Method for quantitative assessment of transformation of non-micellar cholesterol carriers in model bile systems

Abstract

Aggregation and fusion of non-micellar particulate species, such as unilamellar vesicle and phospholipid lamellae, are believed to precede the nucleation of cholesterol crystals in bile. However, little is known about the time sequence relationship between transformation of non-micellar particles and the initial appearance of cholesterol crystals, as no adequate technique is available for assessing such transformations quantitatively. We have developed a novel method for quantitatively estimating vesicle transformation in supersaturated model bile systems, using a spectrophotometric technique to determine the time sequence relationship between such transformations and cholesterol crystal nucleation. We also investigated the potency of a given effector substance on this transformation. This method permits simultaneous quantitative determination of vesicle aggregation and of cholesterol crystal growth. Maximal vesicular aggregation as determined from turbidity, coincided with initiation of cholesterol crystal nucleation. The addition of divalent cations, Ca2+ and Mg2+, to the model bile solutions promoted vesicle aggregation and cholesterol crystal nucleation and growth. In contrast, apolipoproteins A-1 and A-2 retarded such processes. These data were highly reproducible and reliable. The method described is easy to perform, provides reproducible results and permits the determination of the potency of effector substances on vesicle transformation and on the nucleation of cholesterol crystals.