Release of anandamide from blood cells

Background: Endogenous ligands of cannabinoid receptors ( endocannabinoids), in particular anandamide ( arachidonylethanolamide), have been recognized as being of crucial importance in a variety of physiological functions. Plasma concentrations of anandamide have been measured in a number of investigations; however, discrepant data on "normal'' anandamide plasma concentrations were reported. Since this might be caused by pre-analytical variables, we investigated the impact of different sample handling conditions on measured plasma anandamide concentrations. Methods: Blood samples were taken from healthy volunteers in EDTA- or heparin-containing tubes; whole blood samples were kept at +4 degrees C, room temperature, or 37 degrees C, respectively, for up to 120 min before obtaining plasma by centrifugation. Plasma anandamide concentrations were measured by an isotope-dilution liquid chromatography tandem mass spectrometry ( LC-MS/MS) method. Results: A marked time- and temperature-dependent increase in plasma anandamide concentrations ex vivo was observed in both EDTA- and heparin-containing tubes. Mean anandamide concentrations approximately doubled when EDTA samples were kept at 4 degrees C for 60 min before centrifugation {[}immediately centrifuged, 1.3 mg/L ( SD 0.3 mg/L); 2.8 mg/L ( SD 0.5 mg/L) after storage for 60 min; n=12). After storage of heparinized whole-blood samples for 120 min at 37 degrees C, a mean plasma anandamide concentration of 11.9 mg/L ( SD 1.8 mg/L) was found. In cell-free plasma, no increase in anandamide concentrations was found. Conclusion: Anandamide is released from blood cells ex vivo at a very high rate; therefore, strictly standardized pre-analytical protocols have to be applied for plasma anandamide determination