BILE SALT-INDUCED CHOLESTEROL CRYSTAL-FORMATION FROM MODEL BILE VESICLES - A TIME-COURSE STUDY

Precipitation of cholesterol crystals from vesicles is an important st ep in the pathogenesis of cholesterol gallstones. Little is known, how ever, about the kinetics and the mechanisms involved in cholesterol cr ystallization. Therefore, the time course of cholesterol crystal preci pitation and lipid exchange between vesicles and micelles were monitor ed in a model bile system. Vesicles obtained from supersaturated model bile (cholesterol saturation index (CSI) 1.4; 10 g/dl) by KBr density gradient ultracentrifugation, were incubated with various bile salts: deoxycholate (DC), chenodeoxycholate (CDC), cholate (C), ursodeoxycho late (UDC), and their respective taurine and glycine conjugates. Vesic le integrity was assessed in a leakage-assay of carboxyfluorescein-loa ded vesicles (0-15 min) and by the change in optical absorbance at 340 nm of a vesicle solution (0-50 min). Fluorescence increased within 1 m in after addition of bile salt, and was stable within 5-10 min. After addition of bile salt, absorbance fell immediately and stabilized with in 30 min. Fluorescence and absorbance were dependent on bile salt hyd rophobicity and concentration. At several time points after addition o f bile salt to vesicles (from 1 to 72 h), the extent of cholesterol nu cleation was determined semiquantitatively and incubation mixtures wer e again subjected to ultracentrifugation to assess the lipid distribut ion among residual vesicles, de novo formed mixed micelles, and choles terol crystals. Nucleation occurred within 0.5 h after exposure of ves icles to the hydrophobic bile salts DC or CDC, and the cholesterol/pho spholipid (cip) ratio of the vesicles showed a transient rise from 1.4 5 to 3-4 (at t = 0.5 h) that coincided with the appearance of mixed mi celles. Then the vesicular c/p ratio decreased to 0.6-0.8 (at t = 24 h ) concomitantly with increasing precipitation of cholesterol crystals. In the case of UDC, the most hydrophilic bile salt used, < 5% micelli zation, no nucleation, and a constant vesicular cip ratio were observe d. We conclude that under the conditions used in the present model stu dy, the kinetics of cholesterol crystallization are governed by the hy drophobicity of the added bile salts and their capacity to form mixed micelles. The results emphasize the pivotal role of time, and the dyna mic aspects of the processes involved in cholesterol crystal formation .