A LOW-TEMPERATURE FLOTATION METHOD TO RAPIDLY ISOLATE LIPOPROTEINS FROM PLASMA

To minimize oxidative modification, a low temperature, sequential flot ation method was developed to isolate plasma lipoproteins in 18 h usin g a benchtop ultracentrifuge. The protein distributions were character ized using agarose and SDS-polyacrylamide gel electrophoresis, and an SDS-Lowry protein assay, The lipid distributions were assessed using a gas chromatography-mass spectrometric assay for cholesterol and an en zymatic assay for triglycerides, To validate the rapid flotation metho d, lipoproteins were also isolated from the same plasma samples using a modified Havel et al. notation method (J. Clin. Invest. 34: 1345-135 3, 1955), The same lipoproteins and apolipoproteins were present in fr actions of comparable density, and the summed recoveries of protein, c holesterol, and triglyceride were also identical for the Havel et al, and rapid notation procedures. Likewise, the amount of cholesterol and triglyceride in corresponding very low, intermediate, and low density lipoprotein (VLDL/IDL and LDL) fractions was the same for the two not ation procedures, The triglyceride and cholesterol levels in high dens ity lipoprotein (HDL) isolated by rapid flotations, however, were 9-12 % higher than in the HDL as isolated by Havel et al, Because a 9-12% i ncrease in the HDL fraction reflects only 1-4% of the total triglyceri de and cholesterol in plasma, we conclude that, while maintained at 4 degrees C, lipoproteins were quantitatively isolated from human plasma in 1 day.