Distribution of cell-derived cholesterol among plasma lipoproteins: a comparison of three techniques.

Three fractionation procedures (immunofinity chromatography, two-dimensional nondenaturing electrophoresis, and heparin-agarose affinity chromatography) have been compared in determining the kinetics of free and ester cholesterol transfer in normolipemic native plasma. Similar results were obtained in each case. Cell-derived free cholesterol is initially enriched in high density lipoproteins (HDL) (mainly HDL without apoE); at longer time periods (> 10 min) greater proportions are observed in very low density lipoproteins (VLDL) and low density lipoproteins (LDL). The major part of cholesteryl ester (about 90%) was retained in HDL, while VLDL and LDL, which contained about 75% of total cholesteryl ester mass, received only about 10 % of cell-derived cholesteryl ester. Within HDL, almost all cholesteryl ester was in the apoE-free fraction. These data provide evidence that lipoprotein free and esterified cholesterol are not at chemical equilibrium in normal plasma, and that cell-derived cholesterol is preferentially directed to HDL. The techniques used had a comparable effectiveness for the rapid fractionation of labile lipoprotein lipid radioactivity.-FFrancone, 0. L., C. J. Fielding, and P. E. Fielding. Distribution of cell-derived cholesterol among plasma lipoproteins: a comparison of three techniques. J. Lipid Res. 1990. 31: 2195-2200. Supplementary key words HDL LDL VLDL apoE cholesteryl ester cholesterol transfer Free cholesterol from both secreted plasma lipoproteins and cell mem.branes contributes to the pool available for metabolism id plasma (1-3). Studies of the early metabolism of cell-derived cholesterol have been made difficult by the rapid diffusional equilibration of free cholesterol between lipoproteins. For this reason much of our knowledge of cholesterol kinetics comes from synthetic lipid vesicles or incubations of isolated plasma lipoproteins (4, 5 ) . However, lipoprotein fractions not separable by classical centrifugation techniques have been implicated as important intermediates of plasma cholesterol metabolism. Examples include the prebeta-HDL species active in the early metabolism of HDL (6, 7), and HDL containing apoE (HDL + E) which, unlike other HDL, can interact with cellular LDL receptors (8). Nondenaturing two-dimensional electrophoresis has been used previously to separate and identify lipoprotein intermediates of plasma cholesterol metabolism for studies using brief continuous incubation and pulse-chase designs (6, 7). In the present study, this and two other techniques recently adapted to this research that allow rapid lipoprotein fractionation from native plasma have been compared. In this comparison, two factors reflecting the metabolism of cell-derived cholesterol in native plasma have been quantitated: the proportion of cellderived free cholesterol recovered in HDL and the proportion of cholesteryl ester synthesized from this free cholesterol that is transferred to other plasma lipoproteins. MATERIALS AND METHODS Blood was obtained from six normolipemic volunteers who had fasted overnight. It was collected directly into plastic tubes cooled in ice water, containing a final concentration of 10 ng/ml PPACK, a thombin antagonist (CalBiochem, San Diego, CA) or streptokinase (150 units/ml) (Sigma Chemical Co., St. Louis, MO) to inhibit coagulation (9). Plasma was then obtained by centrifugation (2000 g, 30 min) at 0°C and used immediately. ApoE phenotype determined by isoelectric focussing on these plasma samples indicated one donor with E4/E3 phenotype, while the remaining donors had the E3/E3 phenotype (10).