Rapid (1 hour) high performance gel filtration chromatography resolves coexisting simple micelles, mixed micelles, and vesicles in bile.
We describe the use and validation of Superose 6, a high performance gel filtration medium for rapid, high resolution separation and sizing of coexisting simple micelles, mixed micelles, and vesicles in bile. We fractionated model biles (1.7-4.2 g/dl total lipid concentration, 0.15 M NaCl) composed of lecithin (L), cholesterol (Ch), and the common bile salt taurocholate (TC) using Superose 6 gel filtration columns (1.0 cm diameter, 30 cm length, 0.5 ml model bile application, 1.0 ml fractions) pre-equilibrated and eluted with 2.5-10.0 mM TC. Lipid particle sizes were determined by quasielastic light scattering and lipid compositions by conventional analyses. In the absence of L and Ch, pure TC "biles" (32.2 mM), when eluted in the presence of 7.5 mM TC, yielded a single peak of particles (mean hydrodynamic radii, Rh values of 11-15 A), consistent with simple TC micelles. Model biles containing L and TC ([L] = 13.8 mM, [TC] = 32.2 mM) were fractionated with baseline resolution into TC-L mixed micelles, (Rh values of 30-40 A) and simple TC micelles. In agreement with the ternary TC-L-H2O phase diagram (Mazer, N. A., et al. 1980. Biochemistry. 19: 601-615), the proportions of simple and mixed micelles were inversely related to L concentrations ([L] = 0-32.2 mM) and correlated positively with eluant TC concentration. Superose 6 gel fractionation of model biles "super-saturated" with Ch (TC:L:Ch molar ratio 27:63:10, total lipid concentration 3 g/dl) yielded high resolution separation of vesicles (Rh value of 320 A) from mixed micelles of TC-L-Ch (Rh values of 40-50 A) and simple TC micelles (Rh values of 11-15 A). At an eluant TC concentration of 7.5 mM, Ch-rich vesicles (Ch/L molar ratio = 1.6) separated that contained 40% of total Ch, 9% of total L, and no TC, accurately reflecting predictions of the quaternary L-Ch-TC-H2O metastable phase diagram (Mazer, N. A., and M. C. Carey. 1983 Biochemistry. 22: 426-442). This suggested that a 7.5 mM TC concentration approximated the intermicellar concentration under the experimental conditions. We also fractionated an identical model bile using conventional Sephacryl S-300, a medium generally used to study model and native biles. Compared with Superose 6, the Sephacryl S-300 column of equivalent size yielded particle separations with lower resolution and speed (30 h v l h).(ABSTRACT TRUNCATED AT 400 WORDS)
Cohen
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Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, MA 02115.
Carey
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MC
D001646:Bile / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry; D002850:Chromatography, Gel / Q000379:methods*; D008823:Micelles; D010713:Phosphatidylcholines / Q000737:chemistry; D013656:Taurocholic Acid / Q000737:chemistry; D013997:Time Factors
D008823:Micelles; D010713:Phosphatidylcholines; D013656:Taurocholic Acid; D002784:Cholesterol