- Email: [email protected]
Application of electrochemical and spin trapping techniques in the investigation of hydroxyl radicals
Analytica Chimica Acta 389 (1999) 213±218
Application of electrochemical and spin trapping techniques in the investigation of hydroxyl radicals Zhiru Maa, Baolu Zhaob, Zhuobin Yuana,* a
Department of Chemistry, Graduate School, University of Science and Technology of China, Academia Sinica, Beijing 100039, China b Institute of Biophysics, Academia Sinica, Beijing 100101, China
Received 4 December 1998; received in revised form 18 January 1999; accepted 31 January 1999
Abstract An electrochemical method combined with spin trapping was applied in the determination of hydroxyl radicals. Hydroxyl radicals were generated by the Fenton reagent, i.e. an iron±EDTA complex reacting with H2O2 in the presence of ascorbic acid. The spin trap reagent phenyl-tert-butyl nitrone (PBN) was used to trap the hydroxyl radicals, and the free radical adduct of PBN could be detected by single sweep oscillopolarography, with its second order derivative cathodic wave at ÿ0.52 V vs SCE. The electrochemical behavior of the PBN adduct was investigated by single sweep oscillopolarography and cyclic voltammetry. An electron spin resonance (ESR) experiment was made in order to verify the existence of the PBN spin adduct in the Fenton system. The experimental conditions for the measurement were optimized, and then the scavenging effect of some compounds on hydroxyl radicals was also studied. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Electrochemical method; Spin trapping technique; PBN; ESR; Hydroxyl radicals; Scavengers
1. Introduction Active oxygen species (including superoxide anion ÿ O2 , hydroxyl radical OH, singlet oxygen 1 O2 and hydrogen peroxide) have been implicated as being important causative agents of aging and various human diseases [1±3], such as cancer, heart diseases, multiple sclerosis, Parkinson's disease, autoimmune disease, strokes and so on. Among the various radicals, the hydroxyl radical ( OH) is presumed to play a central role due to its strong activity [4]. The importance of removing excessive active oxygen species
*Corresponding author. Fax: +86-10-68210510; e-mail: [email protected]
from living organisms is becoming increasingly recognized, together with a growing interest in ®nding antioxidants that can scavenge such species. These antioxidants may have a potential for therapeutic use. Hydroxyl radicals can be produced by pulse radiolysis [5,6], the Fenton reaction [7,8], UV photolysis of hydrogen peroxide [9], the ascorbic acid±Cu2± H2O2 system [9,10] etc. Many methods have been developed for the measurement of OH, for example, the electron spin resonance (ESR)±spin trapping method [11], UV-Vis, spectrophotometry [11,12], liquid chromatography (LC) [13], and a chemiluminescence method [8,10]. The spin trapping technique has become a powerful tool for investigating generation mechanisms of free
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 9 9 ) 0 0 1 4 2 - 7
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radicals and active oxygen [14±17]. This technique involves the inclusion in the experimental system of a nitrone or nitroso compound (spin trap) that can react with a short lived free radical such as OH to generate a long lived nitroxide free radical (spin adduct). The spin adduct is usually detected by ESR spectroscopy. The two nitrone spin traps that have found wide used are 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and phenyl-tert-butylnitrone (PBN). The mechanisms of spin-trapping can be illustrated by the following scheme [14]:
(1)
In our laboratory, electrochemical analysis was combined with the deoxyribose method to determine the rate constants of reactions of OH [18]. However, several complex steps must be taken in this method. This paper aimed to establish a simple and convenient method to detect the hydroxyl radical generation, so that the method could be used to study the scavenging effect of medicines on OH. In the present work, the spin trapping technique and single sweep oscillopolarography were applied to investigate the hydroxyl radicals which were generated by the Fenton reaction. The PBN adduct can easily be detected by single sweep oscillopolarography. ESR spectroscopy was also used to detect the production of the PBN spin adduct. The proposed method is sensitive, convenient, and could be applied to investigate the scavenging effect of some compounds and substances on hydroxyl radicals.
Single sweep oscillopolarography experiments were made using a Model MP-1 Stripping Voltametric Analyzer (The Seventh Telecommunication Equipment Plant of Shandong, China). A dropping mercury electrode was used as the working electrode, a platinum wire as the counter electrode and a saturated calomel electrode (SCE) as the reference. The potential scan range from ÿ0.20 to ÿ0.80 V, and the scan rate, 300 mV sÿ1. Cyclic voltammetric experiments were made using Model 174A Polarographic Analyzer (EG&G Princeton Applied Research). A model 303 hanging mercury drop electrode (HMDE) was used as working electrode, Ag/AgCl, KCl(s) as reference electrode and platinum wire as counter electrode. The potential scan range from ÿ0.20 to ÿ0.95 V, and the scan rate, 20 mV sÿ1. ESR experiments were performed using a Bruker ER-200D-SRC spectrometer. Hydroxyl radicals were generated by the Fenton reaction, Hydrogen peroxide (0.3 M), (NH4)2Fe(SO4)2 (1.010ÿ4 M) and saturated PBN solution were mixed and transferred into a quartz capillary. The conditions for measurement were: X band, 100 kHz modulation with 1.5 G application, microwave power 10 mW, central magnetic ®eld 3400 G, scan width 200 G. The experiment was made at room temperature. 2.2. Generation and detection of PBN adduct in the Fenton system by an electrochemical method Hydroxyl radicals were generated by the Fenton reaction, which can be described by the following scheme according to Halliwell et al. [19]: Fe2 ÿEDTAH2 O2 ! OHÿ OH Fe3 ÿEDTA (2)
2. Experimental 2.1. Reagents and apparatus The following analytical grade chemicals were used as solutions in twice distilled water: ammonium iron(II) sulfate (10 mM), EDTA (10 mM), ascorbic acid (10 mM), H2O2 (1 M), KH2PO4±NaOH (PBS) buffer solution, PBN (16.5 mM), mannitol (10 mM), thiourea (10 mM), sodium formate (10 mM), benzoic acid (10 mM), citric acid (10 mM). PBN was a gift from Prof. Yaoxing Zhao.
Fe3 ÿEDTA ascorbate ! Fe2 ÿEDTA oxidized ascorbate 2
(3)
The following reagents: Fe , EDTA, ascorbate acid, PBN, H2O2, KH2PO4NaOH buffer solutions were mixed to give a ®nal volume of 10.00 ml. The PBN adduct was determined after mixing all the reagents. Inhibitors were added before the H2O2. The PBN adduct was detected by an electroanalytical method. Every test had at least three replicates.
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3. Results 3.1. Polarographic behavior of the PBN adduct The PBN adduct which was produced by reaction (1) was determined by single-sweep oscilloplarography. In pH 7.4 PBS buffer solution, the spin adduct gave a very sensitive and well shaped second order derivative cathodic wave at ÿ0.52 V (vs SCE) (Fig. 1). The peak of the adduct was relatively stable, and the peak height changed little in 2 h. No such peak was detected in the absence of PBN. The cyclic voltammogram of PBN adduct is shown in Fig. 2. In this ®gure, only a cathodic wave was observed, no correspondingly anodic wave appeared. It is a typical diagram of an irreversible process. 3.2. ESR study of PBN hydroxyl radical adduct generated by the Fenton reaction The ESR spectrum of hydroxyl radicals from the Fenton reaction trapped by PBN is shown in Fig. 3(b). The ESR parameters (g2.0058, aN14.9 G, from N in PBN adduct), aH2.74 G (from H in PBN adduct) are similar to those obtained from the ultra-
Fig. 1. The second order derivative cathodic wave of the PBN adduct. Reaction mixtures contained Fe2 (0.10 mM), EDTA (0.10 mM), ascorbic acid (0.20 mM), PBN (0.165 mM), H2O2 (10 mM) in pH 7.4 PBS buffer solution, the potential scan range from ÿ0.20 to ÿ0.80 V, the scan rate, 300 mV sÿ1.
Fig. 2. Cyclic voltammogram of PBN adduct. Reaction mixtures were as in Fig. 1. The potential scan range from ÿ0.20 to ÿ0.95 V, the scan rate, 20 mV sÿ1.
violet photolysis of aqueous hydrogen peroxide solution, which con®rmed that the adduct (1) was formed. No signal could be detected without PBN (Fig. 3(a)). With the addition of salicylic acid (1 mM) to the system, the ESR signal decreased (Fig. 3(c)). 3.3. Effect of pH on the production of the PBN±OH adduct The peak height was proportional to the amount of adduct. Fig. 4 shows the effect of the pH of the PBS buffer solution on the production of the PBN adduct. The peak height increased with pH from 6.0 to 7.4,
Fig. 3. ESR spectrum of hydroxyl radical from the Fenton reaction trapped by PBN.The system containing saturated PBN solution without (a) and with hydrogen peroxide (0.3 M), and (NH4)2Fe(SO4)2 (0.1 mM) (b); salicylic acid (1 mM) (c) was added to (b).
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Fig. 4. The effect of pH on the peak height of the PBN adduct. Reaction mixtures contained Fe2 (0.10 mM), EDTA (0.10 mM), ascorbic acid (0.20 mM), PBN (0.165 mM), H2O2 (10 mM), and PBS buffer solution.
Fig. 5. The effect of ascorbic acid on the peak height of the PBN adduct. Reaction mixtures contained Fe2 (0.10 mM), EDTA (0.10 mM), ascorbic acid (0.00±0.30 mM), PBN (0.165 mM), H2O2 (10 mM), and pH 7.4 PBS buffer solution.
then decreased. The optimal pH for the production of hydroxyl radicals in the Fenton system was 7.4.
it decreased when the concentration exceeded 0.20 mM, but was still higher than that in the absence of ascorbic acid.
3.4. Influence of concentrations of Fe2 and EDTA on the peak height of the PBN adduct The in¯uence of the concentrations of Fe2 and EDTA on the peak height of the PBN adduct was studied. No signal could be observed in the absence of Fe2. The peak height of the adduct increased with the concentrations of Fe2 in a range from 0.00 to 0.10 mM. Then it decreased with increasing concentration of Fe2. The peak height of the adduct was very small when no EDTA was added. The signal increased with increasing concentration of EDTA until a plateau was reached at 0.10 mM.
3.6. Influence of concentration of PBN on the peak height of the PBN adduct The in¯uence of concentration of PBN on the peak height of the adduct is shown in Fig. 6. The results suggested that the peak height increased with increasing concentrations of PBN in a certain range. No signal could be observed in the absence of PBN. The peak height was a maximum when the concentration of PBN was 0.165 mM, and then decreased with increasing concentrations of PBN.
3.5. Effect of concentration of ascorbic acid on the production of the PBN adduct Fig. 5 shows the effect of ascorbic acid on the peak height of the adduct. From reaction (3), it can be seen that ascorbic acid acts as a reducing agent, which could reduce Fe3 to Fe2 and increase the generation of hydroxyl radicals [19,20]. The peak height of the PBN adduct with added ascorbic acid was higher than that in its absence. In a certain concentration range, the peak height of the adduct increased with increasing concentration of ascorbic acid. The peak height reached a maximum at 0.20 mM ascorbic acid;
Fig. 6. The influence of concentration of PBN on the peak height of the PBN adduct. Reaction mixtures contained Fe2 (0.10 mM), EDTA (0.10 mM), ascorbic acid (0.20 mM), PBN (various concentrations), H2O2 (10 mM) and pH 7.4 PBS buffer solution.
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These compounds (including mannitol, thiourea, sodium formate, benzoic acid, citric acid etc.) are known hydroxyl radical scavengers. The inhibitory effect of these compounds on PBN adduct was concentration dependent. Their IC50 (concentration of half inhibition) were mannitol (1.63 mM), sodium formate (1.12 mM), citric acid (1.37 mM), thiourea (0.24 mM), benzoic acid (0.17 mM). 4. Discussion Fig. 7. Effect of concentration of H2O2 on the peak height of the PBN adduct. Reaction mixtures contained Fe2 (0.10 mM), EDTA (0.10 mM), ascorbic acid (0.20 mM), PBN (0.165 mM), H2O2 (various concentrations), and pH 7.4 PBS buffer solution.
3.7. Effect of concentration of H2O2 on the peak height of the PBN adduct The effect of concentration of H2O2 on the peak height of the adduct is shown in Fig. 7. When no additional H2O2 is present, the signal of the PBN adduct is very weak, and could almost not be observed at the same sensitivity. Because a trace of H2O2 would be produced through the reaction of dissolved oxygen at the electrode: O2 2H 2e ! H2 O2
(4) 2
The trace H2O2 could react with Fe and produce OH. Thus a trace of the PBN adduct would be observed at higher sensitivity. The peak height of PBN adduct increased with increasing concentrations of added H2O2. The peak height increased rapidly from 0.00 to 10 mM, and increased slowly when the concentration of H2O2 exceeded 10 mM. It showed that 10 mM H2O2 was a suitable concentration for subsequent experiments. To sum up, the optimum conditions for measurement are: Fe2 (0.10 mM), EDTA (0.10 mM), ascorbic acid (0.20 mM), PBN (0.165 mM), H2O2 (10 mM), in pH 7.4 PBS buffer solution.
3.8. Effect of some compounds on the production of the PBN adduct The inhibitory effect of some compounds on the production of the PBN±OH adduct was investigated.
The hydroxyl radical is believed to be the most reactive species among the oxygen radical intermediates which damage biological tissues. The spin trapping technique is very useful for stabilizing short-lived free radicals. DMPO is more susceptible to oxygen, heat and light, and thus has a shorter shelf life, while PBN has the advantage of forming very stable spin adducts (14); it is stable to light, heat and oxygen, and therefore has greater shelf life. The electrochemical method (single sweep oscillopolargraphy) has the advantages of being simple, sensitive and accurate. Any electroactive substances can be detected. The PBN±hydroxyl radical adduct is electroactive, and gives a sensitive electrochemical signal. The second order derivative cathodic wave had a good shape, and its peak potential was at ÿ0.52 V (vs SCE). The addition of iron, an ion chelating agent such as EDTA and the reductant ascorbic acid in the Fenton system increased the production of hydroxyl radicals [19,20] and the signal from the PBN adduct is very strong. The ESR experiment also showed the existence of the PBN hydroxyl radical adduct (Fig. 3). No such signal could be detected without addition of PBN (Fig. 3(a)). A hydroxyl radical scavenger such as salicylic acid could inhibit the production of the hydroxyl radical adduct, thus the signal of the hydroxyl radical decreased (Fig. 3(b)). Harbour et al. [14] also trapped hydroxyl radicals with PBN in the ultraviolet photolysis of aqueous of hydrogen peroxide solution. In summary, a novel and convenient method has been established for the determination of hydroxyl radicals produced by the Fenton reaction. This technique did not need expensive instrumentation and the operation was simple. It provided a simple, rapid and
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powerful tool to study the scavenging effect of substances on hydroxyl radicals. Acknowledgements This work was supported by a grant from the National Natural Science Foundation of China and Electroanalytical Chemistry Opening Laboratory Foundation of Changchun Applied Chemistry Institute. References [1] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, Oxford University press, New York, 1985, p. 218. [2] J.L. Mark, Science 235 (1987) 529. [3] G.B. Bulkley, Surgery 94 (1983) 407. [4] J.M. McCord, N. Engl. J. Med. 312 (1985) 159. [5] M. Ebert, J.P. Keene, A.J. Swallow, J.H. Baxendale, Pulse Radiolysis, Academic Press, London, 1965. [6] W.F. Wang, J. Luo, S.D. Yao, Z.R. Lian, J.S. Zhang, N.Y. Lin, Radiat. Phys. Chem. 42 (1993) 985.
[7] A. Puppo, Phytochemistry 31 (1992) 85. [8] Y. Yoshiki, K. Okubo, M. Onuma, K. Igarashi, Phytochemistry 39 (1995) 225. [9] S.R. Husain, J. Cillard, P. Cillard, Phytochemistry 26 (1987) 2489. [10] J.W. Chen, T.X. Hu, Prog. Biochem. Biophys. 19 (1992) 136. [11] J.I. Ueda, N. Saito, Y. Shimazu, T. Ozawa, Arch. Biochem. Biophys. 332 (1996) 377. [12] Z.S. Jia, J.M. Wu, M.C. Tang, Prog. Biochem. Biophys. 23 (1996) 184. [13] S. Fukui, Y. Hanasaki, S. Ogawa, J. Chromatogr. 630 (1993) 187. [14] J.R. Harbour, V. Chow, J.R. Bolton, Can. J. Chem. 52 (1974) 3549. [15] B.E. Britigan, T.J. Coffman, G.R. Buettner, J. Biol. Chem. 265 (1990) 2650. [16] E. Finkelstein, G.M. Rosen, E.J. Rauckman, J. Am. Chem. Soc. 102 (1980) 4994. [17] M. Kohno, M. Yamada, K. Mitsuta, Y. Mizuta, Y. Yoshikawa, Bull. Chem. Soc. Jpn. 64 (1991) 1447. [18] R.M. Gao, Z.B. Yuan, H.B. Ding, F.M. Liu, Bioelectrochem. Bioenerg. 45 (1998) 123. [19] B. Halliwell, J.M.C. Gutterige, O.I. Aruoma, Anal. Biochem. 165 (1987) 215. [20] M.J. Langhton, B. Halliwell, P.J. Evans, J.R.S. Hoult, Biochem. Pharmacol. 38 (1989) 2859.
Application of electrochemical and spin trapping techniques in the investigation of hydroxyl radicals Zhiru Maa, Baolu Zhaob, Zhuobin Yuana,* a
Department of Chemistry, Graduate School, University of Science and Technology of China, Academia Sinica, Beijing 100039, China b Institute of Biophysics, Academia Sinica, Beijing 100101, China
Received 4 December 1998; received in revised form 18 January 1999; accepted 31 January 1999
Abstract An electrochemical method combined with spin trapping was applied in the determination of hydroxyl radicals. Hydroxyl radicals were generated by the Fenton reagent, i.e. an iron±EDTA complex reacting with H2O2 in the presence of ascorbic acid. The spin trap reagent phenyl-tert-butyl nitrone (PBN) was used to trap the hydroxyl radicals, and the free radical adduct of PBN could be detected by single sweep oscillopolarography, with its second order derivative cathodic wave at ÿ0.52 V vs SCE. The electrochemical behavior of the PBN adduct was investigated by single sweep oscillopolarography and cyclic voltammetry. An electron spin resonance (ESR) experiment was made in order to verify the existence of the PBN spin adduct in the Fenton system. The experimental conditions for the measurement were optimized, and then the scavenging effect of some compounds on hydroxyl radicals was also studied. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Electrochemical method; Spin trapping technique; PBN; ESR; Hydroxyl radicals; Scavengers
1. Introduction Active oxygen species (including superoxide anion ÿ O2 , hydroxyl radical OH, singlet oxygen 1 O2 and hydrogen peroxide) have been implicated as being important causative agents of aging and various human diseases [1±3], such as cancer, heart diseases, multiple sclerosis, Parkinson's disease, autoimmune disease, strokes and so on. Among the various radicals, the hydroxyl radical ( OH) is presumed to play a central role due to its strong activity [4]. The importance of removing excessive active oxygen species
*Corresponding author. Fax: +86-10-68210510; e-mail: [email protected]
from living organisms is becoming increasingly recognized, together with a growing interest in ®nding antioxidants that can scavenge such species. These antioxidants may have a potential for therapeutic use. Hydroxyl radicals can be produced by pulse radiolysis [5,6], the Fenton reaction [7,8], UV photolysis of hydrogen peroxide [9], the ascorbic acid±Cu2± H2O2 system [9,10] etc. Many methods have been developed for the measurement of OH, for example, the electron spin resonance (ESR)±spin trapping method [11], UV-Vis, spectrophotometry [11,12], liquid chromatography (LC) [13], and a chemiluminescence method [8,10]. The spin trapping technique has become a powerful tool for investigating generation mechanisms of free
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 9 9 ) 0 0 1 4 2 - 7
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radicals and active oxygen [14±17]. This technique involves the inclusion in the experimental system of a nitrone or nitroso compound (spin trap) that can react with a short lived free radical such as OH to generate a long lived nitroxide free radical (spin adduct). The spin adduct is usually detected by ESR spectroscopy. The two nitrone spin traps that have found wide used are 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and phenyl-tert-butylnitrone (PBN). The mechanisms of spin-trapping can be illustrated by the following scheme [14]:
(1)
In our laboratory, electrochemical analysis was combined with the deoxyribose method to determine the rate constants of reactions of OH [18]. However, several complex steps must be taken in this method. This paper aimed to establish a simple and convenient method to detect the hydroxyl radical generation, so that the method could be used to study the scavenging effect of medicines on OH. In the present work, the spin trapping technique and single sweep oscillopolarography were applied to investigate the hydroxyl radicals which were generated by the Fenton reaction. The PBN adduct can easily be detected by single sweep oscillopolarography. ESR spectroscopy was also used to detect the production of the PBN spin adduct. The proposed method is sensitive, convenient, and could be applied to investigate the scavenging effect of some compounds and substances on hydroxyl radicals.
Single sweep oscillopolarography experiments were made using a Model MP-1 Stripping Voltametric Analyzer (The Seventh Telecommunication Equipment Plant of Shandong, China). A dropping mercury electrode was used as the working electrode, a platinum wire as the counter electrode and a saturated calomel electrode (SCE) as the reference. The potential scan range from ÿ0.20 to ÿ0.80 V, and the scan rate, 300 mV sÿ1. Cyclic voltammetric experiments were made using Model 174A Polarographic Analyzer (EG&G Princeton Applied Research). A model 303 hanging mercury drop electrode (HMDE) was used as working electrode, Ag/AgCl, KCl(s) as reference electrode and platinum wire as counter electrode. The potential scan range from ÿ0.20 to ÿ0.95 V, and the scan rate, 20 mV sÿ1. ESR experiments were performed using a Bruker ER-200D-SRC spectrometer. Hydroxyl radicals were generated by the Fenton reaction, Hydrogen peroxide (0.3 M), (NH4)2Fe(SO4)2 (1.010ÿ4 M) and saturated PBN solution were mixed and transferred into a quartz capillary. The conditions for measurement were: X band, 100 kHz modulation with 1.5 G application, microwave power 10 mW, central magnetic ®eld 3400 G, scan width 200 G. The experiment was made at room temperature. 2.2. Generation and detection of PBN adduct in the Fenton system by an electrochemical method Hydroxyl radicals were generated by the Fenton reaction, which can be described by the following scheme according to Halliwell et al. [19]: Fe2 ÿEDTAH2 O2 ! OHÿ OH Fe3 ÿEDTA (2)
2. Experimental 2.1. Reagents and apparatus The following analytical grade chemicals were used as solutions in twice distilled water: ammonium iron(II) sulfate (10 mM), EDTA (10 mM), ascorbic acid (10 mM), H2O2 (1 M), KH2PO4±NaOH (PBS) buffer solution, PBN (16.5 mM), mannitol (10 mM), thiourea (10 mM), sodium formate (10 mM), benzoic acid (10 mM), citric acid (10 mM). PBN was a gift from Prof. Yaoxing Zhao.
Fe3 ÿEDTA ascorbate ! Fe2 ÿEDTA oxidized ascorbate 2
(3)
The following reagents: Fe , EDTA, ascorbate acid, PBN, H2O2, KH2PO4NaOH buffer solutions were mixed to give a ®nal volume of 10.00 ml. The PBN adduct was determined after mixing all the reagents. Inhibitors were added before the H2O2. The PBN adduct was detected by an electroanalytical method. Every test had at least three replicates.
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3. Results 3.1. Polarographic behavior of the PBN adduct The PBN adduct which was produced by reaction (1) was determined by single-sweep oscilloplarography. In pH 7.4 PBS buffer solution, the spin adduct gave a very sensitive and well shaped second order derivative cathodic wave at ÿ0.52 V (vs SCE) (Fig. 1). The peak of the adduct was relatively stable, and the peak height changed little in 2 h. No such peak was detected in the absence of PBN. The cyclic voltammogram of PBN adduct is shown in Fig. 2. In this ®gure, only a cathodic wave was observed, no correspondingly anodic wave appeared. It is a typical diagram of an irreversible process. 3.2. ESR study of PBN hydroxyl radical adduct generated by the Fenton reaction The ESR spectrum of hydroxyl radicals from the Fenton reaction trapped by PBN is shown in Fig. 3(b). The ESR parameters (g2.0058, aN14.9 G, from N in PBN adduct), aH2.74 G (from H in PBN adduct) are similar to those obtained from the ultra-
Fig. 1. The second order derivative cathodic wave of the PBN adduct. Reaction mixtures contained Fe2 (0.10 mM), EDTA (0.10 mM), ascorbic acid (0.20 mM), PBN (0.165 mM), H2O2 (10 mM) in pH 7.4 PBS buffer solution, the potential scan range from ÿ0.20 to ÿ0.80 V, the scan rate, 300 mV sÿ1.
Fig. 2. Cyclic voltammogram of PBN adduct. Reaction mixtures were as in Fig. 1. The potential scan range from ÿ0.20 to ÿ0.95 V, the scan rate, 20 mV sÿ1.
violet photolysis of aqueous hydrogen peroxide solution, which con®rmed that the adduct (1) was formed. No signal could be detected without PBN (Fig. 3(a)). With the addition of salicylic acid (1 mM) to the system, the ESR signal decreased (Fig. 3(c)). 3.3. Effect of pH on the production of the PBN±OH adduct The peak height was proportional to the amount of adduct. Fig. 4 shows the effect of the pH of the PBS buffer solution on the production of the PBN adduct. The peak height increased with pH from 6.0 to 7.4,
Fig. 3. ESR spectrum of hydroxyl radical from the Fenton reaction trapped by PBN.The system containing saturated PBN solution without (a) and with hydrogen peroxide (0.3 M), and (NH4)2Fe(SO4)2 (0.1 mM) (b); salicylic acid (1 mM) (c) was added to (b).
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Fig. 4. The effect of pH on the peak height of the PBN adduct. Reaction mixtures contained Fe2 (0.10 mM), EDTA (0.10 mM), ascorbic acid (0.20 mM), PBN (0.165 mM), H2O2 (10 mM), and PBS buffer solution.
Fig. 5. The effect of ascorbic acid on the peak height of the PBN adduct. Reaction mixtures contained Fe2 (0.10 mM), EDTA (0.10 mM), ascorbic acid (0.00±0.30 mM), PBN (0.165 mM), H2O2 (10 mM), and pH 7.4 PBS buffer solution.
then decreased. The optimal pH for the production of hydroxyl radicals in the Fenton system was 7.4.
it decreased when the concentration exceeded 0.20 mM, but was still higher than that in the absence of ascorbic acid.
3.4. Influence of concentrations of Fe2 and EDTA on the peak height of the PBN adduct The in¯uence of the concentrations of Fe2 and EDTA on the peak height of the PBN adduct was studied. No signal could be observed in the absence of Fe2. The peak height of the adduct increased with the concentrations of Fe2 in a range from 0.00 to 0.10 mM. Then it decreased with increasing concentration of Fe2. The peak height of the adduct was very small when no EDTA was added. The signal increased with increasing concentration of EDTA until a plateau was reached at 0.10 mM.
3.6. Influence of concentration of PBN on the peak height of the PBN adduct The in¯uence of concentration of PBN on the peak height of the adduct is shown in Fig. 6. The results suggested that the peak height increased with increasing concentrations of PBN in a certain range. No signal could be observed in the absence of PBN. The peak height was a maximum when the concentration of PBN was 0.165 mM, and then decreased with increasing concentrations of PBN.
3.5. Effect of concentration of ascorbic acid on the production of the PBN adduct Fig. 5 shows the effect of ascorbic acid on the peak height of the adduct. From reaction (3), it can be seen that ascorbic acid acts as a reducing agent, which could reduce Fe3 to Fe2 and increase the generation of hydroxyl radicals [19,20]. The peak height of the PBN adduct with added ascorbic acid was higher than that in its absence. In a certain concentration range, the peak height of the adduct increased with increasing concentration of ascorbic acid. The peak height reached a maximum at 0.20 mM ascorbic acid;
Fig. 6. The influence of concentration of PBN on the peak height of the PBN adduct. Reaction mixtures contained Fe2 (0.10 mM), EDTA (0.10 mM), ascorbic acid (0.20 mM), PBN (various concentrations), H2O2 (10 mM) and pH 7.4 PBS buffer solution.
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These compounds (including mannitol, thiourea, sodium formate, benzoic acid, citric acid etc.) are known hydroxyl radical scavengers. The inhibitory effect of these compounds on PBN adduct was concentration dependent. Their IC50 (concentration of half inhibition) were mannitol (1.63 mM), sodium formate (1.12 mM), citric acid (1.37 mM), thiourea (0.24 mM), benzoic acid (0.17 mM). 4. Discussion Fig. 7. Effect of concentration of H2O2 on the peak height of the PBN adduct. Reaction mixtures contained Fe2 (0.10 mM), EDTA (0.10 mM), ascorbic acid (0.20 mM), PBN (0.165 mM), H2O2 (various concentrations), and pH 7.4 PBS buffer solution.
3.7. Effect of concentration of H2O2 on the peak height of the PBN adduct The effect of concentration of H2O2 on the peak height of the adduct is shown in Fig. 7. When no additional H2O2 is present, the signal of the PBN adduct is very weak, and could almost not be observed at the same sensitivity. Because a trace of H2O2 would be produced through the reaction of dissolved oxygen at the electrode: O2 2H 2e ! H2 O2
(4) 2
The trace H2O2 could react with Fe and produce OH. Thus a trace of the PBN adduct would be observed at higher sensitivity. The peak height of PBN adduct increased with increasing concentrations of added H2O2. The peak height increased rapidly from 0.00 to 10 mM, and increased slowly when the concentration of H2O2 exceeded 10 mM. It showed that 10 mM H2O2 was a suitable concentration for subsequent experiments. To sum up, the optimum conditions for measurement are: Fe2 (0.10 mM), EDTA (0.10 mM), ascorbic acid (0.20 mM), PBN (0.165 mM), H2O2 (10 mM), in pH 7.4 PBS buffer solution.
3.8. Effect of some compounds on the production of the PBN adduct The inhibitory effect of some compounds on the production of the PBN±OH adduct was investigated.
The hydroxyl radical is believed to be the most reactive species among the oxygen radical intermediates which damage biological tissues. The spin trapping technique is very useful for stabilizing short-lived free radicals. DMPO is more susceptible to oxygen, heat and light, and thus has a shorter shelf life, while PBN has the advantage of forming very stable spin adducts (14); it is stable to light, heat and oxygen, and therefore has greater shelf life. The electrochemical method (single sweep oscillopolargraphy) has the advantages of being simple, sensitive and accurate. Any electroactive substances can be detected. The PBN±hydroxyl radical adduct is electroactive, and gives a sensitive electrochemical signal. The second order derivative cathodic wave had a good shape, and its peak potential was at ÿ0.52 V (vs SCE). The addition of iron, an ion chelating agent such as EDTA and the reductant ascorbic acid in the Fenton system increased the production of hydroxyl radicals [19,20] and the signal from the PBN adduct is very strong. The ESR experiment also showed the existence of the PBN hydroxyl radical adduct (Fig. 3). No such signal could be detected without addition of PBN (Fig. 3(a)). A hydroxyl radical scavenger such as salicylic acid could inhibit the production of the hydroxyl radical adduct, thus the signal of the hydroxyl radical decreased (Fig. 3(b)). Harbour et al. [14] also trapped hydroxyl radicals with PBN in the ultraviolet photolysis of aqueous of hydrogen peroxide solution. In summary, a novel and convenient method has been established for the determination of hydroxyl radicals produced by the Fenton reaction. This technique did not need expensive instrumentation and the operation was simple. It provided a simple, rapid and
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powerful tool to study the scavenging effect of substances on hydroxyl radicals. Acknowledgements This work was supported by a grant from the National Natural Science Foundation of China and Electroanalytical Chemistry Opening Laboratory Foundation of Changchun Applied Chemistry Institute. References [1] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, Oxford University press, New York, 1985, p. 218. [2] J.L. Mark, Science 235 (1987) 529. [3] G.B. Bulkley, Surgery 94 (1983) 407. [4] J.M. McCord, N. Engl. J. Med. 312 (1985) 159. [5] M. Ebert, J.P. Keene, A.J. Swallow, J.H. Baxendale, Pulse Radiolysis, Academic Press, London, 1965. [6] W.F. Wang, J. Luo, S.D. Yao, Z.R. Lian, J.S. Zhang, N.Y. Lin, Radiat. Phys. Chem. 42 (1993) 985.
[7] A. Puppo, Phytochemistry 31 (1992) 85. [8] Y. Yoshiki, K. Okubo, M. Onuma, K. Igarashi, Phytochemistry 39 (1995) 225. [9] S.R. Husain, J. Cillard, P. Cillard, Phytochemistry 26 (1987) 2489. [10] J.W. Chen, T.X. Hu, Prog. Biochem. Biophys. 19 (1992) 136. [11] J.I. Ueda, N. Saito, Y. Shimazu, T. Ozawa, Arch. Biochem. Biophys. 332 (1996) 377. [12] Z.S. Jia, J.M. Wu, M.C. Tang, Prog. Biochem. Biophys. 23 (1996) 184. [13] S. Fukui, Y. Hanasaki, S. Ogawa, J. Chromatogr. 630 (1993) 187. [14] J.R. Harbour, V. Chow, J.R. Bolton, Can. J. Chem. 52 (1974) 3549. [15] B.E. Britigan, T.J. Coffman, G.R. Buettner, J. Biol. Chem. 265 (1990) 2650. [16] E. Finkelstein, G.M. Rosen, E.J. Rauckman, J. Am. Chem. Soc. 102 (1980) 4994. [17] M. Kohno, M. Yamada, K. Mitsuta, Y. Mizuta, Y. Yoshikawa, Bull. Chem. Soc. Jpn. 64 (1991) 1447. [18] R.M. Gao, Z.B. Yuan, H.B. Ding, F.M. Liu, Bioelectrochem. Bioenerg. 45 (1998) 123. [19] B. Halliwell, J.M.C. Gutterige, O.I. Aruoma, Anal. Biochem. 165 (1987) 215. [20] M.J. Langhton, B. Halliwell, P.J. Evans, J.R.S. Hoult, Biochem. Pharmacol. 38 (1989) 2859.