12 hete d8

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^®.

Rotenone, ADP, antimycin A, L-glutamic acid, L-malic acid, succinate disodium, fatty acid–free BSA, collagenase type IV, Percoll®, and glucose were purchase from Millipore Sigma Corp. Calcium ionophore A23187, N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide, nordihydroguaiaretic acid (NDGA), ibuprofen, oligomycin, cell culture supplements, thromboxane B₂-d₄, prostaglandin E₂-d₄, 12-HETE-d₈, and 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP) were purchased from Cayman Chemical Company. The lactate dehydrogenase (LDH)-cytotoxicity assay kit, HepatoZYME-SFM, and other chemicals for preparation of buffers for mitochondrial isolation, respiration, and swelling were obtained from ThermoFisher Scientific Inc. A rabbit polyclonal anti-iPLA₂γ antibody was generated in our laboratory as described previously (15 (link)).

A comprehensive analysis method for eicosanoids has been described previously (17 ). The method utilizes an LC-8060 device, consisting of a quantum triple-quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) and a software method package for the simultaneous analysis of 196 products with 18 deuterium internal standards (supplemental Table S2): tetranor-PGEM-d6, 6-keto-PGF1α-d4, TXB2-d4, PGF2α-d4, PGE2-d4, PGD2-d4, PGA2-d4, LTB4-d4, 14,15-DiHET-d11, 15-HETE-d8, 12-HETE-d8, 5-HETE-d8, 11,12-EET-d11, LTC4-d5, LTD4-d5, PAF-d4, OEA-d4, and AA-d8 (Cayman Chemical, Ann Arbor, MI). The eicosanoids were identified by multiple reactions monitoring (MRM) and were analyzed using LabSolutions software (Shimadzu).

Eicosanoids in skin blister fluid were quantified by liquid chromatography coupled to electrospray ionization tandem mass spectrometry as described previously (30 (link)–32 (link)). Briefly, skin fluid samples (typically 50–200 μL) were diluted with methanol-water (15% w/w) up to 3 mL. Internal standards (40 ng PGB₂-d4 and 80 ng 12-HETE-d8; Cayman Chemicals) were then added and resultant solutions acidified to pH 3.0, followed by solid-phase extraction (C18-E cartridges; Phenomenex) to reduce matrix effects and semi-purify the lipid mediators. Eicosanoids were analyzed on a C18 column (Luna 5 μm; Phenomenex) by using a Waters Alliance 2695 HPLC pump coupled to a triple-quadrupole mass spectrometer equipped with an electrospray ionization probe (Quattro Ultima; Waters). Instrument control and data acquisition were performed by using MassLynx 4.0 software (Waters). Multiple-reaction monitoring transitions used were as follows: PGE₂, m/z 351 > 271; PGE₁, m/z 353 > 317; PGE₃, m/z 349 > 269; PGJ₂, m/z 333 > 271; PGD₁, m/z 353 > 317; PGD₂, m/z 351 > 271; PGF_2α, m/z 353 > 193; 13,14-dihydro-15-keto PGE₂, m/z 351 > 333; 13,14-dihydro-15-keto-PGE₁, m/z 353 > 335; 12-HETE, m/z 319 > 179; 11-HETE, m/z 319 > 167; 15-HETE, m/z 319 > 175; 15-hydroxyeicosatrienoic acid, m/z 321 > 221; 9-hydroxyoctadecadienoic acid, m/z 295 > 171; and 13-hydroxyoctadecadienoic acid, m/z 295 > 195.

Several hydroxyeicosatetraenoic acids (HETEs), lipid mediators from the metabolism of ARA, were determined in plasma by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using a method modified from Dasilva et al. [28 (link)]. Erythrocyte-free plasma samples (90 µL) were thawed, diluted in the presence of butylated hydroxytoluene (BHT), and spiked with the internal standard (12HETE-d8), Cayman Chemicals, Ann Arbor, MI, USA). Then, the samples were centrifuged (800 g, 10 min), and the lipids in the supernatants were purified by solid-phase extraction (SPE). The LC-MS/MS analyzer consisted of a Dionex UltiMate 3000 Series chromatograph (Thermo Fisher, Rockford, IL, USA) coupled to a dual-pressure linear ion-trap mass spectrometer LTQ Velos Pro (Thermo Fisher, Rockford, IL, USA) operated in negative electrospray ionization (ESI) mode. A C18-Symmetry 150 × 2.1 mm inner diameter, 3.5 µm column (Waters, Milford, MA, USA) with a C18 4 × 2 mm guard cartridge (Phenomenex, Torrance, CA, USA) were used in the separation step. Samples (10 µL) were eluted with a binary system consisting of 0.02% aqueous formic acid [A] and 0.02% formic acid in methanol [B] under gradient conditions of: 0 min, 60% B; 2 min, 60% B; 12 min, 80% B; 13 min, 80% B; 23 min, 100% B; 25 min, 100% B; and 30 min, 60% B, at a flow rate of 0.2 mL/min

Prostanoids and hydroxy fatty acids were extracted from whole lung tissue as described^{41 (link),42 (link)}. Briefly, lung tissue was homogenised in methanol, then diluted in water to a final concentration of 15 % (v/v) methanol/water. Internal standards were added (20 ng each of PGB₂-d4, 12-HETE-d8, 8,9DHET-d11, 8(9)EET-d11 and 12(13)DiHOME-d4; Cayman Chemical, Ann Arbor, USA). Samples were semi-purified using solid-phase extraction (500 mg C18-E cartridges; Phenomenex, Macclesfield, UK). Analytes were eluted in methyl formate, dried under nitrogen and reconstituted in ethanol, then stored at −20 °C until analysis. Analyte separation was performed using ultraperformance liquid chromatography (Acquity; Waters, Wilmslow, UK) and a C18 column (Acquity UPLC BEH; 1.7 μm; 2.1 × 50 mm; Waters, Wilmslow, UK), before multiple reaction monitoring on a triple quadrupole mass spectrometer with electrospray ionisation (Xevo TQ-S; Waters, Wilmslow, UK). Quantitation was performed using calibration lines constructed with commercially available standards (Cayman Chemicals, Ann Arbor, UK).