Separation of bile vesicles and micelles by gel filtration chromatography: The importance of the intermicellar bile salt concentration

Micelles and vesicles coexist in native bile. Mixed micelles are composed of bile salt, phospholipid, and cholesterol. Micellar bile salt is in equilibrium with the aqueous phase bile salt (intermicellar bile salt), and mixed micelles can be converted to cholesterol-phospholipid vesicles by depletion of bile salt. To determine the amount of cholesterol carried in vesicles and micelles, these two populations must be separated without altering the relative proportion of each. Based on the size difference between micelles and vesicles, gel filtration chromatography has been used to accomplish this separation. We reasoned that to maintain the proportion of micelles and vesicles in bile, the column must be equilibrated and eluted with buffer containing the intermicellar bile salt concentration (IMBC) and species. To test this hypothesis we created a model bile composed exclusively of micelles, a solution containing micelles and vesicles, and a model bile containing all vesicles, as demonstrated by quasielastic light scattering. Gel filtration on Sepharose 4B demonstrated that model vesicles and micelles could be separated on a column eluted with buffer containing bile salt at the IMBC. However, a modest decrease in the buffer bile salt concentration (<1 mmol/L) resulted in complete conversion of micelles to vesicles. A comparable increase in the buffer bile salt concentration converted vesicles to micelles. Using only taurocholate in the eluting buffer at the IMBC caused a complete shift of micelles to vesicles, whereas using only taurochenodeoxycholate resulted in conversion of vesicles to micelles. An initial collection of rat bile separated on a column equilibrated with the measured IMBC demonstrated that 94% of the cholesterol was in the micellar fractions. In the same rats depleted of bile salt for a 24-hour period, only 54% of the biliary cholesterol was found in the micellar fractions after column separation, and 46% was contained in vesicles. These results support the concept that the proportion of mixed micelles and vesicles in bile is dependent on the concentration and composition of the intermicellar bile salt.