Rapid determination by centrifugal ultrafiltration of inter-mixed micellar/vesicular (non-lecithin-associated) bile salt concentrations in model bile: influence of Donnan equilibrium effects

Abstract

We have developed the technique of rapid (< 2 h) centrifugal ultrafiltration to measure the inter-mixed micellar/vesicular (non-lecithin-associated) bile salt (BS) concentrations (IMC) of individual BS in model biles. This methodology uses a centrifugal concentrator with a reinforced membrane, through which a small fraction (< 15% total volume) of model biles was ultrafiltered by low centrifugal forces (1500 g 5-60 min). Total and individual BS concentrations in the filtrate were measured by high performance liquid chromatography. However, non-filterable anions, in this case BS/lecithin/cholesterol mixed micelles and unilamellar vesicles, induce an asymmetric distribution of ions across the membrane. Therefore, BS concentrations in the filtrate exceeded the true IMC, which was estimated taking into account Donnan equilibrium effects. To confirm the hypothesis that a correction for Donnan forces was necessary, distributions of BS and another filterable anion, chloride, were measured in systems containing the non-filterable polyanion dextran sulfate as an analogue for non-filterable polyanionic mixed micelles and vesicles. An asymmetric distribution of the monovalent anions chloride and BS monomers as well as polyvalent simple BS micelles was indeed present during centrifugal ultrafiltration. This new methodology was validated by comparing IMC values with those obtained by modified equilibrium dialysis also corrected for Donnan equilibrium effects (Donovan, J.M., et al. 1991. J. Lipid Res. 32: 1501-1512). Centrifugal ultrafiltration, which utilizes < 1 ml of bile, determines the composition in the IMC necessary to separate micelles and vesicles of native biles by techniques that involve dilution of bile such as gel filtration chromatography.