5 hete d8

The following reagents were used for metabolomics analyses: methanol and ultra-pure water (LC-MS grade, EMD Millipore, Gibbstown, NJ); zirconium oxide beads (Next Advance; Averill Park, NY); formic acid, acetic acid (Optima LC/MS grade; Fisher Chemical, Pittsburgh, PA); Deuterium (d) labeled internal standards DHA-d₅, ARA-d₈, EPA-d₅, LA-d₄, ALA-d₁₄, 9(S)-HODE-d₄, and 5-HETE-d₈ (Cayman Chemical, Ann Arbor, MI) and butylated hydroxytoluene (BHT, TCI America; Portland, OR) were used for quantification of total and free fatty acids, and oxidized lipid derivatives, respectively.

Samples were taken to solid-phase extraction (SPE) using Isolute C18 SPE 3 mL cartridges (Biotage, USA), as in Colas et al.^{15 (link)}. Briefly, internal standards (d8-5-HETE, d5-RvD2, d5-LXA₄, d4-LTB₄, d4-PGE₂; 500 pg each; Cayman Chemicals, USA) were added along with four volumes of methanol before SPE, and covered on ice for 30-60 minutes to allow for protein precipitation. During SPE, 6 mL of water was eluted through each cartridge, followed by elution of 6 mL of hexane. Lipid mediators were collected by elution and collection of 6 mL of methyl formate. Methyl formate fractions from SPE were analyzed by a liquid chromatography-tandem mass spectrometry system, QTrap 5500 (AB Sciex) equipped with a Shimadzu LC-20AD HPLC (Tokyo, Japan). A Poroshell 120 EC-18 column (100 mm × 4.6 mm × 2.7 μm; Agilent Technologies, USA) was kept in a column oven maintained at 50°C, and lipid mediators (LMs) were eluted in a gradient of methanol/water/acetic acid from 55:45:0.01 (v/v/v) to 98:2:0.01 at 0.5 mL/min flow rate. In order to monitor and quantify the levels of targeted LMs, multiple reaction monitoring (MRM) was used with MS/MS matching signature ion fragments for each molecule (at least six diagnostic ions; ~0.1 pg limits of detection) and standard curves (r²>0.98 for each lipid mediator and pathway marker).