The effects of bile salt hydrophobicity on model bile vesicle morphology

The addition of bile salts to vesicles supersaturated with cholesterol induces cholesterol precipitation, an important step in the formation of cholesterol gallstones. To investigate the effects of bile salt hydrophobicity on vesicle morphology, vesicles obtained from supersaturated model bile by density gradient ultracentrifugation, were incubated with mixtures of deoxycholate (DC) and ursodeoxycholate (UDC) with a constant total bile salt concentration of 30 mM but with a varying hydrophobicity index ranging from -0.31 (UDC alone) up to +0.72 (DC alone) depending on the composition of the mixture. Five days after addition of bile salts to vesicles, cholesterol precipitation was determined microscopically and incubation samples were again subjected to ultracentrifugation to assess the lipid distribution among residual vesicles, mixed micelles, and cholesterol crystals. Structure and size of the isolated residual vesicles were studied by freeze fracture electron microscopy. The control, and samples exposed to hydrophilic bile salt mixtures, consisted of unilamellar vesicles of which more than 75% had a diameter of 50-80 nm. After addition of increasingly hydrophobic bile salt mixtures, multilamellar vesicles with progressively greater diameters (up to 1300 nm) were found, suggesting that vesicle fusion and aggregation took place and might hence be important in the cholesterol precipitation process. Accordingly, crystallization was positively correlated with bile salt hydrophobicity. We conclude that cholesterol crystallization from vesicles depends on the hydrophobicity of the bile salts added, and apparently occurs from fused or aggregated vesicles of extended magnitude and with a multilamellar constitution.