Short communication Received: 19 August 2013,
Revised: 11 October 2013,
Accepted: 25 November 2013
Published online in Wiley Online Library: 8 January 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2628
In vitro screening of Fe2+-chelating effect by a Fenton’s reaction–luminol chemiluminescence system Mitsuhiro Wada,a* Hiroaki Komatsu,a Rie Ikeda,a Talal A. Aburjai,b Suleiman M. Alkhalil,b Naotaka Kurodaa and Kenichiro Nakashimac ABSTRACT: In vitro screening of a Fe2+-chelating effect using a Fenton’s reaction–luminol chemiluminescence (CL) system is described. The luminescence between the reactive oxygen species generated by the Fenton’s reaction and luminol was decreased on capturing Fe2+ using a chelator. The proposed method can prevent the consumption of expensive seed compounds (drug discovery candidates) owing to the high sensitivity of CL detection. Therefore, the assay could be performed using small volumes of sample solution (150 μL) at micromolar concentrations. After optimization of the screening conditions, the efficacies of conventional chelators such as ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10-phenanthroline were examined. EC50 values for these compounds (except 1,10phenanthroline) were in the range 3.20 ± 0.87 to 9.57 ± 0.64 μM (n = 3). Rapid measurement of the Fe2+-chelating effect with an assay run time of a few minutes could be achieved using the proposed method. In addition, the specificity of the method was discussed. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Fe2+; chelating effect; Fenton’s reaction; luminol; in vitro screening
Introduction
Luminescence 2014; 29: 955–958
Experimental Chemicals EDTA, DETAPAC, deferoxamine, 1,10-phenanthroline, luminol (purity: 99.3%) and FeCl3 were purchased from Sigma Chemical Corp. (St Louis, MO, USA). Deferiprone and trolox were from Tokyo Kasei (Tokyo, Japan). H2O2 (30%), FeCl2, MgCl2, CaCl2, CuSO4, ascorbic acid (ASA), catalase (Cat, from bovine liver, 10,000 U/mg) and superoxide dismutase (SOD, from bovine erythrocyte, 90,000 U/mg), from Wako Pure Chemicals (Osaka, Japan) were used. Distilled water was passed through a Pure Line WL21P * Correspondence to: M Wada, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki, 852-8521, Japan. E-mail: [email protected] a
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyomachi, Nagasaki, 852-8521, Japan
b
Faculty of Pharmacy, University of Jordan, Amman, 11942, Jordan
c
Faculty of Pharmaceutical Science, Nagasaki International University, 2825-7 Sasebo, Nagasaki, 859-3298, Japan
Copyright © 2014 John Wiley & Sons, Ltd.
955
Thalassemia is the most common heterogeneous group of genetic disorders in which the production of normal hemoglobin is partly or completely suppressed due to the defective synthesis of one or more globin chains. Thalassemia varies widely in severity from asymptomatic forms to severe or even fatal. Patients with major thalassemia receive frequent blood transfusions that lead to iron overload, and iron-chelating treatment is necessary to prevent damage to the internal organs. Because of recent advances in iron-chelating treatments, patients can live longer if they have access to proper treatment (1,2). Iron-chelating therapy has dramatically altered the prognosis of this previously fatal disease. Successes have been achieved using conventional medication such as deferoxamine or deferiprone. However, limitations of this treatment such as safety, cost and administration route remain, and the development of a new iron chelator is desired (3–5). To evaluate the in vitro efficiency of the chelators under biological conditions, the pFe value is widely used as a standard (6). pFe is defined as a negative logarithm of the concentration of the free Fe3+ in solution, calculated as: total [ligand] = 10 μM and total [iron] = 1 μM at pH 7.4, and is measured by potentiometric and spectrophotometric titration (7). In our previous study, an assay to evaluate the hydroxyl radical quenching effect using a Fenton’s reaction–luminol chemiluminescence (CL) system was established and applied to rosemary extractions (8). The CL between reactive oxygen species (ROS) generated by the Fenton’s reaction and luminol was determined; the luminescence intensity decreased on capture of Fe2+ by the chelators, and based on this, the chelating effects could be evaluated. The CL method completes the reaction simply and rapidly without the need for complicated instruments,
and owing to its high sensitivity, prevents the consumption of expensive seed compounds (drug discovery candidates). In this report, in vitro screening of the Fe2+-chelating effect by a Fenton’s reaction–luminol CL system was studied. Ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10-phenanthroline were examined as chelators. In addition, the specificity of the proposed method was discussed.
M. Wada et al. system (Yamato Scientific Co., Tokyo, Japan). All other chemicals were of analytical reagent grade.
Procedures for the measurement of CL to evaluate the chelating effect To 150-μL of sample in 50 mM phosphate buffer (pH 7.4), was added 150 μL each of 200 μM FeCl2 and 200 μM of luminol in 50 mM phosphate buffer (pH 7.4) solution. After vortex-mixing for 5 s, 300 μL of H2O2 in 50 mM phosphate buffer (pH 7.4) was added to the mixture. The CL intensity at room temperature was measured for 60 s after addition of H2O2 solution using a Lumat LB9507 (Berthold, Bad Wildbad, Germany).
Specificity of the proposed method To distinguish between the decrease in CL by iron chelating or ROS quenching, CL profiles with or without the ROS quenchers were monitored at 0.05 s intervals for 1 s using the conditions described above. Moreover, the CL profiles obtained on addition of a chelator (EDTA and deferoxamine) and an antioxidant (ASA and Trolox) were monitored. The difference in the CL profiles on Fe2+-chelating and ROS quenching was evaluated. Furthermore, to clarify what types of ROS were generated by the Fenton’s reaction to produce CL, a ROS quencher such as catalase or superoxide dismutase (SOD) was added. Concentrations ranging from 78 to 625 U/mL for catalase and 1.75 to 14 U/mL for SOD were examined.
Results and discussion Effects of chelatable metals on CL intensity The influence of Fe3+ on CL intensity was examined by measuring the CL intensity without a chelator. CL intensities in the range 25–1600 μM Fe3+ or Fe2+ were compared. In addition, to evaluate the effects of metals other than FeCl2, the CL intensity with or without 800 μM EDTA was examined; 200 μM FeCl2, FeCl3, MgCl2 and CaCl2, and 10 μM of CuSO4 were used.
Screening of chelating effects Five chelators, EDTA, DETAPAC, deferoxamine, deferiprone and 1,10-phenanthroline, were used (Fig. 1). The chelating effect was calculated as: Chelating effect % = (CLblank–CLchelator)/CLblank × 100 where CLblank is the CL intensity without chelator and CLchelator is that obtained with chelator. The CL intensities at 16 concentrations (0.05, 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 10, 15, 20, 25, 50, 100, 200 and 800 μM) for each chelator were measured. Measurements were performed in triplicate for each concentration. The chelating effect was indicated as EC50, which was the concentration needed to quench CLblank to half.
Optimization of CL conditions To obtain intense CL and precise measurement, the effects of different concentrations of luminol and H2O2 were examined (Fig. 2). The CL intensity without chelator and its relative standard deviation (RSD) were monitored. Two hundred micromolar FeCl2 was used, which is the FeCl2 concentration used under current guidelines in chelation therapy to remove excess iron (9). A luminol concentration of 25-800 μM was used, and concentrations > 175 μM gave maximum and constant CL intensity with low RSD% (Fig. 2A). Therefore, 200 μM luminol was used in the subsequent experiments. The concentration of H2O2 contributes to ROS generation by the Fenton’s reaction. An H2O2 concentration of 30–940 mM was used. Concentrations > 300 mM H2O2 gave the maximum and constant CL intensity. Therefore, 400 mM H2O2 was selected, owing to its minimum RDS% (Fig. 2B).
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Figure 1. Chemical structures of iron chelators.
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Figure 2. Effects of luminol (A) and H2O2 concentration on blank CL intensity and its precision. Conditions: 200 μM FeCl2 in 50 mM phosphate buffer (pH 7.4); 470 mM H2O2 in 50 mM phosphate buffer (pH 7.4). Precision indicated as RSD%. The white arrow indicates the selected condition.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014; 29: 955–958
Screening of Fe2+-chelating effect by a chemiluminescence method Effects of chelatable metals on CL intensity Fenton’s reaction (10) progresses as: Fe2þ þ H2 O2 →Fe3þ þ OH þ OH Owing to the coexistence of Fe3+ in the system, the effect of Fe3+ on CL intensity was examined (Fig. 3). Instead of Fe2+, Fe3+ in the range 25–1600 μM was used and the CLblank was monitored. No enhancement of CL with Fe3+ could be observed, whereas increasing CL was found with an increase in Fe2+. This indicated that the quenching of CL was caused by the selective chelating of Fe2+. Furthermore, the effects of other chelatable metals on CL intensity were also examined (Fig. 4); 200 μM of FeCl2, FeCl3, MgCl2 and CaCl2, and 10 μM of CuSO4 were used. The CLblank and CLEDTA at 800 μM of EDTA were monitored. All metals other than Fe2+ and Cu2+ did not influence CL, although greater enhancement of CL by Cu2+ compared with Fe2+ was observed. This agrees well with the fact that Cu2+ with H2O2 promotes radical species (10). Using this CL system, the chelating effect of Cu2+ was also evaluated. In the proposed method, quenching of CL intensity was caused selectively by the chelating effect of Fe2+. However, a practical Fe3+-selective chelator for is desirable as a new chelator 6 5 4 3
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Figure 3. Effects of Fe or Fe concentration on CL intensity. Conditions: 200 μM luminol in 50 mM phosphate buffer (pH 7.4); 400 mM H2O2 in 50 mM phosphate buffer (pH 7.4). Data are shown as mean ± SD (n = 3).
for clinical application. Because most tribasic cations such as aluminum and gallium are not essential for living cells. Fe3+ might be a suitable target for a clinical chelator. Designing a Fe2+-selective chelator has some problems because the chelator ordinarily retained appreciable affinity for other biologically important bivalent metals such as copper and zinc. Therefore, the information obtained in this study was useful for iron chelator development. Screening of chelating effects The chelating effects of representative agents, EDTA, DETAPAC, 1,10-phenanthroline, deferoxamine and deferiprone, were evaluated using the proposed method. Dose–response curves for each agent were prepared, and the EC50 values were estimated. Measurements were taken in triplicate, and their resulting precisions, as RSD%, were < 15%. All agents examined (except 1,10-phenanthroline) showed a clear chelating effect. EC50 values for EDTA, DETAPAC, deferoxamine and deferiprone were 9.20 ± 2.88, 3.20 ± 0.87, 8.47 ± 1.68 and 9.57 ± 0.64 μM, respectively (Table 1). The proposed method could complete the assay using small volume of sample (150 μL), whereas the conventional method needed several milliliters of sample (11). Metal affinity constants on Fe2+ for these compounds have been reported previously [DETAPAC (16.5) > EDTA (14.3) > deferiprone (12.1) > deferoxamine (7.2)] (5). The data mostly agree with our results. However, our results might be related to pFe3+ which has been widely used as a standard to evaluate the chelating effect, because chelators sometimes show different affinity for Fe2+ and Fe3+ (3). In addition, essential properties for iron chelators are affinity to iron and the stability of the resulting chelate complex. However, the proposed chelating effect does not indicate the stability of the chelate complex in addition to conventional parameters such as the pFe value. By contrast, enhancement of the CL intensity was observed on addition of 1,10-phenanthroline (data not shown). In a previous study, 1,10-phenanthroline reacted with the O2- generated by decomposing H2O2, and excited 3,3′-diformyl-2,2’-dipyridil was generated, passing through the formation of unstable 1,2dioxetane derivatives (12). This might be the reason for the CL enhancement seen with 1,10-phenanthroline. Specificity of the proposed method
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The quenching effect of ROS by antioxidants such as ASA may decrease CL intensity as well as an iron chelator. To distinguish between a decrease in CL based on iron chelating from that due to ROS quenching, CL profiles with or without a CL quencher were monitored at 0.05 s intervals for 1 s. As shown in Fig. 5, two
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Luminescence 2014; 29: 955–958
3.20 ± 0.87 9.20 ± 2.88 8.41 ± 1.68 9.57 ± 0.64 ND
ND, not determined.
Copyright © 2014 John Wiley & Sons, Ltd.
957
Figure 4. Effects of EDTA-chelatable metals on CL intensity. Conditions: 200 μM luminol in 50 mM phosphate buffer (pH 7.4); 400 mM H2O2 in 50 mM phosphate buffer (pH 7.4); 800 μM EDTA in 50 mM phosphate buffer (pH 7.4). *10 μM of solution was used. Data are shown as mean ± SD (n = 3).
EDTA DETAPAC Deferoxamine Deferiprone 1,10-Phenanthroline
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Figure 5. CL profiles of EDTA (A), deferoxamine (B), ascorbic acid (C) and trolox (D).
In conclusion, the proposed method using the Fenton’s reaction–luminol CL system was simple and rapid for the selective evaluation of the Fe2+-chelating effect. This method might also be useful for screening of drug discovery seed compounds and to give complementary information for the development of medication.
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CL reactions by different ROS with different reactivity on luminol occurred, because the blank CL profile showed a dipolar curve. Furthermore, addition of the iron chelators (EDTA and deferoxamine) selectively quenched the latter CL, whereas ROS quenchers (ASA and trolox) quenched both CL reactions (Fig. 5). The CL profile of deferoxamine appeared to quench both the former and latter CL. This might be due to ROS quenching. Monitoring of the CL profile is useful to distinguish the decreasing in CL based on iron chelating from that due to ROS quenching. The influence of the CL from the first reaction step (appearing within 0.4 s in Fig. 5) on the chelating effect at low chelator concentrations was not so high, because the chelating effect was evaluated using the CL measured for 60 s, just after addition of H2O2 solution. To clarify which ROS contributes to each CL, selective ROS quenchers, Cat and SOD were added to the CL system. No quenching was observed on addition of Cat (data was not shown). On addition of SOD, the latter CL was quenched (Fig. 6), whereas the former was not quenched. As a result, the superoxide anion contributed to the later CL. The ·OH generated by the Fenton’s reaction reacts with H2O2 to generate the superoxide anion (10). After which, luminol reacts with the superoxide anion and emits light. Therefore, an iron chelator apparently quenched the CL due to the superoxide anion. However, which ROS contribute the former CL was not clarified.
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This work was supported in part by a Scientific Research Fund from Ministry of Higher Education and Scientific Research, Jordan.
References 1. Mamtani M, Kulkarni H. Influence of iron chelators on myocardial iron and cardiac function in transfusion-dependent thalassaemia: a systematic review and meta-analysis. Br J Haematol 2008;141:882–90. 2. Rachmilewitz EA, Weizer-Stern O, Adamsky K, Amariglio N, Rechevi G, Breda L, et al. Role of iron in inducing oxidative stress in thalassemia. Ann NY Acad Sci 2005;1054:118–23. 3. Crisponi G, Remelli M. Iron chelating agents for the treatment of iron overload. Coord Chem Rev 2008;252:1225–40. 4. Liu ZD, Hider RC. Design of iron chelators with therapeutic application. Coord Chem Rev 2002;232:151–71. 5. Liu ZD, Hider RC. Design of clinically useful iron(III)-selective chelators. Med Res Rev 2002;22:26–64. 6. Liu ZD, Piyamongkol S, Hider RC. The design and properties of 3+ 3-hydroxypyridin-4-one iron chelators with high pFe values. Transfus Sci 2000;23:269–70. 7. Steinhauser S, Heinz U, Bartholomä M, Weyhermüller T, Nick H, Hegetschweiler K. Complex formation of ICL670 and related ligands III II with Fe and Fe . Eur J Inorg Chem 2004;21:4177–92. 8. Wada M, Kido H, Ohyama K, Kishikawa N, Ohba Y, Kuroda N, et al. Evaluation of quenching effects of non-water-soluble and watersoluble rosemary extracts against active oxygen species by chemiluminescent assay. Food Chem 2004;87:261–7. 9. Gattermann N. Overview of guidelines on iron chelation therapy in patients with myelodysplastic syndromes and transfusional iron overload. Int J Hematol 2008;88:24–9. 10. Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. 4th ed. New York: Oxford University Press, 2007:30–78. 11. Zhu CF, Qiu DH, Kong XL, Hider RC, Zhou T. Synthesis and in vitro antimicrobial evaluation of a high-affinity iron chelator in combination with chloramphenicol. J Pharm Pharmacol 2012;65:512–20. 12. Watanabe K, Yamasaki T, Itagaki M. Chemiluminescence determination of iron(III) with 9,10-phenanthroline. Bunseki Kagaku 1996;45:407–13.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014; 29: 955–958
Revised: 11 October 2013,
Accepted: 25 November 2013
Published online in Wiley Online Library: 8 January 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2628
In vitro screening of Fe2+-chelating effect by a Fenton’s reaction–luminol chemiluminescence system Mitsuhiro Wada,a* Hiroaki Komatsu,a Rie Ikeda,a Talal A. Aburjai,b Suleiman M. Alkhalil,b Naotaka Kurodaa and Kenichiro Nakashimac ABSTRACT: In vitro screening of a Fe2+-chelating effect using a Fenton’s reaction–luminol chemiluminescence (CL) system is described. The luminescence between the reactive oxygen species generated by the Fenton’s reaction and luminol was decreased on capturing Fe2+ using a chelator. The proposed method can prevent the consumption of expensive seed compounds (drug discovery candidates) owing to the high sensitivity of CL detection. Therefore, the assay could be performed using small volumes of sample solution (150 μL) at micromolar concentrations. After optimization of the screening conditions, the efficacies of conventional chelators such as ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10-phenanthroline were examined. EC50 values for these compounds (except 1,10phenanthroline) were in the range 3.20 ± 0.87 to 9.57 ± 0.64 μM (n = 3). Rapid measurement of the Fe2+-chelating effect with an assay run time of a few minutes could be achieved using the proposed method. In addition, the specificity of the method was discussed. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Fe2+; chelating effect; Fenton’s reaction; luminol; in vitro screening
Introduction
Luminescence 2014; 29: 955–958
Experimental Chemicals EDTA, DETAPAC, deferoxamine, 1,10-phenanthroline, luminol (purity: 99.3%) and FeCl3 were purchased from Sigma Chemical Corp. (St Louis, MO, USA). Deferiprone and trolox were from Tokyo Kasei (Tokyo, Japan). H2O2 (30%), FeCl2, MgCl2, CaCl2, CuSO4, ascorbic acid (ASA), catalase (Cat, from bovine liver, 10,000 U/mg) and superoxide dismutase (SOD, from bovine erythrocyte, 90,000 U/mg), from Wako Pure Chemicals (Osaka, Japan) were used. Distilled water was passed through a Pure Line WL21P * Correspondence to: M Wada, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki, 852-8521, Japan. E-mail: [email protected] a
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyomachi, Nagasaki, 852-8521, Japan
b
Faculty of Pharmacy, University of Jordan, Amman, 11942, Jordan
c
Faculty of Pharmaceutical Science, Nagasaki International University, 2825-7 Sasebo, Nagasaki, 859-3298, Japan
Copyright © 2014 John Wiley & Sons, Ltd.
955
Thalassemia is the most common heterogeneous group of genetic disorders in which the production of normal hemoglobin is partly or completely suppressed due to the defective synthesis of one or more globin chains. Thalassemia varies widely in severity from asymptomatic forms to severe or even fatal. Patients with major thalassemia receive frequent blood transfusions that lead to iron overload, and iron-chelating treatment is necessary to prevent damage to the internal organs. Because of recent advances in iron-chelating treatments, patients can live longer if they have access to proper treatment (1,2). Iron-chelating therapy has dramatically altered the prognosis of this previously fatal disease. Successes have been achieved using conventional medication such as deferoxamine or deferiprone. However, limitations of this treatment such as safety, cost and administration route remain, and the development of a new iron chelator is desired (3–5). To evaluate the in vitro efficiency of the chelators under biological conditions, the pFe value is widely used as a standard (6). pFe is defined as a negative logarithm of the concentration of the free Fe3+ in solution, calculated as: total [ligand] = 10 μM and total [iron] = 1 μM at pH 7.4, and is measured by potentiometric and spectrophotometric titration (7). In our previous study, an assay to evaluate the hydroxyl radical quenching effect using a Fenton’s reaction–luminol chemiluminescence (CL) system was established and applied to rosemary extractions (8). The CL between reactive oxygen species (ROS) generated by the Fenton’s reaction and luminol was determined; the luminescence intensity decreased on capture of Fe2+ by the chelators, and based on this, the chelating effects could be evaluated. The CL method completes the reaction simply and rapidly without the need for complicated instruments,
and owing to its high sensitivity, prevents the consumption of expensive seed compounds (drug discovery candidates). In this report, in vitro screening of the Fe2+-chelating effect by a Fenton’s reaction–luminol CL system was studied. Ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10-phenanthroline were examined as chelators. In addition, the specificity of the proposed method was discussed.
M. Wada et al. system (Yamato Scientific Co., Tokyo, Japan). All other chemicals were of analytical reagent grade.
Procedures for the measurement of CL to evaluate the chelating effect To 150-μL of sample in 50 mM phosphate buffer (pH 7.4), was added 150 μL each of 200 μM FeCl2 and 200 μM of luminol in 50 mM phosphate buffer (pH 7.4) solution. After vortex-mixing for 5 s, 300 μL of H2O2 in 50 mM phosphate buffer (pH 7.4) was added to the mixture. The CL intensity at room temperature was measured for 60 s after addition of H2O2 solution using a Lumat LB9507 (Berthold, Bad Wildbad, Germany).
Specificity of the proposed method To distinguish between the decrease in CL by iron chelating or ROS quenching, CL profiles with or without the ROS quenchers were monitored at 0.05 s intervals for 1 s using the conditions described above. Moreover, the CL profiles obtained on addition of a chelator (EDTA and deferoxamine) and an antioxidant (ASA and Trolox) were monitored. The difference in the CL profiles on Fe2+-chelating and ROS quenching was evaluated. Furthermore, to clarify what types of ROS were generated by the Fenton’s reaction to produce CL, a ROS quencher such as catalase or superoxide dismutase (SOD) was added. Concentrations ranging from 78 to 625 U/mL for catalase and 1.75 to 14 U/mL for SOD were examined.
Results and discussion Effects of chelatable metals on CL intensity The influence of Fe3+ on CL intensity was examined by measuring the CL intensity without a chelator. CL intensities in the range 25–1600 μM Fe3+ or Fe2+ were compared. In addition, to evaluate the effects of metals other than FeCl2, the CL intensity with or without 800 μM EDTA was examined; 200 μM FeCl2, FeCl3, MgCl2 and CaCl2, and 10 μM of CuSO4 were used.
Screening of chelating effects Five chelators, EDTA, DETAPAC, deferoxamine, deferiprone and 1,10-phenanthroline, were used (Fig. 1). The chelating effect was calculated as: Chelating effect % = (CLblank–CLchelator)/CLblank × 100 where CLblank is the CL intensity without chelator and CLchelator is that obtained with chelator. The CL intensities at 16 concentrations (0.05, 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 10, 15, 20, 25, 50, 100, 200 and 800 μM) for each chelator were measured. Measurements were performed in triplicate for each concentration. The chelating effect was indicated as EC50, which was the concentration needed to quench CLblank to half.
Optimization of CL conditions To obtain intense CL and precise measurement, the effects of different concentrations of luminol and H2O2 were examined (Fig. 2). The CL intensity without chelator and its relative standard deviation (RSD) were monitored. Two hundred micromolar FeCl2 was used, which is the FeCl2 concentration used under current guidelines in chelation therapy to remove excess iron (9). A luminol concentration of 25-800 μM was used, and concentrations > 175 μM gave maximum and constant CL intensity with low RSD% (Fig. 2A). Therefore, 200 μM luminol was used in the subsequent experiments. The concentration of H2O2 contributes to ROS generation by the Fenton’s reaction. An H2O2 concentration of 30–940 mM was used. Concentrations > 300 mM H2O2 gave the maximum and constant CL intensity. Therefore, 400 mM H2O2 was selected, owing to its minimum RDS% (Fig. 2B).
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Figure 1. Chemical structures of iron chelators.
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Figure 2. Effects of luminol (A) and H2O2 concentration on blank CL intensity and its precision. Conditions: 200 μM FeCl2 in 50 mM phosphate buffer (pH 7.4); 470 mM H2O2 in 50 mM phosphate buffer (pH 7.4). Precision indicated as RSD%. The white arrow indicates the selected condition.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014; 29: 955–958
Screening of Fe2+-chelating effect by a chemiluminescence method Effects of chelatable metals on CL intensity Fenton’s reaction (10) progresses as: Fe2þ þ H2 O2 →Fe3þ þ OH þ OH Owing to the coexistence of Fe3+ in the system, the effect of Fe3+ on CL intensity was examined (Fig. 3). Instead of Fe2+, Fe3+ in the range 25–1600 μM was used and the CLblank was monitored. No enhancement of CL with Fe3+ could be observed, whereas increasing CL was found with an increase in Fe2+. This indicated that the quenching of CL was caused by the selective chelating of Fe2+. Furthermore, the effects of other chelatable metals on CL intensity were also examined (Fig. 4); 200 μM of FeCl2, FeCl3, MgCl2 and CaCl2, and 10 μM of CuSO4 were used. The CLblank and CLEDTA at 800 μM of EDTA were monitored. All metals other than Fe2+ and Cu2+ did not influence CL, although greater enhancement of CL by Cu2+ compared with Fe2+ was observed. This agrees well with the fact that Cu2+ with H2O2 promotes radical species (10). Using this CL system, the chelating effect of Cu2+ was also evaluated. In the proposed method, quenching of CL intensity was caused selectively by the chelating effect of Fe2+. However, a practical Fe3+-selective chelator for is desirable as a new chelator 6 5 4 3
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Figure 3. Effects of Fe or Fe concentration on CL intensity. Conditions: 200 μM luminol in 50 mM phosphate buffer (pH 7.4); 400 mM H2O2 in 50 mM phosphate buffer (pH 7.4). Data are shown as mean ± SD (n = 3).
for clinical application. Because most tribasic cations such as aluminum and gallium are not essential for living cells. Fe3+ might be a suitable target for a clinical chelator. Designing a Fe2+-selective chelator has some problems because the chelator ordinarily retained appreciable affinity for other biologically important bivalent metals such as copper and zinc. Therefore, the information obtained in this study was useful for iron chelator development. Screening of chelating effects The chelating effects of representative agents, EDTA, DETAPAC, 1,10-phenanthroline, deferoxamine and deferiprone, were evaluated using the proposed method. Dose–response curves for each agent were prepared, and the EC50 values were estimated. Measurements were taken in triplicate, and their resulting precisions, as RSD%, were < 15%. All agents examined (except 1,10-phenanthroline) showed a clear chelating effect. EC50 values for EDTA, DETAPAC, deferoxamine and deferiprone were 9.20 ± 2.88, 3.20 ± 0.87, 8.47 ± 1.68 and 9.57 ± 0.64 μM, respectively (Table 1). The proposed method could complete the assay using small volume of sample (150 μL), whereas the conventional method needed several milliliters of sample (11). Metal affinity constants on Fe2+ for these compounds have been reported previously [DETAPAC (16.5) > EDTA (14.3) > deferiprone (12.1) > deferoxamine (7.2)] (5). The data mostly agree with our results. However, our results might be related to pFe3+ which has been widely used as a standard to evaluate the chelating effect, because chelators sometimes show different affinity for Fe2+ and Fe3+ (3). In addition, essential properties for iron chelators are affinity to iron and the stability of the resulting chelate complex. However, the proposed chelating effect does not indicate the stability of the chelate complex in addition to conventional parameters such as the pFe value. By contrast, enhancement of the CL intensity was observed on addition of 1,10-phenanthroline (data not shown). In a previous study, 1,10-phenanthroline reacted with the O2- generated by decomposing H2O2, and excited 3,3′-diformyl-2,2’-dipyridil was generated, passing through the formation of unstable 1,2dioxetane derivatives (12). This might be the reason for the CL enhancement seen with 1,10-phenanthroline. Specificity of the proposed method
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The quenching effect of ROS by antioxidants such as ASA may decrease CL intensity as well as an iron chelator. To distinguish between a decrease in CL based on iron chelating from that due to ROS quenching, CL profiles with or without a CL quencher were monitored at 0.05 s intervals for 1 s. As shown in Fig. 5, two
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Table 1. EC50 values for EDTA, DETAPAC, deferoxamine, deferiprone and 1,10-phenanthroline
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EC50 Mean ± SD, n = 3 (μM)
0 FeCl2
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Luminescence 2014; 29: 955–958
3.20 ± 0.87 9.20 ± 2.88 8.41 ± 1.68 9.57 ± 0.64 ND
ND, not determined.
Copyright © 2014 John Wiley & Sons, Ltd.
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Figure 4. Effects of EDTA-chelatable metals on CL intensity. Conditions: 200 μM luminol in 50 mM phosphate buffer (pH 7.4); 400 mM H2O2 in 50 mM phosphate buffer (pH 7.4); 800 μM EDTA in 50 mM phosphate buffer (pH 7.4). *10 μM of solution was used. Data are shown as mean ± SD (n = 3).
EDTA DETAPAC Deferoxamine Deferiprone 1,10-Phenanthroline
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M. Wada et al.
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Figure 5. CL profiles of EDTA (A), deferoxamine (B), ascorbic acid (C) and trolox (D).
In conclusion, the proposed method using the Fenton’s reaction–luminol CL system was simple and rapid for the selective evaluation of the Fe2+-chelating effect. This method might also be useful for screening of drug discovery seed compounds and to give complementary information for the development of medication.
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Acknowledgements
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Figure 6. Effects of SOD on CL intensity.
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CL reactions by different ROS with different reactivity on luminol occurred, because the blank CL profile showed a dipolar curve. Furthermore, addition of the iron chelators (EDTA and deferoxamine) selectively quenched the latter CL, whereas ROS quenchers (ASA and trolox) quenched both CL reactions (Fig. 5). The CL profile of deferoxamine appeared to quench both the former and latter CL. This might be due to ROS quenching. Monitoring of the CL profile is useful to distinguish the decreasing in CL based on iron chelating from that due to ROS quenching. The influence of the CL from the first reaction step (appearing within 0.4 s in Fig. 5) on the chelating effect at low chelator concentrations was not so high, because the chelating effect was evaluated using the CL measured for 60 s, just after addition of H2O2 solution. To clarify which ROS contributes to each CL, selective ROS quenchers, Cat and SOD were added to the CL system. No quenching was observed on addition of Cat (data was not shown). On addition of SOD, the latter CL was quenched (Fig. 6), whereas the former was not quenched. As a result, the superoxide anion contributed to the later CL. The ·OH generated by the Fenton’s reaction reacts with H2O2 to generate the superoxide anion (10). After which, luminol reacts with the superoxide anion and emits light. Therefore, an iron chelator apparently quenched the CL due to the superoxide anion. However, which ROS contribute the former CL was not clarified.
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This work was supported in part by a Scientific Research Fund from Ministry of Higher Education and Scientific Research, Jordan.
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Luminescence 2014; 29: 955–958
