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vls/2603noah-balancer_mdapi1elekpub.bib.uni-wuppertal.devdCombined untargeted and targeted LC-MS analysis of the fatty acid and oxylipin pattern in phospholipidsCarpanedo, Laura1996-VerfasserautSchebb, Nils HelgeAkademischer BetreuerdgsSteinhilber, Dieter1959-Akademischer BetreuerdgsBornhorst, Julia1984-Akademischer BetreuerdgsBergische Universität WuppertalGrad-verleihende InstitutiondggtextthesisTextHochschulschriftthesisdoctoralThesisgwXA-DE[Wuppertal]2025single unitengelectronic resourceremotetext/htmltext/html1 Online-Ressource (260 Seiten) : Illustrationen, DiagrammeComputerOnline-Ressource(Glycero-)phospholipids (PL) are the main components of the cellular membranes and play a key role in cell signaling. Polyunsaturated fatty acids (PUFA) are usually esterified at the sn-2 position, while saturated fatty acids are bound at the sn-1 position. In mammals, the diet modulates the proportion of n6-PUFA and n3-PUFA in the organism. PUFA can undergo oxidation by non-enzymatic or enzymatic processes, leading to the formation of eicosanoids and other oxylipins. Oxylipins can be present as non-esterified in biological samples, but the major part is esterified to lipids particularly PL. Esterified oxylipins are commonly analyzed indirectly as non-esterified oxylipins by targeted liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) following alkaline hydrolysis. However, this indirect approach does not provide information on the lipid class, lipid species, and sn-position in which the oxylipin is esterified. In this thesis, untargeted LC-high resolution (HR)-MS and targeted LC-MS/MS methods were developed for the analysis of both unoxidized and oxidized PL in biological samples. Optimized MS parameters resulted in sensitive analysis and acquisition of sufficient data points for (semi-)quantification. Chromatographic separation of isobaric and positional isomeric (ox)PL was achieved using reversed-phase LC. Characterization of (ox)PL species was performed based on precursor ions, characteristic product ions, and retention times. Matrix effects were evaluated by detailed ion suppression analysis. Semi-quantification of PL by untargeted LC-HRMS was carried out using one internal standard per lipid class. Key findings were supported by accurate quantification of selected lipids by targeted LC-MS/MS using external calibration with internal standards. Combined untargeted and targeted LC-MS methods revealed alterations in the plasma lipidome following n3-PUFA supplementation, with a general increase in lipids containing 20:5 and a decrease in those containing 22:4. Specifically, lysoPC 20:5_0:0 and PC 16:0_20:5 were found to be strongest elevated, while PE 18:0_22:4 and surprisingly PC 18:2_18:2 were decreased. Direct analysis of oxPL demonstrated a distinct incorporation of positional isomers hydroxy- and epoxy-PUFA into both PL classes and molecular species in HEK293T cells supplemented with oxylipins and overexpressing 15-lipoxygenase-2. Oxylipins with a hydroxy or epoxy functionality at the C15-position and 20 carbon atoms, namely 20:4;15OH, 20:5;15OH, 20:4;14Ep, and 20:5;14Ep, were preferentially incorporated into PI. In contrast, 18:2;13OH, 20:4;12OH, 20:4;11Ep, and 20:5;11Ep, were mostly detected to PC, while 22:6;17OH was predominantly found in PE-P. Specifically, PI 18:0/20:4;15OH, PI 18:0/20:5;15OH, PC 16:0/18:2;13OH, and PE-P 16:0/22:6;17OH were found to be most predominant species. This selective incorporation may affect PI-based signaling pathways and thus could be of high biological relevance.by Laura CarpanedoBergische Universität Wuppertal, Dissertation, 2025Wuppertal540urn:nbn:de:hbz:468-2-605410.25926/BUW/0-88499376090067306441Combined untargeted and targeted LC-MS analysis of the fatty acid and oxylipin pattern in phospholipidsCarpanedo, LauraDruck-AusgabeHT031268432https://dx.doi.org/10.25926/BUW/0-884https://nbn-resolving.org/urn:nbn:de:hbz:468-2-6054Open AccessRS INC 1.02025-08-18oaDoctoralThesispublic25090120260808210440.0CT009000420s2w-hsswupubwgerUBW:C:C.03 https://elekpub.bib.uni-wuppertal.de//domainresource/static/graphics/connectors/viewerLogo.gifWuppertal : Bergische Universität Wuppertalreserved https://ub-wuppertal.digibib.net/search/katalog/record/(DE-605)urn%3Anbn%3Ade%3Ahbz%3A468-2-6054 https://elekpub.bib.uni-wuppertal.de/id/8054158 https://elekpub.bib.uni-wuppertal.de/i3f/v20/8054158/manifest https://elekpub.bib.uni-wuppertal.de/proto_ftsearch/8054158