title: Comprehensive profiling of the human circulating endocannabinoid metabolome: clinical sampling and sample storage parameters.
abstract: 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.
authors.affiliation: Center for Drug Discovery, Northeastern University, Boston, MA 02115, USA. j.wood@neu.edu
authors.forename: JodiAnne T
authors.initials: JT
authors.lastname: Wood
authors.forename: John S
authors.initials: JS
authors.lastname: Williams
authors.forename: Lakshmipathi
authors.initials: L
authors.lastname: Pandarinathan
authors.forename: Amber
authors.initials: A
authors.lastname: Courville
authors.forename: Melissa R
authors.initials: MR
authors.lastname: Keplinger
authors.forename: David R
authors.initials: DR
authors.lastname: Janero
authors.forename: Paul
authors.initials: P
authors.lastname: Vouros
authors.forename: Alexandros
authors.initials: A
authors.lastname: Makriyannis
authors.forename: Carol J
authors.initials: CJ
authors.lastname: Lammi-Keefe
mesh_terms: 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
chemical_list: D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids