Persistent Link:
http://hdl.handle.net/10150/301746
Title:
Developing Detection Methods for Nitroxyl
Author:
Johnson, Gail Marie
Issue Date:
2013
Publisher:
The University of Arizona.
Rights:
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.
Embargo:
Release after 31-Jul-2015
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.
Type:
text; Electronic Dissertation
Keywords:
Chemistry
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry
Degree Grantor:
University of Arizona
Advisor:
Miranda, Katrina

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleDeveloping Detection Methods for Nitroxylen_US
dc.creatorJohnson, Gail Marieen_US
dc.contributor.authorJohnson, Gail Marieen_US
dc.date.issued2013-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © 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.en_US
dc.description.releaseRelease after 31-Jul-2015en_US
dc.description.abstractAccurate 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.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectChemistryen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorMiranda, Katrinaen_US
dc.contributor.committeememberAspinwall, Craigen_US
dc.contributor.committeememberSaavedra, Scotten_US
dc.contributor.committeememberTomat, Elisaen_US
dc.contributor.committeememberLichtenberger, Dennisen_US
dc.contributor.committeememberMiranda, Katrinaen_US
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