Separation and quantitation of cholesterol carriers in native bile by ultracentrifugation

The vesicular and micellar carriers of biliary cholesterol were isolated and quantitated from native bile by a simple and short isopyknic ultracentrifugal method. The method was designed to decrease the potential pitfalls of classic ultracentrifugation: osmotic effects of the centrifugation media and hydrostatic pressure effects generated in the centrifuge tube. This was accomplished by using metrizamide as an inert centrifugation medium for isopyknic separation and a vertical rotor. The buoyant density of vesicles isolated from human native bile varied between 1.010 and 1.030 gm/ml, as determined in preformed bile-metrizamide density gradients after 285 min of centrifugation. When 16% metrizamide was directly dissolved in bile, its density increased to 1.060 gm/ml. After 120 min of centrifugation, it was found that more than 95% of total vesicular cholesterol floated at the top of the centrifuge tube. This fraction appeared as one or two white opalescent bands. The present ultracentrifugal method was validated by gel filtration chromatography. It was found that more than 95% of vesicular cholesterol migrated to the top 0.4 ml of the centrifuge tube after the short-run centrifugation. Approximately 5% of total biliary cholesterol present in the vesicular fractions was in fact solubilized in mixed micelles as assessed by gel filtration chromatography. Although the proportion of vesicles and micelles estimated with the present ultracentrifugal method is in the range reported by other authors using the more common chromatographic method, we believe that our method has two major advantages. First, it eliminates the dilutional effect of buffers necessary for gel filtration chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)