Comprehensive profiling of the human circulating endocannabinoid metabolome: clinical sampling and sample storage parameters.
BACKGROUND
Endogenous cannabinoid-receptor ligands (endocannabinoids) and over a dozen related metabolites now comprise the "endocannabinoid metabolome". The diverse (patho)physiological roles of endocannabinoids, the predictive/diagnostic utility of systemic endocannabinoid levels, and the growing interest in endocannabinoid-related pharmacotherapeutics mandate a valid clinical protocol for processing human blood that does not jeopardize profiling of the circulating endocannabinoid metabolome.


METHODS
We systematically evaluated the potential effect of pre-analytical variables associated with phlebotomy and sample handling/work-up on the human-blood endocannabinoid metabolome as quantified by state-of-the-art liquid chromatography-mass spectrometry.


RESULTS
Neither subject posture during phlebotomy nor moderate activity beforehand influenced the blood levels of the 15 endocannabinoid-system lipids quantified. Storage of fresh blood at 4 degrees C selectively enhanced ethanolamide concentrations artifactually without affecting monoglycerides and nonesterified fatty acids, such as arachidonic acid. In marked contrast, ethanolamides and monoglycerides remained stable through three plasma freeze/thaw cycles, whereas plasma arachidonic acid content increased, probably a reflection of ongoing metabolism.


CONCLUSIONS
Class- and compound-selective pre-analytical influences on circulating human endocannabinoid levels necessitate immediate plasma preparation from fresh blood and prompt plasma apportioning and snap-freezing. Repeated plasma thawing and refreezing should be avoided. This protocol ensures sample integrity for evaluating the circulating endocannabinoid metabolome in the clinical setting.
Wood
JodiAnne T
JT
Center for Drug Discovery, Northeastern University, Boston, MA 02115, USA. j.wood@neu.edu
Williams
John S
JS
Pandarinathan
Lakshmipathi
L
Courville
Amber
A
Keplinger
Melissa R
MR
Janero
David R
DR
Vouros
Paul
P
Makriyannis
Alexandros
A
Lammi-Keefe
Carol J
CJ
D000293:Adolescent; D000328:Adult; D000818:Animals; D001775:Blood Circulation; D001800:Blood Specimen Collection / Q000379:methods*; D063385:Cannabinoid Receptor Modulators / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D063388:Endocannabinoids; D005260:Female; D006801:Humans; D009043:Motor Activity; D011187:Posture; D012016:Reference Values; D012680:Sensitivity and Specificity; D013696:Temperature
D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids