Aea d8

The sample extraction of AEA and 2-AG was adapted from published methods [103 (link)]. Twenty-seven of mouse brain tissue samples were weighed prior to extraction. One milliliter of 1:1 cold methanol (MeOH) (liquid chromatography-mass spectrometry (LC-MS) grade; Sigma-Aldrich, St. Louis, MO, USA) to acetonitrile (high performance liquid chromatography grade; Sigma-Aldrich) were added to each sample, and the sample was spiked with 2.5 fmol/μL of d₈-AEA (Cayman Chemical, Ann Arbor, MI, USA) and 0.25 ρmol/μL of d₈-2AG (Cayman Chemical) to account for any sample loss during the extraction process. Samples were then homogenized using a Branson Ultrasonics Sonifier S-250A homogenizer (Thermo Fisher Scientific, Waltham, MA, USA) for 20 s. After homogenization, sample homogenates were centrifuged for 20 min at 14,000× g at 4 °C. Supernatants were then transferred to a new microcentrifuge tube and evaporated under N₂ gas at room temperature. Then, 200 μL of 30:70 LC-MS grade MeOH to H₂O was added to resuspend each sample before loading them to a SPE cartridge. After samples were loaded on the SPE cartridge, 2 × 250 μL of LC-MS grade H₂O were added to remove salt and other polar materials. After the washing steps, the analytes were eluted with 3 × 200 μL of 100% LC-MS grade MeOH and dried under N₂ gas at room temperature before storage at −80 °C until use.

LC/MS was conducted as in [32 (link)]. Briefly, samples were homogenized in 2 ml of acetonitrile with 5 pmol d8-AEA (Cayman Chemical Company, Ann Arbor, Michigan, USA, #390050) in a borosilicate glass tube with a glass rod. Samples were sonicated, incubated overnight at −20 °C, and centrifuged at 1500 × g. Supernatants containing lipids were isolated, evaporated with nitrogen gas, washed with acetonitrile and evaporated with nitrogen gas again. Final reconstitution for liquid chromatography/tandem mass spectrometry was in 200 μl of acetonitrile.

AA-5HT, 2-AG, AM-251, AM-630, OEA, PEA and URB597 were obtained from Biomol GmbH (Hamburg, Germany). AEA was purchased from Enzo Life Sciences GmbH (Lörrach, Germany). Dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), glycerol, glycine, hydrogen peroxide (H₂O₂), sodium chloride (NaCl), Tris hydrocloride (Tris-HCl) and Tris ultrapure were obtained from AppliChem (Darmstadt, Germany). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was bought from Ferak (Berlin, Germany). Aprotinine, bovine serum albumin, capsazepine, HCl, leupeptine, luminal, orthovanadate, para-coumaric acid, para-formaldehyde, phenylmethylsulfonyl fluoride (PMSF) and Triton^® X-100 were from Sigma (Taufkirchen, Germany). Dulbecco's modified eagle medium (DMEM) with 4.5 g/l glucose and with L-glutamine was provided by Lonza (Cologne, Germany). Fetal calf serum (FCS) and penicillin-streptomycin were purchased from Invitrogen (Darmstadt, Germany) and phosphate-buffered saline (PBS) was provided by PAN Biotech (Aidenbach, Germany). Milk powder was obtained from Bio-Rad Laboratories GmbH (Munich, Germany). Acrylamide (Rotiphorese^® Gel 30) was obtained from Carl Roth GmbH (Karlsruhe, Germany). Pure standards for LC-MS analysis of AEA, 2-AG, OEA, PEA and AEA-d8 were purchased from Cayman Chemical (Ann Arbor, MI, USA).

Plasma AEA was extracted and performed as previously described [15 (link)]. Briefly, 4 mL blood was collected in EDTA tube and placed on ice. After centrifugation at 1200 g/30 min at 22 °C, 2 mL of plasma was transferred to a glass Kimble scintillation vial (Fisher Scientific, Loughborough, UK) and added 2.5 pmol of deuterium-labelled AEA (AEA-d8; Cayman Chemicals, Ann Arbor, MI, USA). Plasma proteins were mixed with an equal volume of ice-cold acetone followed by centrifugation at 1200 g/10 min at 22 °C. Then the supernatant was transferred to a clean Kimble vial and used in the subsequent steps according to the instructions. The reconstituted mixture was performed by the ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) as described [16 (link)].