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Stubiger G.,Medical University of Vienna | Belgacem O.,Shimadzu Biotech | Rehulka P.,University of Hradec Kralove | Bicker W.,University of Vienna | And 3 more authors.
Analytical Chemistry | Year: 2010

6-Aza-2-thiothymine (ATT) is introduced as novel matrix system for the analysis of oxidized phospholipids (OxPLs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A systematic evaluation comparing different established and novel matrix substances, especially 2,4,6-THAP matrix (Stübiger, G.; Belgacem O. Anal. Chem. 2007, 79, 3206-3213) as reference compound for phospholipid analysis, and specific matrix additives was performed. Thereby, ATT turned out to be the reagent of choice for MALDI analysis of major biologically relevant OxPL classes (e.g., OxPC, OxPE, and OxPS) in positive and negative ionization mode. ATT used together with specific chaotropic reagents at low concentration (0.5-2 mM) acting as OxPL ionization enhancers revealed an excellent comatrix system for application with MALDI instrument types employing UV- and Nd:YAG laser systems (337 and 355 nm). Moreover, disposable MALDI targets surfaces with specific physicochemical properties (e.g., metallized glass or polymeric substrates) were revealed as superior over stainless steel in terms of reduced chemical background noise (∼10-fold better S/N ratios), increased mass spectral reproducibility, and enhanced sensitivity (LOD ∼250-500 fg on target). The combination of these parameters offers a significant advantage for highly sensitive OxPL profiling by MALDI-MS of biological samples (e.g., human plasma) at trace levels. © 2010 American Chemical Society. Source

Gruber F.,Medical University of Vienna | Bicker W.,FTC Forensic Toxicological Laboratory Ltd | Oskolkova O.V.,Medical University of Vienna | Tschachler E.,Medical University of Vienna | Bochkov V.N.,Medical University of Vienna
Journal of Lipid Research | Year: 2012

Oxidized phospholipids (OxPLs) are increasingly recognized as signaling mediators that are not only markers of oxidative stress but are also "makers" of pathology relevant to disease pathogenesis. Understanding the biological role of individual molecular species of OxPLs requires the knowledge of their concentration kinetics in cells and tissues. In this work, we describe a straightforward "fingerprinting" procedure for analysis of a broad spectrum of molecular species generated by oxidation of the four most abundant species of polyunsaturated phosphatidylcholines (OxPCs). The approach is based on liquid-liquid extraction followed by reversedphase HPLC coupled to electrospray ionization MS/MS. More than 500 peaks corresponding in retention properties to polar and oxidized PCs were detected within 8 min at 99 m/z precursor values using a single diagnostic product ion in extracts from human dermal fibroblasts. Two hundred seventeen of these peaks were fluence-dependently and statistically significantly increased upon exposure of cells to UVA irradiation, suggesting that these are genuine oxidized or oxidatively fragmented species. This method of semitargeted lipidomic analysis may serve as a simple first step for characterization of specific "signatures"of OxPCs produced by different types of oxidative stress in order to select the most informative peaks for identification of their molecular structure and biological role. Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc. Source

Uderhardt S.,Friedrich - Alexander - University, Erlangen - Nuremberg | Herrmann M.,Friedrich - Alexander - University, Erlangen - Nuremberg | Oskolkova O.V.,Medical University of Vienna | Aschermann S.,Friedrich - Alexander - University, Erlangen - Nuremberg | And 10 more authors.
Immunity | Year: 2012

Noninflammatory clearance of apoptotic cells (ACs) is crucial to maintain self-tolerance. Here, we have reported a role for the enzyme 12/15-lipoxygenase (12/15-LO) as a central factor governing the sorting of ACs into differentially activated monocyte subpopulations. During inflammation, uptake of ACs was confined to a population of 12/15-LO-expressing, alternatively activated resident macrophages (resMΦ), which blocked uptake of ACs into freshly recruited inflammatory Ly6Chi monocytes in a 12/15-LO-dependent manner. ResMΦ exposed 12/15-LO-derived oxidation products of phosphatidylethanolamine (oxPE) on their plasma membranes and thereby generated a sink for distinct soluble receptors for ACs such as milk fat globule-EGF factor 8, which were essential for the uptake of ACs into inflammatory monocytes. Loss of 12/15-LO activity, in turn, resulted in an aberrant phagocytosis of ACs by inflammatory monocytes, subsequent antigen presentation of AC-derived antigens, and a lupus-like autoimmune disease. Our data reveal an unexpected key role for enzymatic lipid oxidation during the maintenance of self-tolerance. © 2012 Elsevier Inc. Source

Stubiger G.,Medical University of Vienna | Wuczkowski M.,Technoclone GmbH | Bicker W.,FTC Forensic Toxicological Laboratory Ltd | Belgacem O.,Shimadzu
Analytical Chemistry | Year: 2014

In this paper we present a pioneering approach exploiting nanoparticles (NPs) for the "on-probe" (i.e., directly from the NP-surface) monitoring of OxPLs by MALDI-MS (i.e., the Nano-MALDI approach). The "electrophilic interaction" with either metal oxide (e.g., ZrO 2) or surface-functionalized Fe3O4 core-shell superparamagnetic NPs (100 nm diameter) was exploited for the direct enrichment of short-chain carboxylic (CARBO)-OxPLs, whereas detection of aldehydic (ALDO)-OxPLs was enabled by prior derivatization with bifunctional carbonyl-reactive reagents containing a negatively charged moiety (e.g., 4-AA) followed by NP-binding. Polyetheramine (PEA)-NPs were found best suited in terms of solvent stability, binding efficiency and compatibility with MALDI-MS analysis. For quantitative analysis of the OxPLs a recently introduced MALDI-QIT-TOF-MS/MS platform (Stübiger et al. Atherosclerosis 2012, 224, 177-186) was employed and cross-validated by LC-ESI-SRM-MS/MS. The sensitivity was found in the sub-nanomolar range (LOD ∼200 pM), which is 1-4 orders of magnitude higher than necessary for detection of individual OxPLs under normal and diseased conditions in vivo (e.g., in mouse plasma or human lipoproteins). Consequently, the Nano-MALDI approach shows the potential to serve as novel platform for the screening of OxPLs in biological samples and the development of clinical diagnostic tests in the future. © 2014 American Chemical Society. Source

Reischl R.J.,University of Vienna | Bicker W.,University of Vienna | Bicker W.,FTC Forensic Toxicological Laboratory Ltd | Keller T.,University of Salzburg | And 2 more authors.
Analytical and Bioanalytical Chemistry | Year: 2012

Acetaldehyde is a strongly electrophilic compound that is endogenously produced as a first intermediate in oxidative ethanol metabolism. Its high reactivity towards biogenic nucleophiles has toxicity as a consequence. Acetaldehyde readily undergoes a non-enzymatic condensation reaction and consecutive ring formation with cysteine to form 2-methylthiazolidine-4- carboxylic acid (MTCA). For analytical purposes, N-acetylation of MTCAwas required for stabilization and to enable its quantification by reversed-phase chromatography combined with electrospray ionization-tandem mass spectrometry. Qualitative screening of post mortem blood samples with negative blood alcohol concentration (BAC) mostly showed low basal levels of MTCA. In BACpositive post mortem samples, but not in corresponding urine specimens, strongly increased levels were present. To estimate the association between ethanol consumption and the occurrence ofMTCA in human blood, the time curves of BAC and MTCA concentration were determined after a single oral dose of 0.5 g ethanol per kilogram of body weight. The blood elimination kinetics ofMTCAwas slower than that of ethanol. The peak concentration of MTCA (12.6 mgL-1) was observed 4 h after ethanol intake (BAC 0.07%) and MTCA was still detectable after 13 h. Although intermediary acetaldehyde scavenging by formation of MTCA is interesting from a toxicological point of view, lack of hydrolytic stability under physiological conditions may hamper the use of MTCA as a quantitative marker of acetaldehyde exposure, such as resulting from alcohol consumption. © Springer-Verlag 2012. Source

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