A Method for Direct Incorporation of Radiolabeled Cholesteryl Ester into Human Plasma Low-Density-Lipoproteins: Preparation of Tracer Substrate for Cholesteryl Ester Transfer Reaction between Lipoproteins1

Abstract

[¹⁴C]Cholesteryl ester was directly incorporated into human plasma low-density lipoproteins (LDL) for the purpose of preparing a tracer substrate for investigation of the cholesteryl ester transfer reaction between plasma lipoproteins. The radiolabeled cholesteryl oleate was sonicated with egg phosphatidyicholine to form cholesteryl ester-containing liposomes. The liposomes were incubated with plasma fraction of density > 1.006 at 37°C in the presence of dithionitroberaoic acid. When the distribution of the radiolabeled cholesteryl ester was equilibrated among liposomes and lipoprotein fractions, the mixture was applied to an affinity chromatog raphy column of dextran sulfate-cellulose (LAO1) (Arteriosclerosis 4, 276–282). LDL was eluted by increasing the NaCl concentration and was finally isolated as a floating fraction by ultracentrifugation at a solvent density of 1.063 (adjusted with NaCl). The chemical composition, electrophoretic mobility and density of the labeled LDL were consistent with those of the native LDL. Radioactivity in this preparation was present exclusively in cholesteryl ester. Apolipoprotein B100 was preserved intact throughout the procedure. When the rate of cholesteryl ester transfer was measured between LDL and high-density lipoproteins by using this labeled LDL, the kinetics was consistent with the equilibrium transfer model, but the apparent rate measured was slightly higher than that measured with the labeled LDL prepared by the method using the intrinsic cholesterol esterification reaction of plasma.