Cholesterol precipitation from cholesterol-supersaturated bile models

Abstract

Bile-model systems containing cholesterol (CH), phosphatidylcholine (PC) and sodium cholate (NaC) at concentrations similar to those found in supersaturated human gall bladder bile ([CH]/[PC] = 0.60 ± 0.01; CH saturation index, CSI = 1.58 ± 0.03) were prepared by mixing PCCH vesicles with NaC micellar solutions. Following mixing, the dispersion became transparent and gave rise to high resolution HNMR spectra typical of mixed micellar systems. Cryo-transmission electron micrographs of specimens vitrified at that stage support the conclusion that the vesicles had become completely micellized. Following micellization, the metastable (cholesterol-supersaturated) bile-models spontaneously underwent a series of reorganizational steps: first, cholesterol-rich vesicles with a [CH]/[PC] ratio of 1.57 ± 0.69 were formed, in co-existence with a mixed micellar system with [CH]/[PC] = 0.43 ± 0.01 and CSI = 1.12 ± 0.03. The resultant cholesterol-rich vesicles subsequently aggregated and cholesterol crystals of varying sizes and shapes appeared within the aggregates: needle-like structures were first observed, followed by clusters of those crystals and of helical crystals. Eventually, typical plate-like cholesterol crystals appeared, at which time some of the PC returned to the non-particulate (isotropic) phase. Consequently, the system contained cholesterol crystals coexisting with mixed micelles, whose composition was close to the limit of saturation (CSI = 1.08). These findings confirm the sequence of events preceding the appearance of cholesterol crystals, as previously proposed in our less detailed studies ((1990) Hepatology 12, 149S) and support the relevance of the morphologically similar results of Konikoff et al. ((1992) J. Clin. Invest. 90, 1155) obtained in a very dilute supersaturated bile-model.