Cyclodextrin-micellar electrokinetic chromatography of apolipoproteins on human very low-density lipoprotein.

The apolipoproteins (APOs) of human very low-density lipoprotein (VLDL) were investigated by an optimized cyclodextrin-micellar electrokinetic chromatography (CD-MEKC) method. The separation buffer consisted of 20 mM sodium phosphate, 40 mM bile salts (50% sodium cholate and 50% sodium deoxycholate), 25 mM carboxymethyl-ß-cyclodextrin (CM-ß-CD) (pH 7.0). For CD-MEKC separation, a sample injection time of 12 s, a separation voltage of 15 KV, and a capillary temperature of 15°C were chosen. The optimal CD-MEKC method showed good resolution and repeatability for VLDL APOs. Identification and quantitation of VLDL APOs CI, CIII, and E were based on comparison with human APO standards. Good linear relationships with correlation coefficient (R2 ) 0.99 were obtained for APOs CI, CIII, and E standards. For these three APOs, the linear ranges were within 0.01-0.54 mg/mL, and the concentration limits of detection (LODs) were lower than 0.02 mg/mL. Moreover, VLDL APOs from four uremic patients and four healthy subjects were compared. The uremic and healthy CD-MEKC profiles showed dramatic difference. The levels of APO CIII were significantly higher for two patients, and the level of APO E was significantly higher for one patient. This study might be helpful for following the disease development of uremia and cardiovascular disease (CVD) in the future.

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

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-ß-CD (CM-ß-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.