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 porcine 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 phosphodiesterase. In this study a sensitive and specific procedure was developed to quantify NAE and N-ArPE, including organic solvent extraction, reverse-phase C-18 cartridge separation, derivatization, and gas chromatography/mass spectrometry (GC/MS) analysis. NAE is converted by a two-step derivatization procedure to a pentafluorobenzoyl ester followed by pentafluoropropionyl acylation. Quantification was performed by isotope dilution GC/MS using deuterium-labeled NAE (NAE-2H8) as an internal standard. The same chemical derivatization was applicable to N-ArPE quantification. The separated N-ArPE fractions were converted by a two-step cleavage/derivatization procedure into the pentafluorobenzoyl ester of NAE and then to its pentafluoropropionyl amide. The derivative was quantified by GC/MS using deuterium-labeled 1,2-[2H8]dioleoyl-sn-glycero-3-phospho(arachidonoyl)ethanolamid e as an internal standard. Using these methods, we have found that endogenous NAE levels in rat brain, spleen, testis, liver, lung, and heart were below the level 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 highest level in the spinal cord. The striatum, hippocampus, and accumbens contain intermediate concentrations of N-ArPE, whereas the value is lowest in the cerebellum.
Yang
H Y
HY
National Institute of Mental Health, Neuroscience Center at St. Elizabeths, Washington, DC.
Karoum
F
F
Felder
C
C
Badger
H
H
Wang
T C
TC
Markey
S P
SP
D000818:Animals; D001095:Arachidonic Acids / Q000032:analysis*; D001923:Brain Chemistry; D063388:Endocannabinoids; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D008297:Male; D010714:Phosphatidylethanolamines / Q000032:analysis*; D053284:Polyunsaturated Alkamides; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000737:chemistry*; D013154:Spleen / Q000737:chemistry*; D013737:Testis / Q000737:chemistry*
D001095:Arachidonic Acids; D063388:Endocannabinoids; D010714:Phosphatidylethanolamines; D053284:Polyunsaturated Alkamides; C078814:anandamide