Gel filtration and quasielastic light scattering studies of human bile

Analyses of cholesterol carriers in the extremely variable biliary samples were performed by chromatography under standard conditions. Sephacryl columns were eluted with buffer containing 10 mmol/L sodium cholate, 150 mmol/L sodium chloride, 50 mmol/L Tris(hydroxymethyl)aminomethane-hydrochloride, pH 8.0, and 1.5 mmol/L ethylenediaminetetraacetate. Moderate changes in temperature, flow rate or sample size produced only minor differences in the results. Increments in bile salt concentration in the elution buffer caused progressive disappearance of vesicles and a rise in their cholesterol/phospholipid ratio. Using the standard chromatographic technique, analyses of the same bile gave reproducible results, and comparisons among various biles were possible. Quantitative light scattering measurements of bile showed that the amount of vesicular cholesterol in whole unprocessed bile was similar to that measured by Sephacryl chromatography. Analyses of human gallbladder biles using a high-resolution Sephacryl column showed four cholesterol-containing peaks: vesicles, lamellae, mixed micelles and an undefined carrier. This long chromatographic column separated the lamellar from the micellar peaks (which had merged on the short column). Phospholipid lamellae solubilized most of the biliary cholesterol. These findings were confirmed by ultracentrifugation of bile.