Journal of Lipid Research, Vol 31, 2103-2112, Copyright © 1990 by Lipid Research, Inc.

ARTICLES

Rapid (1 hour) high performance gel filtration chromatography resolves coexisting simple micelles, mixed micelles, and vesicles in bile

DE Cohen and MC Carey
Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, MA 02115.

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)
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