Identification and quantification of diacylglycerols in HDL and accessibility to lipase

We have investigated the presence of diacylglycerols in lipoproteins and especially in HDL. Lipoprotein diacylglycerols are very difficult to isolate and to quantify using classical enzymatic techniques, as they are measured in the presence of triacylglycerols and monoacylglycerols. Using a rapid and very sensitive method of gas-liquid chromatography, developed for neutral lipid analysis on an Ultra 1 Hewlett-Packard fused silica capillary column, diacylglycerols (DG) were identified in HDL and classified into five groups: DG 14-16, DG 16-16, DG 16-18, DG 18-18, and DG 18-20. However, their quantitation was difficult due to only partial resolution of molecular species. HDL lipids were submitted to preparative gas-liquid chromatography and diacylglycerols were then silylated using trimethylsilyl reagents. The trimethylsilyl ethers were analyzed by gas-liquid chromatography on a Restek 50 capillary column and were resolved on the basis of carbon number, degree of unsaturation, and double bond positions. The amount of HDL diacylglycerols was twice that of triacylglycerols. The major molecular species of diacylglycerols consisted of 16:0-18:2n-6, 18:0-18:2n-6, and 16:0-18:1n-9 as the major molecular species (33.4, 22.2, and 16.1 mol % of total diacylglycerols, respectively). Using guinea pig cationic pancreatic lipase in order to test the accessibility of diacylglycerols at the surface of HDL, we measured 59% of diacylglycerol hydrolysis, whereas no triacylglycerol hydrolysis was obtained. In addition, most of diacylglycerols having long chain fatty acids, such as 18-20, were completely hydrolyzed, whereas 18-18 and 16-18 were only partially hydrolyzed (64 and 46% respectively). This reflects a different partition of diacylglycerol molecular species between the particle's surface and the lipid core in HDL. This is the first analysis of diacylglycerol molecular species and their distribution in native lipoprotein particles.