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

Abstract

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 due to 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 following 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 selectively. GS(O)NH2 was detected in the lysate of cells treated with low micromolar concentrations of HNO donors, verifying that this marker 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.

Signaling and stress: The redox landscape in NOS2 biology

“…Nonetheless, synthetic GS(O)NH 2 has been detected with a reasonable recovery percentage in cell lysate and has been observed after exposure of cells to an HNO donor [193]. …”

“…20) [178], becomes progressively competitive such that a mixture of RS(O)NH 2 and RSSR is produced. The sulfinamide is indicated to be the major product however [193, 194]. $$2\mathrm{RSH}+\mathrm{HNO}\to \mathrm{RSSR}+{\mathrm{NH}}_2\mathrm{OH}$$ Interestingly NH 2 OH can be reoxidized to HNO forming a shunt system for HNO that is initiated by RSNOs [150].…”

Biological signaling by small inorganic molecules

“…18) [229, 253]. The disulfide product represents a biologically reversible process as biological reductants regenerate the thiol while generation of sulfinamide is unique in terms of HNO chemical biology and represents an irreversible modification of thiols [249, 254]. …”

Capillary electrophoresis in the analysis of biologically important thiols

“…A new strategy for detection of GS(O)NH 2 in cultured cells as an HNO‐selective biomarker was presented by Johnson et al. . The authors separated GSH and GS(O)NH 2, which is the reaction product of HNO with GSH, by labeling the thiol compounds with NDA in the presence of CN − anion.…”