Kampschulte, N. | bioRxiv Science

bioRxiv2025

Quantification of esterified oxylipins following HILIC-fractionation of lipid classes

Several oxylipins are lipid mediators derived from the oxidation of polyunsaturated fatty acids (PUFAs). The majority of oxylipins in biological samples occurs esterified in neutral lipids (nLs) and phospholipids (PLs). They are commonly quantified indirectly following alkaline hydrolysis providing excellent sensitivity but the information in which lipid classes the oxylipins occurred in is lost. The direct analysis of oxidized lipids is currently not sensitive enough to detect all esterified oxylipins. Here, a new hydrophilic interaction liquid chromatography (HILIC) based lipid class fractionation using solid-phase extraction (SPE) cartridges was developed separating lipids into nLs and 4 PL fractions using a single column. Esterified oxylipins in the fractions were quantified following alkaline hydrolysis to sensitively pinpoint in which lipid classes they are bound in plasma. The fractionation was extensively characterized for different lipid extracts demonstrating high separation efficiency and recovery using labeled standards and untargeted analysis of endogenous lipids. Esterified oxylipins in the fractions were quantitatively detected. Based on the results from two independent human plasma pools including SRM1950 it is shown that: hydroxy-linoleic acid- and hydroxy--linolenic acid-derived oxylipins are preferably bound to nLs whereas long chain hydroxy-PUFAs and PUFAs (i.e. ARA EPA and DHA) are predominantly esterified to phospholipid classes. Supplementation of n3-PUFAs for 12 months led to an increase in EPA- and -DHA-derived oxylipins in all lipid fractions with the highest increase of hydroxy-PUFAs in nLs. This demonstrates a precursor PUFA-dependent binding of oxylipins and a direct effect of diet on esterified oxylipins in plasma.

biochemistry↗

bioRxiv2024

Ex vivo DHA supplementation suppresses prostaglandin E2 formation in primary human macrophages

ScopeThere is evidence that intake of long-chain n-3 polyunsaturated fatty acids (PUFA) is associated with improved prognosis for inflammatory diseases. However, the underlying mechanisms are still subject of ongoing research. For this purpose, we developed an ex vivo n-3 PUFA supplementation strategy to test n-3 PUFA supplementation under controlled conditions in primary human macrophages. Methods and resultsCells were supplemented with docosahexaenoic acid (DHA). Quality parameters to account for possible confounders were established for a reproducible and reliable supplementation. Following supplementation, PUFA pattern of cells was shifted towards a pattern reflecting that of subjects with a high n-3 PUFA status. This was accompanied by a decrease of arachidonic acid-derived oxylipins in a dose- and time-dependent manner in favor of n-3 PUFA ones. Stimulation with LPS resulted in decreased levels of pro-inflammatory prostaglandins in the DHA-supplemented cells, but no changes in cytokines. ConclusionIn vitro supplementation studies with n-3 PUFA need rigorous controls to exclude background formation of oxylipins. By accounting for these possible confounders the desribed ex vivo approach is a promising tool for the mechanistic investigation of n-3 PUFA in primary human immune cells, offering an alternative for intervention studies in humans.

physiology↗