An in Vitro Method to Assess Oxidative Status of Low-Density Lipoprotein Using Fluorescence-Labeled Heptapeptides

Abstract

Oxidized low density lipoprotein (Ox-LDL) plays an important role in the pathogenesis of atherosclerosis. Recently, we identified two fluorescence-labeled heptapeptides, (FITC)KP6 and (FITC)dKP6, that bind specifically to lyso- and oxidized-phosphatidylcholine (LPC and Ox-PC), both of which are generated during LDL oxidation and are known to be major bioactive lipid components of Ox-LDL. Here, we report an in vitro method to assess oxidative status of LDL by specific binding of (FITC)KP6 and (FITC)dKP6 to LPC and ox-PC in Ox-LDL. Briefly, LDL with a candidate was oxidized by copper ions, and the reaction mixture with (FITC)KP6 or (FITC)dKP6 was subjected to non-denaturing polyacrylamide gel electrophoresis. The gels were visualized and characterized using a luminescent image analyzer. This method may provide important information about the oxidative status of LDL by measuring LPC and Ox-PC generated during LDL oxidation in conjunction with other oxidation assays such as thiobarbituric acid–reactive substances (TBARS) and the production of LDL conjugated dienes and also be useful for searching inhibitors and promoters of atherosclerosis.

Keywords:

• Atherosclerosis
• fluorescence
• oxidative status
• oxidized low density lipoprotein
• synthetic peptide

Conflicts of interest

The authors declare that there are no conflicts of interest.

Additional information

Funding

This work was supported by JSPS KAKENHI Grant-in-Aid for Young Scientists (B) (grant number 16K19201) (to A.S.).