Human lipoproteins were separated on Superose 6 HR 10/30 gel-filtration column using Fast Protein Liquid Chromatography System (FPLC, Pharmacia Fine Chemicals). We used the mobile phase which was composed of 0.15 M sodium chloride, 0.01% EDTA-2Na, 0.02% sodium azide, pH 7.2. The P-500 pump, fraction collector and recorder were controlled by the gradient programmer. A constant-elution flow-rate of 0.75ml/min was achieved and fractions of 0.5ml were collected. Five lipoprotein fractions such as VLDL (d<1.006g/ml), LDL (1.006<d<1.063g/ml), HDL (1.063<d<1.210g/ml), HDL2 (1.063<d<1.125g/ml) and HDL3 (1.125<d<1.210g/ml) and total lipoprotein (d<1.210g/ml) were analyzed. The absorbance of the eluate was monitored at 280 nm using the UV-1 monitor. Concentration of cholesterol, triglyceride and phospholipid in each fraction collected were analyzed by the enzymatic methods and protein concentration was measured according to the method of Lowry et al. Total lipoprotein (d<1.210g/ml) was separated to three peaks. Elution volumes of peak 1, 2 and 3 were 7.7ml, 11.6ml and 15.6ml, respectively. Elution volumes of VLDL, LDL, HDL, HDL2 and HDL3 showed 7.6ml, 11.6ml, 15.5ml, 15.0ml and 15.5ml, respectively. Percent composition of lipids and protein in peak 1, 2 and 3 were comparable to those of VLDL, LDL and HDL fractionated by ultracentrifugation, respectively. Recovery and reproducibility of this method were quite satisfactory. In the present study, a new agarose gel matrix, Superose 6 was used. This gel reduced the separation time and improved the separation of plasma lipoproteins. FPLC system will be a useful method for analysis of plasma lipoproteins.
