Glutathione sulfinamide serves as a selective,endogenous biomarker for nitroxyl after exposure to therapeutic levels of donors

Nitroxyl (HNO) donors exhibit promising pharmacological characteristics for treatment of cardiovascular disorders, cancer, and alcoholism. However, whether HNO also serves as an endogenous signaling agent is currently unknown, largely because of the inability to selectively and sensitively detect HNO in a cellular environment. Although a number of methods to detect HNO have been developed recently, sensitivity and selectivity against other nitrogen oxides or biological reductants remain problematic. To improve selectivity, the electrophilic nature of HNO has been harnessed to generate modifications of thiols and phosphines that are unique to HNO, especially compared to nitric oxide (NO). Given high bioavailability, glutathione (GSH) is expected to be a major target of HNO. As a result, the putative selective product glutathione sulfinamide (GS(O)NH₂) may serve as a high-yield biomarker of HNO production. In this work, the formation of GS(O)NH₂ after exposure to HNO donors was investigated. Fluorescent labeling followed by separation and detection using capillary zone electrophoresis with laser-induced fluorescence allowed quantitation of GS(O)NH₂ with nanomolar sensitivity, even in the presence of GSH and derivatives. Formation of GS(O)NH₂ was found to occur exclusively upon exposure of GSH to HNO donors, thus confirming selectivity. GS(O)NH₂ was detected in the lysate of cells treated with low-micromolar concentrations of HNO donors, verifying that this species has sufficient stability to server as a biomarker of HNO. Additionally, the concentration-dependent formation of GS(O)NH₂ in cells treated with an HNO donor suggests that the concentration of GS(O)NH₂ can be correlated to intracellular levels of HNO.