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Technical Briefs |
1 Department of Neuroscience, Pharmacology Section, University of Siena, Via A. Moro 4, 53100 Siena, Italy;
2
Department of Biology, University of Milan, Via Celoria 26, 20133 Milan, Italy;
3
Department of Laboratory Medicine, Policlinico "Le Scotte", Azienda Ospedaliera Senese, Viale Mario Bracci 16, 53100 Siena, Italy;
aauthor for correspondence: fax 39-0577-234208, e-mail ranieri{at}unisi.it
Analysis of antioxidant molecules is potentially important to understanding the role of oxidative stress (1)(2)(3)(4)(5)(6) in disease, as oxidative damage is accompanied or preceded by their depletion.
Reduced glutathione (GSH) is ubiquitous and abundant; it can be oxidized to its disulfide form (GSSG) in response to an oxidative perturbation. Usually, however, this species is rapidly reduced by the action of glutathione reductase (7). If GSSG accumulates within the cell, it can create protein-glutathione adducts via thiol-disulfide exchange reactions. Thus, in addition to the ratio of GSH to GSSG, the content of glutathionylated proteins (GSSPs) can indicate oxidative stress. The analysis of GSSPs has potential advantages over measurements of GSH and GSSG because GSSPs are more stable than GSSG, being less prone to enzymatic reduction by glutathione reductases (8).
Because blood can be studied as an indicator of the overall body oxidative status, GSSPs and, particularly, glutathionyl hemoglobin (Hb-SSG) could represent useful markers of oxidative stress. The use of Hb-SSG as a clinical marker has been proposed (9)(10). Significant increases in GSSP concentrations have been found in diabetes mellitus, hyperlipidemia, Friedreich ataxia, and chronic renal failure (10)(11)(12)(13).
The technology commonly used for the assays of GSSPs, electrospray ionization mass spectrometry (ESI-MS), is not widely available. In addition, we have recently reported (14) that oxygenated Hb is able to artificially produce large amounts of GSSG and GSSP if not adequately manipulated. We have therefore developed a rapid and sensitive HPLC method to measure GSSPs that could avoid possible pitfalls and artifacts.
Monoclonal anti-GSH antibody was obtained from Virogen. Sheep anti-mouse IgG, horseradish peroxidase conjugate, was obtained from Amersham Pharmacia Biotech. All others chemicals were from Sigma. Human blood was obtained by venipuncture, after consent, from 12 healthy volunteers and from 10 patients with type I diabetes. Rat blood was drawn from the abdominal aorta after pentobarbital anesthesia. Tripotassium EDTA was used as anticoagulant. Blood (1 mL) was collected in tubes containing 130 µL of 310 mmol/L N-ethyl-maleimide (NEM) for SH derivatization (14). Red blood cells (RBCs) were obtained by centrifugation at 10 000g for 20 s and were lysed by the addition of 10 volumes of water. After centrifugation at 15 000g for 10 min to separate membranes, supernatants were passed through gel-filtration columns (Pharmacia PD10, equilibrated with 50 mmol/L phosphate buffer, pH 7.4) to remove low-molecular weight thiols and disulfides. The protein fraction was incubated with 0.5 mmol/L dithiothreitol (DTT) to reduce S–S bonds. After 15 min, 2 mmol/L (final concentration) monobromobimane (mBBr), a fluorescent label for sulfhydryl groups (15), was added. After sitting for 15 min at room temperature in the dark, samples were deproteinized with trichloroacetic acid (final concentration, 50 g/L) and loaded on the HPLC. Membranes were washed of nonspecifically bound Hb (16) and treated with DTT and mBBr as described above.
HPLC separation (Bio-Tek Instruments KromaSystem 2000, equipped with a Kontron SFM 25 spectrofluorometer) was carried out on a Supelco Spherisorb C18 column [250 x 4 mm (i.d.)] (14). Samples were eluted isocratically with 0.25 mL/L acetate buffer, pH 3.09, containing 200 mL/L methanol. Derivatized thiols were analyzed by fluorescence detection (excitation at 390 nm; emission at 480 nm). Calibration curves were constructed with authentic GSH.
Protein samples (5 µg) were slot-blotted on polyvinylidene difluoride membranes, and S-glutathionylation was detected by monoclonal mouse anti-GSH antibody as described previously (17). The Hb concentration was measured spectrophotometrically (18). GSH and GSSG were assessed as described previously (14).
The basal concentrations of GSSPs in both human and rat erythrocytes detected with our method are shown in Fig. 1
. GSSPs were very low for all untreated samples; after induction of oxidative stress with diamide or t-butylhydroperoxide (t-BOOH), GSSPs increased (Fig. 1
and Table 1
). In rat erythrocytes, cytosolic GSSPs reached high values: especially after diamide treatment, almost 100% of available GSH was found as mixed-disulfides. This indicates that cytosolic RBC sulfhydryl groups are potentially able to form protein mixed-disulfides with GSH (particularly in rat RBCs), but in healthy individuals, almost 100% of GSH is in the reduced form. GSSPs in membrane proteins were minimal in both humans and rats. Analysis of the same samples by slot-blot (inset in Fig. 1
for the cytosolic samples) and Western blotting (not shown) with anti-GSH antibodies confirmed the data obtained by HPLC: these methods showed negligible basal concentrations of GSSPs and an increase after oxidant addition. Moreover, Western blotting revealed that almost all cytosolic GSSPs are in a 16-kDa band, thus suggesting that Hb accounts for almost all GSSPs present in RBCs. GSSP values before and after induction of oxidative stress, measured via the HPLC method, are shown in Table 1
. The loss in soluble GSH + GSSG correlated with the concentrations of GSSPs detected in all samples after oxidative stress, thus suggesting that our method is able to quantitatively measure all GSSPs present. The within-run imprecision (CV) was 0.5% at 8.9 nmol/g of Hb (n = 8). The detection limit was 0.5 nmol/g of Hb. In samples from patients with type I diabetes, measured values of Hb-SSG were significantly increased compared with those in healthy controls (64 ± 21 vs 9.9 ± 3.0 nmol/g; P <0.001). In the same samples, we observed higher concentration of glutathionylated membrane proteins (1.75 ± 0.61 vs 0.76 ± 0.151 nmol/g; P <0.01).
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The need to propose a new method to detect the presence of GSSPs in blood samples derives from two considerations: (a) numerous artifacts are generated during sample manipulation (14) because oxyhemoglobin, under various conditions, produces artificially high GSSG and protein mixed-disulfide values (14)(18)(19); and (b) most proposed methods for GSSP determination use HPLC combined with ESI-MS (10)(11)(12)(13)(20)(21)(22), which is specific but has a poorer reproducibility and sensitivity than do HPLC methods coupled with fluorometric detection. In addition, different researchers have reported RBC GSSP concentrations ranging from 30 to 2600 nmol/g of Hb (i.e., 0.6–8% of total Hbß chains) in healthy individuals (10)(11)(12)(13)(19)(20)(21), suggesting that analytical procedures must be improved.
A key factor in our procedure is the rapid quenching of free thiols by the membrane-penetrating agent NEM. In our opinion, it is recommendable not only when the protocol requires acid deproteinization, but under all conditions, as this treatment maintains the blood thiol/disulfide status (14). The separation of proteins by gel filtration is necessary to eliminate glutathione disulfide from the sample, which would be reduced by DTT and detected together with protein mixed-disulfides. This procedure is preferred to the use of washed acid-precipitated proteins to avoid the rapid oxidation of SH groups (14), which takes place after neutral pH is restored for the DTT reduction and mBBr conjugation.
Measured basal GSSPs in human and rat RBCs are very low, and only a few previous reports found relatively close values (23). Conversely, most authors reported higher concentrations; one reason for these discrepancies could be that samples were frozen before the ESI-MS determination. It is possible that ferric Hb, hemicromes, and hemocromes, which can be generated by freezing (24), produce oxidants, increasing the Hb-SSG content. Our measures indicate that human and rat Hbs, under typical conditions, are minimally glutathionylated (9.9 nmol/g; i.e., 0.03% of ß chains). A negligible percentage of GSH was found bound to membrane proteins. Hb-SSG content can increase after oxidative stress produced by diamide or t-BOOH treatments. In any case, human RBC GSSP content accounts for <10% of total cellular glutathione after oxidative stress. Conversely, rat Hb was shown to produce large amounts of Hb-SSG: after treatment with diamide, all GSH was bound to Hb. This is attributable to the peculiarity of rat Hb (25), which is characterized by a highly reactive cysteine in position ß125, in addition to cysteine ß93, which is common to most mammalian Hbs. In diabetes, where oxidative stress is increased, we have measured enhanced Hb-SSG (+648%); in addition, we have also evaluated the correlation of obtained values with other indicators of oxidative stress, i.e., protein carbonyls, isoprostanes, and malondialdehyde. Preliminary results (not shown) indicate a positive correlation with protein carbonyls and isoprostanes but not with lipid peroxidation products.
In conclusion, our studies suggest, as discussed previously (9), that Hb-SSG could be used as a clinical marker of oxidative status and, consequently, of pathologies whose development is strongly associated with oxidative stress. The availability of a simple, sensitive, and reproducible method to detect Hb-SSG could be useful both for diagnostic and therapeutic purposes.
Acknowledgments
We wish to thank Dr. Elena Menegola and Prof. Erminio Giavini (Department of Biology, University of Milan, Milan, Italy) for their support of our work.
References
The following articles in journals at HighWire Press have cited this article:
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