1-arachidonyl-monoglyceride and glyceryl-2-arachidonate

2-Arachidonoylglycerol (2-AG) is the most potent and abundant endocannabinoid that acts as a full agonist at the cannabinoid 1 (CB1) and 2 (CB2) receptors. It serves as a substrate for several serine hydrolases, including monoacylglycerol lipase (MGL), α/β hydrolase domain 6 (ABHD6) and fatty acid amide hydrolase (FAAH). However, 2-AG's rapid conversion to 1-AG (the S stereoisomer) and 3-AG (the R stereoisomer) complicates in vivo signaling. Here, we present the interaction profiles of 2-AG and its isomerization products, 1- and 3-AG, with the endocannabinoid MGL, ABHD6 and FAAH enzymes as well as the CB1 receptor. The 1- and 3-AG enantiomers are less prone to isomerization, and their affinities to endocannabinoid enzymes and potencies at CB1 receptor are quite different compared to 2-AG. Although MGL is the principal hydrolytic enzyme of 2-AG, 3-AG (the R isomer) appears to be the best substrate for hMGL. Contrarily, 1-AG (the S isomer) demonstrates the worst substrate profile, indicating that the stereochemistry of 1(3)-monoacylglycerols is very important for MGL enzyme. On the other hand, both 1- and 3-AG (the sn1 monoacylglycerols) are efficiently hydrolyzed by hABHD6 without preference, while 2-AG (the sn2 monoacylglycerol) has the lowest rate of hydrolysis. FAAH, the principal hydrolytic enzyme for arachidonoylethanolamide (anandamide, AEA), catalyzes the hydrolysis of all three isomers with similar efficiencies. In a functional cAMP assay at CB1 receptor, all three isomers behaved as agonists, with 2-AG being the most potent, followed by 3-AG then 1-AG. The presented data provides stereochemical insights to design chemically stable AG analogs with preferential stability against enzymes of interest.

Topics: Amidohydrolases; Arachidonic Acids; Buffers; Chromatography, High Pressure Liquid; Cyclic AMP; Endocannabinoids; Glycerides; HEK293 Cells; Humans; Hydrolysis; Isomerism; Kinetics; Ligands; Monoacylglycerol Lipases; Receptor, Cannabinoid, CB1; Substrate Specificity

The determination of endocannabinoids and endocannabinoid-like substances in biological human samples is a vibrant field of research with great significance due to postulated relevance of these substances in diseases such as Alzheimer's disease, multiple sclerosis, cancer and cardiovascular diseases. For a possible use as biomarker in early prediction or diagnosis of a disease as well as examination of a successful treatment, the valid determination of the analytes in common accessible human samples, such as plasma or serum, is of great importance. A method for the determination of arachidonoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, 1-arachidonoyl glycerol and 2-arachidonoyl glycerol in human K3EDTA plasma using liquid-liquid-extraction in combination with liquid chromatography-tandem-mass spectrometry has been developed and validated for the quantification of the aforementioned analytes. Particular emphasis was placed on the chromatographic separation of the isomers 1-arachidonoyl glycerol and 2-arachidonoyl glycerol, arachidonoyl ethanolamide and O-arachidonoyl ethanolamine (virodhamine) as well as oleoyl ethanolamide and vaccenic acid ethanolamide. During the validation process, increasing concentrations of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol while storing plasma samples were observed. In-depth investigation of pre-analytical sample handling revealed rising concentrations for both analytes in plasma and for arachidonoyl ethanolamide, oleoyl ethanolamide and palmitoyl ethanolamide in whole blood, dependent on the period and temperature of storage. Prevention of the increase in concentration was not possible, raising the question whether human K3EDTA plasma is suitable for the determination of endocannabinoids and endocannabinoid-like substances. Especially the common practice to calculate the concentration of 2-arachidonoyl glycerol as sum of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol is highly questionable because the concentrations of both analytes increase unequally while storing the plasma samples in the fridge.

Topics: Amides; Anticoagulants; Arachidonic Acids; Chromatography, High Pressure Liquid; Edetic Acid; Endocannabinoids; Ethanolamines; Glycerides; Humans; Liquid-Liquid Extraction; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Specimen Handling; Tandem Mass Spectrometry