Cholesterol gallstone formation in overweight mice establishes that obesity per se is not linked directly to cholelithiasis risk

The relationship between obesity and cholesterol cholelithiasis is not well understood at physiologic or genetic levels. To clarify whether obesity per se leads to increased prevalence of cholelithiasis, we examined cholesterol gallstone susceptibility in three polygenic (KK/H1J, NON/LtJ, NOD/LtJ) and five monogenic [carboxypeptidase E (Cpe (fat)), agouti yellow (A(y)), tubby (tub), leptin (Lep(ob)), leptin receptor (Lepr (db))] murine models of obesity during ingestion of a lithogenic diet containing dairy fat, cholesterol, and cholic acid. At 8 weeks on the diet, one strain of polygenic obese mice was resistant whereas the others revealed low or intermediate prevalence rates of cholelithiasis. Monogenic obese mice showed distinct patterns with either high or low gallstone prevalence rates depending upon the mutation. Dysfunction of the leptin axis, as evidenced by the Lep(ob) and the Lepr (db) mutations, markedly reduced gallstone formation in a genetically susceptible background strain, indicating that in mice with this genetic background, physiologic leptin homeostasis is a requisite for cholesterol cholelithogenesis. In contrast, the Cpe (fat) mutation enhanced the prevalence of cholelithiasis markedly when compared with the background strain. Since CPE converts many prohormones to hormones, a deficiency of biologically active cholecystokinin is a likely contributor to enhanced susceptibility to cholelithiasis through compromising gallbladder contractility and small intestinal motility. Because some murine models of obesity increased, whereas others decreased cholesterol gallstone susceptibility, we establish that cholesterol cholelithiasis in mice is not simply a secondary consequence of obesity per se. Rather, specific genes and distinct pathophysiological pathways are responsible for the shared susceptibility to both of these common diseases.     

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.     

Susceptibility to cholesterol gallstone formation: evidence that LITH genes also encode immune-related factors

Cholesterol gallstones are solid calculi that form in the gallbladder from bile constituents and chiefly comprise cholesterol. Cholesterol gallstones are prevalent and costly for healthcare systems. In addition to various environmental factors, genetic risk contributes substantially to gallstone susceptibility. Candidate gene approaches to identify contributory genes are based on prior knowledge of gene-protein function. Whether selected from the entire genome or from limited genomic regions detected by experimental linkage analyses, thus far, candidate genes predominantly were related to lipid homeostasis. Alternatively, comprehensive review of available data suggests that a fundamental driving force underlying cholesterol gallstone formation is inflammation. Therefore, we predict that Lith genes in mice and LITH genes in humans also encode inflammatory molecules, their receptors and other mediators. Indeed, many Lith loci, defined experimentally using inbred mouse models, co-localise with genes that encode inflammation-related proteins. Systematic review of the literature reveals evidence consistent with inflammatory responses that may dictate each of the three cornerstones of cholesterol gallstone formation: biliary cholesterol supersaturation; cholesterol nucleation; gallbladder hypomotility. Genetically targeted inbred mice represent a powerful tool to interrogate the relationship between immune-related genes and gallstone susceptibility. We urge researchers to consider inflammation-related genes when designing population case-control genetic association studies pertaining to the genetic basis of gallstones. Immune and inflammatory events underlie each criterion necessary for cholesterol gallstone formation, which suggests that variation within the respective genes is fundamental for gallstone formation. In turn, inflammatory mediators may exert a spectrum of effects in response to genetic variation within lipid homeostatic genes.     

Hepatic cholesterol metabolism in cholesterol gallstone disease

Hepatic cholesterol metabolism was examined in 27 Swedish patients with cholesterol gallstone disease and in 13 patients free of gallstones operated for roentgenographically suspect polyps in the gallbladder. All 40 patients underwent cholecystectomy, and a liver biopsy and gallbladder bile were obtained at surgery. The cholesterol saturation of gallbladder bile was significantly higher in patients with gallstones compared to the gallstone-free controls (131 +/- 13 vs. 75 +/- 5%, P less than 0.001). Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, governing cholesterol synthesis, did not differ between gallstone and gallstone-free patients (104 +/- 11 vs. and 109 +/- 22 pmol/min per mg protein, respectively). The activity of cholesterol 7 alpha-hydroxylase, catalyzing the catabolism of cholesterol to bile acids, was not significantly decreased in gallstone patients (6.2 +/- 1.1 vs. 8.0 +/- 2.0 pmol/min per mg protein). The capacity to esterify cholesterol, judged by the activity of acyl coenzyme A:cholesterol acyltransferase (ACAT), was similar in gallstone and gallstone-free patients (5.4 +/- 0.4 vs. 6.7 +/- 1.1 pmol/min per mg protein). In the presence of exogenous cholesterol, ACAT activity increased by more than fourfold in both groups. No correlation was found between the saturation of gallbladder bile and any of the mentioned enzyme activities in gallstone patients. It is concluded that distinct abnormalities in cholesterol metabolizing enzymes are not of major importance for development of gallstones in Swedish patients with cholesterol gallstone disease. The results support the contention that the etiology of cholesterol gallstones is multifactorial.     

Gallstones

Cholesterol saturation of bile has a primary role in the pathogenesis of gallstone formation. Predisposing factors should be considered. The characteristic features of biliary colic are important to keep in mind, as well as the fact that a history of fatty food intolerance is not of value in the diagnosis of gallstones. The technique of endoscopic retrograde cholangiography is useful for the diagnosis of bile duct stones in jaundiced patients and in patients with a strong clinical history, but in whom findings on oral and intravenous cholangiograms are within normal limits. Improved techniques of operative cholangiography to diminish the incidence of retained gallstones have been developed. Also, choledochoscopy provides a remarkable technique for diagnosis and choledocholithotomy. The dissolution of gallstones with chenodeoxycholic acid is an experimental procedure. This bile acid is thought to act by increasing the chenodeoxycholic acid pool size and decreasing cholesterol synthesis and secretion, thereby reversing the defects responsible for gallstone formation.     

Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: integrated activities of hepatic lipid regulatory enzymes.

There is no consensus whether hepatic lipid regulatory enzymes play primary or secondary roles in cholesterol cholelithiasis. We have used inbred mice with Lith genes that determine cholesterol gallstone susceptibility to evaluate the question. We studied activities of regulatory enzymes in cholesterol biosynthesis (HMG-CoA reductase), cholesterol esterification (acyl-CoA:cholesterol acyltransferase) and the "neutral" (cholesterol 7alpha-hydroxylase) and "acidic" (sterol 27-hydroxylase) pathways of bile salt synthesis in strains C57L/J and SWR/J as well as recombinant inbred (AKXL-29) mice, all of which have susceptible Lith alleles, and compared them to AKR/J mice with resistant Lith alleles. We determined hepatic enzyme activities of male mice before and at frequent intervals during feeding a lithogenic diet (15% dairy fat, 1% cholesterol, 0.5% cholic acid) for 12 weeks. Basal activities on chow show significant genetic variations for HMG-CoA reductase, sterol 27-hydroxylase, and acyl-CoA: cholesterol acyltranferase, but not for cholesterol 7alpha-hydroxylase. In response to the lithogenic diet, activities of the regulatory enzymes in the two bile salt synthetic pathways are coordinately down-regulated and correlate inversely with prevalence rates of cholesterol crystals and gallstones. Compared with gallstone-resistant mice, significantly higher HMG-CoA reductase activities together with lower activities of both bile salt synthetic enzymes are hallmarks of the enzymatic phenotype in mice with susceptible Lith alleles. The most parsimonious explanation for the multiple enzymatic alterations is that the primary Lith phenotype induces secondary events to increase availability of cholesterol to supply the sterol to the hepatocyte canalicular membrane for hypersecretion into bile.     

Aging per se is an independent risk factor for cholesterol gallstone formation in gallstone susceptible mice

Cholesterol gallstones occur rarely in childhood and adolescence and increase linearly with age in both genders. To explore whether aging per se increases cholesterol saturation of bile and gallstone prevalence, and to investigate age-related changes in hepatic and biliary lipid metabolism, we studied gallstone-susceptible C57L mice and resistant AKR mice of both genders fed 8 weeks with a lithogenic diet containing 1% cholesterol, 0.5% cholic acid, and 15% butter fat starting at (young adult) 8, (older adult) 36, and (aged) 50-weeks-of-age. After the 8-week feeding, gallstone prevalence, gallbladder size, biliary lipid secretion rate, and HMG-CoA reductase activity were significantly greater but cholesterol 7alpha-hydroxylase activity was lower in C57L mice of both genders compared with AKR mice. Increasing age augmented biliary secretion and intestinal absorption of cholesterol, reduced hepatic synthesis and biliary secretion of bile salts, and decreased gallbladder contractility, all of which increased susceptibility to cholesterol cholelithiasis in C57L mice. We conclude that aging per se is an independent risk factor for cholesterol gallstone formation. Because aging increases significantly biliary cholesterol hypersecretion and gallstone prevalence in C57L mice carrying Lith genes, it is highly like that Longevity (aging) genes can enhance lithogenesis of Lith (gallstone) genes.     

Mice overexpressing hepatic Abcb11 rapidly develop cholesterol gallstones

Cholelithiasis is a polygenic disease, although the genes responsible for gallstone formation have not yet been clearly identified. QTL analysis has identified the Lith 1 loci on mouse Chromosome 2, and the hepatic bile salt transporter Abcb11 maps to the Lith 1 locus. We have used recently developed TTR-Abcb11 transgenic mice that overexpress Abcb11 to determine the effects of Abcb11 overexpression on cholesterol gallstone formation. TTR-Abcb11 and FVB/NJ strain control mice were fed a lithogenic or chow diet and cholesterol crystal and gallstone formation were measured. Biliary lipids in gallbladder bile and gene expression of canalicular lipid transporters were also analyzed. TTR-Abcb11 mice fed a lithogenic diet had an increased rate of cholesterol crystal and gallstone formation. This was associated with an increase in both the hydrophobic bile salt and cholesterol content of gallbladder bile. Expression of Abcb4, Abcg5, and Abcg8 did not change before gallstone formation. These data indicate that hepatic overexpression of Abcb11 increases the rate of cholesterol gallstone formation. This is likely because of increases in bile salt hydrophobicity but not because of alterations of other biliary lipid transporters. These findings strongly support Abcb11 as a Lith 1 gene.     

Gallstone Formation and Weight Loss

Obesity is associated with increased bile stasis and cholesterol saturation, and an increased risk of gallstone development. Conversely, bile composition is normalized following reduction in body weight. It would appear advantageous to promote weight loss in obesity, which would reduce the predisposition to gallstone formation. Despite the potential health benefits of weight reduction, very-low-calorie diets appear to increase the risk for cholesterol crystal and gallstone formation. The incidence of gallstone formation seems to be dependent on the degree of caloric restriction, the rate of weight loss, and the duration of the dietary intervention. Thus, faster rates of weight loss for longer periods of time are associated with increased risk. Available data obtained from prospective studies of subjects during active weight loss suggest that newly formed gallstones occur within 4 weeks and with incidence rates 15 to 25-fold higher than in the general obese population. The stones produce symptoms in approximately one-third of the subjects, of whom approximately one-half will undergo surgery. Proposed mechanisms underlying gallstone formation during weight reduction include bile stasis due to reduced caloric intake, increased biliary cholesterol saturation secondary to increased cholesterol mobilization, and increased nucleation due to changes in bile arachidonate and givcoprotein concentrations. Data are lacking on the effects of gradual rates of weight loss and risk of gallstone formation.     

Hepatic cholesterol transport from plasma into bile: implications for gallstone disease

PURPOSE OF REVIEW: The transhepatic traffic of cholesterol from plasma lipoproteins into the bile is critical for overall cholesterol homeostasis and its alterations may lead to cholesterol gallstone formation. This review summarizes recent progress in understanding the key hepatic cholesterol metabolism-related proteins and pathways that influence biliary secretion of cholesterol. RECENT FINDINGS: In cholesterol-fed apolipoprotein E knockout mice, the availability of dietary cholesterol for biliary disposal is decreased and diet-induced gallstone formation is impaired. Scavenger receptor class B type I is relevant for cholesterol transport from plasma HDL into the bile in chow-fed mice, however its expression is not critical for biliary cholesterol secretion and gallstone formation in lithogenic diet-fed mice. Intrahepatic cholesterol transport proteins (e.g. sterol carrier protein-2, Niemann Pick type C-1 protein) also determine liver cholesterol available for biliary secretion in mice. Genetic manipulation of canalicular ATP-binding cassette transporter G5 and G8 expression in mice has established their essential role for biliary cholesterol secretion. SUMMARY: Recent studies have underscored that different proteins involved in hepatic cholesterol transport regulate the availability of cholesterol for biliary secretion. These advances may provide new avenues for prevention and treatment of various disease conditions linked to abnormal cholesterol metabolism.     

miRNA-223 Suppresses Mouse Gallstone Formation by Targeting Key Transporters in Hepatobiliary Cholesterol Secretion Pathway

miRNA-223 has been previously reported to play an essential role in hepatic cholesterol homeostasis. However, its role in regulation of biliary cholesterol secretion and gallstone formation remains unknown. Hence, mice with conventional knockout (KO), hepatocyte-specific knockout (ΔHepa) / knockdown (KD) or gain expression of miRNA-223 were included in the study and were subjected to lithogenic diet (LD) for various weeks. The gall bladders and liver tissues were harvested for cholesterol crystal imaging, gallstone mass measurement and molecular analysis. Levels of cholesterol, bile salt, phospholipids, and triglyceride were determined in serum, liver tissues, and bile by enzyme color reactive assays. A 3'' UTR reporter gene assay was used to verify the direct target genes for miRNA-223. LD-induced gallstone formation was remarkably accelerated in miRNA-223 KO, ΔHepa, and KD mice with concurrent enhancement in total cholesterol levels in liver tissues and bile. Key biliary cholesterol transporters ABCG5 and ABCG8 were identified as direct targets of miRNA-223. Reversely, AAV-mediated hepatocyte-specific miRNA-223 overexpression prevented gallstone progression with reduced targets expression. Therefore, the present study demonstrates a novel role of miRNA-223 in the gallstone formation by targeting ABCG5 and ABCG8 and elevating miRNA-223 would be a potentially novel approach to overcome the sternness of cholesterol gallstone disease.     

Regulation of expression of the genes encoding steroidogenic enzymes

In recent years it has become apparent that tropic hormones involved in steroidogenesis act to regulate the expression of the enzymes involved in the various steroidogenic pathways. This is particularly evident in the ovary where the episodic secretion of steroids throughout the ovarian cycle is regulated largely by changes in the levels of the particular enzymes involved in each step of the steroid biosynthetic pathways. Recently, the genes for the various cytochrome P450 species involved in ovarian steroidogenesis, namely cholesterol side-chain cleavage P450 (P450SCC), 17 alpha-hydroxylase P450 (P450(17 alpha], and aromatase cytochrome P450 (P450AROM) have been isolated and characterized, making it possible to study the regulation of expression at the molecular level. To this end, a series of chimeric constructs have been prepared in which fragments of the 5'-untranslated region of bovine P450(17 alpha) and P450SCC have been inserted upstream of the chloramphenicol acetyl transferase (CAT) and beta-globin reporter genes. These constructs have been used to transfect primary cultures of bovine luteal and thecal cells. The results indicate that cAMP responsiveness lies within defined regions of genes which do not contain a classical CRE, similar to previous results utilizing adrenal cells in culture. Furthermore, although constructs containing both the P450(17 alpha) and P450SCC 5'-upstream regions are expressed in both luteal and thecal cell cultures, only those containing the P450SCC sequences are expressed in luteal cells. Studies on the expression of P450AROM indicate that the promoter which is responsible for its expression in human placenta is not operative in the corpus luteum. Thus estrogen biosynthesis may be regulated by the differential use of tissue specific promoters, thus accounting for the complexity and multifactorial nature of the expression of this activity.     

The Syrian golden hamster strain LPN: a useful animal model for human cholelithiasis

The purpose of this study was to specify the main mechanisms at the origin of gallstone formation in very young (5-week old) or young adult (9-week old) LPN hamsters fed a sucrose-rich (normal lipid) lithogenic diet for one and four weeks, respectively. It was also to compare these mechanisms in the two strains of hamsters (LPN and Janvier) or when an anti-lithiasic diet was given by substituting 10% of the sucrose by beta cyclodextrin. The LPN strain of hamsters showed a very high incidence of cholesterol gallstones (73%) after receiving the lithogenic diet. The gallstone formation is very rapid and occurs in less than one week in very young hamsters which show a high cholesterol synthesis rate in the liver. The cholesterol and phospholipid concentrations in the bile, cholesterol saturation index (CSI) and hydrophobic index (HI) increased significantly, concomitantly with a higher liver cholesterol synthesis in very young hamsters and with a lower bile acid synthesis (neutral pathway: cholesterol 7alpha-hydroxylase, CYP7A1 and acidic pathway: sterol 27 hydroxylase, CYP27A1) in young adult hamsters. No significant changes in the lipoprotein receptor expression (LDLr, SR-BI) were observed after feeding the lithogenic diet. Adding ten per cent beta-cyclodextrin, a cyclic oligosaccharide that binds cholesterol and bile acids to the lithogenic diet at the expense of sucrose, induced a decrease in cholesterol bile secretion and in the CSI and HI and prevented cholesterol gallstone formation. Similarly, another strain of Syrian Golden hamsters (" Janvier ") which originally exhibited a smaller bile cholesterol concentration, lower liver cholesterol synthesis and higher CYP7A1/CYP27A1 activity ratio did not carry cholesterol gallstones when fed the lithogenic diet. The main parameters always found at the origin of cholelithiasis in the Hamster are discussed: a higher hepatic cholesterogenesis (HMGCoAR), a higher HMGCoAR/CYP7A1 activity ratio, a lower cholesterol ester storage capacity, a higher CYP27A1/CYP7A1 activity ratio correlated to a higher cholesterol secretion in the bile and higher CSI and HI. In LPN hamsters, the incidence of cholesterol gallstones is nil when CSI + HI < 0.8 and positive for CSI + HI > 0.9. An overall comparison of the data obtained in LPN Hamsters and in Man suggests that this hamster strain appears to be an interesting model for human cholelithiasis.     

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.     

Complex segregation analysis of plasma lipid and lipoprotein variables in a Jerusalem sample of nuclear families

Plasma lipid and lipoprotein concentrations from 3,074 nuclear families in the multiethnic Jerusalem Lipid Research Clinic study population were analyzed for possible involvement of major genes in determination of high levels of these traits. Complex segregation analysis under a mixed model including major gene and multifactorial transmissible components was performed on transformed-plasma lipids and lipoproteins after covariance adjustment for age, sex and environmental measures. Likelihood analysis provided evidence for recessive major genes influencing plasma triglyceride, and low-density lipoprotein cholesterol (LDL-C). The estimated gene frequencies for triglyceride and for hyperbetalipoproteinemia in our study population were about 0.1. Our positive results for total cholesterol and high-density lipoprotein cholesterol (HDL-C) were nonconclusive and the major effects could result from causes other than major genes. The mixed-model parameters were homogeneous across origin groups for LDL-C and HDL-C and heterogeneous for total plasma cholesterol and triglyceride. The multifactorial-transmission heritability index was similar in all origin groups for all the traits. The origin heterogeneity in the major gene parameters appeared to be mainly due to the North African group which favored a multifactorial transmission for all traits except for LDL-C.     

High cholesterol absorption efficiency and rapid biliary secretion of chylomicron remnant cholesterol enhance cholelithogenesis in gallstone-susceptible mice

The study of chylomicron pathway through which it exerts its metabolic effects on biliary cholesterol secretion is crucial for understanding how high dietary cholesterol influences cholelithogenesis. We explored a relationship between cholesterol absorption efficiency and gallstone prevalence in 15 strains of inbred male mice and the metabolic fate of chylomicron and chylomicron remnant cholesterol in gallstone-susceptible C57L and gallstone-resistant AKR mice. Our results show a positive and significant (P<0.0001, r=0.87) correlation between percent cholesterol absorption and gallstone prevalence rates. Compared with AKR mice, C57L mice displayed significantly greater absorption of cholesterol from the small intestine, more rapid plasma clearance of chylomicrons and chylomicron remnants, higher activities of lipoprotein lipase and hepatic lipase, greater hepatic uptake of chylomicron remnants, and faster secretion of chylomicron remnant cholesterol from plasma into bile. All of these increased susceptibility to cholesterol gallstone formation in C57L mice. We conclude that genetic variations in cholesterol absorption efficiency are associated with cholesterol gallstone formation in inbred mice and cholesterol absorbed from the intestine provides an important source for biliary hypersecretion. Differential metabolism of the chylomicron remnant cholesterol between C57L and AKR mice clearly plays a crucial role in the formation of lithogenic bile and gallstones.     

Phosphatidylcholine-enriched diet prevents gallstone formation in mice susceptible to cholelithiasis

Cholesterol gallstones affect approximately 10-15% of the adult population in North America. Phosphatidylcholine (PC) is considered to be the main cholesterol solubilizer in bile. This study examined the effect of a PC-enriched diet on gallstone incidence in mice susceptible to cholelithiasis. The result obtained showed that the feeding of a lithogenic (LG) diet for 4 weeks or 8 weeks resulted in cholesterol gallstone incidences of 47% and 89%, respectively. These gallstone incidences were either reduced or prevented when the LG diet was enriched with 2% or 6% PC, respectively. The cholesterol saturation index (CSI) was reduced only in mice fed with LG + 6% PC diet as compared with mice fed the LG diet alone. However, in all groups, the CSI was significantly higher than in mice fed Purina chow diet. The biliary anionic polypeptide fraction (APF) was significantly increased in mice fed the LG + 2% PC diet and was reduced in those fed with LG + 6% PC diet. In conclusion, prevention or delay of gallstone formation was not due to a consistent effect on biliary lipid composition, suggesting a direct effect of PC on cholesterol solubilization and/or the effect of an additional nonlipid biliary component such as APF.