Rapid isolation of vesicular and micellar carriers of biliary lipids by ultracentrifugation.
A simple, rapid, and new method has been developed to isolate and to quantitate the vesicular carrier of biliary lipids by isopycnic ultracentrifugation. The method combines the use of Metrizamide, as an inert centrifugation media to change the density of bile for isopycnic separation of vesicles, and a vertical rotor, to decrease both the time of centrifugation and the pressure of the hydrostatic column in the ultracentrifuge tube. Vesicles harvested from bile-Metrizamide density gradients were identified by negative staining electron microscopy. The buoyant densitites of biliary vesicles varied between 1.010 and 1.030 g/ml. The diameter of vesicles in fractions with d less than 1.020 g/ml was 82 +/- 10 nm and in fraction with d approximately 1.030 g/ml was 57 +/- 8 nm. Gel filtration chromatography with Ultrogel AcA 34 was used to validate the quantitive isolation of vesicles by the ultracentrifugal method. In experiments with bile-Metrizamide continuous preformed density gradients, greater than 93% of vesicular cholesterol was found in fractions with d less than 1.030 g/ml after 285 min of centrifugation at 50,000 rpm in a VTi vertical rotor (Beckman Instruments, Inc.). When 16% Metrizamide was dissolved in bile and centrifuged for 120 min, greater than 96% of total vesicular cholesterol was found in the top 0.4 ml of the 5-ml centrifuge tube, as assessed by gel filtration chromatography. This fraction contained less than 8% of cholesterol carried in micelles, as assessed by gel filtration chromatography. The variation coefficient of this short ultracentrifugal method to isolate biliary vesicles was 4.6%.(ABSTRACT TRUNCATED AT 250 WORDS)
Amigo
L
L
Departmento de Gastroenterología, Facultad de Medicina, Pontifica Universidad Católica de Chile.
Covarrubias
C
C
Nervi
F
F
D001646:Bile / Q000032:analysis*; D002499:Centrifugation, Density Gradient / Q000379:methods*; D002500:Centrifugation, Isopycnic / Q000295:instrumentation / Q000379:methods*; D002850:Chromatography, Gel; D006801:Humans; D008055:Lipids / Q000302:isolation & purification*; D008793:Metrizamide; D008823:Micelles
D008055:Lipids; D008823:Micelles; D008793:Metrizamide