Direct Separation of the Diastereomers of Cholesterol Ester Hydroperoxide Using LC-MS/MS to Evaluate Enzymatic Lipid Oxidation

Abstract

<jats:p>Cholesterol ester hydroperoxide (CEOOH) is one of the main lipid oxidation products contained in oxidized low-density lipoprotein (LDL). Previous studies suggest that CEOOH in oxidized LDL is closely related to several diseases. Of the oxidation mechanisms of cholesterol ester (CE) in vivo, it has been suggested that enzymatic oxidation induced by lipoxygenase (LOX) plays an important role. Thus, we attempted to develop a method that can evaluate the enzymatic oxidation of CE via the diastereoselective separation of CEOOH bearing 13RS-9Z,11E-hydroperoxy-octadecadienoic acid (13(RS)-HPODE CE). Firstly, we synthesized the standard of 13(RS)-HPODE CE. Using this standard, the screening of analytical conditions (i.e., column, mobile phase, and column temperature) was conducted, and separation of the diastereomers of 13(RS)-HPODE CE was achieved. The diastereoselective separation of 13(RS)-HPODE CE was also confirmed by LC-MS/MS. The developed method (column, CHIRALPAK IB N-3; mobile phase, hexane:ethanol (100:1, v/v); column temperature, 0 °C) can distinguish between enzymatic oxidation and other oxidation mechanisms of CE. Thus, the method can be expected to provide a greater understanding of the biochemical oxidation mechanisms in vivo. Such information will be essential to further elucidate the involvement of CEOOH in various diseases.</jats:p>

Analytical and Structural Tools of Lipid Hydroperoxides: Present State and Future Perspectives

“…Cholesterol ester hydroperoxide (CEOOH), one of the main lipid-oxidation products contained in oxidized low-density lipoprotein (LDL), is closely related to several diseases, according to studies in oxidized LDL. Ito et al [ 80 ] achieved diastereoselective separation of CEOOH-bearing 13RS-9 Z ,11 E -hydroperoxy-octadecadienoic acid (13(RS)-HPODE CE) using LC-MS/MS equipped with a chiral column ( Figure 13 ). This method can differentiate between enzymatic oxidation and other oxidation mechanisms (i.e., radical oxidation and singlet-oxygen-induced oxidation) of CE.…”

“…NMR spectroscopy has the advantages of relatively simple sample preparation, high reproducibility and the use of separation techniques not being a requirement for several applications. MS spectroscopy has considerable sensitivity advantages, and the development of Na + /MS techniques provides structure-diagnostic fragment ions that can contribute to the facile identification of the position of the hydroperoxide group and of diastereomers using chiral column [ 79 , 80 ]. MS and NMR spectroscopic methods, therefore, will undoubtedly continue to be developed as primary structural and analytical tools in the field of lipid hydroperoxide research.…”

“…In the field of MS: The use of Na + during MS/MS analysis afforded characteristic fragment ions for diastereomers of cholesterol-ester hydroperoxides (CEOOH) separated using a chiral column. As the main lipid-peroxidation products contained in oxidized low-density lipoprotein (LDL) are cholesterol-ester hydroperoxide (CEOOH) and phosphatidylcholine hydroperoxide (PCOOH), this method can be expected to provide further understanding of the biochemical oxidation mechanisms in vivo and elucidate the involvement of CEOOH in the development of diseases [ 80 ]. The collision-induced dissociation (CID) of the Na + adduct, due to high sensitivity and selectivity, is expected to provide further understanding of the hydroperoxyl group position of esterified lipids (i.e., phospholipids and cholesterol esters) as well as of polyunsaturated fatty acids (PUFAs) including ω-3, ω-6, and ω-9 FA [ 81 ].…”

“…The use of Na + during MS/MS analysis afforded characteristic fragment ions for diastereomers of cholesterol-ester hydroperoxides (CEOOH) separated using a chiral column. As the main lipid-peroxidation products contained in oxidized low-density lipoprotein (LDL) are cholesterol-ester hydroperoxide (CEOOH) and phosphatidylcholine hydroperoxide (PCOOH), this method can be expected to provide further understanding of the biochemical oxidation mechanisms in vivo and elucidate the involvement of CEOOH in the development of diseases [ 80 ].…”

Ozonation products of zidovudine and thymidine in oxidative water treatment

“…It is remarkable that organic hydroperoxides were scarcely identified with direct injection LC-HRMS methods ( Ito et al. 2015 , 2020 ). Furthermore, ozonation transformation processes could result in the same products as naturally occurring oxidation reactions in the human body, as the urea-TP 7a and formamide-TP 10a of THY were found as potential lesion of DNA/RNA resulting from ROS reactions ( Cadet et al., 2012 ; Irvoas et al., 2014 ; Toga et al., 2009 ).…”