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Improved analysis of hydroethidine and 2-hydroxyethidium by HPLC and electrochemical detection
Free Radical Biology & Medicine 43 (2007) 1095 – 1096 www.elsevier.com/locate/freeradbiomed
Letter to the Editor
Improved analysis of hydroethidine and 2-hydroxyethidium by HPLC and electrochemical detection
U
Superoxide anion radical (O2 − ) is implicated in many physiological and pathological processes [1], yet its quantification inside cells remains a challenge [2]. Hydroethidine (HE) is U potentially useful for detection of cellular O2 − [3], because the probe is cell-permeable and the major product of its reaction U with O2 − has been identified as 2-hydroxyethidium (2-OH-E+) [4]. Here we report a simple and robust method for the simultaneous determination of HE and 2-OH-E+. As reported by Zielonka et al. [5], we employed an ether-linked phenol column (250 × 4.6 mm, 4 μm, Synergy Polar-RP; Phenomenex). However, we replaced the gradient [5] with an isocratic system composed of acetonitrile (35%) and water (65%) containing 50 mM phosphate, pH 2.6, and eluted at 1 ml/min. HE and 2OH-E + were detected using an electrochemical detector (BioAnalytical Systems, USA) equipped with a dual glassy carbon working electrode positioned in series, to which we applied potentials of 600 and 750 mV, respectively, relative to a Ag/AgCl reference electrode. Fig. 1 shows a typical chromatogram of an equimolar mixture of HE and 2-OH-E+ and their respective hydrodynamic voltammograms. Both compounds were baseline separated and eluted within 12 min, allowing for faster quantification compared to gradient elution [6]. We applied 600 and 750 mV to the working electrodes to verify the identity of 2U OH-E+ (eluting at ∼ 11 min), as the O2 −-derived product is oxidized at the higher but not the lower potential. The detection limits for HE and 2-OH-E+ were comparable to those reported by Zielonka et al. [6] and nearly an order of magnitude greater than that achieved with HPLC-fluorescence (Table 1). Intraand interassay coefficients of variation for HE and 2-OH-E+ were excellent with the present method (Table 1). To verify the suitability of our method for detection of U cellular O2 −, Chinese hamster ovary cells were exposed to increasing concentrations of menadione (5–40 μM). As U expected, this increased cellular O2 −, as indicated by the menadione concentration-dependent increase in 2-OH-E+ (Fig. 2). Typical chromatograms of cellular extracts are shown in the Fig. 2 inset. Importantly, menadione also increased the ratio of
0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.06.023
U
2-OH-E+/HE (Fig. 2), a measure of O2 − that accounts for any potential difference in the amount of HE that entered cells. We suggest the HPLC–EC method described herein and the use of 2-OH-E+/HE ratio as a simple and robust tool to U accurately and rapidly determine cellular O2 − . References [1] Halliwell, B.; Gutteridge, J. M. C. Free radicals in biology and medicine. Oxford Univ. Press, Oxford; 1999.
Fig. 1. HPLC–EC analyses of HE and 2-OH-E+. (A) Elution profile of a mixture of HE and 2-OH-E+ (5 pmol each) using the Synergy Polar-RP column (Phenomenex). (B) Hydrodynamic voltammograms of HE and 2-OH-E+ constructed from repeated injections of 5 pmol of HE (▲) and 2-OH-E+ (●) with decreasing detector potentials.
1096
Letter to the Editor
Table 1 Limits of detection (LoD) and reproducibility of HE and 2-OH-E+ for HPLC–EC and compared to HPLC–fluorescence detection (HPLC–FL) Analyte
HE 2-OH-E+
LoD (ng on column)
Reproducibility
HPLC–FL [5]
HPLC–EC [6]
HPLC–EC (this method)
Intra-assay (%) HPLC–EC (this method)
Interassay (%) HPLC–EC (this method)
2 0.2
0.01 0.004
0.009 0.005
5.1 4.3
6.6 3.9
[2] Tarpey, M. M.; Fridovich, I. Methods of detection of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite. Circ. Res. 89:224–236; 2001.
Fig. 2. HPLC–EC analysis of HE and 2-OH-E+ in Chinese hamster ovary cells treated with menadione. Cells incubated for 1 h at 37 °C in the presence of the indicated concentrations of menadione were washed twice with 0.9% saline and then incubated with 10 μM HE. After 30 min, cells were washed twice with 0.9% saline and lysed in 300 μl of phosphate-buffered saline, pH 7.4, containing 0.1% Triton X-100; the lysate was mixed vigorously with 500 μl of 1-butanol for 1 min and then centrifuged (10 min at 12,000 g). Butanol was then removed, the samples were dried under vacuum, and the residue was dissolved in 100 μl of 1 mM HCl and then 10 μl of the solution subjected to HPLC. 2-OH-E+/HE ratio (black columns), 2-OH-E+ (gray columns). (Inset) HPLC–EC chromatograms of HE and 2-OH-E+ in untreated cells (top) and cells treated with 40 μM menadione (bottom). Data represent means ± SEM of three independent experiments. p = 0.05, using Mann–Whitney rank sum test.
[3] Rothe, G.; Valet, G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′-dichlorofluorescin. J. Leukocyte Biol. 47:440–448; 1990. [4] Zhao, H.; Joseph, J.; Fales, H. M.; Sokoloski, E. A.; Levine, R. L.; Vasquez-Vivar, J.; Kalyanaraman, B. Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proc. Natl. Acad. Sci. USA 102:5727–5732; 2005. [5] Zielonka, J.; Sarna, T.; Roberts, J. E.; Wishart, J. F.; Kalyanaraman, B. Pulse radiolysis and steady-state analyses of the reaction between hydroethidine and superoxide and other oxidants. Arch. Biochem. Biophys. 456:39–47; 2006. [6] Zielonka, J.; Vasquez-Vivar, J.; Kalyanaraman, B. The confounding effects of light, sonication, and Mn(III)TBAP on quantitation of superoxide using hydroethidine. Free Radic. Biol. Med. 41:1050–1057; 2006.
Ghassan J. Maghzal* Roland Stocker Centre for Vascular Research, Discipline of Pathology, Faculty of Medicine, University of Sydney, Medical Foundation Building, 92-94 Parramatta Road, Camperdown, NSW 2006, Australia E-mail address: [email protected]. ⁎Corresponding author. Fax: +61 2 9036 3038. 6 June 2007
Letter to the Editor
Improved analysis of hydroethidine and 2-hydroxyethidium by HPLC and electrochemical detection
U
Superoxide anion radical (O2 − ) is implicated in many physiological and pathological processes [1], yet its quantification inside cells remains a challenge [2]. Hydroethidine (HE) is U potentially useful for detection of cellular O2 − [3], because the probe is cell-permeable and the major product of its reaction U with O2 − has been identified as 2-hydroxyethidium (2-OH-E+) [4]. Here we report a simple and robust method for the simultaneous determination of HE and 2-OH-E+. As reported by Zielonka et al. [5], we employed an ether-linked phenol column (250 × 4.6 mm, 4 μm, Synergy Polar-RP; Phenomenex). However, we replaced the gradient [5] with an isocratic system composed of acetonitrile (35%) and water (65%) containing 50 mM phosphate, pH 2.6, and eluted at 1 ml/min. HE and 2OH-E + were detected using an electrochemical detector (BioAnalytical Systems, USA) equipped with a dual glassy carbon working electrode positioned in series, to which we applied potentials of 600 and 750 mV, respectively, relative to a Ag/AgCl reference electrode. Fig. 1 shows a typical chromatogram of an equimolar mixture of HE and 2-OH-E+ and their respective hydrodynamic voltammograms. Both compounds were baseline separated and eluted within 12 min, allowing for faster quantification compared to gradient elution [6]. We applied 600 and 750 mV to the working electrodes to verify the identity of 2U OH-E+ (eluting at ∼ 11 min), as the O2 −-derived product is oxidized at the higher but not the lower potential. The detection limits for HE and 2-OH-E+ were comparable to those reported by Zielonka et al. [6] and nearly an order of magnitude greater than that achieved with HPLC-fluorescence (Table 1). Intraand interassay coefficients of variation for HE and 2-OH-E+ were excellent with the present method (Table 1). To verify the suitability of our method for detection of U cellular O2 −, Chinese hamster ovary cells were exposed to increasing concentrations of menadione (5–40 μM). As U expected, this increased cellular O2 −, as indicated by the menadione concentration-dependent increase in 2-OH-E+ (Fig. 2). Typical chromatograms of cellular extracts are shown in the Fig. 2 inset. Importantly, menadione also increased the ratio of
0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.06.023
U
2-OH-E+/HE (Fig. 2), a measure of O2 − that accounts for any potential difference in the amount of HE that entered cells. We suggest the HPLC–EC method described herein and the use of 2-OH-E+/HE ratio as a simple and robust tool to U accurately and rapidly determine cellular O2 − . References [1] Halliwell, B.; Gutteridge, J. M. C. Free radicals in biology and medicine. Oxford Univ. Press, Oxford; 1999.
Fig. 1. HPLC–EC analyses of HE and 2-OH-E+. (A) Elution profile of a mixture of HE and 2-OH-E+ (5 pmol each) using the Synergy Polar-RP column (Phenomenex). (B) Hydrodynamic voltammograms of HE and 2-OH-E+ constructed from repeated injections of 5 pmol of HE (▲) and 2-OH-E+ (●) with decreasing detector potentials.
1096
Letter to the Editor
Table 1 Limits of detection (LoD) and reproducibility of HE and 2-OH-E+ for HPLC–EC and compared to HPLC–fluorescence detection (HPLC–FL) Analyte
HE 2-OH-E+
LoD (ng on column)
Reproducibility
HPLC–FL [5]
HPLC–EC [6]
HPLC–EC (this method)
Intra-assay (%) HPLC–EC (this method)
Interassay (%) HPLC–EC (this method)
2 0.2
0.01 0.004
0.009 0.005
5.1 4.3
6.6 3.9
[2] Tarpey, M. M.; Fridovich, I. Methods of detection of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite. Circ. Res. 89:224–236; 2001.
Fig. 2. HPLC–EC analysis of HE and 2-OH-E+ in Chinese hamster ovary cells treated with menadione. Cells incubated for 1 h at 37 °C in the presence of the indicated concentrations of menadione were washed twice with 0.9% saline and then incubated with 10 μM HE. After 30 min, cells were washed twice with 0.9% saline and lysed in 300 μl of phosphate-buffered saline, pH 7.4, containing 0.1% Triton X-100; the lysate was mixed vigorously with 500 μl of 1-butanol for 1 min and then centrifuged (10 min at 12,000 g). Butanol was then removed, the samples were dried under vacuum, and the residue was dissolved in 100 μl of 1 mM HCl and then 10 μl of the solution subjected to HPLC. 2-OH-E+/HE ratio (black columns), 2-OH-E+ (gray columns). (Inset) HPLC–EC chromatograms of HE and 2-OH-E+ in untreated cells (top) and cells treated with 40 μM menadione (bottom). Data represent means ± SEM of three independent experiments. p = 0.05, using Mann–Whitney rank sum test.
[3] Rothe, G.; Valet, G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′-dichlorofluorescin. J. Leukocyte Biol. 47:440–448; 1990. [4] Zhao, H.; Joseph, J.; Fales, H. M.; Sokoloski, E. A.; Levine, R. L.; Vasquez-Vivar, J.; Kalyanaraman, B. Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proc. Natl. Acad. Sci. USA 102:5727–5732; 2005. [5] Zielonka, J.; Sarna, T.; Roberts, J. E.; Wishart, J. F.; Kalyanaraman, B. Pulse radiolysis and steady-state analyses of the reaction between hydroethidine and superoxide and other oxidants. Arch. Biochem. Biophys. 456:39–47; 2006. [6] Zielonka, J.; Vasquez-Vivar, J.; Kalyanaraman, B. The confounding effects of light, sonication, and Mn(III)TBAP on quantitation of superoxide using hydroethidine. Free Radic. Biol. Med. 41:1050–1057; 2006.
Ghassan J. Maghzal* Roland Stocker Centre for Vascular Research, Discipline of Pathology, Faculty of Medicine, University of Sydney, Medical Foundation Building, 92-94 Parramatta Road, Camperdown, NSW 2006, Australia E-mail address: [email protected]. ⁎Corresponding author. Fax: +61 2 9036 3038. 6 June 2007