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 microg/L (SD 0.3 microg/L); 2.8 microg/L (SD 0.5 microg/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 microg/L (SD 1.8 microg/L) was found. In cell-free plasma, no increase in anandamide concentrations was found.


CONCLUSIONS
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.
Vogeser
Michael
M
Institute of Clinical Chemistry, Hospital of the University of Munich, Munich, Germany. vincent.sapin@u-clermont1.fr
Hauer
Daniela
D
Christina Azad
Shahnaz
S
Huber
Erasmus
E
Storr
Martin
M
Schelling
Gustav
G
D001095:Arachidonic Acids / Q000097:blood* / Q000378:metabolism / Q000592:standards; D001773:Blood Cells / Q000378:metabolism; D001793:Blood Preservation / Q000379:methods; D001800:Blood Specimen Collection / Q000379:methods / Q000592:standards*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D063388:Endocannabinoids; D006801:Humans; D053284:Polyunsaturated Alkamides; D013696:Temperature; D013997:Time Factors
D001095:Arachidonic Acids; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; C078814:anandamide