RESUMO
BACKGROUND: Extracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation. METHODS: Amino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively. RESULTS: Urate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 103 M-1 s-1. Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio. CONCLUSIONS: Our results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence. GENERAL SIGNIFICANCE: These findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.
Assuntos
Peróxidos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Ácido Úrico/análogos & derivados , Domínio Catalítico , Adesão Celular/fisiologia , Membrana Celular/metabolismo , Sobrevivência Celular/fisiologia , Cromatografia Líquida/métodos , Cisteína/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Cinética , Oxirredução , Peroxidases/metabolismo , Agregação Plaquetária , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Isomerases de Dissulfetos de Proteínas/fisiologia , Compostos de Sulfidrila/metabolismo , Espectrometria de Massas em Tandem/métodos , Trombose/metabolismo , Ácido Úrico/metabolismoRESUMO
Several patients experience at least one drug-related problem and Pharmaceutical Care can change this reality. This work describes a model for structuring the pharmaceutical care service at a pharmacy training unit of the Brazilian Public Health System based on pharmacotherapy follow-up program of Parkinson’s disease patients’ results. From the follow-up results (phase 1), a Therapy Management Scheme was designed (phase 2). Of the 57 patients followed-up, 30 presented at least one drug-related problem and 42% were non-adherent to treatment, which supported the need of pharmacotherapy management. The Pharmacotherapy Management Scheme was proposed as a pharmaceutical care service model, which presents 6 steps: first, the pharmacist fills out the dispensing form and assesses patient´s pharmacotherapy, if there is a suspect problem, he is invited to the follow-up (steps 1 and 2) and they agree the first appointment. After that, pharmacist studies the patient’s case (study phase, steps 3 and 4). At the second meeting, the pharmacist proposes the intervention needed, and at the third, assesses the intervention results and new problems (steps 5 and 6, respectively). The process ends when all therapeutics outcomes are reached. This practical model can significantly contributed to the development and organization of pharmaceutical care services.
Muitos pacientes vivenciam pelo menos um problema relacionado ao medicamento e à atenção farmacêutica pode mudar este fato. Este trabalho descreve um modelo para estruturar o serviço de atenção farmacêutica numa farmácia escola do Sistema Único de Saúde brasileiro baseado nos resultados de um programa de seguimento farmacoterapêutico de pacientes com Doença de Parkinson. A partir dos resultados do seguimento, um esquema de gerenciamento da farmacoterapia foi desenhado. Dos 57 pacientes acompanhados, 30 apresentaram um problema relacionado ao medicamento e 42% não aderiram ao tratamento, o que reforça a necessidade de gerenciar a farmacoterapia. O esquema proposto apresenta 6 passos: primeiro, o farmacêutico preenche o formulário de dispensação e avalia a farmacoterapia do paciente; caso haja suspeita de um problema, ele é convidado a participar do seguimento farmacoterapêutico (passos 1 e 2) e marcam a primeira consulta. Após esta, o farmacêutico estuda o caso (fase de estudo, passos 3 e 4). Na segunda consulta, o farmacêutico propõe as intervenções necessárias e, na terceira, avalia seus resultados e novos problemas (passos 5 e 6, respectivamente). O processo termina quando todos os objetivos terapêuticos são alcançados. Este modelo de prática pode contribuir significativamente para o desenvolvimento e organização de serviços de atenção farmacêutica.