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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.

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Figure 1: Schematic representation of the mechanism of the GSH recycling method.
Figure 2: Graphical representation of the preanalytical manipulation of blood samples for GSH and GSSG analyses.
Figure 3: Representative tracings obtained by applying the GSH recycling method for GSSG analysis in blood.
Figure 4: Representative chromatogram of the GS-NEM conjugate in blood samples.

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Acknowledgements

This work was supported in part by grants to P.F. from the US National Institutes of Health (NIH) National Center for Complementary and Alternative Medicine (NCCAM) (grant no. AT004490) and from the US Department of Veterans Affairs (Merit Review no. 1I01CX000264).

Author information

Authors and Affiliations

Authors

Contributions

D.G. performed the experiments and wrote the manuscript; I.D-.D. and A.M. analyzed data; P.F. wrote the manuscript; and R.R. supervised the work and wrote the manuscript.

Corresponding author

Correspondence to Ranieri Rossi.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 GSH titration by spectrophotometer

Spectrophotometer is set at 412 nm wavelength and a continuous reading is performed for a few minutes, until a plateau is reached. 1 mL PB50, 40 µM (final concentration) GSH and 0.1 mM (final concentration) DTNB are added into optical cuvettes in this order. A typical curve obtained by this procedure is shown in the figure. Other details are described in Supplementary Methods.

Supplementary Figure 2 Analysis of GSH by HPLC in rat tissues other than blood

Some tissues present a slight peak around 4.95 min r.t. that may overlap with one of the peaks of GS-NEM. An example is shown in the figure. Rat lungs were removed and rapidly washed. One lung was homogenized in BSAN buffer as described in PROCEDURE, the other one was homogenized in the same buffer omitting NEM. After deproteination with TCA15, both samples were analyzed by HPLC. The elution profile of the sample homogenized in BSAN buffer (blue tracing) is characterized by the presence of both the two symmetrical peaks corresponding to GS-NEM conjugate (4.29 min and 4.95 min r.t.) and the peak corresponding to the excess of NEM (5.8 min r.t.). In the absence of NEM an interfering peak in correspondence of the GS-NEM peak eluting at 4.95 min r.t. can be noted (red tracing). These data enhance the notion that even if, in theory, either peaks relative to GS-NEM conjugate can be used for calculations, the peak eluting at about 4.29 min r.t. is to be preferred since, in our experience, it is always free of interferences.

Supplementary Figure 3 Stability of GS-NEM conjugate

Human blood was processed for GSH analysis by HPLC, i.e. collection into tubes containing K3EDTA/NEM310 and deproteination by addition of TCA15 (as described in PROCEDURE). The blue tracing shows the chromatogram obtained by freshly analyzing the sample. The same sample was then analyzed after 72 hours of storage at room temperature (red tracing). No significant difference was observed between the two tracings and the area of the peak eluting at 4.29 min r.t. was identical. These data indicate that acidified samples are stable for at less 48 hours at room temperature.

Supplementary information

Supplementary Figure 1: GSH titration by spectrophotometer.

The spectrophotometer is set at a 412-nm wavelength and a continuous reading is performed for a few minutes until a plateau is reached. 1 ml of PB50, 40 μM (final concentration) GSH and 0.1 mM (final concentration) DTNB are added into optical cuvettes in this order. A typical curve obtained by this procedure is shown. Other details are described in the Supplementary Methods. (PDF 483 kb)

Supplementary Figure 2: Analysis of GSH by HPLC in rat tissues other than blood.

Some tissues present a slight peak around an r.t. of 4.95 min that may overlap with one of the peaks of GS-NEM. An example is shown. Rat lungs were removed and rapidly washed. One lung was homogenized in BSAN buffer as described in the PROCEDURE; the other one was homogenized in the same buffer omitting NEM. After deproteination with TCA15, both samples were analyzed by HPLC. The elution profile of the sample homogenized in BSAN buffer (blue tracing) is characterized by the presence of both the two symmetrical peaks corresponding to the GS-NEM conjugate (4.29 min and 4.95 min r.t.) and the peak corresponding to the excess of NEM (5.8 min r.t.). In the absence of NEM, an interfering peak corresponding to he GS-NEM peak eluting at 4.95 min r.t. can be noted (red tracing). These data enhance the notion that even if, in theory, either peak relative to the GS-NEM conjugate can be used for calculations, the peak eluting at about 4.29 min r.t. is preferred, as in our experience, it is always free of interference. (PDF 2629 kb)

Supplementary Figure 3: Stability of GS-NEM conjugate.

Human blood was processed for GSH analysis by HPLC (i.e., by collection into tubes containing K3EDTA/NEM310 and deproteination by addition of TCA15 (as described in the PROCEDURE)). The blue tracing shows the chromatogram obtained by freshly analyzing the sample. The same sample was then analyzed after 72 h of storage at room temperature (red tracing). No significant difference was observed between the two tracings, and the area of the peak eluting at 4.29 min r.t. was identical. These data indicate that acidified samples are stable for at least 48 h at room temperature. (PDF 561 kb)

Supplementary Methods

Titration of NADPH, activity of glutathione reductase and preparation of RBCs. (PDF 494 kb)

Supplementary Table 1

Measured levels of GSH and GSSG in human whole blood by applying different methodological protocols. (PDF 513 kb)

Supplementary Table 2

Recovery of GSH and GSSG added to blood. (PDF 471 kb)

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Giustarini, D., Dalle-Donne, I., Milzani, A. et al. Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nat Protoc 8, 1660–1669 (2013). https://doi.org/10.1038/nprot.2013.095

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