the results indicate that crystallization-promoting compounds (mucin) are of great importance in the development of cholesterol crystals and gallstones in obese subjects during weight reduction, probably because of defective gallbladder emptying.

Genetic and environmental influences on symptomatic gallstone disease: a Swedish study of 43,141 twin pairs.

The contribution of hereditary and environmental factors to the pathogenesis of symptomatic gallstone disease is still unclear. We estimated the relative importance of genetic and environmental factors by analyzing a large population of twins. For this purpose, the Swedish Twin Registry was linked with the Swedish inpatient-discharge and causes of death registries for symptomatic gallstone disease and gallstone surgery-related diagnoses in 43,141 twin pairs born between 1900 and 1958. Concordance rates, correlations, and odds ratios were calculated for males, females, monozygotic, and dizygotic twins, respectively, as well as for twin pairs of opposite sex. Structural equation modeling techniques were used to estimate the contributions of genetic effects as well as shared and non-shared environmental factors to the development of symptomatic gallstone disease. We found that concordances and correlations were higher in monozygotic compared with dizygotic twins, both for males and females. Of note, there were no significant sex differences in heritability. In the full model, genetic effects accounted for 25% (95% CI, 9%-40%), shared environmental effects for 13% (95% CI, 1%-25%), and unique environmental effects for 62% (95% CI, 56%-68%) of the phenotypic variance among twins. In conclusion, our results show heritability to be a major susceptibility factor for symptomatic gallstone disease, consistent with results from previous, much smaller studies.