1-arachidonyl-monoglyceride and anandamide

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

Anandamide amidohydrolase (AAH) catalyzes the hydrolysis of arachidonylethanolamide (anandamide), an endogenous cannabinoid receptor ligand. To delineate the structural requirements of AAH substrates, rat brain microsomal AAH hydrolysis of a series of anandamide congeners was studied using two reverse-phase high-performance liquid chromatography (RP-HPLC) assays developed in our laboratory. Arachidonamide (1) was found to be the best substrate with an apparent Km of 2.34 mM and a Vmax of 2.89 nmol/min/mg of protein. Although anandamide (2) has a similar Km value, its Vmax is approximately one-half that of arachidonamide. N, N-Bis(2-hydroxyethyl)arachidonamide (3) was not hydrolyzed, suggesting specificity for unsubstituted or mono-N-substituted arachidonamides. Analogues with a methyl group at the 1'-position of the ethanolamido headgroup were also found to have greater resistance to enzymatic turnover and therefore increased metabolic stability. The enzyme exhibited high stereoselectivity as the rate of hydrolysis of (R)-alpha-methanandamide (2.4%) (anandamide = 100%) was about 10-fold lower than that of its (S)-enantiomer (23%). In contrast, (R)-beta-methanandamide was 6-times more susceptible (121%) than the (S)-beta-enantiomer (21%). Interestingly, an inverse correlation was shown between AAH stereoselectivity and the brain cannabinoid receptor affinity as the enantiomers with high receptor affinity displayed low susceptibility to hydrolysis by AAH. Metabolic stability is also imparted to analogues with a short hydrocarbon headgroup as well as to those possessing 2-monomethyl or 2,2-dimethyl substituents. 2-Arachidonylglycerol and racemic 1-arachidonylglycerol were shown to be excellent AAH substrates. To identify AAH inhibitors, hydrolysis of anandamide was also studied in the presence of a select group of cannabimimetics. Of these, (-)-Delta8-THC and SR141716A, a biarylpyrazole CB1 antagonist, were found to inhibit enzymatic activity. These newly defined enzyme recognition parameters should provide a foundation for the rational development of stable, therapeutically useful anandamide analogues with high receptor affinity.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Brain; Cannabinoids; Endocannabinoids; Hydrolysis; Kinetics; Ligands; Microsomes; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, Drug; Stereoisomerism; Substrate Specificity

Macrophages are the primary cellular targets of bacterial lipopolysaccharide (LPS), but the role of macrophage-derived cytokines in LPS-induced septic shock is uncertain. Recent evidence indicates that activation of peripheral CB1 cannabinoid receptors contributes to hemorrhagic hypotension and that macrophage-derived anandamide as well as unidentified platelet-derived substances may be contributing factors. Here we demonstrate that rat platelets contain the endogenous cannabinoid 2-arachidonyl glyceride (2-AG), as identified by reverse phase high-performance liquid chromatography, gas chromatography, and mass spectrometry, and that in vitro exposure of platelets to LPS (200 microg/ml) markedly increases 2-AG levels. LPS-stimulated, but not control, macrophages contain anandamide, which is undetectable in either control or LPS-stimulated platelets. Prolonged hypotension and tachycardia are elicited in urethane-anesthetized rats treated 1) with LPS (15 mg/kg i.v.); 2) with macrophages plus platelets isolated from 3 ml of blood from an LPS-treated donor rat; or 3) with rat macrophages or 4) platelets preincubated in vitro with LPS (200 microg/ml). In all four cases, the hypotension but not the tachycardia is prevented by pretreatment of the recipient rat with the CB1 receptor antagonist SR141716A (3 mg/kg i.v.), which also inhibits the hypotensive response to anandamide or 2-AG. The hypotension elicited by LPS-treated macrophages or platelets remains unchanged in the absence of sympathetic tone or after blockade of nitric oxide synthase. These findings indicate that platelets and macrophages generate different endogenous cannabinoids, and that both 2-AG and anandamide may be paracrine mediators of endotoxin-induced hypotension via activation of vascular CB1 receptors.

Topics: Animals; Arachidonic Acids; Blood Platelets; Cannabinoids; Cells, Cultured; Dronabinol; Endocannabinoids; Glycerides; Hypotension; Lipopolysaccharides; Macrophages; Male; Nitric Oxide; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant