Analysis of GSH and GSSG after derivatization with N-ethylmaleimide

Daniela Giustarini, Isabella Dalle-Donne, Aldo Milzani, Paolo Fanti, Ranieri Rossi

Research output: Contribution to journalArticle

90 Citations (Scopus)

Abstract

This protocol describes a procedure for determining glutathione (GSH) and glutathione disulfide (GSSG) concentrations in blood and other tissues. Artifactual oxidation to GSSG of 5-15% of the GSH found in a sample can occur during deproteination of biological samples with any of the commonly used acids, with consequent marked overestimation of GSSG. This can be prevented by derivatizing GSH with the alkylating agent N-ethylmaleimide (NEM) to form GS-NEM before acid deproteination, followed by back-extraction of excess NEM from the deproteinized samples with dichloromethane. GSSG concentration is then measured by spectrophotometry with the GSH recycling method, on the basis of conversion of GSSG to GSH by glutathione reductase and NADPH and reaction with 5,5′-dithiobis-(2-nitrobenzoic acid). GSH concentration is instead measured by either of two methods: by analysis of GS-NEM conjugates by HPLC in the same sample that is used to measure GSSG or, alternatively, by analysis of GSH by spectrophotometry (GSH recycling method) on one additional sample aliquot that has not been derivatized with NEM. The procedure can assay GSH and GSSG in blood and other tissues in 30 min or less.

Original languageEnglish (US)
Pages (from-to)1660-1669
Number of pages10
JournalNature Protocols
Volume8
Issue number9
DOIs
StatePublished - Sep 2013

Fingerprint

Ethylmaleimide
Glutathione Disulfide
Spectrophotometry
Recycling
Blood
Nitrobenzoates
Tissue
Acids
Glutathione Reductase
Alkylating Agents
Methylene Chloride
NADP
Glutathione
Assays
High Pressure Liquid Chromatography
Oxidation

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Giustarini, D., Dalle-Donne, I., Milzani, A., Fanti, P., & Rossi, R. (2013). Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nature Protocols, 8(9), 1660-1669. https://doi.org/10.1038/nprot.2013.095

Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. / Giustarini, Daniela; Dalle-Donne, Isabella; Milzani, Aldo; Fanti, Paolo; Rossi, Ranieri.

In: Nature Protocols, Vol. 8, No. 9, 09.2013, p. 1660-1669.

Research output: Contribution to journalArticle

Giustarini, D, Dalle-Donne, I, Milzani, A, Fanti, P & Rossi, R 2013, 'Analysis of GSH and GSSG after derivatization with N-ethylmaleimide', Nature Protocols, vol. 8, no. 9, pp. 1660-1669. https://doi.org/10.1038/nprot.2013.095
Giustarini D, Dalle-Donne I, Milzani A, Fanti P, Rossi R. Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nature Protocols. 2013 Sep;8(9):1660-1669. https://doi.org/10.1038/nprot.2013.095
Giustarini, Daniela ; Dalle-Donne, Isabella ; Milzani, Aldo ; Fanti, Paolo ; Rossi, Ranieri. / Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. In: Nature Protocols. 2013 ; Vol. 8, No. 9. pp. 1660-1669.
@article{12e7b63bed0d4d8cb6b6c0bf9aedd17e,
title = "Analysis of GSH and GSSG after derivatization with N-ethylmaleimide",
abstract = "This protocol describes a procedure for determining glutathione (GSH) and glutathione disulfide (GSSG) concentrations in blood and other tissues. Artifactual oxidation to GSSG of 5-15{\%} of the GSH found in a sample can occur during deproteination of biological samples with any of the commonly used acids, with consequent marked overestimation of GSSG. This can be prevented by derivatizing GSH with the alkylating agent N-ethylmaleimide (NEM) to form GS-NEM before acid deproteination, followed by back-extraction of excess NEM from the deproteinized samples with dichloromethane. GSSG concentration is then measured by spectrophotometry with the GSH recycling method, on the basis of conversion of GSSG to GSH by glutathione reductase and NADPH and reaction with 5,5′-dithiobis-(2-nitrobenzoic acid). GSH concentration is instead measured by either of two methods: by analysis of GS-NEM conjugates by HPLC in the same sample that is used to measure GSSG or, alternatively, by analysis of GSH by spectrophotometry (GSH recycling method) on one additional sample aliquot that has not been derivatized with NEM. The procedure can assay GSH and GSSG in blood and other tissues in 30 min or less.",
author = "Daniela Giustarini and Isabella Dalle-Donne and Aldo Milzani and Paolo Fanti and Ranieri Rossi",
year = "2013",
month = "9",
doi = "10.1038/nprot.2013.095",
language = "English (US)",
volume = "8",
pages = "1660--1669",
journal = "Nature Protocols",
issn = "1754-2189",
publisher = "Nature Publishing Group",
number = "9",

}

TY - JOUR

T1 - Analysis of GSH and GSSG after derivatization with N-ethylmaleimide

AU - Giustarini, Daniela

AU - Dalle-Donne, Isabella

AU - Milzani, Aldo

AU - Fanti, Paolo

AU - Rossi, Ranieri

PY - 2013/9

Y1 - 2013/9

N2 - This protocol describes a procedure for determining glutathione (GSH) and glutathione disulfide (GSSG) concentrations in blood and other tissues. Artifactual oxidation to GSSG of 5-15% of the GSH found in a sample can occur during deproteination of biological samples with any of the commonly used acids, with consequent marked overestimation of GSSG. This can be prevented by derivatizing GSH with the alkylating agent N-ethylmaleimide (NEM) to form GS-NEM before acid deproteination, followed by back-extraction of excess NEM from the deproteinized samples with dichloromethane. GSSG concentration is then measured by spectrophotometry with the GSH recycling method, on the basis of conversion of GSSG to GSH by glutathione reductase and NADPH and reaction with 5,5′-dithiobis-(2-nitrobenzoic acid). GSH concentration is instead measured by either of two methods: by analysis of GS-NEM conjugates by HPLC in the same sample that is used to measure GSSG or, alternatively, by analysis of GSH by spectrophotometry (GSH recycling method) on one additional sample aliquot that has not been derivatized with NEM. The procedure can assay GSH and GSSG in blood and other tissues in 30 min or less.

AB - This protocol describes a procedure for determining glutathione (GSH) and glutathione disulfide (GSSG) concentrations in blood and other tissues. Artifactual oxidation to GSSG of 5-15% of the GSH found in a sample can occur during deproteination of biological samples with any of the commonly used acids, with consequent marked overestimation of GSSG. This can be prevented by derivatizing GSH with the alkylating agent N-ethylmaleimide (NEM) to form GS-NEM before acid deproteination, followed by back-extraction of excess NEM from the deproteinized samples with dichloromethane. GSSG concentration is then measured by spectrophotometry with the GSH recycling method, on the basis of conversion of GSSG to GSH by glutathione reductase and NADPH and reaction with 5,5′-dithiobis-(2-nitrobenzoic acid). GSH concentration is instead measured by either of two methods: by analysis of GS-NEM conjugates by HPLC in the same sample that is used to measure GSSG or, alternatively, by analysis of GSH by spectrophotometry (GSH recycling method) on one additional sample aliquot that has not been derivatized with NEM. The procedure can assay GSH and GSSG in blood and other tissues in 30 min or less.

UR - http://www.scopus.com/inward/record.url?scp=84883383166&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84883383166&partnerID=8YFLogxK

U2 - 10.1038/nprot.2013.095

DO - 10.1038/nprot.2013.095

M3 - Article

VL - 8

SP - 1660

EP - 1669

JO - Nature Protocols

JF - Nature Protocols

SN - 1754-2189

IS - 9

ER -