2h 5 2 ag

Animals were anesthetized with isoflurane at time of tissue harvest (1,500–1,700 h) following ad libitum food and water access. Blood was collected by cardiac puncture and deposited into vacutainers containing EDTA; plasma was collected as supernatant following 10 min centrifugation at 1,500 g (kept at 4°C). Jejunum was quickly removed and washed in phosphate-buffered saline (PBS), opened longitudinally on a stainless steel tray on ice, and contents were removed. Jejunum mucosa was isolated using glass slides to scrape the epithelial layer and was snap-frozen in liquid N₂. Samples were stored at −80°C pending analysis. Frozen tissues were weighed and then homogenized in 1 ml methanol solution containing 500 pmol [²H₅]-2-AG (Cayman Chemicals, Ann Arbor, MI) as an internal standard. Lipids were extracted as previously described (Argueta and DiPatrizio, 2017 (link)) and resuspended in 0.1 ml methanol:chloroform (9:1) and analyzed via ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).

Blood was harvested from vaccinated mice by cardiac puncture and placed into tubes coated with EDTA dipotassium salt (Sarstedt, Nümbrecht, Germany). Blood was separated by centrifugation at 2000 g for 5 minutes at 20 °C and serum collected and stored at −80 °C. Inguinal, parotid, axillary, accessory axillary and submandibular lymph nodes were harvested and stored at −80 °C. Bone marrow dendritic cells and macrophages were harvested by gentle scrapping after rinsing twice with cold PBS. Frozen tissue or cells were homogenized in 1.0 mL of methanol solution containing the internal standard, [²H₅] 2-AG and [²H₄]-AEA (Cayman Chemical, Ann Arbor, MI, USA). Lipids were extracted with chloroform (2 mL) and washed with water (1 mL). Lipids were similarly extracted from serum samples, with the exception of a 0.9% saline wash replacing water (0.1 mL serum at the expense of saline). Organic phases were collected and separated by open-bed silica gel column chromatography as previously described (DiPatrizio et al., 2011, PNAS). Eluate was gently dried under N₂ stream (99.998% pure) and resuspended in 0.1 mL of methanol:chloroform (9:1), with 1 μL injection for ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis.

Lipids were extracted from cell suspensions and 2-AG, anandamide, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) pre-purified and quantified using LC-APCI-MS as described previously^{51 (link)}. First, cells were Dounce-homogenized and extracted with chloroform/methanol/Tris-HCl 50 mM pH 7.5 (2:1:1, v/v) containing internal deuterated standards (5 pmol) for anandamide, 2-AG, PEA and OEA quantification by isotope dilution ([²H]₈ AEA, [²H]₅ 2-AG, [²H]₄ PEA, [²H]₄ OEA (Cayman Chemicals, MI, USA). The lipid-containing organic phase was dried down, weighed and pre-purified by open-bed chromatography on silica gel. 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 anandamide, 2-AG, PEA and OEA quantification by LC-APCI-MS, as previously described and using selected ion monitoring at M + 1 values for the four compounds and their deuterated homologues, as described previously^{52 (link)}.

The acetonitrile (CH₃CN), methanol (CH₃OH), chloroform (CHCl₃), n-hexane (C₆H₁₄), ethanol (C₂H₅OH), and acetic acid (CH₃COOH) were HPLC grade and purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All standards of fatty acid standards were purchased from the same company.
Ascorbic acid, potassium hydroxide (KOH), and hydrochloric acid (HCl) were purchased from Carlo Erba, Milano, Italy. Deferoxamine mesylate was purchased from CIBA-Geigy (Basel, Switzerland). Internal deuterated standards for the AEA, 2-AG, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) quantification by isotope dilution ([2H]8AEA, [2H]52AG, [2H]4 PEA, and [2H]4 OEA) were purchased from Cayman Chemicals (MI, USA).
The total lipids were extracted by the method of Folch^{63 (link)}. Briefly, samples of human plasma (1 ml) or erythrocytes were each homogenized into a 2:1 chloroform-methanol solution containing 2 μg of vitamin E and deuterated AEA (200 ng), 2-AG (300 ng), OEA (200 ng), and PEA (100 ng).
An aliquot of the plasma lipid extract was used for HPLC separation to determine the total free fatty acids as described previously^{64 (link)}. The total lipid quantification was performed by the method of Chiang⁶⁵ .

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)].

Skin biopsies were homogenized in chloroform/methanol/Tris–HCl 50 mM pH 7.4 (2 : 1 : 1, v/v) containing 10 pmol of [2H]₈-AEA, [2H]₄-PEA and [2H]₄-OEA, and 50 pmol of [2H]₅-2-AG as internal deuterated standards (purchased from Cayman Chemicals, Ann Arbor, MI). The lipid-containing organic phase was dried down, weighed and prepurified by open-bed chromatography on silica gel. Fractions obtained by eluting the column with 9 : 1 (by vol) chloroform/methanol were analysed by liquid chromatography– atmospheric pressure chemical ionization–mass spectrometry (LC– APCI–MS) by using a Shimadzu HPLC apparatus (LC-10ADVP) coupled to a Shimadzu (LCMS-2010) quadrupole MS via a Shimadzu APCI interface. LC–APCI–MS analyses were carried out in the selected ion monitoring mode, using m/z values of 356 and 348 (molecular ions +1 for deuterated and undeuterated AEA), 304 and 300 (molecular ions +1 for deuterated and undeuterated PEA), 330 and 326 (molecular ions +1 for deuterated and undeuterated OEA), and 384.35 and 379.35 (molecular ions +1 for deuterated and undeuterated 2-AG). AEA, OEA, PEA and 2-AG concentrations were calculated by isotope dilution and are expressed as pmol per g of wet tissue weight. The concentrations of 2-AG were obtained by adding up to the amounts of the 2-isomer also those of the 1(3)-isomer, which mostly originates from the isomerization of the former during work-up.