Biliary haptoglobin, a potent promoter of cholesterol crystallization at physiological concentrations

BackgroundlAims: Several proteins present in human bile have been reported to promote cholesterol crystalli- zation 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 abun- dant but unexamined biliary proteins based upon their also hav- ing sufficiently high serum concentrations that antibodies were available for ,both their isolation and quantitation. Methods: Protein purification was accomplished by immunoaffinity chro- matography 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 glyco- protein fractions (CABG) was measured by a photometric crys- tal growth assay. A competitive antibody-capture ELISA assay was developed to measure concentrations of al-antitrypsin, transferrin, and haptoglobin in native bile. Results: At their rele- vant physiological concentrations, biliary haptoglobin (15 pg/ml) had a crystallization-promoting activity twice that of the biliary IgM (75 pglml) used as a reference standard (P < 0.05). Biliary transferrin (20 pg/ml) had only modest promoting activity (P < 0.05). Biliary a,-antitrypsin (50 pglml), by contrast, showed no promoting activity. Delipidation of the CABG frac- tion decreased its promoting activity by 75%. Biliary hapto- globin 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 at- tention in understanding gallstone pathogenesis. Biliary lipids associated with CABG account for a major portion of the cholesterol-crystallization-promoting activity of this fraction.