Identification of a Widespread Palmitoylethanolamide Contamination in Standard Laboratory Glassware.

Introduction: Fatty acid ethanolamides (FAEs) are a family of lipid mediators that participate in a host of biological functions. Procedures for the quantitative analysis of FAEs include organic solvent extraction from biological matrices (e.g., blood), followed by purification and subsequent quantitation by liquid chromatography-mass spectrometry (LC/MS) or gas chromatography-mass spectrometry. During the validation process of a new method for LC/MS analysis of FAEs in biological samples, we observed unusually high levels of the FAE, palmitoylethanolamide (PEA), in blank samples that did not contain any biological material. Materials and Methods: We investigated a possible source of this PEA artifact via liquid chromatography coupled to tandem mass spectrometry, as well as accurate mass analysis. Results: We found that high levels of a contaminant indistinguishable from PEA is present in new 5.75″ glass Pasteur pipettes, which are routinely used by laboratories to carry out lipid extractions. This artifact might account for discrepancies found in the literature regarding PEA levels in human blood serum and other tissues. Conclusions: It is recommended to take into account this pitfall by analyzing potential contamination of the disposable glassware during the validation process of any method used for analysis of FAEs

Identification of a Widespread Palmitoylethanolamide Contamination in Standard Laboratory Glassware

Introduction: Fatty acid ethanolamides (FAEs) are a family of lipid mediators that participate in a host of biological functions. Procedures for the quantitative analysis of FAEs include organic solvent extraction from biological matrices (e.g., blood), followed by purification and subsequent quantitation by liquid chromatography-mass spectrometry (LC/MS) or gas chromatography-mass spectrometry. During the validation process of a new method for LC/MS analysis of FAEs in biological samples, we observed unusually high levels of the FAE, palmitoylethanolamide (PEA), in blank samples that did not contain any biological material. Materials and Methods: We investigated a possible source of this PEA artifact via liquid chromatography coupled to tandem mass spectrometry, as well as accurate mass analysis. Results: We found that high levels of a contaminant indistinguishable from PEA is present in new 5.75″ glass Pasteur pipettes, which are routinely used by laboratories to carry out lipid extractions. This artifact might account for discrepancies found in the literature regarding PEA levels in human blood serum and other tissues. Conclusions: It is recommended to take into account this pitfall by analyzing potential contamination of the disposable glassware during the validation process of any method used for analysis of FAEs

Serum Endocannabinoid Levels in Patients With End-Stage Renal Disease.

Context:Previous studies have shown that the endocannabinoid system plays a major role in energy metabolism through the actions of its main mediators, 2-arachidonoyl-sn-glycerol (2-AG) and anandamide (AEA). Objective:We examined serum levels of major endocannabinoid mediators and their association with clinical parameters in patients with end-stage renal disease (ESRD). Design and Setting:Serum concentrations of 2-AG and AEA were measured in patients on maintenance hemodialysis (MHD) and controls, and correlations with various clinical and laboratory indices were examined. 2-AG was also measured in age and sex-matched healthy subjects for comparison of levels in patients undergoing MHD. Main Outcome Measure:Serum 2-AG. Results:Serum 2-AG levels were significantly elevated in patients with ESRD compared with healthy controls. Higher levels of 2-AG were found in patients on MHD compared to healthy subjects, and similar findings were seen in a second set of subjects in independent analyses. Among 96 patients on MHD, 2-AG levels correlated significantly and positively with serum triglycerides (ρ = 0.43; P < 0.0001), body mass index (ρ = 0.40; P < 0.0001), and body anthropometric measures and negatively with serum high-density lipoprotein cholesterol (ρ = -0.33; P = 0.001) following adjustment for demographic and clinical variables. Conclusions:In patients on MHD, levels of serum 2-AG, a major endocannabinoid mediator, were increased. In addition, increasing serum 2-AG levels correlated with increased serum triglycerides and markers of body mass. Future studies will need to evaluate the potential mechanisms responsible for these findings

Spatiotemporal Alterations in Gait in Humanized Transgenic Sickle Mice

Sickle cell disease (SCD) is a hemoglobinopathy affecting multiple organs and featuring acute and chronic pain. Purkinje cell damage and hyperalgesia have been demonstrated in transgenic sickle mice. Purkinje cells are associated with movement and neural function which may influence pain. We hypothesized that Purkinje cell damage and/or chronic pain burden provoke compensatory gait changes in sickle mice. We found that Purkinje cells undergoe increased apoptosis as shown by caspase-3 activation. Using an automated gait measurement system, MouseWalker, we characterized spatiotemporal gait characteristics of humanized transgenic BERK sickle mice in comparison to control mice. Sickle mice showed alteration in stance instability and dynamic gait parameters (walking speed, stance duration, swing duration and specific swing indices). Differences in stance instability may reflect motor dysfunction due to damaged Purkinje cells. Alterations in diagonal and all stance indices indicative of hesitation during walking may originate from motor dysfunction and/or arise from fear and/or anticipation of movement-evoked pain. We also demonstrate that stance duration, diagonal swing indices and all stance indices correlate with both mechanical and deep tissue hyperalgesia, while stance instability correlates with only deep tissue hyperalgesia. Therefore, objective analysis of gait in SCD may provide insights into neurological impairment and pain states

Host- and Helminth-Derived Endocannabinoids That Have Effects on Host Immunity Are Generated during Infection.

Helminths have coevolved with their hosts, resulting in the development of specialized host immune mechanisms and parasite-specific regulatory products. Identification of new pathways that regulate helminth infection could provide a better understanding of host-helminth interaction and may identify new therapeutic targets for helminth infection. Here we identify the endocannabinoid system as a new mechanism that influences host immunity to helminths. Endocannabinoids are lipid-derived signaling molecules that control important physiologic processes, such as feeding behavior and metabolism. Following murine infection with Nippostrongylus brasiliensis, an intestinal nematode with a life cycle similar to that of hookworms, we observed increased levels of endocannabinoids (2-arachidonoylglycerol [2-AG] or anandamide [AEA]) and the endocannabinoid-like molecule oleoylethanolamine (OEA) in infected lung and intestine. To investigate endocannabinoid function in helminth infection, we employed pharmacological inhibitors of cannabinoid subtype receptors 1 and 2 (CB1R and CB2R). Compared to findings for vehicle-treated mice, inhibition of CB1R but not CB2R resulted in increased N. brasiliensis worm burden and egg output, associated with significantly decreased expression of the T helper type 2 cytokine interleukin 5 (IL-5) in intestinal tissue and splenocyte cultures. Strikingly, bioinformatic analysis of genomic and transcriptome sequencing (RNA-seq) data sets identified putative genes encoding endocannabinoid biosynthetic and degradative enzymes in many parasitic nematodes. To test the novel hypothesis that helminth parasites produce their own endocannabinoids, we measured endocannabinoid levels in N. brasiliensis by mass spectrometry and quantitative PCR and found that N. brasiliensis parasites produced endocannabinoids, especially at the infectious larval stage. To our knowledge, this is the first report of helminth- and host-derived endocannabinoids that promote host immune responses and reduce parasite burden

Peripheral Endocannabinoid Control of Feeding Behavior and Obesity

The endocannabinoid (eCB) system is a key modulator of central brain processes that control feeding, however recent evidence points to peripheral mechanisms by which eCB signaling may modulate feeding in various disease states. This body of work provides novel mass spectrometric analyses for components of the eCB system, while describing the pitfalls of currently used techniques. Using the described methods, a role for increased small intestinal eCB ligands signaling via peripheral cannabinoid type 1 receptor (CB1R) in the control of hyperphagia is discussed in the context of obesity. These findings were enhanced by further scrutiny of the intestinal eCB system, which revealed that corn-oil induced secretion of an intestine-derived satiation peptide, cholecystokinin (CCK), is blunted by CB1R activation; activation occurred via pharmacological agonists or enhanced eCB levels following diet induced obesity. Further, administration of a CCKA receptor antagonist, devazepide, blocked the anorexigenic effects of peripheral CB1R restriction, suggesting that peripheral CB1R act via intestine derived CCK to block satiation by impaired gut-brain signaling. Additionally, helminth infection was used to evaluate intestinal and lung eCB levels in a model of hookworm infection. For the first time, it was observed that the helminth Nippostrongylus brasiliensis produces endocannabinoids. Together, this body of work provides novel insight to the varied roles that intestinal and peripheral eCBs have during obesity in the modulation of feeding behaviors and immune responses

Considerations for Cannabis Use to Treat Pain in Sickle Cell Disease.

Pain in Sickle Cell Disease (SCD) is a major comorbidity and unique with acute pain due to recurrent and episodic vaso-occlusive crises as well as chronic pain, which can span an individual’s entire life. Opioids are the mainstay treatment for pain in SCD. Due to recent health crises raised by adverse effects including deaths from opioid use, pain management in SCD is adversely affected. Cannabis and its products are most widely used for pain in multiple conditions and also by patients with SCD on their own. With the availability of “Medical Cannabis” and approval to use cannabis as medicine across majority of States in the United States as well as over-the-counter preparations, cannabis products are being used increasingly for SCD. The reliability of many of these products remains questionable, which poses a major health risk to the vulnerable individuals seeking pain relief. Therefore, this review provides up to date insights into available categories of cannabis-based treatment strategies, their mechanism of action and pre-clinical and clinical outcomes in SCD. It provides evidence for the benefits and risks of cannabis use in SCD and cautions about the unreliable and unvalidated products that may be adulterated with life-threatening non-cannabis compounds