Lipoprotein subclass analysis by immunospecific density

Apolipoprotein C-1 (apo C-1) enriched HDL has been described as an atherogenic form of HDL associated with an increased risk for cardiovascular disease (CVD). The objective of the present study was to develop a rapid method for the separation, purification, and characterization of Apo C-1 from serum. We isolated and characterize HDL subclasses from individuals with and without angiographically-proven CVD who have elevated and normal-to-low HDL-C levels. Ultracentrifugation was linked with immunoaffinity separations for the specific separation of Apo C-1 enriched HDL from other lipoproteins. A 50 ?L sample of serum is diluted in TRIS HCl buffer (pH 7.5) and incubated with CNBr-activated Sepharose (Amersham) containing antibodies to apo C-1 (Academy Bio-medical Company). The apo C-1-depleted serum is removed by centrifugation and all apo C-1-containing lipoproteins are released from the Sepharose beads at pH 2. The apo C-1-depleted sample and the apo C-1-containing sample were ultracentrifuged to obtain a lipoprotein density profile in the absence and presence of apo C-1. Density Lipoprotein Profiling (DLP) gives relevant information of lipoproteins, such as density and subclass characterization, and is a novel approach to purify apo C-1-enriched HDL. An additional advantage of this approach is that lipoprotein-a (Lp(a)), which is often an interfering component in the HDL density region, is eliminated. Results show feasibility that these methods could be used in a clinical setting, was achieved. This measurement may yield a precise and quantitative profile of the distribution of apo C-1 for all lipoprotein particles including HDL.