An analytical free flow capillary isotachophoresis (cITP) procedure for the detailed analysis of lipoproteins on commercially available capillary electrophoresis systems has been developed. The technique is based on the specific staining of lipoproteins with the fluorescent lipophilic dye 7-nitrobenz-2-oxa-1,3-diazole (NBD)-ceramide before separation. Prestained lipoprotein samples are applied between leading and terminating buffer and separated into 9 well-characterized subpopulations according to their electrophoretic mobility in the absence of any molecular sieve effect. High density lipoproteins are separated into three major subpopulations: (i) the fast migrating high density lipoprotein (HDL) subpopulation (alpha-HDL, containing mainly apolipoprotein (apo) A-I and phosphatidylcholine, (ii) the subpopulation with intermediate mobility, consisting of particles rich in cholesterol, apo A-II, apo E and C apolipoproteins, and (iii) the slow migrating HDL subpopulation (pre-beta-HDL), containing particles rich in apo A-I, apo A-IV. The majority of HDL-associated lecithin:cholesterol acyltransferase (LCAT) activity is also associated with the last subpopulation. The apo B-containing lipoproteins can be subdivided into three major functional groups. The first represents chylomicron derived particles and large triglyceride-rich very low density lipoproteins (VLDL). The second group consists of small VLDL and intermediate density lipoprotein (IDL) particles, and the third group represents the low density lipoproteins (LDL). Results obtained by the isotachophoretic lipoprotein analysis revealed a good correlation in the range of HDL with routinely used techniques, like lipoprotein electrophoresis, HDL-cholesterol analysis by a precipitation procedure or turbidimetric determination of apo A-I. Similar correlations with other analytical techniques were found for the quantitation of the apo B-containing lipoproteins. Advantages of the isotachophoretic separation compared to zone electrophoresis are the high resolution combined with small sample volumes. Moreover, lipoprotein analysis can be performed directly from whole serum, plasma, lymph and other biological fluids in a short time. With these characteristics analytical capillary isotachophoresis may be a helpful tool for a fast and reliable automated quantitation of lipoprotein subpopulations in the clinical laboratory.

An analytical free flow capillary isotachophoresis procedure, with a discontinuous electrolyte system, for the detailed analysis of lipoproteins in human body fluids has been developed. The technique is based on prestaining whole serum lipoproteins with a lipophilic dye before separation. Human serum lipoproteins are separated into 14 well-characterized subfractions according to their electrophoretic mobility. High density lipoproteins (fraction 1 to 6) are separated into three major subpopulations, the fast migrating high density lipoprotein (HDL) subpopulation, containing mainly apo AI and phosphatidylcholine, the subpopulation with intermediate mobility, consisting of particles rich in apo AII, apo E, and C apolipoproteins, and the slowly migrating HDL subfraction, containing mainly particles rich in apo AI, apo AIV, and lecithin: cholesterol acyltransferase (LCAT) activity. The apo B containing lipoproteins (fraction 7 to 14) can be subdivided into four major functional groups. The first represents chylomicron derived particles and large triglyceride-rich very low density lipoproteins (VLDL). The second group consists of small VLDL and intermediate density lipoprotein (IDL) particles, anf the third and fourth group represent the low density lipoproteins. The isotachophoretic analysis of human serum samples obtained from patients with hyperlipoproteinemias is compatible with the classification according to the Frederickson phenotypes and reflects the respective biochemical abnormalities. Furthermore, several genetic disorders of lipid and lipoprotein metabolism like HDL deficiency syndromes, familial LCAT deficiency, Fish eye disease, hypobetalipoproteinemia and abetalipoproteinemia can be well characterized by analytical capillary isotachophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

We compared HDL3- and LDL-induced signal transduction in normal and Tangier fibroblasts to elucidate whether impaired signal transduction responses to lipoproteins might contribute to disturbed cellular lipid and lipoprotein metabolism in Tangier disease, a rare autosomal disorder of cellular lipid and lipoprotein metabolism. In several cell types HDL and LDL activate a currently unknown isoform of phosphatidylinositol-specific phospholipase C (PI-PLC) that results in the generation of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Compared with normal fibroblasts, Tangier fibroblasts stimulated with HDL3 or LDL resulted in a significantly reduced accumulation of inositol phosphates and 1,2-diacylglycerol formation. Furthermore, in Tangier fibroblasts both lipoproteins failed to mobilize calcium from internal pools, and the cytosol-to-membrane redistribution of protein kinase C (in both the alpha and epsilon isoforms) was markedly reduced. Thus, the data indicate an impaired PI-PLC activation in response to lipoproteins in Tangier fibroblasts.

An automated free-solution isotachophoresis system (FS-ITP) for preparative fractionation of biopolymers is described, operated in a batch mode. The dimension of the separation chamber allows an up to 1200-fold higher sample load compared to separation in capillaries of 180 microm inner diameter as used in analytical capillary isotachophoresis (C-ITP). The preparative capacity of the system is within the milligram range. The method is fully compatible with analytical C-ITP, which is essential for preparative-scale isotachophoresis with regard to optimization of electrolyte systems and the search for suitable spacers. As a model application the fractionation of human serum proteins is reported. The collected fractions were analyzed by C-ITP and agarose gel electrophoresis.

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