Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol.
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 (r(2) > 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 microL injection volume.
Ates
Burhan
B
Department of Chemistry, Missouri University of Science and Technology, 400 West 11th Street, Rolla, MO 65409, USA.
Ercal
Baris Can
BC
Manda
Kalyan
K
Abraham
Linu
L
Ercal
Nuran
N
D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004229:Dithiothreitol / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000032:analysis* / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D016014:Linear Models; D008099:Liver / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D008301:Maleimides / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D008301:Maleimides; C003944:N-(3-pyrene)maleimide; D005980:Glutathione Reductase; D005978:Glutathione; D004229:Dithiothreitol; D019803:Glutathione Disulfide