BILIARY HAPTOGLOBIN, A POTENT PROMOTER OF CHOLESTEROL CRYSTALLIZATIONAT PHYSIOLOGICAL CONCENTRATIONS

Background/Aims: Several proteins present in human bile have been repo rted to promote cholesterol crystallization and thus are potentially i mportant in the formation of cholesterol crystals as the initial stage in gallstone pathogenesis. To be physiologically relevant, such prote ins must either be present in high concentration in bile or have a pot ent 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 purificat ion was accomplished by immunoaffinity chromatography of bile followed by delipidation. Con A affinity chromatography of bile was used to ob tain the bound fraction, a portion of which was delipidated. Crystalli zation-promoting activity of both the purified proteins and Con A-boun d 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 h aptoglobin in native bile. Results: At their relevant physiological co ncentrations, biliary haptoglobin (15 mu g/ml) had a crystallization-p romoting 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 o nly 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-promotin g activity. Conclusions: Biliary haptoglobin at its physiological conc entration has a highly potent crystallization-promoting activity and t hus 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.