Investigating apolipoproteins of human high-density lipoprotein by cyclodextrin-micellar electrokinetic chromatography

Abstract

A cyclodextrin-micellar electrokinetic chromatography (CD-MEKC) method has been developed to determine the apolipoproteins (apos) of human high-density lipoprotein (HDL). The optimal CD-MEKC conditions included a separation buffer mixture of 5 mM sodium phosphate, 40 mM bile salts (50% sodium cholate and 50% sodium deoxycholate), 25 mM carboxymethyl-β-CD (CM-β-CD) and pH 7.0. The separation voltage was 15 kV, and the capillary temperature was 15℃. The CD-MEKC profiles of human HDL apolipoproteins showed good repeatability and sensitivity.Linear analysis has been performed for human apolipoprotein standards including apos AI, AII, CI, CII, CIII and E. Linear regression lines with coefficients of determination (R2) greater than 0.99 were obtained for apos AI, AII, CI, CII and E. The linear ranges for the six apolipoproteins were within 0.18–0.70 mg/mL, and the concentration limits of detection (LOD) were lower than 0.0617 mg/mL.Apos AI, AII, CI and CIII were identified and quantified in human HDL by comparing with apolipoprotein standards. Furthermore, the CD-MEKC profiles of uremic patients differed significantly from healthy subjects. The concentration ratios of apo AI/apo CIII were significantly lower for uremic patients than healthy subjects.This study demonstrated the feasibility of determining human HDL apolipoproteins by CD-MEKC. In the future, it might help monitor the progression of uremia and cardiovascular disease.