Clinical Science (1992) 82, I 17-1 18 (Printed in Great Britain)
II7
Correspondence
Difficulties in measuring oxidized glutathione in
biological samples
D. SCHOFIELD and J.M. BRAGANZA
Pancreato-Biliary Service, Royal Infirmary, Manchester
M f 3 9WL, U.K.
(Received 3 July 1991)
A recent report by Robertson et al. (Clin. Sci. 1991; 80,
61 1-18) [l]highlights the difficulty in accurately measuring oxidized glutathione (GSSG) in biological material.
The authors found that <75% of glutathione in erythrocytes was present in the active reduced form (GSH). The
implication that as much as 25% was in the inactive
oxidized form of GSSG is contrary to the accepted view,
i.e. that GSSG normally accounts for < 0.2% of the total
glutathione in cells, including erythrocytes [2, 31. The
discrepancy reflects the ease with which GSH autoxidizes
to GSSG during the extraction procedure, as applied to
biological samples [3]. It is recognized that chemical
trapping of GSH before determination of GSSG, e.g. by
the glutathione reductase recyclingassay [3], must be both
fast and quantitative.
The authors used 2-vinylpyridine to trap GSH because
the presence of an excess of this reagent does not interfere
with the subsequent recycling assay for GSSG 141.
However, autoxidation of GSH is very likely in the 20-60
min period that is required for the derivatization reaction
at neutral pH: a sevenfold increase in GSSG has been
recorded within 60 min in untreated rat blood [5]. Therefore the more reactive trapping reagent N-ethylmaleimide
(NEM) is preferred [3], although this method is labourintensive because excess reagent interferes with the
recycling assay and must be removed. The use of NEM
avoids the artifactual high levels of GSSG reported by
Robertson et al. [l]and confirms that intracellular glutathione is almost entirely in the reduced form in healthy
subjects, owing to rapid and efficient reduction of GSSG
by the glutathione reductase/NADPH system in vivo.
Difficulties in measuring oxidized glutathione in
biological samples: authors' reply
G. G. DUTHIE, R. J.MAUGHAN and J. D. ROBERTSON
Rowett Research Institute, Greenburn Road, Bucksburn,
Aberdeen AB2 993, U.K.
In referring to our publication (Clin. Sci. 1991; 80,
611-18) [l], Schofield and Braganza point out that the
determination of the proportion of reduced (GSH) and
oxidized (GSSG) glutathione even by the modified
glutathione reductase recycling assay [2] requires speed
and care to avoid artifactual oxidation of GSH. We are
grateful to them for emphasizing this important point.
Reference is made to an example whereby there is a
seven-fold increase in GSSG in untreated rat blood within
60 min. However, Table 1 indicates that, when stored at
4"C, the proportion of GSSG does not change within 2 h
of the blood being obtained. Consequently, it is also
unlikely that significant autoxidation occurs during the 60
min exposure to 2-vinylpyridine (2-VP). Incubation of
GSH (Sigma, catalogue no. G4251) with 2-VP does not
produce detectable autoxidation. Moreover, the reaction
occurs under acid conditions [2] and not at neutral pH as
suggested by Schofield and Braganza, as the addition of
triethanolamine occurs after this stage of the processing.
We have no experience in the use of N-ethylmaleimide
and cannot comment on the assertion that this method is
to be preferred. However, any disparity between the two
methods does not alter the conclusion from our results,
which indicates that the presumably adaptive increase in
total glutathione in response to training load arises
predominantly from an increase in GSH. Finally, a study
from another laboratory on human subjects [3]using h.p.1.c.
[4] indicates that the pre-exercise concentrations of total
glutathione and GSSG in human blood are 0.6 mmol/l
and 0.15 mmol/l, respectively. These proportions are
similar to those obtained in our study.
Table I. Proportion of GSSG in r a t blood stored a t 4°C. Values
are the means of five samples.
~
~~
REFERENCES
Time taken after sample taken
GSSG (%)
I . Robertson, J.D., Maughan, R.J., Duthie, G.G. & Morrice, P.C. Increased blood
Immediately
2h
I day
3 days
I.8
1.3
4.6
15.8
2.
3.
4.
5.
antioxidant systems of runners in response to training load. Clin. Sci. 1991;
80,611-18.
Srivastava, S.K. & Beutler, E. Accurate measurement of oxidised glutathione
content of human, rabbit, and rat red blood cells and tissues. Anal. Biochem.
1968; 25,70-6.
Akerboom, T.P.M. & Sies, H. Assay of glutathione, glutathione disulfide, and
glutathione mixed disulphides in biological samples. Methods Enzymol. 1981;
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Griffith, O.W. Determination of glutathione and glutathione disulfide using
glutathione reductase and 2-vinylpyridine. Anal. Biochem. 1980; 106, 207-1 2.
Adams, J.D., Lauterburg, B.H. & Mitchell, J.R. Plasma glutathione and glutathione disulfide in the rat; regulation and response to oxidative stress.
J. Pharmacol. Exp. Ther. 1983; 221,749-54.
REFERENCES
I. Robertson, J.D., Maughan, R.J., Duthie. G.G. & Morrice, P.C. Increased blood
antioxidant systems of runners in response t o training load. Clin. Sci. 1991;
80,611-18.
2. Allen, K.G.D. & Arthur, J.R. Inhibition by 5-sulphosalicylic acid of the gluta-
I I8
Correspondence
thione reductase recycling assay for glutathione analysis. Clin. Chim. Acta
1987; 162,237-9.
3. Gohil, K., Viguie, C.. Stanley, W.C.. Brooks, G.A. & Packer, L. Blood glutathione oxidation during human exercise. J. Appl. Physiol. 1988; 64, 115-19.
4. Reed, D.J.. Babson, J.R., Beatty, P.W., Brodie, A.E., Ellis, W.W. & Potter,
D.W. High performance liquid chromatography of nanomol levels of glutathione, glutathione disulphide and related thiols and disulphides. Anal.
Biochem. 1980 106,55-62.