Characterization of crystallization pathways during cholesterol precipitation from human gallbladder biles: Identical pathways to corresponding model biles with three predominating sequences

In model biles, five crystallization sequences are present as functions of bile salt/lecithin (egg yolk) ratio and their position on phase diagrams are influenced by bile salt hydrophobicity, temperature, and total lipid concentration (D. Q.-H., Wang and M. C. Carey, J. Lipid Res. 1996, 37: 606- 630). To determine whether the same pathways occur ex vivo during cholesterol precipitation from human gallbladder biles, we examined 22 cholesterol gallstone (CSI = 1.56 ± 0.26), 4 pigment gallstone (0.69 ± 0.06), and 4 controls biles (0.85 ± 0.22) by microscopy and lipid analytic techniques for 30 days. Temperature was varied (4-45°C) to more relative compositions into adjacent pathways or supersaturated zones to test whether the same bile could be forced to crystallize in different sequences. Sequences in native bile were identical to those in model systems composed of mixed bile salts- lecithin-cholesterol mixtures, and three corresponding pathways (B,C,D; op. cit.) were observed at 37°C. With increasing lecithin content, we found i) B: plate-like cholesterol monohydrate crystals appeared before arc-shaped (putatively anhydrous cholesterol) crystals which transformed via helices and tubules into plate-like crystals and no liquid crystals formed; ii) C: lamellar liquid crystals, typified by birefringent multilamellar vesicles, were detected before cholesterol monohydrate crystals, and subsequently arc, helical and tubular crystals appeared; and iii) D: precipitation of lamellar liquid crystals was followed by cholesterol monohydrate crystals and no arc crystals were detected. Added EDTA prevented calcium bilirubinate formation, but crystallization sequences in these biles were identical to those without EDTA. We conclude that i) cholesterol crystallization pathways and sequences in human gallbladder biles are identical to model biles matched for appropriate physical-chemical conditions: ii) three of the five sequences observed in model biles were found in native bile; and iii) calcium bilirubinates neither promote biliary cholesterol crystallization nor influence crystal growth.