Aea d4

Standards of AEA, 2-AG, PEA, and OEA were obtained from Tocris (Bristol, United Kingdom), AEA-d₄, 2-AG-d₅, PEA-d₄ and OEA-d₄ were from Cayman Chemical (Ann Arbor, MI, USA), acetonitrile and chloroform were from Merck (Darmstadt, Germany), while methanol and formic acid were from POCh (Katowice, Poland). All the stock solutions of the standards, excluding 2-AG and 2-AG-d₅, were prepared in ethanol, whereas 2-AG and 2-AG-d₅ stock solutions were prepared in acetonitrile (concentration 1 mg/mL). All the stock solutions were stored at −80 °C. Further dilutions were prepared using acetonitrile.

Anandamide (AEA), 2-arachidonoyl glycerol (2-AG), arachidonic acid (AA), and their deuterated analogues AEA-d4, 2-AG-d5, and AA-d8 were obtained from Cayman Chemicals (Ann Arbor, MI, USA). Water, acetonitrile (ACN), formic acid (FA), ethyl acetate, and hexane (all from Fluka LC-MS grade) were obtained from Sigma-Aldrich.

The POA region of hypothalamus and pituitary gland were used for analysis of AEA concentrations by using liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Briefly, tissues were weighed and spiked with 2 μL of the internal standard AEA-d4 (2.8 μM; Cayman Chemicals, Ann Arbor, MI, USA) and submitted to a two cycle of 20 s with 5 s in-between homogenization with 1 g of zirconium beads and 500 μL of toluene performed in a Precellys 24 homogenizer (Bertin Technologies, France). After centrifugation at 5000× g for 5 min at 4 °C, the upper phase was collected, dried at constant nitrogen stream, and then reconstituted in 100 μL of phase B (96% methanol (MeOH) and 4% ammonium acetate (AA) 2 mM) and transferred to an HPLC vial ready for UPLC-MS/MS analysis.
Plasma (500 μL) was also spiked with deuterated internal standard solution and 0.4 g of MgSO₄, 0.1 g NaCl and 500 μL of ethyl acetate were added to each sample followed by a vortex homogenization and centrifugation at 3500× g for 10 min at room temperature. The upper phase was collected, dried at constant nitrogen stream, reconstituted in phase B following UPLC–MS/MS analysis. Chromatographic and detection details were reported in a previous work [10 (link)].

All chemical solvents and standards were of analytical grade. Standards of AEA, 2-AG, OEA, and PEA were obtained from Tocris (Bristol, United Kingdom), AEA-d₄, 2-AG-d₅, OEA-d₄, and PEA-d₄ from Cayman Chemical (USA), acetonitrile and chloroform from Merck (Darmstadt, Germany), methanol and formic acid from POCh (Katowice, Poland). Standards stock solutions were prepared in ethanol, except from 2-AG and 2-AG-d₅ which were prepared in acetonitrile. All stock solutions were stored at −80 °C. Further dilutions were carried out appropriately in acetonitrile.

All chemical solvents and standards were of analytical grade. Standards of AEA and 2-AG were obtained from Tocris (Bristol, United Kingdom), AEA-d₄ and 2-AG-d₅ from Cayman Chemical (USA), acetonitrile and chloroform from Merck (Darmstadt, Germany), and methanol and formic acid from POCh (Katowice, Poland). Standard stock solutions were prepared in ethanol (AEA and AEA-d₄) or acetonitrile (2-AG and 2-AG-d₅). All stock solutions were stored at −80 °C. Further dilutions were carried out appropriately in acetonitrile.

FAAH activity (reported in counts per minute, cpm) was quantified from atrial and ventricular homogenates at 37 °C for 30 min in 0.5 mL Tris buffer (50 mM, pH 7.5) containing fatty acid-free bovine serum albumin (BSA) (0.05%, w/v), 50 μg of protein from brain homogenates, 10 μM anandamide and [³H]-anandamide (10000 disintegrations per minute), following previously described procedures28 (link)30 (link). Fatty acid ethanolamides AEA, OEA and PEA were extracted from atrial and ventricular homogenates by organic solvent (ice-cold acetonitrile) addition and quantified by HPLC/MS/MS30 (link). The HPLC/MS/MS analytical standards AEA, OEA, PEA and the deuterated internal standards, AEA-d₄, PEA-d₄, OEA-d₄ were purchased from Cayman Chemicals (Ann Arbor, Michigan) as stock solutions in ethanol.