2.6. Examination of Endocannabinoid System

Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) level was measured using ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) [29]. Octadeuterated endocannabinoids: AEA-d8 and 2-AG-d8 were used as internal standards, and all cannabinoids from samples were isolated using solid-phase extraction (SPE). Analysis was performed using an Agilent 1290 UPLC system with a Zorbax Extend C18 column (2.1 mm × 150 mm, 1.8 mm, Agilent, Santa Clara, CA, USA) and interfaced with an Agilent 6460 triple quadrupole mass spectrometer with electrospray ionization source (ESI). The samples were analyzed in positive-ion mode using multiple reaction monitoring (MRM). Transition of the precursor to the product ion was: m/z 348.3→62.1 for AEA, and m/z 379.3→287.2 for 2-AG. Endocannabinoids concentrations were expressed as femtomoles normalized per milligram of protein.

FAAH (fatty acid amide hydrolase) (EC.3.5.1.99) activity was determined according to the Siegmund procedure [30] based on the spectrophotometric measurement of m-nitroaniline (m-NA) releasing from decanoyl m-nitroaniline. Enzyme activity was expressed as the amount of enzyme metabolizing 1 pmol of substrate per minute normalized per milligram of protein.

MAGL (monoacylglycerol lipase) (EC.3.1.1.23) activity was analyzed basing on spectrophotometric determination of 5′-thio-2-nitrobenzoic acid releasing from arachidonoyl-1-thio-glycerol [31]. Enzyme activity was expressed as the amount of enzyme metabolizing 1 pmol of substrate per minute normalized per milligram of protein.

Endocannabinoid receptors CB1 and CB2 expression were analyzed using Western blot technique described below.