Endocannabinoids: what they are, how they are degraded and how they are measured

Endocannabinoids are bioactive lipids derived from arachidonic acid that activate the cannabinoid receptors CB1 and CB2. The two best characterised are anandamide, or N-arachidonoylethanolamine (AEA), isolated in 1992, and 2-arachidonoylglycerol (2-AG), identified in 1995. Unlike classical neurotransmitters they are not stored in vesicles: they are synthesised on demand from membrane precursors, released by the postsynaptic neuron and act retrogradely on CB1 receptors of the presynaptic terminal, where they reduce neurotransmitter release. CB1 is expressed mainly in the central nervous system, CB2 mainly on immune cells. Together with their synthesising and degrading enzymes they form the endocannabinoid system, studied in neuroscience, pain, inflammation and metabolism. Chemically they belong to the same family as the eicosanoids, with which they share the precursor and many analytical difficulties.

Which enzymes make them and break them down?

The two molecules follow entirely separate routes. Anandamide derives from N-arachidonoyl-phosphatidylethanolamine through NAPE-phospholipase D, and is hydrolysed by fatty acid amide hydrolase (FAAH) into arachidonic acid and ethanolamine. 2-AG derives from diacylglycerol through diacylglycerol lipases α and β, and is hydrolysed mainly by monoacylglycerol lipase (MAGL).

The split has been quantified: in rodent brain about 85% of 2-AG hydrolase activity is attributable to MAGL, while ABHD12 accounts for roughly 9% and ABHD6 for roughly 4%. The three serine hydrolases have different subcellular distributions and probably control distinct pools of 2-AG. This is why selective FAAH and MAGL inhibitors produce different pharmacological effects and are studied with dedicated screening assays.

Why sampling is the critical step of the assay

Tissue endocannabinoid concentrations change quickly after collection, and the two molecules change in opposite directions. In human brain, AEA rises continuously and in a region-dependent way from one hour after death, up to about sevenfold at six hours; 2-AG, which in the same regions is 10 to 100 times more concentrated than AEA, instead falls to 25-35% of its initial value within the first hour and then stays broadly stable. A 2-AG/AEA ratio read without controlling the time and temperature between collection and freezing therefore measures the history of the sample as much as the biology. Hence the practice of immediate freezing or rapid enzyme inactivation, and the need to record the post-collection interval alongside the result.

What makes 2-AG hard to quantify

2-AG spontaneously isomerises into 1-arachidonoylglycerol (1-AG) by acyl migration, mostly during sample preparation and storage, at a rate that depends on pH, temperature and solvent composition. 1-AG activates CB1 with roughly threefold lower potency than 2-AG: mistaking one isomer for the other overstates the biological signal. Two strategies are in use: chromatographically separating the isomers with a longer gradient, or integrating their peaks together and reporting the result as total 2-AG + 1-AG, a frequent choice given the absence of an isomerically pure 2-AG reference standard. Either way, extraction has to be chosen to limit isomerisation: toluene extraction has been proposed for exactly this reason, together with reduced matrix effect in plasma.

Which standards LC-MS/MS requires

Quantification is performed by isotope dilution: before extraction, each sample receives a deuterated analogue of the analyte, which compensates for extraction losses and ion suppression. Both the unlabelled standard for the calibration curve and the matching deuterated compound as internal standard are therefore needed. Solutions of these polyunsaturated lipids must be protected from light and oxygen and stored under the conditions stated in the batch certificate of analysis.

Standards and reagents in the catalogue

The references below come from Cayman Chemical and are listed in the CABRU catalogue. The list is partial: the full range of standards, deuterated standards, enzyme inhibitors and antibodies is in the product catalogue, in the biochemicals category and among the ELISA kits.

To check which reference suits the analytical panel in use, it is enough to state the code or the analyte and write to CABRU.