Separation and quantitation of cholesterol "carriers" in bile

On the basis of phase equilibria theory and experimental data, we discuss the identity, separation and quantitation of cholesterol-solubilizing lipid aggregates in bile, trivially known as cholesterol "carriers." We first describe analogies (as well as lack of correspondence) between equilibrium phase diagrams of aqueous bile salt, lecithin and cholesterol systems and phase equilibria in native biles. At equilibrium, the possible number and types of different cholesterol "carriers" are limited by constraints placed by the phase rule. These "carriers" include simple micelles (bile salts without lecithin), mixed micelles (bile salts plus lecithin), a lecithin plus cholesterol lamellar phase composed of single bilayer structures (vesicles) or multilamellar structures (liquid crystals) and possibly fragments of a hexagonal phase. Clearly, in this context, cholesterol monohydrate crystals, no matter how small, cannot be considered "carriers." We also use a metastable "phase diagram" for aqueous taurocholate-egg yolk lecithin-cholesterol systems to interpret physicochemical studies on the formation and stability of native biles. In biliary lipid systems, bile salt monomers with or without simple bile salt micelles are present in the aqueous "phase" in equilibrium with mixed micelles/or vesicles. This bile salt concentration is designated the intermicellar concentration but actually represents the total nonmixed micellar/nonvesicular concentration of bile salts (i.e., bile salt monomers and simple bile salt micelles when present) and may be a value that falls below, equal to or above the critical micellar concentration of the bile salts. Experimental estimates of the intermicellar-intervesicular bile salt concentration in model systems are compared with the range of bile salt concentrations that has been used in the literature to separate the presently accepted cholesterol "carriers" (mixed micelles and vesicles) in both model and native biles. We also reinterpret published lipid compositions of phases separated from human bile, using both a nonequilibrium "phase diagram" and an equilibrium phase diagram for the model system, taurocholate-egg yolk lecithin-cholesterol. From these analyses, we conclude that precise quantitation of cholesterol "carriers" in bile awaits methods to accurately determine the intermicellar-intervesicular concentration of bile salts in any individual bile, as well as advances in nonperturbing separatory procedures, and methods to control the thermal and temporal history of native bile samples.