Desenvolvimento e validação de um método por CLAE-EM/EM para quantificação de gliotoxina em plasma humano para diagnóstico precoce da aspergilose

Orientador: Prof. Dr. Roberto PontaroloCo-orientadora: Profa. Dra. Francinete Ramos CamposDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmaceuticas. Defesa: Curitiba, 22/02/2013Bibliografia: fls. 117-125Resumo: A aspergilose é uma infecção oportunista causada por fungos do gênero Aspergillus. É uma das principais complicações que acometem pacientes imunossuprimidos, podendo atingir índices de mortalidade de até 80%. O principal agente causador dessa infecção é o A. fumigatus, também considerada a principal espécie produtora de gliotoxina. Esta toxina é um produto do metabolismo secundário fúngico, apresenta comprovada toxicidade frente às células do sistema hematopoético e já foi detectada em soro de pacientes com diagnóstico positivo de aspergilose, podendo ser considerada um potencial biomarcador para o diagnóstico da doença. Atualmente o método de escolha para o diagnóstico da aspergilose é imunoenzimático, mas oferece apenas um indicativo da infecção, sendo necessários três resultados positivos consecutivos para que ela seja confirmada. A cromatografia liquida de alta eficiência acoplada à espectrometria de massas sequencial (CLAE-EM/EM) surge como uma nova alternativa para o diagnóstico, uma vez que é capaz de fornecer resultados com alta precisão e exatidão, e excelente sensibilidade. Nesse sentido, esse trabalho visa o desenvolvimento de um método por CLAE-EM/EM para detecção de gliotoxina em plasma de pacientes imunossuprimidos com suspeita de aspergilose. Para isso, alíquotas de plasma branco foram fortificadas com gliotoxina e submetidas ao processo de extração liquido-liquido, utilizando uma solução de éter etílico/acetato de etila (50:50, v/v), agitada em vórtex por 3 min. A fase orgânica foi recuperada e, em seguida, evaporada. O resíduo obtido foi resuspendido em uma solução de ACN/H2O (98:2, v/v) contendo 0,1% de ácido fórmico (AFO). As análises foram realizadas utilizando um cromatógrafo Agilent 1200 acoplado ao espectrômetro de massas do tipo triplo quadrupolo API 3200 da ABSciex, provido de fonte de ionização por electrospray (ESI), operado no modo negativo de ionização. O experimento de Multiple Reaction Monitoring (MRM) foi utilizado para monitorar as transições dos íons de m/z 325'243 e m/z 325'261 referente à gliotoxina e m/z 301'179 e m/z 301'151 referentes ao padrão interno (quercetina). A separação cromatográfica foi obtida em uma coluna C18 XBridge (150 x 2,1 mm i.d; 5 ?m) e pré-coluna (XBridge C18, 10 x 2,1 mm i.d; 5 ?m). A fase móvel foi constituída de solução de NH4HCO2 1mM (A) e ACN contendo 5% de solução 1mM de NH4HCO2 (B) submetidos ao seguinte gradiente de eluição: t0-0,10 min.: 65%-0% A; t0,10-0,70 min.: 0% A; t0,7-0,71 min.: 0-65% A; t0,71-4,00 min.: 65% A. O tempo de corrida foi de 4 min. em um fluxo de 0,45 mL/min. O volume de injeção foi de 20 ?L. O método desenvolvido foi validado, considerado sensível, seletivo, linear (r>0,99) na faixa de 10 a 120 ng/mL, preciso, exato e livre de efeitos residuais e de matriz. O método foi aplicado com sucesso a 30 amostras reais de pacientes com suspeita de aspergilose, com 70% de concordância com os resultados obtidos pelo método de ELISA. O presente trabalho confirmou que é possível quantificar a gliotoxina em plasma através da técnica de CLAE-EM/EM e sua aplicação como diagnóstico é uma possibilidade real e muito promissora.Abstract: Aspergillosis is an opportunistic infection caused by fungi of the genus Aspergillus. It is a major complication of immunocompromised patients and its mortality can reach up to 80%. The main causative agent of this infection is A. fumigatus, which is also the main gliotoxin-producing species. This toxin is a product of fungal secondary metabolism and has been shown toxicity against hematopoietic system cells. It has already been detected in sera samples of patients with positive diagnosis of aspergillosis and, for this reason, can be considered as a potential biomarker for diagnosis of this disease. Imunoenzimatic assay is the currently gold standard method for diagnosing Aspergillosis but it only provides an indicative of infection, requiring three consecutive positive results to confirm the diagnosis. The high performance liquid chromatography coupled tandem mass spectrometry (HPLC-MS/MS) appears as a new alternative for the diagnosis of aspergillosis, since it is capable to provide high precision and accuracy results with excellent sensitivity. On this way, this work aims to develop an HPLC-MS/MS method for the detection of gliotoxin in plasma of patients with suspected infection by Aspergillus. For this, aliquots of blank plasma were spiked with gliotoxin and submitted to liquid-liquid extraction, using a solution of ethyl ether / ethyl acetate (1:1, v /v). The organic phase was recovered and then evaporated. The residue obtained was resuspended in a solution of ACN/H2O (98:2, v /v) with 1 mM of NH4HCO2. Analyses were performed using an Agilent 1200 liquid chromatograph coupled to mass spectrometer API 3200 triple quadrupole ABSciex provided with electrospray ionization source (ESI) operated in the negative ion mode. The experiment Multiple Reaction Monitoring (MRM) was used to monitor the transition of the ions of m/z 325' 243 and m/z 325 ' 261 corresponding to gliotoxin and m/z 301 ' 179 and m/z 301' 151 related to internal standard (quercetin). The chromatographic separation was obtained in a XBridge C18 column (150 x 2.1 mm id; 5 mm) and guard column (XBridge C18, 10 x 2.1 mm id, 5 mm). The mobile phase was composed of 1 mM NH4HCO2 solution (A) and MeCN containing 5% NH4HCO2 1mM (B) eluted by the following gradient: t0-0, 10 min: 65% -0% A; t0 10 -0.70 min: 0% A; t0 0.7 to 0, 71 min: 0-65% A; t0 0.71 to 4, 00 min: 65% A. The run time was 4 min at a flow rate of 0.45 mL / min. The injection volume was 20 ?L. The method was fully validated, being considered linear (r> 0.99) in the range of 10 to 120 ng/ mL, sensitive, selective, precise, accurate and free of matrix and carry over effects. The method was successfully applied to real samples from 30 patients with suspected aspergillosis, reaching 70% of agreement with ELISA results. This study confirmed that it is possible to quantify gliotoxin in plasma using the technique of HPLC-MS/MS and the present method can be potentially applied for aspergillosis diagnosis

Efficacy and Safety of Chagas Disease Drug Therapy and Treatment Perspectives

Chagas disease, also known as American trypanosomiasis, is a neglected disease caused by the protozoan parasite Trypanosoma cruzi. The disease affects about 6–7 million people worldwide, mostly in Latin America. Although Chagas disease was discovered more than 100 years ago, and the first treatments over 40, only 2 drugs were used to treat this pathology, it is still considered one of the neglected diseases. In this chapter, the subjects related to conventional etiological therapies, benznidazole and nifurtimox, such as the drug, the mechanism of action, the therapy schedule for treatment, efficacy and safety and their adverse effects will be discussed. Additionally, it will address alternative therapies of comorbidities related to the progression of Chagas’ disease in patients with chronic disease, such as heart disease and dysfunction of the digestive system. Finally, novel pharmacological strategies and their related compounds will be reviewed accounting for their progression in pharmacological studies and their success rate

Endothelium-Dependent Vasorelaxant Effect of Butanolic Fraction from Caryocar brasiliense Camb. Leaves in Rat Thoracic Aorta

Caryocar brasiliense Camb. "pequi" is a native plant from the Cerrado region of Brazil that contains bioactive components reported to be antioxidant agents. Previous work has demonstrated that dietary supplementation with pequi decreased the arterial pressure of volunteer athletes. We found that the crude hydroalcoholic extract (CHE) of C. brasiliense leaves relaxed, in a concentration-dependent manner, rat aortic rings precontracted with phenylephrine, and that the butanolic fraction (BF) produced an effect similar to that of the CHE. Aortic relaxation induced by BF was abolished by endothelium removal, by incubation of the nitric oxide synthase inhibitor L-NAME, or the soluble guanylatecyclase inhibitor ODQ. However, incubation with atropine and pyrilamine had no effect on the BF-induced vasorelaxation. Moreover, this effect was not inhibited by indomethacin and tetraethylammonium. The concentration-response curve to calcium in denuded-endothelium rings was not modified after incubation with BF, and the vasorelaxation by BF in endothelium-intact rings precontracted with KCl was abolished after incubation with L-NAME. In addition, administration of BF in anesthetized rats resulted in a reversible hypotension. The results reveal that C. brasiliense possesses both in vivo and in vitro activities and that the vascular effect of BF involves stimulation of the nitric oxide/cyclic GMP pathway