A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma.

Free arachidonic acid is functionally interlinked with different lipid signaling networks including those involving prostanoid pathways, the endocannabinoid system, N-acylethanolamines, as well as steroids. A sensitive and specific LC-MS/MS method for the quantification of arachidonic acid, prostaglandin E2, thromboxane B2, anandamide, 2-arachidonoylglycerol, noladin ether, lineoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, steroyl ethanolamide, aldosterone, cortisol, dehydroepiandrosterone, progesterone, and testosterone in human plasma was developed and validated. Analytes were extracted using acetonitrile precipitation followed by solid phase extraction. Separations were performed by UFLC using a C18 column and analyzed on a triple quadrupole MS with electron spray ionization. Analytes were run first in negative mode and, subsequently, in positive mode in two independent LC-MS/MS runs. For each analyte, two MRM transitions were collected in order to confirm identity. All analytes showed good linearity over the investigated concentration range (r>0.98). Validated LLOQs ranged from 0.1 to 190ng/mL and LODs ranged from 0.04 to 12.3ng/mL. Our data show that this LC-MS/MS method is suitable for the quantification of a diverse set of bioactive lipids in plasma from human donors (n=32). The determined plasma levels are in agreement with the literature, thus providing a versatile method to explore pathophysiological processes in which changes of these lipids are implicated.

Sulla proposta di un collegio rabbinico italiano : osservazioni

del M. A. Salom

Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla.

The endocannabinoid system (ECS) comprises the cannabinoid receptors CB1 and CB2 and their endogenous arachidonic acid-derived agonists 2-arachidonoyl glycerol and anandamide, which play important neuromodulatory roles. Recently, a novel class of negative allosteric CB1 receptor peptide ligands, hemopressin-like peptides derived from alpha hemoglobin, has been described, with yet unknown origin and function in the CNS. Using monoclonal antibodies we now identified the localization of RVD-hemopressin (pepcan-12) and N-terminally extended peptide endocannabinoids (pepcans) in the CNS and determined their neuronal origin. Immunohistochemical analyses in rodents revealed distinctive and specific staining in major groups of noradrenergic neurons, including the locus coeruleus (LC), A1, A5 and A7 neurons, which appear to be major sites of production/release in the CNS. No staining was detected in dopaminergic neurons. Peptidergic axons were seen throughout the brain (notably hippocampus and cerebral cortex) and spinal cord, indicative of anterograde axonal transport of pepcans. Intriguingly, the chromaffin cells in the adrenal medulla were also strongly stained for pepcans. We found specific co-expression of pepcans with galanin, both in the LC and adrenal gland. Using LC-MS/MS, pepcan-12 was only detected in non-perfused brain (∼40 pmol/g), suggesting that in the CNS it is secreted and present in extracellular compartments. In adrenal glands, significantly more pepcan-12 (400-700 pmol/g) was measured in both non-perfused and perfused tissue. Thus, chromaffin cells may be a major production site of pepcan-12 found in blood. These data uncover important areas of peptide endocannabinoid occurrence with exclusive noradrenergic immunohistochemical staining, opening new doors to investigate their potential physiological function in the ECS. This article is part of a Special Issue entitled 'Fluorescent Neuro-Ligands'.

Elevated levels of endocannabinoids in chronic hepatitis C may modulate cellular immune response and hepatic stellate cell activation.

The endocannabinoid (EC) system is implicated in many chronic liver diseases, including hepatitis C viral (HCV) infection. Cannabis consumption is associated with fibrosis progression in patients with chronic hepatitis C (CHC), however, the role of ECs in the development of CHC has never been explored. To study this question, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) were quantified in samples of HCV patients and healthy controls by gas and liquid chromatography mass spectrometry. Fatty acid amide hydrolase (FAAH) and monoaclyglycerol lipase (MAGL) activity was assessed by [3H]AEA and [3H]2-AG hydrolysis, respectively. Gene expression and cytokine release were assayed by TaqMan PCR and ELISpot, respectively. AEA and 2-AG levels were increased in plasma of HCV patients, but not in liver tissues. Hepatic FAAH and MAGL activity was not changed. In peripheral blood mononuclear cells (PBMC), ECs inhibited IFN-γ, TNF-α, and IL-2 secretion. Inhibition of IL-2 by endogenous AEA was stronger in PBMC from HCV patients. In hepatocytes, 2-AG induced the expression of IL-6, -17A, -32 and COX-2, and enhanced activation of hepatic stellate cells (HSC) co-cultivated with PBMC from subjects with CHC. In conclusion, ECs are increased in plasma of patients with CHC and might reveal immunosuppressive and profibrogenic effects.

Pharmacokinetics and pharmacodynamics of γ-hydroxybutyrate in healthy subjects.

AIMS γ-Hydroxybutyrate (GHB) is used as a treatment for narcolepsy and alcohol withdrawal and as recreational substance. Nevertheless, there are limited data on the pharmacokinetics and pharmacokinetic-pharmacodynamic relationship of GHB in humans. We characterized the pharmacokinetic profile and exposure-psychotropic effect relationship of GHB in humans. METHODS Two oral doses of GHB (25 and 35 mg/kg) were administered to 32 healthy male subjects (16 for each dose) using a randomized, placebo-controlled, cross-over design. RESULTS Maximal concentrations of GHB were (geometric mean and 95%CI): 218 (176-270) nmol/ml and 453 (374-549) nmol/ml for the 25 and 35 mg/kg GHB doses, respectively. The elimination half-lives (mean ± SD) were 36 ± 9 and 39 ± 7 min and the AUC∞ values (geometric mean and 95%CI) were 15,747 (12,854-19,290) and 40,113 (33,093-48,622) nmol∙min/ml for the 20 and 35 mg/kg GHB doses, respectively. Thus, plasma GHB exposure (AUC0-∞ ) rose disproportionally (+40%) with the higher dose. γ-Hydroxybutyrate produced mixed stimulant-sedative effects, with a dose-dependent increase in sedation and dizziness. It did not alter heart rate or blood pressure. A close relationship between plasma GHB exposure and its psychotropic effects was found, with higher GHB concentrations associated with higher subjective stimulation, sedation, and dizziness. No clockwise hysteresis was observed in the GHB concentration effect plot over time (i.e., no acute pharmacological tolerance). CONCLUSION Evidence was found of a non-linear dose-exposure relationship (i.e., no dose proportionality) at moderate doses of GHB. The effects of GHB on consciousness were closely linked to its plasma exposure and exhibited no acute tolerance. This article is protected by copyright. All rights reserved.

Quantitative analysis of arachidonic acid, endocannabinoids, N-acylethanolamines and steroids in biological samples by LCMS/MS: Fit to purpose.

Arachidonic acid (5Z,8Z,11Z,14Z-eicosatetraenoic acid; C20:4) (arachidonate, AA) is a vital polyunsaturated omega-6 fatty acid (PUFA) without its presence the mammalian brain, muscles, and possibly other organs cannot develop or function [1] and [2]. AA fulfils numerous known and possibly yet unknown functions as integral part of mammalian phospholipid membranes and as free AA which also acts as a precursor of a variety of biologically active lipid mediators generally referred to as eicosanoids (e.g., prostaglandins, leukotrienes). A more recent class of eicosanoids is composed of the endogenous cannabinoids (endocannabinoids) 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide (anandamide, AEA), which act on cannabinoid CB1 and CB2 receptors but also modulate ion channels and nuclear receptors [3] and [4]. In recent years, the role of endocannabinoids as prominent anti-inflammatory and neuromodulatory eicosanoids has been shown by numerous studies [5].

Gamma-hydroxybutyrate enhances mood and prosocial behavior without affecting plasma oxytocin and testosterone.

Gamma-hydroxybutyrate (GHB) is a GHB-/GABAB-receptor agonist. Reports from GHB abusers indicate euphoric, prosocial, and empathogenic effects of the drug. We measured the effects of GHB on mood, prosocial behavior, social and non-social cognition and assessed potential underlying neuroendocrine mechanisms. GHB (20mg/kg) was tested in 16 healthy males, using a randomized, placebo-controlled, cross-over design. Subjective effects on mood were assessed by visual-analogue-scales and the GHB-Specific-Questionnaire. Prosocial behavior was examined by the Charity Donation Task, the Social Value Orientation test, and the Reciprocity Task. Reaction time, memory, empathy, and theory-of-mind were also tested. Blood plasma levels of GHB, oxytocin, testosterone, progesterone, dehydroepiandrosterone (DHEA), cortisol, aldosterone, and adrenocorticotropic-hormone (ACTH) were determined. GHB showed stimulating and sedating effects, and elicited euphoria, disinhibition, and enhanced vitality. In participants with low prosociality, the drug increased donations and prosocial money distributions. In contrast, social cognitive abilities such as emotion recognition, empathy, and theory-of-mind, and basal cognitive functions were not affected. GHB increased plasma progesterone, while oxytocin and testosterone, cortisol, aldosterone, DHEA, and ACTH levels remained unaffected. GHB has mood-enhancing and prosocial effects without affecting social hormones such as oxytocin and testosterone. These data suggest a potential involvement of GHB-/GABAB-receptors and progesterone in mood and prosocial behavior.

4'-O-methylhonokiol increases levels of 2-arachidonoyl glycerol in mouse brain via selective inhibition of its COX-2-mediated oxygenation

BACKGROUND AND PURPOSE 4'-O-methylhonokiol (MH) is a natural product showing anti-inflammatory, anti-osteoclastogenic, and neuroprotective effects. MH was reported to modulate cannabinoid CB2 receptors as an inverse agonist for cAMP production and an agonist for intracellular [Ca2+]. It was recently shown that MH inhibits cAMP formation via CB2 receptors. In this study, the exact modulation of MH on CB2 receptor activity was elucidated and its endocannabinoid substrate-specific inhibition (SSI) of cyclooxygenase-2 (COX-2) and CNS bioavailability are described for the first time. METHODS CB2 receptor modulation ([35S]GTPγS, cAMP, and β-arrestin) by MH was measured in hCB2-transfected CHO-K1 cells and native conditions (HL60 cells and mouse spleen). The COX-2 SSI was investigated in RAW264.7 cells and in Swiss albino mice by targeted metabolomics using LC-MS/MS. RESULTS MH is a CB2 receptor agonist and a potent COX-2 SSI. It induced partial agonism in both the [35S]GTPγS binding and β-arrestin recruitment assays while being a full agonist in the cAMP pathway. MH selectively inhibited PGE2 glycerol ester formation (over PGE2) in RAW264.7 cells and significantly increased the levels of 2-AG in mouse brain in a dose-dependent manner (3 to 20 mg kg(-1)) without affecting other metabolites. After 7 h from intraperitoneal (i.p.) injection, MH was quantified in significant amounts in the brain (corresponding to 200 to 300 nM). CONCLUSIONS LC-MS/MS quantification shows that MH is bioavailable to the brain and under condition of inflammation exerts significant indirect effects on 2-AG levels. The biphenyl scaffold might serve as valuable source of dual CB2 receptor modulators and COX-2 SSIs as demonstrated by additional MH analogs that show similar effects. The combination of CB2 agonism and COX-2 SSI offers a yet unexplored polypharmacology with expected synergistic effects in neuroinflammatory diseases, thus providing a rationale for the diverse neuroprotective effects reported for MH in animal models.

La Matriz Conservadora en el Peronismo Cordobés y el Intervencionismo Sistemático

El tema que recorre nuestro estudio es el nacimiento del Partido Peronista en la Provincia de Córdoba. Entendemos que se trata de un caso particular pues dicha provincia trae consigo los resabios y las huellas de gobiernos como el de Amadeo Sabattini, en el cual se destacaba un radicalismo popular e yrigoyenista que apostaba a la industrialización de la provincia y sostenía que la tierra debía ser para quien la trabajara. Analizaremos también la relación que se observa entre el Partido Laborista y el naciente peronismo. Allí observaremos la división que provoca en los laboristas la orden de unificación, emanada por Perón, dentro del Partido Peronista. Esta situación nos llevará a relevar la posición de los sindicatos y su relación con los partidos políticos y con el Estado. A su vez entendemos que las reacciones que ha tenido el Partido Laborista en la década de los 40, puede compararse con ciertas actitudes y respuestas que ha dado la agrupación político-militar 'Montoneros' en los años 70 ante circunstancias similares. Otro aspecto que nos ayuda a comprender el peronismo cordobés es el intervencionismo que se observa por parte de Perón en su Partido Político, inteverncionismo emanado desde la cúpula porteña

Nacimiento del Clasismo Sindical

El tema que hemos elegido para nuestra ponencia es el nacimiento del clasismo sindical. El mismo será observado y analizado desde la provincia de Córdoba, luego del Golpe de Estado de 1955. El exponente máximo de esta corriente político-sindical es Agustín Tosco, líder del sindicato de Luz y Fuerza, como él han surgido distintas expresiones sindicales que convergen o suplantan expresiones políticas. El sindicalismo, luego del Golpe de Estado, se encuentra frente a una situación desconocida, ya que debe resolver problemas políticos que antes se relegaban a los partidos. El cambio implica la imposibilidad de seguir manteniéndose dentro del aspecto puramente administrativo, esto es, abandonar su lugar de burócratas sindicales. Los trabajadores de 1945 encontraron en Perón, el 17 de octubre, la expresión de su unidad como actor político; sin embargo bajo el nuevo contexto dicha unidad se ve trastocada, dando lugar al nacimiento de un conjunto basado en lazos horizontales. Es decir, las decisiones se encontraban fuera de ellos en un líder externo y en esta situación política resurgen en el interior del movimiento obrero los nuevos dirigentes que analizaremos

La Matriz Conservadora en el Peronismo Cordobés y el Intervencionismo Sistemático

El tema que recorre nuestro estudio es el nacimiento del Partido Peronista en la Provincia de Córdoba. Entendemos que se trata de un caso particular pues dicha provincia trae consigo los resabios y las huellas de gobiernos como el de Amadeo Sabattini, en el cual se destacaba un radicalismo popular e yrigoyenista que apostaba a la industrialización de la provincia y sostenía que la tierra debía ser para quien la trabajara. Analizaremos también la relación que se observa entre el Partido Laborista y el naciente peronismo. Allí observaremos la división que provoca en los laboristas la orden de unificación, emanada por Perón, dentro del Partido Peronista. Esta situación nos llevará a relevar la posición de los sindicatos y su relación con los partidos políticos y con el Estado. A su vez entendemos que las reacciones que ha tenido el Partido Laborista en la década de los 40, puede compararse con ciertas actitudes y respuestas que ha dado la agrupación político-militar 'Montoneros' en los años 70 ante circunstancias similares. Otro aspecto que nos ayuda a comprender el peronismo cordobés es el intervencionismo que se observa por parte de Perón en su Partido Político, inteverncionismo emanado desde la cúpula porteña

Nacimiento del Clasismo Sindical

El tema que hemos elegido para nuestra ponencia es el nacimiento del clasismo sindical. El mismo será observado y analizado desde la provincia de Córdoba, luego del Golpe de Estado de 1955. El exponente máximo de esta corriente político-sindical es Agustín Tosco, líder del sindicato de Luz y Fuerza, como él han surgido distintas expresiones sindicales que convergen o suplantan expresiones políticas. El sindicalismo, luego del Golpe de Estado, se encuentra frente a una situación desconocida, ya que debe resolver problemas políticos que antes se relegaban a los partidos. El cambio implica la imposibilidad de seguir manteniéndose dentro del aspecto puramente administrativo, esto es, abandonar su lugar de burócratas sindicales. Los trabajadores de 1945 encontraron en Perón, el 17 de octubre, la expresión de su unidad como actor político; sin embargo bajo el nuevo contexto dicha unidad se ve trastocada, dando lugar al nacimiento de un conjunto basado en lazos horizontales. Es decir, las decisiones se encontraban fuera de ellos en un líder externo y en esta situación política resurgen en el interior del movimiento obrero los nuevos dirigentes que analizaremos

La Matriz Conservadora en el Peronismo Cordobés y el Intervencionismo Sistemático

El tema que recorre nuestro estudio es el nacimiento del Partido Peronista en la Provincia de Córdoba. Entendemos que se trata de un caso particular pues dicha provincia trae consigo los resabios y las huellas de gobiernos como el de Amadeo Sabattini, en el cual se destacaba un radicalismo popular e yrigoyenista que apostaba a la industrialización de la provincia y sostenía que la tierra debía ser para quien la trabajara. Analizaremos también la relación que se observa entre el Partido Laborista y el naciente peronismo. Allí observaremos la división que provoca en los laboristas la orden de unificación, emanada por Perón, dentro del Partido Peronista. Esta situación nos llevará a relevar la posición de los sindicatos y su relación con los partidos políticos y con el Estado. A su vez entendemos que las reacciones que ha tenido el Partido Laborista en la década de los 40, puede compararse con ciertas actitudes y respuestas que ha dado la agrupación político-militar 'Montoneros' en los años 70 ante circunstancias similares. Otro aspecto que nos ayuda a comprender el peronismo cordobés es el intervencionismo que se observa por parte de Perón en su Partido Político, inteverncionismo emanado desde la cúpula porteña

Nacimiento del Clasismo Sindical

El tema que hemos elegido para nuestra ponencia es el nacimiento del clasismo sindical. El mismo será observado y analizado desde la provincia de Córdoba, luego del Golpe de Estado de 1955. El exponente máximo de esta corriente político-sindical es Agustín Tosco, líder del sindicato de Luz y Fuerza, como él han surgido distintas expresiones sindicales que convergen o suplantan expresiones políticas. El sindicalismo, luego del Golpe de Estado, se encuentra frente a una situación desconocida, ya que debe resolver problemas políticos que antes se relegaban a los partidos. El cambio implica la imposibilidad de seguir manteniéndose dentro del aspecto puramente administrativo, esto es, abandonar su lugar de burócratas sindicales. Los trabajadores de 1945 encontraron en Perón, el 17 de octubre, la expresión de su unidad como actor político; sin embargo bajo el nuevo contexto dicha unidad se ve trastocada, dando lugar al nacimiento de un conjunto basado en lazos horizontales. Es decir, las decisiones se encontraban fuera de ellos en un líder externo y en esta situación política resurgen en el interior del movimiento obrero los nuevos dirigentes que analizaremos