BILIARY HAPTOGLOBIN, A POTENT PROMOTER OF CHOLESTEROL CRYSTALLIZATION AT PHYSIOLOGICAL CONCENTRATIONS

Background/Aims: Several proteins present in human bile have been reported to promote cholesterol crystallization and thus are potentially important in the formation of cholesterol crystals as the initial stage in gallstone pathogenesis. To be physiologically relevant, such proteins must either be present in high concentration in bile or have a potent promoting activity. The current study explored several of the more abundant but unexamined biliary proteins based upon their also having sufficiently high serum concentrations that antibodies were available for both their isolation and quantitation. Methods: Protein purification was accomplished by immunoaffinity chromatography of bile followed by delipidation. Con A affinity chromatography of bile was used to obtain the bound fraction, a portion of which was delipidated. Crystallization-promoting activity of both the purified proteins and Con A-bound glycoprotein fractions (CABG) was measured by a photometric crystal growth assay. A competitive antibody-capture ELISA assay was developed to measure concentrations of alpha(1)-antitrypsin, transferrin, and haptoglobin in native bile. Results: At their relevant physiological concentrations, biliary haptoglobin (15 mu g/ml) had a crystallization-promoting activity twice that of the biliary IgM (75 mu g/ml) used as a reference standard (P < 0.05). Biliary transferrin (20 mu g/ml) had only modest promoting activity (P < 0.05). Biliary alpha(1)-antitrypsin (50 mu g/ml), by contrast, showed no promoting activity. Delipidation of the CABG fraction decreased its promoting activity by 75%. Biliary haptoglobin accounts for about 30% of delipidated total CABG-promoting activity. Conclusions: Biliary haptoglobin at its physiological concentration has a highly potent crystallization-promoting activity and thus becomes a candidate for major attention in understanding gallstone pathogenesis. Biliary lipids associated with CABG account for a major portion of the cholesterol-crystallization-promoting activity of this fraction

Biliary alpha 1-acid glycoprotein concentrations in gallstone-free controls and in patients with multiple or solitary cholesterol gallstones.

Abstract: We recently identified a promoting glycoprotein in the concanavalin A-bound fraction of gallbladder bile as a biliary form of alpha 1-acid glycoprotein (AAG), The concentration of biliary AAG appears to exert an important promoting effect on the speed of cholesterol nucleation in many patients with cholesterol gallstone disease, In the current study, we provide information about the biliary concentration of AAG as well as the amount and comparative potency of its subfractions in patients with and without cholesterol gallstone disease, The amount of total biliary AAG and the amounts of its different isoforms separated by concanavalin A affinity chromatography were measured by ELISA, Estimates of absolute concentrations of AAG for each sample were normalized to the sample total protein content to give relative AAG values. The promoting activity (potency) of immunopurified biliary AAG from gallstone patients and gallstone-free controls on cholesterol crystallization was compared by a crystal growth assay. The mean absolute concentration of AAG in gallstone-free controls was not significantly different from multiple stone patients. The relative concentration of AAG (micrograms per milligram total protein) was significantly increased in patients with multiple stones when compared to controls (P < 0.05), and both the absolute and relative concentrations of AAG (micrograms per milligram bile), were three- and to five fold higher in a number of these patients. The functional activity and distribution of AAG in different subfractions was similar in gallstone patients and gallstone-free controls, The relative concentration of biliary AAG is significantly greater in cholesterol gallstone patients with multiple stones than in gallstone-free controls, These observations suggest that raised levels of AAG may be of pathogenetic importance in a subgroup of patients with multiple gallstones

Correlation between biliary alfa1-acid glycoprotein concentration and cholesterol crystal nucleation time in gallstone disease.

Abstract: 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 (less than or equal to 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