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The liver secretes cholesterol and lecithin in the form of mixed vesicles during the formation of bile. When exposed to bile salts, these metastable vesicles undergo various structural rearrangements. We have examined the effects of three different bile salts, taurocholate (TC), tauroursodeoxycholate (TUDC), and taurodeoxycholate (TDC), on the stability of sonicated lecithin vesicles containing various amounts of cholesterol. Vesicle growth was probed by turbidity measurements, quasi-elastic light scattering, and a resonance energy transfer lipid-mixing assay. Leakage of internal contents was monitored by encapsulation of fluorescence probes in vesicles. At low bile salt-to-lecithin ratios (TC/L or TUDC/L1), pure lecithin vesicles do not grow, but exhibit slow intervesicular mixing of lipids as well as gradual leakage. At high BS/L (TC/L or TUDC/L5), pure lecithin vesicles are solubilized into mixed micelles with a concomitant decrease in the overall particle size. In this regime, extensive leakage and lipid mixing occur instantaneously after exposure to bile salt. At intermediate BS/L (1TC/L or TUDC/L5), vesicles grow with time, and the rates of both leakage and lipid mixing are rapid. The data suggest that vesicles grow by the transfer of lecithin and cholesterol via diffusion in the aqueous medium. The addition of cholesterol to lecithin vesicles reduces leakage dramatically and increases the amount of BS required for complete solubilization of vesicles. The more hydrophobic TDC induces vesicle growth at a lower BS/L than does TC or TUDC. These results demonstrate the physiologic forms of lipid microstructures during bile formation and explain how the hydrophilic-hydrophobic balance of BS mixtures may profoundly affect the early stages of CH gallstone formation.

We have used small-angle neutron scattering (SANS) to probe the structure and interparticle interactions of lecithin-taurodeoxycholate mixed micelles. The data are fit to a core-shell model that provides the micelle composition and dimensions. The effects on the scattering spectra of electrostatic and excluded-volume interactions are explored in terms of the decoupling approximation and the random phase approximation. We found the TDC-lecithin micelles are cylindrical particles with an average cross-sectional radius of 26.7 [+-] 0.4 A. The core-shell structure is found to be an appropriate model for the highly hydrated micelles. The micelle length increases dramatically with an increase in added electrolyte, but not with decreasing concentration as previously reported. The SANS data analysis shows that particles in 0.05 M NaCl grow by less than 15% with a 3-fold decrease in the total surfactant concentration. This is in contrast to the simple interpretation of dynamic light scattering of the same samples that shows an apparent doubling of the micelle length with the same decrease in surfactant concentration. This discrepancy is attributed to neglect of the thermodynamic and hydrodynamic interactions in the analysis of the dynamic light scattering data. 59 refs., 5 figs., 5 tabs.

We have investigated the effects of the Ca(2+)-requiring enzyme phospholipase C on the stability of sonicated vesicles made with different molar ratios of cholesterol to lecithin. Vesicle aggregation is detected by following turbidity with time. Upon the addition of phospholipase C and after a short lag period, the turbidity of a vesicle dispersion increases continuously with time. The rate of increase of turbidity increases with both the enzyme-to-vesicle ratio and the cholesterol content of the vesicles. Vesicle fusion and leakage of contents are monitored by a contents-mixing fusion assay using 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and p-xylylenebis(pyridinium bromide) (DPX) as the fluorescence probes [Ellens, H., Bentz, J. & Szoka, F.C. (1985) Biochemistry 24, 3099-3106]. The results clearly show that phospholipase C induces vesicle fusion. The rate of vesicle fusion correlates with the enzyme-to-vesicle ratio but not with the cholesterol content of the membrane. Negligible aggregation and fusion of vesicles occurs when the experiment is repeated with buffer free of Ca2+. The membrane-destabilizing diacylglycerol, a product of lecithin hydrolysis by phospholipase C, is speculated to play a major role in driving the observed vesicle aggregation and fusion. The kinetics of vesicle aggregation and vesicle fusion can be predicted by linking Michaelis-Menten enzyme kinetics to a mass-action model.