A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum.

Abstract

A density gradient ultracentrifugal procedure is described for the rapid and reproducible isolation of the major lipoprotein classes, VLDL, LDL, HDL2, and HDL3, from human serum. A step gradient is constructed from four NaCl/KBr solutions varying in density from 1.006 to 1.24 g/ml and from 3 ml of serum adjusted to d 1.21 g/ml. Separation is achieved after a single ultracentrifugation for some 56 x 10(7) gavg min at 15 degrees C in a swinging bucket rotor, at which time the lipoproteins band isopycnically and albumin and other serum proteins are sedimented. Densitometric scanning of gradients revealed a lipoprotein mass profile distinguished by four absorption maxima which fell within the hydrated density ranges of VLDL (d less than 1.016 g/ml), LDL (1.028-1.050 g/ml), HDL2 (1.066-1.100 g/ml), and HDL3 (1.100-1.153 g/ml). Fractionation of gradients on the basis of band distribution, followed by chemical, physical, and immunological analyses of the four principal fractions (i.e., bands) provided data on their electrophoretic mobility, chemical composition, morphology and size distribution, immunological reactivity and apolipoprotein content, thereby confirming their identities as VLDL, LDL, HDL2, and HDL3. The validity of this separation was supported by the quantitative distribution of apo B and apo A-I as assessed by radial immunodiffusion. Lipoprotein quantitation based on chemical analysis of gradient fractions was compared with that by analytical ultracentrifugation for a group of normolipidemic males; results concorded well, giving a similar HDL2:HDL3 ratio (0.35-0.36). Our procedure thus provides a simple and precise manner in which to assess the lipoprotein and apolipoprotein profile of human serum quantitatively and qualitatively.