Supersaturated Bile Research Articles

The cholecystokinin-1 receptor antagonist devazepide increases cholesterol cholelithogenesis in mice.

A defect in gallbladder contraction function plays a key role in the pathogenesis of gallstones. The cholecystokinin-1 receptor (CCK-1R) antagonists have been extensively investigated for their therapeutic effects on gastrointestinal and metabolic diseases in animal studies and clinical trials. However, it is still unknown whether they have a potential effect on gallstone formation. To study whether the CCK-1R antagonists enhance cholelithogenesis, we investigated cholesterol crystallization, gallstone formation, hepatic lipid secretion, gallbladder emptying function and intestinal cholesterol absorption in male C57BL/6J mice treated by gavage with devazepide (4 mg/day/kg) or vehicle (as controls) twice per day and fed the lithogenic diet for 21 days. During 21 days of feeding, oral administration of devazepide significantly accelerated cholesterol crystallization and crystal growth to microlithiasis, with 40% of mice forming gallstones, whereas only agglomerated cholesterol monohydrate crystals were found in mice receiving vehicle. Compared to the vehicle group, fasting and postprandial residual gallbladder volumes in response to the high-fat meal were significantly larger in the devazepide group during cholelithogenesis, showing reduced gallbladder emptying and bile stasis. Moreover, devazepide significantly increased hepatic secretion of biliary cholesterol, but not phospholipids or bile salts. The percentage of intestinal cholesterol absorption was higher in devazepide-treated mice, increasing the bioavailability of chylomicron-derived cholesterol in the liver for biliary hypersecretion into bile. These abnormalities induced supersaturated bile and rapid cholesterol crystallization. The potent CCK-1R antagonist devazepide increases susceptibility to gallstone formation by impairing gallbladder emptying function, disrupting biliary cholesterol metabolism and enhancing intestinal cholesterol absorption in mice.

Prevalence of hypothyroidism in patients with biliary stones: a prospective study

Background: Biliary stones (gall bladder stones and common bile duct stones) are hardened concretions that develop due to supersaturation of bile. The known risk factors for development of biliary stones include genetics, body weight, high fat diet, diabetes, women taking estrogen containing pills and decreased motility of gall bladder. Of late there has been discussion whether hypothyroidism could lead to formation of biliary stones. Aim of our study is to analyze the prevalance of hypothyroidism in patients of biliary stones and subsequently finding a correlation between hypothyroidism and formation of biliary stones. Methods: The present study was conducted on 100 patients of biliary stones admitted in inpatient department of Surgery at SGRDIMSAR, Amritsar. Their fasting thyroid and lipid profiles were tested to study the prevalence of hypothyroidism in these patients of biliary stones. Results: Percentage of males with gall stones who were diagnosed as hypothyroid, euthyroid and hyperthyroid is 24%, 64% and 12% respectively. Whereas males with common bile duct (CBD) stones who were hypothyroid, euthyroid and hyperthyroid were 18%, 62.5% and 18.75% respectively. Percentage of females with gall stones diagnosed hypothyroid, euthyroid and hyperthyroid was 24.4%, 65.85% and 1% respectively. Percentage of females with common bile duct stones who were hypothyroid, euthyroid and hyperthyroid were 27.7%, 61.1% and 1.1% respectively. Conclusions: We therefore conclude that the role of hypothyroidism with respect to biliary stone formation in human beings is currently not well investigated and further research is needed.

Estrogen induces two distinct cholesterol crystallization pathways by activating ERα and GPR30 in female mice

To distinguish the lithogenic effect of the classical estrogen receptor α (ERα) from that of the G protein-coupled receptor 30 (GPR30), a new estrogen receptor, on estrogen-induced gallstones, we investigated the entire spectrum of cholesterol crystallization pathways and sequences during the early stage of gallstone formation in gallbladder bile of ovariectomized female wild-type, GPR30((-/-)), ERα((-/-)), and GPR30((-/-))/ERα((-/-)) mice treated with 17β-estradiol (E2) at 6 µg/day and fed a lithogenic diet for 12 days. E2 disrupted biliary cholesterol and bile salt metabolism through ERα and GPR30, leading to supersaturated bile and predisposing to the precipitation of cholesterol monohydrate crystals. In GPR30((-/-)) mice, arc-like and tubular crystals formed first, followed by classical parallelogram-shaped cholesterol monohydrate crystals. In ERα((-/-)) mice, precipitation of lamellar liquid crystals, typified by birefringent multilamellar vesicles, appeared earlier than cholesterol monohydrate crystals. Both crystallization pathways were accelerated in wild-type mice with the activation of GPR30 and ERα by E2. However, cholesterol crystallization was drastically retarded in GPR30((-/-))/ERα((-/-)) mice. We concluded that E2 activates GPR30 and ERα to produce liquid crystalline versus anhydrous crystalline metastable intermediates evolving to cholesterol monohydrate crystals from supersaturated bile. GPR30 produces a synergistic lithogenic action with ERα to enhance E2-induced gallstone formation.

Nutritional factors (nutritional aspects) in biliary disorders: Bile acid and lipid metabolism in gallstone diseases and pancreaticobiliary maljunction

Nutritional factors play a key role in the pathogenesis of biliary diseases such as gallstones and pancreaticobiliary maljunction. Gallstones are primarily classified into cholesterol stone and pigment stone according to the major composition. Cholesterol gallstone formation is very likely based upon supersaturated bile formation, and pigment stones are formed in bile rich in bilirubin. Thus, defects of hepatic metabolism of lipids and organic anions lead to biliary stones. Here, the recent understanding of cholesterol gallstone pathogenesis is elaborated. On the other hand, there is another important link of biliary lipid degradation to serious biliary disease, namely pancreaticobiliary maljunction. Lysophosphatidylcholine (lysoPC), a derivative of phosphatidylcholine hydrolysis by phospholipase A2, is a highly abundant bioactive lipid mediator present in circulation as well as in bile. Increases in bile of lysoPC and phospholipase A2 have been reported in pancreaticobiliary maljunction and considered to be the major risk factor for biliary tract cancers. Further, oxidized fatty acids have been established as a potent ligand for G2A, a member of G protein-coupled receptor family that mediates a diverse array of biological processes including cell growth and apoptosis. Thus, both of lysoPC and free fatty acids are supposed to play an important role through G2A in biliary inflammation and carcinogenesis of pancreaticobiliary maljunction. Taken together, nutritional factors, especially lipid compounds, are seemingly crucial in the pathogenesis of biliary diseases, and such a causal relationship is reviewed by mainly authors' previous publications.

To study serum iron levels in patients of gall bladder stone disease and to compare with healthy individuals.

Gallstone disease is a common gastrointestinal problem in day-to-day practice. The Old concept that a typical gallstone sufferer is fat, fertile, flatulent female of 50. This is partially true as the disease has been found in women soon after their first delivery who are thin and underweight and in males also. Conditions that favor the formation of cholesterol gallstones are super saturation of bile with cholesterol, kinetically favorable nucleation and presence of cholesterol crystals in the gall bladder long enough to agglomerate into a stone. Recent studies have defined the role of trace elements (Fe, Ca, Zn and Cu) and defective pH in the formation of gallstones. The aim of the study was to correlate iron-deficiency anemia with gallstone disease. To estimate the serum ferritin level as a diagnostic tool of iron deficiency anemia in patients with gallstone disease, this prospective study of 100 patients was conducted over a period of 24months in the Department of General Surgery, Jaipur Golden Hospital, New Delhi, India. Serum cholesterol, iron and ferritin levels were compared in patients having gallstones and healthy individuals. A low serum iron level is a factor in bile super saturation with respect to cholesterol, leading to gallstone formation.

Rafting for gallstones by slowing mass transit

Rafting for gallstones by slowing mass transit

Fibroblast growth factor receptor 4 Gly388Arg polymorphism associated with severity of gallstone disease in a Chinese population

The etiology of gallstone disease is multifactorial; supersaturation of bile with cholesterol is a primary cause for gallstone formation. In previous studies, we found that fibroblast growth factor receptor 4 (FGFR4) plays an important role in maintaining bile acid homeostasis by regulating the expression of cholesterol 7α-hydroxylase (CYP7A1), a rate-limiting enzyme for bile acid biosynthesis. The Gly388Arg (G-388R) polymorphism of FGFR4 affects stabilization and activation of FGFR4. Consequently, we studied the FGFR4 gene as a candidate gene for genetic susceptibility to gallstone disease. We found that overexpression of FGFR4, especially the G-388R mutant of FGFR4, inhibits luciferase activity of CYP7A1 reporter in HepG2 cells, indicating that the G-388R mutant of FGFR4 may have greater inhibitory activity against bile acid biosynthesis. To investigate the association of FGFR4 polymorphism with gallstone disease, 117 patients with gallstone disease and 457 controls were genotyped for FGFR4 polymorphism G-388R by PCR-RFLP. Although the incidence of gallstone disease was not greater in patients with the FGFR4 RR genotype, the ratio of gallstone patients with acute cholecystitis in the FGFR4 RR genotype (42%) was significantly higher than that in other genotypes of FGFR4 (P = 0.019). In conclusion, the FGFR4 polymorphism is a genetic risk factor contributing to aggravation of gallstone disease.

Quantitative analysis of cholesterol nucleation with time in supersaturated model bile

The amount of cholesterol (Ch) crystals formed in supersaturated taurochenodeoxycholate (TCDC) - lecithin (L) solutions of the same Ch saturation index (CSI) but at different Ch thermodynamic activities (Ch AT) was quantified at different time intervals. The initial Ch nucleation rate (i.e., amount of Ch crystals formed with respect to time) in a Ch AT=1.73 and TCDC to L molar ratio (TCDC:L)=5.1 system was faster than that in a Ch AT=1.42 and TCDC:L=3.4 system. Shaking could enhance the early appearance of Ch crystals and cause the fast initial Ch nucleation rates for the TCDC:L=5.1 and the TCDC:L=3.4 systems. The final Ch nucleation rates were faster than the initial Ch nucleation rates for the TCDC:L=5.1 and the TCDC:L=3.4 systems. According to a light scattering analysis of vesicle concentration in supersaturated TCDC–L solutions, vesicles provide nucleation sites only in the Ch nucleation process and the vesicle concentration may not be an important factor for the Ch nucleation rate. A model of a mixed TCDC–L micelle releasing Ch molecules together with the surface area of Ch crystals formed was used in the interpretation of the Ch nucleation.

Decreased NPC1L1 expression in the liver from Chinese female gallstone patients

BackgroundCholesterol gallstone disease is a very common disease in both industrialized and developing countries. Many studies have found that cholesterol gallstones are more common in women than men. The molecular mechanisms underlying the relationship between female gallstone disease and hepatic sterol transporters are still undergoing definition and have not been evaluated in humans.AimsThe aim of this study is to probe for underlying hepatic molecular defects associated with development of gallstones in female.Methods/ResultsFifty-seven nonobese, normolipidemic Chinese female gallstone patients (GS) were investigated with 12 age- and body mass index-matched female gallstone-free controls (GSF). The bile from the female GS had higher cholesterol saturation than that from the female GSF. The hepatic NPC1L1 mRNA levels were lower in female GS, correlated with SREBP2 mRNA. NPC1L1 downregulation was confirmed at protein levels. Consistently, immunohistochemistry showed decreased NPC1L1 expression in female GS.ConclusionsThe decreased hepatic NPC1L1 levels in female GS might indicate a downregulated reabsorption of biliary cholesterol in the liver, which, in turn, leads to the cholesterol supersaturation of bile. Our data are consistent with the possibility that hepatic NPC1L1 may be mediated by SREBP2.

Macronutrients and Insulin Resistance in Cholesterol Gallstone Disease

Cholelithiasis is a major source of digestive morbidity worldwide. Cholesterol stones account for the majority of gallstones in the United States and other Western countries. The pathogenesis of cholesterol gallstone disease is multifactorial with key factors including cholesterol supersaturation of bile, altered biliary motility, and nucleation and growth of cholesterol crystals. Increasing evidence suggests that many, but not all, causative factors of cholesterol gallstones are related to insulin resistance which, in association with obesity, has reached an epidemic level worldwide. Experimental studies show that hyperinsulinemia, a key feature of insulin resistance, may cause increased hepatic cholesterol secretion and cholesterol supersaturation of bile and gallbladder dysmotility, and thereby may enhance gallstone formation. Insulin resistance syndrome can be modified by environmental factors, including dietary factors. The impact of diet on insulin sensitivity is mediated by both dietary composition and its energy content. The contribution of specific dietary elements to the prevalence and incidence of cholesterol gallstone disease has been explored in animal and human studies. There is considerable evidence to suggest that different types of fatty acids, independent of the total amount of fat consumption, affect insulin sensitivity and cholesterol gallstone disease differently. The effects of salt intake, consumption of protein and carbohydrates, and alcohol drinking on insulin resistance are controversial. Additional intervention trials and controlled experimental feeding studies are needed to further clarify these relationships and to provide useful prophylactic and therapeutic strategies.

Effects of Aramchol on in vitro bile cholesterol crystallization and bile acid detergency

AbstractThe hydrophilic bile acid ursodeoxycholic acid may dissolve cholesterol gallstones and is beneficial in cholestatic liver diseases. The C20 fatty acid‐bile acid conjugate arachidyl amido cholanoic acid (Aramchol) could be a more effective option. We therefore studied its effects on cholesterol crystallization and on bile salt‐induced cytotoxicity. Effects of Aramchol at therapeutically relevant concentrations on crystallization in supersaturated model biles (by microscopy and chemical measurement), on the ternary cholesterol‐taurocholate‐phosphatidylcholine phase diagram, and on micelle ↔ vesicle transitions (by serial dilution or by incubation of cholesterol‐phosphatidylcholine vesicles with taurocholate) were evaluated. Effects on bile salt‐induced cytotoxicity were determined in erythrocytes and CaCo2 cells. Incorporation of Aramchol in model biles did not change micellar cholesterol solubilization, induced a small rightward shift of crystal‐containing zones of the ternary phase diagram, exerted no appreciable effects on vesicle ↔ micelle transitions and had only minor effects on cholesterol crystallization. Bile salt‐induced cytotoxicity was increased by Aramchol in all models. Since Aramchol does not affect cholesterol crystallization, its previously reported beneficial effects in animal gallstone models should relate to other mechanisms. Since Aramchol increases bile salt detergency, it is not likely to be beneficial in cholestatic liver disease.

Increased expression of LXRα, 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.

Correlation Of Gallstone Disease With Iron-Deficiency Anaemia: A Prospective Study

Gallstone disease is a common clinical entity affecting the adult population of both sexes. Conditions that favor the formation of cholesterol gallstones are super-saturation of bile with cholesterol, kinetically favorable nucleation and the presence of cholesterol crystals in the gall bladder long enough to agglomerate into a stone. Recent studies have defined the role of trace elements (Fe, Ca, Zn, and Cu) and defective pH in the formation of gallstones. The aim of the study was to correlate irondeficiency anemia with gallstone disease. This prospective study of 100 patients was conducted over a period of 12 months in the Department of Surgery and Department of Biochemistry, Himalayan Institute of Medical Sciences, Dehradun, India. Serum and gallbladder biliary cholesterol levels were compared with gallstone patients having normal and low serum ferritin level. Gallbladder cholesterol level was significantly higher in anemic, than in non-anemic patients. Low serum iron level is a factor in bile super-saturation with respect to cholesterol leading to gallstone formation.

Gender and Gallstone Disease

Gallstone disease is a common disorder all over the world. In the Western societies about 80 % of the gallstones are composed primarily of cholesterol. Several risk factors for gallstone formation have been identified. One of the most important risk factors is female gender. Rates of gallstones are two to three times higher among women than men. But this is primarily a phenomenon of the childbearing age. Pregnancy is also a major risk factor for gallstone formation. The risk is related to the number of pregnancies. Sex hormones are most likely to be responsible for the increased risk. Estrogen increases biliary cholesterol secretion causing cholesterol supersaturation of bile. Thus, hormone replacement therapy in postmenopausal women and oral contraceptives have also been described to be associated with an increased risk for gallstone disease. However, the effect of estrogen is dose-dependent and new oral contraceptives with a low estrogen dose do not seem to increase the rate of gallstone formation. The present article focuses on the mentioned risk factors associated with female sex hormones.

Apical sodium bile acid transporter and ileal lipid binding protein in gallstone carriers

Although a cholesterol supersaturation of gallbladder bile has been identified as the underlying pathophysiologic defect, the molecular pathomechanism of gallstone formation in humans remains poorly understood. A deficiency of the apical sodium bile acid transporter (ASBT) and ileal lipid binding protein (ILBP) in the small intestine may result in bile acid loss into the colon and might promote gallstone formation by reducing the bile acid pool and increasing the amount of hydrophobic bile salts. To test this hypothesis, protein levels and mRNA expression of ASBT and ILBP were assessed in ileal mucosa biopsies of female gallstone carriers and controls. Neither ASBT nor ILBP levels differed significantly between gallstone carriers and controls. However, when study participants were subgrouped by body weight, ASBT and ILBP protein were 48% and 67% lower in normal weight gallstone carriers than in controls (P < 0.05); similar differences were found for mRNA expression levels. The loss of bile transporters in female normal weight gallstone carriers was coupled with a reduction of protein levels of hepatic nuclear factor 1alpha and farnesoid X receptor. In conclusion, in normal weight female gallstone carriers, the decreased expression of ileal bile acid transporters may form a molecular basis for gallstone formation.

Bile composition and osmolarity in the interpretation of formation of bile stones and their classification

Summary We studied the bile and gallstone composition of 38 surgical patients. According to their composition, the stones were classified as: 1, cholesterol stones, when they have more than 60 per cent cholesterol, or 2, pigment or pigment-mixed stones, when they have more than 40 per cent pigments (including stones with more than 30 per cent calcium). In the cholesterol-stone-forming group (24 cases) a ‘supersaturation’ in the cholesterol component of the bile and a concomitant reduction in bile salts was found. No such bile supersaturation was found in those with pigment or pigment-mixed stones (14 cases), or in the control group (8 cases). The osmolarity of bile in patients with pigment or pigment-mixed stones is significantly higher than in those with cholesterol stones or in controls. These results suggest a different mechanism for gallstone formation in the two groups: a ‘supersaturation’ of cholesterol with respect to bile salts and phospholipids in the formation of cholesterol stones and an ‘overconcentration’ of all bile components as a whole in the formation of pigment stones. In an attempt to clarify the aetiology of gallstone formation, we measured in the bile of patients operated on for cholelithiasis the following parameters: cholesterol, bile salts, phospholipids and, for the first time, osmolarity. Chemical analysis of the gallstones was conducted to ascertain the content of cholesterol, pigments and calcium salts. We report the results of our study and the conclusions we have drawn.

Effects of Leptin on Biliary Lipids: Potential Consequences for Gallstone Formation and Therapy in Obesity

Gallstone disease is exceptionally common, occurring especially in Western populations, with cholesterol gallstones predominating. Currently, it is believed that obesity is the most consistent and important risk factor for the development of cholesterol gallstones. Obesity has been shown to be associated with the supersaturation of bile with cholesterol because of increased hepatic secretion of the sterol. In accord with current information from experimental studies, leptin appears to be involved in biliary cholesterol secretion and cholesterol gallstone formation in humans. This review summarizes the current information on the role of obesity in biliary lipid secretion as well as the effect of leptin and its potential consequences for gallstone formation and therapy in the obese.

Solubility of cholesterol in the crystal-free gallbladder bile of gallstone patients

Supersaturation of bile with cholesterol is generally considered the driving force of cholesterol precipitation. However, in most investigations the amount of cholesterol crystals is included in the calculation of the cholesterol saturation index (CSI). We therefore studied the solubility of cholesterol in crystal-free gallbladder bile from gallstone patients. Our results demonstrate significantly ( P <.05) higher CSIs (1.4 +/- 0.5 and 1.4 +/- 0.4 vs 1.1 +/- 0.4, mean +/- SD) in crystal-free gallbladder bile from 66 patients with cholesterol stones and 21 patients with mixed stones compared with those in 30 patients with pigment stones and a significant difference ( P <.001) in the amount of cholesterol in vesicles (19.2% +/- 13.7% and 14.3% +/- 11.6 % vs 4.2% +/- 5.9%) and of the crystal-observation time (COT; 1-21 days, median 2 days and 1-21 days, median 3 days, vs 3-21 days, median 21 days). We detected a positive correlation ( r =.24, P <.01) between the percentage of cholesterol in vesicles and the CSI and a negative correlation between COT and CSI ( r = -.23, P <.02 ) and COT and the percentage of cholesterol in vesicles ( r = -.52, P <.001 ). However, in 14 of 30 gallbladder-bile specimens from patients with pigment stones but in just 5 of 21 specimens from patients with mixed stones patients and 12 of 66 specimens from patients with cholesterol stones, the distribution of cholesterol in different phases (mixed micelles, vesicles, and crystals) was within the limits of solubility determined in previous studies of model bile. Therefore, in addition to the relative composition of biliary lipids, nonlipid components exert considerable influence on the solubility of cholesterol in the gallbladder bile of gallstone patients.

Gallstones

Gallstones are common, with a reported prevalence of 8% in the UK population aged over 40. The incidence increases with age, and women are more often affected than men, particularly before the menopause. The factor which links all the F words (fat, fair, fertile, over 40) is increased exposure to female sex hormones, which stimulate cholesterol excretion by the liver. In a population predisposed to gallstones (e.g. due to consumption of a “Western diet”), the resulting cholesterol supersaturation of bile favours precipitation and crystal growth (Figure 1). Although most gallstones are cholesterol-based, some stones form as the result of bacterial colonization of cholesterol crystals or other particulate matter in the gallbladder. The bacteria deconjugate bilirubin, leading to its precipitation and the growth of mixed or soft brown stones. These stones are usually seen in the bile duct, and are particularly associated with chronic biliary infection and the presence of parasitic worms, a pattern often seen in South-East Asia. Bile pigment stones are small black hard deposits which occur when excess bile pigment is excreted. They are found in patients with haemolytic anaemia, such as hereditary spherocytosis. Although gallstones are common, the majority of affected individuals have no symptoms. About 10% of gallstones become symptomatic. This poses a diagnostic problem when gallstones are found in patients with atypical symptoms (see below). Cholecystectomy is the most common abdominal procedure in the UK. This reflects the very high prevalence of gallstones, but also gives an indication of the severe discomfort they can cause, sufficient to require surgical treatment. With the introduction of laparoscopic cholecystectomy, a shift occurred in the balance between benefit of operation, and the associated risks and discomfort. This led to an increase in the number of cholecystectomies performed, with a corresponding reduction in the numbers of patients treated by alternative techniques.

Inhibition of cholesterol crystallization under bilirubin deconjugation: partial characterization of mechanisms whereby infected bile accelerates pigment stone formation

Pigment gallstones have been reported to be closely associated with biliary tract infection. We previously reported that addition of unconjugated bilirubin (UCB), which is deconjugated by β-glucuronidase in infected bile, could enhance cholesterol crystal formation in supersaturated model bile (MB). The present study evaluated the effect of β-glucuronidase on the processes of pigment gallstone formation and cholesterol crystallization. Supersaturated MB (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 10.0 g/dl and a cholesterol saturation index (CSI) of 2.0) and native rat bile were mixed at a ratio of 3:1. Then, mixed bile was incubated with or without β-glucuronidase and changes of the following parameters were investigated over time: (1) the UCB/total bilirubin ratio; (2) cholesterol crystal formation; (3) the precipitate weight and the cholesterol concentration in the precipitate and supernatant; and (4) the lipid distribution of vesicles in the supernatant. Compared with β-glucuronidase-free bile, (1) β-glucuronidase-containing bile showed a significant increase of the UCB/total bilirubin ratio, (2) as well as a significantly longer nucleation time (96±17.0 vs. 114±20.0) and fewer cholesterol crystals. (3) The precipitate weight and the cholesterol concentration in the precipitate were significantly increased, while the cholesterol concentration in supernatant was decreased. (4) When mixed bile was incubated with β-glucuronidase, the cholesterol concentration in the vesicles was lower than in bile without β-glucuronidase. The precipitate weight and the cholesterol concentration in the precipitate was increased by incubation with β-glucuronidase, while cholesterol concentration was decreased in the supernatant (especially in the vesicles). This means that bile vesicles were more stable and it was more difficult for cholesterol crystals to form. Thus, the presence of β-glucuronidase may inhibit the formation of pure cholesterol stones even in the presence of cholesterol supersaturation.