Quantitative Method for the Profiling of the Endocannabinoid Metabolome by LC-Atmospheric Pressure Chemical Ionization-MS

Abstract

The endocannabinoid system's biological significance continues to grow as novel endocannabinoid metabolites are discovered. Accordingly, a myopic view of the system that focuses solely on one or two endocannabinoids, such as anandamide or 2-arachidonoyl glycerol, is insufficient to describe the biological responses to perturbations of the system. Rather, the endocannabinoid metabolome as a whole must be analyzed. The work described here is based on liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry. This method has been validated to quantify, in a single chromatographic run, the levels of 15 known or suspected metabolites of the endocannabinoid system in the rat brain and is applicable to other biological matrixes. We have obtained an endocannabinoid profile specifically for the frontal cortex of the rat brain and have determined anandamide level differences following the administration of the fatty acid amide hydrolase inhibitor AM374.

Dietary docosahexaenoic acid supplementation alters select physiological endocannabinoid-system metabolites in brain and plasma

“…Plasma, immediately isolated by centrifugation, and brain samples were fl ash frozen in liquid nitrogen and stored at Ϫ 80°C until analysis. This method of euthanization, plasma/tissue collection, sample processing, and storage prevents artifactual changes in the lipid analytes, as reported for AEA and AG and validated by our laboratory for the other metabolites quantifi ed ( 35,36 ).…”

“…Ethanol (200 proof), HPLC-grade acetone, and PBS powder were from Sigma-Aldrich (St. Louis, MO). Other reagents, including deuterated standards for LC-MS/MS analysis, were purchased from commerc ial sources or synthesized in-house, as previously described ( 36 ).…”

“…As previously detailed ( 35,36 ), frozen plasma was rapidly thawed, and a known amount of deuterated internal standard mixture was added. Plasma proteins were precipitated with one volume ice-cold acetone and centrifugation (20,000 g , 5 min, 4°C).…”

“…The impact of DHA consumption on the various endocannabinoid-related metabolites in compartments where endocannabinoid signaling carries particular (patho)physiological and/or diagnostic import, such as brain and the blood circulation, has not been comprehensively examined in vivo ( 3,29,34,35 ). To address this issue, we have applied our targeted LC-MS/MS lipidomics approach ( 35,36 ) in a mouse model to quantify the coordinate effect of short-term (2-week) dietary DHA supplementation on 15 fatty acid, N -acyl-ethanolamine, and glycerol-ester constituents of the physiological brain and plasma endocannabinoid metabolomes. The results defi ne in vivo metabolite-and compartment-selective responses of diverse endocannabinoid-related lipid species to dietary DHA enhancement that may be of functional and therapeutic signifi cance.…”

“…A total of 15 fatty acid, N -acyl-ethanolamine, and acylglycerol species currently recognized as constituents of the endocannabinoid metabolome were analyzed simultaneously by our quantitative LC-MS/MS-based metabolomics approach ( 35,36 ). Standard curves for all 15 analytes were linear with regression values of >0.98, except those for DHA and AA, which had linear regression values of 0.85 (data not shown).…”