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James M. Hempe1,2 Jeannine Ory-Ascani1 1 Research
Institute for Children, Children’s Hospital, New Orleans, LA, USA 2 Department of Pediatrics, Louisiana State University Health Sciences Center, New Orleans, LA, USA
Received September 13, 2013 Revised November 21, 2013 Accepted November 26, 2013
Research Article
Simultaneous analysis of reduced glutathione and glutathione disulfide by capillary zone electrophoresis This report describes modifications to a CZE method developed by Serru et al. (Clinical Chemistry 2001, 47, 1321–1324) for the simultaneous analysis of reduced glutathione (GSH) and glutathione disulfide (GSSG). Lowering the pH of the run buffer (75 mmol/L boric acid, 25 mmol/L bis-Tris) from pH 8.4 to 7.8 markedly improved GSH peak area reproducibility and allowed multiple samples to be analyzed without changing run buffers due to ion depletion. Sample preparation using red blood cells (RBC) instead of whole blood, combined with glutathione extraction at a lower concentration of metaphosphoric acid (5%), increased assay sensitivity and decreased interference. CZE assay results for clinical samples containing 1000 to 3200 mol GSH/L RBC and 100 to 400 mol GSSG/L RBC were highly correlated (r2 0.95) with results obtained using a commercial dithionitrobenze-based glutathione assay. The modified CZE assay has proven useful for the analysis of glutathione in both mouse and human RBC. Keywords: Capillary electrophoresis / Erythrocytes / Glutathione DOI 10.1002/elps.201300450
1 Introduction The normal balance between prooxidant and antioxidant forces are altered in a wide variety of pathological states, such as sickle cell disease, infection, cancer, diabetes, and other chronic conditions [1–6]. Biomarkers of redox status are widely used in clinical research to assess oxidative stress (e.g. C-reactive protein, malondialdehyde) and antioxidant status (e.g. vitamins C and E) and are increasingly used for the routine clinical management of patients with chronic disease [7,8]. Red blood cell (RBC) glutathione is an endogenous antioxidant that plays an important role in the detoxification of hydrogen peroxide and in the maintenance of intracellular redox homeostasis [9, 10]. Glutathione also plays important roles in metabolic regulation via glutathionylation [11] and transnitrosylation [12] of protein cysteine residues. We adapted a CZE assay reported by Serru et al. [13] and have used the assay for the measurement of glutathione status in both mouse and human studies [14–16]. Advantages of the original assay included relatively rapid, precise, specific and
Correspondence: Dr. James M. Hempe, Research Institute for Children, Children’s Hospital, 200 Henry Clay Avenue, New Orleans, LA 70118, USA E-mail: [email protected] Fax: +1-504-896-2722
Abbreviations: DTNB, 5,5 -dithiobis-2-nitrobenzoic acid; GSH, reduced glutathione; GSSG, glutathione disulfide; MPA, metaphosphoric acid; RBC, red blood cell; TFG, total free glutathione C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
simultaneous analysis of both reduced glutathione (GSH) and glutathione disulfide (GSSG). A major disadvantage was poor peak area reproducibility in repeated runs which the authors attributed to oxidation of GSH. The present report describes modifications to the method of Serru et al. [13] that improve both assay sensitivity and assay reproducibility.
2 Materials and methods The recommended method for simultaneous analysis of GSH and GSSG as modified from the CZE method of Serru et al. [13] is described below. Departures from the recommended analytical conditions during development and validation experiments are described as needed in the results.
2.1 Chemicals and reagents Unless otherwise noted, all chemicals and reagents were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO). A 5% w/v stock solution of metaphosphoric acid (MPA) was prepared weekly in deionized water and stored at 4°C. The stock MPA solution was used in sample preparation and was also diluted daily to make a 0.5% MPA working solution that was used to prepare calibrators. The CZE run buffer contained 75 mmol/L boric acid and 25 mmol/L bis-Tris in deionized water adjusted to pH 7.8 with NaOH. Run buffer can be stored at room temperature for at least 6 months. www.electrophoresis-journal.com
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2.2 Samples
2.4 Capillary zone electrophoresis
Discarded blood samples collected in EDTA were obtained from short-stay and diabetes clinics at Children’s Hospital under protocols approved by the Research Institute for Children and LSU Health Sciences Center Institutional Review Boards. Samples used in the correlation study were stored as whole blood at 4°C for up to 6 h prior to analysis. To process blood samples for glutathione analysis, RBCs were first separated from plasma by centrifuging 0.5 mL of whole blood at 4°C and 1500 × g for 10 min. A 1:4 hemolysate was then prepared by adding 100 L of RBC (pipetted from beneath the buffy coat) to 400 L of hemolyzing reagent (10 mmol/L KCN and 5 mmol/L EDTA in deionized water). Proteins in the hemolysate were precipitated by adding 100 L of 5% MPA to an equal volume of hemolysate in a 0.5 mL tube. The acidified samples were vigorously mixed on a vortex machine, left at room temperature for 5 min, then centrifuged at 10 000 × g for 5 min at 4°C. The supernatant fraction (MPA extract) was transferred to labeled 0.5 mL tubes and stored at −70°C prior to analysis. For CZE analysis, the MPA extracts were thawed and diluted 1:4 by adding 20 L of MPA extract to a 0.5 mL centrifuge tube containing 80 L of deionized water. Under these conditions the diluted MPA extract constituted a 50-fold dilution of RBC in 0.5% MPA and results are reported in mol/L of RBC.
CZE was performed using a P/ACE 5000 capillary electrophoresis system with UV detection and System Gold Software (Beckman Instruments, Fullerton, CA). The instrument was operated in normal polarity with anode and cathode at the inlet and outlet reservoirs, respectively. All separations were performed in a 57 cm long × 75 m (id) fused silica capillary (MicroSolv Technology Corporation, Long Branch, NJ) with capillary cooling at 20°C and detection at 200 nm. Before each assay, the capillary was pressure-rinsed (138 kPa) for 1 min with 0.1 M NaOH solution followed by a 1 min rinse with run buffer. Calibrators or MPA extracts were introduced into the capillary by low-pressure (3.5 kPa) injection for 20 s followed by low-pressure injection of a deionized water plug for 2 s. Both ends of the capillary were then immersed in vials containing 4 mL of run buffer and the current was ramped to 20 kV (25 A) over 0.2 min. Under these conditions, all peaks of interest eluted within 7 min. The capillary was pressure-rinsed for 1 min with deionized water between analyses. When not in use the capillary was stored on the instrument with both ends in water. For best results run buffer was replaced after five analyses to avoid ion depletion.
2.3 Analytical quality control Calibrator solutions were prepared in a three-step process from commercially available GSH and GSSG. First, primary stock GSH (20 mmol/L GSH) and GSSG (10 mmol/L GSSG) calibrator solutions were prepared in 0.5% MPA and stored at −70°C in 200 L aliquots. A secondary stock calibrator solution (80 mol/L GSH and 16 mol/L GSSG) was prepared by adding 100 L of GSH primary stock solution and 40 L of GSSG primary stock solution to 25 mL of 0.5% MPA. Secondary stock calibrator solution was stored at −70°C in 200 L aliquots. Six working calibrator solutions were prepared daily for each analytical run by thawing one aliquot of secondary stock calibrator and using it to prepare solutions containing 5, 10, 20, 40, 60, and 80 mol/L GSH and 1, 2, 4, 8, 12, and 16 mol/L GSSG in 0.5% MPA. Calibrator concentrations and corrected peak areas were used to generate a linear response curve to estimate the molar concentrations of GSH and GSSG in unknown samples. GSH/GSSG ratio was calculated as the molar ratio of GSH to GSSG. Total free glutathione (TFG) is expressed in molar equivalents of GSH (TFG = GSH + (2 × GSSG)). Pooled MPA extract was prepared as described for patient samples and used as an analytical quality control sample to assess assay performance over time. Control MPA extracts were stored in 50 L aliquots at −70°C and the amounts of GSH and GSSG in a control sample were determined with each analytical run. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.5 Assay development and validation experiments The sensitivity and linear range of the CZE method were determined by measuring dilutions of a standard containing 4 mmol/L each of GSH and GSSG. Intraassay imprecision was assessed by measuring GSH and GSSG in ten different aliquots of the control MPA extract in the same analytical run. Interassay imprecision was assessed by analyzing the control in 50 different analytical runs over a period of 18 wk. To assess the effect of sample preparation on assay imprecision, including pipetting and MPA extraction, ten different aliquots of the same blood sample were processed separately and analyzed in the same analytical run. The stability of glutathione was determined by repeat analysis of blood samples stored at 4°C for 0 or 6 h. The stability of glutathione in stored MPA extracts was determined by repeat analysis prior to and after storage of MPA extracts at −20 or −70°C.
2.6 Correlation study A correlation study was conducted to compare results obtained by CZE with those obtained using a commercial glutathione assay kit (Cayman Chemical Company, Ann Arbor, MI). The commercial kit uses glutathione reductase enzymatic recycling and 5,5 -dithiobis-2-nitrobenzoic acid (DTNB) to quantify GSH and GSSG. End point determination was performed at 25 min as described by the manufacturer. Both the CZE and DTNB methods were used to analyze 22 patient samples with a wide range of glutathione concentrations. All samples were extracted with 5% MPA as described above. This sample extraction protocol was identical to that www.electrophoresis-journal.com
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Figure 1. CZE of GSH and GSSG in RBC hemolysate. Run buffer was 75 mmol/L boric acid and 25 mmol/L bis-Tris, pH 7.8. Separation was performed in a 57 cm long × 75 m id fused silica capillary using a P/ACE 5000 CE system at 20 kV and normal polarity. The identities of the numbered peaks are unknown.
suggested by the manufacturer except 5% MPA replaced 10% MPA. Results were compared by linear regression analysis.
2.7 Statistical analysis Results were statistically analyzed using PC-SAS (SAS Institute, Cary, NC). Linear regression analysis, Pearson correlation, and paired t-tests were used to compare the results of the CZE and commercial DTNB glutathione assays. Differences between groups were considered significant at p ⬍ 0.05.
3 Results 3.1 Glutathione analysis by CZE Figure 1 shows the CZE separation of compounds that have absorbance at 200 nm and are present in MPA extract prepared from RBC hemolysate. Sample preparation and CZE analytical conditions were as described above. GSH eluted before GSSG and was more abundant. The identities of the numbered peaks remain unknown. Analysis of a purified nitrosoglutathione standard produced a peak that migrated in the vicinity of peak 1. Peak 2 was abundant in plasma MPA extracts analyzed under the same CZE conditions. The migration time of peak 2 was markedly influenced by run buffer pH such that it co-eluted and interfered with GSSG under some run conditions.
3.2 Effect of run buffer pH on CZE assay performance Figure 2A shows that GSH peak area progressively decreased over time when the pH 8.4 run buffer of Serru et al. [13] was used to consecutively analyze a standard containing 80 mol/L GSH and 16 mol/L GSSG in 0.5% MPA. GSH peak area returned to approximately the starting value when run buffers were replaced after 10 consecutive runs. The fact that GSSG did not increase as GSH decreased argues against oxidation of GSH as an explanation for decreasing GSH peak C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Effect of buffer pH on peak area reproducibility. CZE run parameters were as described in Fig. 1 except for differences in run buffer pH. Panel (A): Standard sample containing 80 mol/L GSH and 16 mol/L GSSG analyzed 20 times in a row using run buffer pH 8.4 with buffer change after 10 runs. Panels (B) (GSH), (C) (GSSG), and (D) (GSH/GSSG peak area): Standard sample containing 200 mol/L GSH and 200 mol/L GSSG analyzed 10 times in a row using run buffer pH 8.4, 8.1 or 7.8.
area. The restorative effect of the buffer change strongly suggests that the decreased GSH peak area observed in consecutive runs at pH 8.4 was due to buffer ion depletion. We next determined the effect of run buffer pH on corrected peak areas for a standard containing 200 mol/L GSH and 200 mol/L GSSG in 0.5% MPA. Consecutive analysis of this standard at pH 8.4 again resulted in a progressive decrease in GSH peak area (Fig. 2B). The effect was less pronounced at pH 8.1 and was essentially eliminated at pH 7.8. In contrast, the corrected peak area of GSSG was constant in consecutive analyses and unaffected by run buffer pH (Fig. 2C). The GSH/GSSG ratio (Fig. 2D) reflected the disparate effect of buffer pH and buffer ion depletion on www.electrophoresis-journal.com
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GSH versus GSSG. Run buffers with pH lower than 7.8 or different concentrations of borate or bis-Tris produced less effective separations. Unless otherwise indicated, all validation experiments described below were conducted using pH 7.8 run buffer.
3.3 Effect of sample preparation on CZE assay performance In a series of pilot studies we found that CZE analytical performance was markedly influenced by the ion concentration of the MPA extract and the volume injected into the capillary. Extracts containing more than 1% MPA markedly diminished analytical performance as assessed based on GSH and GSSG peak separation and peak areas. Analytical performance was also diminished when larger injection volumes were used with samples where the MPA concentration was constant. These results indicated that assay performance could be maximized by minimizing the amount of MPA required for protein precipitation while also minimizing sample injection volumes. We empirically compared different combinations of sample extraction procedures (e.g. comparing whole blood versus RBC hemolysates, and varying the MPA content of extraction solutions) and CZE run parameters (e.g. different injection volumes, capillary temperatures, voltages, etc.) to identify an optimal combination of sample preparation and CZE assay conditions. The result was a sample preparation procedure that differs from that described by Serru et al. [13] in that 5% rather than 10% MPA solution was used to precipitate protein. Glutathione extraction from RBC hemolysates using lower MPA concentration resulted in an increased GSSG S/N and improved peak area reproducibility. Using RBC rather than whole blood also minimized potential interference by acid-soluble plasma constituents.
3.4 Sample stability A series of experiments were conducted to determine how long whole blood or blood extracts could be stored for batch processing. Pilot studies showed that GSH and GSSG levels were similar in whole blood processed immediately or stored for up to 6 h at 4°C. We next evaluated how long samples could be stored at different stages of processing and the effect of storage temperature. RBC hemolysates stored overnight at −20 or −70°C contained significantly more GSSG than hemolysate that was processed and analyzed immediately without freezing. In contrast, the GSH and GSSG levels of MPA extracts analyzed immediately were not significantly different from those stored for long periods of time at −70°C. MPA extracts cannot be stored at −20°C as evidenced by increased GSSG levels within 24 h. However, MPA extracts can be stored at −70°C for at least 3 months without detectable changes in GSH and GSSG levels. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2014, 35, 967–971
3.5 Linearity and sensitivity Analysis of a serial dilution of a standard sample containing 4 mmol/L each of GSH and GSSG showed that the relationship between GSH and GSSG concentration and corrected peak area was linear for each analyte (r2 ⬎ 0.98, p ⬍ 0.001) throughout the range tested (31.25 mol/L to 4 mmol/L). Calibration standards for GSH (5 to 80 mol/L) and GSSG (1 to 16 mol/L) were selected for use in this assay because these levels best cover the range encountered in samples prepared from RBC and diluted 50-fold as described in Section 2. Elution profiles and migration times of standards prepared in 0.5% MPA were similar to those observed in samples prepared from RBC. 3.6 Assay imprecision Interassay reproducibility was assessed by analyzing aliquots of the control MPA extract in 20 different analytical runs performed on different days. The estimated glutathione composition of the control (mean ± SEM) was 1342 ± 74 mol/L GSH (CV = 5.5%), 118 ± 10 mol/L GSSG (CV = 8.3%), 1577 ± 86 mol/L TFG (CV = 6.5%), and 11.4 ± 0.7 GSH/GSSG (CV = 5.5%). Intraassay reproducibility was assessed by extracting and analyzing ten separate aliquots of the same RBC in the same analytical run. The estimated glutathione composition of the blood was 1610 ± 73 mol/L GSH (CV = 4.5%), 78 ± 11 mol/L GSSG (CV = 13.6%), 1766 ± 70 mol/L TFG (CV = 3.9%), and 21.0 ± 2.8 GSH/GSSG (CV = 13.3%). 3.7 Correlation study Figure 3 shows that the GSH, GSSG and total glutathione concentrations of blood samples analyzed by CZE were highly correlated (r2 0.95, p ⬍ 0.0001) with concentrations measured using the commercial DTNB assay kit. GSH levels were not significantly different between assays as determined by paired t-test. GSSG and total glutathione levels were both significantly higher when measured using the DTNB assay.
4 Discussion Two major modifications to the CZE method described by Serru et al. [13] significantly improved the ease of assay use and assay reproducibility. The first important modification was changing the method used to prepare blood samples for analysis. We used RBC hemolysates instead of whole blood with MPA extraction in 5% MPA rather than 10% MPA. These changes in sample preparation had a marked influence on assay performance because it increased the amount of analyte that could be injected, reduced interference by plasma constituents present in whole blood, and enhanced GSH and GSSG separation. The second important modification was decreasing run buffer pH from 8.4 to 7.8 which markedly improved the reproducibility of both peak area and migration times. www.electrophoresis-journal.com
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interpreted the data. Both authors contributed to the drafting of the manuscript and are responsible for the intellectual content and final approval of the published version. This research was supported by the Louisiana State University Health Sciences Center Department of Pediatrics and the Research Institute for Children at Children’s Hospital of New Orleans. We would like to express our appreciation to the staff of Children’s Hospital laboratory for their assistance in sample collection. The authors have declared no conflict of interest.
5 References [1] Chan, A. C., Chow, C. K., Chiu, D., Proc. Soc. Exp. Biol. Med. 1999, 222, 274–282. [2] Gizi, A., Papassotiriou, I., Apostolakou, F., Lazaropoulou, C., Papastamataki, M., Kanavaki, I., Kalotychou, V., Goussetis, E., Kattamis, A., Rombos, I., Kanavakis, E., Blood Cells Mol. Dis. 2011, 46, 220–225. [3] Pan, H. Z., Zhang, L., Guo, M. Y., Sui, H., Li, H., Wu, W. H., Qu, N. Q., Liang, M. H., Chang, D., Acta Diabetol. 2010, 47(Suppl 1), 71–76. [4] Cruz, K. K., Fonseca, S. G., Monteiro, M. C., Silva, O. S., Andrade, V. M., Cunha, F. Q., Romao, P. R., Parasite Immunol. 2008, 30, 171–174. [5] Matthews, G. M., Butler, R. N., Helicobacter. 2005, 10, 298–306. Figure 3. Correlation between GSH, GSSG, and total glutathione measured by CZE and a commercial DTNB assay. CZE run parameters were as described in Fig. 1. Results depict the linear regression equations with 99% confidence intervals. Total glutathione is expressed in GSH molar equivalents (GSH + (2 × GSSG)).
CZE assay results were highly correlated with results obtained using a commercial DTNB assay. A major advantage of the CZE assay is that both GSH and GSSG can be analyzed in a single run. In contrast, each blood sample must be analyzed twice with two different standard curves to measure total glutathione and GSSG in the DTNB assay. GSH is then determined as the difference between these total glutathione and GSSG levels. Although GSH levels were comparable when measured by CZE or the DTNB assay, total glutathione and GSSG levels were significantly different. As DTNB is nonspecific and reacts with cysteine and other thiols it seems likely that the higher total glutathione and GSSG levels observed with the DTNB assay were due to interference by nonglutathione constituents. Contribution statement: JMH and JOA were responsible for the conception and design of the study and performed the experiments and analytical measurements. JMH analyzed and
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[6] Locigno, R., Castronovo, V., Int. J. Oncol. 2001, 19, 221–236. [7] Lushchak, V. I., J. Amino. Acids 2012, 2012, 736837. [8] Lang, C. A., Mills, B. J., Mastropaolo, W., Liu, M. C., J. Lab. Clin. Med. 2000, 135, 402–405. [9] Flohe, L., Meth. Enzymol. 2010, 473, 1–39. [10] Wu, G., Fang, Y. Z., Yang, S., Lupton, J. R., Turner, N. D., J. Nutr. 2004, 134, 489–492. [11] Dalle-Donne, I., Rossi, R., Colombo, G., Giustarini, D., Milzani, A., Trends Biochem. Sci. 2009, 34, 85–96. [12] Hogg, N., Annu. Rev. Pharmacol. Toxicol. 2002, 42, 585–600. [13] Serru, V., Baudin, B., Ziegler, F., David, J. P., Cals, M. J., Vaubourdolle, M., Mario, N., Clin. Chem. 2001, 47, 1321–1324. [14] Hempe, J. M., McGehee, A. M., Stoyanov, A. V., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009, 877, 3462–3466. [15] Thom, G. G., Kallanagowdar, C., Somjee, S. S., Velez, M. C., Yu, L. C., Hempe, J. M., J. Pediatr. Hematol. Oncol. 2009, 31, 895–900. [16] Hempe, J. M., Ory-Ascani, J., Hsia, D., Exp. Biol. Med. 2007, 232, 437–444.
www.electrophoresis-journal.com
Electrophoresis 2014, 35, 967–971
James M. Hempe1,2 Jeannine Ory-Ascani1 1 Research
Institute for Children, Children’s Hospital, New Orleans, LA, USA 2 Department of Pediatrics, Louisiana State University Health Sciences Center, New Orleans, LA, USA
Received September 13, 2013 Revised November 21, 2013 Accepted November 26, 2013
Research Article
Simultaneous analysis of reduced glutathione and glutathione disulfide by capillary zone electrophoresis This report describes modifications to a CZE method developed by Serru et al. (Clinical Chemistry 2001, 47, 1321–1324) for the simultaneous analysis of reduced glutathione (GSH) and glutathione disulfide (GSSG). Lowering the pH of the run buffer (75 mmol/L boric acid, 25 mmol/L bis-Tris) from pH 8.4 to 7.8 markedly improved GSH peak area reproducibility and allowed multiple samples to be analyzed without changing run buffers due to ion depletion. Sample preparation using red blood cells (RBC) instead of whole blood, combined with glutathione extraction at a lower concentration of metaphosphoric acid (5%), increased assay sensitivity and decreased interference. CZE assay results for clinical samples containing 1000 to 3200 mol GSH/L RBC and 100 to 400 mol GSSG/L RBC were highly correlated (r2 0.95) with results obtained using a commercial dithionitrobenze-based glutathione assay. The modified CZE assay has proven useful for the analysis of glutathione in both mouse and human RBC. Keywords: Capillary electrophoresis / Erythrocytes / Glutathione DOI 10.1002/elps.201300450
1 Introduction The normal balance between prooxidant and antioxidant forces are altered in a wide variety of pathological states, such as sickle cell disease, infection, cancer, diabetes, and other chronic conditions [1–6]. Biomarkers of redox status are widely used in clinical research to assess oxidative stress (e.g. C-reactive protein, malondialdehyde) and antioxidant status (e.g. vitamins C and E) and are increasingly used for the routine clinical management of patients with chronic disease [7,8]. Red blood cell (RBC) glutathione is an endogenous antioxidant that plays an important role in the detoxification of hydrogen peroxide and in the maintenance of intracellular redox homeostasis [9, 10]. Glutathione also plays important roles in metabolic regulation via glutathionylation [11] and transnitrosylation [12] of protein cysteine residues. We adapted a CZE assay reported by Serru et al. [13] and have used the assay for the measurement of glutathione status in both mouse and human studies [14–16]. Advantages of the original assay included relatively rapid, precise, specific and
Correspondence: Dr. James M. Hempe, Research Institute for Children, Children’s Hospital, 200 Henry Clay Avenue, New Orleans, LA 70118, USA E-mail: [email protected] Fax: +1-504-896-2722
Abbreviations: DTNB, 5,5 -dithiobis-2-nitrobenzoic acid; GSH, reduced glutathione; GSSG, glutathione disulfide; MPA, metaphosphoric acid; RBC, red blood cell; TFG, total free glutathione C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
simultaneous analysis of both reduced glutathione (GSH) and glutathione disulfide (GSSG). A major disadvantage was poor peak area reproducibility in repeated runs which the authors attributed to oxidation of GSH. The present report describes modifications to the method of Serru et al. [13] that improve both assay sensitivity and assay reproducibility.
2 Materials and methods The recommended method for simultaneous analysis of GSH and GSSG as modified from the CZE method of Serru et al. [13] is described below. Departures from the recommended analytical conditions during development and validation experiments are described as needed in the results.
2.1 Chemicals and reagents Unless otherwise noted, all chemicals and reagents were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO). A 5% w/v stock solution of metaphosphoric acid (MPA) was prepared weekly in deionized water and stored at 4°C. The stock MPA solution was used in sample preparation and was also diluted daily to make a 0.5% MPA working solution that was used to prepare calibrators. The CZE run buffer contained 75 mmol/L boric acid and 25 mmol/L bis-Tris in deionized water adjusted to pH 7.8 with NaOH. Run buffer can be stored at room temperature for at least 6 months. www.electrophoresis-journal.com
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2.4 Capillary zone electrophoresis
Discarded blood samples collected in EDTA were obtained from short-stay and diabetes clinics at Children’s Hospital under protocols approved by the Research Institute for Children and LSU Health Sciences Center Institutional Review Boards. Samples used in the correlation study were stored as whole blood at 4°C for up to 6 h prior to analysis. To process blood samples for glutathione analysis, RBCs were first separated from plasma by centrifuging 0.5 mL of whole blood at 4°C and 1500 × g for 10 min. A 1:4 hemolysate was then prepared by adding 100 L of RBC (pipetted from beneath the buffy coat) to 400 L of hemolyzing reagent (10 mmol/L KCN and 5 mmol/L EDTA in deionized water). Proteins in the hemolysate were precipitated by adding 100 L of 5% MPA to an equal volume of hemolysate in a 0.5 mL tube. The acidified samples were vigorously mixed on a vortex machine, left at room temperature for 5 min, then centrifuged at 10 000 × g for 5 min at 4°C. The supernatant fraction (MPA extract) was transferred to labeled 0.5 mL tubes and stored at −70°C prior to analysis. For CZE analysis, the MPA extracts were thawed and diluted 1:4 by adding 20 L of MPA extract to a 0.5 mL centrifuge tube containing 80 L of deionized water. Under these conditions the diluted MPA extract constituted a 50-fold dilution of RBC in 0.5% MPA and results are reported in mol/L of RBC.
CZE was performed using a P/ACE 5000 capillary electrophoresis system with UV detection and System Gold Software (Beckman Instruments, Fullerton, CA). The instrument was operated in normal polarity with anode and cathode at the inlet and outlet reservoirs, respectively. All separations were performed in a 57 cm long × 75 m (id) fused silica capillary (MicroSolv Technology Corporation, Long Branch, NJ) with capillary cooling at 20°C and detection at 200 nm. Before each assay, the capillary was pressure-rinsed (138 kPa) for 1 min with 0.1 M NaOH solution followed by a 1 min rinse with run buffer. Calibrators or MPA extracts were introduced into the capillary by low-pressure (3.5 kPa) injection for 20 s followed by low-pressure injection of a deionized water plug for 2 s. Both ends of the capillary were then immersed in vials containing 4 mL of run buffer and the current was ramped to 20 kV (25 A) over 0.2 min. Under these conditions, all peaks of interest eluted within 7 min. The capillary was pressure-rinsed for 1 min with deionized water between analyses. When not in use the capillary was stored on the instrument with both ends in water. For best results run buffer was replaced after five analyses to avoid ion depletion.
2.3 Analytical quality control Calibrator solutions were prepared in a three-step process from commercially available GSH and GSSG. First, primary stock GSH (20 mmol/L GSH) and GSSG (10 mmol/L GSSG) calibrator solutions were prepared in 0.5% MPA and stored at −70°C in 200 L aliquots. A secondary stock calibrator solution (80 mol/L GSH and 16 mol/L GSSG) was prepared by adding 100 L of GSH primary stock solution and 40 L of GSSG primary stock solution to 25 mL of 0.5% MPA. Secondary stock calibrator solution was stored at −70°C in 200 L aliquots. Six working calibrator solutions were prepared daily for each analytical run by thawing one aliquot of secondary stock calibrator and using it to prepare solutions containing 5, 10, 20, 40, 60, and 80 mol/L GSH and 1, 2, 4, 8, 12, and 16 mol/L GSSG in 0.5% MPA. Calibrator concentrations and corrected peak areas were used to generate a linear response curve to estimate the molar concentrations of GSH and GSSG in unknown samples. GSH/GSSG ratio was calculated as the molar ratio of GSH to GSSG. Total free glutathione (TFG) is expressed in molar equivalents of GSH (TFG = GSH + (2 × GSSG)). Pooled MPA extract was prepared as described for patient samples and used as an analytical quality control sample to assess assay performance over time. Control MPA extracts were stored in 50 L aliquots at −70°C and the amounts of GSH and GSSG in a control sample were determined with each analytical run. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.5 Assay development and validation experiments The sensitivity and linear range of the CZE method were determined by measuring dilutions of a standard containing 4 mmol/L each of GSH and GSSG. Intraassay imprecision was assessed by measuring GSH and GSSG in ten different aliquots of the control MPA extract in the same analytical run. Interassay imprecision was assessed by analyzing the control in 50 different analytical runs over a period of 18 wk. To assess the effect of sample preparation on assay imprecision, including pipetting and MPA extraction, ten different aliquots of the same blood sample were processed separately and analyzed in the same analytical run. The stability of glutathione was determined by repeat analysis of blood samples stored at 4°C for 0 or 6 h. The stability of glutathione in stored MPA extracts was determined by repeat analysis prior to and after storage of MPA extracts at −20 or −70°C.
2.6 Correlation study A correlation study was conducted to compare results obtained by CZE with those obtained using a commercial glutathione assay kit (Cayman Chemical Company, Ann Arbor, MI). The commercial kit uses glutathione reductase enzymatic recycling and 5,5 -dithiobis-2-nitrobenzoic acid (DTNB) to quantify GSH and GSSG. End point determination was performed at 25 min as described by the manufacturer. Both the CZE and DTNB methods were used to analyze 22 patient samples with a wide range of glutathione concentrations. All samples were extracted with 5% MPA as described above. This sample extraction protocol was identical to that www.electrophoresis-journal.com
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Figure 1. CZE of GSH and GSSG in RBC hemolysate. Run buffer was 75 mmol/L boric acid and 25 mmol/L bis-Tris, pH 7.8. Separation was performed in a 57 cm long × 75 m id fused silica capillary using a P/ACE 5000 CE system at 20 kV and normal polarity. The identities of the numbered peaks are unknown.
suggested by the manufacturer except 5% MPA replaced 10% MPA. Results were compared by linear regression analysis.
2.7 Statistical analysis Results were statistically analyzed using PC-SAS (SAS Institute, Cary, NC). Linear regression analysis, Pearson correlation, and paired t-tests were used to compare the results of the CZE and commercial DTNB glutathione assays. Differences between groups were considered significant at p ⬍ 0.05.
3 Results 3.1 Glutathione analysis by CZE Figure 1 shows the CZE separation of compounds that have absorbance at 200 nm and are present in MPA extract prepared from RBC hemolysate. Sample preparation and CZE analytical conditions were as described above. GSH eluted before GSSG and was more abundant. The identities of the numbered peaks remain unknown. Analysis of a purified nitrosoglutathione standard produced a peak that migrated in the vicinity of peak 1. Peak 2 was abundant in plasma MPA extracts analyzed under the same CZE conditions. The migration time of peak 2 was markedly influenced by run buffer pH such that it co-eluted and interfered with GSSG under some run conditions.
3.2 Effect of run buffer pH on CZE assay performance Figure 2A shows that GSH peak area progressively decreased over time when the pH 8.4 run buffer of Serru et al. [13] was used to consecutively analyze a standard containing 80 mol/L GSH and 16 mol/L GSSG in 0.5% MPA. GSH peak area returned to approximately the starting value when run buffers were replaced after 10 consecutive runs. The fact that GSSG did not increase as GSH decreased argues against oxidation of GSH as an explanation for decreasing GSH peak C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Effect of buffer pH on peak area reproducibility. CZE run parameters were as described in Fig. 1 except for differences in run buffer pH. Panel (A): Standard sample containing 80 mol/L GSH and 16 mol/L GSSG analyzed 20 times in a row using run buffer pH 8.4 with buffer change after 10 runs. Panels (B) (GSH), (C) (GSSG), and (D) (GSH/GSSG peak area): Standard sample containing 200 mol/L GSH and 200 mol/L GSSG analyzed 10 times in a row using run buffer pH 8.4, 8.1 or 7.8.
area. The restorative effect of the buffer change strongly suggests that the decreased GSH peak area observed in consecutive runs at pH 8.4 was due to buffer ion depletion. We next determined the effect of run buffer pH on corrected peak areas for a standard containing 200 mol/L GSH and 200 mol/L GSSG in 0.5% MPA. Consecutive analysis of this standard at pH 8.4 again resulted in a progressive decrease in GSH peak area (Fig. 2B). The effect was less pronounced at pH 8.1 and was essentially eliminated at pH 7.8. In contrast, the corrected peak area of GSSG was constant in consecutive analyses and unaffected by run buffer pH (Fig. 2C). The GSH/GSSG ratio (Fig. 2D) reflected the disparate effect of buffer pH and buffer ion depletion on www.electrophoresis-journal.com
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GSH versus GSSG. Run buffers with pH lower than 7.8 or different concentrations of borate or bis-Tris produced less effective separations. Unless otherwise indicated, all validation experiments described below were conducted using pH 7.8 run buffer.
3.3 Effect of sample preparation on CZE assay performance In a series of pilot studies we found that CZE analytical performance was markedly influenced by the ion concentration of the MPA extract and the volume injected into the capillary. Extracts containing more than 1% MPA markedly diminished analytical performance as assessed based on GSH and GSSG peak separation and peak areas. Analytical performance was also diminished when larger injection volumes were used with samples where the MPA concentration was constant. These results indicated that assay performance could be maximized by minimizing the amount of MPA required for protein precipitation while also minimizing sample injection volumes. We empirically compared different combinations of sample extraction procedures (e.g. comparing whole blood versus RBC hemolysates, and varying the MPA content of extraction solutions) and CZE run parameters (e.g. different injection volumes, capillary temperatures, voltages, etc.) to identify an optimal combination of sample preparation and CZE assay conditions. The result was a sample preparation procedure that differs from that described by Serru et al. [13] in that 5% rather than 10% MPA solution was used to precipitate protein. Glutathione extraction from RBC hemolysates using lower MPA concentration resulted in an increased GSSG S/N and improved peak area reproducibility. Using RBC rather than whole blood also minimized potential interference by acid-soluble plasma constituents.
3.4 Sample stability A series of experiments were conducted to determine how long whole blood or blood extracts could be stored for batch processing. Pilot studies showed that GSH and GSSG levels were similar in whole blood processed immediately or stored for up to 6 h at 4°C. We next evaluated how long samples could be stored at different stages of processing and the effect of storage temperature. RBC hemolysates stored overnight at −20 or −70°C contained significantly more GSSG than hemolysate that was processed and analyzed immediately without freezing. In contrast, the GSH and GSSG levels of MPA extracts analyzed immediately were not significantly different from those stored for long periods of time at −70°C. MPA extracts cannot be stored at −20°C as evidenced by increased GSSG levels within 24 h. However, MPA extracts can be stored at −70°C for at least 3 months without detectable changes in GSH and GSSG levels. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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3.5 Linearity and sensitivity Analysis of a serial dilution of a standard sample containing 4 mmol/L each of GSH and GSSG showed that the relationship between GSH and GSSG concentration and corrected peak area was linear for each analyte (r2 ⬎ 0.98, p ⬍ 0.001) throughout the range tested (31.25 mol/L to 4 mmol/L). Calibration standards for GSH (5 to 80 mol/L) and GSSG (1 to 16 mol/L) were selected for use in this assay because these levels best cover the range encountered in samples prepared from RBC and diluted 50-fold as described in Section 2. Elution profiles and migration times of standards prepared in 0.5% MPA were similar to those observed in samples prepared from RBC. 3.6 Assay imprecision Interassay reproducibility was assessed by analyzing aliquots of the control MPA extract in 20 different analytical runs performed on different days. The estimated glutathione composition of the control (mean ± SEM) was 1342 ± 74 mol/L GSH (CV = 5.5%), 118 ± 10 mol/L GSSG (CV = 8.3%), 1577 ± 86 mol/L TFG (CV = 6.5%), and 11.4 ± 0.7 GSH/GSSG (CV = 5.5%). Intraassay reproducibility was assessed by extracting and analyzing ten separate aliquots of the same RBC in the same analytical run. The estimated glutathione composition of the blood was 1610 ± 73 mol/L GSH (CV = 4.5%), 78 ± 11 mol/L GSSG (CV = 13.6%), 1766 ± 70 mol/L TFG (CV = 3.9%), and 21.0 ± 2.8 GSH/GSSG (CV = 13.3%). 3.7 Correlation study Figure 3 shows that the GSH, GSSG and total glutathione concentrations of blood samples analyzed by CZE were highly correlated (r2 0.95, p ⬍ 0.0001) with concentrations measured using the commercial DTNB assay kit. GSH levels were not significantly different between assays as determined by paired t-test. GSSG and total glutathione levels were both significantly higher when measured using the DTNB assay.
4 Discussion Two major modifications to the CZE method described by Serru et al. [13] significantly improved the ease of assay use and assay reproducibility. The first important modification was changing the method used to prepare blood samples for analysis. We used RBC hemolysates instead of whole blood with MPA extraction in 5% MPA rather than 10% MPA. These changes in sample preparation had a marked influence on assay performance because it increased the amount of analyte that could be injected, reduced interference by plasma constituents present in whole blood, and enhanced GSH and GSSG separation. The second important modification was decreasing run buffer pH from 8.4 to 7.8 which markedly improved the reproducibility of both peak area and migration times. www.electrophoresis-journal.com
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interpreted the data. Both authors contributed to the drafting of the manuscript and are responsible for the intellectual content and final approval of the published version. This research was supported by the Louisiana State University Health Sciences Center Department of Pediatrics and the Research Institute for Children at Children’s Hospital of New Orleans. We would like to express our appreciation to the staff of Children’s Hospital laboratory for their assistance in sample collection. The authors have declared no conflict of interest.
5 References [1] Chan, A. C., Chow, C. K., Chiu, D., Proc. Soc. Exp. Biol. Med. 1999, 222, 274–282. [2] Gizi, A., Papassotiriou, I., Apostolakou, F., Lazaropoulou, C., Papastamataki, M., Kanavaki, I., Kalotychou, V., Goussetis, E., Kattamis, A., Rombos, I., Kanavakis, E., Blood Cells Mol. Dis. 2011, 46, 220–225. [3] Pan, H. Z., Zhang, L., Guo, M. Y., Sui, H., Li, H., Wu, W. H., Qu, N. Q., Liang, M. H., Chang, D., Acta Diabetol. 2010, 47(Suppl 1), 71–76. [4] Cruz, K. K., Fonseca, S. G., Monteiro, M. C., Silva, O. S., Andrade, V. M., Cunha, F. Q., Romao, P. R., Parasite Immunol. 2008, 30, 171–174. [5] Matthews, G. M., Butler, R. N., Helicobacter. 2005, 10, 298–306. Figure 3. Correlation between GSH, GSSG, and total glutathione measured by CZE and a commercial DTNB assay. CZE run parameters were as described in Fig. 1. Results depict the linear regression equations with 99% confidence intervals. Total glutathione is expressed in GSH molar equivalents (GSH + (2 × GSSG)).
CZE assay results were highly correlated with results obtained using a commercial DTNB assay. A major advantage of the CZE assay is that both GSH and GSSG can be analyzed in a single run. In contrast, each blood sample must be analyzed twice with two different standard curves to measure total glutathione and GSSG in the DTNB assay. GSH is then determined as the difference between these total glutathione and GSSG levels. Although GSH levels were comparable when measured by CZE or the DTNB assay, total glutathione and GSSG levels were significantly different. As DTNB is nonspecific and reacts with cysteine and other thiols it seems likely that the higher total glutathione and GSSG levels observed with the DTNB assay were due to interference by nonglutathione constituents. Contribution statement: JMH and JOA were responsible for the conception and design of the study and performed the experiments and analytical measurements. JMH analyzed and
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[6] Locigno, R., Castronovo, V., Int. J. Oncol. 2001, 19, 221–236. [7] Lushchak, V. I., J. Amino. Acids 2012, 2012, 736837. [8] Lang, C. A., Mills, B. J., Mastropaolo, W., Liu, M. C., J. Lab. Clin. Med. 2000, 135, 402–405. [9] Flohe, L., Meth. Enzymol. 2010, 473, 1–39. [10] Wu, G., Fang, Y. Z., Yang, S., Lupton, J. R., Turner, N. D., J. Nutr. 2004, 134, 489–492. [11] Dalle-Donne, I., Rossi, R., Colombo, G., Giustarini, D., Milzani, A., Trends Biochem. Sci. 2009, 34, 85–96. [12] Hogg, N., Annu. Rev. Pharmacol. Toxicol. 2002, 42, 585–600. [13] Serru, V., Baudin, B., Ziegler, F., David, J. P., Cals, M. J., Vaubourdolle, M., Mario, N., Clin. Chem. 2001, 47, 1321–1324. [14] Hempe, J. M., McGehee, A. M., Stoyanov, A. V., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009, 877, 3462–3466. [15] Thom, G. G., Kallanagowdar, C., Somjee, S. S., Velez, M. C., Yu, L. C., Hempe, J. M., J. Pediatr. Hematol. Oncol. 2009, 31, 895–900. [16] Hempe, J. M., Ory-Ascani, J., Hsia, D., Exp. Biol. Med. 2007, 232, 437–444.
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