Lipid Molecular Geometry, Phase Behaviour, and Nucleation Kinetics in Cholesterol Gallstone Formation:Toward an Integrated Physicochemical Framework

ABSTRACT Cholesterol gallstone formation requires supersaturation of bile with cholesterol, commonly quantified by the cholesterol saturation index (CSI). While supersaturation of bile with cholesterol is a necessary prerequisite for cholesterol precipitation, bile samples with comparable cholesterol saturation index (CSI) values can exhibit markedly different crystallization behaviours. This inconsistency indicates that the cholesterol saturation index alone cannot predict crystallization pathways or nucleation kinetics. Here, we synthesize experimental findings from native and model bile systems to propose an integrated physicochemical framework for interpreting cholesterol gallstone formation. Evidence from the literature indicates that lipid molecular geometry—associated with phospholipid acyl-chain composition and bile acid structure—is associated with differences in how cholesterol is accommodated within lipid aggregates. These molecular-scale properties are expressed at the mesoscale through distinct phase behaviours, including micellar, vesicular, and liquid-crystalline states, which are associated with different, reproducible cholesterol crystallization pathways described in bile systems. Experimental observations further indicate that nucleation kinetics plays a decisive role in shaping crystallization outcome. Even within similar phase regimes, differences in vesicle stability, aggregation dynamics, and interfacial organization are associated with substantial variability in nucleation behaviour, including delayed or absent crystal formation despite thermodynamically permissive conditions. Within this framework, the established cholesterol crystallization pathways can be interpreted as alternative trajectories that emerge from differences in lipid organization and kinetic accessibility under comparable degrees of supersaturation. By explicitly integrating lipid molecular geometry, phase behaviour, and nucleation kinetics, this synthesis provides a coherent physicochemical context for interpreting variability in cholesterol crystallization behaviour and situates cholesterol supersaturation within a broader, experimentally grounded framework for understanding cholesterol gallstone formation.