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A new program for lipoprotein characterization is outlined where capillary electrophoresis (CE) plays a central role in the analysis of intact lipoprotein serum components and the apoprotein domains. The first characterization step involves separation and particle density analysis of very low-, low-, and high-density lipoprotein fractions (VLDL, LDL, HDL) by ultracentrifugation and image analysis. VLDL, HDL, and LDL fractions are analyzed by capillary electrophoresis. Sodium dodecyl sulfate (SDS) at low concentrations in the background electrolyte used in the CE analysis is incorporated into the lipoprotein particle without appreciable delipidation, as determined by ultracentrifuge particle density analysis. Increasing the concentration of SDS results in extensive delipidation, resulting in the release of apoproteins (apo) which are detected as components of the electropherogram. Apo B-100 is detected in the delipidated VLDL and LDL fractions along with micelles of the lipids. Micelles from LDL delipidation have uniform charge densities. Apo A-I and A-II are detected in the HDL fraction. A new method for lipoprotein delipidation is introduced where the lipoprotein fraction is adsorbed on a reversed-phase hydrophobic cartridge. Delipidation and recovery of the apoprotein fractions is made by serial elutions with acetonitrile. CE of the lipid-free apoprotein mixture shows the presence of apoC-I,II,III and apoE in the VLDL fraction, and apoA-I,II apoC-I and apoE in the HDL fraction. Electrospray ionization mass spectrometry analysis gives the isoform distribution for each apoprotein. The identification of the apoproteins in the electropherograms is the first step in developing a CE-based quantitation method for measuring serum levels of these apoproteins and their distribution between the lipoprotein fractions. The assay described in this paper is being used as a level 2 and 3 cardiac risk profile analysis for individuals with normal lipid profiles who have a documented or family history of cardiovascular disease.

New isoforms of apolipoprotein (apo)C-I and apoC-III have been detected in delipidated fractions from very low density lipoprotein (VLDL) using matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI) mass spectrometry (MS). The cleavage sites of truncated apoC-III isoforms have also been identified. The VLDL fractions were isolated by fixed-angle single-spin ultracentrifugation using a self-generating sucrose density gradient and delipidated using a newly developed C18 solid phase extraction protocol. Fifteen apoC isoforms and apoE were identified in the MALDI spectra and the existence of the more abundant species was verified by ESI-MS. The relative intensities of the apoCs are closely correlated in three normolipidemic subjects. A fourth subject with type V hyperlipidemia exhibited an elevated apoC-III level and a suppressed level of the newly discovered truncated apoC-I isoform. ApoC-II was found to be particularly sensitive to in vitro oxidation. The dynamic range and specificity of the MALDI assay shows that the complete apoC isoform profile and apoE phenotype can be obtained in a single measurement from the delipidated VLDL fraction.-Bondarenko,

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