RESUMO
BACKGROUND: Vascular endothelial injury during ischemia generates apoptotic cell death and precedes apoptosis of underlying tissues. We aimed at studying the role of extracellular adenosine triphosphate (ATP) on endothelial cells protection against hypoxia injury. METHODS: In a hypoxic model on endothelial cells, we quantified the extracellular concentration of ATP and adenosine. The expression of mRNA (ectonucleotidases, adenosine, and P2 receptors) was measured. Apoptosis was evaluated by the expression of cleaved caspase 3. The involvement of P2 and adenosine receptors and signaling pathways was investigated using selective inhibitors. RESULTS: Hypoxic stress induced a significant increase in extracellular ATP and adenosine. After a 2-h hypoxic injury, an increase of cleaved caspase 3 was observed. ATP anti-apoptotic effect was prevented by suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and CGS15943, as well as by selective A2A, A2B, and A3 receptor antagonists. P2 receptor-mediated anti-apoptotic effect of ATP involved phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinases (ERK1/2), mitoKATP, and nitric oxide synthase (NOS) pathways whereas adenosine receptor-mediated anti-apoptotic effect involved ERK1/2, protein kinase A (PKA), and NOS. CONCLUSIONS: These results suggest a complementary role of P2 and adenosine receptors in ATP-induced protective effects against hypoxia injury of endothelial. This could be considered therapeutic targets to limit the development of ischemic injury of organs such as heart, brain, and kidney.
Assuntos
Trifosfato de Adenosina/metabolismo , Apoptose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Hipóxia/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina/metabolismo , Apoptose/genética , Biomarcadores , Espaço Extracelular/metabolismo , Expressão Gênica , Humanos , Hipóxia/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/genética , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P2/genética , Transdução de Sinais , Estresse Fisiológico/genéticaRESUMO
During ischemia, numerous effective endogenous extracellular mediators have been identified, particularly, nucleosides such as adenosine as well as purinergic and pyrimidinergic nucleotides. They may play important regulatory roles within the cardiovascular system and notably as cardio-protectants. Indeed, the distribution of the P2Y receptors in mammalian heart includes several cellular constituents relevant for the pathophysiology of myocardial ischemia. Beside the well-known cardioprotective effect of adenosine, the additional protective role of P2Y receptors has emerged. However, interpretation of experimental results may be sometimes perplexing. This is due to the variability of: the experimental models, the endpoints criteria, the chemical structure of agonist and antagonist ligands and their concentrations, the sequences of drug administration with respect to the model used (before and/or during and/or after ischemia). The net effect may be in the opposite direction after a transient or a prolonged stimulation. Nevertheless, the overall reading of published data highlights the beneficial role of the P2Y2/4 receptor stimulation, the useful and synergistic role of P2Y6/11 receptor activation and even of the P2Y11 receptor alone in cardioprotection. More, the P2Y11 receptor could be involved in counter-regulation of profibrotic processes. Paradoxically, transient P2X7 receptor stimulation could contribute to the net cardioprotective effect of ATP. Recently, experimental data have shown that blocking the P2Y12 receptor after ischemia confers cardioprotection independently of platelet antiaggregatory effect. This suggests for P2Y receptors an important role in primary prevention and as a therapeutic target in myocardial protection during ischemia and reperfusion.
Assuntos
Traumatismo por Reperfusão Miocárdica/prevenção & controle , Receptores Purinérgicos P2Y/fisiologia , Cálcio/metabolismo , Humanos , Receptores Purinérgicos P2/fisiologia , Transdução de Sinais , Uridina Trifosfato/fisiologiaRESUMO
Adrenocortical carcinoma (ACC) is an aggressive malignancy of the adrenal gland. Mitotane (o,p'-DDD) is the most effective chemotherapy for ACC. According to the literature, mitotane plasma trough concentrations within 14-20 mg L-1 are correlated with a higher response rate with acceptable toxicity. Therapeutic drug monitoring (TDM) of mitotane is therefore recommended. The aim of this study was to propose a robust and simple method for mitotane quantification in plasma. The validation procedures were based on international guidelines. Sample preparation consisted of a single protein precipitation with methanol using 100 µL of plasma. The supernatant was submitted to liquid chromatography coupled with ultra-violet detection at 230 nm. Mitotane retention time was 7.1 min. The limit of detection was 0.1 mg L-1 and the limit of quantification was 0.78 mg L-1 . The assay demonstrated a linear range of 0.78-25 mg L-1 with correlation coefficients (r2 ) at 0.999. Inter- and intra-assay precision was <4.85%. Evaluation of accuracy showed a deviation <13.69% from target concentration at each quality control level. This method proved easy and rapid to perform mitotane TDM and required a small volume of sample. It was successfully applied to routine TDM in our laboratory.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Monitoramento de Medicamentos/métodos , Mitotano/sangue , Adulto , Feminino , Humanos , Limite de Detecção , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Mitotano/química , Mitotano/farmacocinética , Reprodutibilidade dos TestesRESUMO
AIMS: Nefopam is a nonmorphinic central analgesic, for which no recommendation exists concerning adaptation of regimen in aged patients with or without renal impairment. The objective was to describe the pharmacology of nefopam in aged patients to obtain guidelines for practical use. METHODS: Elderly patients (n = 48), 65-99 years old, with severe or moderate renal impairment or with normal renal function, were recruited. Nefopam (20 mg) was administered as a 30 min infusion postoperatively. Simultaneously, a 1 min intravenous infusion of iohexol was performed, in order to calculate the glomerular filtration rate. Blood samples were drawn to determine nefopam, desmethyl-nefopam and iohexol plasma concentrations. Nefopam and desmethyl-nefopam concentrations were analysed using a nonlinear mixed-effects modelling approach with Monolix version 4.1.3. The association between pharmacokinetic parameters and treatment response was assessed using logistic regression. RESULTS: A two-compartment open model was selected to describe the pharmacokinetics of nefopam. The typical population estimates (between-subject variability) for clearance, volume of distribution, intercompartmental clearance and peripheral volume were, respectively, 17.3 l h(-1) (53.2%), 114 l (121%), 80.7 l h(-1) (79%) and 208 l (63.6%). Morphine requirement was related to exposure of nefopam. Tachycardia and postoperative nausea and vomiting were best associated with maximal concentration and the rate of increase in nefopam plasma concentration. CONCLUSIONS: We identified the nefopam pharmacokinetic predictors for morphine requirement and side-effects, such as tachycardia and postoperative nausea and vomiting. In order to maintain morphine sparing and decrease side-effects following a single dose of nefopam (20 mg), simulations suggest an infusion time of >45 min in elderly patients with or without renal impairment.
Assuntos
Analgésicos não Narcóticos/farmacocinética , Nefopam/farmacocinética , Insuficiência Renal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Feminino , Taxa de Filtração Glomerular , Humanos , Modelos Logísticos , Masculino , Modelos Biológicos , Método de Monte Carlo , Morfina/administração & dosagem , Morfina/efeitos adversosRESUMO
Methylglyoxal (MGO) is an endogenous, highly reactive dicarbonyl metabolite generated under hyperglycaemic conditions. MGO plays a role in developing pathophysiological conditions, including diabetic cardiomyopathy. However, the mechanisms involved and the molecular targets of MGO in the heart have not been elucidated. In this work, we studied the exposure-related effects of MGO on cardiac function in an isolated perfused rat heart ex vivo model. The effect of MGO on calcium homeostasis in cardiomyocytes was studied in vitro by the fluorescence indicator of intracellular calcium Fluo-4. We demonstrated that MGO induced cardiac dysfunction, both in contractility and diastolic function. In rat heart, the effects of MGO treatment were significantly limited by aminoguanidine, a scavenger of MGO, ruthenium red, a general cation channel blocker, and verapamil, an L-type voltage-dependent calcium channel blocker, demonstrating that this dysfunction involved alteration of calcium regulation. MGO induced a significant concentration-dependent increase of intracellular calcium in neonatal rat cardiomyocytes, which was limited by aminoguanidine and verapamil. These results suggest that the functionality of various calcium channels is altered by MGO, particularly the L-type calcium channel, thus explaining its cardiac toxicity. Therefore, MGO could participate in the development of diabetic cardiomyopathy through its impact on calcium homeostasis in cardiac cells.
Assuntos
Cálcio , Miócitos Cardíacos , Aldeído Pirúvico , Ratos Wistar , Animais , Aldeído Pirúvico/toxicidade , Ratos , Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Masculino , Guanidinas/farmacologia , Canais de Cálcio Tipo L/metabolismo , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Verapamil/farmacologia , Contração Miocárdica/efeitos dos fármacosRESUMO
The aim of the study was to identify a signalling pathway allowing NAADP-induced intracellular NAADP increase and involving the P2Y11-like receptor. P2Y11-like and ß-adrenergic receptors may play important regulatory roles within the cardiovascular system. Both receptors have been shown to be involved in triggering myocardial preconditioning. Using a Langendorff model we report a positive inotropic response induced by extracellular NAADP via P2Y11-like receptor stimulation. In cardiomyocyte cultures, P2Y11-like receptor stimulation by extracellular NAADP ([NAADP]e) increased intracellular cADP-ribose and NAADP concentration as evidenced by direct measurements. NF546, a new selective P2Y11 receptor agonist, increased intracellular cAMP, cADP-ribose and NAADP concentration confirming the involvement of the P2Y11-like receptor in this signalling pathway. NF157, a P2Y11 receptor antagonist, suppressed the increase in intracellular cADPr, NAADP and NAAD induced by either [NAADP]e or NF546. The response profile for intracellular cADP-ribose and NAADP concentration following P2Y11-like stimulation with NF546 was similar to reported data relating ß-adrenergic stimulation with isoprenaline. This response represents the signature of the Gs/ADP-ribosyl cyclase activity. Moreover, this study provides a signalling pathway: intracellular NAADP increase induced by extracellular NAADP via metabotropic activity of P2Y11-like receptor. This pathway implying P2Y11-like could take part in the intracellular calcium rise reported for extracellular NAADP.
Assuntos
ADP-Ribose Cíclica/metabolismo , NADP/análogos & derivados , Receptores Purinérgicos P2Y/metabolismo , ADP-Ribosil Ciclase/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , AMP Cíclico/metabolismo , Difosfonatos/farmacologia , Relação Dose-Resposta a Droga , Coração/efeitos dos fármacos , Coração/fisiologia , Técnicas In Vitro , Masculino , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NADP/metabolismo , NADP/farmacologia , Naftalenossulfonatos/farmacologia , Ratos , Ratos Wistar , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suramina/análogos & derivados , Suramina/farmacologia , Fatores de TempoRESUMO
BACKGROUND AND PURPOSE: Extracellular nucleotides may play important regulatory roles within the cardiovascular system and notably in cardioprotection. We aimed to look for a possible pharmacological preconditioning effect of extracellular NAADP ([NAADP]e) against ischemia/reperfusion injury. [NAADP]e has been recently reported to be a full agonist of the P2Y11 receptor. Therefore, we characterized the involvement of the P2Y11-like receptor in mediating ischemic/reperfusion tolerance induced by [NAADP]e. EXPERIMENTAL APPROACH: The cardioprotective effects of [NAADP]e were evaluated in a model of ischemia/reperfusion carried out on Langendorff perfused rat hearts. This model was also instrumented with a microdialysis probe. Furthermore, using isolated cardiomyocytes, we assessed cAMP, inositol phosphate accumulation and prosurvival protein kinases activation induced by [NAADP]e pretreatement. RESULTS: Pretreatment with 1µM [NAADP]e induced cardioprotective effects with regards to functional recovery, necrosis and arrhythmogenesis (p<0.05). These effects were completely suppressed with NF157, an antagonist of the P2Y11 receptor. Moreover, global ischemia induced a time-dependent increase in interstitial concentration of adenosine, NAADP and UTP. In cardiomyocyte cultures, NF157 suppressed cAMP and inositol phosphate accumulation induced by [NAADP]e. [NAADP]e induced phosphorylation of ERK 1/2, AKT and its downstream target GSK-3ß (p<0.05). These activations were also suppressed by NF157. CONCLUSIONS: Evidence suggests that NAADP signalling at the P2Y11-like receptor affords significant cardioprotection against ischemia/reperfusion injury. Besides adenosine and UTP, microdialysis study supports a potential endogenous role of [NAADP]e.
Assuntos
Cardiotônicos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , NADP/análogos & derivados , Receptores Purinérgicos P2/fisiologia , Animais , Arritmias Cardíacas/prevenção & controle , Western Blotting , AMP Cíclico/metabolismo , Ativação Enzimática , Fosfatos de Inositol/metabolismo , NADP/fisiologia , Proteínas Quinases/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Several studies have reported the beneficial effects of anti-platelet drugs in cardioprotection against ischaemia-reperfusion injuries. To date, no studies have focused on the indirect cytoprotective effects of ticagrelor via adenosine receptor on the endothelium. METHOD: By evaluating cell viability and cleaved caspase 3 expression, we validated a model of endothelial cell apoptosis induced by hypoxia. In hypoxic endothelial cells treated with ticagrelor, we quantified the extracellular concentration of adenosine, and then we studied the involvement of adenosine pathways in the cytoprotective effect of ticagrelor. RESULTS: Our results showed that 10 µM ticagrelor induced an anti-apoptotic effect in our model associated with an increase of extracellular adenosine concentration. Similar experiments were conducted with cangrelor but did not demonstrate an anti-apoptotic effect. We also found that A2B and A3 adenosine receptors were involved in the anti-apoptotic effect of ticagrelor in endothelial cells exposed to 2 h of hypoxia stress. CONCLUSION: we described an endothelial cytoprotective mechanism of ticagrelor against hypoxia stress, independent of blood elements. We highlighted a mechanism triggered mainly by the increased extracellular bioavailability of adenosine, which activates A2B and A3 receptors on the endothelium.
Assuntos
Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/patologia , Transdução de Sinais , Ticagrelor/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Biomarcadores/metabolismo , Hipóxia Celular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Óxido Nítrico Sintase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacosRESUMO
Elastin peptides (EPs) generated by hydrolysis of elastic fibers by elastinolytic enzymes display a wide spectrum of biological activities. Here, we investigated their influence on rat heart ischemia-mediated injury using the Langendorff ex vivo model. EPs, i.e., kappa elastin, at 1.32- and 660-nM concentrations, when administered before the ischemia period, elicited a beneficial influence against ischemia by accelerating the recovery rate of heart contractile parameters and by decreasing significantly creatine kinase release and heart necrosis area when measured at the onset of the reperfusion. All effects were S-Gal-dependent, as being reproduced by (VGVAPG)3 and as being inhibited by receptor antagonists, such as lactose and V14 peptide (VVGSPSAQDEASPL). EPs interaction with S-Gal triggered NO release and activation of PI3-kinase/Akt and ERK1/2 in human coronary endothelial cells (HCAECs) and rat neonatal cardiomyocytes (RCs). This signaling pathway, as designated as RISK, for reperfusion injury salvage kinase pathway, was shown to be responsible for the beneficial influence of EPs on ischemia/reperfusion injury on the basis of its inhibition by specific pharmacological inhibitors. EPs survival activity was attained at a concentration averaging that present into the blood circulation, supporting the contention that these matrikines might offer a natural protection against cardiac injury in young and adult individuals. Such protective effect might be lost with aging, since we found that hearts from 24-month-old rats did not respond to EPs.
Assuntos
Cardiotônicos/farmacologia , Elastina/farmacologia , Precondicionamento Isquêmico Miocárdico/métodos , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/efeitos dos fármacos , beta-Galactosidase/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cardiotônicos/uso terapêutico , Circulação Coronária/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Avaliação Pré-Clínica de Medicamentos , Elastina/química , Elastina/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Coração/crescimento & desenvolvimento , Humanos , Masculino , Dados de Sequência Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Ligação Proteica , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Pressão Ventricular/efeitos dos fármacosRESUMO
Ischemic heart disease in diabetic patients might be linked to the accumulation of advanced-glycation end products (AGEs). In ischemic rat hearts, expression of receptor for AGEs and its ligands is significantly enhanced and involved in cardiac ischemia/reperfusion (I/R) injury even in the absence of diabetes. It has recently been reported that diabetic human myocardium cannot be protected by preconditioning. In this context, our hypothesis was that beta1-adrenergic preconditioning might be altered in the presence of AGEs. Using an isolated non-working rat heart model, this study investigated the effect of AGEs on cardioprotection induced by transient beta1-adrenoceptor (beta1-AR) stimulation with xamoterol (Xa). After 6-hydroxydopamine (6-OHDA) pre-treatment and a 20-min stabilization period, hearts were perfused at constant pressure for 20 min, then subjected to 40 min of global ischemia and 30 min of reperfusion (I/R, Ctrl); and exposed to 0.01 microm Xa for 5 min framed with or without 15.2 microm albumin (Alb) or glycated albumin (Gly Alb). The main endpoints were the mean coronary flow (MCF), the left ventricular end-diastolic pressure (LVEDP), rate-pressure product (RPP) and creatine kinase (CK) release and necrosis area. XA induced an increase in the MCF after I/R (t = 85 min), a protective effect on the LVEDP, an improvement in RPP, a decrease of CK release during reperfusion and a reduction of necrotic area. The beneficial effects induced by Xa during reperfusion were suppressed by the administration of Gly Alb during Xa infusion, whereas Alb did not hamper Xa-induced protection. These results suggest that AGEs suppress the cardioprotection resulting from the activation of beta1-ARs and thus might contribute to cardiovascular damages seen in diabetic patients.
Assuntos
Agonistas de Receptores Adrenérgicos beta 1 , Agonistas Adrenérgicos beta , Precondicionamento Isquêmico Miocárdico , Albumina Sérica/farmacologia , Xamoterol , Albuminas/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Circulação Coronária/efeitos dos fármacos , Creatina Quinase/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , Coração/efeitos dos fármacos , Coração/fisiopatologia , Técnicas In Vitro , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Ratos , Ratos Wistar , Albumina Sérica GlicadaRESUMO
Children aged between 1 to 4 years are the most at risk of unintentional poisonings. Benzodiazepines are the most medicine often cause of the poisoning. Among the twenty-two most prescribed benzodiazepines in France, lorazepam ranks fifth behind zolpidem, alprazolam, bromazepam and zopiclone. However the automated assay currently available does not allow to detect and/or to quantify lorazepam. The alternative to the immunoassay is the liquid chromatography coupled with mass spectrometry (HPLC/MS). This technique, highly sensitive and specific, requires a pre-treatment phase and a good technical proficiency, justifying specialized staff. The clinical cases presented here illustrate the major interest of availability to this type of technology in routine and 24h/24h.
Assuntos
Análise Química do Sangue/métodos , Lorazepam/intoxicação , Análise Química do Sangue/normas , Maus-Tratos Infantis/diagnóstico , Pré-Escolar , Cromatografia Líquida , Reações Falso-Negativas , Feminino , França , Humanos , Masculino , Espectrometria de Massas , Síndromes Neurotóxicas/sangue , Síndromes Neurotóxicas/diagnóstico , IrmãosRESUMO
Trivialization of cannabis consumption goes hand in hand with a growing exposure of children and the number of cannabis poisoning cases is steadily increasing. As clinical presentation can be different from what is currently seen in adults, added to the fact that it is not always suspected, diagnosis of cannabis intoxication in children is often delayed or missed. A 16-month-old girl was admitted to the pediatric emergency unit for an important drowsiness combined to moderate fever. After elimination of infectious causes, a toxic origin was considered and biological analyses led to the diagnosis of involuntary acute cannabis intoxication. In conclusion, cannabis intoxication in child has uncommon presentations compared to that seen in adults. In this context, biological analyses have a great importance for a rapid diagnosis and also for the understanding intoxication circumstance. This is of paramount importance because it may lead to consider child protection measures.
Assuntos
Cannabis/intoxicação , Febre/induzido quimicamente , Febre/diagnóstico , Fases do Sono/efeitos dos fármacos , Diagnóstico Diferencial , Feminino , Febre/patologia , Humanos , LactenteRESUMO
A specific and sensitive liquid chromatography-tandem mass spectrometric (LC-MS-MS) method using an ion trap spectrometer was developed for quantitation of nefopam and desmethyl-nefopam in human plasma. Nefopam, desmethyl-nefopam and the internal standard (ethyl loflazepate) were extracted in a single step with diethyl ether from 1 mL of alkalinized plasma. The mobile phase consisted of acetonitrile with 0.1% formic acid (50:50, v:v). It was delivered at a flow-rate of 0.3 mL/min. The effluent was monitored by MS-MS in positive-ion mode. Ionisation was performed using an electrospray ion source operating at 200 degrees C. Nefopam and desmethyl-nefopam were identified and quantified in full scan MS-MS mode using a homemade MS-MS library. Calibration curves were linear over the concentration range of 0.78-100 ng/mL with determination coefficients >0.996. This method was fast (total run time<6 min), accurate (bias<12.5%), and reproducible (intra- and inter-assay precision<17.5%) with a quantitation limit of 0.78 ng/mL. The high specificity and sensitivity achieved by this method allowed the determination of nefopam and desmethyl-nefopam plasma levels in patients following either intermittent or continuous intravenous administration of nefopam.
Assuntos
Cromatografia Líquida , Nefopam/análogos & derivados , Espectrometria de Massas em Tandem , Calibragem , Humanos , Injeções Intravenosas , Nefopam/sangue , Nefopam/metabolismo , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: Using an isolated non-working rat heart model, this study investigated the mechanisms of pharmacological preconditioning (PC) induced by transient beta1-adrenoreceptor (beta1-AR) stimulation with xamoterol (XA). METHODS: After 6-hydroxydopamine (6-OHDA) pretreatment and a 20-min stabilization period, hearts were perfused at constant pressure for 20 min then subjected to 40 min of global ischemia and 30 min of reperfusion (I/R, Ctrl); exposed to 0.01 microM XA for 5 min with or without 10 microM atenolol (ATE), a specific antagonist of beta1-AR, followed by a 15-min XA-free perfusion before I/R (PC, ATE-PC, respectively); treated during 20 min with either phosphoinositide (PI) 3-kinase inhibitors, LY-294002 (LY, 15 microM), or wortmaninn (WO, 0.1 microM); protein kinase C (PKC) inhibitor, GF-109203X (GF, 4 nM); or protein kinase A (PKA) inhibitor, H89 (H89, 1 microM), with an infusion starting 3 min before XA (LY-PC, WO-PC, GF-PC, and H89-PC, respectively). The main endpoints were the mean coronary flow (MCF), the left ventricular end-diastolic pressure (LVEDP), rate-pressure product (RPP), and creatine kinase (CK) release. RESULTS: XA induced an increase in the MCF after I/R (t 105 min) and a protective effect on the LVEDP, which were blocked by ATE and abolished with the different inhibitors. The transient increase in RPP following XA infusion was blocked by ATE and was not modified by the inhibitors except for H89. Recovery of RPP, measured 25 min after reperfusion, was improved by XA, blocked by ATE, and decreased with the different inhibitors. Fifteen minutes after the end of ischemia, CK release reached maximal values in all groups. XA provided significant protection whereas ATE and the four inhibitors suppressed XA-induced protection. CONCLUSION: The transient preischemic exposure to nanomolar concentrations of a beta1-AR agonist is protective against I/R. PI 3-kinase, PKC, and PKA are implicated in the trigger phase of PC. These observations were confirmed by Western blots.
Assuntos
Agonistas Adrenérgicos beta/uso terapêutico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Precondicionamento Isquêmico Miocárdico , Miocárdio/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , Androstadienos/farmacologia , Animais , Western Blotting/métodos , Cromonas/farmacologia , Circulação Coronária/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Isoquinolinas/farmacologia , Masculino , Morfolinas/farmacologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Oxidopamina/farmacologia , Perfusão , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Wistar , Sulfonamidas/farmacologia , Wortmanina , Xamoterol/uso terapêuticoRESUMO
High-dose of methotrexate chemotherapy is used in the treatment of some tumors. It presents several side effects that required therapeutic drug monitoring, which is commonly performed on 24, 48 and 72h after the beginning of the methotrexate infusion. Treatment of overexposure to methotrexate is based on injection of carboxypeptidase G2, which specifically degrades methotrexate into inactive metabolite: DAMPA. FPIA immunoassay on TDx automated analyzer (Abbott™) was used for therapeutic drug monitoring of methotrexate. This immunoassay presented a significant cross-reactivity between methotrexate and DAMPA, which widely overestimate the residual concentration compared to the gold standard HPLC/MS. TDx automated analyzer was substituted by a new immunoassay on Architect automated analyzer (Abbott™). However, this immunoassay has the same cross-reactivity, which needs to be careful when monitoring methotrexate after an injection of carboxypeptidase G2. In order to determine the most suitable assay for the therapeutic drug monitoring of methotrexate, the knowledge of injection of carboxypeptidase G2 remains essential.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Monitoramento de Medicamentos , Metotrexato/farmacocinética , Metotrexato/uso terapêutico , Osteossarcoma/tratamento farmacológico , Adolescente , Neoplasias Ósseas/sangue , Neoplasias Ósseas/metabolismo , Contraindicações , Reações Cruzadas , Monitoramento de Medicamentos/instrumentação , Monitoramento de Medicamentos/métodos , Monitoramento de Medicamentos/normas , Substituição de Medicamentos , Imunoensaio de Fluorescência por Polarização/métodos , Humanos , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Masculino , Osteossarcoma/sangue , Osteossarcoma/metabolismoRESUMO
BACKGROUND: Homocitrulline (HCit) is a carbamylation-derived product (CDP) that has been identified as a valuable biomarker of morbidity and mortality in patients with chronic kidney disease (CKD). The aim of this study was to determine whether initiation of hemodialysis therapy (HD) could induce variations of HCit concentrations in CKD patients. METHODS: Serum HCit concentrations were determined by LC-MS/MS in CKD patients (n=108) just before (M0) and six months (M6) after the initiation of HD therapy. RESULTS: Mean HCit concentrations reached 1000µmol/mol Lysine before initiation of HD therapy and decreased by 50% within 6months after HD onset. HCit concentrations remained stable over time as assessed during a 24-months follow-up period. HCit was mostly found in its protein-bound form in HD patients. HCit concentrations obtained at M0 were positively correlated with urea (r=0.58) and carbamylated hemoglobin (r=0.41), and are likely to be promising predictive markers of mortality. However, no correlations were found between HCit concentrations and Kt/V values, suggesting that HCit is not a marker of HD efficiency. CONCLUSION: HCit concentrations reflect the intensity of protein carbamylation and are stable over time during HD treatment, making HCit a reliable biomarker in the follow-up of CKD patients.
Assuntos
Carbamatos/metabolismo , Citrulina/análogos & derivados , Insuficiência Renal Crônica/sangue , Biomarcadores/sangue , Citrulina/sangue , Hemoglobina A/análogos & derivados , Hemoglobina A/análise , Humanos , Estudos Longitudinais , Diálise Renal , Insuficiência Renal Crônica/metabolismoRESUMO
A sensitive high-performance liquid chromatography-tandem mass spectrometric (HPLC-MS-MS) method, using an ion trap spectrometer, was developed for quantitation of bupivacaine in human plasma. Bupivacaine and an internal standard (ropivacaine) were extracted in a single step from 100 microL of alkalinized plasma with diethyl-ether. The mobile phase consisted of acetonitrile with 0.1% formic acid (50:50, v/v), and was delivered at a flow rate of 0.3 mL/min. The effluent was detected by MS-MS in positive ion mode. Ionisation was performed, using an electrospray ion source, operating at 200 degrees C. The selected reaction monitoring transitions m/z 289-->m/z 140 and m/z 275-->m/z 126 were chosen for bupivacaine and ropivacaine, respectively. Calibration curves were linear over the concentration range of 3.90-500 microg/L with determination coefficients >0.996. The method is accurate (bias <10%) and reproducible (intra-assay and inter-assay precision <15%), with a quantitation limit of 3.90 microg/L, using only 100 microL of plasma. The high specificity and sensitivity, achieved by this fast method (total run-time <3 min), allowed the determination of bupivacaine plasma levels in pediatric patients, following epidural administration of bupivacaine.
Assuntos
Bioensaio/métodos , Bupivacaína/análise , Bupivacaína/química , Bupivacaína/farmacocinética , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Acetonitrilas/química , Amidas/análise , Amidas/farmacocinética , Anestésicos Locais/análise , Anestésicos Locais/química , Calibragem , Química Farmacêutica/métodos , Indústria Farmacêutica/métodos , Formiatos/química , Humanos , Ropivacaina , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray , Temperatura , Fatores de TempoRESUMO
In several circumstances, determination and quantification of illicit drugs in biological fluids are determinant. Contexts are varied such as driving under influence, traffic accident, clinical and forensic toxicology, doping analysis, chemical submission. Whole blood is the favoured matrix for the quantification of illicit drugs. Gas chromatography coupled with mass spectrometry (GC-MS) is the gold standard for these analyses. All methods developed must be at least equivalent to gas chromatography coupled with a mass spectrometer. Nowadays, new technologies are available to biologists and clinicians: liquid chromatography coupled with a mass spectrometry (LC/MS) or coupled with a tandem mass spectrometer (LC/MS/MS). The aim of this paper is to describe the state of the art regarding techniques of confirmation by mass spectrometry used for quantification of conventional drugs except cannabis.
Assuntos
Anfetamina/análise , Cocaína/análise , Drogas Ilícitas/análise , Entorpecentes/análise , Detecção do Abuso de Substâncias/métodos , Toxicologia/métodos , Anfetamina/sangue , Análise Química do Sangue/métodos , Cromatografia Líquida , Cocaína/sangue , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Drogas Ilícitas/sangue , Entorpecentes/sangue , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Conventional methods for analysis of drugs of abuse require multiple assays which can be both expensive and time-consuming. This work describes a novel, rapid, simple and sensitive method for the quantification of 14 illicit drugs and their metabolites in whole blood. Results/methodology: This method employed a rapid liquid-liquid sample extraction of whole blood followed by UPLC-MS/MS analysis. Calibration curves were validated for analysis of appropriate concentrations. Inter- and intra-assay variations were <14.8%. Deviation of accuracy was <14.9% from target concentration for each quality control level. CONCLUSION: This work described the development and the full validation of a precise, sensitive and accurate assay. After validation, this new assay was successfully applied to routine toxicological analysis.
Assuntos
Anfetaminas/sangue , Analgésicos Opioides/sangue , Análise Química do Sangue/métodos , Cromatografia Líquida de Alta Pressão/normas , Cocaína/sangue , Espectrometria de Massas em Tandem/métodos , Anfetaminas/isolamento & purificação , Anfetaminas/metabolismo , Analgésicos Opioides/isolamento & purificação , Analgésicos Opioides/metabolismo , Cocaína/isolamento & purificação , Cocaína/metabolismo , Humanos , Extração Líquido-Líquido , Controle de Qualidade , Espectrometria de Massas em Tandem/normas , Estudos de Validação como AssuntoRESUMO
A simple and accurate high-performance liquid chromatographic method with ultraviolet detection at 220 nm has been validated for the simultaneous determination of amoxicillin and clavulanic acid in human plasma. Plasma samples were pretreated by direct deproteinization with methanol. A good chromatographic separation between both compounds was achieved using a reversed phase C8 column and a mobile phase, consisting of acetonitrile-phosphate solution-tetramethyl ammonium chloride solution. The calibration curves were linear over the concentration range of 0.625-20 mg l(-1) for amoxicillin and 0.3125-10 mg l(-1) for clavulanic acid with determination coefficients > 0.998. The method is accurate (bias < 7%) and reproducible (intra- and inter-day R.S.D. < 15%), with a quantitation limit of 0.625 and 0.3125 mg l(-1) for amoxicillin and clavulanic acid, respectively. Analytical recoveries from human plasma ranged from 91 to 102% for both components. This fully validated method, which allows the simultaneous measurement of amoxicillin and clavulanic acid in biological samples, is rapid (total run time < 10 min) and requires only a 100 microl sample. This assay is suitable for biomedical applications and was successfully applied to a pilot pharmacokinetics study in healthy volunteers after a single-oral administration of amoxicillin/clavulanic acid combination (500/125 mg).