| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1
Service de Biochimie A, Hôpital Saint-Antoine, AP-HP, 184 rue du Fbg Saint-Antoine, 75571 Paris Cedex 12, France;
2
Service de Biochimie and
3
Service de Gérontologie, Centre Hospitalier Emile Roux, AP-HP, 34456 Limeil-Brévannes, France;
4
Laboratoire de Biologie, Hôpital Corentin Celton, AP-HP, 32133 Issy-les-Moulineaux, France;
aauthor for correspondence: fax 33-1-49-28-20-77, e-mail valerie.serru{at}sat.ap-hop-paris.fr
In the past few years, there has been increasing interest in the measurement of thiols, glutathione in particular, as indicators of oxidative stress. The reduced glutathione/oxidized glutathione ratio (GSH/GSSG) is used to evaluate oxidative stress status in biological systems, and alterations of this ratio have been demonstrated in aging, cancer, HIV replication, and cardiovascular diseases (1)(2)(3)(4)(5). Surprisingly, glutathione is usually studied in plasma, which contains only 0.5% of the blood content, whereas erythrocytes contain 99.5%. We were interested in direct measurement of GSH and GSSG in whole blood samples to evaluate overall glutathione status.
Numerous methods are available for measuring GSH and GSSG (6), but few of these are suitable for direct analysis in routine use. HPLC is the most widely used, but it requires a long sample preparation with pre- or postcolumn derivatization (7)(8) or a special apparatus for electrochemical detection (1)(9). Alternative methods based on GSH conjugation with a chromophore or a fluorophore have been used, but they need GSSG calculation from total glutathione measured after reduction of GSSG (10)(11). Capillary zone electrophoresis (CZE) coupled with laser-induced fluorescence has been proposed recently (12). The separation of GSH and GSSG by CZE with direct ultraviolet detection in tissues and mitochondria (13)(14) and by micellar electrokinetic capillary electrophoresis in plasma (15) has also been reported.
The aim of the present work was to develop and validate a rapid CZE method suitable for a direct measurement of GSH and GSSG in whole blood. Moreover, we describe the distribution of blood GSH and GSSG concentrations in a healthy adult population and in elderly subjects.
All chemicals used were of analytical-reagent grade. bis-Tris, boric acid, GSH, and GSSG were obtained from Sigma Chemical, and metaphosphoric acid was obtained from Prolabo. We used a PACE 5000 System equipped with an ultraviolet detector set at 200 nm and PACE Station software from Beckman Instruments. Data were quantified on the basis of corrected peak areas with migration times. The separations were performed on a fused-silica capillary [75 µm (i.d.) x 57 cm (total length)/50 cm (length to detector)] purchased from Beckman Instruments. The instrument was set up with the anode at the inlet end of the capillary and the cathode at the outlet. The capillary was thermostated at 28 °C. Before each analytical run, the capillary was pressure-rinsed and filled with the buffer [75 mmol/L boric acid, 25 mmol/L bis-Tris (pH 8.4)] for 3 min at 138 kPa. The sample was injected by low pressure for 10 s at 3.5 kPa. The electrophoresis was performed with a constant voltage of 20 kV (
26 µA) for 7 min. Between analytical runs, the capillary was rinsed with 1 mol/L NaOH and deionized water (138 kPa; 1 min each).
The GSH and GSSG calibrators were prepared quantitatively, 40 µmol/L each, in 10 g/L metaphosphoric acid. Blood samples were collected by venipuncture after an overnight fast into EDTA-containing tubes (Terumo). We immediately added 1200 µL of metaphosphoric acid, 50 g/L, to 400 µL of whole blood. The sample was vortex-mixed for 15 s and centrifuged at 4 °C for 10 min at 3000g. The supernatant was stored at -70 °C until analysis. A 40-µL aliquot was diluted with 160 µL of deionized water before injection (final dilution, 1:20).
We studied 47 apparently healthy volunteers (28 females, 19 males), 19–51 years of age (mean, 35 years) to estimate reference values. We assessed 64 subjects >75 years of age (age range, 75–102 years; mean, 86) for their glutathione status; this group included in- and outpatients of a geriatric hospital who were carefully selected with a good nutritional status. All subjects gave informed consent before blood sampling.
Typical electrophoregrams of a whole blood sample and a calibrator are shown in Fig. 1
. Blood GSH and GSSG were eluted at 6.3 and 6.7 min, respectively. An unknown peak that eluted last was found in all blood samples. The intra- (n = 20) and interassay (n = 10) CVs for migration times were 1.4% and 1.1% for GSH and 0.4% and 1.4% for GSSG, respectively. The imprecision study, evaluated on a blood sample adjusted to 5 µmol/L GSH and GSSG and another to 20 µmol/L each, yielded CVs of 2.6–11% for GSH and 1.3–6.3% for GSSG (Table 1
). The measurement of GSH was linear between 1 and 40 µmol/L (measured GSH = 0.88 x theoretical GSH + 0.53; r = 0.998) and of GSSG between 0.5 and 40 µmol/L (measured GSSG = 1.20 x theoretical GSSG + 0.22; r = 0.999). The percentages of recovery evaluated on blood samples supplemented with 1, 2, 5, 10, 20, and 40 µmol/L GSH and GSSG were 82%, 105%, 92%, 89%, 102%, and 84%, respectively, for GSH and 94%, 101%, 99%, 97%, 118%, and 97%, respectively, for GSSG. To verify the selectivity of the method, we used N-ethylmaleimide (NEM) to convert GSH into GSH-NEM, which led to a shift of the GSH peak, and dithiothreitol to reduce GSSG, which led to the disappearance of GSSG and proportional increase of GSH (data not shown).
|
|
Concentrations of GSH and GSSG were measured simultaneously on blood samples assayed as duplicates, and the total glutathione and the GSH/GSSG ratio were calculated. The mean values for healthy adults (n = 47), expressed as mean ± SD, were 486 ± 85 µmol/L for GSH, 68 ± 26 µmol/L for GSSG, 553 ± 90 µmol/L for total glutathione, and 8.1 ± 2.7 for the GSH/GSSG ratio. The results showed considerable interindividual variability with concentration ranges of 343–728 µmol/L for GSH and 25–151 µmol/L for GSSG, and a GSH/GSSG ratio of 2.3–15.5. The mean values for elderly subjects (n = 64) were of 337 ± 138 µmol/L for GSH, 129 ± 77 µmol/L for GSSG, 466 ± 122 µmol/L for total glutathione, and 4.1 ± 3.6 for the GSH/GSSG ratio. The distribution of glutathione concentrations in each group passed the Kolmogorov-Smirnov test for normality. Elderly people showed significantly lower GSH and higher GSSG, yielding decreased total glutathione and GSH/GSSG ratio (unpaired Student t-test; P <0.0001).
The CZE operating conditions were optimized to allow simultaneous measurement of GSH and GSSG in whole blood samples. The time for a complete CE analysis was 12 min, including rinses, making it extremely rapid compared with HPLC or conjugation-based methods. Reproducibility (CV) was <6.5% for GSSG, but the intraassay CV for GSH at the low concentration was 11%. This could be explained by oxidation of GSH during the analysis, leading to a loss of GSH in a series and yielding interassay CVs (<8%) lower than the intraassay CVs. The poor stability of GSH, well documented for HPLC analysis, could be prevented by addition of NEM or potassium borohydride to trap thiols or reduce disulfide bonds (1)(7). However, we found that a decrease in GSH occurred within a low proportion of GSH in freshly thawed samples and decided to analyze biological samples as duplicates in short series.
The linearity range was compatible with the analysis of whole blood samples, a procedure that includes an acid deproteinization step to avoid GSH and GSSG degradation catalyzed by 
-glutamyltransferase and to prevent oxidation of GSH (16) and an 1:5 dilution in distilled water to minimize the ionic strength of the sample. This produced a final dilution factor of 20 at the injection time with a measurement range equivalent to 10–800 µmol/L in the blood. This range allows the analysis of pathologic blood samples, such as in aging or HIV infection (3)(5). However, the assay could not be used for plasma analysis where GSH and GSSG concentrations are 0.5–10 µmol/L (1)(16)(17).
No reference method has yet been defined, either for sample preparation or for glutathione measurement in whole blood, that leads to a great variability in reference values reported in healthy subjects (17)(18)(19). We used a protocol designed to minimize error in sample collection, processing, and storage. Immediately after blood sampling, samples were put on ice and treated by metaphosphoric acid, an approach described as among the most reliable for glutathione storage (2)(20). The satisfactory recovery of known amounts of GSH or GSSG added to whole blood indicated that no glutathione was lost during sample preparation (data not shown). Our reference values for GSH were in agreement with those reported by Matsubara et al. (18), who used an enzymatic method, although their GSSG range was lower. Conversely, our reference values were lower by 30% for GSH and total glutathione in comparison with results of Michelet et al. (17) who used HPLC after derivatization by o-phthalaldehyde. However, their GSH/GSSG ratio was near that in our study (9.1 vs 8.1). Derivatization for HPLC analysis is a well-known source of overestimation, in particular o-phthalaldehyde reacts with glutathione, and to some extent, with other sulfhydryl and amino compounds. Indeed, we developed a direct method to simultaneously measure GSH and GSSG free of analytic deviation from derivatizating and reducing agents. Therefore, it seemed important that reference values had to be determined for each analytic method.
Physiologic variations of blood glutathione concentrations (17)(21) might also add to interindividual variability. Our results showed no significant gender difference in young healthy subjects, as reported previously (17). We found decreased GSH and total glutathione concentrations in elderly subjects (>75 years of age) as described previously (19). Moreover, we demonstrated for the first time an increase in GSSG concentration in elderly subjects. Further investigations of GSSG in elderly people would contribute to the understanding of the pathogenic role of oxidative stress in the course of aging (3)(22).
In conclusion, CZE is rapid, precise, and specific for GSH and GSSG measurement in whole blood. This assay requires only a small amount of biologic sample and brief sample preparation and thus could be used for routine measurement of glutathione concentrations, even in pathologic conditions where the GSH/GSSG is altered.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  J. M. Hempe, J. Ory-Ascani, and D. Hsia Genetic Variation in Mouse Beta Globin Cysteine Content Modifies Glutathione Metabolism: Implications for the Use of Mouse Models Experimental Biology and Medicine, March 1, 2007; 232(3): 437 - 444. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. T. Hsu, L. Y. Tsai, S. K. Lin, J. K. Hsiao, and B. H. Chen Effects of diabetes duration and glycemic control on free radicals in children with type 1 diabetes mellitus. Ann. Clin. Lab. Sci., March 1, 2006; 36(2): 174 - 178. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Rossi, A. Milzani, I. Dalle-Donne, D. Giustarini, L. Lusini, R. Colombo, and P. Di Simplicio Blood Glutathione Disulfide: In Vivo Factor or in Vitro Artifact? Clin. Chem., May 1, 2002; 48(5): 742 - 753. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
