13 hode d4

Whole frozen lungs and spleens were pulverized using a CP02 automated cryoPREP® (Covaris). Briefly, the frozen tissue was transferred into a tissue tube (Covaris, extra thick) and cooled down by dipping in liquid nitrogen for 60 s. Next, the sample was pulverized with an impact level of four. This step was repeated. 50 mg of powder were immediately extracted with 500 µl ice cold methanol (Roth®) containing 0.1% 3,5-Di-tert-4-butylhydroxytoluene (Sigma-Aldrich) and 500 µl ice cold water. Next, 100 µl internal standard consisting of 12-HETE-d₈ and 13-HODE-d₄ (both 100 ng/ml in acetonitrile; Cayman chemicals) was added. Alkaline hydrolysis was performed by adding 300 µl of sodium hydroxide (10 mol/l; Sigma-Aldrich) followed by an incubation for 30 min at 60 °C. Immediately after hydrolysis, the pH was adjusted to a value of 6 using acetic acid (10 mol/l, VWR). For EDTA plasma samples, an aliquot of 100 µl was hydrolyzed and extracted using 500 µl ice cold methanol with 0.1% 3,5-Di-tert-4-butylhydroxytoluene, 500 µl ice cold water, and 100 µl internal standard solution. Samples were hydrolyzed with sodium hydroxide as described for the tissue material. Afterwards, solid phase extraction was done for all samples types as previously described (Schultz et al., 2019 ).

All eicosanoid standard compounds including the deuterated internal standards 12-HETE-d8, 13-HODE-d4, PG E2-d4, Resolvin D1-d5 and AA-d11 were purchased from Cayman chemicals. Solid phase extraction cartridges Bond Elut Certify II (200 mg, 3 mL) were obtained from Agilent^®. Acetonitrile (99.97%, HPLC-MS grade) was purchased from Th. Geyer^®, methanol from Roth^® and acetic acid (glacial, HPLC grade) from VWR^®. Butylated hydroxytoluene (≥99.0%) and all other chemicals including hexane, ethyl acetate and sodium hydroxide were obtained from Sigma-Aldrich^®.

Lipid mediators and the deuterated standards 12-HETE-d8 and 13-HODE-d4 were purchased from Cayman chemicals. 12-HETE-d8 and 13-HODE-d4 were dissolved in acetonitrile (both 100 ng/ml as internal standard) on ice and aliquots were stored at −80 °C until usage. Acetonitrile (MS grade) was purchased from Th. Geyer^®, methanol from Roth^®, and acetic acid (HPLC grade) from VWR^®. Solid-phase extraction cartridges Bond Elut Certify II (3 mL, 200 mg) were obtained from Agilent^®. BHT and other chemicals, including hexane, ethyl acetate, and sodium hydroxide were purchased from Sigma-Aldrich.