Ribeiro Marta F T, Dias Ana C B, Santos João L M, Fernandes Eduarda, Lima José L F C, Zagatto Elias A G
Departamento de Química-Física, Faculdade de Farmácia da Universidade do Porto, Porto, Portugal.
J Biomol Screen. 2007 Sep;12(6):875-80. doi: 10.1177/1087057107305402.
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.
过氧亚硝酸盐阴离子是一种活性氮物种，它在体内由一氧化氮和超氧自由基之间快速、可控的扩散反应形成。通过与多种生物分子反应，过氧亚硝酸盐可能会导致重要的细胞和组织有害效应，这些效应与许多疾病有关。在这项工作中，开发了一种基于流动的自动化程序，用于体外生成过氧亚硝酸盐并随后筛选选定化合物的清除活性。该程序涉及一个多泵流动系统（MPFS），并利用硫辛酸、二氢硫辛酸、半胱氨酸、还原型谷胱甘肽、氧化型谷胱甘肽、舒林酸和舒林酸砜等化合物在生理模拟条件下抑制鲁米诺与过氧亚硝酸盐化学发光反应的能力。过氧亚硝酸盐在MPFS中通过酸化过氧化氢与亚硝酸盐的在线反应生成，随后与氢氧化钠溶液合并以快速淬灭正在进行的反应，从而实现稳定。MPFS提供的脉冲流以及样品和试剂溶液的定时同步插入确保仅在流通池中建立反应区，从而实现最大化学发光发射检测。对所检测化合物获得的结果表明，除了氧化型谷胱甘肽外，所有化合物在所研究的浓度下都是过氧亚硝酸盐的高效清除剂。
