Poster de conférence
Résumé : The oxidation of low- and high-density lipoproteins (LDL and HDL) is important in the development of atherosclerosis. Myeloperoxidase (MPO) oxidation is one of the most relevant oxidation processes. By oxidizing apoA-1, MPO leads to dysfunctional HDL and cholesterol efflux deficiency. Modifications on apoB-100 promote atherosclerosis by foam cell formation and pro-inflammatory properties of oxidized-LDL. Several modifications of apoA-1 and apoB-100 have already been described in the literature and seem relevant in patients with CVD [1,2]. However, it is not clear yet whether new lipoprotein biomarkers can provide better predictability of CVD risk. It is then necessary to update knowledge about new biomarkers, such as apolipoproteins A-1 and B and their quality.The aim is to develop a method to quantify the native and the oxidized forms of apoA-1 and apoB-100 in plasma. The analytical method is based on liquid chromatography coupled to mass spectrometry (LC-MS/MS) [3]. In vitro oxidations of purified HDLs and LDLs were performed to previously optimize LC-MS parameters, to detect relevant oxidized peptides from apolipoproteins. Sample preparation was optimized with isolation of lipoproteins (LDLs, HDLs…) from plasma samples by lipid removal agent (LRA) to increase the sensitivity of the method.The LRA has affinity for phospholipid carrying particles and has been tested to separate lipoproteins from plasma. A volume of plasma has been directly added on LRA. Then, trypsin digestion has been applied directly on the resin to extract in the supernatant the peptides from apolipoproteins. Plasma from healthy volunteers (n=3) and patients (n=3) where analysed. When regarding the ratio of oxidized peptides, we observe a difference between volunteers and patients for peptides from apoA-1: respectively 2.36 and 5.14 % for Trp72, and 0.97 and 1.44 % for Trp108. In contrast, no oxidized peptide from apoB-100 were detected. If we can identify relevant oxidized peptides from apoB-100 in purified LDL from patients, their detection directly in plasma by our method needs further investigations. The method is currently being optimized to detect oxidized residues of methionine (Met4, M785, M3719) and tryptophan (Trp1114, Met1207-Trp1210, Trp2546 and Trp4369). Oxidized plasma with HOCl (10 mM) were tested to assess our method. The formation of oxidized peptides was relevant with the proportion of oxidized plasma for both apoA-1 and B-100. Then, we performed supplementation of characterized plasma from volunteer with in vitro oxidized-LDL. Difference between plasma without and with supplementation of 2.5, 5, 10, 15 and 20% of oxidized-LDL was not significant, even if ratio of oxidized peptides tended to increase with supplementation.The isolation of lipoproteins with LRA allowed us to detect apoA-1 oxidized peptides directly in plasma of patients with cardiovascular diseases. However, oxidized peptides from apoB-100 were not detected. Future investigations will be carried out to increase the sensitivity of the method.Acknowledgements: We would like to thank Prof. A. Kontush for his help and the project was funded by the FRS-FNRS (Belgium). [1] Huang, Y., et al., Nature Medicine, 2014. 20(2): p. 193-203.[2] Delporte, C., et al., Journal of Lipid Research, 2014. 55(4): p. 747-57.[3] Smit, N.P., et al., Journal of proteomics, 2014. 109: p. 143-61.