Developing Detection Methods for Nitroxyl

Abstract

Accurate detection of nitroxyl (HNO) is essential to understanding its bioactivity, metabolism, and relation to health and disease. However, 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. In this work we have used thiols to efficiently and selectively trap HNO. We then used fluorescence-based detection methods for highly sensitive and selective detection. An in vitro assay was developed for quantifying HNO from donors. HNO was trapped by glutathione (GSH), a commercially and biologically available thiol. GSH was then selectively labeled with the fluorogenic agent, naphthalene-2,3-dicarboxaldehyde (NDA), which removes the need for separation prior to analysis, enabling high throughput analysis. HNO released from micromolar doses of donors was then quantified by measuring changes in GSH concentration. GSH reacts with HNO to produce glutathione sulfinamide (GS(O)NH₂), which is a selective biomarker of HNO. We developed an in vivo assay that indirectly detects HNO by quantifying the production of GS(O)NH₂. The free amine on GS(O)NH₂ was fluorescently labeled with NDA, and capillary zone electrophoresis with laser-induced fluorescence (CZE-LIF) was used for separation and detection. CZE-LIF provided high analytical sensitivity for GS(O)NH₂, with a limit of detection (LOD) of 1.4 ± 0.1 nM. Cultured cells were treated with micromolar doses of HNO donor, and the concentrations of GS(O)NH₂ increased in a dose-dependent manner showing that the concentration of GS(O)NH₂ can be correlated to the intracellular concentration of HNO. Another in vivo assay was developed using an exogenous fluorescein thiol dye (CFS) to simultaneously trap and fluorescently label HNO as a fluorescent sulfinamide adduct (CFS(O)NH₂). CZE-LIF was used to separate and detect CFS(O)NH₂, and provided a LOD of 0.40 ± 0.01 nM for CFS(O)NH₂. Cultured cells treated with micromolar doses of HNO donor showed a dose-dependent response for CFS(O)NH₂. Therefore, quantitation CFS(O)NH₂ can be indirectly correlated to the concentration of HNO in cells. Additionally, this assay decreases analysis time by removing the need for a labeling reaction, and increases the overall sensitivity of the assay by minimizing dilution.