Lipidomic metabolism analysis of the endogenous cannabinoid anandamide (N-arachidonylethanolamide)

Elucidation of pathways involved with lipid metabolism has been limited by analytical challenges associated with detection and structure identification. A discovery-based mass spectrometry lipidomic approach has been applied to identify metabolites of the endogenous cannabinoid anandamide (N-arachidonylethanolamide). Previously, a model system was established to show that anandamide can be recycled by cells to form new endocannabinoids suggesting recycling of the arachidonate carbon chain. We hypothesized that distinct cellular pathways exist to direct the anandamide-derived arachidonate chain into a specific set of metabolites, different from the metabolite pool that is comprised of non-anandamide-derived arachidonic acid. Using stable isotope encoding and liquid chromatography-mass spectrometry, we identified a distinct pool of lipid metabolites derived from exogenous anandamide or arachidonic acid in RBL-2H3 cells. We discovered that arachidonic acid-derived metabolites were primarily comprised of the eicosanoid lipid class, whereas anandamide-derived arachidonic acid, in addition to eicosanoids, was metabolized into diradylglycerols, fatty acid amides, sterols, and glycerophospholipids. From the list of anandamide metabolites of particular interest was 1-O-arachidonyl-sn-glycero-3-phosphocholine. Furthermore, we determined that while 1-O-arachidonyl-sn-glycero-3-phosphocholine may be a metabolite of anandamide, the sn-2 compound was more abundant in mouse brain tissue. Overall, our results provide a novel approach to study the metabolic fate of endocannabinoids and fatty acid-derived signaling molecules.