Harmonization of OF LC‐MS oxylipin analysis to investigate lipid mediator formation and signaling in immune cells
Authors: Mainka Malwina , Schebb Nils 
Published: June 2023

The oxidation products of polyunsaturated fatty acids (PUFA) ‐ oxylipins ‐ are formed via enzymatic conversion by cyclooxygenases, lipoxygenases (LOX) and cytochrome P450 monooxygenases or by autoxidation. Together with multiple PUFA substrates this leads to a large variety of oxylipins, whose physiological functions are ‐ besides those of well‐studied prostaglandins and leukotrienes ‐ little understood. The interplay of several PUFA‐oxidizing enzymes, as in the case of multihydroxylated oxylipins, additionally complicates the investigation of their biosynthesis. Due to regulatory crosstalk and complex interactions of different oxylipin classes during physiological processes, such as inflammation, a study of the whole oxylipin profile rather than individual compounds is necessary. Therefore, in order to understand the functions of these lipid mediators potent and reliable biochemical methods and (molecular) biological models are required, in combination with sensitive and sound preanalytical, analytical and post‐analytical procedures for their investigation.The first part of this thesis investigated the relevance of harmonizing such analytical methods for the quantitative determination of oxylipins in biological samples. Based on the outcome of an international laboratory comparison, comparable and reproducible results were obtained across independent laboratories when these harmonized methods were used for sample preparation and analysis by means of liquid chromatography coupled with tandem mass spectrometry. The examination of the effect of different storage times and temperatures as a parameter of pre‐analytics demonstrated that oxylipin levels in plasma samples are robust to long‐term storage and, furthermore, to minor variations during plasma generation. As accurate quantification of oxylipin concentrations rises and falls with the quality of analytical standards, a strategy for the verification of oxylipin standard concentrations with mass‐ spectrometry and UV spectroscopy based approaches using certified standard compounds was presented.In the second part of the thesis the established methods were applied to study the biosynthesis of oxylipins in immune cells with a special attention to the involved LOX. Stimulation of primary human M2‐like macrophages with the liver X receptor (LXR) agonist T09 led to a massive induction of ALOX15 gene expression and thus, 15‐LOX abundance and activity. Cholesterol derivatives were identified as potent endogenous ligands of LXR yielding increased 15‐LOX abundance and activity. The examination of 5‐LOX induced synthesis of multihydroxylated metabolites through supplementation of neutrophils with 15‐HETE, 18‐ HEPE and 17‐HDHA primary leads to formation of double oxygenated oxylipins. In intact cells 5‐LOX preferred DHA‐derived 17‐HDHA as substrate, whereas upon cell integrity destruction the formation of 15‐HETE derived multihydroxylated oxylipins increased.With the combination of harmonized protocols for sampling and analysis this thesis sets the basis for the reliable quantification of oxylipins. The use of the optimized methodologies allowed to further characterize regulatory pathways of the ARA cascade in human immune cells, contributing to a more thorough understanding of inflammation regulation.