Comparison of various methods for in vitro cholesteryl ester labeling of lipoproteins from hypercholesterolemic rabbits

Abstract

Little or no information is available on biologically valid labeling of hypercholesterolemic plasma lipoproteins with cholesteryl ester. The esterification of labeled unesterified cholesterol in hypercholesterolemic rabbit plasma by the lecithin: cholesterol acyltransferase reaction is inefficient. The use of the d > 1.063 plasma fraction for this reaction greatly improves the efficiency, but some labeled unesterified cholesterol remains in the end products. The latter disadvantage can be avoided by the addition to whole plasma of labeled cholesteryl ester dissolved in DMSO or acetone. However, in hypercholesterolemic rabbit plasma only a small fraction of the added cholesteryl ester was associated with lipoproteins. When phosphatidylcholine/ cholesteryl ester liposomes were incubated with hypercholesterolemic rabbit plasma for 18–24 h at 37°C the labeled cholesteryl ester was quantitatively incorporated into lipoproteins. Chylomicron-like, cholesteryl ester-rich particles were removed by centrifugation (10 6 g · min) and the subsequently isolated d < 1.019 and d = 1.019–1.063 (LDL) fractions were injected intravenously into normal and hypercholesterolemic rabbits. The disappearance of d < 1.019 and LDL cholesteryl ester and the appearance of cholesteryl ester in other lipoprotein fractions was indistinguishable from that of in vivo-labeled lipoproteins. In vivo and in vitro cholesteryl ester-labeled lipoproteins were also compared by measuring the exchangeability of their cholesteryl ester with HDL cholesteryl ester in vitro. Equal exchangeability of the two labels was observed in the d < 1.019 fraction from which the chylomicron-like particles had been removed. These findings demonstrate that when cholesteryl ester is incorporated by the liposome procedure, the distribution of labeled cholesteryl ester within the lipoprotein complex corresponds closely to that of the in vivo-incorporated labeled cholesteryl ester.