Filamentous, helical, and tubular microstructures during cholesterol crystallization from bile. Evidence that cholesterol does not nucleate classic monohydrate plates.

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Abstract
Precipitation of cholesterol in gallbladder bile is believed to produce platelike cholesterol monohydrate crystals directly. We report complementary time-lapse microscopic studies of cholesterol crystallization from model bile that reveal initial assembly of filamentous cholesterol crystals covered by a monomolecular layer of lecithin. Over a few days, the filaments evolved through needle, helical, and tubular microstructures to form classical platelike cholesterol monohydrate crystals. Similar crystallization phenomena were observed in human gallbladder biles from cholesterol but not pigment stone patients. Synchrotron x-ray diffraction of the earliest filaments suggested a cholesterol monohydrate polymorph or admixture with an anhydrous cholesterol precursor. However, density gradient centrifugation of filamentous crystals revealed that their density was 1.032 g/ml, consistent with anhydrous cholesterol. Conventional x-ray diffraction of transitional crystalline forms was consistent with pure cholesterol monohydrate crystals, as were the equilibrium platelike crystals. These novel findings suggest that crystalline cholesterol in bile may not be completely mature or hydrated initially, but undergoes a series of transformations to become thermodynamically stable monohydrate plates. These observations have important implications for understanding the control of cholesterol crystallization in bile, as well as explaining putative crystal cytotoxicity during gallstone formation.