Endocannabinoids are lipid mediators thought to modulate central and peripheral neural functions. We report here gas chromatography±electron impact mass spectrometry analysis of human brain, showing that lipid extracts contain anandamide and 2-arachidonoylglycerol (2-AG), the most active endocannabinoids known to date. Human brain also contained the endocannabinoid-like compounds N-oleoylethanolamine, N-palmitoylethanolamine and N-stearoylethanolamine. Anandamide and 2-AG (0.16^0.05 and 0.10^0.05 nmol/mg protein, respectively) represented 7.7% and 4.8% of total endocannabinoid-like compounds, respectively. N-Palmitoyethanolamine was the most abundant (50%), followed by N-oleoyl (23.6%) and N-stearoyl (13.9%) ethanolamines. A similar composition in endocannabinoid-like compounds was found in human neuroblastoma CHP100 and lymphoma U937 cells, and also in rat brain. Remarkably, human meningioma specimens showed an approximately six-fold smaller content of all N-acylethanolamines, but not of 2-AG, and a similar decrease was observed in a human glioblastoma. These ex vivo results fully support the purported roles of endocannabinoids in the nervous system.

CD1 mice lacking the CB 1 receptors (knockout, KO) were compared with wild-type littermates for their ability to degrade N-arachidonoylethanolamine (anandamide, AEA) through a membrane transporter (AMT) and a fatty acid amide hydrolase (FAAH). The regional distribution and age-dependence of AMT and FAAH activity were investigated. Anandamide membrane transporter and FAAH increased with age in knockout mice, whereas they showed minor changes in wild-type animals. Remarkably, they were higher in all brain areas of 6-month-old knockout versus wild-type mice, and even higher in 12-month-old animals. The molecular mass (<67 kDa) and isoelectric point (<7.6) of mouse brain FAAH were determined and the FAAH protein content was shown to parallel the enzyme activity. The kinetic constants of AMT and FAAH in the cortex of wild-type and knockout mice at different ages suggested that different amounts of the same proteins were expressed. The cortex and hippocampus of wild-type and knockout mice contained the following N-acylethanolamines: AEA (8% of total), 2-arachidonoylglycerol (5%), N-oleoylethanolamine (20%), N-palmitoylethanolamine (53%) and N-stearoylethanolamine (14%). These compounds were twice as abundant in the hippocampus as in the cortex. Minor differences were observed in AEA or 2-arachidonoylglycerol content in knockout versus wild-type mice, whereas the other compounds were lower in the hippocampus of knockout versus wild-type animals.

A direct experimental approach based on the evaluation of the ligand exchange equilibrium H20-N02+ + CH30H CH3(H)O-N02+ + H 2 0 has been exploited for the determination of APA = PA(HN03) -PA(CH30N02) at 298 K. The result, APA = 4.0 f 1.2 kcal mol-', is sufficiently accurate to provide firm experimental support to the counterintuitive prediction, based on high-level ab initio calculations, that the PA of HN03 exceeds that of CH3-ON02 by 5.6 f 5 kcal mol-'. Combination of the experimental APA with the known PA of CH30N02 gives PA(HNO3) = 182.0 f 2.3 kcal mol-', in excellent agreement with the theoretically computed value, 182.5 f 3 kcal mol-'. An explanation of the considerably lower PA(HN03) value derived from earlier ICR bracketing experiments is offered, based on the R(H)O-N02+ vs RON02H+ isomerism. whose role in determining the observed PA trend along the RON02 series (R = H, CH3, C2H5) is discussed.

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