MONITORING CHOLESTEROL CRYSTALLIZATION FROM LITHOGENIC MODEL BILE BY TIME-LAPSE DENSITY GRADIENT ULTRACENTRIFUGATION

Background/Aims: Cholesterol crystallization in a dilute, bile salt-ri ch model bile is a multiphase process in which early filamentous cryst als gradually transform to classical cholesterol monohydrate plates, T he pertinence of similar transformations in more complex model systems or native bile is, however, unclear, The aim of the present study was to characterize and monitor cholesterol crystallization in a model bi le of physiological relevance. Methods: A supersaturated model bile wa s prepared with a lipid composition (18 mM cholesterol, 37 mM lecithin , 120 mM taurocholate) that was derived from analyzing 10 gallbladder biles from cholesterol gallstone patients, Cholesterol crystallization was followed by light and electron microscopy, and sequential density gradient analysis of cholesterol-containing precipitates. Results: Du ring cholesterol crystallization a reproducible sequence of events was recorded, First (T<18 h), cholesterol-rich vesicular and multilamella r structures (density 1.005-1.015 g/ml) were observed, Later, (T>60 h) filamentous, helical, tubular (density 1.015-1.04 g/ml) and plate-lik e (density 1.04-1.06 g/ml) cholesterol crystals appeared, The concentr ation of crystals increased gradually, while bilayer structures became desaturated with cholesterol and disappeared, and early crystal forms were replaced by plates, Eventually (T>25 days) only classical plate- like cholesterol monohydrate crystals were present, Exposure of choles terol-containing precipitates to micellar (100 mM) deoxycholate dissol ved the bilayer structures but not the crystals. Conclusions: These da ta demonstrate that cholesterol crystallization in a physiologically r elevant model bile is a multiphase process consisting of a sequence of transitions from vesicular and multilamellar structures to early crys tal forms and to classical plate-like cholesterol monohydrate crystals , These transitions are associated with increasing density and decreas ing phospholipid content of cholesterol precipitates, We suggest that time-lapse density gradient ultracentrifugation is a useful method for investigating and quantitating the process of cholesterol crystalliza tion and factors that influence this process in bile.