Clinical Science (1992) 83; 637 (Printed in G r e a t Britain)
637
Correspondence
Oxidation state of glutathione in the erythrocyte M. GARNER, J. REGLINSKI, W. E. SMITH and 1. McMURRAY* Department of Pure and Applied Chemistry, Strathclyde University, Glasgow GI IXL, U.K., and *Department of Medicine, Western Infirmary, Glasgow GI1 6NT, U.K. (Received II June 1992)
The oxidation-reduction status of erythrocyte glutathione (GSH:GSSG) is central to many studies of disease involving oxidative stress and free-radical pathologies. A prerequisite of many assay procedures is that the cell must be ruptured before analysis. Many reports suggest that erythrocyte glutathione is predominantly in the reduced state. More recently this position has been the focus of some attention [l, 21. A technique that can assay glutathione directly in the intact erythrocyte is 'H spin echo n.m.r. We have been using this technique within a clinical framework for our studies on diseases involving oxidative stress [3, 41. It works by comparing two resonances in the spectrum. One is derived from the hydrogen environment adjacent to the thiol function and is therefore susceptible to changes in the oxidation state of glutathione. This resonance is compared with another proton resonance in glutathione which is remote from the thiol function and invariant to oxidation. In 18 normal healthy subjects we found that erythrocyte glutathione is approximately 46+3% reduced. However, when the cell is ruptured and the assay is repeated on the lysate the concentration of the reduced form of glutathione rises to 60_+3%, indicating that a major shift in the oxidation-reduction balance occurs on lysis. Lysis is common to most other methods of assessment of glutathione oxidation-reduction status in cells. Researchers should be aware that they are analysing a situation which is, in part, created by the preparation technique. In addition, the n.m.r. data demonstrate that the premise that the initial oxidation-reduction status of glutathione inside the cell is that it is totally in the reduced state is not always correct.
REFERENCES I. Schofield, D. & Braganza, J.M. Difficulties in measuring oxidized glutathione in biological samples. Clin. Sci. 1992; 82, 117-18.
2. Duthie, G.G., Maughan, R.J. 81 Robertson, J.D. Difficulties in measuring oxidized glutathione in biological samples: authors' reply. Clin. Sci. 1992; 82,
117-18. 3. Reglinski, I., Smith, W.E., Wilson, R. et al. Clinical analysis in intact
-
.
erythrocytes using 'H spin echo NMR. Clin. Chim. Acta 1991; 201, 45-58. 4. Reglinski, J., Smith, W.E., Marabani, M., Breuki, M. & Sturrock, R.D. Clinical analysis using 'H spin echo NMR. II:Oxidation of intracellular glutathione as a consequenceof penicillamine therapy. J. Med Chem. 1992; 35, 2134-7.
637
Correspondence
Oxidation state of glutathione in the erythrocyte M. GARNER, J. REGLINSKI, W. E. SMITH and 1. McMURRAY* Department of Pure and Applied Chemistry, Strathclyde University, Glasgow GI IXL, U.K., and *Department of Medicine, Western Infirmary, Glasgow GI1 6NT, U.K. (Received II June 1992)
The oxidation-reduction status of erythrocyte glutathione (GSH:GSSG) is central to many studies of disease involving oxidative stress and free-radical pathologies. A prerequisite of many assay procedures is that the cell must be ruptured before analysis. Many reports suggest that erythrocyte glutathione is predominantly in the reduced state. More recently this position has been the focus of some attention [l, 21. A technique that can assay glutathione directly in the intact erythrocyte is 'H spin echo n.m.r. We have been using this technique within a clinical framework for our studies on diseases involving oxidative stress [3, 41. It works by comparing two resonances in the spectrum. One is derived from the hydrogen environment adjacent to the thiol function and is therefore susceptible to changes in the oxidation state of glutathione. This resonance is compared with another proton resonance in glutathione which is remote from the thiol function and invariant to oxidation. In 18 normal healthy subjects we found that erythrocyte glutathione is approximately 46+3% reduced. However, when the cell is ruptured and the assay is repeated on the lysate the concentration of the reduced form of glutathione rises to 60_+3%, indicating that a major shift in the oxidation-reduction balance occurs on lysis. Lysis is common to most other methods of assessment of glutathione oxidation-reduction status in cells. Researchers should be aware that they are analysing a situation which is, in part, created by the preparation technique. In addition, the n.m.r. data demonstrate that the premise that the initial oxidation-reduction status of glutathione inside the cell is that it is totally in the reduced state is not always correct.
REFERENCES I. Schofield, D. & Braganza, J.M. Difficulties in measuring oxidized glutathione in biological samples. Clin. Sci. 1992; 82, 117-18.
2. Duthie, G.G., Maughan, R.J. 81 Robertson, J.D. Difficulties in measuring oxidized glutathione in biological samples: authors' reply. Clin. Sci. 1992; 82,
117-18. 3. Reglinski, I., Smith, W.E., Wilson, R. et al. Clinical analysis in intact
-
.
erythrocytes using 'H spin echo NMR. Clin. Chim. Acta 1991; 201, 45-58. 4. Reglinski, J., Smith, W.E., Marabani, M., Breuki, M. & Sturrock, R.D. Clinical analysis using 'H spin echo NMR. II:Oxidation of intracellular glutathione as a consequenceof penicillamine therapy. J. Med Chem. 1992; 35, 2134-7.
