Preparative free-solution isotachophoresis for separation of human plasma lipoproteins: apolipoprotein and lipid composition of HDL subfractions

We have previously shown that plasma lipoproteins can be separated by analy tical capillary isotachophoresis (ITP) according to their electrophoretic m obility in a defined buffer system, As in lipoprotein electrophoresis, HDL show the highest mobility followed by VLDL, LDL, and LDL, Chylomicrons migr ate according to their net-charge between HDL and VLDL, because ITP has neg ligible molecular sieve effects. Three HDL subfractions were obtained which were designated fast-, intermediate-, and slow-migrating HDL, To further c haracterize these HDL subfractions, a newly developed free-solution ITP (FS -ITP)-system was used, that allows micro-preparative separation of human li poproteins directly front whole plasma (Bottcher, A. et al, 1998, Electroph oresis. 19: 1110-1116), The fractions obtained by FS-ITP were analyzed for their lipid and apolipoprotein composition and by two-dimensional nondenatu ring polyacrylamide gradient gel electrophoresis (2D-GGE) with subsequent i mmunoblotting. fHDL are characterized by the highest proportion of esterifi ed cholesterol of all three subfractions and are relatively enriched in LpA -I, Together with iHDL they contain the majority of plasma apoA-I, while sH DL contain the majority of plasma apoA-IV, apoD, apoE, and apoJ, Pre-beta-H DL were found in separate fractions together with triglyceride-rich fractio ns between sHDL and LDL, jlr In summary, ITP can separate the bulk of HDL i nto lipoprotein subfractions, which differ in apolipoprotein composition an d electrophoretic mobility, While analytical ITP permits rapid separation a nd quantitation for diagnostic purposes, FS-ITP can be used to obtain these lipoprotein subfractions on a preparative scale for functional analysis. A s FS-ITP is much better suited for preparative purposes than gel electropho resis, it represents an important novel tool for the functional analysis of lipoprotein subclasses.