Separation of the main lipoprotein density classes from human plasma by rate-zonal ultracentrifugation

Abstract

The major lipoprotein density classes (chylomicrons-VLDL, LDL, HDL2 and HDL3) were isolated from human plasma in a two-step ultracentrifugal procedure using the Ti-14 zonal rotor. The isolation of the two major high density lipoprotein subclasses (HDL2 and HDL3) was achieved in a 24-hr run using a nonlinear NaBr gradient in the density range of 1.00-1.40. The lipoproteins with a density < 1.063 found in the rotor's center were isolated in a second run of 140 min duration using a continuous linear NaBr gradient in the density range of 1.00-1.30. The isolated lipoproteins were analyzed for chemical composition and for electrophoretic mobility; purity of isolated fractions was checked by immunochemistry. The lipoproteins exhibited flotation rates, chemical compositions, and molecular weights similar to those found with the common sequential procedures in angle-head rotors. The amount of lipoprotein lipids in the bottom fraction of the zonal rotor was comparable to that of the angle-head rotor. The described method yields the main lipoprotein density classes free from albumin in a very short running time; compared with the rate-zonal techniques already in use, this method allows the quantitative separation of an additional lipoprotein density class (HDL2) without increasing the running time. Furthermore, this procedure proved to be suitable for isolation of plasma lipoproteins from subjects with various types and varying degrees of hyperlipoproteinemia.

Keywords

• nonlinear density gradient
• continuous linear density gradient
• lipoprotein protein
• lipoprotein lipids