Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721.
Free Radic Biol Med. 2013 Oct;63:476-84. doi: 10.1016/j.freeradbiomed.2013.05.011. Epub 2013 May 16.
Donors of nitroxyl (HNO) have shown promise for treatment of stroke, heart failure, alcoholism and cancer. However, comparing the pharmacological capacities of various donors is difficult without first quantifying the amount of HNO released from each donor. Detection and quantitation of HNO has been complicated by the rapid self-consumption of HNO through irreversible dimerization, poor selectivity of trapping agents against other nitrogen oxides, and/or low sensitivity towards HNO. Here, an assay is described for the trapping of HNO by glutathione (GSH) followed by labeling of GSH with the fluorogenic agent, naphthalene-2,3-dicarboxaldehyde (NDA), and subsequent quantitation by fluorescence difference. The newly developed assay was used to validate the pH-dependence of HNO release from isopropylamine NONOate (IPA/NO), which is a dual donor of HNO and NO at physiological pH. Furthermore, varied assay conditions were utilized to suggest the ratios of the products of the reaction of GSH with HNO. At intracellular concentrations of GSH, the disulfide (GSSG) was the major product, but significant concentrations of glutathione sulfinamide (GS(O)NH₂) were also detected. This suggests that GS(O)NH₂, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis.
硝普盐（HNO）供体在治疗中风、心力衰竭、酒精中毒和癌症方面显示出巨大的潜力。然而，如果不首先量化每种供体释放的 HNO 量，就很难比较各种供体的药理学能力。由于 HNO 会通过不可逆的二聚化迅速自我消耗、捕获剂对其他氮氧化物的选择性差、以及/或对 HNO 的灵敏度低，因此 HNO 的检测和定量一直很复杂。这里描述了一种使用谷胱甘肽（GSH）捕获 HNO 的测定方法，然后用荧光试剂萘-2,3-二羧酸醛（NDA）标记 GSH，并通过荧光差异进行定量。新开发的测定方法用于验证生理 pH 下异丙胺硝普酸盐（IPA/NO）释放 HNO 的 pH 依赖性，IPA/NO 是 HNO 和 NO 的双重供体。此外，还利用不同的测定条件来推测 GSH 与 HNO 反应产物的比例。在细胞内 GSH 浓度下，二硫化物（GSSG）是主要产物，但也检测到相当浓度的谷胱甘肽亚磺酰胺（GS(O)NH₂）。这表明 HNO 的选择性生物标志物 GS(O)NH₂可能以适合体内分析的浓度产生。
