Analysis of cholesterol levels in lipoprotein(a) with anion-exchange chromatography

• Published in: Journal of lipid research Vol. 51; no. 5; pp. 1237 - 1243
• Main Authors: Hirowatari, Yuji, Yoshida, Hiroshi, Kurosawa, Hideo, Shimura, Yuko, Yanai, Hidekatsu, Tada, Norio
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2010; American Society for Biochemistry and Molecular Biology; The American Society for Biochemistry and Molecular Biology; Elsevier
• Subjects: Adult; atherosclerotic risk factor; Blotting, Western; Cholesterol - analysis; Cholesterol - blood; Cholesterol - isolation & purification; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange - methods; Chylomicrons - analysis; Chylomicrons - isolation & purification; Female; high-performance liquid chromatography; Humans; Linear Models; lipoprotein (a); Lipoprotein(a) - chemistry; Male; Methods; Middle Aged; Nephelometry and Turbidimetry; Reproducibility of Results; Ultracentrifugation; high-performance liquid chromatography; atherosclerotic risk factor; lipoprotein (a)
• ISSN: 0022-2275; 1539-7262
• DOI: 10.1194/jlr.D003624

We previously established an analysis method for determining the cholesterol levels of five major lipoprotein classes [HDL, LDL, intermediate density lipoprotein (IDL), VLDL, and chylomicrons] in serum by an anion-exchange (AEX)-HPLC method, but lipoprotein(a) [Lp(a)], a well-known risk factor for atherosclerotic diseases, was not determinable. Therefore, we established new AEX-HPLC separation conditions for analyzing the cholesterol levels of six lipoprotein classes, including Lp(a). Serum lipoproteins were separated by HPLC with a diethylaminoethyl-ligand nonporous polymer-based column by elution with a stepwise gradient of the sodium perchlorate concentration. In this improved method, HDL, LDL, IDL, VLDL, chylomicrons, and Lp(a) were each eluted from the column. The cholesterol levels of the eluted lipoproteins were measured enzymatically by a postcolumn reaction. The within-day assay and between-day assay coefficients of variation for the lipoprotein cholesterol levels were in the ranges of 0.29–11.86% and 0.57–11.99%, respectively. The Lp(a) cholesterol levels determined by AEX-HPLC were significantly correlated with the amounts of Lp(a) protein measured by an immunoturbidimetry method available commercially (r = 0.9503, P < 0.0001). Taken together, this AEX-HPLC method may be effectively applied to the analysis of serum lipoproteins with high levels of Lp(a).