Potent Method for the Simultaneous Determination of Glutathione and

Anal. Chem. 2010, 82, 2006–2012

Potent Method for the Simultaneous Determination of Glutathione and Hydrogen Peroxide in Mitochondrial Compartments of Apoptotic Cells with Microchip Electrophoresis-Laser Induced Fluorescence Zhenzhen Chen,† Qingling Li,† Xu Wang,† Zhiyuan Wang,† Ruirui Zhang,† Miao Yin,‡ Lingling Yin,† Kehua Xu,† and Bo Tang*,† College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Shandong Normal University, and College of Life Sciences, Shandong Normal University, Jinan 250014, China The first application of microchip electrophoresis with laser-induced fluorescence (MCE-LIF) detection to simultaneously determine glutathione (GSH) and hydrogen peroxide (H2O2) in mitochondria was described. Organoselenium probe Rh-Se-2 and bis(p-methylbenzenesulfonate)dichlorofluorescein (FS) synthesized in our laboratory were utilized as fluorescent probes for GSH and H2O2, respectively. Rh-Se-2, which is nonfluorescent, reacts with GSH to produce rhodamine 110 (Rh110) with high quantum yield. Similarly, nonfluorescent FS reacts with H2O2 and produces dichlorofluorescein (DCF) accompanied by drastic fluorescence enhancement. Both probes exhibit good sensitivity toward their respective target molecule determination. Fast, simple, and sensitive determination of GSH and H2O2 was realized within 37 s using a running buffer of 50 mM mannitol, 40 mM HEPES (pH 7.4), and an electric field of 360 V/cm for separation. The linear ranges of the method were 3.3 × 10-9-1.0 × 10-7 M/2.9 × 10-7-1.0 × 10-4 M and 2.7 × 10-9-4.0 × 10-7 M with detection limits (signal-tonoise ratio ) 3) of 1.3 nM (0.16 amol) and 1.0 nM (0.12 amol) for GSH and H2O2, respectively. The relative standard deviations (RSDs) of migration time and peak area were less than 1.0% and 4.0%, respectively. The MCE-LIF assay was utilized to investigate the levels of GSH and H2O2 in mitochondria isolated from HepG2 cells and were found to be 2.01 ( 0.21 mM and 5.36 ( 0.45 µM, respectively. The method was further extended to observe situations of the two species in mitochondria of HepG2 cells experiencing cell apoptosis that were induced by doxorubicin and photodynamic therapy (PDT). During cellular metabolism, mitochondrial electron transport results in the formation of superoxide anion (O2-•) and subse* To whom correspondence should be addressed. E-mail: [email protected]. Fax: 86-531-86180017. † College of Chemistry, Chemical Engineering and Materials Science. ‡ College of Life Sciences.

2006

Analytical Chemistry, Vol. 82, No. 5, March 1, 2010

quently hydrogen peroxide (H2O2) which can be reduced by glutathione (GSH) under the catalysis of glutathione peroxidase.1 Under certain conditions, H2O2 exhibits rapid alterations in concentration and exerts reverse effects which are commensurate with metabolic requirements. On the one hand, endogenously generated H2O2 is involved in various biological functions and has been proved to act as a second messenger in the regulation of cellular redox poise, bioenergy output,2 gene expression,3 and cell differentiation.4 On the other hand, when overproduced (e.g., due to exogenous stimulation), H2O2 can react with Fe2+ to produce the damaging hydroxyl radical ( · OH).5 Both H2O2 and the produced · OH can cause oxidative stress through the oxidation of biomolecules, leading to cellular damage that may become irreversible and eventually, cause cell death. GSH is the most abundant low-molecular-weight thiol in cells and is involved in many cellular processes including antioxidant defense, cell signaling, and cell proliferation.6 GSH is found mainly in cytosol where it is synthesized from its constituent amino acids and in mitochondria where it plays a key protective role in helping to maintain the delicate mitochondrial redox balance. A case in point is the detoxification of H2O2,7 as mentioned above. In mitochondria of most animal cells, catalase, which can metabolize H2O2, is absent.8 H2O2 is mainly metabolized by glutathione peroxidase, whose substrate is GSH. Therefore, (1) Prabhakar, R.; Vreven, T.; Morokuma, K.; Musaev, D. G. Biochemistry 2005, 44, 11864–11871. (2) Linnane, A. W.; Kios, M.; Vitetta, L. Biogerontology 2007, 8, 445–467. (3) Jin, N.; Hatton, N. D.; Harrington, M. A.; Xia, X.; Larsen, S. H.; Rhoades, R. A. Free Radical Biol. Med. 2000, 29, 736–746. (4) Yamamoto, T.; Sakaguchi, N.; Hachiya, M.; Nakayama, F.; Yamakawa, M.; Akashi, M. Leukemia 2009, 23, 761–769. (5) Costa, N. J.; Dahm, C. C.; Hurrell, F.; Taylor, E. R.; Murphy, M. P. Antioxid. Redox. Signal 2003, 5, 291–305. (6) Franco, R.; Panayiotidis, M. I.; Cidlowski, J. A. J. Biol. Chem. 2007, 282, 30452–30465. (7) Jo, S. H.; Son, M. K.; Koh, H. J.; Lee, S. M.; Song, I. H.; Kim, Y. O.; Lee, Y. S.; Jeong, K. S.; Kim, W. B.; Park, J. W.; Song, B. J.; Huhe, T. L. J. Biol. Chem. 2001, 276, 16168–16176. (8) Esworthy, R. S.; Ho, Y. S.; Chu, F. F. Arch. Biochem. Biophys. 1997, 340, 59–63. 10.1021/ac902741r  2010 American Chemical Society Published on Web 02/09/2010

GSH required for the activity of mitochondrial glutathione peroxidase has been proven to be the best defense against the potential toxicity of H2O2 in the mitochondria. In recent years, there has been increasing demand for the detection and quantitation of GSH and H2O2 because of their importance in a wide range of physiological and pathogenic circumstances.9 Owing to their close relationship, simultaneous determination of them is more desirable, especially for their analysis in subcellular compartments such as mitochondria, the central site of oxygen consumption, and regulator of apoptosis,10 whose function and content analysis has attracted scientific interest recently.11-13 In the study of their functions in cellular milieu, GSH and H2O2 are often detected individually by different methods,14,15 which are complicated, time-consuming, and need to be simplified. What’s more, the data obtained from two different detection systems cannot track the concentration of these two compounds at the same time exactly, bring errors in the evaluation of their physiological functions. Fortunately, microchip electrophoresis (MCE) has risen as an efficient platform for molecular and cellular analysis of biological systems because of its advantages, including reduced reagent consumption and analysis time, ease of integration, and the potential for parallel processing.16-18 When coupled with ultrasensitive detection schemes, such as laser induced fluorescence (LIF), the MCELIF detection system has exhibited great potential in accurate biomarker analysis with a sample volume of only ∼10 µL.19 The MCE-LIF methodology, associated with the use of suitable fluorescent probes, is an excellent approach to measure GSH and H2O2 concurrently from the perspective of both scientific interest and technological validity. But first of all, the selection of proper fluorescent probes is extremely necessary. Naphthalene-2,3-dicarboxaldehyde (NDA) was commonly used in MCELIF for the determination of GSH.20-22 However, in most cases, an alkalescent borate buffer medium of pH 9.2 was utilized to ensure higher efficiency. It is well-known that a neutral medium is much closer to the cell growth environment, which is recommended when it comes to the determination of GSH in cultured (9) Armstrong, J. S.; Steinauer, K. K.; Hornung, B.; Irish, J. M.; Lecane, P.; Birrell, G. W.; Peehl, D. M.; Knox, S. J. Cell Death Differ. 2002, 9, 252– 263. (10) Mayer, B.; Oberbauer, R. News Physiol. Sci. 2003, 18, 89–94. (11) Allen, P. B.; Doepker, B. R.; Chiu, D. T. Anal. Chem. 2009, 81, 3784– 3791. (12) Kanno, A.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2006, 78, 8076–8081. (13) Meany, D. L.; Thompson, L.; Arriga, E. A. Anal. Chem. 2007, 79, 4588– 4594. (14) Shen, D.; Dalton, T. P.; Nebert, D. W.; Shertzer, H. G. J. Biol. Chem. 2007, 280, 25305–25312. (15) Han, D.; Canali, R.; Rettori, D.; Kaplowitz, N. Mol. Pharmacol. 2003, 64, 1136–1144. (16) Wheeler, A. R.; Throndset, W. R.; Whelan, R. J.; Leach, A. M.; Zare, R. N.; Liao, Y. H.; Farrell, K.; Manger, I. D.; Daridon, A. Anal. Chem. 2003, 75, 3581–3586. (17) Easley, C. J.; Karlinsey, J. M.; Bienvenue, J. M.; Legendre, L. A.; Roper, M. G.; Feldman, S. H.; Hughes, M. A.; Hewlett, E. L.; Merkel, T. J.; Ferrance, J. P.; Landers, J. P. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19272–19277. (18) Huebner, A.; Olguin, L. F.; Bratton, D.; Whyte, G.; Huck, W. T.; de Mello, A. J.; Edel, J. B.; Abell, C.; Hollfelder, F. Anal. Chem. 2008, 80, 3890– 3896. (19) Yang, W.; Sun, X.; Wang, H. Y.; Woolley, A. T. Anal. Chem. 2009, 81, 8230–8235. (20) Gao, J.; Yin, X.; Fang, Z. Lab Chip 2004, 4, 47–52. (21) Qin, J.; Ye, N.; Yu, L.; Liu, D.; Fung, Y.; Wang, W.; Ma, X.; Lin, B. Electrophoresis 2005, 26, 1155–1162. (22) Ling, Y.; Yin, X.; Fang, Z. Electrophoresis 2005, 26, 4759–4766.

cells. As for H2O2 analysis, Amplex Red, 2′,7′-dichlorofluorescin, and dihydrorhodamine have been used in MCE-LIF. But, limitations in the detection reliability also exist because of the low stability and selectivity of the probes. Fortunately, bis(pmethylbenzenesulfonate) dichlorofluorescein (FS) was developed as a fluorescent probe in the determination of H2O2 in cell extracts using MCE-LIF by our group.23,24 In this study, two fluorescent probes, including Rh-Se-2, a rhodamine-based organoselenium fluorescent probe for GSH25 and FS for H2O2, were used in the simultaneous determination of GSH and H2O2 in mitochondria by MCE-LIF. The levels of GSH and H2O2 in mitochondria isolated from HepG2 cells were investigated and validated by depletion and recovery experiments. To further testify about the feasibility of the proposed method, the MCE-LIF assay was extended to observe variation of GSH and H2O2 in mitochondria during cell apoptosis, since mitochondria are intimately involved with apoptotic cell death,26,27 and more importantly, both mitochondrial GSH and H2O2 play key roles in redox disturbance and signaling pathways in mitochondria-related apoptosis, the levels of them in mitochondria are closely linked with the execution of apoptosis. 28,29 Doxorubicin, a chemotherapeutic drug, and multifunctional nanoparticles as photodynamic therapy (PDT) reagent were introduced to induce oxidative stress and apoptosis of HepG2 cells, respectively. During these two processes, GSH and H2O2 were simultaneously determined by the present MCE-LIF method. Results showed that the concentration of GSH decreased, while that of H2O2 increased, giving evidence to the H2O2 generation and depletion of GSH, which showed mitochondrial redox environment alteration during apoptosis. To our best knowledge, this is the first report of simultaneous determination of one antioxidant and one reactive oxygen species in biological environments with high resolution. The results proved that the method can reliably detect subcellular content changes of a pair of antioxidant/oxidant species in apoptotic cells. EXPERIMENTAL SECTION Reagents. All chemicals and solvents used were of analytical grade. Water was purified with a Sartorius Arium 611 VF system (Sartorius AG, Germany) to a resistivity of 18.2 MΩ · cm. FS and Rh-Se-2 were synthesized in our laboratory. Dichlorofluorescein (DCF), Rh110, N-ethylmaleimide (NEM), catalase, GSH, and HEPES were obtained from Sigma-Aldrich Chemicals (St. Louis, MO). Stock solutions of FS, Rh-Se-2, DCF, and Rh110 (1.0 mM) were prepared in DMSO and stored at 4 °C in darkness separately. NEM was dissolved in DMSO at a concentration of 20 mM and stored at 4 °C before use. A stock solution of catalase (10 U/mL) was prepared in 0.10 M phosphate buffer (pH 7.0) and stored at 4 °C. GSH (1.0 mM) and H2O2 (1.0 mM) in ultrapure water were (23) Li, H.; Li, Q.; Wang, X.; Xu, K.; Chen, Z.; Gong, X.; Liu, X.; Tong, L.; Tang, B. Anal. Chem. 2009, 81, 2193–2198. (24) Gong, X.; Li, Q.; Xu, K.; Liu, X.; Li, H.; Chen, Z.; Tong, L.; Tang, B. Electrophoresis 2009, 30, 1983–1990. (25) Tang, B.; Yin, L.; Wang, X.; Chen, Z.; Tong, L.; Xu, K. Chem. Commun. 2009, 5293–5295. (26) Ricci, J. E.; Gottlieb, R. A.; Green, D. R. J. Cell Biol. 2003, 160, 65–75. (27) Green, D. R.; Kroemer, G. Science 2004, 305, 626–629. (28) Circu, M. L.; Rodriguez, C.; Maloney, R.; Moyer, M. P.; Aw, T. Y. Free Radic. Biol. Med. 2008, 44, 768–778. (29) Quillet-Mary, A.; Jaffre’zou, J. P.; Mansat, V.; Bordier, C.; Naval, J.; Laurent, G. J. Biol. Chem. 1997, 272, 21388–21395.


2007

freshly prepared before use. Borate, HEPES, and phosphate buffer used for electrophoretic migration were prepared by dissolving an appropriate amount of Na2B4O7, HEPES, and NaH2PO4 in ultrapure water, respectively. The required pH was adjusted by adding an appropriate amount of HCl or NaOH. Doxorubicin (DOX, Yuancheng Sci & Tech Co. LTD, Wuhan) was dissolved in DMSO and kept in the dark. Core-shell nanocomposite material (SiO2@HP; for a transmission electron microscopry (TEM) image, see the Supporting Information) was synthesized in our laboratory according to the procedure in ref 30. The obtained nanocomposite was diluted with ultrapure water to a concentration of 200 µg/mL. All solutions were filtered through a 0.22 µm nylon syringe filter before being added into the chip. Fluorescence Experiments. Fluorescence experiments were performed on FLS-920 Edingburgh fluorescence spectrofluorimeter (Edinburgh Analytical Instruments, Edinburgh, UK) with a Xenon lamp and 1.0 cm quartz cells. The emission wavelengths were determined at an excitation wavelength of 493 or 495 nm by scanning the emission monochromator between 500 and 600 nm. Cell Culture, Incubation, and Irradiation. HepG2 cells (American Type Culture Collection, Manassas) were grown in cell culture media and incubated at 37 °C in a 5% CO2/95% air humidified incubator (MCO-15AC, SANYO). The cell culture medium was RPMI-1640 (Hyclone, USA) supplemented with 10% newborn calf serum (Gibco, Invitrogen), 1% penicillin, and 1% streptomycin. Cell viability was determined by the trypanblue exclusion assay. When cells were in a logarithmic growth phase, DOX at concentrations of 0.5, 1, and 2 µM was administered into the cell culture medium. As for the PDT induced apoptosis, core-shell nanocomposite was added to the culture medium and incubated for 4 h. After that, cells were rinsed with PBS three times. Then, light irradiation was administered at a dose of 4.5 mW/cm2 to the sample surface. The light-emmiting diode (LED) light source for irradiation had a wavelength of 405 nm (J-li LED, Shenzhen, China). Preparation of Mitochondria. Mitochondria were prepared by differential centrifugation31 using traditional Dounce homogenization with a commercially available Beyotime mitochondria isolation kit (Beyotime Inst. Biotech, Haimen, China). Briefly, before and after treatment of HepG2 cells with DOX or PDT using nanocomposite SiO2@HP, cells were washed twice with phosphate-buffered saline, trypsinized, and suspended in cold mitochondrial isolation reagent for 15 min. Cells were then disrupted with a Dounce homogenizer, and the homogenate was centrifuged at 1000g (Sigma, 3K15, Germany) for 10 min. The resulting supernatant was centrifuged at 3500g for 15 min, and the pellet was washed with uptake buffer (70 mM sucrose, 1 mM KH2PO4, 5 mM sodium succinate, 5 mM HEPES, 220 mM mannitol, 0.1 mM EDTA, pH 7.4) before it is centrifuged at 10 000g for 5 min. After centrifugation, the mitochondria pellet was suspended in uptake buffer to a final concentration of 0.5 mg/mL protein. All steps were performed below 4 °C to

minimize the oxidation of GSH. The isolated mitochondrial sample was kept on ice until being used in the experiments. MCE-LIF Detector System. The schematic diagram of the experimental setup is shown in Figure 1. The glass-based microfluidic chip (Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China), the injection channel, S-SW, and the separation channel, B-BW, formed a “cross”, and the distance was 15 mm between the detection point and the cross, C. The channel cross-section was close to a rectangle structure (70 µm width × 25 µm depth). The four reservoirs were the same column structure with 3 mm diameter and 1.5 mm depth. The glass microchip assembly was mounted on the X-Y-Z translational stage that also served as a platform of laser induced fluorescence detection (LIFD). A versatile programmable eight-path-electrode power supply (PEPS) and chip-based MCE-LIFD were made in-house.32,33 PEPS was used for sample injection and MCE separation. The connecting interface between the PEPS and the chip was also shown in Figure 1, by dipping the 4-Pt-electrode (0.5 mm) of the PEPS into the 4-reservoir of the microfluidic chip, respectively. The optics collection system of LIFD was in confocal optics mode.23 The semiconductor double-pumped solid state laser was used as the exciting light source (SDPSS, MBL-20, λex 473 nm, power stability < 2%, Changchun Xinchanye Guangdianjishu Co. Ltd., China). The emission light filter was a narrowband filter with a wavelength in the range of 525 ± 5 nm (Omega Optical, Brattleboro, VT). A photomultiplier (PMT, Hamamatsu, R928, Japan) was used as the fluorescence detector. The sampling frequency of a CT-22 data acquisition card (Shanghai Qianpu Shuju Co. Ltd., China) was 20 Hz. Microfluidic Electrophoresis Procedure. The microchannels were rinsed sequentially with 0.1 M NaOH and ultrapurewater for 10 min, respectively, before flushing with electrophoresis buffer for 5 min. Prior to the MCE separation, sample waste (SW), buffer

(30) Zhang, R.; Wu, C.; Tong, L.; Tang, B.; Xu, Q. Langmuir 2009, 25, 10153– 10158. (31) Lluis, J. M.; Colell, A.; Garcia-Ruiz, C.; Kaplowitz, N.; Fernandez-Checa, J. Gastroenterology 2003, 124, 708–724.

(32) Li, Q.; Zhang, H.; Wang, Y.; Tang, B.; Liu, X.; Gong, X. Sens. Actuators B 2009, 136, 265–274. (33) Liu, X.; Li, Q.; Gong, X.; Li, H.; Chen, Z.; Tong, L.; Tang, B. Electrophoresis 2009, 30, 1077–1083.

2008


Figure 1. Schematic diagrams of the MCE-LIF experimental setup. S, sample reservoir; SW, sample waste reservoir; B, buffer reservoir; BW, buffer waste reservoir; PEPS, programmable eight-path-electrode power supply; LIFD, laser induced fluorescence detector.

Figure 2. Chemical structures and fluorescence spectra of Rh-Se-2 and FS. (lines 1 and 2) Fluorescence spectra of Rh-Se-2 (20 µM), recorded before and after GSH (10 µM) addition, respectively (λex/λem ) 495/522 nm). (line 3 and 4) Fluorescence spectra of FS (10 µM), recorded before and after H2O2 (5 µM) addition, respectively (λex/λem ) 493/525 nm). Buffer: 100 mM HEPES (pH 7.4).

(B), and buffer waste (BW) reservoirs were all filled with 10 µL electrophoresis running buffer and the sample reservoir (S) was filled with 10 µL of sample solution. The electrokinetic pinched sample injection and electrophoresis separation were controlled by the voltage output of the PEPS for each reservoir. During the pinched injection, 400 V was applied to the sample reservoir for 20 s, 280 and 600 V were applied to reservoirs B and BW, respectively, while the SW reservoir was grounded. A separation was performed by applying 1800 V to the B reservoir, 1200 V to S and SW reserviors with the BW reservoir grounded. Analysis of GSH and H2O2 Produced in the Mitochondria of HepG2 Cells. Mitochondria in uptake buffer were treated with 5% trichloroacetic acid at 4 °C for 10 min. Then, the mixture was centrifuged at 14 000 rpm for 15 min. After that, the protein pellet was discarded and the supernatant was diluted and incubated at 37 °C for 30 min in the presence of 5 µM FS and 20 µM Rh-Se-2.

Depletion Experiments. For GSH depletion, NEM was added into the mitochondria extract to a final concentration of 5 mM, which reacts with thiol groups to form a stable S-(N-ethylsuccinimido) cysteine derivative. Depletion of hydrogen peroxide was performed by addition of catalase to the mitochondrial extract. Statistical Analysis. Data were expressed as the mean ± standard deviation. All experiments were repeated three times, and the data were calculated with Microsoft Excel. For significance testing, the student t-test was used (a p < 0.05 is considered significant). RESULTS AND DISCUSSION Fluorescence Properties of Rh-Se-2 and FS. In this work, Rh-Se-2 and FS were employed as labeling reagents for GSH and H2O2, respectively. Upon reaction with GSH, the Se-N bond of the nonfluorescent organoselenium probe Rh-Se-2 is cleaved and subsequently generates fluorescent product rhodamine 110 (Rh110, Figure 2 lines 1 and 2).25 As for FS, previous studies in Analytical Chemistry, Vol. 82, No. 5, March 1, 2010

2009

Figure 4. A typical electropherogram of standard solutions of 100 nM Rh110 and 20 nM DCF. MCE conditions: running buffer, 50 mM mannitol, 40 mM HEPES, pH 7.4; injection time, 20 s; separation electric field, 360 V/cm; effective separation distance, 15 mm.

Figure 3. Effect of (A) buffer pH on the relative fluorescence intensity (RFU) of 50 nM Rh110 and 50 nM DCF in 40 mM HEPES buffer and (B) concentration on migration time of 50 nM Rh110 and 50 nM DCF in HEPES buffer (pH 7.4) with a separation electric field of 300 V/cm. Effect of (C) applied electric field on the migration times and (D) separation efficiency of Rh110 and DCF in 50 mM mannitol, 40 mM HEPES buffer, pH 7.4. Separation conditions: injection time, 20 s; effective separation distance, 15 mm. Each point represents an average of experiments repeated three times. The error bar superimposed on each marker means standard deviation (SD).

our laboratory23,24 demonstrated that nonfluorescent FS exhibits good reactivity to H2O2, and the prompt fluorescence enhancement of dichlorofluorescein (DCF) can be observed (Figure 2 lines 3 and 4). Rhodamine 110 and dichlorofluorescein have similar fluorescence properties (Rh110, Exmax 495 nm and Emmax 522 nm; DCF, Exmax 493 nm and Emmax 525 nm). Therefore, they can be separated by MCE-LIF and determined under the same excitation/emission wavelength. Cross reaction was tested for Rh-Se-2 with H2O2 and FS with GSH. As expected, no fluorescence change occurred in both occasions (data not shown). Optimization of MCE. Negatively charged dichlorofluorescein (DCF) and neutral rhodamine 110 (Rh110) can be easily separated by the MCE-LIF detector system. Three kinds of buffers (i.e., HEPES, phosphate, and borate buffers) were tested. It was found that a better baseline was obtained with the use of HEPES buffer. Then, the effect of pH was examined in the range from pH 7.0 to 8.0. pH-dependent DCF showed an increase in fluorescence intensity, but that of Rh110 almost remained constant (Figure 3A). Hence, a pH of 7.4 was selected to obtain a compromise between signal responses and a suitable medium for biological analysis. An increase in buffer concentration will enhance the peak area but at the same time prolong the migration time of the analytes (Figure 3B), a concentration of 40 mM HEPES was selected with the consideration of a better peak shape. Buffer additive mannitol was utilized to suppress the adsorption of analytes onto the surface of microchannels, and as a result, resolution and sensitivity can be greatly enhanced.24 A buffer solution of 50 mM mannitol and 40 mM HEPES (pH 7.4) was therefore chosen as the electrophoresis running buffer. In the MCE system, separation electric field influences migration time and separation efficiency. Increasing the separation 2010


electric field shortens the migration time (Figure 3C) and elevates the theoretical plates for the two analytes (Figure 3D). But, a higher voltage generates Joule heating and higher current. Considering both the analysis time and Joule heating, an electric field of 360 V/cm was employed. Figure 4 shows the typical electropherogram of Rh110 and DCF at the migration times of 30 and 36 s under the optimized separation conditions, respectively. A separation can be completed within 37 s, indicating that simultaneous and rapid determination of GSH and H2O2 can be realized. Linear Regression, Reproducibilities, Detection Limit, and Method Validation. The standard solutions of GSH and H2O2 were treated with Rh-Se-2 and FS solutions to prepare six-point calibration curves. The calibration curves were tested over the concentration range of 3.3 × 10-9-1.0 × 10-7 and 2.9 × 10-7-1.0 × 10-4 M for GSH and 2.7 × 10-9-4.0 × 10-7 M for H2O2. The analytical characteristics of the MCE-LIF system for GSH and H2O2 are summarized in Table 1. Reproducibilities obtained from migration time and peak area measurements were studied by six injections of a standard solution consecutively. The results were given in terms of peak areas. In pinched sample injection mode, the volume and the shape of the injected analyte is dependent on the pinched and pushback intensities.34 Considering an electrokinetic pinched injection volume of 123 pL experimentally measured using an inverted fluorescent microscope (DMIL, Leica, Germany) and a a charge-coupled device (CCD) camera (DFC300FX, Leica, Germany),24 the calculated mass limits of detection (LODs) of GSH and H2O2 were 0.16 and 0.12 amol, respectively. The MCE-LIF system was developed to separate Rh110 and DCF, which resulted from the reaction of mitochondrial GSH with Rh-Se-2 and H2O2 with FS, respectively. Peaks 1 and 2 in Figure 5A were identified as Rh110 and DCF based on their electrophoretic mobilities. So, the two peaks are representatives of GSH and H2O2, respectively. To verify the selectivity of the probes, catalase was first added to the mitochondrial extract before Rh-Se-2 and FS probe were added. The products were then analyzed by MCE-LIF. As expected, the H2O2 peak vanished as a result of catalase activity and the GSH peak remained unchanged. On the other hand, NEM as a scavenger of GSH was added to the same amount of mitochondrial pellet to verify the selectivity of Rh-Se-2. As (34) Fu, L. M.; Lin, C. H. Anal. Chem. 2003, 75, 5790–5796.

Table 1. Linearity, Reproducibilities of Migration Time and Peak Area, and LODs for Rh110 and DCF analyte

regression equationa

r2

LODb

linear range (M)

GSH

y ) 0.1943 + 11.90x y ) 2.820 + 4.562x y ) 0.2836 + 39.51x

0.9994 0.9993 0.9990

1.3 nM

3.3 × 10-9-1.0 × 10-7 M 2.9 × 10-7-1.0 × 10-4 M 2.7 × 10-9-4.0 × 10-7M

H2O2 a

1.0 nM

RSD (%, n ) 6) (migration time)

RSD (%, n ) 6) (peak area)

0.30

2.5

0.70

3.1

b

y, peak area; x, concentration of the analyte in micromolar. Concentration limits of detection measured for a signal/noise ratio of 3.

Figure 5. (A) Typical microchip electropherograms of Rh110 and DCF in mitochondria from HepG2 cells: peak 1, Rh110; peak 2, DCF. (B) Electropherograms of Rh110 and DCF in mitochondria of HepG2 cells in the presence of of NEM and catalase. MCE conditions were as in Figure 4.

illustrated in Figure 5B, the GSH peak was observed to disappear when NEM was added. In addition, the H2O2 peak showed a slight increase, partly because of a significant depletion of the mitochondrial pool of GSH which is required for the enzymatic metabolism of mitochondrial hydrogen peroxides. It should be noted that, although thiol-specific, Rh-Se-2 has shown good selectivity for mitochondria GSH in the present system. This can be attributed to two points. First, the elimination of mitochondrial proteins through precipitating with 5% trichloroacetic acid and subsequent isolation by centrifugation excludes the interference from thiol proteins. Second, Rh-Se-2 showed the highest response to GSH than any other nonprotein thiols,25 such as cysteine, whose content was about 3 orders of magnitude lower than that of GSH.35 Standard addition protocol was used to validate the two peaks. Known amounts of GSH and H2O2 were added to the mitochondrial extracts, which were then diluted and analyzed under the same conditions by MCE-LIF. The concentrations and recoveries of GSH and H2O2 in the mitochondrial extract were shown in Table 2. The method is thus adequate for detection of GSH and H2O2 levels in mitochondrial samples, although a large (35) Rebrin, I.; Bayne, A. C. V.; Mockett, R. J.; Orr, W. C.; Sohal, R. S. Biochem. J. 2004, 382, 131–136.

stoichiometric difference between GSH and H2O2 in mitochondria exists (∼103-fold).15 Monitoring Mitochondrial GSH and H2O2 in Apoptotic HepG2 Cells. Apoptosis is a ubiquitous, evolutionary conserved process involved in numerous biological systems. It is an important phenomenon in cytotoxicity induced by anticancer drugs and other therapeutic methods for disease treatment.36 Numerous studies are interested in exploring the possible roles of GSH and H2O2 involved in cell apoptosis and the possible link between the two species.37,38 Here, we studied the process of apoptosis of HepG2 cells induced by DOX and photodynamic therapy (PDT) using a core-shell nanocomposite material. The proposed MCELIF assay was applied to observe variation of GSH and H2O2 levels in mitochondria during cell apoptosis. Anthracycline antibiotic DOX can rapidly localize to the nucleus in tumor cells,39 inducing DNA damage and cell apoptosis.40 At low concentrations (0.5-2 µM), DOX was also observed to cause site-specific DNA fragmentation in HepG2 cells (Figure S-1 of the Supporting Information). Studies indicated that DOX induced apoptosis in HepG2 cells in a time-dependent manner. Hochest 33258 staining for chromatin condensation assessment demonstrated a nuclear marginalization within 6 h after DOX treatment (Figure S-2 of the Supporting Information) as an early sign of apoptosis. At the same time, H2O2 showed no significant change. However, a ∼5% depletion of GSH was observed according to the analysis (Figure 6A). Mitochondrial membrane potential (MMP) collapse (Figure S-3 of the Supporting Information) observation using Rhodamine 123 (Rh-123) staining indicated an apparent MMP decrease after incubation with DOX for 8 h, and an increase in H2O2 levels occurred thereafter (Figure 6B). The depletion of GSH concentration and increase in H2O2 concentration in mitochondrial continued until DOX treatment for 24 h, accompanied by the total disruption of MMP. We next applied the MCE-LIF method to the detection of mitochondrial GSH and H2O2 during HepG2 cells apoptosis induced by PDT with core-shell nanocomposite synthesized in-house.30 The silica nanovehicle acts as not only a carrier for the photosensitizers, but also as a nanoreactor to facilitate the photo-oxidation reaction, which exhibits excellent photooxidation efficiency. Both mitochondrial GSH depletion and H2O2 level enhancement was significant 6 h after PDT (Figure (36) Kim, R.; Tanabe, K.; Uchida, Y.; Emi, M.; Inoue, H.; Toge, T. Cancer Chemother. Pharmacol. 2002, 50, 343–352. (37) Wang, S.; Konorev, E. A.; Kotamraju, S.; Joseph, J.; Kalivendi, S.; Kalyanaraman, B. J. Biol. Chem. 2004, 279, 25535–25543. (38) Magi, B.; Ettorre, A.; Liberatori, S.; Bini, L.; Andreassi, M.; Frosali, S.; Neri, P.; Pallini, V.; Di. Stefano, A. Cell Death Differ. 2004, 11, 842–852. (39) Kotamraju, S.; Konorev, E. A.; Joseph, J.; Kalyanaraman, B. J. Biol. Chem. 2000, 275, 33585–33592. (40) L’Ecuyer, T.; Sanjeev, S.; Thomas, R.; Novak, R.; Das, L.; Campbell, W.; Heide, R. V. Am. J. Physiol. Heart Circ. Physiol. 2006, 291, H1273–H1280.


2011

Table 2. Concentrations and Recoveries of GSH and H2O2 in Mitochondrial Extracts of HepG2 Cells (n ) 3)a

a

analyte

concentration

added

found

mean

average recovery (%)

GSH H2O2

2.01 ± 0.21mM 5.36 ± 0.45 µM

2.0 mM 5.0 µM

3.88, 3.75, 3.97 mM 10.02, 10.33, 9.91 µM

3.87 ± 0.11mM 10.09 ± 0.22 µM

93.0 94.6

Conditions were as in Figure 4.

Figure 6. Time course of mitochondrial GSH depletion (A) and H2O2 generation (B) after DOX or PDT induced HepG2 cell apoptosis. GSH and H2O2 were determined with MCE-LIF methods. Cells were exposed to a PBS solution as a control (O). MCE conditions were as in Figure 4. Each point represents an average of experiments repeated three times. The error bar superimposed on each marker means SD.

6) as determined by MCE-LIF. The histomorphologic evaluation by Giemsa staining showed cell shrinkage and deepened cytoplasmic stain as the early sign of apoptosis (Figure S-5 of the Supporting Information), while gel electrophoresis showed no DNA fragmentation at the same time (Figure S-6 of the Supporting Information). The processes, including GSH depletion, H2O2 enhancement, cell shrinkage, cell number decrease, and DNA fragmentation, continued progressively. At 18 and 24 h intervals, the appearance of characteristic DNA ladder revealed that the degree of apoptosis were significant, where GSH shows a ∼38% depletion and H2O2 features a 4-fold increase. Taken together, both types of apoptosis are associated with increased mitochondrial GSH depletion and H2O2 generation. In total, a reduction of almost 40% of GSH and an 4-5-fold enhancement of H2O2 were observed. Interestingly, time-course measurements indicated that mitochondrial GSH depletion is an early hallmark in the progression of cell death in response to a variety of apoptotic stimuli. The findings suggest the important role of mitochondrial GSH in the regulation of apoptosis,41 and H2O2 generation might well be just an epiphenomena associated with the depletion of GSH. CONCLUSION There is a real interest in the simultaneous monitoring of antioxidant and oxidant species when the intracellular redox homeostasis is investigated. In this work, mitochondrial GSH and H2O2 were simultaneously determined for the first time using MCE-LIF. The results demonstrated the feasibility of sampling, (41) Franco, R.; Cidlowsk, J. A. Cell Death Differ. 2009, 16, 1303–1314.

2012


simultaneous detection, and quantification of GSH and H2O2 in subcellular oragnelles, which could be a useful resource for analyzing their concentration and distribution in other biological systems. The procedure is promising for characterizing the molecular mechanism and exploring the possible role of GSH and H2O2 involved in apoptosis. Searching for more selective, more sensitive, and site-specific fluorescent probes, to realize high-throughput and simultaneous determination of antioxidant and oxidant species in single cells with the MCE-LIF method is now underway. ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (973 Program, 2007CB936000), the National Natural Science Funds for Distinguished Young Scholar, (No. 20725518), the Major Program of National Natural Science Foundation of China, (No. 90713019), the National Natural Science Foundation of China (No. 20875058), and Science and Technology Development Programs of Shandong Province of China (No. 2008GG30003012), and Natural Science Foundation of Shandong Province in China (No. Y2008B15). SUPPORTING INFORMATION AVAILABLE Agarose gel electrophoresis, morphological changes, mitochondrial membrane potential, and Figures S-1-S-6. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 2, 2009. Accepted January 22, 2010. AC902741R

Potent Method for the Simultaneous Determination of Glutathione and

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