HDL Subfraction Separation by Ion Exchange

Dialyzed HDL-c were separated to different subfractions by using UnoQ6 anion-exchange columns (Bio-Rad, Hercules, CA, USA) with an NGC Quest 10 chromatography system (Bio-Rad, Hercules, CA, USA) as described [43 (link)]. HDL-c in 3 mL was injected onto a UnoQ6 column and eluted with a multistep gradient of buffer B (1M NaCl in buffer A) at 2 mL/min rate. Five HDL-c subfractions were eluted with a multistep gradient of buffer B according to electronegativity. H1 was the effluent collected between fractions 8 to 11 (14–22 min) and H5 fractions 30 to 35 (58–70 min). The respective fractions were then concentrated with Vivaspin Turbo (Sartorius, Göttingen, Germany) and sterilized by passage through 0.22 μm syringe filters (Fintech, Changhua, Taiwan).