Differential density lipoprotein profiling for the characterization of Lipoprotein(a)



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Texas A&M University


Lipoprotein(a) (Lp(a)) has been described as an emerging risk factor for cardiovascular disease. The complexity of the Lp(a) molecule sets a challenge for the determination of the risk it represents for the cardiovascular system. The objective of the present study was to develop a rapid method for the separation, purification, density measurement, and characterization of Lp(a) from serum using a procedure that is isoform independent. The objective was met by linking ultracentrifugation with affinity separations for the specific separation of Lp(a) from other lipoproteins. The mean density distribution of Lp(a) was determined by a differential density lipoprotein profile (DDLP). For DDLP, the lipoprotein density distribution of a serum sample with elevated Lp(a) levels was determined by ultracentrifugation using NaBiEDTA complex as a density gradient. Lp(a) was removed from a second aliquot of the same serum sample by carbohydrate affinity using wheat germ agglutinin (WGA). WGA was demonstrated to have high specificity for Lp(a) in serum. The Lp(a)-depleted sample was ultracentrifuged to obtain a lipoprotein density distribution in the absence of Lp(a). A DDLP was obtained after subtracting the Lp(a)-depleted lipoprotein density profile from the untreated lipoprotein density profile. DDLP gives relevant information of the lipoproteins in serum such as density, Lp(a) isoform, and subclass characteristics. Lp(a) was quantitatively removed from serum with a recovery efficiency of more than 80%. Lp(a) was purified by ultracentrifugation. Lp(a) obtained in this way retained its inherent density and immunoreactivity. Lp(a) was further characterized by gel electrophoresis and Western blot as well as by capillary electrophoresis. Capillary electrophoresis demonstrated to be a powerful analytical technique for the characterization of Lp(a) and apoprotein(a) isoforms. The major outcome of this research was the effectiveness of using affinity separations coupled with density ultracentrifugation for the isolation of pure Lp(a) from serum and its isoform characterization based on density and electromobility. The methodology developed and described here are relevant in a clinical setting for the analysis of Lp(a).