Abstract
We have previously shown that plasma lipoproteins can be separated by analytical capillary isotachophoresis (ITP) according to their electrophoretic mobility in a defined buffer system. As in lipoprotein electrophoresis, HDL show the highest mobility followed by VLDL, IDL, and LDL. Chylomicrons migrate according to their net-charge between HDL and VLDL, because ITP has negligible molecular sieve effects. Three HDL subfractions were obtained which were designated fast-, intermediate-, and slow-migrating HDL. To further characterize these HDL subfractions, a newly developed free-solution ITP (FS-ITP)-system was used, that allows micro-preparative separation of human lipoproteins directly from whole plasma (Böttcher, A. et al. 1998. Electrophoresis. 19: 1110–1116). The fractions obtained by FS-ITP were analyzed for their lipid and apolipoprotein composition and by two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-GGE) with subsequent immunoblotting. fHDL are characterized by the highest proportion of esterified cholesterol of all three subfractions and are relatively enriched in LpA-I. Together with iHDL they contain the majority of plasma apoA-I, while sHDL contain the majority of plasma apoA-IV, apoD, apoE, and apoJ. Pre-β-HDL were found in separate fractions together with triglyceride-rich fractions between sHDL and LDL.In summary, ITP can separate the bulk of HDL into lipoprotein subfractions, which differ in apolipoprotein composition and electrophoretic mobility. While analytical ITP permits rapid separation and quantitation for diagnostic purposes, FS-ITP can be used to obtain these lipoprotein subfractions on a preparative scale for functional analysis. As FS-ITP is much better suited for preparative purposes than gel electrophoresis, it represents an important novel tool for the functional analysis of lipoprotein subclasses.—Böttcher, A., J. Schlosser, F. Kronenberg, H. Dieplinger, G. Knipping, K. J. Lackner, and G. Schmitz. Preparative free-solution isotachophoresis for separation of human plasma lipoproteins: apolipoprotein and lipid composition of HDL subfractions. J. Lipid Res. 2000. 41: 905–915.
Highlights
We have previously shown that plasma lipoproteins can be separated by analytical capillary isotachophoresis (ITP) according to their electrophoretic mobility in a defined buffer system
We report here the use of free-solution ITP (FS-ITP) to further characterize the composition of high density lipoprotein (HDL) fractions separated by analytical ITP
By addition of purified lipoproteins, these fractions can be further identified: peaks 1–3 represent HDL subfractions, peak 4 represents chylomicrons, peak 5 represents very low density lipoprotein (VLDL)/IDL, and peaks 6–8 represent low density lipoprotein (LDL) (Fig. 1B–D). According to their mobility on analytical ITP, the HDL subfractions have been defined as fast-migrating HDL, intermediate-migrating HDL, and slow-migrating HDL
Summary
We have previously shown that plasma lipoproteins can be separated by analytical capillary isotachophoresis (ITP) according to their electrophoretic mobility in a defined buffer system. ITP can separate the bulk of HDL into lipoprotein subfractions, which differ in apolipoprotein composition and electrophoretic mobility. Preparative freesolution isotachophoresis for separation of human plasma lipoproteins: apolipoprotein and lipid composition of HDL subfractions. A major drawback of 2D-GGE is its poor suitability for preparative purposes Another method for separation of lipoproteins by their electrophoretic mobility is capillary isotachophoresis, which is a support-free technique with negligible molecular sieve effects. It is performed in a discontinuous buffer system with the advantages of concentration and self-sharpening effects. ITP can be automated for the analysis of large sample series
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