Analysis of anandamide, an endogenous cannabinoid substance, and of other natural N-acylethanolamines

Fontana, Angelo; Marzo, Vincenzo Di; Cadas, Hugues; Piomelli, Daniele

doi:10.1016/0952-3278(95)90130-2

Cited by 72 publications

(76 citation statements)

References 19 publications

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“…Initial efforts to derivatize directly organic solvent extracts indicated that the complex mixture of lipids in rat brain extracts precluded successful G C M S analyses without additional separation, as previously noted (Fontana et al, 1995). We investigated solid-phase cartridge separations as a means of separating N-ArPE from NAE.…”

Section: Discussionmentioning

confidence: 94%

“…Two groups (Kempe et al, 1996;Schmid et al, 1996) have reported the postmortem generation of NAE. Others have noted the generation of NAE-like artifacts (Fontana et al, 1995) that could be separated by HPLC and have recommended the use of chlorofordmethanol in preference to ethyl acetate or diethyl ether and the use of silica gel minicolumns for cleanup before GCMS analyses.…”

Section: Discussionmentioning

confidence: 99%

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GC/MS Analysis of Anandamide and Quantification of N‐Arachidonoylphosphatidylethanolamides in Various Brain Regions, Spinal Cord, Testis, and Spleen of the Rat

Yang

Karoum

Felder

et al. 1999

Journal of Neurochemistry

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Abstract: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-arachidonoylphosphatidylethanolarnides (N-ArPEs) by phosphodiesterase. In this study a sensitive and specific procedure was developed to quantify NAE and N-ArPE, including organic solvent extraction, reversephase C-18 cartridge separation, derivatization, and gas chromatography/mass spectrometry (GUMS) 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 deuteriumlabeled NAE (NAE-*H,) 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-[2H,]dioleoyl-sn-glycero-3-phospho(arachidonoyl)ethanolamide 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 striaturn, hippocampus, and accumbens contain intermediate concentrations of N-ArPE, whereas the value is lowest in the cerebellum. Key Words: Anandamide-N-Arachidonoylphosphatidylethanolamides-Endogenous cannabinoidGas chromatography/mass spectrometry-Deuterium labeling-Reverse-phase C-18 cartridge.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

GC/MS Analysis of Anandamide and Quantification of N‐Arachidonoylphosphatidylethanolamides in Various Brain Regions, Spinal Cord, Testis, and Spleen of the Rat

Yang

Karoum

Felder

et al. 1999

Journal of Neurochemistry

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“…The pellets as well as the supernatant from the last centrifugation were incubated, unless otherwise stated, in 0.5 ml of 50 mM Tris-HCl, pH 7.4 at 37 °C for 30 min in the presence of 10,000 cpm (2.4 ArM) of either [~4C]anandamide (5.3 mCi/mmol, labelled on the ethanolamine moiety) or [~4C]oleoyl-amide (5.3 mCi/mmol, labelled on the oleic acid moiety). These were synthesized as described previously [5,10] [6]. Thermal stability of the 10,000 x g pellet enzymatic activities was determined by keeping identical aliquots of the same 10,000 x g pellet suspension at various temperature for 5 min, and then conducting the incubation as usual [7].…”

Section: Methodsmentioning

confidence: 99%

Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma ‘anandamide amidohydrolase’

et al. 1995

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“…This mutant is referred to as "FAAH." Enzyme activity was assayed by measuring the release of [ 14 C]ethanolamine from [ 14 C]anandamide (5 mCi/mmol; ARC, St. Louis, MO), using liquid scintillation counting (16).…”

Section: Methodsmentioning

confidence: 99%

Closing the Gate to the Active Site

Mei

Venere

Gasperi

et al. 2007

Journal of Biological Chemistry

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Fatty acid amide hydrolase (FAAH) is a dimeric, membranebound enzyme that degrades neuromodulatory fatty acid amides and esters and is expressed in mammalian brain and peripheral tissues. The cleavage of ≈30 amino acids from each subunit creates an FAAH variant that is soluble and homogeneous in detergent-containing buffers, opening the avenue to the in vitro mechanistic and structural studies. Here we have studied the stability of FAAH as a function of guanidinium hydrochloride concentration and of hydrostatic pressure. The unfolding transition was observed to be complex and required a fitting procedure based on a three-state process with a monomeric intermediate. The first transition was characterized by dimer dissociation, with a free energy change of ≈11 kcal/mol that accounted for ≈80% of the total stabilization energy. This process was also paralleled by a large change in the solvent-accessible surface area, because of the hydration occurring both at the dimeric interface and within the monomers. As a consequence, the isolated subunits were found to be much less stable (⌬G ≈ 3 kcal/mol). The addition of methoxyarachidonyl fluorophosphonate, an irreversible inhibitor of FAAH activity, enhanced the stability of the dimer by ≈2 kcal/mol, toward denaturant-and pressure-induced unfolding. FAAH inhibition by methoxyarachidonyl fluorophosphonate also reduced the ability of the protein to bind to the membranes. These findings suggest that local conformational changes at the level of the active site might induce a tighter interaction between the subunits of FAAH, affecting the enzymatic activity and the interaction with membranes.Fatty acid amide hydrolase (FAAH) 3 is a dimeric, integral membrane protein (1) that brings about the degradation of important fatty acid neurotransmitters, such as oleamide and anandamide (2). These fatty acid amides (FAAs), collectively termed "endocannabinoids," modulate several aspects of mammalian pathophysiology, both at central and peripheral level (3). It is now widely recognized that the biological activity of FAAs depends on the "metabolic control" of their endogenous tone (3), and in the last few years evidence has accumulated that points to FAAH as the key regulator of FAAs concentration in vivo (4, 5). Therefore, this enzyme is becoming the subject of intense investigation, and the design of ad hoc inhibitors able to tune its catalytic activity is a hot spot in medicinal chemistry (for a recent review see Ref. 6). In fact, FAAH inhibitors might represent next-generation therapeutics for the treatment of drug and alcohol abuse (7), anxiety (8), cancer (9), as well as neurodegenerative (10, 11) and vascular (12) diseases. In addition, interest toward FAAH was boosted by the fact that this enzyme is the first and, to date, the only characterized mammalian member of a large group, referred to as the "amidase signature" (AS) family (13). More than 100 members of this class have been reported in the literature, mostly from bacteria or fungi. Despite its quite remarkable size, struct...

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Analysis of anandamide, an endogenous cannabinoid substance, and of other natural N-acylethanolamines

Cited by 72 publications

References 19 publications

GC/MS Analysis of Anandamide and Quantification of N‐Arachidonoylphosphatidylethanolamides in Various Brain Regions, Spinal Cord, Testis, and Spleen of the Rat

GC/MS Analysis of Anandamide and Quantification of N‐Arachidonoylphosphatidylethanolamides in Various Brain Regions, Spinal Cord, Testis, and Spleen of the Rat

Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma ‘anandamide amidohydrolase’

Closing the Gate to the Active Site

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