A. Bottcher et al., Preparative free-solution isotachophoresis for separation of human plasma lipoproteins: apolipoprotein and lipid composition of HDL subfractions, J LIPID RES, 41(6), 2000, pp. 905-915
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.