Isolation of low density lipoprotein (LDL) with its modification by Copper ion and Malondialdehyde (MDA)

Abstract

Oxidation of low density lipoproteins (LDLs) is belived to be an important step in the pathogenesis of atherosclerosis. During oxidation, LDL particle undergoes a large number of structural changes that alters its biological properties, so it becomes atherogenic. To study atherogenic proteins, usually two forms of modified LDLs, including Cu2+-oxidized LDL (ox-LDL) and malondialdehyde (MDA) modified LDL (mal-LDL) are used. In this study, LDL was isolated from 72 ml freshly prepared plasma by sequential Floatation Ultracentrifugation (SFU), which resulted in separation of 12.5 mg LDL protein. LDL oxidation was accomplished in Phosphate Buffered Saline (PBS) with 2µM cupric sulfate, and mal-LDL was prepared by incubating LDL in PBS with 0.5 M solution of freshly prepared MDA. These modifications were evaluated by measuring optical density at 234 nm, Thiobarbitoric Acid Reactive Substances (TBARS), and electrophoretic mobility at pH 8.6. The increase of 234 nm absorption reflected initiation of LDL oxidation. TBARS of ox-LDL and mal-LDL was 80 Nm MAD/mg LDL protein and 400 nm MDA/mg LDL protein, respectively. Electrophoretic mobility of ox-LDL and mal-LDL, in respect to native LDL (n-LDL), were increased.

Keywords

• Modified LDL
• Cu ion