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. 2006 May;385(1):105-13.
doi: 10.1007/s00216-006-0375-8. Epub 2006 Mar 18.

Rapid determination of reduced and oxidized glutathione levels using a new thiol-masking reagent and the enzymatic recycling method: application to the rat liver and bile samples

Imam H Shaik  1 , Reza Mehvar
Affiliations

Rapid determination of reduced and oxidized glutathione levels using a new thiol-masking reagent and the enzymatic recycling method: application to the rat liver and bile samples

Imam H Shaik et al. Anal Bioanal Chem. 2006 May.

Abstract

A microtiter plate assay for quantitation of reduced (GSH) and oxidized (GSSG) glutathione in the rat liver tissue and bile is described. The assay is based on the established enzymatic recycling method and a new thiol-masking reagent, 1-methyl-4-vinyl-pyridinium trifluoromethane sulfonate (M4VP). Samples were first processed by homogenization with (liver) or addition of (bile) sulfosalicylic acid. The total glutathione and GSSG were then determined before and after rapid (< or = 2 min) and efficient (100%) masking of the GSH content of the samples with M4VP followed by the enzymatic recycling assay. The percentages of error and coefficient of variation of the assay were within the accepted guidelines, indicating the accuracy and precision of the assay in the range of 6.25-100 pmol GSH per microplate well and 2.17-140 pmol GSSG per well, with lower limit of quantitation of 6.25 and 2.17 pmol per well for GSH and GSSG, respectively. Furthermore, the recoveries of added GSH or GSSG from the liver and bile samples were accurate and precise. The assay was applied to measurement of GSH, GSSG, and GSH:GSSG ratio in the liver and serially collected bile samples in sham-operated and ischemic rat livers, demonstrating a depletion of glutathione and a decrease in the GSH:GSSG ratio as a result of ischemia. The developed assay is rapid, sensitive, accurate, and precise and is suitable for studies of the redox status of liver under physiologic and pathophysiologic conditions.

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Figures

Figure 1
Figure 1
The time courses of the reaction of M4VP with GSH under different pH values, resulting in masking GSH. The M4VP: GSH ratio was fixed at 3. Symbols and bars represent the mean and SD values, respectively (n = 3).
Figure 2
Figure 2
The time courses of the reaction of M4VP with GSH at M4VP:GSH molar ratios of 1, 2, 3, 6, or 9 in the presence of 6 mM GSH (top) and at M4VP:GSH molar ratios of 1 and 9 in the presence of 1 or 6 mM of GSH (bottom) in the sample. The pH was fixed at 10. Symbols and bars represent the mean and SD values, respectively (n = 3).
Figure 3
Figure 3
The time courses of the reaction of M4VP with GSH present in the liver homogenate or bile samples using the final method (see the text for details). Symbols and bars represent the mean and SD values, respectively (n = 3).
Figure 4
Figure 4
Typical time courses of the change in the absorbance versus time in the enzymatic recycling method (top) and the resulting calibration curve (bottom) for GSH concentrations in the range of 6.25–100 pmol per well (0.375–6.00 mM in the original samples). Each point is an average of triplicate measurements.
Figure 5
Figure 5
The mean (columns) and SD (bars) of liver GSSG levels analyzed using M4VP as explained in this article and also using 2VP as described in an established method using cuvette [10] or microtiter plate [11]. *, Significantly different from the M4VP method (ANOVA followed by post-hoc analysis using Scheffe F test).
Figure 6
Figure 6
The bile flow rates (top) and biliary concentrations of GSH (solid bars) and GSSG (open bars) and %GSSG contents of total glutathione in bile (line, right y axes) in the sham-operated (middle) and ischemic (bottom) livers.
Scheme 1
Scheme 1
The enzymatic recycling method for quantitation of GSH and/or GSSG. GSSG, oxidized glutathione; GSH, reduced glutathione; GR, glutathione reductase; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); TNB, 5-thio-2-nitrobenzoic acid; GSTNB, the disulfide product of reaction of GSH with DTNB.
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