Anal. Chem. 2007, 79, 4588-4594
Simultaneously Monitoring the Superoxide in the Mitochondrial Matrix and Extramitochondrial Space by Micellar Electrokinetic Chromatography with Laser-Induced Fluorescence Danni L. Meany,† LaDora Thompson,‡ and Edgar A. Arriaga*,†
Department of Chemistry and Department of Physical Medicine and Rehabilitation, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455
Respiring mitochondria produce superoxide and release it into both sides of the mitochondrial inner membrane: the mitochondrial matrix and the extramitochondrial space. These two pools of superoxide are expected to have very distinctive effects on cellular function. Currently, separate measurements are required to measure superoxide in both pools, which complicates the comparison of superoxide’s effects and roles in physiology and pathology. In this study, we describe a highly sensitive strategy to monitor simultaneously these two pools of superoxide in respiring isolated rat skeletal muscle mitochondria using hydroethidine. The oxidation of hydroethidine by superoxide forms the membrane-impermeable 2-hydroxyethidium in these two superoxide pools that can be separated by differential centrifugation. Two technical limitations in using 2-hydroxyethidium as a superoxide reporter are (i) the uncontrolled fluorescence enhancement due to intercalation of 2-hydroxyethidium with variable amounts of mitochondrial DNA and (ii) the spectral interference of ethidium fluorescence. These complications were eliminated by digestion of mitochondrial DNA with DNase and by separation of ethidium and 2-hydroxyethidium using cationic micellar electrokinetic capillary chromatography with laser-induced fluorescence, respectively. Using this method, which has subattomole limits of detection, we compared the levels of 2-hydroxyethidium in normally respiring and antimycin A-treated mitochondria and demonstrated that the strategy can be extended to observe how menadione induces superoxide generation in mitochondria. Some biological processes occur asymmetrically at both sides of biomembranes. An example of such a process is the release of different amounts of superoxide by complexes I and III of the electron transport chain (ETC) to both sides of the mitochondrial inner membrane.1,2 Because the mitochondrial inner membrane * Corresponding author. Tel: 612-624-8024. Fax: 612-626-7541. E-mail:
[email protected]. † Department of Chemistry. ‡ Department of Physical Medicine and Rehabilitation. (1) St-Pierre, J.; Buckingham, J. A.; Roebuck, S. J.; Brand, M. D. J. Biol. Chem. 2002, 277, 44784-44790.
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is not permeable to superoxide, this membrane defines two separate pools of superoxide. The first pool is found at the mitochondrial matrix where most of the superoxide is quickly dismutated by manganese superoxide dismutase (Mn-SOD).2 The second pool (i.e., extramitochondrial pool) starts at the inter membrane space and extends beyond the outer membrane because superoxide in the intermembrane space can cross the outer membrane through voltage-dependent anionic channels (VDAC).3 Methods for the simultaneous determination of these two pools of superoxide would be beneficial to better understand the biogenesis, roles, and toxicities associated with the production of superoxide under normal and pathological conditions. Current methods for detecting superoxide are based on its reaction with electron spin traps,4 inactivation of aconitase activity,2 or its redox reactions with probes such as lucigenin,5 cytochrome c,6 and hydroethidine (HE),7-9 The ideal probe that simultaneously detects superoxide in the mitochondrial matrix and extramitochondrial space should do the following: (i) permeate biological membranes so it reaches both superoxide pools, (ii) generate a reporter that is specific to superoxide or is easily distinguishable from interferences, and (iii) report a measurable property that is not affected by the subcellular environment. The first criterion excludes assays based on lucigenin, cytochrome c, electron spin traps, or aconitase; only HE fulfills this criterion. HE has been used on multiple occasions for measuring the superoxide production.7,8,10,11 Unfortunately, bulk spectrofluorometric assays based on HE do not fulfill the second requirement, because HE forms two oxidation products, 2-hydroxyethidium (2(2) Muller, F. L.; Liu, Y.; Van Remmen, H. J. Biol. Chem. 2004, 279, 4906449073. (3) Han, D.; Antunes, F.; Canali, R.; Rettori, D.; Cadenas, E. J. Biol. Chem. 2003, 278, 5557-5563. (4) Du, G.; Mouithys-Mickalad, A.; Sluse, F. E. Free Radic. Biol. Med. 1998, 25, 1066-1074. (5) Kruglov, A. G.; Yurkov, I. S.; Teplova, V. V.; Evtodienko, Y. V. Biochemistry (Mosc) 2002, 67, 1262-1270. (6) Barbacanne, M. A.; Souchard, J. P.; Darblade, B.; Iliou, J. P.; Nepveu, F.; Pipy, B.; Bayard, F.; Arnal, J. F. Free Radic. Biol. Med. 2000, 29, 388-396. (7) Budd, S. L.; Castilho, R. F.; Nicholls, D. G. FEBS Lett. 1997, 415, 21-24. (8) Bucana, C.; Saiki, I.; Nayar, R. J. Histochem. Cytochem. 1986, 34, 11091115. (9) Zhao, H.; Kalivendi, S.; Zhang, H.; Joseph, J.; Nithipatikom, K.; VasquezVivar, J.; Kalyanaraman, B. Free Radic. Biol. Med. 2003, 34, 1359-1368. (10) Bindokas, V. P.; Jordan, J.; Lee, C. C.; Miller, R. J. J. Neurosci. 1996, 16, 1324-1336. 10.1021/ac062252+ CCC: $37.00
© 2007 American Chemical Society Published on Web 05/11/2007
OH-E+) and ethidium (E+), with similar fluorescence properties (2-OH-E+, Exmax 494 nm and Emmax 567 nm; E+, Exmax 490 nm and Emmax 590 nm); only 2-OH-E+ is specific to the oxidation of HE by superoxide. Additionally, HE does not meet the third requirement in mitochondrial samples because 2-OH-E+ experiences a 10-fold fluorescence enhancement upon intercalation with DNA.11 Usually, the fluorescence increase is beneficial when high sensitivity is needed; however, it is detrimental to the quantification of 2-OH-E+, as the mitochondrial DNA (mtDNA) copy number varies widely in mitochondria (from 1 to 15 mtDNA copies/ organelle).12 In this study, we enzymatically eliminated mtDNA and then used micellar electrokinetic capillary chromatography (MEKC) with cetyltrimethylammonium bromide (CTAB) to separate 2-OHE+ and E+. This separation technique has been widely used for compounds such as anti-inflammatory drugs,13 but no reports have yet appeared concerning compounds that are superoxide reporters.14 Because of the fluorescent nature of both 2-OH-E+ and E+, we employed a laser-induced fluorescence (LIF) detection scheme, which previously has been used to detect zeptomole levels of fluorescent analytes.15 Using the MEKC-LIF separation method, we determined the amounts of 2-OH-E+ found in both the matrix and the extramitochondrial space of the rat skeletal muscle mitochondria, which were used to compare the residual superoxide in the corresponding pools. Under normal respiration, the residual superoxide in the extramitochondrial space was ∼5 times greater than that in the matrix; under the antimycin A treatment, the former was tripled while the latter remained unchanged, resulting in a greater difference between them (∼14 times). Menadione is an antitumor prodrug that induces apoptosis.16 The mechanism of the menadione-induced apoptosis has been potentially linked to its capability to induce superoxide generation in mitochondria.17-19 Applying this method to study the menadione-induced superoxide generation in mitochondria, we found a ∼40% increased level of residual superoxide in the extramitochondrial space of the menadione-treated mitochondria than that of the control. In the future, this procedure may be useful for investigating the role of superoxide radicals in oxidative stress and aging. EXPERIMENTAL SECTION Chemicals. All the reagents were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise noted. HE was from Invitrogen (11) Zhao, H.; Joseph, J.; Fales, H. M.; Sokoloski, E. A.; Levine, R. L.; VasquezVivar, J.; Kalyanaraman, B. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 57275732. (12) Satoh, M.; Kuroiwa, T. Exp. Cell Res. 1991, 196, 137-140. (13) Martinez-Algaba, C.; Escuder-Gilabert, L.; Sagrado, S.; Villanueva-Camanas, R. M.; Medina-Hernandez, M. J. J. Pharm. Biomed. Anal. 2004, 36, 393399. (14) Furtos-Matei, A.; Day, R.; St-Pierre, S. A.; St-Pierre, L. G.; Waldron, K. C. Electrophoresis 2000, 21, 715-723. (15) Anderson, A. B.; Ciriacks, C. M.; Fuller, K. M.; Arriaga, E. A. Anal. Chem. 2003, 75, 8-15. (16) Rooseboom, M.; Commandeur, J. N.; Vermeulen, N. P. Pharmacol. Rev. 2004, 56, 53-102. (17) Dussmann, H.; Kogel, D.; Rehm, M.; Prehn, J. H. J. Biol. Chem. 2003, 278, 12645-12649. (18) Criddle, D. N.; Gillies, S.; Baumgartner-Wilson, H. K.; Jaffar, M.; Chinje, E. C.; Passmore, S.; Chvanov, M.; Barrow, S.; Gerasimenko, O. V.; Tepikin, A. V.; Sutton, R.; Petersen, O. H. J. Biol. Chem. 2006, 281, 40485-40492. (19) Gerasimenko, J. V.; Gerasimenko, O. V.; Palejwala, A.; Tepikin, A. V.; Petersen, O. H.; Watson, A. J. J. Cell Sci. 2002, 115, 485-497.
(Eugene, OR). Synthesis of 2-OH-E+ followed the procedure from Zielonka et al.20 and 2-OH-E+ was purified using reversed-phase HPLC as described by Zhao et al.11 The 4-15% SDS-PAGE gel and silver stain kit were purchased from BioRad (Hercules, CA). The DNA ladder (λ DNA/EcoRI + HindIII markers) was from Promega (Fitchburg, WI). The mitochondrial isolation buffer was made of 100 mM sucrose, 100 mM KCl, 50 mM Tris-HCl, 1 mM KH2PO4, 100 µM EGTA, and 0.2% (w/v) BSA (pH 7.4). The respiration buffer contained 125 mM KCl, 10 mM HEPES, 5 mM MgCl2, 2 mM K2HPO4, and 9 mM succinate (pH 7.4). TM buffer was made of 0.5% w/v Triton X-100, 40 mM Tris HCl, and 6 mM MgCl2 (pH 9.4). MECK running buffer consisted of 10 mM borate and 1 mM CTAB (pH 9.4). XO buffer for xanthine and xanthine oxidase reaction contained 0.1 mM EDTA and 50 mM potassium phosphate buffer (pH 7.4). All buffers were filtered through a 0.22µm filter before use and stored for up to 1 month at 4 °C. Isolation of Mitochondria from Rat Skeletal Muscle. Mitochondria were isolated from skeletal muscle tissue obtained from the hind limbs of Fischer 344 young male rats as described by Madsen et al.21 The quality of the mitochondrial preparation was assessed by determining the respiratory control ratio (RCR) calculated using the rate of oxygen consumption of state 3/state 2 with a FOXY-R Oxygen Sensor (Ocean Optics Inc. Dunedin, FL). State 2 respiration was determined using the respiration buffer. State 3 respiration was determined after addition of ADP to a final concentration of 1 mM.2 The RCR value for mitochondria (1 mg of protein/mL in respiration medium) respiring on succinate, a substrate for complex II, was ∼5, which indicated that the isolated mitochondria were actively respiring. Analysis of Oxidation Products Produced by Respiring Mitochondria. Mitochondria in respiration buffer (200 µL, 0.5 mg/mL protein) were incubated at 37 °C for 40 min in the presence of 50 µM HE and 9 mM succinate. Some control samples also had 300 U/mL SOD, 10 µM antimycin A, or 10 µM rotenone. After incubation, the mitochondria were centrifuged at 10000g for 5 min. The supernatant was analyzed by MEKC-LIF to determine the amount of 2-OH-E+ in the extramitochondrial space. The mitochondrial pellet was washed twice to eliminate crosscontamination from residual supernatant. To release 2-OH-E+ and E+ formed in the matrix and remove mtDNA, the mitochondrial pellet was dissolved in 200 µL of TM buffer, followed by treatment with 2 mg/mL proteinase K (45 min at 37 °C)m and subsequently with 400 U/mL DNase 1 (20 min at 37 °C). An aliquot of the dissolved mitochondria without the proteinase K treatment was used as a positive control of proteinase K activity (Supporting Information Figure S1). The digestion of mtDNA-binding proteins facilitates the digestion of mtDNA with DNase 1 (Supporting Information Figure S2), thereby eliminating variations in fluorescence response due to uncontrollable intercalation of 2-OH-E+ with mtDNA (Supporting Information Figure S3). Samples prepared from both the supernatant and mitochondrial pellet were diluted 50 times in MEKC running buffer and analyzed by MEKC-LIF. Three independent measurements were made for each sample. Analysis of Oxidation Products Formed upon Menadione Treatment. Respiring isolated mitochondria (diluted to 0.25 mg (20) Zielonka, J.; Zhao, H.; Xu, Y.; Kalyanaraman, B. Free Radic Biol Med 2005, 39, 853-863. (21) Madsen, K.; Ertbjerg, P.; Djurhuus, M. S.; Pedersen, P. K. Am. J. Physiol. 1996, 271, E1044-1050.
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of protein/mL in respiration buffer with additional 5 mM glutamate and 5 mM malate) was incubated with 50 µM menadione22 and 50 µM HE at 37 °C for 30 min. A control was prepared in the same way except for the inclusion of menadione. The use of 50 µM menadione did not affect the mitochondrial respiration (data not shown). After incubation, we initially used the same procedure described above for the analysis of oxidation products of respiring mitochondria. After an observation that there was no significant difference between the levels of 2-OH-E+ in the mitochondrial matrix of the menadione-treated sample and control, we decided to use the procedures without the centrifugation step for analyzing the total 2-OH-E+ formed upon treatment with menadione. CTAB-MEKC-LIF. Separations were carried out using a custom-built capillary electrophoresis instrument similar to the setup described by Poe et al.23 with a 150-µm-o.d., 50-µm-i.d. fusedsilica capillary (Polymicro Technologies Inc., Phoenix, AZ) at -400 V/cm in borate-CTAB buffer. Samples were injected hydrodynamically for 2 s at 3.6 kPa, which introduced 3.1 nL into the capillary. Due to adsorption of CTAB onto the capillary surface, the direction of electroosmotic flow was reversed, which required negative polarity applied to the inlet of the capillary.24 The fluorescence emissions of 2-OH-E+ and E+ were spectrally selected with an interference filter (635DF55, Omega Optical). Data Analysis. The data were collected at 10 Hz using a NiDaq I/O board (PCI-MIO-16XE-50, National Instruments, Austin, TX), saved as binary files, and analyzed using Igor Pro software (Wavemetrics, Lake Oswego, OR). In addition, in order to estimate the error associated with the MEKC-LIF procedure, the same isolate containing the oxidation products was analyzed in triplicate. The average relative standard deviation (RSD) of the 2-OH-E+ peak area in the MEKC-LIF analysis was 3.1% (n ) 3). In a separate experiment, we estimated the error associated with the procedure used to isolate oxidation products by analyzing the same sample three times using MEKC-LIF. The total error (i.e., isolation procedure plus MEKC-LIF), represented as the RSD of the 2-OHE+ peak area (δT), was attributed to the error in the MEKC-LIF analysis (δm) and the error in the isolation procedure (δsp). From the equation δT2 ) δm2 + δsp2, the δsp values in the measurement of 2-OH-E+ in the matrix and extramitochondrial space of samples treated with antimycin are 5.7 and 9.0%, respectively. The significance (P < 0.05) was determined by a Student’s unpaired t-test. RESULTS AND DISCUSSION Separation of 2-OH-E+ and E+ by CTAB-MEKC. In order to separate 2-OH-E+ from the interfering E+, we developed a CTAB-MEKC-based separation system. The presence of CTAB above its critical micelle concentration (i.e., 1 mM at 25 °C)25 in the MEKC running buffer causes micelle formation, which serves as the pseudostationary separation phase.26 Figure 1 shows complete resolution of 2-OH-E+and E+ at the migration times of 102.4 ( 0.1 (n ) 3) and 124.1 ( 0.1 s (n ) 3), respectively. Although the only structural difference between 2-OH-E+ and E+ (22) Thor, H.; Smith, M. T.; Hartzell, P.; Bellomo, G.; Jewell, S. A.; Orrenius, S. J. Biol. Chem. 1982, 257, 12419-12425. (23) Poe, B. G.; Navratil, M.; Arriaga, E. A. J. Chromatogr. A 2006. (24) Wang, C.; Lucy, C. A. Electrophoresis 2004, 25, 825-832. (25) Beckers, J. L.; Bocek, P. Electrophoresis 2002, 23, 1947-1952. (26) Ackermans, M. T.; Everaerts, F. M.; Beckers, J. L. J. Chromatogr. 1992, 606, 229-235.
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Figure 1. CTAB-MEKC separation of 2-OH-E+ (5 nM) and E+ (50 nM) standards. The net electrophoretic mobilities of 2-OH-E+ and E+ are -(9.76 ( 0.01) × 10-4 and -(8.06 ( 0.01) × 10 -4 cm2 V-1 s-1 (average ( SD; n ) 3), respectively. Each sample (3.1 nL) was hydrodynamically injected into a 40-cm-long capillary and then the separation was carried out at -400 V/cm in borate-CTAB buffer. The fluorescence emission of 2-OH-E+ and E+ was detected in the range of 607-663 nm.
is a hydroxyl group on the aromatic ring, it is sufficient to reduce the partition of 2-OH-E+ into the CTAB cationic micelles compared to E+. Separation is completed in less than 3 min, which is ∼10 times faster than the reported HPLC method.11 The calibration curve for E+ and 2-OH-E+ is shown in eqs 1 and 2, respectively, where y is the amount of E+ or 2-OH-E+ in attomoles and x is the peak area.
y ) (135 ( 2)x + (4 ( 12); y ) (44 ( 1)x + (0 ( 1);
(R2 ) 0.999) (R2 ) 0.999)
(1) (2)
The concentration and mass limits of detection (LOD), calculated based on signals equal to 3 times the standard deviation of the background, are 0.23 ( 0.01 nM and 0.45 ( 0.02 amol (n ) 3) for E+ and 0.08 ( 0.01 nM and 0.15 ( 0.01 amol (n ) 3) for 2-OHE+, which are the lowest LOD values reported to date for these compounds. In order to confirm that 2-OH-E+ is the only product of HE oxidation by superoxide, superoxide was produced with the xanthine/xanthine oxidase reaction, which is a standard procedure for generating superoxide.27 The products were then analyzed by MEKC-LIF (Figure 2). The 2-OH-E+ signal increased gradually with incubation time. Conversely, the E+ signal remained unchanged (Figure 2A). Superoxide dismutase, an enzyme that dismutates superoxide to hydrogen peroxide,28 was used to confirm that the increase of 2-OH-E+ peak is not due to hydrogen peroxide, a byproduct of the xanthine/xanthine oxidase reaction (Figure 2B). 2-OH-E+ in the Extramitochondrial Space. Superoxide found in the extramitochondrial space includes the superoxide both in the intermembrane space and outside the mitochondria. (27) Benov, L.; Sztejnberg, L.; Fridovich, I. Free Radic. Biol. Med. 1998, 25, 826-831. (28) Beckman, K. B.; Ames, B. N. Physiol. Rev. 1998, 78, 547-581.
Table 1. Properties of 2-OH-E+ and E+ Detected in the Extramitochondrial Spacea
HE + succinate Mito + HE + succinate Mito + HE + succinate + SOD Mito + HE + succinate + rotenone + antimycin A a
2-hydroxyethidium
ethidium
migration time (s)
electrophoretic mobility (-10-4 cm2 V-1 s-1)
peak area (AU × s)
migration time (s)
electrophoretic mobility (-10-4 cm2 V-1 s-1)
peak area (AU × s)
100.6 ( 0.2 98.7 ( 0.2 98.5 ( 0.1 99.3 ( 0.2
9.94 ( 0.02 10.13 ( 0.02 10.15 ( 0.01 10.07 ( 0.02
0.51 ( 0.04 1.04 ( 0.03 0.05 ( 0.01 2.1 ( 0.2
123.9 ( 0.3 120.9 ( 0.2 120.4 ( 0.1 121.8 ( 0.2
8.07 ( 0.02 8.27 ( 0.01 8.30 ( 0.01 8.21 ( 0.01
0.44 ( 0.02 0.80 ( 0.07 0.58 ( 0.04 0.76 ( 0.05
Three independent measurements were made for each sample and the results were reported as mean ( SD (n ) 3).
Figure 3. Electropherograms of the HE oxidation products formed in the extramitochondrial space. Traces B-D are consecutively offset by 1.2 AU on the y-axis. Separation and detection conditions were similar to those described in Figure 1. Succinate provides electrons to ETC from complex II, which flow to both complexes I and III. To make the electrons flow exclusively to complex III, rotenone, a complex I inhibitor, was used to stop the electron flow through complex I (aka, reverse electron transfer).2 The total error in 2-OHE+ area of the treatments associated with the traces A, B, and D is 10.3% RSD. Mito, mitochondria; SOD, superoxide dismutase. Figure 2. Electropherograms of the time-dependent oxidation of HE to 2-OH-E+ by the xanthine/xanthine oxidase reaction. (A) The reaction mixture contained 50 µM HE, 375 µM xanthine, and 5 µg/ mL xanthine oxidase in XO buffer. (B) The reaction mixture was the same as in (A) plus 300 U/mL SOD. The time above each trace represents the incubation time of HE with xanthine/xanthine oxidase. The mixtures were diluted 50 times in MEKC running buffer before MEKC-LIF analysis. The separation and detection conditions were similar to those described in Figure 1.
The latter results from diffusion of the superoxide in the intermembrane space across the outer membrane through VDAC.3 The extramitochondrial superoxide reacts with HE, resulting in the formation of 2-OH-E+. The 2-OH-E+ formed in the intermembrane space leaks through the outer membrane (i.e., this membrane is permeable for MW
