Quantitative detection of nitroxyl upon trapping with glutathione and labeling with a specific fluorogenic reagent.

Abstract

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.

• Authors: Gail M Johnson, Tyler J Chozinski, Debra J Salmon, Alan D Moghaddam, Hsin Chih Chen, Katrina M Miranda
• Journal: Free radical biology & medicine (Free Radic Biol Med) Vol. 63 Pg. 476-84 (Oct 2013) ISSN: 1873-4596 [Electronic] United States
• PMID: 23685286 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: Copyright © 2013 Elsevier Inc. All rights reserved.

Chemical References

• 1-isopropyldiazen-1-ium-1,2-diolate
• Amines
• Fluorescent Dyes
• Hydrazines
• Naphthalenes
• Nitric Oxide Donors
• Nitrogen Oxides
• glutathione sulfinamide
• 2,3-naphthalenedicarboxaldehyde
• Glutathione
• nitroxyl

Topics

• Amines (chemistry)
• Dimerization
• Fluorescent Dyes
• Glutathione (analogs & derivatives, chemistry, metabolism)
• Humans
• Hydrazines (chemistry, metabolism)
• Naphthalenes (chemistry)
• Nitric Oxide Donors
• Nitrogen Oxides (chemistry, metabolism)
• Spectrum Analysis
• Staining and Labeling