RESUMO
Endocannabinoid system, including endocannabinoid neurotransmitters (eCBs), has gained much attention over the last years due to its involvement with the pathophysiology of diseases and the potential use of Cannabis sativa (marijuana). The identification of eCBs and phytocannabinoids in biological samples for forensic, clinical, or therapeutic drug monitoring purposes constitutes a still significant challenge. In this scoping review, the recent advantages, and limitations of the eCBs and phytocannabinoids quantification in biological samples are described. Published studies from 2018-2023 were searched in 8 databases, and after screening and exclusions, the selected 38 articles had their data tabulated, summarized, and analyzed. The main characteristics of the eCBs and phytocannabinoids analyzed and the potential use of each biological sample were described, indicating gaps in the literature that still need to be explored. Well-established and innovative sample preparation protocols, and chromatographic separations, such as GC, HPLC, and UHPLC, are reviewed highlighting their respective advantages, drawbacks, and challenges. Lastly, future approaches, challenges, and tendencies in the quantification analysis of cannabinoids are discussed.
Assuntos
Canabinoides , Cannabis , Endocanabinoides , Endocanabinoides/análise , Endocanabinoides/metabolismo , Humanos , Canabinoides/análise , Cannabis/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Gasosa/métodos , AnimaisRESUMO
The increasing interest in endocannabinoid-like compounds (ECL) in food stems from their important physiological roles, including energy metabolism and satiety. In this study, the effect of fermentation or alkalisation on the formation of ECL compounds in table olives was investigated. N-acylethanolamines, monoacylglycerols, N-acylamino acids, and N-acylneurotransmitters were monitored. Results revealed that alkaline treatment led to a significant increase in the concentrations of N-oleoylethanolamide (80%), N-palmitoylethanolamide (93%), N-linoleoylethanolamide (51%), and 1-oleoylglycerol (679%) compared to control. While N-oleoylethanolamide, N-palmitoylethanolamide, N-linoleoylethanolamide, 1- and 2-oleoylglycerol, 1- and 2-linoleoylglycerol, and oleoylphenylalanine were initially absent or present in trace amounts, their levels significantly rose during fermentation. The formation rate of these compounds was higher in olives fermented in water than those in brine. The study provides detailed information on how specific ECL compounds respond to different processing methods, offering valuable information for optimising table olive production to enhance its nutritional benefits.
Assuntos
Endocanabinoides , Fermentação , Olea , Olea/química , Olea/metabolismo , Endocanabinoides/metabolismo , Endocanabinoides/análise , Frutas/química , Frutas/metabolismo , Concentração de Íons de Hidrogênio , Manipulação de AlimentosRESUMO
Analysis of endocannabinoids (ECs) and N-acylethanolamines (NAEs) in hair is assumed to retrospectively assess long-term EC/NAE concentrations. To inform their use, this study investigated stability of EC/NAE hair concentrations in mothers, fathers, and their children across the perinatal period as well as associations between family members. In a prospective cohort study, EC (AEA, 1-AG/2-AG) and NAE (SEA, PEA, OEA) levels were quantified in hair samples taken four times in mothers (n = 336) and their partners (n = 225) from pregnancy to two years postpartum and in offspring (n = 319) from shortly after birth to two years postpartum. Across the perinatal period, maternal and paternal hair ECs/NAEs showed poor multiple-test consistency (16-36%) and variable relative stability, as well as inconsistent absolute stability for mothers. Regarding children, hair ECs/NAEs evidenced poor multiple-test consistency (4-19%), no absolute stability, and either no or variable relative stability. Hair ECs/NAEs showed small to medium significant associations across the perinatal period within couples and parent-child dyads. Findings suggest hair ECs/NAEs during the perinatal period possess variable stability in adults, albeit more stability in fathers than mothers in this time. This highlights the need to further investigate factors associated with changes in hair ECs/NAEs across time. The first two years of life may be a dynamic phase for the endocannabinoid system in children, potentially characterized by complex within-family correspondence that requires further systematic investigation.
Assuntos
Endocanabinoides , Etanolaminas , Pai , Cabelo , Mães , Humanos , Endocanabinoides/metabolismo , Endocanabinoides/análise , Feminino , Cabelo/química , Cabelo/metabolismo , Masculino , Etanolaminas/metabolismo , Etanolaminas/análise , Adulto , Gravidez , Pré-Escolar , Lactente , Estudos Prospectivos , Recém-Nascido , CriançaRESUMO
Cannabis use has increased sharply in the last 20 y among adults, including reproductive-aged women. Its recent widespread legalization is associated with a decrease in risk perception of cannabis use during breastfeeding. However, the effect of cannabis use (if any) on milk production and milk composition is not known. This narrative review summarizes current knowledge related to maternal cannabis use during breastfeeding and provides an overview of possible pathways whereby cannabis might affect milk composition and production. Several studies have demonstrated that cannabinoids and their metabolites are detectable in human milk produced by mothers who use cannabis. Due to their physicochemical properties, cannabinoids are stored in adipose tissue, can easily reach the mammary gland, and can be secreted in milk. Moreover, cannabinoid receptors are present in adipocytes and mammary epithelial cells. The activation of these receptors directly modulates fatty acid metabolism, potentially causing changes in milk fatty acid profiles. Additionally, the endocannabinoid system is intimately connected to the endocrine system. As such, it is probable that interactions of exogenous cannabinoids with the endocannabinoid system might modify release of critical hormones (e.g., prolactin and dopamine) that regulate milk production and secretion. Nonetheless, few studies have investigated effects of cannabis use (including on milk production and composition) in lactating women. Additional research utilizing robust methodologies are needed to elucidate whether and how cannabis use affects human milk production and composition.
Assuntos
Canabinoides , Cannabis , Adulto , Feminino , Humanos , Animais , Lactação , Leite Humano/química , Aleitamento Materno , Endocanabinoides/análise , Endocanabinoides/metabolismo , Endocanabinoides/farmacologia , Leite/química , Canabinoides/farmacologia , Canabinoides/análise , Canabinoides/metabolismo , Ácidos Graxos/farmacologiaRESUMO
BACKGROUND: The reasons for developing depression are not fully understood. However, it is known that the serotonergic system plays a role in the etiology, but the endocannabinoid system receives attention. METHOD: In this study, 161 patients with a depressive disorder and 161 healthy participants were examined for the distribution of the CNR1 rs4940353, 5-HT2A rs6311, and 5-HT1A rs6295 by high-resolution melting genotyping. The concentration of arachidonoyl ethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) in the blood was measured by liquid chromatography-tandem mass spectrometry. Additionally, depression and anxiety symptoms were evaluated based on self-questionnaires. Fifty-nine patients participated in a second appointment to measure the concentration of AEA, 2-AG, and symptoms of depression and anxiety. RESULTS: We observed higher AEA and decreased 2-AG concentrations in patients with depression compared to healthy participants. During the treatment, the concentrations of AEA and 2-AG did not change significantly. In patients higher symptoms of anxiety correlated with lower concentrations of 2-AG. Gender differences were found concerning increased 2-AG concentration in male patients and increased anxiety symptoms in female patients. Genotypic variations of 5-HT1A rs6295 and 5-HT2A rs6311 are associated with altered serotonergic activity and serotonin content in patients. CONCLUSION: In conclusion, it seems that the endocannabinoid system, especially the endocannabinoids 2-AG and AEA, and genetic variations of the 5-HT1A and 5-HT2A could play a role in patients with depression and may be involved in a depressive disorder.
Assuntos
Endocanabinoides , Alcamidas Poli-Insaturadas , Feminino , Humanos , Masculino , Cromatografia Líquida , Endocanabinoides/análise , Variação Genética , Receptor CB1 de Canabinoide/genéticaRESUMO
Cyclic vomiting syndrome (CVS) is an underdiagnosed disorder of the gut-brain interaction. Our understanding of the pathophysiology of CVS is evolving. Here, we tested the hypotheses that: (1) the levels of endocannabinoids and related lipids are altered in CVS, and (2) cephalic-vagal stimulation drive changes in endolipid levels. Ten adult patients with CVS and eight healthy controls were included. Indirect measurements of parasympathetic (RFa) functions were performed with spectral analysis of heart rate variability and respiratory activity. Plasma levels of endocannabinoids and related lipids were measured at baseline and during a sham feeding. Values are reported as mean ± standard error of the mean and compared using t-test or ANOVA. CVS patients had a lower parasympathetic tone and response to the Valsalva maneuver and deep breathing than the controls. The baseline 2-Arachidonoylglycerol (2-AG) had a significantly higher concentration in CVS (5.9e-008 ± 3.7e-008 mol/L) than control (3.7e-008 ± 1.3e-008 mol/; p < 0.05). Sham feeding did not change the concentration of 2-AG. 2-oleoylglycerol (2-OG) was significantly higher in CVS than control and did not change with sham feeding. Levels of N-acylethanolamines, including anandamide (AEA), were not different in CVS vs control. After sham feeding, AEA showed a trend toward increasing (p = 0.08) in CVS, but not in control. With sham feeding, palmitoyl ethanolamine significantly increased in both CVS and control groups; oleoyl ethanolamine in CVS only, and stearoyl ethanolamine in the control group. Levels of endocannabinoids and related lipids are altered in CVS patients. Sham feeding affects endogenous signaling lipids in a disease and time-dependent manner.
Assuntos
Endocanabinoides , Etanolaminas , Adulto , Humanos , Endocanabinoides/análiseRESUMO
BACKGROUND: Predicting viral suppression early is crucial to improving treatment outcomes among people living with HIV/AIDS (PLWH) in clinics. Viral suppression is affected by stress, making stress indicators a potential predictive factor. Most of previous studies used the self-report questionnaire as stress indicators, but there were great drawbacks due to its subjective. In contrast, end products of neuroendocrine systems such as hypothalamic-pituitaryadrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes and endogenous cannabinoid system (ECS) that involved in regulating stress as objective stress indicators are urgently needed to predict viral suppression. Therefore, this study aimed to investigate whether neuroendocrine indictors can strongly predict viral suppression among PLWH in China. METHODS: This cross-sectional study recruited 1198 PLWH on antiretroviral therapy (ART) in Guangxi, China. The concentrations of steroids (i.e., cortisol, cortisone, dehydroepiandrosterone, testosterone and progesterone) and endocannabinoids (i.e., N-arachidonoyl-ethanolamine and 1-arachidonyl glycerol) in hair were quantitated using the LC-APCI+-MS/MS method. To screen biomarkers that were used to predict viral suppression, association between hair biomarkers and viral suppression was examined by Mann-Whitney U test and partial correlation analyses. Receiver operating characteristic (ROC) curves and binary logistic regression based on the optimal classification threshold determined with ROC curves were used to estimate the prediction effects of the screened biomarkers on viral suppression (HIV-1 RNA < 200 copies/mL). RESULTS: Hair levels of dehydroepiandrosterone (DHEA), and N-arachidonoyl-ethanolamine (AEA), and the cortisol to DHEA ratio exhibited significant intergroup differences (ps < 0.05) and were correlated with HIV viral load (ps < 0.05). Hair DHEA concentrations strongly predicted viral suppression, showing good classification performance (area under the ROC curve = 0.651, p < 0.01) and strong predictive utility (adjusted odd ratio = 2.324, 95 % confidence interval = 1.211-4.899, p < 0.05) with an optimal threshold of 10.5 pg/mg. A hair AEA concentration of 2.4 pg/mg was the optimal threshold for predicting viral suppression based on good classification performance (area under the ROC curve = 0.598, p < 0.05) and predictive power (adjusted odd ratio = 2.124, 95 % confidence interval = 1.045-4.244, p < 0.05). In hair levels of cortisol to DHEA, viral suppression was observed to be highly predictive, with a threshold of 10.5 pg/mg being optimal for classification (area under the ROC curve = 0.624, p < 0.05) and prediction (adjusted odd ratio = 0.421, 95 % confidence interval = 0.201-0.785, p < 0.05). CONCLUSION: Hair levels of DHEA, and AEA and the cortisol to DHEA ratio were screened and verified to have significant predictive power with optimal thresholds for predicting viral suppression in a large-scale cohort. The data may provide new insights into predictors of successful virological outcomes and inform public health intervention and clinical practice to assist PLWH in achieving and sustaining viral suppression.
Assuntos
Síndrome da Imunodeficiência Adquirida , Infecções por HIV , Humanos , Endocanabinoides/análise , Endocanabinoides/uso terapêutico , Hidrocortisona/análise , Espectrometria de Massas em Tandem , Estudos Transversais , China , Infecções por HIV/tratamento farmacológico , Esteroides , Cabelo/química , Desidroepiandrosterona/análise , Desidroepiandrosterona/uso terapêutico , Biomarcadores/análise , Etanolaminas/uso terapêutico , Carga ViralRESUMO
A common method to quantify chronic stress is the analysis of stress markers in keratinized matrices such as hair or nail. In this study, we aimed to validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the combined quantification of steroid hormones and endocannabinoids (eCBs) in the keratinized matrix nail. Furthermore, we aimed to investigate the suitability of the nail matrix for the detection of these stress markers in a pilot study. An LC-MS/MS method was used for the simultaneous identification and quantification of four eCBs (2-arachidonoylglycerol (2-AG), anandamide (AEA), oleoylethanolamide (OEA), palmitoylethanolamide (PEA)) and five steroid hormones (cortisol, cortisone, androstenedione, progesterone, testosterone) in human nails using a surrogate analyte method for each analyte. The method was validated in terms of selectivity, response factor, linearity, limit of quantification (LOQ), precision, accuracy, matrix effect, recovery, robustness, and autosampler stability. Nail samples were extracted for 1 h with methanol following a clean-up with a fully automated supported liquid extraction (SLE). The influence of nail weight on the quantification was investigated by using 0.5-20 mg of nail sample. As a proof of concept, nail samples (N = 57) were analyzed from a cohort representing newborns (1 month old), children (between 1 and 10 years), and adults (up to 43 years). It could be shown that the established workflow using a 1 hour extraction and clean-up by SLE was very robust and resulted in a short sample preparation time. The LC-MS/MS method was successfully validated. Matrix effects with ion enhancement occurred mainly for 2-AG. Sample weights below 5 mg showed variations in quantification for some analytes. Certain analytes such as PEA and progesterone could be accurately quantified at a sample weight lower than 5 mg. This is the first study where steroids and eCBs could be simultaneously detected and quantified in infant and adult nails. These results show that nails may serve as an alternative keratinized matrix (compared to hair) for the retrospective monitoring of cumulative eCB and steroid hormone levels. The combined assessment of eCBs and steroids from nails could provide a new approach to gain new insights into stress exposure in newborns and adults.
Assuntos
Endocanabinoides , Esteroides , Adulto , Criança , Humanos , Lactente , Recém-Nascido , Cromatografia Líquida/métodos , Endocanabinoides/análise , Hidrocortisona/análise , Unhas/química , Projetos Piloto , Progesterona/análise , Estudos Retrospectivos , Esteroides/análise , Espectrometria de Massas em Tandem/métodosRESUMO
Analysis of bioactive lipids is increasingly useful in clinical studies, and there is a need for non-invasive and easy-to-use sampling methods that meet the demands of reliability. Samples that can be taken by a non-professional and that can be taken repeatedly so as to provide more detailed information about the inflammatory process are often desired. In this study, the feasibility of non-invasive sampling of nasal mucosa and saliva for the analysis of bioactive lipid mediators (e.g. oxylipins and endocannabinoids) was evaluated in a pilot study (n = 10). In a second study, the reliability (relative and absolute) of sampling of these lipid mediators derived from nasal mucosa and from plasma was assessed by calculation of the intraclass correlation coefficient and Bland-Altman's limit of agreement. Samples were taken at the same time of day on two occasions from a cohort of individuals with and without building-related intolerance (n = 37). Nasal mucosa proved to be a suitable matrix for the analysis of bioactive lipids and was therefore included in the study on reliability together with the plasma samples. Relative reliability varied among the identified oxylipins and endocannabinoids. Arachidonic acid derivatives showed generally better reliability. Absolute reliability measures also varied indicating that only a subset of the oxylipins and endocannabinoids were suitable as biomarkers in either nasal mucosa or plasma and should therefore be used with caution for that purpose.
Assuntos
Endocanabinoides , Oxilipinas , Endocanabinoides/análise , Estudos de Viabilidade , Humanos , Mucosa Nasal/química , Oxilipinas/análise , Projetos Piloto , Reprodutibilidade dos TestesRESUMO
BACKGROUND: The brain endocannabinoid system is believed to play significant roles in anti-nociception, fear response, anxiety, and stress. This study investigated the effects of rat inguinal surgery on the levels of endocannabinoids in the cerebral cortex. AIM: The aim of this study was to investigate the effects of acute post-surgical pain on the levels of endocannabinoids in the cerebral cortex. METHODS: Quantitation of endocannabinoids in the rat cerebral cortex was performed by liquid chromatography-tandem mass spectrometry. RESULTS: There was no significant difference in the cerebral cortical levels of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) between the sham and surgery experimental groups. However, there were lateralized differences in the levels of these endocannabinoids between the right and left cerebral cortices irrespective of the two groups. The concentrations of AEA and 2-AG were significantly higher in the right cerebral cortex compared to the contralateral cerebral cortex. CONCLUSION: Acute post-surgical pain did not induce significant alterations in the cerebral cortical levels of endocannabinoids in this study, but the phenomenon of lateralization of the cerebral cortical AEA and 2-AG levels was observed; this latter finding may be related to the role played by endocannabinoids in fear conditioning.
Assuntos
Endocanabinoides , Espectrometria de Massas em Tandem , Animais , Córtex Cerebral/química , Cromatografia Líquida/métodos , Endocanabinoides/análise , Humanos , Dor Pós-Operatória , Ratos , Espectrometria de Massas em Tandem/métodosRESUMO
Hair analysis of endocannabinoids and N-acylethanolamines presents a promising methodological advancement for the retrospective assessment of long-term cumulative endocannabinoids and N-acylethanolamines secretion over extended periods of time. A main assumption of this method application that hair endocannabinoid and N-acylethanolamine concentrations show intra-individual stability has not been confirmed yet. Thus, in the current study hair endocannabinoid and N-acylethanolamine levels were measured over a period of two and a half years with six months between each hair sample collection in 100 female participants. We found strong test-retest associations of hair endocannabinoid and N-acylethanolamine levels with intraclass correlation coefficients between 0.79 and 0.92. Furthermore, no correlations between perceived stress and hair endocannabinoids or N-acylethanolamines was observed. The current findings support the notion that endocannabinoids and N-acylethanolamines in hair are rather trait biomarkers that are stable over a considerable period of time rather than rapidly changing state markers.
Assuntos
Endocanabinoides , Etanolaminas , Cabelo , Biomarcadores/análise , Endocanabinoides/análise , Etanolaminas/análise , Feminino , Cabelo/química , Humanos , Estudos RetrospectivosRESUMO
The endocannabinoid system is involved in the regulation of a variety of physiological and cognitive processes. While the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA) have been found in breast milk, their role(s) have yet to be determined. This study determined the normal concentration ranges of endocannabinoids (2-AG and AEA) in breast milk and the influences, if any, of obesity and diurnal rhythms on their levels. Milk samples were collected from 36 breastfeeding mothers at 4-8 weeks postpartum at each feed over a 24-h period, and further stratified into three groups based on body mass index (BMI). The samples were analyzed using liquid chromatography mass spectrometry. AEA was below the limit of detection and 2-AG levels averaged 59.3 ± 18.3 ng/mL (± SD) in women with normal BMI. Wide-ranging 2-AG concentrations in the overweight (65.5 ± 41.9 ng/mL) /obese (66.1 ± 40.6 ng/mL) groups suggest BMI may be a contributing factor influencing its levels. Following a diurnal pattern, there was a significantly higher 2-AG concentration observed during the day, as compared to night time samples. In conclusion, our study clearly suggests that appropriate milk collection and storage conditions are critical. Further, body weight and diurnal rhythm appear to influence levels of 2-AG. Based on these results, future studies are underway to determine what specific roles endocannabinoids may play in human milk and how elevated levels of 2-AG may modulate infant appetite and health.
Assuntos
Ácidos Araquidônicos/análise , Ritmo Circadiano/fisiologia , Endocanabinoides/análise , Glicerídeos/análise , Leite Humano/química , Obesidade/metabolismo , Alcamidas Poli-Insaturadas/análise , Adulto , Índice de Massa Corporal , Cromatografia Líquida , Feminino , Humanos , Estudos Longitudinais , Espectrometria de Massas , Fenômenos Fisiológicos da Nutrição Materna , Sobrepeso/fisiopatologiaRESUMO
In this study, a novel approach was developed to quantify endocannabinoids (eCBs), and was based on the liquid biosensor BIONOTE. This device is composed of a probe that can be immersed in a solution, and an electronic interface that can record a current related to the oxy-reductive reactions occurring in the sample. The two most representative members of eCBs have been analysed in vitro by BIONOTE: anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG). Bovine serum albumin was used to functionalize the probe and improve the sensibility of the whole analytical system. We show that BIONOTE is able to detect both AEA and 2-AG at concentrations in the low nanomolar range, and to discriminate between these eCBs and their moieties arachidonic acid, ethanolamine and glycerol. Notably, BIONOTE distinguished these five different molecules, and it was also able to quantify AEA in human plasma. Although this is just a proof-of-concept study, we suggest BIONOTE as a cheap and user-friendly prototype sensor for high throughput quantitation of eCB content in biological matrices, with an apparent diagnostic potential for tomorrow's medicine.
Assuntos
Técnicas Biossensoriais/métodos , Endocanabinoides/análise , Ácidos Araquidônicos/análise , Ácidos Araquidônicos/sangue , Técnicas Biossensoriais/instrumentação , Endocanabinoides/sangue , Glicerídeos/análise , Glicerídeos/sangue , Humanos , Alcamidas Poli-Insaturadas/análise , Alcamidas Poli-Insaturadas/sangueRESUMO
BACKGROUND: The endocannabinoid system has been suggested to modulate energy metabolism and stress response and could be an important factor in the pathophysiology of anorexia nervosa (AN). In the context of AN, excessive physical activity may influence endocannabinoid concentrations. The objective of this study was to investigate hair endocannabinoid concentrations at different stages of the disorder. Measurement in hair allows for a cumulative assessment of endocannabinoid concentrations independent of circadian rhythms. METHODS: In a combined cross-sectional and longitudinal design, we measured hair concentrations of the endocannabinoids anandamide and 2-arachidonoylglycerol and the endocannabinoid-related compounds palmitoylethanolamide, oleoylethanolamide, and stearoylethanolamide in female underweight patients with acute AN (n = 67, reassessment of n = 47 after short-term weight restoration with a body mass index increase of at least 14%), individuals long-term recovered from AN (n = 27), and healthy control participants (n = 84). RESULTS: Hair concentrations of anandamide and all endocannabinoid-related compounds were elevated in acute AN and decreased over the course of short-term weight restoration. Anandamide concentrations remained elevated in long-term recovered AN patients. In long-term recovered patients, physical activity correlated positively with the concentrations of all endocannabinoid-related compounds. CONCLUSION: The current study provides evidence for a significant alteration of the endocannabinoid system in acute AN, which may partly persist into long-term recovery. The endocannabinoid system may be a possible target for pharmaceutical interventions in AN, which should be explored in further preclinical and subsequently clinical randomized controlled trials.
Assuntos
Anorexia Nervosa/metabolismo , Peso Corporal/fisiologia , Endocanabinoides/metabolismo , Cabelo/metabolismo , Recuperação de Função Fisiológica/fisiologia , Doença Aguda , Adolescente , Adulto , Anorexia Nervosa/diagnóstico , Anorexia Nervosa/psicologia , Criança , Estudos Transversais , Endocanabinoides/análise , Exercício Físico/fisiologia , Exercício Físico/psicologia , Feminino , Cabelo/química , Humanos , Estudos Longitudinais , Adulto JovemRESUMO
To evaluate the endocannabinoid system in an animal model of Parkinson's disease, in-tube solid-phase microextraction (in-tube SPME) was directly coupled to a tandem mass spectrometry (MS/MS) system for determination of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples. In-tube SPME-which consisted of a microtube of restricted access material (RAM) with a hydrophilic diol external surface and a hydrophobic octyl inner surface-efficiently excluded (up to 95%) macromolecules from the biological samples and selectively pre-concentrated the analytes. In-tube SPME parameters, such as sample volume, mobile phases, flow rate, and pre-concentration time, were evaluated to improve the extraction efficiency and throughput performance. The selectivity of the in-tube SPME and MS/MS (MRM mode) techniques allowed them to be directly coupled online, which dismissed the need for the chromatographic separation step. The in-tube SPME-MS/MS method was validated and shown to be linear from 6.0 to 30.0 ng mL-1 for AEA and from 10.0 to 100.0 ng mL-1 for 2-AG; the intra- and inter-assay accuracy and precision were lower than 15%. Parallelism between the calibration curves constructed in the matrix and aqueous solution confirmed that there was no matrix effect. The method allowed endogenous concentrations of AEA and 2-AG to be determined in rat brain striatum from unilaterally 6-hydroxydopamine-lesioned animals. The concentrations of these endocannabinoids in striatum ipsilateral and contralateral to the lesion differed significantly (p<0.001).
Assuntos
Ácidos Araquidônicos/análise , Encéfalo/metabolismo , Endocanabinoides/análise , Glicerídeos/análise , Alcamidas Poli-Insaturadas/análise , Espectrometria de Massas em Tandem/métodos , Animais , Ácidos Araquidônicos/isolamento & purificação , Ácidos Araquidônicos/normas , Encéfalo/efeitos dos fármacos , Calibragem , Cromatografia Líquida de Alta Pressão , Endocanabinoides/isolamento & purificação , Endocanabinoides/normas , Glicerídeos/isolamento & purificação , Glicerídeos/normas , Interações Hidrofóbicas e Hidrofílicas , Masculino , Oxidopamina/farmacologia , Alcamidas Poli-Insaturadas/isolamento & purificação , Alcamidas Poli-Insaturadas/normas , Ratos , Ratos Wistar , Microextração em Fase Sólida , Espectrometria de Massas em Tandem/normasRESUMO
The arachidonic acid derivatives N-arachidonoylethanolamine (anandamide; AEA), 2-arachidonoylglycerol (2-AG), N-arachidonoyldopamine (NADA), 2-arachidonoylglycerol ether (noladin ether; 2-AGE) and O-arachidonoylethanolamine (virodhamine; VA) were identified as physiological components of the endocannabinoid (EC) system. In order to gain further profound knowledge about the different EC-induced physiological and pathophysiological effects, appropriate analytical methods are required. The method described here uses liquid chromatography in combination with positive electrospray ionization mass spectrometry (LC-MS/MS) to quantify the concentrations of the above-mentioned EC compounds in cells. Sample preparation prior to LC-MS/MS analysis was performed by means of two liquid extractions with ethyl acetate. The method has been validated according to the bioanalytical guidelines of the Food and Drug Administration (FDA). The lower limits of quantification were 0.03 ng/mL for AEA, 2 ng/mL for 2-AG, 0.03 ng/mL for NADA, 0.3 ng/mL for 2-AGE and 0.15 ng/mL for VA. Linearity was demonstrated up to 10 ng/mL (AEA, NADA, 2-AGE and VA) and 50 ng/mL (2-AG). The values for intra- and inter-day precision and accuracy were within the guideline recommended acceptance criteria for assay validation. Low matrix effects and good recovery were found for AEA, 2-AG and 2-AGE, while a higher matrix effect was observed for NADA and VA. Extraction yields were lowest for VA. The method was used for EC measurement in different cell lines and in mouse brains.
Assuntos
Ácidos Araquidônicos/análise , Cromatografia Líquida/métodos , Endocanabinoides/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Células A549 , Animais , Química Encefálica , Feminino , Humanos , Limite de Detecção , Modelos Lineares , Camundongos , Camundongos Nus , Reprodutibilidade dos TestesRESUMO
BACKGROUND: The development and maintenance of anxiety disorders is not fully understood. There is consensus in the literature that in addition to genetic factors, social, psychological and neurobiological factors are of crucial importance. The present exploratory study investigates the influence of the endocannabinoids (EC) and related N-acylethanolamines (NA) on the maintenance of panic disorder (PD). METHODS: A total of n = 36 PD and n = 26 healthy controls (HC) were included in the study. Baseline characteristics showed no differences between the two groups. The participants were exposed to the Trier Social Stress Test (TSST) for reliable laboratory stress induction. Blood samples were taken during the TSST by an intravenous catheter to examine the endocannabinoid (EC) stress response. Repeated measures ANOVA was conducted to test for main effects of time and group as well as the respective interaction. RESULTS: Participants with PD consistently had significantly higher EC and NA blood levels than HC. The consistently high EC and NA levels barely showed any reactivity as indicated by a lack of statistical variance. In line with these findings no reaction to the psychosocial stressor TSST could be detected. CONCLUSION: Our main results show significant differences in EC concentrations between participants with PD and HC. These findings suggest that an imbalance in the ECS contributes to the maintenance of PD. Increased endocannabinoid levels may have important implications for organic diseases such as cardiovascular disorders. The limitations of the study as well as implications for further investigations are discussed.
Assuntos
Endocanabinoides/análise , Transtorno de Pânico/metabolismo , Adulto , Endocanabinoides/sangue , Etanolaminas/análise , Etanolaminas/sangue , Feminino , Frequência Cardíaca/fisiologia , Humanos , Hidrocortisona/sangue , Sistema Hipotálamo-Hipofisário/metabolismo , Masculino , Pessoa de Meia-Idade , Transtorno de Pânico/sangue , Sistema Hipófise-Suprarrenal/metabolismo , Saliva/química , Estresse Psicológico/sangue , Estresse Psicológico/psicologiaRESUMO
Endocannabinoid signaling plays a regulatory role in various (neuro)biological functions. 2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid, and although its canonical biosynthetic pathway involving phosphoinositide-specific phospholipase C and diacylglycerol lipase α is known, alternative pathways remain unsettled. Here, we characterize a noncanonical pathway implicating glycerophosphodiesterase 3 (GDE3, from GDPD2 gene). Human GDE3 expressed in HEK293T cell membranes catalyzed the conversion of lysophosphatidylinositol (LPI) into monoacylglycerol and inositol-1-phosphate. The enzyme was equally active against 1-acyl and 2-acyl LPI. When using 2-acyl LPI, where arachidonic acid is the predominant fatty acid, LC-MS analysis identified 2-AG as the main product of LPI hydrolysis by GDE3. Furthermore, inositol-1-phosphate release into the medium occurred upon addition of LPI to intact cells, suggesting that GDE3 is actually an ecto-lysophospholipase C. In cells expressing G-protein-coupled receptor GPR55, GDE3 abolished 1-acyl LPI-induced signaling. In contrast, upon simultaneous ex-pression of GDE3 and cannabinoid receptor CB2, 2-acyl LPI evoked the same signal as that induced by 2-AG. These data strongly suggest that, in addition to degrading the GPR55 LPI ligand, GDE3 can act as a switch between GPR55 and CB2 signaling. Coincident with a major expression of both GDE3 and CB2 in the spleen, spleens from transgenic mice lacking GDE3 displayed doubling of LPI content compared with WT mice. Decreased production of 2-AG in whole spleen was also observed, supporting the in vivo relevance of our findings. These data thus open a new research avenue in the field of endocannabinoid generation and reinforce the view of GPR55 and LPI being genuine actors of the endocannabinoid system.
Assuntos
Diester Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Ácidos Araquidônicos/análise , Ácidos Araquidônicos/metabolismo , Ácidos Araquidônicos/farmacologia , Endocanabinoides/análise , Endocanabinoides/metabolismo , Endocanabinoides/farmacologia , Feminino , Glicerídeos/análise , Glicerídeos/metabolismo , Glicerídeos/farmacologia , Células HEK293 , Humanos , Hidrólise , Fosfatos de Inositol/metabolismo , Lisofosfolipídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoglicerídeos/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/deficiência , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo , Receptores de Canabinoides/metabolismo , Alinhamento de Sequência , Transdução de Sinais/efeitos dos fármacos , Baço/metabolismoRESUMO
A simple and reliable method was developed and validated to determine the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples by micro salting-out assisted liquid-liquid extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry (SALLLE/UHPLC-MS/MS). The SALLE parameters (brain homogenate volume, salting-out agent, salt concentration, salt solution volume, organic solvent, organic solvent volume, and centrifugation temperature) were optimized to improve sensitivity and selectivity of the method. The SALLE/UHPLC-MS/MS method presented linear ranges from 2.00 to 20.00 ng mL-1 for AEA and from 0.300 to 10.00 µg mL-1 for 2-AG, no significant matrix effect, and inter- and intra-assay precision and accuracy with CV and RSE values lower than 15%, respectively. This innovative method was successfully applied to determine AEA and 2-AG in brain hemispheres from a 6-OHDA animal model of Parkinson's disease (PD).
Assuntos
Ácidos Araquidônicos/análise , Química Encefálica/fisiologia , Endocanabinoides/análise , Glicerídeos/análise , Extração Líquido-Líquido/métodos , Alcamidas Poli-Insaturadas/análise , Animais , Ácidos Araquidônicos/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Modelos Animais de Doenças , Endocanabinoides/isolamento & purificação , Glicerídeos/isolamento & purificação , Limite de Detecção , Modelos Lineares , Masculino , Doença de Parkinson/metabolismo , Alcamidas Poli-Insaturadas/isolamento & purificação , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodosRESUMO
Endogenous cannabinoids are an increasingly intriguing target for biological research, given the changing legal status of medicinal cannabinoid-based products throughout the world. However, studying the endogenous cannabinoid system is a relatively new field, with few research teams attempting to develop quantitative methods for these important modulatory analytes in human matrices, other than blood. Here we develop and validate simultaneous methods for quantifying arachidonoyl-ethanolamide, 2-arachidonoyl glycerol, oleoylethanolamide, cortisol and progesterone in human plasma and saliva using liquid-liquid extraction combined with ultra-high performance liquid chromatography coupled to tandem mass spectrometry. The method was fully validated over the linear concentration range 1-20 ng/mL for each analyte in plasma (R2 = 0.98-0.99) and saliva (R2 = 0.99). We find that salivary endogenous cannabinoids and cortisol are acutely responsive to exercise, suggesting that targeting the saliva system may present a convenient way for future research of endogenous cannabinoids. This finding also encourages a broader understanding of the endogenous cannabinoid system during stress responses, and our method may consequently lead to a better understanding of the role of endogenous cannabinoids in peripheral tissues.