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.