RESUMO
PURPOSE: We developed and validated a method for quantitative analysis of ten synthetic cathinones in oral fluid (OF) samples, using microextraction by packed sorbent (MEPS) for sample preparation followed by liquid chromatographyâtandem mass spectrometry (LCâMS/MS). METHOD: OF samples were collected with a Quantisal™ device and 200 µL was extracted using a C18 MEPS cartridge installed on a semi-automated pipette and then analyzed using LCâM/SMS. RESULTS: Linearity was achieved between 0.1 and 25 ng/mL, with a limit of detection (LOD) of 0.05 ng/mL and a limit of quantification (LOQ) of 0.1 ng/mL. Imprecision (% relative standard deviation) and bias (%) were better than 11.6% and 7.5%, respectively. The method had good specificity and selectivity against 9 different blank OF samples (from different donors) and 68 pharmaceutical and drugs of abuse with concentrations varying between 400 and 10,000 ng/mL. No evidence of carryover was observed. The analytes were stable after three freeze/thaw cycles and when kept in the autosampler (10 °C) for up to 24 h. The method was successfully applied to quantify 41 authentic positive samples. Methylone (mean 0.6 ng/mL, median 0.2 ng/mL), N-ethylpentylone (mean 16.7 ng/mL, median 0.35 ng/mL), eutylone (mean 39.1 ng/mL, median 3.6 ng/mL), mephedrone (mean 0.5 ng/mL, median 0.5 ng/mL), and 4-chloroethcathinone (8.1 ng/mL) were quantified in these samples. CONCLUSION: MEPS was an efficient technique for Green Analytical Toxicology purposes, which required only 650 µL organic solvent and 200 µL sodium hydroxide, and the BIN cartridge had a lifespan of 100 sample extractions.
Assuntos
Catinona Sintética , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Microextração em Fase Sólida/métodos , Limite de DetecçãoRESUMO
BACKGROUND: Epilepsy is a neurological disease affected by an imbalance of inhibitory and excitatory signaling in the brain. INTRODUCTION: In this disease, the targets are active in pathophysiology and thus can be used as a focus for pharmacological treatment. METHODS: Several studies demonstrated the antiepileptic effect of drugs acting on the following targets: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, voltage-gated calcium channel (Cav), Gamma aminobutyric acid transporter type 1 (GAT1), voltage-gated sodium channels (Nav), voltage-gated potassium channel of the Q subfamily (KCNQ) and Gamma aminobutyric acid type A (GABAA) receiver. RESULTS: These studies highlight the importance of molecular docking. CONCLUSION: Quantitative Structure-Activity Relationship (QSAR) and computer aided drug design (CADD) in predicting of possible pharmacological activities of these targets.
Assuntos
Epilepsia , Humanos , Simulação de Acoplamento Molecular , Epilepsia/tratamento farmacológico , Receptores de AMPA/fisiologia , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Ácido gama-Aminobutírico/uso terapêuticoRESUMO
The GATs are the membrane proteins responsible for the uptake of GABA in the central nervous system. Alterations in GAT activity are implicated in several neurological diseases, including retinopathies. The present study describes an alternative method to determine GAT activity in tissue preparations of the central nervous system, using high performance liquid chromatography (HPLC) with fluorescence detection. The GABA concentration in the medium was determined using the o-phthaldehyde (OPA)-derivation protocol validated by the Brazilian Health Regulatory Agency (ANVISA) and the United States Food and Drug Administration (US-FDA). The GAT activity in the retinal preparations was determined through the evaluation of the GABA uptake, which was measured by assessing the difference between the initial and final concentrations of GABA in the incubation medium. The evaluation of the GAT kinetics returned values of Km = 382.5 ± 32.2 µM and Vmax = 34 nmol/mg of protein. The data also demonstrated that the GABA uptake was predominantly Na+- and temperature-dependent, and was also inhibited by incubation with nipecotic acid, a substrate of GABA transporters. Taken together, these findings confirm that our approach provided a specific measure of GAT activity in retinal tissue. The data presented here thus validate, for the first time, an alternative, simple and sensitive method for the evaluation of GAT activity using high performance chromatography on preparations of the central nervous system.
Assuntos
Retina , Ácido gama-Aminobutírico , Sistema Nervoso Central/metabolismo , Cromatografia Líquida de Alta Pressão , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Retina/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
The spontaneously hypertensive rat (SHR) is an excellent animal model that mimics the behavioral and neurochemical phenotype of attention-deficit/hyperactivity disorder (ADHD). Here, we characterized the striatal GABA transport of SHR and investigated whether caffeine, a non-selective antagonist of adenosine receptors, could influence GABAergic circuitry. For this purpose, ex vivo striatal slices of SHR and Wistar (control strain) on the 35th postnatal day were dissected and incubated with [3H]-GABA to quantify the basal levels of uptake and release. SHR exhibited a reduced [3H]-GABA uptake and release, suggesting a defective striatal GABAergic transport system. GAT-1 appears to be the primary transporter for [3H]-GABA uptake in SHR striatum, as GAT-1 selective blocker, NO-711, completely abolished it. We also verified that acute exposure of striatal slices to caffeine improved [3H]-GABA uptake and release in SHR, whereas Wistar rats were not affected. GABA-uptake increase and cAMP accumulation promoted by caffeine was reverted by A1R activation with N6-cyclohexyl adenosine (CHA). As expected, the pharmacological blockade of cAMP-PKA signaling by H-89 also prevented caffeine-mediated [3H]-GABA uptake increment. Interestingly, a single caffeine exposure did not affect GAT-1 or A1R protein density in SHR, which was not different from Wistar protein levels, suggesting that the GAT-1-dependent transport in SHR has a defective functional activity rather than lower protein expression. The current data support that caffeine regulates GAT-1 function and improves striatal GABA transport via A1R-cAMP-PKA signaling, specifically in SHR. These results reinforce that caffeine may have therapeutic use in disorders where the GABA transport system is impaired.
Assuntos
Cafeína/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Corpo Estriado/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Western Blotting , Corpo Estriado/metabolismo , Feminino , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos WistarRESUMO
The abuse of stimulants such as amphetamine, methamphetamine, ecstasy (MDMA), and their analogues (MDEA and MDA) has been increasing considerably worldwide since 2009. In this work, an analytical method using dispersive liquid-liquid microextraction (DLLME) to determine amphetamine and derivatives in oral fluid samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. Linearity was achieved between 20 to 5000 ng/mL (r>0.992, 1/x² weighted linear regression), with a limit of quantification (LOQ) of 20 ng/mL. Imprecision (%relative standard deviation) and bias (%) were not higher than 9.1 and -12.3%, respectively. The matrix effect was lower than 14.6%, with no carryover observed up to 5000 ng/mL and no interference with 10 different oral fluid matrix sources and against 14 pharmaceuticals and other common drugs of abuse. MDMA, MDA, and MDEA in processed samples were stable up to 24 h at autosampler (10°C); and amphetamine and methamphetamine up to 18 h. The developed method was successfully applied to authentic oral fluid analyses (n = 140). The proposed method is an example of the Green Analytical Toxicology, since it reduces both the amount of solvent required in samples preparation and the quantity of solvents and reagents used in analytical-instrumental stage, as well as requires a minimal sample volume, being a cheaper, quicker and more ecological alternative to conventional methods. Obtained results showed that DLLME extraction combined with LC-MS/MS is a fast and simple method to quantify amphetamine derivatives in oral fluid samples.
Assuntos
Estimulantes do Sistema Nervoso Central , Microextração em Fase Líquida , Anfetamina , Cromatografia Líquida , Espectrometria de Massas em TandemRESUMO
Cocaine (COC) is a psychostimulant that acts by increasing catecholaminergic neurotransmission mainly due to its effects on the dopamine transporter (DAT). However, other neurotransmitter systems may also be regulated by COC, including the GABAergic system. Since the effect of COC in modulating gamma-aminobutyric acid (GABA) reuptake is not defined, we investigated the molecular mechanisms related to the increase in GABA uptake induced by acute COC exposure and its effects on locomotor activity in adolescent mice. Behavioral experiments showed that COC increased locomotor activity and decreased immobilization time in mice. A single COC exposure reduced both GABA uptake and GAT-1 protein levels. On the other hand, cyclic adenosine monophosphate (cAMP) levels increased after a COC challenge. The major changes induced by acute COC on behavioral and neurochemical assays were avoided by previous treatment with the selective D1 receptor antagonist SCH-23390 (0.5 mg/kg). Our findings suggest that GABA uptake naturally decreases during mice development from preadolescence until adulthood and that dopamine (DA) D1-like receptors are key players in the regulation of GABA uptake levels following a single COC exposure in adolescent mice.
Assuntos
Cocaína/farmacologia , Dopamina/metabolismo , Lobo Frontal/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/efeitos dos fármacos , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/administração & dosagem , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Lobo Frontal/metabolismo , Camundongos , Atividade Motora/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismoRESUMO
GABA transporters regulate synaptic GABA levels and dysfunctions in this system might result in psychiatric disorders. The endocannabinoid system (ECS) is the main circuit breaker in the nervous system and may alter noradrenaline (NA) communication, which in turn modulates the release of GABA. However, a close relationship between these systems has not been recognized. We asked whether NA and ECS might control extracellular GABA levels in slices of frontal cortex (FC) of adolescent Swiss mice with 40 days after birth (PN40). Here we show that NA and isoproterenol (ISO), a beta-adrenergic agonist, increased [3H]-GABA uptake in mice FC, while alpha1-adrenergic agonist phenylephrine had no effect. As GAT-1 is expressed and fully functional at the FC, addition of NO-711, a GAT-1 inhibitor, dose dependently blocked [3H]-GABA uptake. The increase of [3H]-GABA uptake induced by ISO was also blocked by NO-711. [3H]-GABA release induced by 80â¯mM KCl was reduced by NO-711, but not by removal of Ca2+. ISO also increased cyclic AMP (cAMP) levels and addition of WIN 55,212-2, a mixed CB1/CB2 receptor agonist, inhibited the effect of ISO in GABA uptake increase, GAT-1 expression and cAMP levels compared to control. Our data show that GABA transport increased by NA and ISO is negatively regulated by cannabinoid receptor agonist WIN55,212-2.
Assuntos
Benzoxazinas/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Lobo Frontal/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de GABA/efeitos dos fármacos , Morfolinas/farmacologia , Naftalenos/farmacologia , Animais , Endocanabinoides/metabolismo , Lobo Frontal/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Camundongos , Receptor CB1 de Canabinoide/efeitos dos fármacos , Receptor CB1 de Canabinoide/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/efeitos dos fármacosRESUMO
BACKGROUND: Zika virus (ZIKV) and dengue virus (DENV) are mosquito-borne flaviviruses prevalent throughout tropical regions. Currently, management of ZIKV and DENV centers on control of the primary vector Aedes aegypti. This vector is highly anthropophilic and is therefore prevalent throughout densely urbanised landscapes. A new passive trap for gravid Ae. aegypti (Gravid Aedes Trap - GAT) was developed for mosquito surveillance. Here the different killing agents and the level of transmission of arboviruses that may occur in mosquitoes sampled by GATs are assessed for the first time. METHODS: Gravid Aedes traps (GATs) were deployed at the Federal University of Minas Gerais campus, in Belo Horizonte, Brazil to sample Ae. aegypti. Three different killing agents were evaluated within the GATs: sticky cards, long-lasting insecticide-impregnated nets (LLINs) and canola oil. Traps were monitored weekly for 14 weeks then mosquito specimens were identified to the species level and Ae. aegypti catches were pooled and submitted to qRT-PCR assays for to DENV and ZIKV virus detection, followed by Bayesian phylogenetic analysis of the ZIKV. Additionally, comparisons of means were performed on transformed weekly catch data (P = 0.05, t-tests) with the stats package of the R statistical software. RESULTS: In total, 1506 female Ae. aegypti were captured using GATs, with traps using sticky cards catching more mosquito than those using either LLINs or canola oil. Both ZIKV and DENV were detected in Ae. aegypti females captured over several weeks suggesting that this highly populated university campus may have served as a significant transmission hub. The infection rate for ZIKV was present in seven (8.5%) pools from four weeks while DENV was detected in four (4.9%) pools from four weeks. Phylogenetic analysis of ZIKV classified the strain as Asian genotype. CONCLUSIONS: The Federal University of Minas Gerais and similar organizations must strongly consider monitoring Ae. aegypti populations and reinforcing personal protection of staff and students during seasons of high mosquito activity.