Quantitative assays for esterified oxylipins generated by immune cells

Abstract

Phospholipid-esterified oxylipins are newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several, in particular hydro(pero)xyeicosatetraenoic acids (H(p)ETEs), hydroxyoctadecadienoic acids (HODEs), hydroxydocosahexaenoic acids (HDOHEs) and ketoeicosatetraenoic acids (KETEs), attached to either phosphatidylethanolamine (PE) or phosphatidylcholine (PC). Lipids are isolated from cells or tissue using a liquid phase organic

Docosahexaenoic Acid–Derived Fatty Acid Esters of Hydroxy Fatty Acids (FAHFAs) With Anti-inflammatory Properties

“…Other derivatives of HDHA were detected, but only 14-DHAHDHA was upregulated in humans in response to n-3 PUFA supplementation. The exceptional role of DHA hydroxylated at position 14 was also observed in oxidized phospholipids (39). Due to the demanding organic synthesis of the 14-HDHA backbone, HDHA derivatives were not explored further.…”

“…MS/MS/MS analysis revealed 14-HDHA-specific fragments (205.2 and 161.2 m/z) (39,42) in the corresponding peak. Importantly, oxidized phospholipids containing predominantly the 14-HDHA positional isomer were identified in human platelets (39). Besides the 14-HDHA backbone, smaller peaks of DHAHDHA were detected in murine samples.…”

“…Organic synthesis of 13-DHAHLA was performed according to Steglich esterification from DHA and 13-HODE (38,39). Details are provided in the Supplementary Data.…”

A simplified procedure for semi-targeted lipidomic analysis of oxidized phosphatidylcholines induced by UVA irradiation

“…I ). The chemical diversity of oxidation products accumulating in vivo depends on many factors, including the relative impact of nonenzymatic versus enzymatic oxidation and variable tissue expression of enzymes oxidizing PLs directly (e.g., 15-LOX [ 4 ]) or enzymes oxidizing free fatty acids followed by esterifi cation ( 5,6 ), as well as enzymes reducing PL hydroperoxides (GPx4 [ 7 ] and peroxiredoxin 6 [ 8 ]). Different molecular species of OxPLs often demonstrate nonidentical biological activities.…”

“…Previous studies described procedures for focused analysis of OxPCs containing fragmented -terminal ( 27 ), fragmented ␣ , ␤ -unsaturated ( 28,29 ), fragmented terminal furan-containing ( 30 ), and nonfragmented linear ( 5,24 ) and prostane ring-containing ( 31 ) residues. Here we show that a variety of full-length and fragmented OxPCs can be detected at 99 m/z precursor values using m/z 184 as a diagnostic fragment ion within one HPLC-electrospray ionization (ESI)-MS/MS run having a total run time of 20 min (including column re-equilibration).…”