Bioactive lipid mediators derived from arachidonic acid constitute an attractive pool of metabolites that reflect cellular function and signaling, as well as potential biomarkers that may respond quantitatively to disease progression or pharmacological treatment. Their quantitative measurement in biological samples is complicated by the number of isomers that share common structural features, which are not easily distinguished by immunoassays or reverse phase chromatography-tandem mass spectrometry. Here, we present a method that enables the rapid analysis of a panel of over 25 biologically important eicosanoids in a 96-well format for cell culture supernatants, plasma, and organ tissues using convergence chromatography-tandem mass spectrometry to resolve these analytes of interest.
© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Pulmonary hypertension (PH) is a life-threatening disease featuring pulmonary vessel remodelling and perivascular inflammation. The effect, if any, of eosinophils (EOS) on the development of PH remains unclear.
EOS infiltration and chemotaxis were investigated in peripheral blood and lung tissues from pulmonary arterial hypertension (PAH) patients without allergic history and from sugen/hypoxia-induced PH mice. The role of EOS deficiency in PH development was investigated using GATA1-deletion (ΔdblGATA) mice and anti-interleukin 5 antibody-treated mice and rats. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was conducted to identify the critical oxylipin molecule(s) produced by EOS. Culture supernatants and lysates of EOS were collected to explore the mechanisms in co-culture cell experiments.
There was a lower percentage of EOS in peripheral blood but higher infiltration in lung tissues from PAH patients and PH mice. PAH/PH lungs showed increased EOS-related chemokine expression, mainly C-C motif chemokine ligand 11 derived from adventitial fibroblasts. EOS deficiency aggravated PH in rodents, accompanied by increased neutrophil and monocyte/macrophage infiltration. EOS highly expressed arachidonate 15-lipoxygenase (ALOX15). 14-hydroxy docosahexaenoic acid (14-HDHA) and 17-HDHA were critical downstream oxylipins produced by EOS, which showed anti-inflammatory effects on recruitment of neutrophils and monocytes/macrophages through N-formyl peptide receptor 2. They also repressed pulmonary artery smooth muscle cell (PASMC) proliferation by activating peroxisome proliferator-activated receptor γ and blunting Stat3 phosphorylation.
In PH development without external stimuli, peripheral blood exhibits a low EOS level. EOS play a protective role by suppressing perivascular inflammation and maintaining PASMC homeostasis via 14/17-HDHA.
Copyright ©The authors 2023.

Bioactive lipids, including oxylipins, endocannabinoids, and related compounds may function as specific biochemical markers of certain aspects of inflammation. However, the postprandial responsiveness of these compounds is largely unknown; therefore, changes in the circulating oxylipin and endocannabinoid metabolome in response to a challenge meal were investigated at six occasions in a subject who freely modified her usual diet. The dietary change, and especially the challenge meal itself, represented a modification of precursor fatty acid status, with expectedly subtle effects on bioactive lipid levels. To detect even the slightest alteration, highly sensitive ultra-performance liquid chromatography (UPLC) coupled to electrospray ionization (ESI) tandem mass spectrometry (MS/MS) methods for bioactive lipid profiling was employed. A previously validated UPLC-ESI-MS/MS method for profiling the endocannabinoid metabolome was used, while validation of an UPLC-ESI-MS/MS method for oxylipin analysis was performed with acceptable outcomes for a majority of the parameters according to the US Food and Drug Administration guidelines for linearity (0.9938 < R2 < 0.9996), limit of detection (0.0005-2.1 pg on column), limit of quantification (0.0005-4.2 pg on column), inter- and intraday accuracy (85-115%) and precision (< 5%), recovery (40-109%) and stability (40-105%). Forty-seven of fifty-two bioactive lipids were detected in plasma samples at fasting and in the postprandial state (0.5, 1, and 3 hours after the meal). Multivariate analysis showed a significant shift of bioactive lipid profiles in the postprandial state due to inclusion of dairy products in the diet, which was in line with univariate analysis revealing seven compounds (NAGly, 9-HODE, 13-oxo-ODE, 9(10)-EpOME, 12(13)-EpOME, 20-HETE, and 11,12-DHET) that were significantly different between background diets in the postprandial state (but not at fasting). The only change in baseline levels at fasting was displayed by TXB2. Furthermore, postprandial responsiveness was detected for seven compounds (POEA, SEA, 9(10)-DiHOME, 12(13)-DiHOME, 13-oxo-ODE, 9-HODE, and 13-HODE). Hence, the data confirm that the UPLC-ESI-MS/MS method performance was sufficient to detect i) a shift, in the current case most notably in the postprandial bioactive lipid metabolome, caused by changes in diet and ii) responsiveness to a challenge meal for a subset of the oxylipin and endocannabinoid metabolome. To summarize, we have shown proof-of-concept of our UPLC-ESI-MS/MS bioactive lipid protocols for the purpose of monitoring subtle shifts, and thereby useful to address lipid-mediated postprandial inflammation.

Lipid mediators are produced from the oxidation of polyunsaturated fatty acids through enzymatic and free radical-mediated reactions. When subject to oxygenation via cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases, polyunsaturated fatty acids give rise to an array of metabolites including eicosanoids, docosanoids, and octadecanoids. These potent bioactive lipids are involved in many biochemical and signaling pathways, with inflammation being of particular importance. Moreover, because they are produced by more than one pathway and substrate, and are present in a variety of biological milieus, their analysis is not always possible with conventional assays. Liquid chromatography coupled to electrospray mass spectrometry offers a versatile and sensitive approach for the analysis of bioactive lipids, allowing specific and accurate quantitation of multiple species present in the same sample. Here we explain the principles of this approach to mediator lipidomics and present detailed protocols for the assay of enzymatically produced oxygenated metabolites of polyunsaturated fatty acids that can be tailored to answer biological questions or facilitate assessment of nutritional and pharmacological interventions.
Copyright © 2012 Elsevier Inc. All rights reserved.

Oxidized fatty acids formed via lipid peroxidation are implicated in pathological processes such as inflammation and atherosclerosis. A number of methods may be used to detect specific oxidized fatty acids containing a single or multiple combinations of epoxide, hydroxyl, ketone, and hydroperoxide moieties on varying carbon chain lengths from C8 up to C30. Some of these methods are nonspecific and their use in biological systems is fraught with difficulty. Measures of specific oxidized fatty acid derivatives help in identifying oxidation pathways in pathological processes. We used liquid chromatography coupled with electrospray ionization tandem mass spectrometry as an efficient, selective, and sensitive method for identifying and analyzing multiple specific fatty acid peroxidation products in human plasma and other biological matrices. We then distilled the essential components of a number of these analyses to provide an efficient protocol by which fatty acid oxidation products and their parent compounds can be determined. In this protocol, addition of a synthetic internal standard to the sample, followed by base hydrolysis at elevated temperature and liquid-liquid phase sample extraction with lighter-than-water solvents, facilitates isolation of the oxidized fatty acid species. These species can be identified and accurately quantified using stable-isotope dilution and multiple-reaction monitoring. Use of a coupled multiplexed gradient HPLC system on the front end enables high-throughput chromatography and more efficient use of mass spectrometer time.
Copyright © 2013 Elsevier Inc. All rights reserved.