Simultaneous determination of endocannabinoids (arachidonylethanolamide and 2-arachidonylglycerol) and isoprostane (8-epiprostaglandin F2alpha) by gas chromatography-mass spectrometry-selected ion monitoring for medical samples

This article describes the overall procedure for the simultaneous determination of endocannabinoids (arachidonylethanolamide and 2-arachidonyglycerol) and isoprostane by gas chromatography-mass spectrometry in the selected-ion monitoring SIM mode (GC-MS-SIM) for medical samples. It also describes the general points of this method which a scientist who wants to assay a new, unidentified prostanoids and related compounds in medical samples would need to be clarified. The similar structures of prostaglandins, thromboxane, their metabolites, isoprostane, and arachidonyl compounds, allow them to be assayed after the simultaneous preparation of a single sample. The dimethyl isopropylsilyl ether forms of derivatized compounds are suitable for multiple GC-MS-SIM assay because of their molecular stability, and because they produce positive, strong, and large fragments on MS.     

Metabolism of the endocannabinoids, 2-arachidonylglycerol and anandamide,into prostaglandin,thromboxane, and prostacyclin glycerol esters and ethanolamides

Cyclooxygenase-2 (COX-2) action on the endocannabinoids, 2-arachidonylglycerol (2-AG) and anandamide (AEA), generates prostaglandin glycerol esters (PG-G) and ethanolamides (PG-EA), respectively. The diversity of PG-Gs and PG-EAs that can be formed enzymatically following COX-2 oxygenation of endocannabinoids was examined in cellular and subcellular systems. In cellular systems, glycerol esters and ethanolamides of PGE(2), PGD(2), and PGF(2alpha) were major products of the endocannabinoid-derived COX-2 products, PGH(2)-G and PGH(2)-EA. The sequential action of purified COX-2 and thromboxane synthase on AEA and 2-AG provided thromboxane A(2) ethanolamide and glycerol ester, respectively. Similarly, bovine prostacyclin synthase catalyzed the isomerization of the intermediate endoperoxides, PGH(2)-G and PGH(2)-EA, to the corresponding prostacyclin derivatives. Quantification of the efficiency of prostaglandin and thromboxane synthase-directed endoperoxide isomerization demonstrated that PGE, PGD, and PGI synthases catalyze the isomerization of PGH(2)-G at rates approaching those observed with PGH(2). In contrast, thromboxane synthase was far more efficient at catalyzing PGH(2) isomerization than at catalyzing the isomerization of PGH(2)-G. These results define the in vitro diversity of endocannabinoid-derived prostanoids and will permit focused investigations into their production and potential biological actions in vivo.     

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid 2-arachidonylglycerol

The endocannabinoid, 2-arachidonylglycerol (2-AG), is an endogenous ligand for the central (CB1) and peripheral (CB2) cannabinoid receptors and has been shown to be efficiently and selectively oxygenated by cyclooxygenase (COX)-2. We have investigated 2-AG/COX-2 interactions through site-directed mutagenesis. An evaluation of more than 20 site-directed mutants of murine COX-2 has allowed for the development of a model of 2-AG binding within the COX-2 active site. Most strikingly, these studies have identified Arg-513 as a critical determinant in the ability of COX-2 to efficiently generate prostaglandin H(2) glycerol ester, explaining, in part, the observed isoform selectivity for this substrate. Mutational analysis of Leu-531, an amino acid located directly across from Arg-513 in the COX-2 active site, suggests that 2-AG is shifted in the active site away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Despite this difference, aspirin-treated COX-2 oxygenates 2-AG to afford 15-hydroxyeicosatetraenoic acid glycerol ester in a reaction analogous to the C-15 oxygenation of arachidonic acid observed with acetylated COX-2. Finally, the differences in substrate binding do not alter the stereospecificity of the cyclooxygenase reaction; 2-AG-derived and arachidonic acid-derived products share identical stereochemistry.     

Biochemistry, pharmacology and physiology of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand

2-Arachidonoylglycerol (2-AG) is a unique molecular species of monoacylglycerol isolated in 1995 from rat brain and canine gut as an endogenous ligand for the cannabinoid receptors. 2-AG is rapidly formed from arachidonic acid-containing phospholipids through increased phospholipid metabolism, such as enhanced inositol phospholipid turnover, in various tissues and cells upon stimulation. 2-AG binds to the cannabinoid receptors (CB1 and CB2) and exhibits a variety of cannabimimetic activities in vitro and in vivo. Notably, anandamide, another endogenous ligand for the cannabinoid receptors, often acts as a partial agonist at these cannabinoid receptors, whereas 2-AG acts as a full agonist in most cases. The results of structure-activity relationship studies suggested that 2-AG rather than anandamide is the true natural ligand for both the CB1 and the CB2 receptors. Evidence is gradually accumulating which shows that 2-AG plays physiologically essential roles in diverse biological systems. For example, several lines of evidence indicate that 2-AG plays an important role as a retrograde messenger molecule in the regulation of synaptic transmission. 2-AG has also been shown to be involved in the regulation of various types of inflammatory reactions and immune responses. In this review, we focused on 2-AG, and summarized information concerning its biosynthesis, metabolism, bioactions and physiological significance, including our latest experimental results.     

Chemical stability of 2-arachidonylglycerol under biological conditions

Recent evidence indicates that 2-arachidonylglycerol (2-AG) is a potent and specific ligand for the central and peripheral cannabinoid receptors. Therefore, the chemical stability of this molecule under biological conditions is of interest. A method for the isolation and detection of 2-AG using HPLC with evaporative light scattering detection is described. The method provides an extraction recovery from aqueous media of 78%, and a limit of detection of 60 ng on column. Incubation of 2-AG in culture medium or biological buffers indicated that it is stable to oxidation and ester hydrolysis for up to 6 h at 37 degrees C. However, gradual disappearance of the compound was noted due to adherence to glass and plastic surfaces. During incubation in RPMI culture medium, 2-AG rearranged to 1(3)-arachidonylglycerol (1(3)-AG) in a first order process with a half-life of 10 min in the absence of serum and 2.3 min in the presence of 10% fetal calf serum. Further studies indicated that the acyl migration reaction is base catalyzed (k(cat)=78,000/min M), and that the reaction is affected slightly by changes in buffer (Tris) concentration and not at all by changes in ionic strength. The results indicate that 2-AG is readily converted to 1(3)-AG under conditions commonly used to study receptor-ligand interactions, findings that have significant implications for the interpretation of relative ligand potency between the two isomers.     

Selective oxygenation of the endocannabinoid 2-arachidonylglycerol by leukocyte-type 12-lipoxygenase

The endogenous cannabinoid system appears to serve vascular, neurological, immunological, and reproductive functions. The identification of 2-arachidonylglycerol (2-AG) as an endogenous ligand for the central (CB1) and peripheral (CB2) cannabinoid receptors has prompted interest in enzymes capable of modifying or inactivating this endocannabinoid. Porcine leukocyte 12-liopoxygenase (12-LOX) oxygenated 2-AG to the 2-glyceryl ester of 12(S)-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12-HPETE-G). The k(cat)/K(M) for oxygenation of 2-AG was 40% of the value for arachidonic acid. In contrast to the results with leukocyte 12-LOX, 2-AG oxygenation was not detected with platelet-type 12-LOX. Among a series of structurally related arachidonyl esters, 2-AG served as the preferential substrate for leukocyte 12-LOX. 12(S)-Hydroxyeicosa-5,8,10,14-tetraenoic acid glyceryl ester (12-HETE-G) was produced following addition of 2-AG to COS-7 cells transiently transfected with leukocyte 12-LOX. These results demonstrate that leukocyte-type 12-LOX efficiently oxidizes 2-AG in vitro and in intact cells, suggesting a role for this oxygenase in the endogenous cannabinoid system.     

Mechanisms for recycling and biosynthesis of endogenous cannabinoids anandamide and 2-arachidonylglycerol

The mechanisms of endogenous cannabinoid biosynthesis are not completely understood. We hypothesized that anandamide could be recycled by the cell to form new endocannabinoid molecules and released into the extracellular space. We determined that new endocannabinoids derived from exogenous anandamide or arachidonic acid were synthesized and released from RBL-2H3 cells in response to ionomycin. Treatment of RBL-2H3 cells with nystatin and progesterone, agents that disrupt organization of lipid raft/caveolae, resulted in the attenuation of anandamide and 2-arachidonyl glycerol synthesis and/or release in response to stimulation with ionomycin suggesting a role for these membrane microdomains in endocannabinoid biosynthesis. Furthermore, anandamide synthesis may be independent of N-acyl phosphatidylethanolamine phospholipase D as expression of the enzyme was not detected in RBL-2H3 cells. We also established that extracellular calcium is necessary for endocannabinoid biosynthesis because release of intracellular calcium stores alone does not promote endocannabinoid biosynthesis. Next, we examined the role of calcium as a 'switch' to activate the synthesis of anandamide and simultaneously reduce uptake. Indeed, [(3)H] anandamide uptake was reduced in the presence of calcium. Our findings suggest a mechanism indicative of calcium-modulated activation of anandamide synthesis and simultaneous termination of uptake.     

Oxygenation of the endocannabinoid, 2-arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2

Cyclooxygenases (COX) play an important role in lipid signaling by oxygenating arachidonic acid to endoperoxide precursors of prostaglandins and thromboxane. Two cyclooxygenases exist which differ in tissue distribution and regulation but otherwise carry out identical chemical functions. The neutral arachidonate derivative, 2-arachidonylglycerol (2-AG), is one of two described endocannabinoids and appears to be a ligand for both the central (CB1) and peripheral (CB2) cannabinoid receptors. Here we report that 2-AG is a substrate for COX-2 and that it is metabolized as effectively as arachidonic acid. COX-2-mediated 2-AG oxygenation provides the novel lipid, prostaglandin H(2) glycerol ester (PGH(2)-G), in vitro and in cultured macrophages. PGH(2)-G produced by macrophages is a substrate for cellular PGD synthase, affording PGD(2)-G. Pharmacological studies reveal that macrophage production of PGD(2)-G from endogenous sources of 2-AG is calcium-dependent and mediated by diacylglycerol lipase and COX-2. These results identify a distinct function for COX-2 in endocannabinoid metabolism and in the generation of a new family of prostaglandins derived from diacylglycerol and 2-AG.     

In vivo pharmacology of endocannabinoids and their metabolic inhibitors: Therapeutic implications in Parkinson's disease and abuse liability

This review focuses on the behavioral pharmacology of endogenous cannabinoids (endocannabinoids) and indirect-acting cannabinoid agonists that elevate endocannabinoid tone by inhibiting the activity of metabolic enzymes. Similarities and differences between prototype cannabinoid agonists, endocannabinoids and inhibitors of endocannabinoid metabolism are discussed in the context of endocannabinoid pharmacokinetics in vivo. The distribution and function of cannabinoid and non-CB₁/CB₂ receptors are also covered, with emphasis on their role in disorders characterized by dopamine dysfunction, such as drug abuse and Parkinson's disease. Finally, evidence is presented to suggest that FAAH inhibitors lack the abuse liability associated with CB₁ agonists, although they may modify the addictive properties of other drugs, such as alcohol.     

Human adipose tissue binds and metabolizes the endocannabinoids anandamide and 2-arachidonoylglycerol

Endocannabinoids are a group of biologically active endogenous lipids that have recently emerged as important mediators in energy balance control. The two best studied endocannabinoids, anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the endogenous ligands of the central and peripheral cannabinoid receptors. Furthermore, AEA binds to the transient receptor potential vanilloid type-1 (TRPV1), a capsaicin-sensitive, non-selective cation channel. The synthesis of these endocannabinoids is catalyzed by the N-acylphosphatidylethanolamine-selective phospholipase D (NAPE-PLD) and the sn-1-selective diacylglycerol lipase (DAGL), whereas their degradation is accomplished by the fatty acid amide hydrolase (FAAH) and the monoglyceride lipase (MGL), respectively. We investigated the presence of a functional endocannabinoid system in human adipose tissue from seven healthy subjects. Subcutaneous abdominal adipose tissue underwent biochemical and molecular biology analyses, aimed at testing the expression of this system and its functional activity. AEA and 2-AG levels were detected and quantified by HPLC. Real time PCR analyzed the expression of the endocannabinoid system and immunofluorescence assays showed the distribution of its components in the adipose tissue. Furthermore, binding assay for the cannabinoid and vanilloid receptors and activity assay for each metabolic enzyme of the endocannabinoid system gave clear evidence of a fully operating system. The data presented herein show for the first time that the human adipose tissue is able to bind AEA and 2-AG and that it is endowed with the biochemical machinery to metabolize endocannabinoids.     

Production of the endocannabinoids anandamide and 2-arachidonoylglycerol by endothelial progenitor cells

Recent studies have highlighted the importance of paracrine growth factors as mediators of pro-angiogenic effects by endothelial progenitor cells (EPCs), but little is known about the release of lipid-based factors like endocannabinoids by EPCs. In the current study, the release of the endocannabinoids anandamide and 2-arachidonoylglycerol by distinct human EPC sub-types was measured using HPLC/tandem mass-spectrometry. Anandamide release was highest by adult blood colony-forming EPCs at baseline and they also demonstrated increased 2-arachidonoylglycerol release with TNF-alpha stimulation. Treatment of mature endothelial cells with endocannabinoids significantly reduced the induction of the pro-inflammatory adhesion molecule CD106 (VCAM-1) by TNF-alpha.     

Biochemistry and pharmacology of 7α-substituted androstenediones as aromatase inhibitors

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7α-substituted androstenediones. Numerous 7α-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7α-(4′-aminophenylthio)-androsta-1,4-diene-3,17-dione (7α-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7α-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7α-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7α position with a carbon-carbon linkage. Several 7α-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent K_is ranging between 13 and 19 nM. Extension of the research on these 7α-arylaliphatic androgens includes the introduction of a C₁---C₂ double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7α-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7α-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k_inacts ranging from 4.4 × 10⁻⁴ to 1.90 x 10⁻³ s⁻¹. The best inactivator of the series was 7α-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T_1/2 of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC₅₀ values of 64-328 nM. The 7α-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7α-position of the steroid in the active site of aromatase. The size of the 7α-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.     

Lipoxygenase-catalyzed oxygenation of arachidonylethanolamide,a cannabinoid receptor agonist

Various purified lipoxygenases were incubated with [¹⁴C]arachidonylethanolamide which is an endogenous ligand for cannabinoid receptors. When radioactive products were analyzed by thin-layer chromatography, porcine leukocyte 12-lipoxygenase and rabbit reticulocyte and soybean 15-lipoxygenases produced polar compounds at about the same reaction rates as that of oxygenation of free arachidonic acid. In contrast, the reaction of human platelet 12-lipoxygenase proceeded at a much lower rate, and porcine leukocyte 5-lipoxygenase was totally inactive. The result indicated that the lipoxygenases, which had been shown previously to be capable of oxygenating esterified polyunsaturated fatty acids, were also active with the arachidonylethanolamide. High-performance liquid chromatography, ultraviolet and mass spectrometry and nuclear magnetic resonance spectroscopy identified the major product by leukocyte 12-lipoxygenase as 12-hydroperoxy-5,8,10,14-eicosatetraenoylethanolamide and that by 15-lipoxygenases as 15-hydroperoxy-5,8,11,13-eicosatetraenoylethanolamide. The 15-hydroxy derivative inhibited electrically-evoked contraction of mouse vas deferens with an IC₅₀ of 0.63 μM as well as arachidonylethanolamide (0.17 μM), but the 12-hydroxy derivative was much less effective.     

Oxidative metabolism of endocannabinoids

There is increasing evidence that endocannabinoids play roles in a number of physiological and pathological processes ranging from the regulation of food intake to the inhibition of cancer cell proliferation. Consequently, multiple investigations into endocannabinoid metabolic disposition have been initiated. Such studies have begun to shed light on the mechanisms that regulate the endogenous cannabinoid system. In addition, they have identified a number of novel, endocannabinoid-derived lipids. In the future, these studies may form the foundation of efforts designed to subtly manipulate endocannabinoid tone in vivo to achieve therapeutic benefits without the profound side-effects observed with synthetic cannabinoid treatment.In addition to the well-studied hydrolytic mode of endocannabinoid metabolism, accumulating data suggest that these lipids are also susceptible to oxidative metabolism by a number of fatty acid oxygenases. These include the cyclooxygenases, lipoxygenases, and cytochrome P450s known to be involved in eicosanoid production from arachidonic acid. The available evidence concerning endocannabinoid oxidation is reviewed and the potential biological significance of this mode of metabolism is considered.     

15-Lipoxygenase metabolism of 2-arachidonylglycerol. Generation of a peroxisome proliferator-activated receptor alpha agonist

The recent demonstrations that cyclooxygenase-2 and leukocyte-type 12-lipoxygenase (LOX) efficiently oxygenate 2-arachidonylglycerol (2-AG) prompted an investigation into related oxygenases capable of metabolizing this endogenous cannabinoid receptor ligand. We evaluated the ability of six LOXs to catalyze the hydroperoxidation of 2-AG. Soybean 15-LOX, rabbit reticulocyte 15-LOX, human 15-LOX-1, and human 15-LOX-2 oxygenate 2-AG, providing 15(S)-hydroperoxyeicosatetraenoic acid glyceryl ester. In contrast, potato and human 5-LOXs do not efficiently metabolize this endocannabinoid. Among a series of structurally related arachidonyl esters, arachidonylglycerols serve as the preferred substrates for 15-LOXs. Steady-state kinetic analysis demonstrates that both 15-LOX-1 and 15-LOX-2 oxygenate 2-AG comparably or preferably to arachidonic acid. Furthermore, 2-AG treatment of COS-7 cells transiently transfected with human 15-LOX expression vectors or normal human epidermal keratinocytes results in the production and extracellular release of 15-hydroxyeicosatetraenoic acid glyceryl ester (15-HETE-G), establishing that lipoxygenase metabolism of 2-AG occurs in an eukaryotic cellular environment. Investigations into the potential biological actions of 15-HETE-G indicate that this lipid, in contrast to its free-acid counterpart, acts as a peroxisome proliferator-activated receptor alpha agonist. The results demonstrate that 15-LOXs are capable of acting on 2-AG to provide 15-HETE-G and elucidate a potential role for endocannabinoid oxygenation in the generation of peroxisome proliferator-activated receptor alpha agonists.     

Endothelium-derived 2-arachidonylglycerol: an intermediate in vasodilatory eicosanoid release in bovine coronary arteries

Acetylcholine stimulates the release of endothelium-derived arachidonic acid (AA) metabolites including prostacyclin and epoxyeicosatrienoic acids (EETs), which relax coronary arteries. However, mechanisms of endothelial cell (EC) AA activation remain undefined. We propose that 2-arachidonylglycerol (2-AG) plays an important role in this pathway. An AA metabolite isolated from bovine coronary ECs was identified as 2-AG by mass spectrometry. In ECs pretreated with the fatty acid amidohydrolase inhibitor diazomethylarachidonyl ketone (DAK; 20 micromol/l), methacholine (10 micromol/l)-stimulated 2-AG release was blocked by the phospholipase C inhibitor U-73122 (10 micromol/l) or the diacylglycerol lipase inhibitor RHC-80267 (40 micromol/l). In U-46619-preconstricted bovine coronary arterial rings, 2-AG relaxations averaging 100% at 10 micromol/l were inhibited by endothelium removal, by DAK, by the hydrolase inhibitor methyl arachidonylfluorophosphate (10 micromol/l), by the cyclooxygenase inhibitor indomethacin (10 micromol/l), but not by the CB1 cannabinoid receptor antagonist SR-141716 (1 micromol/l). The cytochrome P-450 inhibitor SKF-525a (10 micromol/l) and the 14,15-epoxyeicosa-5Z-enoic acid EET antagonist (14,15-EEZE; 10 micromol/l) further attenuated the indomethacin-resistant relaxations. The nonhydrolyzable 2-AG analogs noladin ether, 2-AG amide, and 14,15-EET glycerol amide did not induce relaxation. N-nitro-L-arginine-resistant relaxations to methacholine were also inhibited by U-73122, RHC-80267, and DAK. 14,15-EET glycerol ester increased opening of large-conductance K(+) channels 12-fold in cell-attached patches of isolated smooth muscle cells and induced relaxations averaging 95%. These results suggest that methacholine stimulates EC 2-AG production through phospholipase C and diacylglycerol lipase activation. 2-AG is further hydrolyzed to AA, which is metabolized to vasoactive eicosanoids. These studies reveal a role for 2-AG in EC AA release and the regulation of coronary tone.