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Technical Notes
Dynamic Monitoring of Glutathione in Erythrocytes, without a Separation Step, in the Presence of an Oxidant Insult Madushi Raththagala, Paul D. Root, and Dana M. Spence*
Department of Chemistry, Wayne State University, Detroit, Michigan 48202
Numerous studies have associated alterations in red blood cell (RBC) glutathione (GSH) concentrations with various diseases including Alzheimer’s,1 HIV,2 multiple sclerosis,3 several types of cancers,4-6 and diabetes.7-9 Upon oxidant insult, RBCs, maintain homeostatic levels of GSH by the activity of glutathione reductase
(GR), which catalyzes the conversion of the oxidized dimeric form of glutathione (GSSG) to GSH, using nicotinamide dinucleotide monohydrogenphosphate (NADPH) as a substrate. In diabetes, the lower GSH/GSSG ratio may be due, in part, to a known decrease in activity of the GR found in the RBCs of diabetics. In other words, the decreased amounts of GSH in diabetics may not be due to lesser amounts of the tripeptide but, rather, the inability of the RBCs to maintain the levels of the GSH upon oxidant attack. It is thus evident that the ability to successfully monitor the GSH oxidation to GSSG, and its return to the reduced form of the thiol in RBCs, is important. In the case of diabetics, it has been known for nearly two decades that the erythrocytes, or RBCs, of people with both type I and type II diabetes are less deformable than healthy, nondiabetics.7-9 It has been postulated that the decrease in deformability may be due to oxidative damage to the protein spectrin. The spectrin is somewhat protected from excessive oxidative stress by various antioxidants in the cell, such as the reduced form of the GSH, the most abundant nonenzymatic antioxidant found in RBCs. Recently, we reported that the ability of the RBC to release adenosine triphosphate (ATP), a recognized stimulus of nitric oxide production in endothelial cells, was correlated with GSH levels in erythrocytes subjected to oxidative stress.10 There are several methods that have been employed, including colorimetric and fluorescence-based assays using DTNB and monochlorobimane (MCB) or monobromobimane (MBB), respectively, to measure GSH in various cell types. Many of the methods employed to quantify intracellular GSH have coupled separation and detection techniques such as HPLC11 and capillary electrophoresis (CE)12 with laser-induced fluorescence or CE with
* To whom correspondence should be addressed. Phone: 313.577.8660. Fax: 313.577.2942. E-mail:
[email protected]. (1) Liu, H.; Wang, H.; Shenvi, S.; Hagen, T. M.; Liu, R.-M. Ann. N. Y. Acad. Sci. 2004, 1019, 346-349. (2) Repetto, M.; Reides, C.; Gomez, Carretero, M. L.; Costa, M.; Griemberg, G.; Llesuy, S. Clin. Chim. Acta 1996, 255, 107-117. (3) Polidoro, G.; Di Ilio, C.; Arduini, A.; La, Rovere, G.; Federici, G. Int. J. Biochem. 1984, 16, 505-509. (4) Gromadzinska, J.; Wasowicz, W.; Andrijewski, M.; Sklodowska, M.; Quispe, O. Z.; Wolkanin, P.; Olborski, B.; Pluzanska, A. Neoplasma 1997, 44, 4551. (5) Upadhya, S.; Upadhya, S.; Mohan, S. K.; Vanajakshamma, K.; Kunder, M.; Mathias, S. Indian J. Clin. Biochem. 2004, 19, 80-83.
(6) Subapriya, R.; Kumaraguruparan, R.; Ramachandran, C. R.; Nagini, S. Clin. Biochem. 2002, 35, 489-493. (7) Costagliola, C. Clin. Physiol. Biochem. 1991, 8, 204-210. (8) Aaseth, J.; Stoa-Birketvedt, G. J. Trace Elem. Exp. Med. 2000, 13, 105111. (9) Beard, K. M.; Shangari, N.; Wu, B.; O’Brien, P. J. Mol. Cell. Biochem. 2003, 252, 331-338. (10) Carroll, J. S.; Subasinghe, W.; Raththagala, M.; Baguzis, S.; Oblak, T.; Root, P. D.; Spence, D. M. Mol. Biosyst. 2006, 2, 305-311. (11) Fujita, M.; Sano, M.; Takeda, K.; Tomita, I. Analyst (Cambridge, United Kingdom) 1993, 118, 1289-1292. (12) Wong, K.-S.; Yeung, E. S. Mikrochim. Acta 1995, 120, 321-327.
A method for the quantitative determination of the antioxidant form of glutathione (GSH) in red blood cells (RBCs) is described that does not require separation of the analyte of interest from the complex cellular matrix. The measurement portion of the analysis is performed using fluorescence spectrophotometry after monochlorobimane (a recognized probe for GSH) is added to a mixture containing RBCs and glutathione transferase (GST). This method was employed to determine the GSH concentration (0.042 ( 0.002 mM) in a solution of 1% RBCs obtained from rabbits (n ) 6). When spiked with authentic GSH (0.50 µmol), 99.8% of the GSH was recovered. Addition of GST to the sample mixture enabled most measurements to be made after 5-10 min of reaction time. Importantly, a decrease in GSH was measured upon the addition of a recognized oxidant (diamide) to the RBC sample followed by a subsequent return to normal levels of GSH. The ability of the GSH to recover from the oxidant attack occurred in a dose-dependent manner, requiring 30 and 90 min to recover from oxidant insults of 20 and 40 µM diamide, respectively. The antioxidant capabilities of the GSH were able to be monitored in real time, thus providing a method to dynamically monitor the ability of the RBC to maintain homeostasis in a complex matrix.
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amperometry using dual electrodes.13 While these methods have been successful in measuring GSH, and in the case of those employing HPLC and CE, the GSH/GSSG ratio, each of these above methods are not without limitations. Methods that employ DTNB are often limited by its lack in specificity for GSH. Fluorescence-based assays using bimane derivatives in erythrocytes have required the separation of cellular constituents. Such a separation may be required because the highest molar extinction coefficient of hemoglobin coincides with the excitation wavelength (λ ) 390 nm) of the probes. Another reason for separation prior to measurement is simply due to the complex matrix of the cellular debris interfering with the optical measurement. Those methods employing HPLC and CE have provided the separation necessary to quantify GSH in erythrocytes; however, such methods are not amenable to continuous monitoring of GSH oxidation due to the requirement of discrete sample loading associated with separation techniques. Here, we describe a fluorescence-based assay using MCB and the standard addition method to quantify GSH in erythrocytes. The use of MCB as a fluorescence probe for GSH determinations has been previously reported.14,15 However, when used in conjunction with the method of standard additions to overcome the complex matrix of the erythrocyte, it is possible to perform a quantitative determination of the GSH levels in the cells. Importantly, this measurement can be performed without any sample preparation beyond isolation of the RBCs from the whole blood. Moreover, because no physical separation of GSH from the cell matrix is required, this method can be used to determine the GSH redox status in a dynamic manner. Thus, this technique enables a constant monitoring of a small portion of the overall cellular metabolism in real time. EXPERIMENTAL SECTION Generation of Washed Red Blood Cells. RBCs were prepared on the day of use. To obtain rabbit RBCs, male New Zealand White rabbits (2.0-2.5 kg) were anesthetized with ketamine (8.0 mg/kg) and xylazine (1.0 mg/kg) followed by pentobarbital sodium (15 mg/kg iv). After tracheotomy, the rabbits were mechanically ventilated (tidal volume 20 mL/kg, rate 20 breaths/min; Harvard ventilator). A catheter was placed into a carotid artery, heparin (500 units, iv) was administered, and after 10 min, animals were exsanguinated. Blood was collected into vials, and the RBCs were separated from other formed elements and plasma by centrifugation at 500g at 4 °C for 10 min. The supernatant and buffy coat were removed by aspiration. Packed RBCs were resuspended and washed three times in PSS [in mM; 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 140.5 NaCl, 21.0 tris(hydroxymethyl)aminomethane, and 11.1 dextrose with 5% bovine serum albumin, pH adjusted to 7.4]. Fluorescence Determination of GSH Using Standard Additions. The standard addition method was employed to perform quantitative determinations of GSH. The procedure for preparing the standards is shown in Figure 1. The standards were prepared by combining 100 µL of a 1.0% hematocrit of RBCs and varying amounts (0, 4.0, 8.0, 12.0, 16.0, and 20.0 µL) of a 1.0 mM (13) Zhong, M.; Lunte, S. M. Anal. Chem. 1999, 71, 251-255. (14) Cook, J. A.; Iype, S. N.; Mitchell, J. B. Cancer Res. 1991, 51, 1606-1612. (15) Scott, R. B.; Collins, J. M.; Matin, S.; White, F.; Swerdlow, P. S. J. Clin. Lab. Anal. 1990, 4, 324-327.
Figure 1. Order of reagent addition for GSH determination using the method of standard additions. Varying volumes of water (730750 µL) are added to a standard cuvette, followed by 100 µL of a 1.0% RBC sample. Next, 100 µL of MCB and varying amounts of GSH (resulting in a 1.0-mL mixture) were added simultaneously, and the reagents were allowed to react for 10 min prior to obtaining the fluorescence emission at 478 nm.
stock solution of standard GSH in separate vials. The GSH stock was prepared by dissolving 0.0307 g of GSH (Sigma Aldrich, St. Louis, MO) in 18.0 MΩ distilled/deionized water (DDW). The reaction mixtures were prepared to a final volume1.0 mL using varying volumes of the DDW, at which point the RBCs were lysed. Also added to the mixture to aid in the labeling of the MCB probe was 50 µL of glutathione transferase (GST; 50 units mL-1). A volume of 100 µL of 250 µM MCB was added simultaneously with the GST. After the addition of the MCB/GST to the mixture, a 10-min incubation period was allotted to allow the MCB to react with GSH. Following the incubation period, the GSH-MCB fluorescence was measured (ex 370 nm, em 478 nm) for each of the prepared vials. The fluorescence intensity was plotted as a function of volume of GSH stock added to each vial. The slope and intercept, along with the volume of red cell sample and concentration of the GSH stock solution, were used in eq 1 (see below) to determine the concentration of GSH in the red cell sample. Monitoring GSH upon Oxidant Attack. To demonstrate the ability of MCB to monitor the GSH redox status in erythrocytes following an oxidant attack, 20 µM diamide was added to 5 mL of RBCs (1% hematocrit) to intentionally oxidize GSH to GSSG.16 Aliquots of RBCs were removed every 5 min and used to prepare samples containing a 0.1% hematocrit of RBCs, GST, and MCB. The changes in fluorescence were monitored every 5 min for up to 90 min (except for the first reading; this reading was taken at 10 min because the MCB-GSH reaction was given a minimum of ∼10 min before a signal was measured). RESULTS AND DISCUSSION Fluorescence Determination of GSH with MCB. Following the 10-min incubation period, the MCB fluorescence in the presence of RBCs was measured (ex 390 nm, em 478 nm) and found to be lower than the fluorescence of MCB without any RBCs (Figure 2a, b). The RBCs contain GSH; thus, the fluorescence signal would be expected to be higher in the presence of RBCs and lower when MCB was measured in the absence of GSH or a GSH source. Mixtures (1 mL total volume) containing 250 µM MCB and 100 µM standard GSH were prepared with and without 100 µL of a 1.0% hematocrit of RBCs (Figure 2c, d). Once again, samples that contained RBCs resulted in fluorescence emission that was lower than samples without any RBCs. It was determined that the high molar extinction coefficient of hemoglobin at 390 nm may be interfering with the excitation of MCB. By changing the excitation wavelength of MCB from 390 to 370 nm, the (16) Becker, P. S.; Cohen, C. M.; Lux, S. E. J. Biol. Chem. 1986, 261, 46204628.
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Figure 2. Initial attempts to quantify GSH in erythrocytes using MCB. In (a), the fluorescence of the MCB probe in a 100 µM GSH standard solution has a higher intensity than MCB in the presence of a 0.1% solution of RBCs that were prepared in 100 µM GSH standard (b). The MCB probe fluorescence in the absence of GSH standard or RBCs is shown in (c); as shown in (d), the fluorescence intensity of the MCB probe also decreases in the presence of a 0.1% RBC solution containing no added GSH standard. Such data imply that the RBCs are interfering with the fluorescence measurements.
absorbance of hemoglobin was significantly decreased while still providing adequate excitation of the probe. Due to the complex biological matrix found in erythrocytes, and the complicating presence of heme in quantifying intracellular GSH in RBCs, the standard addition method was used to determine the GSH levels in the RBC sample. Standards were prepared as described above and incubated for 10 min prior to acquiring the fluorescence spectra (ex 370 nm, em 478 nm). A standard curve was prepared from the peak emission intensities in the spectra in Figure 3. The GSH concentration was then quantified using the standard addition method (1).
GSH RBC ) bCstock/mVRBC
(1)
The GSH concentration in the RBCs can be determined from the y-intercept (b) multiplied by the concentration of the GSH stock
solution of 1 mM (Cstock) divided by the product of the slope (m) and volume of RBCs added to each standard that was measured (VRBC). Preparing the standards directly in the complex matrix of the sample, the hallmark feature of the standard addition method, allowed for the quantitative determination of GSH in erythrocytes without the need for separation of cellular constituents or any sample pretreatment. The GSH concentration (per RBC) from the data in Figure 3 was determined to be 0.047 ( 0.003 mM. The overall standard deviation was calculated using the standard deviations about the slope and intercept, respectively. This value is within the range of concentrations previously published for GSH in mammalian erythrocytes. By incorporating the volume of a red blood cell (87 fL), it was also determined that the amount of GSH per cell was ∼ 400 amol. The average GSH concentration found in the bulk sample for the RBCs from n ) 6 different rabbits was 0.042 ( 0.002 mM. This number translates to a cellular concentration of 3.99 ( 0.25 mM/RBC in the sample and 362 ( 20 amol/ cell. The precision in these results are presented as the standard error of the mean. The values reported here are somewhat higher than previously reported for GSH in human erythrocytes.17 However, it is important to note that the values reported here are for erythrocytes obtained from rabbits and not humans. In addition, other reports exist that suggest GSH and GSSG values are overestimated and underestimated depending on sample handling methods and agonists or antagonists used in the studies. Here, the measurements are made within 2-3 h after obtaining the whole blood sample from the rabbit. Interestingly, we have found that quantitative determinations for GSH in the obtained RBCs after 24 h result in GSH levels that are well below those reported in the literature. There are other general concerns in the literature that the MCB probe used to measure GSH is actually measuring total thiol content in the RBCs. It is established that MBB is a general thiol probe. However, there is also literature detailing the ability of the chlorine derivative of this bimane (MCB) to specifically label only GSH in rodent models.14,15 In order to provide evidence that the signal measured was actually due to the MCB reacting with GSH,
Figure 3. Standard addition method used to quantify GSH in erythrocytes by adding increasing volumes of 1.0 mM GSH to samples containing 0.1% HCT of RBCs. The resulting equation of the line resulting from the emission intensities of each standard was y ) 0.651x + 3.08 (r2 ) 0.9948) was used to determine the GSH concentration in the RBCs. 8558
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Figure 4. Kinetic study demonstrating the importance of GST in quantifying GSH in erythrocytes. In (a), the change in fluorescence intensity as a function of incubation time is shown for mixtures with (+ GST) and without (- GST) the enzymatic catalyst. For samples containing GST, 100 µL of a 1.0% RBC solution and 50 µL of 50 units/mL GST were added to 100 µL of the MCB probe and 750 mL of water. The changes in fluorescence were monitored for 10 min. In (b), the fluorescence signals for samples with and without GST are shown at 1-min incubation with the MCB probe.
and not other thiols found in the cellular matrix, an RBC sample was spiked with a known amount of authentic GSH prior to preparing the standards used in the standard addition determination. The amount of GSH found in one of the RBC samples was 0.046 mM. However, upon addition of 0.50 µmol of authentic GSH to the sample, it was determined that 99.8% of the spiked GSH was recovered. These results suggest that the standard addition method can be employed to successfully determine GSH levels in the RBCs without any sample preparation steps other than separating the RBCs from the whole blood. Determination of GSH Redox Status upon Oxidant Insult. To demonstrate the ability of MCB to monitor the glutathione redox status in erythrocytes following an oxidant attack, a sample of RBCs were prepared in 20 µM diamide by addition of 50 µL of 2 mM diamide to 5 mL of RBCs (1% hematocrit) to intentionally oxidize GSH to GSSG.16 Aliquots of RBCs (100 µL) were removed every 5 min and used to prepare 1-mL samples containing 0.1% hematocrit of RBCs in the presence of GST and MCB. As shown in Figure 4, the GST is added to increase the reaction speed between the GSH and the fluorescence probe. The changes in fluorescence were monitored every 5 min for up to 90 min. The first measurement was not obtained until 10 min due to the reaction time allotted for the MCB/GSH complex to generate a strong, measurable signal. Figure 5 shows the initial decrease in fluorescence due to the GSH being oxidized to GSSG in the RBCs. However, after ∼20 min, the GSH is slowly regenerated due to the RBCs’ ability to maintain proper intracellular oxidant status. As in the quantitative determination of the GSH described above, there may be concern that the measured decrease in fluorescence is not due to the diamide-induced oxidation of GSH to the GSSG dimer. However, diamide is known to be rather specific for GSH over other larger thiols, having a much higher rate constant for GSH over thiols other than GSH and does not react well with protein thiols due to steric hindrances.18,19 Due to the reported specificity of MCB for GSH, the specificity of diamide for GSH, and the increased time required for recovery when diamide (17) Hogan, B. L.; Yeung, E. S. TrAC, Trends Anal. Chem. 1993, 12, 4-9. (18) Kosower, E. M.; Correa, W.; Kinon, B. J.; Kosower, N. S. Biochim. Biophys. Acta 1972, 264, 39-44. (19) Kosower, N. S.; Kosower, E. M. Methods Enzymol. 1995, 251, 123-133.
Figure 5. Ability of MCB to monitor changes in reduced glutathione concentration in erythrocytes upon the addition of 20 µM diamide to a 1% HCT sample of RBCs. Aliquots were removed every 5 min after the addition of diamide and reacted with 250 µM MCB for 10 min. Changes in fluorescence were monitored for 45 min (ex 370 nm, em 478 nm). The first bar in the graph is the reading for no diamide added and thus represents the amount of GSH originally present before the addition of the oxidant.
concentrations are increased, the likelihood that the signals are due to GSH (and not other larger or protein-based thiols) is high. Finally, to provide further evidence that the signal is due to GSH and not other thiols, a calibration curve prepared from external standards of GSH results in the same slope as that obtained using the standard addition method on a 1% hematocrit of RBCs. A difference in these slopes is a classic indicator of analyte interferences that are not accounted for by the standard addition method. No difference was found between the slopes in this method, thus suggesting that the determined value for the GSH does not contain any measurable signal from other thiols in the RBC sample. CONCLUSIONS GSH, the most abundant nonenzymatic antioxidant in RBCs, has been determined using a recognized fluorescence probe. Although GSH has been determined using numerous techniques, Analytical Chemistry, Vol. 78, No. 24, December 15, 2006
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the work presented here represents the first quantitative determination of GSH using a bimane probe without any prior separation step. The ability to perform such a determination without a prior separation step is important due to the dynamic status of the cell. For example, once subjected to an oxidant attack, the GSH in the RBC will be oxidized to the GSSG dimer. However, cells are dynamic and homeostatic systems and will attempt to return to their preoxidative attack conditions. Therefore, it is imperative that the measurement of GSH oxidation to the GSSG dimer occur in near real time with a method that is fast and simple. This type of monitoring would not be possible with any type of system that required a discrete sampling method (injection) like HPLC or CE. For example, consider an investigation of GSH levels every 5 min for up to 60 min using a technique (such as HPLC) that required a discrete injection. One would be required to add diamide (the oxidant) to 12 different aliquots of the RBC sample, lyse and prep (dilutions, centrifugation, sample loading) each for the column, and then wait anywhere from 5 to 15 min for the separation to occur. At ∼30 min total analysis time per aliquot, it
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would take roughly 4-6 h to investigate the effects of the oxidant. At that rate, a triplicate analysis in 12-18 h would probably not be possible since the RBCs are generally functional (in terms of deformability and ATP release) for about 6-8 h. Such a dynamic measurement scheme is important when attempting to measure the effect of oxidant stressors on RBCs, especially considering that patients with type II diabetes have been reported to have lower levels of GSH in their RBCs than the RBCs of healthy nondiabetic controls. Moreover, recently, it has been shown that oxidative stress has a direct effect on the ability of erythrocytes to release nitric oxide-stimulating ATP.10 In fact, the trends in the ability of the erythrocytes (subjected to oxidant stress) to release ATP upon mechanical deformation were similar to the ability of GSH to remain in its antioxidant, reduced form.
Received for review June 27, 2006. Accepted September 15, 2006. AC061163U
