Oxldl

Changes in interleukin-1β (IL-1β; BD Biosciences, Franklin Lakes, NJ, USA), thiobarbituric acid reactive substances (TBARS; R&D Systems, Minneapolis, MN, USA), and oxidized low-density lipoprotein (oxLDL; MyBioSource, San Diego, CA, USA) at months 0, 3, and 6 were measured in heparin plasma (for IL-1β) and EDTA plasma (for TBARS and oxLDL) using commercially available ELISA kits. High-sensitivity C-reactive protein (hs-CRP) changes were measured at months 0, 3, and 6 in serum using a Cobas c111 analyzer (Roche Diagnostics, Indianapolis, IN, USA).

Lipoprotein separation was performed using the iodixanol gradient centrifugation method [17 (link),18 (link),19 (link)]. In brief, EDTA plasma was mixed with iodixanol and PBS, and centrifuged in a near-vertical rotor at 65,000 rpm for 3 h and 10 min at 16 °C. The generated lipoprotein fractions were subsequently fractionated into 20 different parts of equal volume using an Auto Densi-Flow machine (Labconco, Kansas City, MO, USA) and an FC 203 B fraction collector (Gilson, Middleton, WI, USA). High-precision measurement of the index of refraction for each lipoprotein fraction was performed using an Abbe refractometer. The lipoprotein density was calculated from the index of refraction and pooled low-density lipoprotein (LDL), large buoyant low-density lipoprotein (lbLDL) and small dense LDL (sdLDL) subclasses were prepared from the fractions based on density.
Lipoprotein characterisation for total cholesterol (TC), LDL-C and sdLDL-C concentrations was performed for whole plasma, total LDL, lbLDL and sdLDL subclasses using the corresponding kit for an RX Daytona Plus analyser (Randox, Manchester, UK). A commercially available enzyme-linked immunosorbent assay (ELISA) test was used to measure the OxLDL concentration (MyBioSource, San Diego, CA, USA).