Ursodeoxycholic acid reduces lipid peroxidation and mucin secretagogue activity in gallbladder bile of patients with cholesterol gallstones.

BACKGROUND: Recently it has been postulated that gallbladder mucin hypersecretion observed in the pathogenesis of cholesterol gallstone disease may be induced by biliary lipid peroxidation. Ursodeoxycholic acid treatment reduces mucin concentration and the formation of cholesterol crystals in the gallbladder bile of patients with cholesterol gallstones and this effect might be mediated by a decrease of biliary lipid peroxidation. MATERIAL AND METHODS: In a double-blind, placebo-controlled trial patients with symptomatic cholesterol gallstones received either ursodeoxycholic acid (750 mg daily) (n = 10) or placebo (n = 12) 10-12 days prior to cholecystectomy. As a marker for lipid peroxidation malondialdehyde was measured in bile together with mucin concentration. In addition, the mucin secretagogue activity of the individual bile samples was assessed in cultured dog gallbladder epithelial cells. RESULTS: Ursodeoxycholic acid therapy resulted in a significant reduction of lipid peroxidation in bile as determined by the biliary malondialdehyde concentration (1.36 +/- 0.28 vs. 2.05 +/- 0.38 micromol L(-1); P < 0.005) and the malondialdehyde (micromol L(-1))/total bile acid (mmol L(-1)) ratio (0.02 +/- 0.005 vs. 0.06 +/- 0.01; P < 0.001). Furthermore, a decrease in mucin concentrations (0.7 +/- 0.3 vs. 1.3 +/- 0.5 mg mL(-1); P < 0.005) and of the mucin secretagogue activity of gallbladder bile (0.9 +/- 0.2 vs. 2.2 +/- 0.3 times control; P < 0.001) was observed. CONCLUSIONS: The reduction of lipid peroxidation and mucin secretagogue activity of gallbladder bile induced by ursodeoxycholic acid treatment may contribute to the beneficial effects of this drug on gallbladder bile composition and symptoms in cholesterol gallstone patients.

Lipid peroxidation and mucin secretagogue activity in bile of gallstone patients.

BACKGROUND: Chronic inflammation of the gallbladder wall and mucin hypersecretion are considered to be important factors in the pathogenesis of cholesterol gallstone disease. The aim of the study was to compare mucin concentration and mucin secretagogue activity with lipid peroxidation in gallbladder bile of patients with cholesterol or pigment stones. MATERIAL AND METHODS: We studied mucin concentration and, as a marker of lipid peroxidation, malondialdehyde concentration in 11 rapid (1 to 3 days) and eight non-nucleating (> 21 days) gallbladder biles of patients with cholesterol or pigment stones. Furthermore, the mucin secretagogue activity of rapid and non-nucleating gallbladder biles, as well as 1-5 micromol L(-1) malondialdehyde on cultured gallbladder epithelial cells, was determined. RESULTS: Our data show an increased malondialdehyde (7.2 +/- 1.8 vs. 3.8 +/- 0.5 micromol L(-1), P = 0.01) and mucin concentration (0.9 +/- 0.09 vs. 0.41 +/- 0.03 mg mL(-1), P = 0.01) and an increased mucin secretagogue activity (2.0 +/- 0.5 vs. 1.1 +/- 0.3 mucin secretion/control, P = 0.04) and cholesterol saturation index (1.2 +/- 0.1 vs. 08 +/- 0.1, P = 0.04) in rapid as compared to non-nucleating gallbladder biles. Malondialdehyde stimulated mucin secretion of cultured gallbladder epithelial cells in a concentration dependent manner. CONCLUSIONS: Our results support a promoting effect of gallbladder mucin hypersecretion by lipid peroxidation leading to rapid formation of cholesterol crystals in gallbladder bile. These findings suggest that besides hypersecretion of cholesterol in bile, chronic inflammation of the gallbladder wall is implicated in the pathogenesis of cholesterol gallstone disease.

Apolipoprotein E polymorphism and lithogenic factors in gallbladder bile.

BACKGROUND: Associations between the polymorphism of apolipoprotein E, which plays an important role in cholesterol metabolism and cholesterol gallstone formation, have been reported recently. Patients with the apo E4 isoform showed increased numbers and cholesterol contents of their stones, a higher frequency of cholesterol crystals in bile, increased susceptibility to gallstone fragmentation by extracorporeal shock-wave lithotripsy and an increase in recurrence rate after dissolution. A recent study, however, showed that fast cholesterol crystallization in bile is associated with multiple stones but not with apo E4. Therefore the mechanism for an increased risk of gallstone formation in patients with the apo E4 isoform still remains under debate. DESIGN: To clarify this issue we investigated 37 patients with gallstones (10 with the apo E4 allele and 27 without the allele). Gallbladder biles were examined for total cholesterol and other lipids, cholesterol saturation index, crystal observation time, crystal mass, total protein and mucin. Moreover, number of gallstones and cholesterol in gallstones was compared in both groups. RESULTS: The crystal observation time (2.5 vs. 2.0 days, median) and the cholesterol saturation index (1.34 +/- 0.45 vs. 1.43 +/- 0.74) did not differ significantly between the apo E4 and the non apo E4 group. Total biliary lipids (11.6 +/- 3.8 vs. 9.3 +/- 3.9 g 100 mL-1, P = 0.126) and total biliary cholesterol (21.8 +/- 9.7 vs. 15.7 +/- 7 mmol L-1, P = 0.067) tended to be elevated in the apo E4 group. Crystal mass (3.60 +/- 4.10 vs. 2.38 +/- 2.70 mmol L-1), biliary total protein (8.6 +/- 3.5 vs. 8.3 +/- 6.6 mg mL-1) and mucin (0.55 +/- 0.38 vs. 0.66 +/- 0.67 mg mL-1), number (solitary/multiple) of gallstones and cholesterol in gallstones were not different in both groups of patients. CONCLUSIONS: In comparison to the non apo E4 patients the apo E4 group showed a trend to elevated biliary cholesterol whereas crystal observation time, cholesterol saturation index, crystal mass, number of gallstones, cholesterol content of gallstones and total protein and mucin were not different. These findings do not suggest an association of the apo E isoform and the formation of cholesterol gallstones

Mucin and phospholipids determine viscosity of gallbladder bile in patients with gallstones.

AIM: An increased viscosity of gallbladder bile has been considered an important factor in the pathogenesis of gallstone disease. Besides lipids and proteins, mucin has been suggested to affect the viscosity of bile. To further clarify these issues we compared mucin, protein and the lipid componEnts of hepatic and gallbladder bile and its viscosity in patients with gallstones. METHODS: Viscosity of bile (mPa.s) was measured using rotation viscosimetry in regard to the non Newtonian property of bile at low shear rates. RESULTS: Biliary viscosity was markedly higher in gallbladder bile of patients with cholesterol (5.00 +/- 0.60 mPa.s, mean +/- SEM, r= 28) and mixed stones (3.50 +/- 0.68 mPa.s; r= 8) compared to hepatic bile (0.92 +/- 0.06 mPa.s, r= 6). A positive correlation between mucin and viscosity was found in gallbladder biles (r = 0.65; P < 0.001) but not in hepatic biles. The addition of physiologic and supraphysiologic amounts of mucin to gallbladder bile resulted in a dose dependent non linear increase of its viscosity. A positive correlation was determined between phospholipid concentration and viscosity (r = 0.34, P < 0.005) in gallbladder biles. However, no correlation was found between total protein or the other lipid concentrations and viscosity in both gallbladder and hepatic biles. CONCLUSION: The viscosity of gallbladder bile is markedly higher than that of hepatic bile in patients with gallstones. The concentration of mucin is the major determinant of biliary viscosity and may contribute by this mechanism to the role of mucin in the pathogenesis of gallstones.

[75-year-old patient with persistent abdominal complaints and vomiting during a cruise]. / 75-jährige Patientin mit persistierenden abdominellen Beschwerden und Erbrechen während einer Kreuzfahrt.

BACKGROUND: In elderly patients with gallstone disease, a gallstone ileus must be considered for unexplained abdominal pain. This is demonstrated in the following case report. CASE REPORT: A 75-year-old female patient presented with a 72-hour history of abdominal pain, nausea and vomiting. The patient's abdomen was mildly distended, although soft and nontender with bowel sounds present. Plain radiographs and ultrasound investigation of the abdomen were compatible with small bowel obstruction. To clarify the etiology, an abdominal computed tomography scan was obtained. These examinations disclosed air in the biliary tree, dilated small bowel and an impacted intraluminal abnormality in the terminal ileum compatible with a gallstone. Operative intervention confirmed the presence of a 3 cm obstructing calculus in the terminal ileum that was removed by an enterolithotomy. A two-step cholecystectomy and closure of the cholecystoduodenal fistula were performed 8 weeks later. The patient's recovery was uneventful. CONCLUSIONS: Although rare in a general population, gallstone ileus accounts for 25% of nonstrangulated small bowel obstructions in patients over the age of 65. The radiographic picture and ultrasound of small bowel obstruction and the presence of air in the biliary tree are suggestive for the diagnosis of a gallstone ileus. In our patient, the computed tomography and ultrasound findings confirmed the diagnosis and led to a prompt and directed surgical intervention. In patients with comorbid factors a two-step approach with enterolithotomy in a first and cholecystectomy in a second operation should be the therapeutic strategy of choice.

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.

Effects of phospholipase A2, free fatty acids and 2-lysolecithin on the crystallization of cholesterol in gallbladder bile.

BACKGROUND: Phospholipase A2 (PLA2) and its enzymatic products free fatty acids (FFAs) and 2-lysolecithin are physiological constituents of bile. Their role in the crystallization of cholesterol in gallbladder bile of patients with cholesterol gallstones is still controversial. DESIGN: To clarify this issue we evaluated the activity of PLA2 and the concentration and pattern of FFAs in the gallbladder bile of cholesterol stone patients. We furthermore added PLA2, FFAs and 2-lysolecithin to isotropic gallbladder bile, determined the crystal observation time (COT) and counted the cholesterol crystals in a crystal growth assay for up to 21 days. RESULTS: A PLA2 activity of 1.8 +/- 1.2 U L(-1) and total FFA concentrations of 1.32 +/- 0.71 mmol L(-1) were determined. After incubation of bile for 24 h at 37 degrees C total FFAs increased to 2.72 +/- 1.29 mmol L(-1) (P<0.005). Biliary saturated and unsaturated FFAs were found in equal proportions before and after incubation, pointing to an additional presence of lipases other than PLA2. A COTof 1 day was observed in all gallbladder biles and increased to 1.7 +/- 0.5 days after addition of 5 U L(-1) of PLA2 (P<0.01). An even higher COT of 2.5 +/- 0.8 days was seen after addition of 5 mmol L(-1) of a 'biliary' mixture of FFAs (P<0.005) but the COT remained unchanged after addition of 5 mmol L(-1) of 2-lysolecithin. However, in the crystal growth assay in gallbladder bile addition of 5 U L(-1) of PLA2, of 5 mmol L(-1) of 'biliary' FFAs and of 5 mmol L(-1) of 2-lysolecithin decreased significantly the number of cholesterol crystals formed during follow-up. CONCLUSION: An elevated activity of PLA2 in gallbladder bile may counteract the formation of cholesterol crystals through increased formation of FFAs and 2-lysolecithin. However, regarding the comparatively low activity of PLA2 in gallbladder bile PLA2 seems to be of only minor pathophysiological importance in the formation of cholesterol gallstones.

CA19-9: a pedictor of response in pancreatic cancer treated with gemcitabine and cisplatin.

The question was asked whether kinetics of CA19-9 would serve as a predictor of chemotherapeutic outcome in advanced pancreatic cancer treated with gemcitabine and cisplatin. Twenty one patients, 5 females and 16 males (median age 56 yrs, range 36-71 yrs) suffering from adenocarcinoma of the exocrine pancreas were analysed. Chemotherapy was applied for a median of 6 courses (range 2-21). Four patients achieved a complete remission, four a partial remission (OR = 38%), while stable disease was documented in 8 and progressive disease in 5 patients. Among 4 CR patients, all demonstrated a significant decline of CA 19-9 levels during the initial three treatment courses with apparent half-lifes of 15, 18, 24, and 33 days. At a cut-off level of 37 U/mL, all CR patients reached normal values in the course of treatment. All patients achieving PR showed a decrease of CA 19-9 values at apparent half-lifes of 9, 16, 88 and 89 days. Among patients with stable disease, CA19-9 transiently decreased in 7/8 patients and remained stable in 1 patient. However, values increased later in all patients after a median of 3 treatment courses (range 2-9). In patients with disease progression, CA 19-9 initially increased in 4/5 patients, while a further patient did so only beyound 100 days of treatment. In conclusion, kinetics of CA19-9 serum concentration may serve as an early indicator of response to gemcitabine/cisplatin chemotherapy in advanced pancreatic cancer.

Deoxycholic acid is not related to lithogenic factors in gallbladder bile.

The influence of deoxycholic acid (DCA) on the factors in gallbladder bile responsible for cholesterol gallstone disease has been a controversial subject of discussion. This might be partially due to patient selection or inappropriate methods. Therefore, we investigated the relationship between the percentage of DCA and lithogenic factors in the gallbladder bile of patients with cholesterol gallstones and with normal or moderately impaired gallbladder contractility. Patients with pigment stones served as a control group. The percentage of DCA in the gallbladder bile of 20 patients with cholesterol stones (23.2%+/-6.5%; mean+/-SD) was comparable to the DCA percentage in the gallbladder bile of 11 patients with pigment stones (26.5%+/-8.5%). No correlation was seen between the DCA percentage of total bile acids and the crystal observation time, cholesterol saturation index (CSI), total protein value, mucin level, and amount of cholesterol in vesicles or crystals in the total group of patients or in the subgroups with cholesterol or pigment stones, respectively. The lack of correlation between DCA percentage and CSI was determined in native bile (r = 0.048) as well as in crystal-free bile after ultracentrifugation (r = 0.107). Our findings demonstrate that in patients with gallstones, the percentage of DCA in gallbladder bile is not related to any of the known biliary factors associated with cholesterol gallstone disease. We conclude that in patients with normal or moderately impaired gallbladder function, an elevated DCA level in the gallbladder bile is of minor pathophysiologic significance for the formation of cholesterol gallstones.

Carbohydrate-deficient transferrin is not a useful marker for the detection of chronic alcohol abuse.

BACKGROUND: The role of carbohydrate-deficient transferrin (CDT) as a reliable marker for the detection of chronic alcohol abuse has been discussed controversially. METHODS: Therefore, we investigated CDT in the sera from 405 subjects with different alcohol intake. Besides healthy control subjects (n = 42), inpatients and outpatients in a department of gastroenterology (n = 325) and patients admitted to a department of otorhinolaryngology (n = 38) were studied. A total of 213 patients suffered from various forms of liver diseases, and 89 patients had liver transplantation. CDT values were determined by a double-antibody radioimmunoassay. RESULTS: In the 241 alcohol-abstinent subjects, CDT levels ranged from 3 to 90 units L-1 (median = 12); the 92 moderate drinkers (20-60 g of alcohol per day) showed values from 3 to 40 units L-1 (median = 12), and the 72 subjects with chronic alcohol abuse (> 60 g per day) revealed CDT levels from 3 to 100 units L-1 (median = 16). The diagnostic specificity for alcohol abuse was 86.8% for men (sensitivity 36.9%) and 95% for women (sensitivity 0%). CONCLUSION: Our data indicate that measurement of CDT does not reach clinical use in the detection of chronic alcohol abuse in an unselected population because of its insufficient specificity and sensitivity.

Two-dimensional electrophoretic analysis of vesicular and micellar proteins of gallbladder bile.

Proteins associated with lipid vesicles or mixed micelles of human gallbladder bile were separated by Sepharose-2B gel filtration chromatography followed by protein concentration and delipidation. After two-dimensional polyacrylamide gel electrophoresis and silver staining up to 59 and 471 polypeptide spots were counted in vesicular and micellar fractions, respectively. As major components the plasma proteins transferrin, albumin, alpha-fibrinogen, beta-fibrinogen, gamma-immunoglobulin G, immunoglobulin light chains, alpha-1 antitrypsin and haptoglobin alpha-2 chain were identified in the lipid vesicles by comparison with human protein reference maps. However, most biliary proteins including the anionic polypeptide fraction are associated with mixed micelles. The pathophysiological significance of these proteins associated with lipids needs to be investigated further.

Biliary mucin secreted by cultured human gallbladder epithelial cells carries the epitope of CA 19-9.

Serum CA 19-9 is increased in patients with different gastrointestinal malignancies. Unfortunately, CA 19-9 is also detected in high concentrations in normal bile causing unspecific serum elevations during inflammatory disease of the biliary tract and cholestasis. In order to identify the source of CA 19-9 in bile, the capacity of cultured human gallbladder epithelial cells (HGBEC) to secrete CA 19-9 was investigated. Cells were harvested from gallbladders removed by cholecystectomy and cultured for up to 14 days in collagen I coated 24-well culture dishes. CA 19-9 was measured in the culture medium by a solid-phase CA 19-9 EIA (Boehringer). In addition, culture medium was separated by Sepharose 4B-Cl, Concanavalin-A (Con-A) and CA 19-9 affinity chromatography. Significant CA 19-9 activity was measured in the culture medium after a 24 hour incubation period. Following separation of the culture medium by Sepharose 4B-Cl and Con-A affinity chromatography, 90% of the CA 19-9 activity was recovered in the exclusion volume (> 2000 kDa) from which 90% were identified as Con-A negative. A close correlation was found between CA 19-9 and concentrations of mucin purified from human gallbladder bile. Furthermore, CA 19-9 affinity chromatography selectively extracted mucins from the culture medium of HGBEC. Finally, addition of the mucin secretagogue bethanechol (6 mM) to the culture medium increased CA 19-9 activity in the medium. In conclusion, normal HGBEC secrete mucins carrying the epitope of CA 19-9. During inflammatory biliary disease unspecific elevation of CA 19-9 in serum may reflect both inflammatory hypersecretion and leakage of biliary mucins into serum.

Rapid formation of cholesterol crystals in gallbladder bile is associated with stone recurrence after laparoscopic cholecystotomy.

Laparoscopic cholecystotomy (LCT) with subsequent extraction of gallstones and primary closure of the gallbladder has been introduced as an alternative therapy for patients with cholecystolithiasis and preserved gallbladder function. However, stone recurrence has to be considered as a major drawback that might be related to lithogenic factors of gallbladder bile or the composition of gallbladder stones. Therefore, these were studied in relation to stone recurrence within an observation period of 1 to 5 years (median, 3.6 years) in 50 patients after LCT. The concentrations of total and individual bile acids, phospholipids, cholesterol, total lipids, mucin, protein, and the cholesterol saturation indices in gallbladder bile were not significantly different between 10 patients with and 40 patients without stone recurrence. However, the crystal observation time was significantly (P < .02) shorter (range, 1-2 days; median, 1.5) in the bile of patients with stone recurrence compared to those without (range, 1-21 days, median 3.5). Moreover, all 10 stone recurrences were observed in the 28 patients with a crystal observation time in the bile of less than or equal to 2 days (approximate annual risk: 12%-15%), and no recurrences were observed in the 22 patients with a crystal observation time greater than 2 days (P < .0001) or in patients with pigment stones. The rapid formation of cholesterol monohydrate crystals in bile seems to be the major risk factor for recurrent stones after LCT. These are most likely cholesterol stones and, therefore, are amenable to oral bile-acid prevention or treatment.

Sedimentation of biliary sludge: effect on composition of gallbladder bile from patients with cholesterol, mixed, or pigment stones.

BACKGROUND: Ultracentrifugation of bile has been used extensively to remove insoluble material such as sludge from bile before further studies of cholesterol nucleation. Although it has been recognized that this procedure may affect the composition of gallbladder bile, it has not been studied systematically in different gallstone populations. Therefore, we investigated the concentration of biliary lipids, protein, mucin, and bilirubin before and after ultracentrifugation. METHODS: Gallbladder bile samples were aspirated during laparoscopic surgery from 66 patients (35 with cholesterol, 16 with mixed, and 15 with pigment stones). RESULTS: Whereas the concentrations of bile acids, phospholipids, protein, and bilirubin in gallbladder bile did not change significantly after ultracentrifugation, cholesterol (20.6 +/- 1.6 to 14.8 +/- 1.2 mmol/l) and mucin concentrations (0.99 +/- 0.2 to 0.67 +/- 0.1 mg/ml) and the cholesterol saturation index (1.68 +/- 0.12 to 1.31 +/- 0.10) decreased significantly in gallbladder bile from patients with cholesterol stones. CONCLUSIONS: Sedimentation of biliary sludge may profoundly affect the composition of gallbladder bile, which has to be considered in studies of cholesterol saturation and nucleation. The cholesterol concentration difference between native and ultracentrifuged bile reflects the insoluble crystalline fraction of cholesterol and may be useful for quantitation of the mass of cholesterol crystals in gallstone-associated bile samples.

Human chorionic gonadotropin-beta in the differentiation of malignancy-related and nonmalignant ascites.

The clinical value of the tumor marker human chorionic gonadotropin-beta (hCG-beta) in ascitic fluid for the differentiation of malignancy-related and non-malignant ascites was evaluated. Ascitic fluid protein, cholesterol and cytological examination were determined for comparison. Thirty-six patients with malignancy-related ascites (27 peritoneal carcinomatosis, 9 miscellaneous malignant causes without peritoneal carcinomatosis) and 69 patients with nonmalignant ascites (55 with liver cirrhosis, 14 with miscellaneous nonmalignant causes) were investigated. hCG-beta concentrations were elevated in malignant samples and with a cut-off value of 10 mIU/ml hCG-beta yielded a sensitivity of 61%, specificity of 94% and efficiency of 83%. Ascitic fluid protein (cut-off value 3.0 g/100 ml) and cholesterol (cut-off value 45 mg/100 ml) concentrations showed a sensitivity of 64%/83%, specificity of 77%/81% and efficiency of 72%/82%. The combination of hCG-beta and cytological examination yielded 89.5% differential diagnostic efficiency, superior to the combinations of protein and cytology or protein and hCG-beta. hCG-beta tended to be superior to protein/cholesterol determination regarding sensitivity (44% vs. 11%/33%) and specificity (79% vs. 50%/57%) in the subgroups of patients with miscellaneous causes of ascites. In conclusion, hCG-beta is frequently elevated in malignancy-related ascites and seems to be as useful a parameter as total protein for the differentiation of malignancy-related from nonmalignant ascites.

Correlation between biliary alpha 1-acid glycoprotein concentration and cholesterol crystal nucleation time in gallstone disease.

A biliary form of the alpha 1-acid glycoprotein (AAG) promotes cholesterol crystallization in the lower-molecular-weight, concanavalin A-bound fraction of gallbladder bile. In addition, bile AAG concentration is higher in cholesterol gallstone patients with multiple stones than in control patients without gallstone disease. In this study we sought to determine whether the increased biliary concentration of AAG in cholesterol gallstone patients is accompanied by a more rapid nucleation time in patients with multiple stones. AAG concentration in native biles was measured by ELISA. Nucleation time was measured using a standard microscopy method. The concentration of biliary AAG was then related to nucleation time in biles from the same patients. Nucleation times were significantly shorter (< or = 5 days) in cholesterol gallstone patients with raised AAG concentrations (P < 0.03). There was a significant (P = 0.004) negative correlation (r = -0.53) between nucleation time and the AAG concentration in cholesterol gallstone patients with multiple stones. The concentration of biliary AAG appears to exert an important influence on the speed of cholesterol nucleation in bile in many patients with cholesterol gallstone disease.