Several oxylipins are potent lipid mediators. They can be formed by enzymatic and non-enzymatic oxidation of polyunsaturated fatty acids (PUFA) such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. The enzymatic oxidation of PUFAs is catalyzed by lipoxygenases, cyclooxygenases and CYP450 monooxygenases leading to a wide range of different biologically active oxylipins such as prostaglandins and leukotrienes. A dysregulation of the endogenous oxylipin formation has been implicated in the pathogenesis of several inflammatory diseases, including asthma and atherosclerosis. These effects were attributed to free i.e., non-esterified oxylipins. However, the majority of oxylipins in biological samples is present in esterified form, being bound to phospholipids or other lipids. Not much is known about esterified oxylipins in polar lipids and it’s unclear in which phospholipid class they are bound.
The best option for quantification of esterified oxylipins in biological samples is currently the targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) based analysis of free, i.e. non-esterified oxylipins and total oxylipins after saponification. Our established method allows the quantification of more than 230 oxylipins in a single run using LC-MS/MS in multiple reaction monitoring mode1,2. However, the information in which lipid class the esterified oxylipins are bound is lost in the saponification step. In this project solid phase extraction (SPE) in is used to fractionate phospholipid classes before quantifying oxylipins to pinpoint in which lipid class the oxylipins are bound.
In the presentation a lipid fractionation is shown which allows the separation of phospholipid classes i.e. phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in individual fractions. The separation efficacy was evaluated using spiking experiments with one representative phospholipid for each class. In combination with non-targeted lipidomics3 and feature detection using MS-DIAL, the fractionation for all fractionated lipids was evaluated.
This method was applied on the analysis of human cells. The pattern of oxylipins in the phospholipid classes was analyzed to investigate and compare the incorporation of endogenously formed and exogenously supplemented oxylipins.
Literature
- Hartung, N. M., et al., Bioanal. Chem., 2023, 415, 913.
- Ostermann A. I., et al., Prostaglandins Other Lipid Mediat . 2020, 146, 106384.
- Rund, K.M., et al., Anal Bioanal Chem 2024, 416, 925–944.