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°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. Clin Chem Lab Med 2008;46:1289–95.

[1]  B. Gorzalka,et al.  Serum endocannabinoid content is altered in females with depressive disorders: a preliminary report. , 2008, Pharmacopsychiatry.

[2]  Z. Vogel,et al.  Structural requirements for binding of anandamide-type compounds to the brain cannabinoid receptor. , 1997, Journal of medicinal chemistry.

[3]  J. Klosterkötter,et al.  Determination of anandamide and other fatty acyl ethanolamides in human serum by electrospray tandem mass spectrometry. , 2007, Analytical biochemistry.

[4]  K. Mackie,et al.  The emerging functions of endocannabinoid signaling during CNS development. , 2007, Trends in pharmacological sciences.

[5]  H. Schuel.  Tuning the oviduct to the anandamide tone. , 2006, The Journal of clinical investigation.

[6]  H. Zhang,et al.  Fatty acid amide hydrolase deficiency limits early pregnancy events. , 2006, The Journal of clinical investigation.

[7]  A. Makriyannis,et al.  Targeted modulators of the endogenous cannabinoid system: Future medications to treat addiction disorders and obesity , 2007, Current psychiatry reports.

[8]  A. Saghatelian,et al.  A FAAH-regulated class of N-acyl taurines that activates TRP ion channels. , 2006, Biochemistry.

[9]  D. Gibson,et al.  Isolation and structure of a brain constituent that binds to the cannabinoid receptor. , 1992, Science.

[10]  S. Ben-Shabat,et al.  An endogenous cannabinoid (2-AG) is neuroprotective after brain injury , 2001, Nature.

[11]  S. Dey,et al.  Jekyll and Hyde : Two Faces of Cannabinoid Signaling in Male and Female Fertility , 2006 .

[12]  R. Pertwee,et al.  Influence of the degree of unsaturation of the acyl side chain upon the interaction of analogues of 1-arachidonoylglycerol with monoacylglycerol lipase and fatty acid amide hydrolase. , 2005, Biochemical and biophysical research communications.

[13]  H. Homma,et al.  Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric determination of anandamide and its analogs in rat brain and peripheral tissues. , 1997, Journal of chromatography. B, Biomedical sciences and applications.

[14]  M. Gómez,et al.  Effects on development. , 2005, Handbook of experimental pharmacology.

[15]  H. Bradshaw,et al.  The expanding field of cannabimimetic and related lipid mediators , 2005, British journal of pharmacology.

[16]  M. Mitchell,et al.  Characterization of the endocannabinoid system in early human pregnancy. , 2004, The Journal of clinical endocrinology and metabolism.

[17]  M. Yamakuchi,et al.  Endogenous Cannabinoids are Candidates for Lipid Mediators of Bone Cement Implantation Syndrome , 2004, Shock.

[18]  Alexandros Makriyannis,et al.  Pharmacotherapeutic targeting of the endocannabinoid signaling system: Drugs for obesity and the metabolic syndrome , 2008, Physiology & Behavior.

[19]  N. Ueda,et al.  Purification and Characterization of an Acid Amidase Selective for N-Palmitoylethanolamine, a Putative Endogenous Anti-inflammatory Substance* , 2001, The Journal of Biological Chemistry.

[20]  Terry J. Smith,et al.  Cannabinoids and the human uterus during pregnancy. , 2004, American journal of obstetrics and gynecology.

[21]  Y. Ben-Ari,et al.  Altering cannabinoid signaling during development disrupts neuronal activity. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[22]  P. Sparling,et al.  Exercise activates the endocannabinoid system , 2003, Neuroreport.

[23]  F. Fezza,et al.  New insights into endocannabinoid degradation and its therapeutic potential. , 2006, Mini reviews in medicinal chemistry.

[24]  A. Makriyannis,et al.  Endocannabinoid metabolomics: A novel liquid chromatography-mass spectrometry reagent for fatty acid analysis , 2006, The AAPS Journal.

[25]  Y. Tanifuji,et al.  Simultaneous determination of endocannabinoids (arachidonylethanolamide and 2-arachidonylglycerol) and isoprostane (8-epiprostaglandin F2alpha) by gas chromatography-mass spectrometry-selected ion monitoring for medical samples. , 2003, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[26]  M. Randall.  Endocannabinoids and the haematological system , 2007, British journal of pharmacology.

[27]  S. Petrosino,et al.  Endocannabinoids and the regulation of their levels in health and disease , 2007, Current opinion in lipidology.

[28]  D. Piomelli,et al.  Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry. , 2000, Analytical biochemistry.

[29]  V. Marzo,et al.  Circulating endocannabinoid levels, abdominal adiposity and related cardiometabolic risk factors in obese men , 2007, International Journal of Obesity.

[30]  D. Barrett,et al.  Quantitative profiling of endocannabinoids and related compounds in rat brain using liquid chromatography-tandem electrospray ionization mass spectrometry. , 2007, Analytical biochemistry.

[31]  N. Ueda,et al.  Molecular Characterization of N-Acylethanolamine-hydrolyzing Acid Amidase, a Novel Member of the Choloylglycine Hydrolase Family with Structural and Functional Similarity to Acid Ceramidase* , 2005, Journal of Biological Chemistry.

[32]  M. Cooke,et al.  Plasma levels of the endocannabinoid anandamide in women--a potential role in pregnancy maintenance and labor? , 2004, The Journal of clinical endocrinology and metabolism.

[33]  T. Fukushima,et al.  Sensitive determination of anandamide in rat brain utilizing a coupled-column HPLC with fluorimetric detection. , 2000, Biomedical chromatography : BMC.

[34]  J. Folch,et al.  A simple method for the isolation and purification of total lipides from animal tissues. , 1957, The Journal of biological chemistry.

[35]  N. Ueda,et al.  N-cyclohexanecarbonylpentadecylamine: a selective inhibitor of the acid amidase hydrolysing N-acylethanolamines, as a tool to distinguish acid amidase from fatty acid amide hydrolase. , 2004, The Biochemical journal.

[36]  A. Makriyannis,et al.  AM1241, a cannabinoid CB2 receptor selective compound, delays disease progression in a mouse model of amyotrophic lateral sclerosis. , 2006, European journal of pharmacology.

[37]  A. Makriyannis,et al.  Molecular probes for the cannabinoid receptors. , 2000, Chemistry and physics of lipids.

[38]  K. Waku,et al.  Rapid generation of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, in rat brain after decapitation , 2001, Neuroscience Letters.

[39]  M. Maccarrone.  CB2 receptors in reproduction , 2008, British journal of pharmacology.

[40]  J. Turk,et al.  Isotope Dilution Mass Spectrometric Measurements Indicate That Arachidonylethanolamide, the Proposed Endogenous Ligand of the Cannabinoid Receptor, Accumulates in Rat Brain Tissue Post Mortem but Is Contained at Low Levels in or Is Absent from Fresh Tissue* , 1996, The Journal of Biological Chemistry.

[41]  L. Marnett,et al.  Differential regulation of endocannabinoid synthesis and degradation in the uterus during embryo implantation. , 2007, Prostaglandins & other lipid mediators.

[42]  B. Bahr,et al.  Quantitative method for the profiling of the endocannabinoid metabolome by LC-atmospheric pressure chemical ionization-MS. , 2007, Analytical chemistry.

[43]  M. Storr,et al.  Release of anandamide from blood cells , 2006, Clinical chemistry and laboratory medicine.