Oxidation of apolipoprotein A-I and B-100: LC-MS detection in plasma samples by LC-MS/MS (2018)

The oxidative modifications of HDLs and LDLs are regularly mentioned in the context of atherosclerosis. Myeloperoxidase (MPO) dependent oxidation of lipoproteins is one of the factors involved in lipoprotein oxidations. It adsorbs on the surface of apolipoproteins and targets moieties by local production of oxidizing agents. Oxidized ApoA-I leads to dysfunctional HDLs and cholesterol-efflux deficiency and oxidized ApoB-100 leads to LDLs unrecognized by the LDL receptor and are absorbed by macrophages. There are several modifications to ApoA-1 and ApoB-100 described in literature, which seem relevant in patients with cardiovascular risk [1, 2]. The aim is to develop a simple and fast method that enables to quantify in plasma of patients the oxidized forms of ApoA-I and ApoB-100, in parallel to a quantification of these apolipoproteins. The analytical method is based on liquid chromatography coupled to tandem low resolution mass spectrometry (LC-MS/MS) [3]. In vitro oxidations of purified HDLs and LDLs were performed to previously optimize LC and MS parameters to detect the most relevant oxidized peptides in ApoA-I and ApoB-100. Sample preparation was optimized and a separation of LDLs and HDLs from plasma samples was developed to increase the sensitivity. The identification of the most relevant residues in ApoA-I was done on isolated HDLs using a comparison between hemodialyzed patients and healthy volunteers. The residues of interest were oxidized Trp72 and Tyr192 that bring a specific information about the activity of MPO, but the oxidized Met112 and Met148 were more sensitive. Moreover, we have observed that specific residues increased significantly in patients with cardiovascular risk compared to healthy volunteers. Improvement of the sample preparation by precipitation of LDLs with dextran sulphate and magnesium chloride was tested, in order to increase the sensitivity of the analytical method. The best separation of LDLs and HDLs in plasma was obtained with 20 g/L and 0.4 M, respectively. LDLs are precipitated and HDLs are in the supernatant. The separated lipoproteins are therefore treated separately. They are digested by trypsin before analysis by LC-MS/MS. Results show significant signals of oxidized Trp72 and oxidized Met112 of ApoA-I in the plasma of healthy volunteers even in healthy volunteers. The key oxidation sites on ApoB100 have been confirmed and the method of analysis has been optimized to be more sensitive. Potentially significant residues, from a clinical and analytical point of view, have been selected for further work (including Met 4, Trp1207, Trp3126, and Trp4369).The method is sufficiently sensitive to detect HDLs and its oxidized forms in the plasma of healthy volunteers. It enables the early detection of baseline oxidation and modified ApoA-I in patients with cardiovascular risk. Trp72 and Met112 of ApoA-I were selected due to their relevance. The next step is to provide the same sensitivity for ApoB-100 analysis. The difference in oxidized states of lipoproteins between healthy volunteers and patients with cardiovascular risk will allow us to assess the quality of lipoproteins, with the hope to better characterize cardiovascular risk associated to lipoprotein oxidations.