N oleoylethanolamine

We injected 4 μL [¹⁴C₁₆]ceramide (55 mCi/mmol, #ARC-0831, ARC) into the lumen of the trachea of anesthetized mice. Before injection, [¹⁴C₁₆]ceramide was dried, resuspended in 0.05% octylglucopyranoside in 150 mM sodium acetate (pH 7.4 or 5.0), and bath sonicated for 10 min. After 20 min, mice were sacrificed, the trachea was carefully removed and extracted in H₂O and CHCl₃:CH₃OH:HCl (100:100:1, v/v/v). The lower phase was dried, and samples were resuspended in CHCl₃:CH₃OH (1:1, v/v), separated by TLC with CHCl₃:CH₃OH:NH₄OH (90:20:0.5, v/v/v), and analyzed with a Fuji Imager. Injection of 4 μL of the acid ceramidase inhibitors N-oleoylethanolamine (0.5 mM, #00383, Sigma) or carmofur (1 μM, #14243, Cayman Chemicals) onto the trachea was performed 15 min before the injection of [¹⁴C₁₆]-ceramide and served to demonstrate the contribution of acid ceramidase to the observed ceramide consumption.

Commercial BM powder was kindly donated by Reny-Picot (Asturias, Spain). The sample was kept frozen at −35 °C until the analysis.
All solvents were at least HPLC grade and MS grade when available. Chloroform, hexane, methanol, isooctane, isopropanol, ammonium hydroxide, arsenic formate, and acetonitrile were purchased from LABSCAN (Dublin, Ireland). methanol (F.C.C.) ADITIO for industrial food use, sea sand, potassium hydroxide, and sodium carbonate were from PANREAC (Barcelona, Spain). Formic acid (98%) and triethylamine (99.5%), the TAG standards trinanoin and tridecanoin, the FFA standards pelargonic (C9), tridecanoic (C13), myristic (C14), palmitic (C16), estearic (C18), arachidonic (AA, 20:4), eicosapentaenoic (EPA, 20:5), and docosahexaenoic (DHA, 22:6) acids, the sterols 5α-cholestane, cholesterol (CHOL), cholesterol ester (CE), desmosterol, campesterol, β-sitosterol and lanosterol, monostearin, diolein and tripalmitin, as well as PI, PS, PE, SM, PC, and N-oleoylethanolamine standards were from Sigma-Aldrich (St. Louis, MO, USA). Reference samples with known composition as butter fat BCR-164 and BCR-519 (EU Com 121 missions; Brussels, Belgium) were from Fedelco Inc. (Madrid, Spain).