Chiral lipidomics of monoepoxy and monohydroxy metabolites derived from long-chain polyunsaturated fatty acids

Abstract

chidonic acid (AA), EPA, and DHA. Oxygenated PUFAs have also been termed "oxylipins" and comprise metabolites formed by cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450 (CYP) enzymes as well as nonenzymatic oxidation reactions (1)(2)(3)(4). Current methods of targeted lipidomics allow high-throughput, comprehensive, and highly sensitive quantitation of oxylipins in biological and clinical samples (5). Most of these analytical approaches rely on LC coupled with MS/MS. Thereby, LC is performed on achiral stationary phases under reversedphase conditions and MS mostly uses ESI for efficient ionization of the analytes. These advanced methods can measure more than one hundred different oxylipin species in one analytical run, but are unable to distinguish between the enantiomers (5).The lack of enantiomeric resolution is an inherent property of the achiral-LC-ESI-MS/MS approaches. This feature limits the conclusions that can be drawn regarding the enzymatic versus nonenzymatic origin of oxylipin species or the biological significance of changes in the endogenous oxylipin profile, e.g., in the course of cardiovascular and inflammatory diseases. To address these questions, different strategies of targeted chiral lipidomics are under investigation (6). Chiral-LC has been primarily developed for normal-phase conditions using apolar solvent systems that preclude ESI application for efficient ionization. A way out of this problem is provided by electron capture atmospheric Abstract A chiral lipidomics approach was established for comprehensive profiling of regio-and stereoisomeric monoepoxy and monohydroxy metabolites of long-chain PUFAs as generated enzymatically by cytochromes P450 (CYPs), lipoxygenases (LOXs), and cyclooxygenases (COXs) and, in part, also unspecific oxidations. The method relies on reversedphase chiral-LC coupled with ESI/MS/MS. Applications revealed partially opposing enantioselectivities of soluble and microsomal epoxide hydrolases (mEHs). Ablation of the soluble epoxide hydrolase (sEH) gene resulted in specific alterations in the enantiomeric composition of endogenous monoepoxy metabolites. For example, the (R,S)/(S,R)-ratio of circulating 14,15-EET changed from 2.1:1 in WT to 9.7:1 in the sEH-KO mice. Studies with liver microsomes suggested that CYP/mEH interactions play a primary role in determining the enantiomeric composition of monoepoxy metabolites during their generation and release from the ER. Analysis of human plasma showed significant enantiomeric excess with several monoepoxy metabolites. Monohydroxy metabolites were generally present as racemates; however, Ca 2+ -ionophore stimulation of whole blood samples resulted in enantioselective increases of LOX-derived metabolites (12S-HETE and 17S-hydroxydocosahexaenoic acid) and COX-derived metabolites (11R-HETE). Our chiral approach may provide novel opportunities for investigating the role of bioactive lipid mediators that generally exert their physiological functions in a highly regio-and stereospecific manner.-Blum, M., I. Dog...

EPHX1 mutations cause a lipoatrophic diabetes syndrome due to impaired epoxide hydrolysis and increased cellular senescence

“…To evaluate the impact of the loss of enzyme activity in vivo, we measured by liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) circulating levels of a panel of EpFAs and corresponding diols in plasma samples of patient 1. EPHX1 was previously shown to catalyze the hydrolysis of several EpFAs, also called oxylipins ( Edin et al, 2018 ; Snider et al, 2007 ; Marowsky et al, 2017 ; Blum et al, 2019 ). These profiles, which result from the combined action of several EHs, were compared to the patterns determined in eleven sex- and age-matched control women with normal BMI.…”

“…On the other hand, EPHX2 was shown to hydrolyze endogenous terpenoid epoxides and EpFAs ( El-Sherbeni and El-Kadi, 2014 ). More recently, EPHX1 was also shown in animal models to play a significant role in the hydrolysis of different endogenous EpFAs derived from polyunsaturated fatty acids ( Edin et al, 2018 ; Snider et al, 2007 ; Marowsky et al, 2017 ; Blum et al, 2019 ). These EpFAs, including epoxyeicosatrienoic acids (EETs) and epoxyoctadecenoic acids (also called EpOMEs), are formed by ER-attached CYP450s and hydrolyzed by EPHX1 to their respective diols, the so-called dihydroxyeicosatrienoic acids (DHETs) and dihydroxyoctadecenoic acids (DiHOMEs), respectively.…”

MS-based targeted metabolomics of eicosanoids and other oxylipins: Analytical and inter-individual variabilities

“…In particular, independent detection and quantification of enantiomers can aid in interpretation of results in a biologically relevant context. For instance, a variety of oxylipins can be produced by the direct interaction of reactive oxygen species with unsaturated lipids, with the resulting products having no enantiomeric enrichment, while enzymatic reactions generally have high degrees of enantiomeric enrichment (95)(96)(97)(98)(99)(100)(101)(102). Notably, the clotting process yields dramatic shifts in oxylipin enantiomeric patterns (99).…”

“…Historically, these techniques have relied upon normal phase chromatography of pentaflurobenzyl ester derivatives, however new reverse phase chiral materials are opening the door to routine application of these methods in the future. (100)(101)(102). Recently developed approaches couple a non-chiral reversed phase-column with a chiral amylose-based column that can be used with reversed phase solvents (101,105).…”

“…(100)(101)(102). Recently developed approaches couple a non-chiral reversed phase-column with a chiral amylose-based column that can be used with reversed phase solvents (101,105). These were used e.g.…”