2h 2 oea

Unless otherwise stated, all compounds used in this study were purchased from Sigma-Aldrich Company Ltd. (Poole, Dorset, United Kingdom). [³H]-PEA was purchased from Hartmann Analytic GmbH (Germany). [¹⁴C]-AEA was purchased from ARC (St. Louis, MO, United States). Unlabelled AEA was purchased from Tocris Bioscience (Avonmouth, Bristol, United Kingdom). Ultramicronized PEA and PEA-OXA were obtained from Epitech Group SpA, (Saccolongo, Italy). The immortalized HEK-293 cells over-expressing HEK-NAAA were obtained as reported previously (Saturnino et al., 2010 (link)). HEK-293 wild-type (HEK-WT) cells were purchased from LGC Standards (Milano, Italy). Deuterated standards - [²H]₄-PEA, [²H]₈-AEA, [²H]₅-2-AG and [²H]₂-OEA - were purchased from Cayman Chemical (Cabru, Arcore, Italy). All solutions used for in vivo infusions were prepared using non-pyrogenic saline (0.9% wt/vol NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, United Kingdom).

The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) and endocannabinoid-related molecules N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA) were extracted from tissues and then purified and quantified as previously described [27 (link),34 (link)]. First, the tissues were dounce-homogenized and extracted with chloroform/methanol/Tris-HCl 50 mM, pH 7.5 (2:1:1, v/v) containing internal deuterated standards for AEA, 2-AG, PEA, and OEA quantification by isotope dilution (5 pmol of [2H]8 AEA, 50 pmol of [2H]5 2-AG, [2H]4PEA, [2H]2 OEA (Cayman Chemicals, MI, USA). The lipid-containing organic phase was dried down, weighed, and pre-purified by open bed chromatography on silica gel. The fractions were obtained by eluting the column with 99:1, 90:10 and 50:50 (v/v) chloroform/methanol. The 90:10 fraction was used for AEA, 2-AG, PEA, and OEA quantification by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) and using selected ion monitoring at M + 1 values for the four compounds and their deuterated homologues, as previously described [19 (link),28 (link)].

The acetonitrile, methanol, chloroform, n-hexane, ethanol, acetic acid, and fatty acids standards were HPLC grade, and like deferoxamine mesylate, were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Ascorbic acid, potassium hydroxide, and hydrochloric acid were purchased from Carlo Erba (Milano, Italy). Internal deuterated standards for the AEA, 2-AG, and OEA quantification by isotope dilution ([²H]₈ AEA, [²H]₅ 2-AG, [²H]₂ OEA) were purchased from Cayman Chemicals (MI, USA).