Together, these results suggest a role for endogenous endocannabinoid signalling in regulation of endocrine secretion in the human pancreas.

The endocannabinoid N-arachidonoyl ethanolamine (anandamide), found both in the CNS and in the periphery, plays a role in numerous physiological systems. One might expect that the chemically related N-arachidonoyl-L-serine (ARA-S) could also be formed alongside anandamide. We have now isolated ARA-S from bovine brain and elucidated its structure by comparison with synthetic ARA-S. Contrary to anandamide, ARA-S binds very weakly to cannabinoid CB 1 and CB2 or vanilloid TRPV1 (transient receptor potential vanilloid 1) receptors. However, it produces endothelium-dependent vasodilation of rat isolated mesenteric arteries and abdominal aorta and stimulates phosphorylation of p44͞42 mitogen-activated protein (MAP) kinase and protein kinase B͞Akt in cultured endothelial cells. ARA-S also suppresses LPS-induced formation of TNF-␣ in a murine macrophage cell line and in wild-type mice, as well as in mice deficient in CB 1 or CB2 receptors. Many of these effects parallel those reported for abnormal cannabidiol (Abn-CBD), a synthetic agonist of a putative novel cannabinoidtype receptor. Hence, ARA-S may represent an endogenous agonist for this receptor.abnormal cannabidiol ͉ anandamide ͉ cannabinoids ͉ endothelium ͉ reactive oxygen intermediates T he identification, structural elucidation, and syntheses of the plant cannabinoids in the early 1960s led to thorough investigations of the chemistry, metabolism, and pharmacology of these compounds, in particular of the psychoactive constituent ⌬ 9 -tetrahydrocannabinol (1, 2). However, until the late 1980s and early 1990s, when specific receptors were identified and shortly thereafter cloned, the mechanism of the numerous cannabinoid actions remained an enigma (3-5). Two main receptors are now known: the CB 1 receptor, found in the CNS, as well as in some peripheral tissues, and the CB 2 receptor, found predominantly in the immune system (6-8). Additional, not yet fully identified receptors are present both in the CNS and in the periphery (6, 9, 10).Because receptors in mammals are not formed to encounter a plant constituent, research was initiated to discover endogenous ligands. In the 1990s two endogenous cannabinoids (endocannabinoids) were identified, N-arachidonoyl ethanolamine (anandamide) (11) and 2-arachidonoyl-glycerol (12, 13). Additional endocannabinoids have been reported, but their biological roles are yet obscure (6, 14). Anandamide and 2-arachidonoyl-glycerol have large spectrum of physiological actions, most of which are associated with the neural and immune systems. However, cardiovascular effects, which are in part CB 1 -mediated (15), are also well established (9,14,16).Anandamide is a product of phosphatidylethanolamine (17). Because phosphatidylserine is found alongside phosphatidylethanolamine in body tissues, one might expect that arachidonoyl-Lserine (ARA-S) is also an endogenous constituent (see Fig. 1A for the structures of anandamide and ARA-S). We report that we have isolated ARA-S from bovine brain and have evaluated some of its biological propertie...

BACKGROUND We measured Δ9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), and cannabinol (CBN) disposition in oral fluid (OF) following controlled cannabis smoking to evaluate whether monitoring multiple cannabinoids in OF improved OF test interpretation. METHODS Cannabis smokers provided written informed consent for this institutional review board–approved study. OF was collected with the Quantisal™ device following ad libitum smoking of one 6.8% THC cigarette. Cannabinoids were quantified by 2-dimensional GC-MS. We evaluated 8 alternative cutoffs based on different drug testing program needs. RESULTS 10 participants provided 86 OF samples −0.5 h before and 0.25, 0.5, 1, 2, 3, 4, 6, and 22 h after initiation of smoking. Before smoking, OF samples of 4 and 9 participants were positive for THC and THCCOOH, respectively, but none were positive for CBD and CBN. Maximum THC, CBD, and CBN concentrations occurred within 0.5 h, with medians of 644, 30.4, and 49.0 μg/L, respectively. All samples were THC positive at 6 h (2.1–44.4 μg/L), and 4 of 6 were positive at 22 h. CBD and CBN were positive only up to 6 h in 3 (0.6–2.1 μg/L) and 4 (1.0–4.4 μg/L) participants, respectively. The median maximum THCCOOH OF concentration was 115 ng/L, with all samples positive to 6 h (14.8–263 ng/L) and 5 of 6 positive at 22 h. CONCLUSIONS By quantifying multiple cannabinoids and evaluating different analytical cutoffs after controlled cannabis smoking, we determined windows of drug detection, found suggested markers of recent smoking, and minimized the potential for passive contamination.

Development and validation of a method for simultaneous identification and quantification of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), and metabolites 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) in oral fluid. Simultaneous analysis was problematic due to different physicochemical characteristics and concentration ranges. Neutral analytes, such as THC and CBD, are present in ng/mL, rather than pg/mL concentrations, as observed for the acidic THCCOOH biomarker in oral fluid. THCCOOH is not present in cannabis smoke, definitively differentiating cannabis use from passive smoke exposure. THC, 11-OH-THC, THCCOOH, CBD, and CBN quantification was achieved in a single oral fluid specimen collected with the Quantisal™ device. One mL oral fluid/buffer solution (0.25mL oral fluid and 0.75mL buffer) was applied to conditioned CEREX ® Polycrom™ THC solid phase extraction (SPE) columns. After washing, THC, 11-OH-THC, CBD, and CBN were eluted with hexane/acetone/ethyl acetate (60:30:20, v/v/v), derivatized with N, O-bis-(trimethylsilyl) trifluoroacetamide and quantified by two-dimensional gas chromatography electron ionization mass spectrometry (2D-GCMS) with cold trapping. Acidic THCCOOH was separately eluted with hexane/ethyl acetate/acetic acid (75:25:2.5, v/v/v), derivatized with trifluoroacetic anhydride and hexafluoroisopropanol, and quantified by the more sensitive 2D-GCMS-electron capture negative chemical ionization (NCI-MS). Linearity was 0.5-50ng/mL for THC, 11-OH-THC, CBD and 1-50ng/mL for CBN. The linear dynamic range for THCCOOH was 7.5-500pg/mL. Intra-and inter-assay imprecision as percent RSD at three concentrations across the linear dynamic range were 0.3%-6.6%. Analytical recovery was within 13.8% of target. This new SPE 2D-GCMS assay achieved efficient quantification of five cannabinoids in oral fluid, including pg/mL concentrations of THCCOOH by combining differential elution, 2D-GCMS with electron ionization and negative chemical ionization. This method will be applied to quantification of cannabinoids in oral fluid specimens from individuals participating in controlled cannabis and Sativex ® (50% THC and 50% CBD) administration studies, and during cannabis withdrawal.

Background Δ9-Tetrahydrocannabinol (THC) in oral fluid (OF) implies cannabis intake, but eliminating passive exposure and improving interpretation of test results requires additional research. Methods Ten adult cannabis users smoked ad libitum one 6.8% THC cigarette. Expectorated OF was collected for up to 22h, and analyzed within 24 h of collection. THC, 11-nor-9-carboxy-THC (THCCOOH), cannabidiol, and cannabinol were quantified by 2-dimensional-GCMS. Results Eighty specimens were analyzed; 6 could not be collected due to dry mouth. THC was quantifiable in 95.2%, cannabidiol in 69.3%, cannabinol in 62.3%, and THCCOOH in 94.7% of specimens. Highest THC, cannabidiol, and cannabinol concentrations were 22370, 1000, and 1964 μg/l, respectively, 0.25 h after the start of smoking; THCCOOH peaked within 2 h (up to 560 ng/l). Concentrations 6h after smoking were THC (0.9-90.4 μg/l) and THCCOOH (17.0-151 ng/l) (8 of 9 positive for both); only 4 were positive for cannabidiol (0.5-2.4 μg/l) and cannabinol (1.0-3.0 μg/l). By 22h, there were 4 THC (0.4-10.3 μg/l), 5 THCCOOH (6.0-24.0 ng/l), 1 cannabidiol (0.3 μg/l), and no cannabinol positive specimens. Conclusions THCCOOH in OF suggests no passive contamination, and CBD and CBN suggest recent cannabis smoking. Seventeen alternative cutoffs were evaluated to meet the needs of different drug testing programs.

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BACKGROUND Oral fluid (OF) is an accepted alternative biological matrix for drug treatment, workplace, and DUID (driving under the influence of drugs) investigations, but establishing the cannabinoid OF detection window and concentration cutoff criteria are important. METHODS Cannabinoid concentrations were quantified in OF from chronic, daily cannabis smokers during monitored abstinence. Δ9-tetrahydrocannabinol (THC)3, cannabidiol (CBD), cannabinol (CBN), and 11-nor-9-carboxy-THC (THCCOOH) were determined in daily OF samples collected with the Quantisal™ device. GC-MS limits of quantification (LOQ) were 0.5 μg/L for THC and CBD, 1 μg/L for CBN, and 7.5 ng/L for THCCOOH. RESULTS After providing written informed consent for this institutional review board–approved study, 28 participants resided from 4 to 33 days on the secure research unit and provided 577 OF specimens. At the LOQ, THC was generally quantifiable for 48 h, whereas CBD and CBN were detected only at admission. Median THCCOOH detection time was 13 days (CI 6.4–19.6 days). Mean THC detection rates decreased from 89.3% at admission to 17.9% after 48 h, whereas THCCOOH gradually decreased from 89.3% to 64.3% within 4 days. Criteria of THC ≥2 μg/L and THCCOOH ≥20 ng/L reduced detection to <48 h in chronic cannabis smokers. An OF THCCOOH/THC ratio ≤4 ng/μg or presence of CBD or CBN may indicate more recent smoking. CONCLUSIONS THC, THCCOOH, CBD, and CBN quantification in confirmatory OF cannabinoid testing is recommended. Inclusion of multiple cannabinoid cutoffs accounted for residual cannabinoid excretion in OF from chronic, daily cannabis smokers and could reduce the potential for positive test results from passive cannabis smoke exposure and lead to greatly improved test interpretation.

Bone mass is determined by a continuous remodeling process, whereby the mineralized matrix is being removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. Here we report the presence of endogenous amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) in mouse bone. Of these compounds, N-oleoyl-L-serine (OS) had the highest activity in an osteoblast proliferation assay. In these cells, OS triggers a Gi-protein-coupled receptor and Erk1/2. It also mitigates osteoclast number by promoting osteoclast apoptosis through the inhibition of Erk1/2 phosphorylation and receptor activator of nuclear-κB ligand (RANKL) expression in bone marrow stromal cells and osteoblasts. In intact mice, OS moderately increases bone volume density mainly by inhibiting bone resorption. However, in a mouse ovariectomy (OVX) model for osteoporosis, OS effectively rescues bone loss by increasing bone formation and markedly restraining bone resorption. The differential effect of exogenous OS in the OVX vs. intact animals is apparently a result of an OVX-induced decrease in skeletal OS levels. These data show that OS is a previously unexplored lipid regulator of bone remodeling. It represents a lead to antiosteoporotic drug discovery, advantageous to currently available therapies, which are essentially either proformative or antiresorptive.fatty acyl amides I n mammals, including humans, bone mass is determined by an unremitting remodeling process whereby the mineralized matrix is continuously removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. This process is regulated by autocrine/paracrine factors, such as receptor activator of nuclear-κB ligand (RANKL), osteoprotegerin (OPG), bone morphogenetic proteins, and Wnt, as well as circulating hormones (e.g., sex steroids, parathyroid hormone) and brain-derived signals (e.g., sympathetic, pituitary) (1-3). Imbalanced bone remodeling leads to skeletal pathologies, mainly osteoporosis, the most common degenerative disorder in affluent societies, which results from a net increase in bone resorption (4). Identification of endogenous constituents, which regulate bone remodeling and skeletal mass, contributes to the elucidation of the mechanisms involved in this process and offers promise for developing novel antiosteoporotic pharmacotherapy.Amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) represent a major group of endogenous lipids. In mammalian tissues they have numerous physiological functions. For example, anandamide (arachidonoyl ethanolamide) is the first identified endogenous psychoactive ligand of cannabinoid receptors (5); arachidonoyl serine is an endogenous vasodilator, which does not bind to the cannabinoid receptors (6); and oleoyl ethanolamide is an endogenous structural analog to anandamide that regulates food intake through the activation of GPR 119 (7, 8). The well-established biosynthetic tendency to follow existing p...