Affiliation:
1. Department of Health Sciences, Saitama Prefectural University, Saitama, Japan
2. Bioscience Division, TOSOH Corporation, Kanagawa, Japan
3. Department of Diabetes, Endocrinology and Metabolism, National Center for Global Health and Medicine Kohnodai Hospital, Chiba, Japan
Abstract
Background Relationships between the subclasses of high-density lipoprotein (HDL) or low-density lipoprotein (LDL) and the risk of atherosclerotic cardiovascular disease have been studied, and using various methods, such as ultracentrifugation, electrophoresis, and nuclear magnetic resonance, for analysing lipoprotein subclasses. We established a method for HDL and LDL subclasses using anion-exchange high-performance liquid chromatography (AEX-HPLC) with a linear concentration gradient of sodium perchlorate (NaClO4). Method In the AEX-HPLC, the subclasses of HDL and LDL were separated, and detected using a post-column reactor with an enzymatic cholesterol reagent, that contained cholesterol esterase, cholesterol oxidase, and peroxidase as major ingredients. LDL subclasses were divided based on the absolute value of first-derivative chromatogram. Result Three HDL subclasses, HDL-P1, HDL-P2, and HDL-P3, and three LDL subclasses, LDL-P1, LDL-P2, and LDL-P3, were separated by AEX-HPLC, and detected in order. The major components of HDL-P2 and HDL-P3 were HDL3 and HDL2, respectively. The linearity was determined for each lipoprotein subclass. The coefficients of variation of cholesterol concentration of the subclasses for within-day assay ( n = 10) and between-day assay ( n = 10) ranged between 3.08–8.94% and 4.52–9.97%, respectively. Cholesterol levels in HDL-P1 of diabetic patients were positively correlated with oxidized LDL levels (r = 0.409, p = 0.002). Moreover, cholesterol levels in LDL-P2 and LDL-P3 were positively correlated with oxidized LDL levels (r = 0.393, p = 0.004 and r = 0.561, p < 0.001, respectively). Conclusion AEX-HPLC may be highly suitable as an assay to clinically assess lipoprotein subclasses.
Funder
National Center for Global Health and Medicine Research Fund
Tosoh Research Fund
Subject
Clinical Biochemistry,General Medicine