Abstract
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°C, room temperature, or 37°C, respectively, for up to 120min 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°C for 60min before centrifugation [immediately centrifuged, 1.3μg/L (SD 0.3μg/L); 2.8μg/L (SD 0.5μg/L) after storage for 60min; n=12). After storage of heparinized whole-blood samples for 120min at 37°C, a mean plasma anandamide concentration of 11.9μg/L (SD 1.8μg/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.
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