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Journal of Biomolecular Screening
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A Multipumping Flow System for In Vitro Screening of Peroxynitrite Scavengers

Marta F.T. Ribeiro

Departamento de Química-Física, Faculdade de Farmácia da Universidade do Porto, Porto, Portugal, joaolms{at}ff.up.pt

Ana C.B. Dias

Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, São Paulo, Brazil

Joao L.M. Santos

Departamento de Química-Física, Faculdade de Farmácia da Universidade do Porto, Porto, Portugal

Eduarda Fernandes

Departamento de Química-Física, Faculdade de Farmácia da Universidade do Porto, Porto, Portugal

José L.F.C. Lima

Departamento de Química-Física, Faculdade de Farmácia da Universidade do Porto, Porto, Portugal

Elias A.G. Zagatto

Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, São Paulo, Brazil

Peroxynitrite anion is a reactive nitrogen species formed in vivo by the rapid, controlled diffusion reaction between nitric oxide and superoxide radicals. By reacting with several biological molecules, peroxynitrite may cause important cellular and tissue deleterious effects, which have been associated with many diseases. In this work, an automated flow-based procedure for the in vitro generation of peroxynitrite and subsequent screening of the scavenging activity of selected compounds is developed. This procedure involves a multipumping flow system (MPFS) and exploits the ability of compounds such as lipoic acid, dihydrolipoic acid, cysteine, reduced glutathione, oxidized glutathione, sulindac, and sulindac sulfone to inhibit the chemiluminescent reaction of luminol with peroxynitrite under physiological simulated conditions. Peroxynitrite was generated in the MPFS by the online reaction of acidified hydrogen peroxide with nitrite, followed by a subsequent stabilization by merging with a sodium hydroxide solution to rapidly quench the developing reaction. The pulsed flow and the timed synchronized insertion of sample and reagent solutions provided by the MPFS ensure the establishment of the reaction zone only inside the flow cell, thus allowing maximum chemiluminescence emission detection. The results obtained for the assayed compounds show that, with the exception of oxidized glutathione, all are highly potent scavengers of peroxynitrite at the studied concentrations. (Journal of Biomolecular Screening 2007:875-880)

Key Words: peroxynitrite • scavenging activity • flow analysis • multipumping • chemiluminescence

Journal of Biomolecular Screening, Vol. 12, No. 6, 875-880 (2007)
DOI: 10.1177/1087057107305402
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