[Rapid ultracentrifugal isolation and imaging detection of cholesterol-phospholipid vesicles]

We have utilized ultracentrifugation of bile-metrizamide density gradients to isolate and to quantitate the cholesterol-phospholipid vesicles (CPV) and identified them by transmission electron microscopy and scanning electron microscopy metrizamide was dissolved in bile and continuous density gradients were performed. The distribution of biliary cholesterol, phospholipid and bile salt was studied. The distribution of biliary phospholipid is similar in different fraction of the gradients. Approximately 70% of total biliary cholesterol was concentrated in the lightest fraction of the gradients (density < 1.035g/ml) and 70% of bile salt was found in the heaviest fraction of the gradients (density > 1.035g/ml). CPV harvested in fractions with density < 1.035g/ml and their diameter was 70-90nm. When 15% Metrizamide was dissolved in bile and centrifuged (50,000rpm, 100min, 37 degrees C), two thirds of total biliary cholesterol was concentrated in the top 0.4ml of the 5ml centrifuge tube. A lot of CPV was found in this part of bile and their diameter was 85nm. The advantage of the procedure described in this study is simple, rapid and accurate for pathological and physiological studies of CPV.