Figure 2: (a) The figure shows the physical states of lipids in human bile [21]. Bile is composed mainly of water (more than 90%) [30]. Bile acids are highly soluble, while cholesterol and phospholipids are highly insoluble in water. In bile, bile acids are found as monomers up to the critical micellar concentration (1–3 mM), a cut-off value after which bile acids can self-aggregate as simple micelles, binding a molecule of cholesterol. This step leads to increased aqueous solubility of cholesterol. Phospholipids in an aqueous environment can self-aggregate to form stable bilayer vesicles containing also a trace amount of bile acids, if any. A large amount of the cholesterol molecules is inserted into these bilayers of vesicles between the hydrophobic acyl chains of phospholipids. With typical gallbladder lipid concentrations and compositions, simple bile acid and mixed bile acid-lecithin micelles coexist in a ratio of 1 : 5. Unilamellar vesicles are larger spherical carriers in which even more cholesterol is solubilized into the bilayers of phospholipids. The ratio of unilamellar vesicles to micelles depends on the bile acid and phospholipid concentrations of bile, which is the greatest in bile with low bile acid and high phospholipid concentrations. Furthermore, at low bile acid concentrations and high phospholipid concentrations, these biliary phospholipids often form large multilamellar layers of vesicles. High concentrations of bile acids can dissolve these vesicles to form mixed micelles. (b) The picture depicts the ternary bile salt-cholesterol-phospholipid phase diagram in which the different pathways of cholesterol solubilization and/or precipitation in bile are shown [17]. The concentrations of three biliary lipids (bile acids, cholesterol, and phospholipids) are shown as percentages on the three axes of the triangle with a total lipid concentration of 7.2 g/dL, pH 7, and a temperature of 37°C [31, 32]. Different zones occupying areas within the triangle are shown, with each one containing different cholesterol carriers. The one-phase (φ) zone under the saturation curve contains only micelles and represents the bile being unsaturated with cholesterol. Above, three other zones exist with cholesterol supersaturation: a right two-phase (R 2-φ) zone containing saturated micelles and vesicles; a central three-phase (C 3-φ) zone containing saturated micelles, vesicles, and solid cholesterol crystals; and a left two-phase (L 2-φ) zone containing saturated micelles and solid cholesterol crystals. Whereas cholesterol precipitation is rapid in case of excess bile acids, at increasing amounts of phospholipids, cholesterol may reside in vesicles with phospholipids. At this moment, solid cholesterol crystal formation is slower or absent. Cholesterol crystallization, the first key step in cholesterol gallstone disease, is increasing at increasing concentrations of cholesterol, in the central and left zones, above the “safe” and physiological micellar zone.