Characterization of high density lipoprotein subspecies: structural studies by single vertical spin ultracentrifugation and immunoaffinity chromatography.

Affinity columns containing anti-apolipoprotein A-I or A-II were used to fractionate plasma into subpopulations of lipoprotein particles containing: a) apoA-I [Lp(A-I)], b) apoA-I and A-II [Lp(A-I with A-II)], and c) apoA-I but no A-II [Lp(A-I without A-II)]. Single vertical spin and electron microscopy analyses of these HDL subpopulations demonstrated that acid elution from the affinity columns caused no detectable change in size and density of the three subpopulation particles. Analysis by nondenaturing gradient gel electrophoresis of the three subpopulations found in four normal subjects identified nine HDL subspecies, designated [1] through [9] in order of increasing size; [3-7] were the major subspecies. Lp(A-I with A-II) is composed primarily of subspecies [3],[5], and [6], and may contain some subspecies [2] and [7], while Lp(A-I without A-II) represents mainly [4] and [7] and the minor subspecies [1],[2],[8], and [9]. HDL subspecies [4],[5], and [6] are found in the standard sequential flotation density cuts for both HDL3 and HDL2, which illustrates the limitations of the latter terminology. Using single vertical spin ultracentrifugation, HDL fractions were located and isolated for physical and chemical analyses, including immunoassay for apoA-I, A-II, and C-II. The distribution of the Lp(A-I without A-II) particles corresponded closely to the apoC-II distribution. An apoA-I-rich, cholesteryl ester- and phospholipid-poor subspecies was identified in the dense HDL fractions. HDL subspecies [7] was found to contain at least three separate subspecies designated [7a], [7b], and [7c]. Based on these and previously published results (Brouillette, C. G., et al. 1984. Biochemistry. 23: 359-367), we propose that the HDL subspecies adjacent in size generally differ by the association/lack of association of a hinge-like domain of amphipathic helixes in a single molecule of apoA-I.

Computerized rapid high resolution quantitative analysis of plasma lipoproteins based upon single vertical spin centrifugation.

A method has been developed for rapidly quantitating the cholesterol concentration of normal and certain variant lipoproteins in a large number of patients (over 240 in one week). The method employs a microcomputer interfaced to the vertical autoprofiler (VAP) described earlier (Chung et al. 1981. J. Lipid Res. 22: 1003-1014). Software developed to accomplish rapid on-line analysis of the VAP signal uses peak shapes and positions derived from prior VAP analysis of isolated authentic lipoproteins HDL, LDL, and VLDL to quantitate these species in a VAP profile. Variant lipoproteins VHDL (a species with density greater than that of HDL(3)), MDL (a species, most likely Lp(a), with density intermediate between that of HDL and LDL), and IDL are subsequently quantitated by a method combining difference calculations with curve shapes. The procedure has been validated qualitatively by negative stain electron microscopy, gradient gel electrophoresis, strip electrophoresis, chemical analysis of the lipids, radioimmunoassay of the apolipoproteins, and measurement of the density of the peak centers. It has been validated quantitatively by comparison with Lipid Research Clinic methodology for HDL-, LDL-, and VLDL-cholesterol, and for MDL- and IDL-cholesterol by comparison of the amounts of MDL or IDL predicted to be present by the method with that known to be present following standard addition to whole plasma. These validations show that the method is a rapid and accurate technique of lipoprotein analysis suitable for the routine screening of patients for abnormal amounts of normal or variant lipoproteins, as well as for use as a research tool for quantitation of changes in cholesterol content of six or seven different plasma lipoprotein fractions.-Cone, J. T., J. P. Segrest, B. H. Chung, J. B. Ragland, S. M. Sabesin, and A. Glasscock. Computerized rapid high resolution quantitative analysis of plasma lipoproteins based upon single vertical spin centrifugation.

High resolution plasma lipoprotein cholesterol profiles by a rapid, high volume semi-automated method.

A new rapid and sensitive method, the single vertical spin autoprofiler (VAP), has been developed for quantitative profiling of the major plasma lipoproteins. The method involves a combination of single vertical spin separation of plasma and continuous on-line analysis of cholesterol. Plasma lipoproteins are first separated by a 45-min spin in a vertical rotor, after which the amount of cholesterol in the effluent of each tube is monitored continuously by a modification of the BMC automated enzymatic cholesterol method; simultaneously, 80% of the sample is diverted by stream-splitting to a fraction collector for further analysis, if desired. VAP not only resolves very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) peaks quantitatively but also detects the presence of intermediate density lipoprotein (IDL) and other lipoprotein variants. VAP was highly reproducible; the inter-run coefficient of variation for cholesterol concentration in VLDL, LDL, and HDL was 4.8%, 2.9%, and 2.4%, respectively. Cholesterol recovery using VAP was 98.5 +/- 3.5%. Lipoprotein-cholesterol profiles of plasma from three major hyperlipoproteinemia phenotypes examined by VAP were qualitatively and quantitatively different from each other and from profiles of normolipidemic individuals. One significant finding was that IDL could be detected in the plasma of all type IV hyperlipoproteinemic subjects examined thus far. Several variant lipoprotein profiles which did not correspond to known phenotypes have also been detected by VAP using plasma from hyperlipidemic as well as from normolipidemic subjects. We submit that VAP is an accurate and rapid method for lipoprotein analysis, either for routine clinical screening or for detailed experimental studies. In addition, VAP provides a visual display of partially to completely resolved lipoprotein classes that is suitable for computer-assisted analysis.-Chung, B. H., J. P. Segrest, J. T. Cone, J. Pfau, J. C. Geer, and L. A. Duncan. High resolution plasma lipoprotein cholesterol profiles by a rapid, high volume semi-automated method.