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Clinical Chemistry 52: 1406-1414, 2006. First published May 11, 2006; 10.1373/clinchem.2006.067793
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(Clinical Chemistry. 2006;52:1406-1414.)
© 2006 American Association for Clinical Chemistry, Inc.

General Clinical Chemistry

Oxidized Forms of Glutathione in Peripheral Blood as Biomarkers of Oxidative Stress

Ranieri Rossi1,a, Isabella Dalle-Donne2, Aldo Milzani2 and Daniela Giustarini1

1 Department of Neuroscience, Pharmacology Section, University of Siena, Siena, Italy.
2 Department of Biology, University of Milan, Milan, Italy.

aAddress correspondence to this author at: Department of Neuroscience, Pharmacology Section, University of Siena, via A. Moro 4, 53100 Siena, Italy. Fax 39-0577-234208; e-mail ranieri{at}unisi.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Reduced glutathione (GSH) and its redox forms, glutathione disulfide (GSSG) and glutathionylated proteins (PSSG), are biomarkers of oxidative stress, but methodologic artifacts can interfere with their measurement. We evaluated the importance of correct sample handling during the preanalytical phase for GSH, GSSG, and PSSG measurement.

Methods: We used human blood for in vitro experiments with oxidants [tert-butylhydroperoxide (t-BOOH), diamide, and menadione]. For in vivo experiments, we used rats in which we cannulated the jugular and femoral veins for both oxidant administration and blood collection. We measured GSH, GSSG, and PSSG with HPLC with or without sample pretreatment with N-ethylmaleimide (NEM) to prevent artifacts. We also measured malondialdehyde (MDA) with HPLC, and protein carbonyls (PCO) with spectrophotometric procedures.

Results: When methodologic artifacts were prevented by pretreatment with NEM, GSSG results increased up to 3-fold over the basal concentrations, even in the presence of 5 µmol/L t-BOOH or diamide and 20 µmol/L menadione. PSSG increased by ~50% at 20 µmol/L t-BOOH or diamide and at 50 µmol/L menadione. PCO and MDA remained unchanged. In vivo oxidation treatments elicited immediate and significant increases in GSSG and PSSG over basal values (up to 200-fold), whereas PCO and MDA showed only slight variation 120 or 180 min after treatment.

Conclusions: With the use of artifact-free measurement methods, GSH, GSSG, and PSSG are potentially powerful and reliable biomarkers of oxidative stress status and can be used to evaluate whether, and to what extent, oxidative stress may be involved in various diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Unbalanced production of reactive oxygen species (ROS)1 is thought to be related to the pathogenesis of several human diseases, such as ischemic stroke, atherosclerosis, cardiovascular disease, and neurodegenerative diseases, as well as diabetes mellitus and its complications. Despite the plausibility of this relationship, demonstrating a direct link between oxidative stress and specific disease onset/progression has been difficult, partly because of the diversity of analytical procedures used in the field.

Biological macromolecules can be damaged by oxidative insult, leading to oxidized products that act as biomarkers of oxidative stress status. Methods have been developed to measure these biomarkers as a means of evaluating oxidative stress (1)(2). The commonly studied markers are products of lipid peroxidation [such as malondialdehyde (MDA), 4-hydroxynonenal, and isoprostanes (3)], oxidized amino acidic residues (such as cystine, methionine sulfoxide, 3-nitrotyrosine, and 3-Cl-tyrosine), or protein carbonyls (PCO) (1)(4).

A different approach to evaluation of oxidative stress is the analysis of antioxidant concentrations. An ROS attack can lead to a major depletion of antioxidants such as vitamin E, vitamin C, reduced glutathione (GSH), and urate (5). GSH can be oxidized, mainly to glutathione disulfide (GSSG), or can form glutathionylated proteins (PSSG). The measurement of GSH, GSSG, PSSG, and their relative ratios may therefore give fundamental information on the intracellular redox status (6).

Analytical methods based on spectrophotometry, HPLC, capillary electrophoresis, nuclear magnetic resonance, and mass spectrometry have been reported for the determination of glutathione in biological samples (7). Nevertheless, some artifacts still plague the exact evaluation of GSH and, particularly, GSSG and PSSG in blood samples. After showing that sample acidification for protein precipitation artifactually increases GSSG and PSSG concentrations, we developed methods for GSH, GSSG, and PSSG measurements that avoid these pitfalls (8)(9)(10). Sample acidification, in the presence of large quantities of heme-containing proteins, leads to production of large amounts of ROS (8), which in turn increases GSSG and PSSG more than 10-fold and decreases GSH by 20%–30%. We believe that only a few [see, for example, Refs. (8)(9)(10)(11)(12)(13)] of the hundreds of studies of blood glutathione have reported the true concentrations of GSH, GSSG, and PSSG. Here we investigate the ability of GSH, GSSG, and PSSG, when correctly measured in blood, to reflect oxidative stress status.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
blood collection
Sprague-Dawley rats (400–450 g) were purchased from Charles River. A valve was implanted in each animal, and the jugular and femoral veins of each animal were cannulated for both drug administration and blood collection, as described previously (14). The valve was implanted under pentobarbital anesthesia the day before the experiment. Animals were allowed to freely move and fed ad libitum before and during the experiments. Rats received infusions of diamide (25 µmol · kg–1 · h–1), tert-butylhydroperoxide (t-BOOH; 40 µmol · kg–1 · h–1), or menadione bisulfite (40 µmol · kg–1 · h–1) in saline (1.5 mL/h) via the cannula implanted in the femoral vein. The infusion was started immediately after blood withdrawal for zero-time measurements and lasted 2 h. Blood aliquots (200 µL each) were collected through the valve and immediately processed as described below. All animal manipulations were made in accordance with the European Community guidelines for the use of laboratory animals.

Blood samples were provided by healthy volunteers who had given informed consent. Samples were obtained by puncture of the antecubital vein and collected into heparin-containing tubes.

blood processing
Nine parts of blood were immediately (or at the indicated times) diluted with 1 part of a solution containing 1.5 g/L tripotassium EDTA and 500 mmol/L N-ethylmaleimide (NEM), after which GSH and GSSG were determined as described previously (10). Briefly (for the standardized procedure and further details see the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol52/issue7), samples were deproteinized with trichloroacetic acid (TCA), and the excess of NEM was extracted from the supernatant with dichloromethane. After alkalinization, supernatants were reacted with 2,4-dinitrofluorobenzene, acidified, and analyzed by HPLC (Agilent series 1100) (10).

Blood samples in which NEM was omitted to investigate the artifactual acid-derived increases in GSSG and PSSG (see Results) were immediately (or at the indicated times) diluted 10:1 with a solution containing 1.5 g/L tripotassium EDTA and 250 mmol/L NaCl. After sample acidification (see above), 50 mmol/L NEM was added to the deproteinized supernatants. Samples were alkalinized for 30 s; the NEM was then extracted and both GSH and GSSG were measured as above described.

S-Glutathionylated proteins were measured by a previously described method with slight modifications (9). Briefly, the protein pellets obtained from TCA-treated samples were washed 3 times with 5 g/L TCA. GSH was formed by incubation with dithiothreitol, conjugated with fluorescent monobromobimane (Calbiochem), and detected by HPLC (for the standardized procedure and further details, see the online Data Supplement).

PCO and MDA were measured by HPLC and spectrophotometry (Jasco; V-530) in the same blood samples collected in tripotassium EDTA and NEM (15)(16).

statistical analysis
Values are reported as the mean (SD). Statistical significance was evaluated using the Student t-test with P <0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
widespread artifact
Most methods for titration of GSH, GSSG, and PSSG require removal of proteins from samples before analysis. This is commonly achieved by acid addition, e.g., TCA. However, during acid denaturation, the presence of hemoglobin within erythrocytes can lead to oxidant production (8)(17). This artifact affects measured concentrations of GSH, GSSG, and PSSG, slightly decreasing the GSH concentration and greatly increasing GSSG and PSSG (Fig. 1 ). We divided the blood samples into 2 aliquots, one of which was treated with the thiol alkylating agent NEM before acidification. Samples were then analyzed by HPLC for simultaneous detection of both GSH and GSSG (10). Acid treatment without blocking of the free sulfhydryl groups of glutathione decreased the GSH peak area by 15%–20% and greatly increased the GSSG peak area. A similar large increase was detected in PSSG (tracings not shown).


Figure 1
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  		Figure 1. HPLC tracings of GSH and GSSG in human blood.

Blood samples from healthy donors were collected into tubes with or without NEM. We measured GSH and GSSG concentrations by HPLC after derivatization of the amino groups with 2,4-dinitrofluorobenzene. ABS, absorbance; mAU, milliabsorbance units. Inset shows an enlargement of the GSSG peaks.

Both GSSG and PSSG increased almost 100-fold in blood samples from healthy donors when thiols were not protected (by NEM) from oxidation before acidification of the sample (Table 1 ). The data suggest that the acid-induced artifactual GSH oxidation can be influenced by several factors, e.g., hemoglobin binding state and sample dilution. Thus, the acid-derived artifact can produce a large random bias of measured GSSG and PSSG values from their true concentrations.


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  		Table 1. Concentrations of GSH, GSSG, and PSSG in human blood under different conditions.a

effect of the artifact on the evaluation of oxidative stress
Treatment of blood samples with the oxidants diamide, t-BOOH, or menadione, which have different mechanisms of action (18)(19)(20), generated GSSG and PSSG (Figs. 2 and 3 ). The increases in GSSG and PSSG were evident at low concentrations of the oxidants only when the preanalytical artifact was prevented by pretreatment with NEM (bottom panels in Figs. 2 and 3 ). The measured GSSG concentration increased significantly when as little as 5 µmol/L t-BOOH or diamide or 20 µmol/L menadione was added (Fig. 2 ). Oxidant-induced increases in PSSG were similar to those in GSSG (Fig. 3 ). Sample acidification in the absence of NEM (Figs. 2 and 3 , top panels) falsely increased GSSG and PSSG values, which can obscure oxidative stress–induced increases in GSSG and PSSG at low micromolar concentrations of oxidants.


Figure 2
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  		Figure 2. Blood GSSG concentrations after in vitro oxidative stress.

Blood from healthy donors was treated, within 30–60 min from collection, with t-BOOH (5 mmol/L stock solution dissolved in saline), diamide (5 mmol/L stock solution dissolved in saline), or menadione (5 mmol/L stock solution dissolved in saline) at different final concentrations. After 2.5 min, a solution containing 1.5 g/L tripotassium EDTA and 0.5 mol/L NEM or 250 mmol/L NaCl (9:1 blood/solution) was added and mixed well. Samples were deproteinized with TCA, and GSSG in the supernatant was measured by HPLC. The experiments were carried out under gentle stirring at 37 °C. (Top panels), samples without NEM; (bottom panels), samples with NEM. Data points are the mean (SD; error bars); n = 5. *, P <0.05 vs control value.


Figure 3
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  		Figure 3. Blood PSSG concentration after in vitro oxidative stress.

Blood from healthy donors was treated, within 30–60 min from collection, with t-BOOH (5 mmol/L stock solution dissolved in saline), diamide (5 mmol/L stock solution dissolved in saline), or menadione (5 mmol/L stock solution dissolved in saline) at different final concentrations. After 2.5-min, a solution containing 1.5 g/L tripotassium EDTA and 0.5 mol/L NEM or 250 mmol/L NaCl (9:1 blood/solution) was added and mixed well. Samples were deproteinized with TCA, and PSSG in the protein pellet was measured by HPLC. The experiments were carried out under gentle stirring at 37 °C. (Top panels), samples without NEM; (bottom panels), samples with NEM. Data points are the mean (SD; error bars); n = 5. *, P <0.05 vs control value.

PSSG decreased dramatically at near-millimolar t-BOOH concentrations in samples without NEM (Fig. 4 , left panel). This is a paradoxical effect because treatment with an oxidant should increase rather than decrease the PSSG concentration. In confirmation of this, when the same samples were pretreated with NEM before acidification, PSSG increased in parallel with t-BOOH concentration (Fig. 4 , right panel). The results obtained without pretreatment with NEM can be explained by considering that t-BOOH at higher doses completely (and rapidly) oxidizes GSH to GSSG and (scarcely) to PSSG, making it unavailable for the artifactual formation of PSSG. Conversely, at lower t-BOOH concentrations (or in untreated samples), much of the GSH can be artifactually oxidized as a consequence of the acid treatment.


Figure 4
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  		Figure 4. Rapid t-BOOH-induced PSSG changes in human blood.

Blood from healthy donors was treated, within 30–60 min from collection, with t-BOOH (50 mmol/L stock solution dissolved in saline) at different final concentrations. After 15 s, a solution containing 1.5 g/L tripotassium EDTA and 0.5 mol/L NEM or 250 mmol/L NaCl (9:1 blood/solution) was added and mixed well. Samples were deproteinized with TCA, and PSSG was then measured in the protein pellet by HPLC. The experiments were carried out under gently stirring at 37 °C. (Left), samples without NEM; (right), samples with NEM. Data points are the mean (SD; error bars); n = 5.

measurement of gssg and pssg to reveal oxidative stress
In vitro experiments.
When methodologic artifacts were avoided as much as possible, measurement of GSH, GSSG, and/or PSSG indicated the occurrence of oxidative stress. To evaluate the effectiveness of these molecules compared with other "fingerprinting molecules" of oxidative stress, we treated human blood with increasing concentrations of t-BOOH, diamide, and menadione and verified the effect on GSSG and PSSG as well as on PCO and MDA concentrations. PCO and MDA were chosen because they are among the most widely used indicators of oxidative stress (21). Our data (Fig. 5 ) indicated that for oxidant concentrations <100 µmol/L, only GSSG and PSSG were significantly increased over basal concentrations, whereas PCO and MDA remained unchanged.


Figure 5
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  		Figure 5. Percentage increases of GSSG, PSSG, MDA, and PCO in blood over basal concentrations after in vitro oxidative stress.

Blood from healthy donors was treated, within 30–60 min from collection, with t-BOOH (5 mmol/L stock solution dissolved in saline), diamide (5 mmol/L stock solution dissolved in saline), or menadione (5 mmol/L stock solution dissolved in saline) at different final concentrations. After 2.5 min, a solution containing 1.5 g/L tripotassium EDTA and 0.5 mol/L NEM (9:1 blood/solution) was added and mixed well. Sample aliquots were deproteinized with TCA. GSSG and MDA in the supernatants and PSSG in the protein pellets were measured by HPLC. PCO concentrations were determined by spectrophotometry in plasma obtained after blood centrifugation. The experiments were carried out under gentle stirring at 37 °C. Data points are the mean (SD; error bars); n = 5 for each oxidant treatment.

In vivo experiments.
We evaluated oxidant-induced changes in GSSG, PSSG, MDA, and PCO concentrations in vivo after intravenous administration of t-BOOH, diamide, and menadione. Rats were treated with the oxidants through the femoral vein with a low flux to simulate (relatively) slight and (relatively) long-lasting oxidative stress. At the indicated times, blood aliquots were drawn from the jugular vein. In analogy with the in vitro experiments, the in vivo treatments also elicited immediate and significant increases in GSSG and PSSG over basal concentrations, whereas PCO and MDA increased slightly only after 120 or 180 min of menadione or t-BOOH treatment (Fig. 6 ). Only PSSG concentrations increased after diamide treatment, a finding that is consistent with previous findings in rat blood (14)(22) and is attributable to the presence of highly reactive sulfhydryl groups in rat hemoglobin, which react first with diamide and then with GSH, forming PSSG.


Figure 6
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  		Figure 6. Percentage increase over basal concentrations of blood GSSG, PSSG, MDA, and PCO after in vivo oxidative stress.

Rats received 2-h infusions containing diamide (25 µmol · kg–1 · h–1; A), t-BOOH (40 µmol · kg–1 · h–1; B), or menadione (40 µmol · kg–1 · h–1; C) in saline (1.4–1.6 mL/h, according to rat weight) via the cannula implanted in a femoral vein. For each analysis, 200 µL of blood was collected. A solution containing 1.5 g/L tripotassium EDTA and 0.5 mol/L NEM (9:1 blood/solution) was added and mixed well. Sample aliquots were deproteinized with TCA; GSSG and MDA in the supernatants and PSSG in the protein pellets were measured by HPLC. PCO concentrations were determined by spectrophotometry in plasma obtained after blood centrifugation. Data points are the mean (SD; error bars); n = 4 for each oxidant treatment. Saline stock solutions contained 7 mmol/L diamide, 11 mmol/L t-BOOH, or 7 mmol/L menadione.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Information regarding the existence, severity, time course, and features of oxidative stress status may be gleaned from the analysis of discrete biomarkers isolated from tissues and biological fluids (2)(10). The usefulness of the ideal biomarker of oxidative damage lies in its ability to provide a valid and early indication of disease and/or its progression [for reviews, see Dalle-Donne and coworkers (1)(21)].

Glutathione is the major low–molecular-mass thiol in mammals, and it plays a key role in cell resistance against oxidative and nitrosative damage by providing enzymes involved in the metabolism of ROS with reducing equivalents, by eliminating potentially toxic oxidation products, and by reducing oxidized or nitrosated protein thiols. The availability of GSH under oxidative conditions is ensured by GSH recycling and biosynthetic pathways, which can be up-regulated when oxidative and/or nitrosative stress occurs (5).

Measurement of GSH (and its disulfide forms, i.e., GSSG and GSSP) in blood has been considered an index of the whole-organism oxidative status and a useful indicator of disease risk in humans (7)(8). However, the usefulness of GSH and, particularly, of GSSG and PSSG as powerful biomarkers of oxidative stress is still hampered by some methodologic artifacts, as highlighted by 2 findings: (a) measurements by different research groups of the concentrations of GSH and, even more, of GSSG and PSSG span a more than 100-fold range in healthy persons (8); and (b) in individuals with the same pathology, GSH, GSSG, and PSSG have frequently been observed to increase by one research group and decrease by another group, e.g., in diabetes (23)(24) or HIV (25)(26).

Blood and other tissues contain millimolar concentrations of GSH and low-micromolar concentrations of its oxidized forms. Therefore, minimal artifactual oxidation can cause, on one hand, a slight decrease in GSH concentrations with an acceptable bias from the real values and, on the other hand, dramatic effects on GSSG and PSSG concentrations, with increases of more than 100-fold. Hence, handling any tissue without immediate blocking of free thiols may lead to misleading results. In handling blood, the situation is further complicated because hemoglobin can deliver electrons to oxygen or other molecules, yielding free radicals, particularly during sample acidification, artificially increasing the real values of GSSG and PSSG (8)(9). Techniques for PSSG detection are available that do not include sample acidification, but other pitfalls seem to occur, mainly attributable to sample hemolysis, freezing and thawing, or washing blood cells with saline without adequate glucose supplementation (23)(27). Thus, most values reported in the literature are largely discordant and higher than those measured by us and others (9)(28)(29) in samples in which NEM was immediately added after blood collection. Furthermore, because blood can reduce considerable amounts of hydroperoxides in a few minutes (10), it rapidly reduces both GSSG and PSSG back to GSH if free thiols are not immediately blocked. To avoid this, the thiol/disulfide status must be "frozen" immediately after blood withdrawal. Because NEM inhibits the enzymes, the addition of NEM immediately after blood collection appears to prevent both acid-induced GSH oxidation and reduction of GSSG and PSSG by reductases (8). When NEM is omitted, artifactually higher GSSG and PSSG concentrations (as well as lower GSH values) are measured (Table 1Up ) to different extents, depending on the type of acid used for deproteinization, sample dilution before acidification, and hemoglobin-binding status. Consequently, actual oxidant effects on GSH, GSSG, and PSSG concentrations cannot be measured without thiol blocking because the artifactual acidification-induced oxidation of GSH masks the real increase in its oxidized forms, as demonstrated by our in vitro experiments in which GSH, GSSG, and PSSG were measured after blood treatment with t-BOOH, diamide, and menadione (Figs. 2Up and 3Up , top panels). Only in the presence of NEM did even minimal concentrations of oxidants elicit significant increases in GSSG and PSSG (Figs. 2Up and 3Up , bottom panels). Additionally, without NEM, a paradoxical event may occur when oxidants are administered at higher concentrations. Given the rapid oxidation of GSH to GSSG, the former is not prone to artifactual oxidation to form PSSG; therefore, the concentrations of the latter seem to decrease rather than increase (Fig. 4Up ).

Interestingly, neither PCO nor MDA concentrations changed measurably after treatment with oxidants at concentrations of 5–100 µmol/L (in vitro experiments) or 25–40 µmol · kg–1 · h–1 (in vivo experiments), whereas GSSG and PSSG concentrations increased substantially (Figs. 5Up and 6Up ). Although this in vivo model has some limitations, being essentially indicative of a short exposure to a substantial oxidative stress, similar to other studies (2), it gives some basic information on what could be the best biomarkers for evaluating oxidative stress (1). These data indicate that, when measured with artifact-free methods, GSSG and PSSG are extremely sensitive biomarkers of oxidative stress status.

Other authors have also identified artifacts that invalidate GSSG and PSSG analyses and, after controlling these variables, obtained measurements close to the actual concentrations of GSSG and PSSG (11)(12)(30)(31). Unfortunately, those reports seem to be exceptions. No large-scale population studies on GSH status in blood have applied artifact-free methods. Such studies could give important information on the occurrence of oxidative stress and its possible link with the primary or secondary pathophysiologic mechanisms in multiple acute and chronic human diseases. For example, glutathione peroxidase activity was reported to be independently associated with an increased risk of cardiovascular events and may have prognostic value in addition to traditional risk factors (32)(33). Analogous studies on the variation of GSH, GSSG, and PSSG in blood, performed with artifact-free procedures, should be encouraged.

Whether ROS activity has a causal or propagating role in human diseases associated with oxidative stress remains an unresolved question (34)(35). Moreover, even if the alteration of one or more biomarkers of oxidative stress has been reported to occur virtually in all diseases, data on the outcome of preventive strategies based on an antioxidant therapy are still extremely scarce (36)(37). It is time for researchers in this field to pay more attention to the choice of the right/valid biomarkers of oxidative stress and to attribute the proper value to the contribution of analytical (bio)chemistry.


   Acknowledgments
 
This work was supported by grants from FIRST 2005 (Fondo Interno Ricerca Scientifica e Tecnologica), the University of Milan, and by Fondazione Monte dei Paschi di Siena.


   Footnotes
 
1 Nonstandard Abbreviations: ROS, reactive oxygen species; MDA, malondialdehyde; PCO, protein carbonyls; GSH, reduced glutathione; GSSG, glutathione disulfide; PSSG, protein mixed disulfides with glutathione; t-BOOH, tert-butylhydroperoxide; NEM, N-ethylmaleimide; and TCA, trichloroacetic acid.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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