Quantification of HDL2 and HDL3 cholesterol by the Vertical Auto Profile-II (VAP-II) methodology.
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.
Kulkarni
K R
KR
Department of Medicine, The University of Alabama at Birmingham, 35294, USA.
Marcovina
S M
SM
Krauss
R M
RM
Garber
D W
DW
Glasscock
A M
AM
Segrest
J P
JP
D001774:Blood Chemical Analysis / Q000379:methods*; D008076:Cholesterol, HDL / Q000097:blood*; D016264:Dextran Sulfate / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood*; D012044:Regression Analysis; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D014461:Ultracentrifugation
D008076:Cholesterol, HDL; D008075:Lipoproteins, HDL; D016264:Dextran Sulfate