Cholesterol Enhances Membrane-Damaging Properties of Model Bile by Increasing the Intervesicular-Intermixed Micellar Concentration of Hydrophobic Bile Salts

Abstract

Bile salts are potent detergents that, at concentrations attained in bile and intestine, can disrupt cell membranes. Hepatic secretion of vesicles containing lecithin and cholesterol appears to be critical in preventing bile salt damage to hepatobiliary epithelia. We hypothesize that the protective effect of biliary lipids results from lowering of the bile salt intervesicular intermixed micellar bile salt concentration (IMMC) to which epithelial membranes are exposed. We further hypothesize that increases in biliary cholesterol, by reducing association of bile salts with vesicles and mixed micelles, may increase bile toxicity by raising the bile salt IMMC.Method.Large unilamellar lecithin vesicles (100 nm) with varying cholesterol:lecithin molar ratios (C:L) of 0, 0.5, and 1 were added to taurochenodeoxycholate (TCDCA), taurocholate (TCA), or taurodeoxycholate (TDCA) in Tris-buffered saline, pH 7.4. Human erythrocyte ghosts (model target membrane), prepared by osmotic hemolysis and resealed with [14C]inulin trapped inside, were added and incubated at 37°C for 30 min and 4 h. Plasma membrane disruption was quantified by [14C]inulin release and bile salt IMMC was determined by ultrafiltration.Results.Membrane disruption started at a concentration of 0.5 mM for TDCA, 1 mM for TCDCA, and 2 mM for TCA and was complete within 4 h at concentrations of 1, 2, and 4 mM, respectively. Addition of 2 mM lecithin to 2 mM TDCA, 4 mM TCDCA, or 5 mM TCA reduced or eliminated membrane leakage and lowered the IMMC. For TDCA and TCDCA, the protective effect of vesicles was entirely attributable to reduction in IMMC; in contrast for TCA, the protective effect exceeded that which would have been expected based solely on reduction of the IMMC. Inclusion of cholesterol attenuated the binding of bile salts to vesicles and raised the IMMC, thereby reducing the protective effect of lecithin over the time course of these studies. Although there was loss of phospholipid and cholesterol from the erythrocyte membranes on addition of bile acids even in the presence of vesicles, the ratio of cholesterol to phospholipid in the erythrocyte membrane did not change.Conclusion.Lecithin protects against membrane disruption by hydrophobic bile salts by lowering the IMMC. Cholesterol added to lecithin raises the bile salt IMMC and reduces or eliminates this protective effect. This mechanism of potentiation of bile salt toxicity by cholesterol may be an important contributor to the pathogenesis of gallbladder disease in cholesterol cholelithiasis.