Composition and immunofluorescence studies of biliary "sludge" in patients with cholesterol or mixed gallstones

BACKGROUND/AIMS: Gallbladder bile from patients with cholesterol or mixed gallstones frequently contains biliary "sludge", a suspension of cholesterol monohydrate crystals and pigment granules embedded in mucin and proteins. The composition of biliary "sludge" and the preferential localization of mucin and proteins could be an indicator for its potential role in gallstone formation. METHODS: Ultracentrifugation (100000 g/l h) was used to precipitate "sludge" from bile, and the concentration difference of its main components between native bile and ultracentrifuged bile samples was calculated. After purification of the sediment, immunolocalization was performed for the detection of mucin, IgA, albumin, aminopeptidase, and anionic polypeptide fraction using polyclonal and monoclonal antibodies. RESULTS: The amount of sludge in gallbladder bile was 4.26 mg/ml-0.78 (mean+/-SEM) in patients with cholesterol and 2.51 mg/ml+/-0.39 in patients with mixed stones and cholesterol was the main component (48.9+/-4.6% and 44.4+/-7.1%). The sediment appeared as a mixture of vesicular aggregates and pigment particles which were linked by a gel matrix of mucin containing cholesterol crystals. While anionic polypeptide fraction and aminopeptidase were associated to pigments, IgA was uniformly spread in the crystalline parts of "core-like" structures, and albumin, when it was present, appeared as randomly located small spots. CONCLUSIONS: Our study demonstrates that the cholesterol content and the distribution pattern of mucin and different proteins is similar in the sediments of biliary "sludge" to that previously shown in cholesterol and mixed gallstones. This suggests that biliary "sludge" represents an early stage of gallstone formation in these patients.     

Supersaturated bile from obese patients without gallstones supports cholesterol crystal growth but not nucleation

Cholesterol crystal formation was studied in gallbladder bile samples collected from 18 patients with cholesterol gallstones, 6 patients with black pigment stones, and 14 obese patients without gallstones. In the absence of seed crystals, bile from patients with cholesterol stones showed a much greater tendency to form cholesterol crystals in vitro than bile of similar cholesterol saturation from patients without cholesterol stones. The ability to form crystals was not related to the biliary hexosamine concentration, an indicator of mucin content. When small seed crystals of cholesterol monohydrate were added to each bile, the seed crystals dissolved in all biles (n = 8) with a cholesterol saturation index less than 0.76. In contrast, 29 of 30 biles with a cholesterol saturation index greater than 0.76 supported crystal growth, even when collected from patients without gallstones. These results indicate that the difference between supersaturated biles in the ability to form cholesterol crystals resides at the nucleation, rather than the growth, stage of crystal formation.     

Polarized light microscopic examination of human bile in the diagnosis of microlithiasis of the gallbladder

Of the 20 cases with biliary colics who had normal OCG and ultrasound, 11 (55%) showed microlithiasis in the form of cholesterol monohydrate crystals and/or calcium bilirubinate granules on polarized light microscopy of the duodenal bile. Microlithiasis was noted in gallbladder bile of all (100%) the cases with proven gallstones but in none of the duodenal bile samples from healthy subjects. This study suggests that polarized microscopy may be a useful method to detect microlithiasis in patients with repeated biliary colics who have normal OCG and ultrasound examination.     

The usefulness of microscopic bile examination in patients with suspected microlithiasis: a prospective evaluation

Gallbladder bile collected by duodenal intubation or during surgery was examined microscopically in patients who were free of stones and in patients with proven stones. None of the 16 patients free of stones had cholesterol monohydrate crystals or calcium bilirubinate granules in bile. Among the 17 patients with proven cholelithiasis, 13 with cholesterol stones had cholesterol monohydrate crystals in their bile, but only 2 of the 4 patients with pigment stones had calcium bilirubinate granules. These data confirm that cholesterol monohydrate crystals are sensitive and specific for cholesterol stones, whereas calcium bilirubinate granules lack sensitivity for the diagnosis of pigment stones. From these results, the diagnostic usefulness of microscopic examination of bile collected from the duodenum was studied prospectively in 46 patients with symptoms suggestive of cholelithiasis but in whom stones had not been visualized at cholecystography and ultrasonography. In 15 of them, bile was found to be abnormal: cholesterol monohydrate crystals were seen in 11, cholesterol crystals + calcium bilirubinate granules in 2 and calcium bilirubinate granules in 2. To date, nine of these patients have been operated on: 6 (all with cholesterol monohydrate crystals) had small cholesterol gallstones and 3 (2 with cholesterol monohydrate crystals and 1 with calcium bilirubinate granules) had signs strongly suggestive of the recent migration of gallstones. One patient refused operation, but minute pigment stones were found to be associated with calcium bilirubinate granules at duodenal intubation. In the other 31 patients, bile contained neither cholesterol monohydrate crystals nor calcium bilirubinate granules. They were not operated on and were followed up with repeated investigations for 12 to 24 months.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol crystal morphology in acalculous gallbladder disease

BACKGROUND: Biliary crystal morphology is best described in patients with gallbladder stones, but most patients undergoing bile collection for microscopy have a clinical diagnosis of acalculous gallbladder disease. We investigated the morphology of biliary crystals in such patients. STUDY: Bile was obtained for polarizing microscopy from fresh cholecystectomy specimens of patients with a clinical diagnosis of acalculous or calculous gallbladder disease. Slides for microscopy were prepared by touch contact with bile in freshly opened gallbladder specimens, and following aspiration of gallbladder bile through a 5-French cannula. RESULTS: Bile was examined from five patients with a clinical diagnosis of acalculous gallbladder disease and five patients with known gallstones. Needle-like cholesterol crystals predominated in most patients without gallstones, whereas plate-like and dot-like crystals were more common in patients with gallstones. All three crystal types were seen in most patients. Crystal morphology was not affected by aspiration of bile through a 5-French cannula. CONCLUSIONS: Birefringent needles and dots should be recognized as cholesterol crystals during bile microscopy. These crystal morphologies may predominate in some patients with a clinical diagnosis of acalculous gallbladder disease.     

Biliary microlithiasis,sludge,crystals,microcrystallization,and usefulness of assessment of nucleation time

BACKGROUND:The process of microcrystallization,its sequel and the assessment of nucleation time is ignored.This systematic review aimed to highlight the importance of biliary microlithiasis,sludge,and crystals,and their association with gallstones,unexplained biliary pain,idiopathic pancreatitis, and sphincter of Oddi dysfunction.DATA SOURCES:Three reviewers performed a literature search of the PubMed database.Key words used were"biliary microlithiasis","biliary sludge","bile crystals","cholesterol crystallisation","bile microscopy","microcrystal formation of bile","cholesterol monohydrate crystals","nucleation time of cholesterol","gallstone formation","sphincter of Oddi dysfunction"and"idiopathic pancreatitis".Additional articles were sourced from references within the studies from the PubMed search.RESULTS:We found that biliary microcrystals account for almost all patients with gallstone disease,7%to 79%with idiopathic pancreatitis,83%with unexplained biliary pain, and 25%to 60%with altered biliary and pancreatic sphincter function.Overall,the detection of biliary microcrystals in gallstone disease has a sensitivity ranging from 55%to 87%and a specificity of 100%.In idiopathic pancreatitis,the presence of microcrystals ranges from 47%to 90%.A nucleation time less than 10 days in hepatic bile or ultra-filtered gallbladder bile has a specificity of 100%for cholesterol gallstone disease.CONCLUSIONS:Biliary crystals are associated with gallstone disease,idiopathic pancreatitis,sphincter of Oddi dysfunction, unexplained biliary pain,and post-cholecystectomy biliary pain.Pathways of cholesterol super-saturation,crystallisation, and gallstone formation have been described with scientificsupport.Bile microscopy is a useful method to detect microcrystals and the assessment of nucleation time is a good method of predicting the risk of cholesterol crystallisation.     

Association between cholesterol-phospholipid vesicles and cholesterol crystals in human gallbladder bile

Rapid aggregation of cholesterol-phospholipid vesicles in gallbladder bile seems to be the first event in the production of cholesterol crystals, a prerequisite for cholesterol gallstone formation. We examined the amount of these vesicles in 33 human gallbladder biles in relation to biliary lipid composition and to the presence of cholesterol crystals. Biliary microscopy detected cholesterol crystals in all 19 biles from patients with cholesterol gallstones but in none of 14 biles from patients with pigment stones. Gel chromatography was used to separate vesicles and micelles in the native bile with an eluting buffer containing 10 mM sodium cholate to prevent disruption of micellar lipids. Cholesterol, phospholipid and bile salt concentrations were measured in every fraction collected. Bile acid, phospholipid, cholesterol and total lipid concentrations were not significantly different in samples with and without cholesterol crystals. The cholesterol saturation index (1.4 +/- 0.11 vs. 1.0 +/- 0.08) was significantly (p less than 0.01) higher in biles with crystals than without crystals. Gel filtration revealed a vesicular peak in addition to micellar fraction in 18 (23.1 +/- 3.2% of total cholesterol) of the 19 biles with crystals but only in three (15.7 +/- 2.4% of total cholesterol) of 14 biles without crystals. There was no relation between biliary lipid concentration or the cholesterol saturation index and the percentage of vesicular cholesterol in biles with or without crystals. The close association of vesicles and crystals in human gallbladder bile supports the contention that vesicles are important in the initial nucleation of cholesterol monohydrate crystals.     

Sensitivity and specificity of microscopic examination of gallbladder bile for gallstone recognition and identification

During cholecystectomy, gallbladder bile and gallstones were obtained from 77 patients and gallbladder bile was obtained from 39 patients free of stones (11 patients had biliary stenosis). According to their chemical composition, gallstones were classified as cholesterol (n = 46) or pigment (n = 31) stones. In patients with gallstones (a) cholesterol crystals better helped to identify cholesterol gallstones (sensitivity, 87%; specificity, 97%; positive predictive value, 97%) than did an abnormal cholesterol saturation index of bile (sensitivity, 93%; specificity, 48%; positive predictive value, 73%); (b) the presence of cholesterol crystals was significantly related to the cholesterol content of gallstones and the bile cholesterol saturation index; and (c) bilirubinate crystals, when present alone (without cholesterol crystals), were good predictors of pigment gallstones (sensitivity, 71%; specificity, 93%; positive predictive value, 88%). In the absence of stones, bilirubinate crystals were present in 9 of 28 patients without biliary stenosis (4 with alcoholic cirrhosis and 2 with alcoholic pancreatitis) and 8 of 11 patients with biliary stenosis. In the absence of stones, cholesterol crystals were present in 2 of 28 patients without biliary stenosis and in 4 of 11 patients with biliary stenosis, suggesting that bile stasis can induce cholesterol crystal formation.     

Cholesterol crystals, cholesterol saturation of bile and biliary lithiasis

It has been repeatedly shown that normal human gallbladder bile is commonly supersaturated wih cholesterol. It has been therefore suggested that the crucial step of the formation of cholesterol gallstones might be the nucleation and growth of cholesterol monohydrate crystals. Consequently this work was aimed at determining: 1) if cholesterol crystal formation is really a typical feature of gallbladder bile with cholesterol gallstones; 2) the influence of the degree of cholesterol saturation of bile on the formation of cholesterol crystals. Gallbladder bile from 89 patients (23 from patients with cholesterol gallstones, 7 from patients with non-cholesterol gallstones and 59 from patients free of gallstones) and hepatic bile from 17 previously cholecystectomized patients were studied. Four of these patients had cholesterol stones of the common bile duct. Results: (a) gallbladder bile: cholesterol crystals were present on immediate examination in 19 of the 23 bile samples with cholesterol stones, in 2 of the 7 bile samples with non-cholesterol stones and in 1 of the 59 bile samples without stones. Only 1 bile sample with cholesterol stone developed crystals. Cholesterol saturation of bile with or without crystals did not differ significantly; (b) hepatic bile: cholesterol crystals were detected on immediate examination in one of the 17 bile samples and subsequently appeared in one of the remaining samples. Cholesterol saturation of hepatic bile (2.10 +/- 0.43) was significantly higher (p less than 0.01) than that of gallbladder bile containing cholesterol stones (1.32 +/- 0.43).(ABSTRACT TRUNCATED AT 250 WORDS)     

High vesicular cholesterol and protein in bile are associated with formation of cholesterol but not pigment gallstones

To examine the differentiating parameters between cholesterol and pigment gallstones, we compared the nucleation times, concentrations of biliary lipid and protein, and the distribution of vesicular cholesterol in gallbladder bile of 16 patients with cholesterol, eight patients with black pigment gallstones, and nine gallstone-free control patients. Cholesterol monohydrate crystals were present in the fresh bile of only the cholesterol gallstone group. The nucleation time was significantly faster in the cholesterol stone group (3.3±3.2 days) than in the other two groups (pigment stone: 15.8±6.6, control: 16.9±5.7). The cholesterol saturation indices and the distribution of vesicular cholesterol were significantly higher in the cholesterol gallstone group than those in the other two groups. The total biliary protein concentration was significantly (P<0.01) higher in the cholesterol gallstone group [2.57±1.91 (sd) mg/ml] than that in the black pigment stone group (1.09±0.59). All parameters in patients with black pigment gallstone were essentially similar to the controls. We conclude that the presence of cholesterol crystals, rapid nucleation time, high vesicular cholesterol distribution, elevated cholesterol saturation index, and high protein concentration are associated with cholesterol gallstones but not with black pigment gallstones.     

Gallstones in cystic fibrosis: a critical reappraisal

Radiolucent gallstones are common in young adults with cystic fibrosis. In the mid-1970s, it was suggested that gallstones are made of cholesterol, but this hypothesis has never been tested. Several recent studies have shown that the detection of cholesterol monohydrate crystals in bile has high sensitivity and specificity for the diagnosis of cholesterol gallstones. We therefore used this approach to study 17 young adults with cystic fibrosis, 10 of whom had radiolucent gallstones. The two groups of patients were comparable in age and gender (all patients but one were male). Duodenal bile was obtained after gallbladder contraction with intravenous cerulein; it was used for lipid and protein chemistry studies and for polarizing microscopy. The latter was performed both in whole bile and in the postultracentrifugation (100,000 g) sediment. Bile cholesterol saturation did not significantly differ between patients with (1.21 +/- 0.28) or without gallstones (0.99 +/- 0.54). Slight cholesterol supersaturation was found in 7 of 10 gallstone and three of seven nongallstone patients. At no time were cholesterol crystals detected in either the group, even after bile ultracentrifugation. Two more cystic fibrosis patients with gallstones died of severe bronchopneumopathy, and small pigment gallstones were obtained at autopsy. At stone analysis, cholesterol content was 44% and 28% of dry weight, respectively. Infrared spectroscopy of stone powder was compatible with the presence of calcium bilirubinate and proteins as major components. We conclude that radiolucent gallstones of cystic fibrosis are not of the conventional cholesterol type.     

Intermittency of cholesterol crystals in duodenal bile from gallstone patients

The detection of cholesterol crystals in duodenal bile is of clinical value in the diagnosis of cholesterol gallstone disease; however, not all patients with cholesterol gallstones have crystals detected in their duodenal bile, thus limiting the value of examination of duodenal bile. The aims of this study were to (i) determine whether the lack of crystals in some patients with cholesterol gallstones was due to (a) the intermittent presence of crystals, (b) spontaneous crystal dissolution, or (c) changes in dietary cholesterol intake; (ii) determine whether incubation of duodenal bile for 24 h would result in crystal formation. Sixteen patients with radiolucent gallstones each underwent three duodenal biliary drainages. Thirty-one percent of patients had crystals in all three bile specimens, 12% in two specimens, 25% in one specimen, and 32% in no specimen. One of 31 specimens with small numbers of crystals initially had no crystals at 24 h, and five specimens initially devoid of crystals developed crystals by 24 h. Despite a significant increase in biliary cholesterol saturation index with increasing cholesterol intake, the prevalence of crystals in bile did not increase in gallstone patients. No crystals were identified in 18 specimens from normal subjects examined initially or in the 15 specimens that were examined after 24 h. We conclude that the intermittent presence of cholesterol crystals in duodenal bile is probably not due to dissolution of crystals or varying dietary cholesterol intake and that the frequency with which crystals are found increases with incubation. Determination of the diagnostic value of multiple duodenal biliary drainages or incubation of bile in patients with normal oral cholecystograms or gallbladder ultrasonograms, however, will require examinations of large numbers of patients.     

Inhibitory effects of pravastatin, a competitive inhibitor of hydroxymethylglutaryl coenzyme A reductase, on cholesterol gallstone formation in prairie dogs.

The effects of pravastatin on cholesterol gallstone formation were determined in prairie dogs. We fed 10 prairie dogs 1% cholesterol with or without 0.05% (w/w) pravastatin (n = 5, each) for 4 weeks. In addition, another 5 prairie dogs were fed a standard rodent chow as a control. Only the animals fed 1% cholesterol without pravastatin treatment formed cholesterol gallstones. Gallbladder bile from cholesterol-fed animals contained cholesterol monohydrate crystals, whereas those treated with pravastatin contained no crystal. Furthermore, marked increases in tissue cholesterol levels (serum, liver and bile), and in biliary mucous glycoprotein levels were evident in cholesterol-fed animals, whereas pravastatin treatment normalized these levels. These findings raise the possibility that such inhibitors might have a future role to play in the prevention of cholesterol gallstone formation and/or recurrence.     

Ceftriaxone-associated gallbladder sludge. Identification of calcium-ceftriaxone salt as a major component of gallbladder precipitate

Ceftriaxone, a third-generation cephalosporin, is partially excreted into bile. With its clinical use, the formation of gallbladder sludge detected by ultrasonography has been reported. Four surgical specimens were examined and no gallstones were found. Instead, fine precipitates of 20-250 microns were present. Microscopically, there was a small number of cholesterol monohydrate crystals and bilirubin granules among an abundant amount of granular-crystalline material that was not morphologically cholesterol monohydrate crystals. The chemical composition of the precipitates (n = 4) was determined. There was a small amount of cholesterol (1.7% +/- 0.8%) and bilirubin (13.9% +/- 0.74%). The major component of the precipitate was a residue. On further analysis using thin-layer chromatography, high-performance liquid chromatography, and electron microprobe analysis, the residue was identified as a calcium salt of ceftriaxone. The residue also had identical crystal morphology and chromatographic elution profile as authentic calcium-ceftriaxone standards. It is concluded that ceftriaxone, after excretion and being concentrated in the gallbladder bile, can form a precipitate. The major constituent has been identified as a ceftriaxone-calcium salt.     

Frequency of biliary crystals in patients with suspected sphincter of Oddi dysfunction

BACKGROUND: The passage of gallstones (macro- or microlithiasis) is theorized to play a role in inducing sphincter of Oddi dysfunction. This study examined the frequency at which biliary crystals are found in patients with suspected type II and type III sphincter of Oddi dysfunction. METHODS: A total of 85 patients (66 women, 19 men; mean age 38 years) with unexplained abdominal pain of suspected pancreatobiliary origin and no prior episode of pancreatitis underwent ERCP with sphincter of Oddi manometry and bile collection for crystal analysis. Eighty-one patients had a gallbladder in situ. No patient had evidence of stones or sludge on prior abdominal imaging. Sphincter of Oddi manometry was performed in standard retrograde fashion by using an aspirating catheter. Patients were classified by sphincter of Oddi dysfunction type by using a modified Hogan-Geenen classification system. Patients with type I sphincter of Oddi dysfunction were excluded. Bile was collected directly from the gallbladder (n=23) or common bile duct (n=62) after an infusion of 3.5 microg of cholecystokinin and was examined by light and polarizing microscopy for cholesterol crystals or calcium bilirubinate granules. RESULTS: The proportion of patients with crystals was 3.5% (3/85). Thirty-five patients (41%) had elevated biliary and/or pancreatic sphincter pressure (type II, 16; type III, 19), of whom one (3%) had cholesterol crystals. Fifty patients had normal sphincter pressure, of whom two (4%) had cholesterol crystals (p=0.6). All 3 patients with cholesterol crystals had a gallbladder in situ. Calcium bilirubinate granules were not found in any patient. CONCLUSIONS: Microlithiasis appears to be rare in patients suspected to have type II or type III sphincter of Oddi dysfunction. Evaluation of bile for crystals appears unproductive in this group of patients.     

Pigment gallstone formation in the cholesterol-fed guinea pig

Female Hartley guinea pigs fed a 0.5% cholesterol-supplemented diet were found to form pigmented gallstones after 6 weeks (17/23) and 12 weeks (11/11), while only 2 of 44 animals fed a trace cholesterol diet formed gallstones over a comparable period. The light brown stones consisted primarily of aggregates of fine granular crystals, morphologically similar to calcium bilirubinate crystals. The stones were soluble in 0.1 N sodium hydroxide and were found to contain a substance which co-migrated with unconjugated bilirubin during thin-layer chromatography. Despite hypercholesterolemia (202 +/- 34 vs. 59 +/- 22 mg per dl in controls, p less than 0.05) and fatty infiltration of the liver, cholesterol-fed animals had a lithogenic index of only 0.22 +/- 0.04 in gallbladder bile as compared to a lithogenic index of 0.02 +/- 0.01 in animals fed the trace cholesterol diet. Accordingly, no cholesterol monohydrate crystals were found in any animals. Hematocrits among cholesterol-fed animals (47.6 +/- 1.2%) were lower than those of controls (54.8 +/- 1.3%, p less than 0.05) probably as a result of the cholesterol-induced hemolytic anemia which has been reported by others in this species. Fasting gallbladder volume was greater in cholesterol-fed animals (2.4 +/- 0.18 ml) than in controls (1.7 +/- 0.11, p less than 0.0025), and a comparable increase in gallbladder dry tissue mass was found. There was no evidence of biliary obstruction, however, and the gallbladder contractile response to octapeptide cholecystokinin was comparable in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-mucin proteins in the matrix of human cholesterol gallstones

Human cholesterol gallstones contain a pigmented organic matrix that may originate from biliary sludge. The cholesterol gallstone matrix contains mucin, bile pigments, and calcium salts. The goal of this study was to examine whether non-mucin proteins are present in the matrix of cholesterol gallstones. Matrix was prepared from cholesterol gallstones from 18 patients. Proteins were identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by molecular sieve high-performance liquid chromatography (HPLC). Two proteins were present in each gallstone and migrated with or just adjacent to standards of bovine serum albumin on SDS-PAGE. Several additional lower molecular weight proteins were identified, but not in every gallstone. Protein fractions contained visible pigment after chloroform extraction, and pigment co-eluted with proteins on HPLC, suggesting binding of pigments to proteins in the matrix. We conclude that low molecular weight proteins are present in the cholesterol gallstone matrix. The major protein appears to be serum albumin, although definitive identification has not been established. The origin of these matrix proteins and their possible significance in the pathogenesis of cholesterol cholelithiasis is unknown.     

Clofibrate, caloric restriction, supersaturation of bile, and cholesterol crystals

Lipid composition, cholesterol saturation, and cholesterol crystal formation of gallbladder bile were studied in seven type-IV hyperlipoproteinemic subjects who did not have gallstones. Thereafter, biliary cholesterol solubilization was overloaded, first by clofibrate and then by caloric restriction treatment. Initially increased cholesterol saturation was still increased by both clofibrate and caloric restriction treatment, but none of the subjects developed cholesterol crystals in bile, indicating that they had a mechanism to maintain cholesterol in solution in the bile despite remarkable supersaturation. This suggests that the patients who are at risk of developing gallstones can be better selected by cholesterol crystal analysis of bile samples than by analysis of lipid composition of bile.     

Estrogen receptor alpha, but not beta, plays a major role in 17beta-estradiol-induced murine cholesterol gallstones

BACKGROUND & AIMS: Cholesterol gallstones are more common in women than men, and exposure to oral contraceptive steroids and conjugated estrogens increases the risk for gallstones. It is hypothesized that estrogen enhances cholesterol cholelithogenesis by augmenting functions of hepatic estrogen receptors (ERs). METHODS: To investigate molecular mechanisms of how estrogen influences cholesterol gallstones, we studied gonadectomized AKR/J mice of both genders that were implanted subcutaneously with pellets releasing 17beta-estradiol at 0, 3, or 6 microg/day and that were fed a lithogenic diet for 12 weeks. To test the hypothesis that ERs play a pivotal role in mediating lithogenic actions of estrogen and to dissect the potential pathophysiologic roles of each receptor subtype, ERalpha and ERbeta, in the formation of gallstones, we investigated gonadectomized mice treated with synthetic ER subtype-selective agonists or antagonists. RESULTS: 17beta-estradiol promoted gallstone formation by up-regulating hepatic expression of ERalpha but not ERbeta, and the lithogenic actions of estrogen can be blocked completely by the antiestrogenic ICI 182,780. The ERalpha-selective agonist propylpyrazole, but not the ERbeta-selective agonist diarylpropionitrile, augmented hepatic cholesterol output that resulted in cholesterol supersaturated bile and gallstones. Similar to the 17beta-estradiol treatment, tamoxifen significantly increased biliary cholesterol secretion and gallstone prevalence in both gonadectomized females and males. CONCLUSIONS: The hepatic ERalpha, but not ERbeta, plays a critical role in 17beta-estradiol-induced cholesterol gallstones. Our findings may offer a new approach to treat gallstones by inhibiting hepatic ER activity with a liver-specific, ERalpha-selective antagonist.     

Comparison of haptoglobin and apolipoprotein A-I on biliary lipid particles involved in cholesterol crystallization

Several proteins are known to modulate cholesterol crystallization. We recently demonstrated that haptoglobin has cholesterol crystallization promoting activity. However, this effect is still not well understood mechanistically. The current study examined the distribution of haptoglobin compared to apolipoprotein A-I (apo A-I) to micelles, vesicles and crystals as an initial step in providing a focus for further studies of the mechanism of cholesterol crystallization activity. Specific protein purification was accomplished by immunoaffinity chromatography. The crystallization-promoting activity of biliary haptoglobin, albumin and commercial apo A-I was measured by a photometric crystal growth assay. The distribution of micelles, vesicles and proteins in model bile was determined by Sepharose CL-6B column chromatography. Detection of the presence of test proteins in cholesterol crystals was determined using specific ¹²⁵I-radiolabelled proteins. Haptoglobin (20 μg/mL) showed a significant crystallization promoting-activity, whereas apo A-I (30 μg/mL) only tended to show a slight inhibitory activity. The cholesterol crystal-bound protein in each case was found to be less than 1% of the total concentration of that protein that had been added to the model bile system. The elution profile of commercial apo A-I from a Sepharose CL-6B column was strikingly altered when it was added to model bile prior to elution. In contrast, the column elution profiles for both haptoglobin and albumin were unchanged when model bile was similarly added to the sample. Haptoglobin increased the amount of cholesterol found in the vesicular fraction when compared to apo A-I. Haptoglobin does not bind tightly to either biliary lipid particles or to cholesterol crystals but does increase the amount of cholesterol in vesicles by inducing a shift from micellar cholesterol (P=0.046). This shift appears to explain in part its promoting effect on cholesterol crystallization.