GC/MS analysis of anandamide and quantification of N-arachidonoylphosphatidylethanolamides in various brain regions, spinal cord, testis, and spleen of the rat

Anandamide [N-arachidonoylethanolamide (NAE)] was initially isolated from p orcine brain and proposed as an endogenous ligand for cannabinoid receptors in 1992. Accumulating evidence has now suggested that, in the tissue, NAE is generated from N-arachidonoylphosphatidylethanolamides (N-ArPEs) by phos phodiesterase, In this study a sensitive and specific procedure was develop ed to quantify NAE and N-ArPE, including organic solvent extraction, revers e-phase C-18 cartridge separation, derivatization, and gas chromatography/m ass spectrometry (GC/MS) analysis, NAE is converted by a two-step derivatiz ation procedure to a pentafluorobenzoyl ester followed by pentafluoropropio nyl acylation, Quantification was performed by isotope dilution GC/MS using deuterium-labeled NAE (NAE-H-2(8)) as an internal standard. The same chemi cal derivatization was applicable to N-ArPE quantification. The separated N -ArPE fractions were converted by a two-step cleavage/derivatization proced ure into the pentafluorobenzoyl ester of NAE and then to its pentafluoropro pionyl amide, The derivative was quantified by GC/MS using deuterium-labele d 1,2[H-2(8)]dioleoyl-sn-glycero-3-phospho(arachidonoyl)ethanolamide as an internal standard. Using these methods, we have found that endogenous NAE l evels in rat brain, spleen, testis, liver, lung, and heart were below the l evel of quantification achievable (0.1 pmol/mg of protein) but that N-ArPE is readily quantifiable and is widely distributed in the rat CNS with the h ighest level in the spinal cord. The striatum, hippocampus, and accumbens c ontain intermediate concentrations of N-ArPE, whereas the value is lowest i n the cerebellum.