Developing Detection Methods for Nitroxyl

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 ag...

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Main Author: Johnson, Gail Marie
Other Authors: Miranda, Katrina
Language:en
Published: The University of Arizona. 2013
Subjects:
Online Access:http://hdl.handle.net/10150/301746
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spelling ndltd-arizona.edu-oai-arizona.openrepository.com-10150-3017462015-10-23T05:25:39Z Developing Detection Methods for Nitroxyl Johnson, Gail Marie Miranda, Katrina Aspinwall, Craig Saavedra, Scott Tomat, Elisa Lichtenberger, Dennis Miranda, Katrina Chemistry 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. 2013 text Electronic Dissertation http://hdl.handle.net/10150/301746 en Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. The University of Arizona.
collection NDLTD
language en
sources NDLTD
topic Chemistry
spellingShingle Chemistry
Johnson, Gail Marie
Developing Detection Methods for Nitroxyl
description 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.
author2 Miranda, Katrina
author_facet Miranda, Katrina
Johnson, Gail Marie
author Johnson, Gail Marie
author_sort Johnson, Gail Marie
title Developing Detection Methods for Nitroxyl
title_short Developing Detection Methods for Nitroxyl
title_full Developing Detection Methods for Nitroxyl
title_fullStr Developing Detection Methods for Nitroxyl
title_full_unstemmed Developing Detection Methods for Nitroxyl
title_sort developing detection methods for nitroxyl
publisher The University of Arizona.
publishDate 2013
url http://hdl.handle.net/10150/301746
work_keys_str_mv AT johnsongailmarie developingdetectionmethodsfornitroxyl
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