QUANTIFICATION OF CHOLESTEROL IN ALL LIPOPROTEIN CLASSES BY THE VAP-II METHOD

We have developed a high resolution microvolume Vertical Auto Profile (VAP) method for the simultaneous measurement of cholesterol in all li poprotein classes, including lipoprotein[a] (Lp[a]) and intermediate d ensity lipoprotein (IDL). This method, designated as VAP-II, uses a no nsegmented continuous flow (controlled-dispersion flow) analyzer for t he enzymatic analysis of cholesterol in lipoprotein classes separated by a short spin (47 min) single vertical ultracentrifugation. Choleste rol concentrations of high (HDL), low (LDL), very low (VLDL), and inte rmediate (IDL) density lipoproteins, as well as Lp[a], are determined by decomposing the spectrophotometric absorbance curve, obtained from the continuous analysis of the centrifuged sample, into its components using software developed in this laboratory. Analysis by VAP-II is ra pid and sensitive (as little as 40 mu l plasma is required per assay). The resolution of lipoprotein peaks is considerably enhanced in the p resent analyzer compared to the previous analyzer (VAP-I, which used t he Technicon AutoAnalyzer); improvement is especially noticeable for L p[a] and IDL. Total and lipoprotein cholesterol values obtained by VAP -II correlated well with the values obtained by Northwest Lipid Resear ch Laboratories (NWLRL). VAP-II Lp[a] cholesterol values also correlat ed well with the Lp[a] mass values obtained by an immunoassay techniqu e performed at NWLRL (r = 0.907). The reproducibility and accuracy of the method are within the requirements of the CDC-NHLBI (Centers for D isease Control-National Heart, Lung, and Blood Institute) Lipid Standa rdization Program.