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

Gail M. Johnson, Tyler J. Chozinski, Debra J Salmon, Alan D. Moghaddam, Hsin Chih Chen, Katrina M. Mirandan

Research output: Contribution to journalArticle

16 Citations (Scopus)

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)NH 2, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis.

Original languageEnglish (US)
Pages (from-to)476-484
Number of pages9
JournalFree Radical Biology and Medicine
Volume63
DOIs
StatePublished - Jan 1 2013

Fingerprint

Labeling
Glutathione
Assays
Nitrogen Oxides
Heart Neoplasms
Glutathione Disulfide
Dimerization
Disulfides
Alcoholism
Heart Failure
Biomarkers
Fluorescence
Stroke
Pharmacology
nitroxyl
glutathione sulfinamide
2,3-naphthalenedicarboxaldehyde
1-isopropyldiazen-1-ium-1,2-diolate

Keywords

  • Angeli's salt
  • Free radicals
  • Glutathione
  • Isopropylamine NONOate
  • Naphthalene-2,3-dicarboxaldehyde
  • Nitric oxide
  • Nitroxyl
  • Sulfinamide

Cite this

Quantitative detection of nitroxyl upon trapping with glutathione and labeling with a specific fluorogenic reagent. / Johnson, Gail M.; Chozinski, Tyler J.; Salmon, Debra J; Moghaddam, Alan D.; Chen, Hsin Chih; Mirandan, Katrina M.

In: Free Radical Biology and Medicine, Vol. 63, 01.01.2013, p. 476-484.

Research output: Contribution to journalArticle

Johnson, Gail M. ; Chozinski, Tyler J. ; Salmon, Debra J ; Moghaddam, Alan D. ; Chen, Hsin Chih ; Mirandan, Katrina M. / Quantitative detection of nitroxyl upon trapping with glutathione and labeling with a specific fluorogenic reagent. In: Free Radical Biology and Medicine. 2013 ; Vol. 63. pp. 476-484.
@article{8c4419c299a6467faa13fc36307edcac,
title = "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)NH 2, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis.",
keywords = "Angeli's salt, Free radicals, Glutathione, Isopropylamine NONOate, Naphthalene-2,3-dicarboxaldehyde, Nitric oxide, Nitroxyl, Sulfinamide",
author = "Johnson, {Gail M.} and Chozinski, {Tyler J.} and Salmon, {Debra J} and Moghaddam, {Alan D.} and Chen, {Hsin Chih} and Mirandan, {Katrina M.}",
year = "2013",
month = "1",
day = "1",
doi = "10.1016/j.freeradbiomed.2013.05.011",
language = "English (US)",
volume = "63",
pages = "476--484",
journal = "Free Radical Biology and Medicine",
issn = "0891-5849",
publisher = "Elsevier Inc.",

}

TY - JOUR

T1 - Quantitative detection of nitroxyl upon trapping with glutathione and labeling with a specific fluorogenic reagent

AU - Johnson, Gail M.

AU - Chozinski, Tyler J.

AU - Salmon, Debra J

AU - Moghaddam, Alan D.

AU - Chen, Hsin Chih

AU - Mirandan, Katrina M.

PY - 2013/1/1

Y1 - 2013/1/1

N2 - 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)NH 2, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis.

AB - 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)NH 2, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis.

KW - Angeli's salt

KW - Free radicals

KW - Glutathione

KW - Isopropylamine NONOate

KW - Naphthalene-2,3-dicarboxaldehyde

KW - Nitric oxide

KW - Nitroxyl

KW - Sulfinamide

UR - http://www.scopus.com/inward/record.url?scp=84884887175&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84884887175&partnerID=8YFLogxK

U2 - 10.1016/j.freeradbiomed.2013.05.011

DO - 10.1016/j.freeradbiomed.2013.05.011

M3 - Article

VL - 63

SP - 476

EP - 484

JO - Free Radical Biology and Medicine

JF - Free Radical Biology and Medicine

SN - 0891-5849

ER -