The objective of this project is to determine whether the major cholesteryl ester-rich lipid particle in plasma, low density lipoprotein (LDL), can transform by treatment with cholesterol esterase into a larger unesterified cholesterol-rich particle similar to those we have isolated from atherosclerotic lesions. When LDL was incubated with cholesterol esterase, the LDL cholesteryl ester was completely hydrolyzed during 4 hrs of incubation. Interestingly, only when LDL was treated with trypsin prior to incubation with cholesterol esterase was LDL cholesteryl ester hydrolyzed. When LDL was incubated in a weak buffer in which pH gradually changed from 7.2 to 6.0 during the incubation, the LDL cholesteryl ester was initially hydrolyzed from 75% to 12% (molar % of total cholesterol). However, upon prolonged incubation, a reverse reaction occurred in which re-esterification of cholesterol slowly proceeded. After 24 hrs of CEase incubation, the degraded LDL contained 55% of its cholesterol as ester. The addition of human serum albumin prevented cholesterol re-esterification indicating that removal of free fatty acids from hydrolyzed LDL attenuated the cholesterol re-esterification process probably due to lack of substrate. The structure of the LDL particle changed following hydrolysis of LDL cholesteryl ester. Small projections extended from the surface of LDL during early cholesteryl ester hydrolysis. These projections transformed into films of various sizes and shapes, some still associated with the native LDL. With complete hydrolysis, flattened vesicles of irregular shape and aggregates of flat or curled films were present. In LDL incubated with the weak buffer the cholesterol re-esterification resulted in disruption of the films and the formation of lipid droplet-like spherical particles. Our results demonstrate that LDL transforms into a larger vesicular structure during hydrolysis of its cholesteryl ester core. This newly-formed larger lipid particle may become trapped in the vessel wall and lead to vascular cholesterol accumulation.
