Lipoprotein separation and low density lipoprotein molecular weight determination using high performance...

Plasma lipoproteins were isolated at d less than 1.225 g/ml from nonhuman primates of three species, cynomolgus, rhesus, and African green (vervet) monkeys. Individual lipoprotein classes were separated by high performance gelfiltration chromatography and low density lipoprotein (LDL) molecular weight was determined. A comparison was made using column configurations including TSK 3000 SW, 4000 SW, and 5000 PW columns. Due to its relative simplicity, stability, and economy, a single 5000 PW column was selected for most of the work. The recovery of lipoprotein cholesterol from the column averaged 91 +/- 2.5%. A comparison of the immunologic, chemical, and electrophoretic properties of high density lipoproteins (HDL) and LDL isolated by this technique with those of HDL and LDL isolated by conventional agarose column chromatography indicated that lipoproteins isolated by high performance gel-filtration chromatography were intact and reasonably free of cross contamination. A standard preparation of 125I-labeled LDL was added to the d less than 1.225 g/ml lipoprotein fraction just prior to separation and a relative size index, r1, was determined. When r1 values for a large number of samples were compared with the log of the LDL molecular weight (determined by agarose column chromatography) a linear relationship was found with a correlation coefficient, r = 0.85. The regression equation for this relationship could be used to calculate LDL molecular weights from the r1 value. These values agreed with LDL molecular weight determined by flotation equilibrium analysis in the analytical ultracentrifuge. We conclude that high performance gel-filtration chromatography using the TSK 5000 PW column provides an analytical and preparative technique for simultaneous separation of individual lipoproteins and determination of LDL molecular weight.