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| Title: | Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol |
| Author (s): | Ates, Burhan Ercal, Baris Can Manda, Kalyan Abraham, LInu Ercal, Nuran |
| Department/Lab Affiliations: | Center for Environmental Science and Technology (CEST) Chemistry Environmental Research Center |
| Keywords: | DTT GSH GSSG HPLC thiol-disulfide exchange |
| Issue Date: | 2008-07 |
| Publisher: | John Wiley & Sons |
| Citation: | Ates, Burhan, Baris Can Ercal, Kalyan Manda, Linu Abraham, and Nuran Ercal. "Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol." Biomedical Chromatography, Jul 2008 |
| Abstract: | A reverse-phase HPLC method incorporating dithiothreitol (DTT) reduction for quantitative determination of oxidized glutathione (GSSG) in biological samples is described here. This method is based on our previous enzymatic reduction technique that uses N-1-(pyrenyl) maleimide (NPM) as a derivatizing agent. In our earlier method, glutathione disulfide (GSSG) was measured by first reducing it to GSH with glutathione reductase (GR) in the presence of NADPH. However, this is a very costly and time-consuming technique. The method described here employs a common and inexpensive thiol-disulfide exchanging agent, DTT, for reduction of GSSG to GSH, followed by derivatization with NPM. The calibration curves are linear over a concentration range of 25-1250 nm (r2 > 0.995). The coefficients of variations for intra-run precision and inter-run precision range from 0.49 to 5.10% with an accuracy range of 1.78-6.15%. The percentage of relative recovery ranges from 97.3 to 103.2%. This new method provides a simple, efficient, and cost-effective way of determining glutathione disulfide levels with a 2.5 nm limit of detection per 5 µL injection volume. |
| Type: | Article - Journal text |
| In Title: | Biomedical Chromatography |
| Copyright Notice: | This material is presented to ensure timely dissemination of scholarly and technical work. Copyright and all rights therein are retained by authors or by other copyright holders. All persons copying this information are expected to adhere to the terms and constraints invoked by each author's copyright. In most cases, these works may not be reposted without the explicit permission of the copyright holder. Pre-print: author can archive; Post-print: author can archive; FULL COPYRIGHT INFORMATION: |
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| title | Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol |
| contributor.author | Ates, Burhan |
| contributor.author | Ercal, Baris Can |
| contributor.author | Manda, Kalyan |
| contributor.author | Abraham, LInu |
| contributor.author | Ercal, Nuran |
| contributor.deptlab | Center for Environmental Science and Technology (CEST) |
| contributor.deptlab | Chemistry |
| contributor.deptlab | Environmental Research Center |
| contributor.sponsor | National Institute of Health |
| contributor.sponsor | Turkish Scientific Technical Research Council |
| subject | DTT |
| subject | GSH |
| subject | GSSG |
| subject | HPLC |
| subject | thiol-disulfide exchange |
| date.issued | 2008-07 |
| publisher | John Wiley & Sons |
| identifier.citation | Ates, Burhan, Baris Can Ercal, Kalyan Manda, Linu Abraham, and Nuran Ercal. "Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol." Biomedical Chromatography, Jul 2008 |
| identifier.pub.URI | |
| description.abstract | A reverse-phase HPLC method incorporating dithiothreitol (DTT) reduction for quantitative determination of oxidized glutathione (GSSG) in biological samples is described here. This method is based on our previous enzymatic reduction technique that uses N-1-(pyrenyl) maleimide (NPM) as a derivatizing agent. In our earlier method, glutathione disulfide (GSSG) was measured by first reducing it to GSH with glutathione reductase (GR) in the presence of NADPH. However, this is a very costly and time-consuming technique. The method described here employs a common and inexpensive thiol-disulfide exchanging agent, DTT, for reduction of GSSG to GSH, followed by derivatization with NPM. The calibration curves are linear over a concentration range of 25-1250 nm (r2 > 0.995). The coefficients of variations for intra-run precision and inter-run precision range from 0.49 to 5.10% with an accuracy range of 1.78-6.15%. The percentage of relative recovery ranges from 97.3 to 103.2%. This new method provides a simple, efficient, and cost-effective way of determining glutathione disulfide levels with a 2.5 nm limit of detection per 5 µL injection volume. |
| type | Article - Journal |
| type.DCMIType | text |
| type.status | Postprint |
| relation.isPartOf | Biomedical Chromatography |
| rights | This material is presented to ensure timely dissemination of scholarly and technical work. Copyright and all rights therein are retained by authors or by other copyright holders. All persons copying this information are expected to adhere to the terms and constraints invoked by each author's copyright. In most cases, these works may not be reposted without the explicit permission of the copyright holder. |
| rights | Pre-print: author can archive; Post-print: author can archive; |
| rights.URI | |
| rights.URI | |
| identifier.persist.URI | |
| date.available | 2008-12-22T17:45:17Z |