Separation of Lipoproteins by Sucrose Density Gradient

Abstract

Ultracentrifugal separation of plasma lipoproteins by discontinuous sucrose gradient was tried. Lipid and apolipoprotein composition of each fraction was compared with those obtained by the method of Havel. 0.5gm of sucrose, 0.4gm of KBr and 0.1gm of NaCl were added to 2.0ml serum to make final d=1.21 and 1.0ml of d=1.21 solution with same composition was piled gently. After the centrfugation for 20 hours at 50, 000rpm, 2.0ml of d=1.063 solution (6w/v% in sucrose) and 2.5ml of d=1.006 solution (2.5w/v% in sucrose) were overlayered and recentribuged for 4 hours. Each lipoprotein floated in the solvent corresponding to each density class. Samples were collected by capillary pipette and dialyzed to physiological saline containing 1mg/ml EDTA (pH 7.2) for more than 12 hours.
Polyacrylamide gel electrophoresis demonstrated distinct separation of each lipoprotein class without contamination. Contents of apolipoproteins, total lipids, total cholesterol and triglycerides were measured. When compared with those obtained by the Havel's method, lipid and apolipoprotein contents were higher in VLDL and lower in LDL in our method. These changes suggest that VLDL contains the “remnants” of d<1.019. Recovery of lipids and apolipoproteins in HDL was higher in the present method. Shortening of ultracentrifugal time was considered to have prevented degeneration of HDL.
Although our method would provide more rapid ultracentrifugal separation of plasma lipoproteins, more detail examinations of its physicochemical and structural properties should be required.