Quantification of HDL2 and HDL3 cholesterol by the Vertical Auto Profile-II (VAP-II) methodology

Abstract

Of the several existing methods for quantification of major subspecies of high density lipoprotein (HDL), HDL2 and HDL3, the methods based upon double precipitation are particularly useful for large-scale studies or for routine assay because of their high speed and low cost. The Vertical Auto Profile-II (VAP-II) method developed in our laboratory primarily for the direct single test measurement of cholesterol (C) in all major lipoproteins, including Lp[a] and IDL, is rapid, highly sensitive, and suitable for large-scale studies. Here we describe the modification of this procedure so as to be able to quantify both HDL2- and HDL3-C in addition to all major lipoproteins without any additional assay steps, time, or cost. The VAP-II procedure was validated by comparison with four other methods using plasma samples obtained from 35 healthy subjects: 1) HDL-VAP-II (a variation of the VAP-II procedure designed specifically to separate HDL subspecies); 2) dextran sulfate (DS)/Mg2+ double precipitation method performed at Northwest Lipid Research Laboratories (NWLRL), Seattle, WA; 3) 4-30% polyacrylamide-agarose (4/30 PAA) nondenaturing gradient gel electrophoresis (GGE); and 4) analytical ultracentrifugation (AUC), with both GGE and AUC performed at the Donner Laboratory, University of California at Berkeley. Both HDL2- and HDL3-C measurements by VAP-II correlated well with the measurements by all comparison methods (r for HDL3-C: HDL-VAP-II, 0.948; NWLRL, 0.947; GGE, 0.861; and AUC, 0.706, and r for HDL2-C: HDL-VAP-II, 0.867; NWLRL, 0.854; GGE, 0.885; and AUC, 0.721). The measurements of HDL2- and HDL3-C by the VAP-II method are reproducible, with the long-term between-rotor CV of 5.0% for HDL3-C and 9.0% for HDL2-C.