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Dietary antioxidants interfere with Amplex Red-coupled-fluorescence assays
Biochemical and Biophysical Research Communications 388 (2009) 443–449
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Dietary antioxidants interfere with Amplex Red-coupled-fluorescence assays José Serrano *, Mariona Jové, Jordi Boada, María Josep Bellmunt, Reinald Pamplona, Manuel Portero-Otín Department of Experimental Medicine, Faculty of Medicine, University of Lleida-IRBLleida, Lleida, Spain
a r t i c l e
i n f o
Article history: Received 21 July 2009 Available online 11 August 2009 Keywords: Amplex Red Dietary antioxidants Horseradish peroxidase Interference
a b s t r a c t Oxidation of Amplex Red by hydrogen peroxide in the presence of horseradish peroxidase (HRP) gives rise to an intensely colour product, resorufin. This reaction has been frequently employed for measurements based on enzyme-coupled reactions that detect hydrogen peroxide as a final reaction product. In the current study, we show that the presence of dietary antioxidants at biological concentrations in the reaction medium produced interferences in the Amplex Red/HRP catalyzed reaction that result in an over quantification of the hydrogen peroxide produced. The interference observed showed a dose-dependent manner, and a possible mechanism of interaction of dietary antioxidants with HRP in the Amplex Red-coupled-fluorescent assay is proposed. Ó 2009 Elsevier Inc. All rights reserved.
Introduction 10-Acetyl-3,7-dihydroxyphenoxazine, Amplex Red, is a nonfluorescent molecule that when oxidized by hydrogen peroxide in the presence of horseradish peroxidase (HRP) originates resorufin, a highly fluorescent product (kex = 563 nm; kem = 587 nm). This reaction has been frequently employed for measurements of low concentrations of hydrogen peroxide in biological samples, since Amplex Red oxidation consumes stoichiometric amounts of hydrogen peroxide. As its advantages, Amplex Red presents low background fluorescence (this probe does not emit fluorescences, only its oxidation products) as well as stability and high fluorescence intensity of its oxidation product, all this resulting in a sensibility increase for detecting hydrogen peroxide [1]. The assay is highly sensitive, allowing measurements of hydrogen peroxide concentrations as low as 50 nM [2]. However, the accuracy of such determinations also depends on the specificity of the assay system. When considering biological fluids, there are several compounds that could potentially interfere with the Amplex Red reaction catalyzed by HRP. The molecular mechanism of oxidation by HRP is unusually complex as it also catalyzes oxidation of several products in the absence of hydrogen peroxide (oxidation pathway). HRP exhibits oxidase activity toward a number of substrates in the absence of hydrogen peroxide under aerobic conditions. Among various substrates, dihydroxyfuAbbreviation: HRP, horseradish peroxidase. * Corresponding author. Address: Department of Experimental Medicine, Faculty of Medicine, University of Lleida-IRBLleida, C/Montserrat Roig 2, 25008 Lleida, Spain. Fax: +34 973 70 2426. E-mail address: [email protected] (J. Serrano). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.08.041
merate, indole-3-acetic acid, indole butyrate, oxalate, dihydroxytartarate, phenylacetaldehydes, NADH, and NADPH may be mentioned as a few which are oxidized by the HRP oxidase system [3]. Unpublished data from our group, demonstrate an over estimation of resorufin (measured by fluorescence) in the determination of peroxide hydrogen production by an Amplex Red enzymatic assay kit (Molecular Probes A12216) when a polyphenol extract was added to the reaction medium. This suggested that polyphenols contained in the extracts can promote the formation of resorufin from Amplex Red, and may thus be an important source of unspecific signals when using Amplex Red for the quantification of hydrogen peroxide production from enzymatic-substrate determination. The aim of this study was to demonstrate a dose-dependent interaction between well-known dietary antioxidants (gallic acid, epicatechin, quercetin, vanillic acid and ascorbic acid) with the HRP–Amplex Red assay systems. A serial of five experiments were followed to confirm the interference produced. The observed interactions with dietary antioxidants must be taken into account in further experiments when using the Amplex Red assay for measuring hydrogen peroxide. A potential molecular mechanism to explain this phenomenon is proposed. Materials and methods Chemicals. Amplex Red and horseradish peroxidase (HRP) were obtained from Molecular Probes from the Cholesterol Assay kit (A12216). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water soluble analogue of vitamin E (238813), gallic acid (398225), ascorbic acid (A4544), ( )-epicatechin (E4018), quercetin (Q3001), glucose (G8270), myeloperoxidase (M6908),
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lactoperoxidase (L8257) and catalase (C9322) were purchased from Sigma–Aldrich. 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) (93285) and vanillic acid (94770) were from Fluka Chemicals (Sigma–Aldrich). All reagents used were of analytical grade. Amplex Red assay. The Amplex Red assay was developed in a 96 well plate by the reaction of 50 lL of Amplex Red working solution with 50 lL of assay sample. Five milliliters of working solution, prepared prior the analysis, contained 75 lL of a 300 lM of Amplex Red reagent and 2 U/mL of HRP, lactoperoxidase or myeloperoxidase. The working solution volume was adjusted to 5 mL with reaction buffer, which contained 25 mM potassium phosphate, pH 7.4, 12.5 mM NaCl, 1.25 mM cholic acid and 0.025% TritonÒ X-100. The reactions were incubated for 30 min at 37 °C, protected from light. After incubation, fluorescence was measured in a fluorescence microplate reader (Tecan Infinite M200, Grödig, Austria) using excitation wavelength at 560 nm and emission detection at 590 nm. FRAP assay. For the FRAP assay [4], 900 lL of the FRAP reagent, containing TPTZ, FeCl3, and acetate buffer, was mixed with 90 lL of distilled water and 30 lL of the test sample of the blank (solvents used for extraction). Maximum absorbance values at 595 nm were taken every 15 s at 37 °C, using a Beckman DU-640 spectrophotometer (Beckman Instruments Inc., Fullerton, CA). The readings at 30 min were selected for calculations of FRAP values. Solutions of known Trolox concentration were used for antioxidant capacity equivalents.
Red. Deserves an especial attention the different degree of reactivity between the dietary antioxidant tested at equimolar concentrations. The observed differences may be due to different molecular interactions between antioxidants and HRP. Oxidation of Amplex Red depends on the antioxidant capacity of dietary antioxidants The previous task showed some relationship between the oxidation of Amplex Red with the antioxidant capacity of dietary antioxidants. The aim of this task was to demonstrate that the interaction observed is related to the antioxidant capacity of dietary antioxidant. To do this, Amplex Red reaction was carried on with two different degrees of antioxidant capacity of gallic and ascorbic acid (in normal conditions prepared immediately after the reaction and in an oxidized form produced at room temperature during 1 week) the lost of the antioxidant capacity was evaluated by the FRAP antioxidant capacity assay. The results are shown in Fig. 1, where the ability of gallic and ascorbic acids to induce the oxidation of Amplex Red to resorufin (measured by the intensity of fluorescence) was reduced considerably with the reduction of the antioxidant capacity (measured by FRAP) of the dietary antioxidant under study. However, although the compounds under study showed a lower antioxidant capacity, they were able to induce the oxidation of Amplex Red, but to a lesser degree. Interactions between Amplex Red and dietary antioxidants in the presence or absence of HRP
Results Dose-dependent manner interference The first task was to determine if the observed interference was also induced by well-known dietary antioxidants (gallic acid, ascorbic acid, epicatechin, quercetin, vanillic acid) in a concentration dependent manner. Glucose was used as a negative control, as a non-antioxidant molecule. All dietary antioxidant tested were able to induce the oxidation of Amplex Red to resorufin catalyzed by HRP, being dietary polyphenols (( )-epicatechin, gallic acid, quercetin) the species which showed the highest reactivity in the tested conditions. Table 1 shows the slope and the correlation coefficient of the calculated linear regression of the dose dependent behaviour observed. No hydrogen peroxide was added or indented to be produced in the reaction medium, and no fluorescence was observed by dietary antioxidants in the reaction medium without Amplex Red, demonstrating the ability of dietary antioxidants to induce Amplex Red oxidation in the absence of hydrogen peroxide. Glucose used as a negative control do not produced resorufin. Antioxidant capacity, measured by FRAP assay, was performed in parallel to evaluate the possible correlation between the antioxidant capacity and the rate of resorufin formation. No statistical correlation was observed, despite dietary antioxidants with higher antioxidant capacity showed a higher oxidation rate of Amplex
With the above tasks it has been shown that dietary antioxidants are able of inducing oxidation of Amplex Red catalyzed by HRP. It has also been shown that the interaction can be observed due to the antioxidant capacity of the compounds under study. The next question to resolve was whether the observed reaction is due to direct interactions between dietary antioxidants and Amplex Red. However, the ‘‘oxidation” of Amplex Red by an ‘‘antioxidant” seems to be difficult to explain. Therefore, it was considered necessary the presence of HRP in the reaction to catalyze the reaction. To demonstrate this hypothesis, the degree of oxidation of Amplex Red by gallic and ascorbic acid was assessed in the presence and absence of HRP in the reaction medium. The results of this task are shown in Fig. 2, where no formation of resorufin was observed in the reaction wells without HRP, showing that the enzymatic activity of HRP is essential in the oxidation reaction of Amplex Red. Oxidation of Amplex Red catalyzed by different peroxidases in the presence of dietary antioxidants The forth task was to demonstrate that other Haem-containing redox enzymes like lactoperoxidase (found in milk) and myeloperoxidase (most abundantly present in neutrophil granulocytes) are able to work as catalyst in the oxidation of Amplex Red, also in
Table 1 Dose-dependent reactivity of different dietary antioxidants when added in the Amplex Red reaction mixture. Antioxidants tested shown linear correlation between the amount of antioxidants and the fluorescence emitted (excitation wavelength 560 nm, emission wavelength 590 nm), while glucose (without antioxidant capacity, demonstrated by the FRAP assay) showed almost non-fluorescence. The slope represents the increase in fluorescence per microM of antioxidant in the reaction media (Amplex Red) and the Trolox (a water soluble analogue of vitamin E) equivalency in the FRAP antioxidant capacity assay. Antioxidant
Gallic acid Ascorbic acid ( )-Epicatechin Quercetin Vanillic acid Glucose
Amplex Red reaction
Antioxidant capacity – FRAP 2
Slope
R
p-Value
Slope
R2
p-Value
23.686 3.485 55.390 20.462 15.545 0.203
0.9574 0.9721 0.8478 0.8767 0.8576 0.2379
0.000 0.000 0.009 0.001 0.002 0.220
0.0012 0.0009 0.0023 0.0035 0.0012 0.00005
0.9766 0.9535 0.9962 0.9732 0.9931 0.2889
0.000 0.000 0.000 0.000 0.000 0.109
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A Fluorescence intensity
8000
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0 25
125
225
Concentration microM Gallic acid
Oxidized Gallic acid
Fluorescence intensity
2400 2000 1600 1200 800 400 0 25
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B
Oxidized Ascorbic acid
FRAP assay. Gallic acid Slope
R2
Ascorbic Acid p-value
Slope
R2
p-value
Normal
8.6329 0.9999
0.000
2.7761 0.9967
0.000
Oxidized
0.2661 0.9451
0.000
0.0215 0.0706
0.320
Fig. 1. The oxidation of resorufin or resorufin catalyzed by dietary antioxidants and HRP is related to the antioxidant capacity of dietary antioxidants. (A) Fluorescence intensity produced after the reaction of Amplex Red catalyzed by HRP in the presence of gallic and ascorbic acid () and oxidized gallic and ascorbic acid (j). (B) Antioxidant capacity measured by FRAP of normal and oxidized gallic and ascorbic acid. Data is presented as slope and correlation coefficients of the regression curves.
the absence of hydrogen peroxide and in the presence of dietary antioxidants. Results from this task are shown in Fig. 3. The three peroxidases tested showed the same behaviour and in a dosedependent manner observed in previous experiments. It was interesting to observed, however, different catalytic levels (measured by fluorescence intensity) between peroxidases. HRP showed the highest Amplex Red oxidation rate and lactoperoxidase the lowest; whose different reactivity levels may be modulate by the different protein environments between peroxidases.
Evaluation of the spontaneous formation of hydrogen peroxide and its implications in the reaction system Results from previous experiments demonstrate an interaction between dietary antioxidants and peroxidases that promotes the oxidation of Amplex Red in the absence of added hydrogen peroxide. However, some authors have described spontaneous formation of hydrogen peroxide under cell culture conditions, resulting from the reaction of polyphenols with yet unknown cell culture media
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A
Gallic acid 10000
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1 0
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Gallic acid concentration (microM)
Gallic acid + HRP
B
Gallic acid - HRP
Ascorbic acid.
Fluorescence intensity
10000
1000
100
10
1 0
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Ascorbic acid concentration (microM) Ascorbic acid + HRP
Ascorbic acid - HRP
Fig. 2. Interactions between Amplex Red and dietary antioxidants in the presence or absence of HRP. Fluorescence due to the oxidation of Amplex Red to resorufin with a linear dose-dependent manner (up to a concentration of 1 mM) was observed only in the presence of HRP. No fluorescence was observed in reaction wells without HRP. Reactions in the presence of HRP are presented as () and without HRP as (j).
constituents [5]. It has been suggested that metal-ion catalysed oxidation, especially involving iron ions, is responsible for the hydrogen peroxide generation. In this way, the observed interference could be due to the indirect production of hydrogen peroxide in the media. To evaluate the possible spontaneous hydrogen peroxide formation, catalase at different concentrations (1–7 mg/dL) was added to the reaction system, results are shown in Fig. 4. Spontaneous hydrogen peroxide formation may produce an increase in the rate of oxidation of Amplex Red, the incorporation of catalase to the system would reduce hydrogen peroxide in the medium and therefore reduce the oxidation of Amplex Red. However, it was observed that catalase was unable to inhibit the oxidation of Amplex Red to resorufin, showing that hydrogen peroxide is not produced in the reaction medium and therefore not involved as a catalyst in the oxidation of Amplex Red. Moreover, an increase in the rate of oxidation of Amplex Red was observed at higher concentrations of catalase, maybe because of the inhibition of the oxidation of dietary antioxidants.
Discussion Amplex Red/HRP enzymatic coupled assays have been employed in several biochemical studies for the quantification of low concentrations of hydrogen peroxide. In this work, it was demonstrated that some well-known dietary antioxidants enhance the oxidation of Amplex Red catalyzed by HRP. First, it was suggested a possible direct interaction, via redox reaction, between dietary antioxidants and Amplex Red. However, oxidation of Amplex Red by dietary antioxidants is difficult to explain and no Amplex Red oxidation was observed in the absence of peroxidase, indicating the essentiality of peroxidase to catalyze Amplex Red oxidation. In this context a direct interaction between dietary antioxidants and peroxidases was suggested. Peroxidases are often described as enzymes with ‘‘broad substrate specificity” because of the relative small number of restrictions limiting the choice of reducing substrates [6]. Phenolics are known substrates for peroxidases [7–10]. A typical mechanism of
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Fluorescence intensity
A
Gallic acid 10000
8000
6000
4000
2000
0 25
75
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Gallic acid concentration (microM) HRP
Fluorescence intensity
B
Lactoperoxidase
Myeloperoxidase
Ascorbic acid 4000
3000
2000
1000
0 25
75
125
175
225
Ascorbic acid concentration (microM) HRP
Lactoperoxidase
Myeloperoxidase
Fig. 3. Amplex Red reaction catalyzed by HRP (d) in the presence of dietary antioxidants is also catalyzed by myeloperoxidase (j) and lactoperoxidase (N).
Fluorescence intensity
14000
12000
10000
8000
6000
4000 0
1
2
3
4
5
6
7
8
Catalase (mg/dL) Gallic acid
Ascorbic acid
Fig. 4. Oxidation of Amplex Red to resorufin catalyzed by HRP and gallic acid (j) or ascorbic acid () (2 mM) in the presence of different concentrations of catalase (mg/dL). No inhibition of Amplex Red oxidation was observed; moreover the oxidation of Amplex Red is increased with the addition of catalase.
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O2
dietary antioxidant
Fe(II)
Fe(III) Ferric enzyme ground state
(1)
H+
Ferrous enzyme
O-
.O
H+
Fe(III) (2)
Compound III
(3)
Fe(III)
+
O2-
Ferric enzyme ground state
dietary antioxidant
(4) Antioxidant radical
+ Resorufin
HRP Compound III
Amplex Red
(Ex/Em 571/585 nm)
(5) Fig. 5. Possible mechanism of interaction of dietary antioxidants with HRP in the Amplex Red-coupled-fluorescent assay. Dietary antioxidants may directly reduce ferric HRP to the ferrous form (1), which, being highly unstable because for its very high affinity for O2 [15,16] gives rise to compound III (2). Compound III can undergo spontaneous decay to ferriperoxidase [16] with the generation of O2 (3) [13,15,17]. The plausible interaction of O2 with ferro or ferriperoxidases is prevented by high concentrations of dietary antioxidants in the system (4). Where dietary antioxidants may react with O2 and an antioxidant radical may be produced.
their oxidation involves a catalytic pathways in which the enzyme in the native (ferric) state is oxidized by hydrogen peroxide to a highly reactive form, peroxidase compound I, containing two electron-deficient centres. In the subsequent two steps, compound I is converted back to its ferric form at the expense of two molecules of substrate, which undergo oxidation to free radicals. However, the reaction observed in this system was produced in the absence of hydrogen peroxide. In the absence of hydrogen peroxide or organic hydroperoxides, peroxidases show oxidase activity. Although the mechanism of this reaction is not completely understood, it is apparently initiated by the reduction of ferric HRP to ferrous HRP, which then reacts with molecular oxygen to form compound III [11]. One example of these types of reactions is the indole-3-acetic acid peroxidase catalyzed reaction that take place without added hydrogen peroxide [12], where the ferrous enzyme/compound III shuttle (native enzyme ? ferrous enzyme ? compound III ? ferrous enzyme) [13] is produced. This reaction is exclusively produced in aerobic conditions [14]. It is also described a peroxidase–oxidase reaction with other substrates such as NADH and dihydroxy fumarate, where utilization of oxygen results in the formation of superoxide rather than an organic hydroperoxide. Taking into account this information, its appears that the reaction observed in the Amplex Red system employed in the presence of dietary antioxidants, follows the peroxidase oxidative pathways, principally due to the lack of hydrogen peroxide in the medium. Fig. 5 shows a possible mechanism of action of the interaction observed between dietary antioxidants with HRP in the Amplex Red-coupled-fluorescent assay. In the first step, dietary antioxidants may directly reduce ferric HRP to the ferrous form (1), which, being highly unstable because for its very high affinity for O2 [15,16] gives rise to compound III (2). Compound III can undergo spontaneous decay to ferriperoxidase [16] with the generation of O2 (3) [13,15,17]. The plausible interaction of O2 with ferro or ferriperoxidases could be prevented by high concentrations of dietary antioxidants in the system (4). Where dietary antioxidants may react with O2 and an antioxidant radical may be produced which may act as hydrogen peroxide in the peroxidative oxidation of Amplex Red (5) described in the presence of hydrogen peroxide.
The observed oxidation rate of Amplex Red to resorufin in the presence of dietary antioxidants at physiological doses was higher than that produced by hydrogen peroxide and may interfere with the quantification of hydrogen peroxide if antioxidants are present in the medium resulting in its over quantification. Therefore, particular attention must be taken when employing this assay, especially when low concentrations of hydrogen peroxide are expected. Acknowledgments This study was supported by Grants from the Spanish Ministry of Science and Innovation (CENIT MET-DEV-FUN) to M. PorteroOtín, the Spanish Instituto de Salud Carlos III (FIS PI081238) to J. Boada and (FIS PI081843) to M. Portero-Otin and by the COST action B35. We thank S. Arenas-Soria, M. Martí and D. Argiles for their skillful technical assistance. References [1] A. Gomes, E. Fernandes, J.L.F.C. Lima, Fluorescent probes used for detection of reactive oxygen species, J. Biochem. Biophys. Methods 65 (2005) 45–80. [2] M. Zhou, Z. Diwu, N. Panchuk-Voloshina, R.P. Haugland, A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases, Anal. Biochem. 253 (1997) 162–168. [3] I. Yamazaki, L.H. Piette, The mechanism of aerobic oxidase reaction catalyzed by peroxidase, Biochim. Biophys. Acta 77 (1963) 47–64. [4] I.F.F. Benzie, J.J. Strain, The ferric reducing ability of plasma (FRAP) as a measure of ‘antioxidant power’: the FRAP assay, Anal. Biochem. 239 (1996) 70–76. [5] L.H. Long, M.V. Clement, B. Halliwell, Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of ( )-epigallocatechin, ( )-epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media, Biochem. Biophys. Res. Commun. 273 (2000) 50–53. [6] N.C. Veich, Structural determinants of plant peroxidases function, Phytochem. Rev. 3 (2004) 3–18. [7] D. Job, H.B. Dunford, Substituent effect on the oxidation of phenols and aromatic amines by horseradish peroxidase compound I, Eur. J. Biochem. 66 (1976) 607–614. [8] E. Monzani, A.L. Gatti, A. Profumo, L. Casella, M. Gullotti, Oxidation of phenolic compounds by lactoperoxidase: evidence for the presence of a low-potential compound II during catalytic turnover, Biochemistry 36 (1997) 1918–1926.
J. Serrano et al. / Biochemical and Biophysical Research Communications 388 (2009) 443–449 [9] A.J. Kettle, C.C. Winterbourn, Oxidation of hydroquinone by myeloperoxidase: mechanism of stimulation by benzoquinone, J. Biol. Chem. 267 (1992) 8319– 8324. [10] U. Burner, G. Krapfenbauer, P.G. Furtmuller, G. Regelsberger, C. Obinger, Oxidation of hydroquinone, 2,3-dimethylhydroquinone, and 2,3,5-trimethylhydroquinone by human myeloperoxidase, Redox Rep. 5 (2000) 185–190. [11] G.I. Berglund, G.H. Carlsson, A.T. Smith, H. Szöke, A. Henriksen, J. Hajdu, The catalytic pathway of horseradish peroxidase at high resolution, Nature 417 (2002) 463–468. [12] N.C. Veich, Horseradish peroxidase: a modern view of a classic enzyme, Phytochemistry 65 (2004) 249–259. [13] A.M. Smith, W.L. Morison, P.J. Milham, Oxidation of indole-3-acetic acid by peroxidase: involvement of reduce peroxidase and compound III with superoxide as a product, Biochemistry 21 (1982) 4414–4419.
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[14] I.G. Gazaryan, L.M. Lagrimini, G.A. Ashby, N.F. Thorneley, Mechanism of indole3-acetic acid oxidation by plant peroxidases: anaerobic stopped-flow spectrophotometric studies on horseradish and tobacco peroxidases, Biochem. J. 313 (1996) 841–847. [15] C.F. Phepls, E. Antonini, G. Giacometti, M. Brunori, The kinetics of oxidation of ferroperoxidase by molecular oxygen. A model of a terminal oxidase, Biochem. J. 141 (1975) 265–272. [16] J.B. Wittenberg, R.W. Noble, B.A. Wittenberg, E. Antonini, M. Brunori, J. Wyman, Studies on the equilibria and kinetics of the reactions of peroxidase with ligands. II. The reaction of ferroperoxidase with oxygen, J. Biol. Chem. 242 (1967) 626–634. [17] I. Yamazaki, K. Yokota, Conversion of ferrous peroxidase into compound III in the presence of NADH, Biochem. Biophys. Res. Commun. 19 (1965) 249–254.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Dietary antioxidants interfere with Amplex Red-coupled-fluorescence assays José Serrano *, Mariona Jové, Jordi Boada, María Josep Bellmunt, Reinald Pamplona, Manuel Portero-Otín Department of Experimental Medicine, Faculty of Medicine, University of Lleida-IRBLleida, Lleida, Spain
a r t i c l e
i n f o
Article history: Received 21 July 2009 Available online 11 August 2009 Keywords: Amplex Red Dietary antioxidants Horseradish peroxidase Interference
a b s t r a c t Oxidation of Amplex Red by hydrogen peroxide in the presence of horseradish peroxidase (HRP) gives rise to an intensely colour product, resorufin. This reaction has been frequently employed for measurements based on enzyme-coupled reactions that detect hydrogen peroxide as a final reaction product. In the current study, we show that the presence of dietary antioxidants at biological concentrations in the reaction medium produced interferences in the Amplex Red/HRP catalyzed reaction that result in an over quantification of the hydrogen peroxide produced. The interference observed showed a dose-dependent manner, and a possible mechanism of interaction of dietary antioxidants with HRP in the Amplex Red-coupled-fluorescent assay is proposed. Ó 2009 Elsevier Inc. All rights reserved.
Introduction 10-Acetyl-3,7-dihydroxyphenoxazine, Amplex Red, is a nonfluorescent molecule that when oxidized by hydrogen peroxide in the presence of horseradish peroxidase (HRP) originates resorufin, a highly fluorescent product (kex = 563 nm; kem = 587 nm). This reaction has been frequently employed for measurements of low concentrations of hydrogen peroxide in biological samples, since Amplex Red oxidation consumes stoichiometric amounts of hydrogen peroxide. As its advantages, Amplex Red presents low background fluorescence (this probe does not emit fluorescences, only its oxidation products) as well as stability and high fluorescence intensity of its oxidation product, all this resulting in a sensibility increase for detecting hydrogen peroxide [1]. The assay is highly sensitive, allowing measurements of hydrogen peroxide concentrations as low as 50 nM [2]. However, the accuracy of such determinations also depends on the specificity of the assay system. When considering biological fluids, there are several compounds that could potentially interfere with the Amplex Red reaction catalyzed by HRP. The molecular mechanism of oxidation by HRP is unusually complex as it also catalyzes oxidation of several products in the absence of hydrogen peroxide (oxidation pathway). HRP exhibits oxidase activity toward a number of substrates in the absence of hydrogen peroxide under aerobic conditions. Among various substrates, dihydroxyfuAbbreviation: HRP, horseradish peroxidase. * Corresponding author. Address: Department of Experimental Medicine, Faculty of Medicine, University of Lleida-IRBLleida, C/Montserrat Roig 2, 25008 Lleida, Spain. Fax: +34 973 70 2426. E-mail address: [email protected] (J. Serrano). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.08.041
merate, indole-3-acetic acid, indole butyrate, oxalate, dihydroxytartarate, phenylacetaldehydes, NADH, and NADPH may be mentioned as a few which are oxidized by the HRP oxidase system [3]. Unpublished data from our group, demonstrate an over estimation of resorufin (measured by fluorescence) in the determination of peroxide hydrogen production by an Amplex Red enzymatic assay kit (Molecular Probes A12216) when a polyphenol extract was added to the reaction medium. This suggested that polyphenols contained in the extracts can promote the formation of resorufin from Amplex Red, and may thus be an important source of unspecific signals when using Amplex Red for the quantification of hydrogen peroxide production from enzymatic-substrate determination. The aim of this study was to demonstrate a dose-dependent interaction between well-known dietary antioxidants (gallic acid, epicatechin, quercetin, vanillic acid and ascorbic acid) with the HRP–Amplex Red assay systems. A serial of five experiments were followed to confirm the interference produced. The observed interactions with dietary antioxidants must be taken into account in further experiments when using the Amplex Red assay for measuring hydrogen peroxide. A potential molecular mechanism to explain this phenomenon is proposed. Materials and methods Chemicals. Amplex Red and horseradish peroxidase (HRP) were obtained from Molecular Probes from the Cholesterol Assay kit (A12216). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water soluble analogue of vitamin E (238813), gallic acid (398225), ascorbic acid (A4544), ( )-epicatechin (E4018), quercetin (Q3001), glucose (G8270), myeloperoxidase (M6908),
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J. Serrano et al. / Biochemical and Biophysical Research Communications 388 (2009) 443–449
lactoperoxidase (L8257) and catalase (C9322) were purchased from Sigma–Aldrich. 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) (93285) and vanillic acid (94770) were from Fluka Chemicals (Sigma–Aldrich). All reagents used were of analytical grade. Amplex Red assay. The Amplex Red assay was developed in a 96 well plate by the reaction of 50 lL of Amplex Red working solution with 50 lL of assay sample. Five milliliters of working solution, prepared prior the analysis, contained 75 lL of a 300 lM of Amplex Red reagent and 2 U/mL of HRP, lactoperoxidase or myeloperoxidase. The working solution volume was adjusted to 5 mL with reaction buffer, which contained 25 mM potassium phosphate, pH 7.4, 12.5 mM NaCl, 1.25 mM cholic acid and 0.025% TritonÒ X-100. The reactions were incubated for 30 min at 37 °C, protected from light. After incubation, fluorescence was measured in a fluorescence microplate reader (Tecan Infinite M200, Grödig, Austria) using excitation wavelength at 560 nm and emission detection at 590 nm. FRAP assay. For the FRAP assay [4], 900 lL of the FRAP reagent, containing TPTZ, FeCl3, and acetate buffer, was mixed with 90 lL of distilled water and 30 lL of the test sample of the blank (solvents used for extraction). Maximum absorbance values at 595 nm were taken every 15 s at 37 °C, using a Beckman DU-640 spectrophotometer (Beckman Instruments Inc., Fullerton, CA). The readings at 30 min were selected for calculations of FRAP values. Solutions of known Trolox concentration were used for antioxidant capacity equivalents.
Red. Deserves an especial attention the different degree of reactivity between the dietary antioxidant tested at equimolar concentrations. The observed differences may be due to different molecular interactions between antioxidants and HRP. Oxidation of Amplex Red depends on the antioxidant capacity of dietary antioxidants The previous task showed some relationship between the oxidation of Amplex Red with the antioxidant capacity of dietary antioxidants. The aim of this task was to demonstrate that the interaction observed is related to the antioxidant capacity of dietary antioxidant. To do this, Amplex Red reaction was carried on with two different degrees of antioxidant capacity of gallic and ascorbic acid (in normal conditions prepared immediately after the reaction and in an oxidized form produced at room temperature during 1 week) the lost of the antioxidant capacity was evaluated by the FRAP antioxidant capacity assay. The results are shown in Fig. 1, where the ability of gallic and ascorbic acids to induce the oxidation of Amplex Red to resorufin (measured by the intensity of fluorescence) was reduced considerably with the reduction of the antioxidant capacity (measured by FRAP) of the dietary antioxidant under study. However, although the compounds under study showed a lower antioxidant capacity, they were able to induce the oxidation of Amplex Red, but to a lesser degree. Interactions between Amplex Red and dietary antioxidants in the presence or absence of HRP
Results Dose-dependent manner interference The first task was to determine if the observed interference was also induced by well-known dietary antioxidants (gallic acid, ascorbic acid, epicatechin, quercetin, vanillic acid) in a concentration dependent manner. Glucose was used as a negative control, as a non-antioxidant molecule. All dietary antioxidant tested were able to induce the oxidation of Amplex Red to resorufin catalyzed by HRP, being dietary polyphenols (( )-epicatechin, gallic acid, quercetin) the species which showed the highest reactivity in the tested conditions. Table 1 shows the slope and the correlation coefficient of the calculated linear regression of the dose dependent behaviour observed. No hydrogen peroxide was added or indented to be produced in the reaction medium, and no fluorescence was observed by dietary antioxidants in the reaction medium without Amplex Red, demonstrating the ability of dietary antioxidants to induce Amplex Red oxidation in the absence of hydrogen peroxide. Glucose used as a negative control do not produced resorufin. Antioxidant capacity, measured by FRAP assay, was performed in parallel to evaluate the possible correlation between the antioxidant capacity and the rate of resorufin formation. No statistical correlation was observed, despite dietary antioxidants with higher antioxidant capacity showed a higher oxidation rate of Amplex
With the above tasks it has been shown that dietary antioxidants are able of inducing oxidation of Amplex Red catalyzed by HRP. It has also been shown that the interaction can be observed due to the antioxidant capacity of the compounds under study. The next question to resolve was whether the observed reaction is due to direct interactions between dietary antioxidants and Amplex Red. However, the ‘‘oxidation” of Amplex Red by an ‘‘antioxidant” seems to be difficult to explain. Therefore, it was considered necessary the presence of HRP in the reaction to catalyze the reaction. To demonstrate this hypothesis, the degree of oxidation of Amplex Red by gallic and ascorbic acid was assessed in the presence and absence of HRP in the reaction medium. The results of this task are shown in Fig. 2, where no formation of resorufin was observed in the reaction wells without HRP, showing that the enzymatic activity of HRP is essential in the oxidation reaction of Amplex Red. Oxidation of Amplex Red catalyzed by different peroxidases in the presence of dietary antioxidants The forth task was to demonstrate that other Haem-containing redox enzymes like lactoperoxidase (found in milk) and myeloperoxidase (most abundantly present in neutrophil granulocytes) are able to work as catalyst in the oxidation of Amplex Red, also in
Table 1 Dose-dependent reactivity of different dietary antioxidants when added in the Amplex Red reaction mixture. Antioxidants tested shown linear correlation between the amount of antioxidants and the fluorescence emitted (excitation wavelength 560 nm, emission wavelength 590 nm), while glucose (without antioxidant capacity, demonstrated by the FRAP assay) showed almost non-fluorescence. The slope represents the increase in fluorescence per microM of antioxidant in the reaction media (Amplex Red) and the Trolox (a water soluble analogue of vitamin E) equivalency in the FRAP antioxidant capacity assay. Antioxidant
Gallic acid Ascorbic acid ( )-Epicatechin Quercetin Vanillic acid Glucose
Amplex Red reaction
Antioxidant capacity – FRAP 2
Slope
R
p-Value
Slope
R2
p-Value
23.686 3.485 55.390 20.462 15.545 0.203
0.9574 0.9721 0.8478 0.8767 0.8576 0.2379
0.000 0.000 0.009 0.001 0.002 0.220
0.0012 0.0009 0.0023 0.0035 0.0012 0.00005
0.9766 0.9535 0.9962 0.9732 0.9931 0.2889
0.000 0.000 0.000 0.000 0.000 0.109
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A Fluorescence intensity
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Concentration microM Gallic acid
Oxidized Gallic acid
Fluorescence intensity
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B
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Ascorbic Acid p-value
Slope
R2
p-value
Normal
8.6329 0.9999
0.000
2.7761 0.9967
0.000
Oxidized
0.2661 0.9451
0.000
0.0215 0.0706
0.320
Fig. 1. The oxidation of resorufin or resorufin catalyzed by dietary antioxidants and HRP is related to the antioxidant capacity of dietary antioxidants. (A) Fluorescence intensity produced after the reaction of Amplex Red catalyzed by HRP in the presence of gallic and ascorbic acid () and oxidized gallic and ascorbic acid (j). (B) Antioxidant capacity measured by FRAP of normal and oxidized gallic and ascorbic acid. Data is presented as slope and correlation coefficients of the regression curves.
the absence of hydrogen peroxide and in the presence of dietary antioxidants. Results from this task are shown in Fig. 3. The three peroxidases tested showed the same behaviour and in a dosedependent manner observed in previous experiments. It was interesting to observed, however, different catalytic levels (measured by fluorescence intensity) between peroxidases. HRP showed the highest Amplex Red oxidation rate and lactoperoxidase the lowest; whose different reactivity levels may be modulate by the different protein environments between peroxidases.
Evaluation of the spontaneous formation of hydrogen peroxide and its implications in the reaction system Results from previous experiments demonstrate an interaction between dietary antioxidants and peroxidases that promotes the oxidation of Amplex Red in the absence of added hydrogen peroxide. However, some authors have described spontaneous formation of hydrogen peroxide under cell culture conditions, resulting from the reaction of polyphenols with yet unknown cell culture media
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Gallic acid 10000
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Gallic acid concentration (microM)
Gallic acid + HRP
B
Gallic acid - HRP
Ascorbic acid.
Fluorescence intensity
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Ascorbic acid concentration (microM) Ascorbic acid + HRP
Ascorbic acid - HRP
Fig. 2. Interactions between Amplex Red and dietary antioxidants in the presence or absence of HRP. Fluorescence due to the oxidation of Amplex Red to resorufin with a linear dose-dependent manner (up to a concentration of 1 mM) was observed only in the presence of HRP. No fluorescence was observed in reaction wells without HRP. Reactions in the presence of HRP are presented as () and without HRP as (j).
constituents [5]. It has been suggested that metal-ion catalysed oxidation, especially involving iron ions, is responsible for the hydrogen peroxide generation. In this way, the observed interference could be due to the indirect production of hydrogen peroxide in the media. To evaluate the possible spontaneous hydrogen peroxide formation, catalase at different concentrations (1–7 mg/dL) was added to the reaction system, results are shown in Fig. 4. Spontaneous hydrogen peroxide formation may produce an increase in the rate of oxidation of Amplex Red, the incorporation of catalase to the system would reduce hydrogen peroxide in the medium and therefore reduce the oxidation of Amplex Red. However, it was observed that catalase was unable to inhibit the oxidation of Amplex Red to resorufin, showing that hydrogen peroxide is not produced in the reaction medium and therefore not involved as a catalyst in the oxidation of Amplex Red. Moreover, an increase in the rate of oxidation of Amplex Red was observed at higher concentrations of catalase, maybe because of the inhibition of the oxidation of dietary antioxidants.
Discussion Amplex Red/HRP enzymatic coupled assays have been employed in several biochemical studies for the quantification of low concentrations of hydrogen peroxide. In this work, it was demonstrated that some well-known dietary antioxidants enhance the oxidation of Amplex Red catalyzed by HRP. First, it was suggested a possible direct interaction, via redox reaction, between dietary antioxidants and Amplex Red. However, oxidation of Amplex Red by dietary antioxidants is difficult to explain and no Amplex Red oxidation was observed in the absence of peroxidase, indicating the essentiality of peroxidase to catalyze Amplex Red oxidation. In this context a direct interaction between dietary antioxidants and peroxidases was suggested. Peroxidases are often described as enzymes with ‘‘broad substrate specificity” because of the relative small number of restrictions limiting the choice of reducing substrates [6]. Phenolics are known substrates for peroxidases [7–10]. A typical mechanism of
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Fluorescence intensity
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Gallic acid 10000
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Gallic acid concentration (microM) HRP
Fluorescence intensity
B
Lactoperoxidase
Myeloperoxidase
Ascorbic acid 4000
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0 25
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125
175
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Ascorbic acid concentration (microM) HRP
Lactoperoxidase
Myeloperoxidase
Fig. 3. Amplex Red reaction catalyzed by HRP (d) in the presence of dietary antioxidants is also catalyzed by myeloperoxidase (j) and lactoperoxidase (N).
Fluorescence intensity
14000
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1
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3
4
5
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Catalase (mg/dL) Gallic acid
Ascorbic acid
Fig. 4. Oxidation of Amplex Red to resorufin catalyzed by HRP and gallic acid (j) or ascorbic acid () (2 mM) in the presence of different concentrations of catalase (mg/dL). No inhibition of Amplex Red oxidation was observed; moreover the oxidation of Amplex Red is increased with the addition of catalase.
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O2
dietary antioxidant
Fe(II)
Fe(III) Ferric enzyme ground state
(1)
H+
Ferrous enzyme
O-
.O
H+
Fe(III) (2)
Compound III
(3)
Fe(III)
+
O2-
Ferric enzyme ground state
dietary antioxidant
(4) Antioxidant radical
+ Resorufin
HRP Compound III
Amplex Red
(Ex/Em 571/585 nm)
(5) Fig. 5. Possible mechanism of interaction of dietary antioxidants with HRP in the Amplex Red-coupled-fluorescent assay. Dietary antioxidants may directly reduce ferric HRP to the ferrous form (1), which, being highly unstable because for its very high affinity for O2 [15,16] gives rise to compound III (2). Compound III can undergo spontaneous decay to ferriperoxidase [16] with the generation of O2 (3) [13,15,17]. The plausible interaction of O2 with ferro or ferriperoxidases is prevented by high concentrations of dietary antioxidants in the system (4). Where dietary antioxidants may react with O2 and an antioxidant radical may be produced.
their oxidation involves a catalytic pathways in which the enzyme in the native (ferric) state is oxidized by hydrogen peroxide to a highly reactive form, peroxidase compound I, containing two electron-deficient centres. In the subsequent two steps, compound I is converted back to its ferric form at the expense of two molecules of substrate, which undergo oxidation to free radicals. However, the reaction observed in this system was produced in the absence of hydrogen peroxide. In the absence of hydrogen peroxide or organic hydroperoxides, peroxidases show oxidase activity. Although the mechanism of this reaction is not completely understood, it is apparently initiated by the reduction of ferric HRP to ferrous HRP, which then reacts with molecular oxygen to form compound III [11]. One example of these types of reactions is the indole-3-acetic acid peroxidase catalyzed reaction that take place without added hydrogen peroxide [12], where the ferrous enzyme/compound III shuttle (native enzyme ? ferrous enzyme ? compound III ? ferrous enzyme) [13] is produced. This reaction is exclusively produced in aerobic conditions [14]. It is also described a peroxidase–oxidase reaction with other substrates such as NADH and dihydroxy fumarate, where utilization of oxygen results in the formation of superoxide rather than an organic hydroperoxide. Taking into account this information, its appears that the reaction observed in the Amplex Red system employed in the presence of dietary antioxidants, follows the peroxidase oxidative pathways, principally due to the lack of hydrogen peroxide in the medium. Fig. 5 shows a possible mechanism of action of the interaction observed between dietary antioxidants with HRP in the Amplex Red-coupled-fluorescent assay. In the first step, dietary antioxidants may directly reduce ferric HRP to the ferrous form (1), which, being highly unstable because for its very high affinity for O2 [15,16] gives rise to compound III (2). Compound III can undergo spontaneous decay to ferriperoxidase [16] with the generation of O2 (3) [13,15,17]. The plausible interaction of O2 with ferro or ferriperoxidases could be prevented by high concentrations of dietary antioxidants in the system (4). Where dietary antioxidants may react with O2 and an antioxidant radical may be produced which may act as hydrogen peroxide in the peroxidative oxidation of Amplex Red (5) described in the presence of hydrogen peroxide.
The observed oxidation rate of Amplex Red to resorufin in the presence of dietary antioxidants at physiological doses was higher than that produced by hydrogen peroxide and may interfere with the quantification of hydrogen peroxide if antioxidants are present in the medium resulting in its over quantification. Therefore, particular attention must be taken when employing this assay, especially when low concentrations of hydrogen peroxide are expected. Acknowledgments This study was supported by Grants from the Spanish Ministry of Science and Innovation (CENIT MET-DEV-FUN) to M. PorteroOtín, the Spanish Instituto de Salud Carlos III (FIS PI081238) to J. Boada and (FIS PI081843) to M. Portero-Otin and by the COST action B35. We thank S. Arenas-Soria, M. Martí and D. Argiles for their skillful technical assistance. References [1] A. Gomes, E. Fernandes, J.L.F.C. Lima, Fluorescent probes used for detection of reactive oxygen species, J. Biochem. Biophys. Methods 65 (2005) 45–80. [2] M. Zhou, Z. Diwu, N. Panchuk-Voloshina, R.P. Haugland, A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases, Anal. Biochem. 253 (1997) 162–168. [3] I. Yamazaki, L.H. Piette, The mechanism of aerobic oxidase reaction catalyzed by peroxidase, Biochim. Biophys. Acta 77 (1963) 47–64. [4] I.F.F. Benzie, J.J. Strain, The ferric reducing ability of plasma (FRAP) as a measure of ‘antioxidant power’: the FRAP assay, Anal. Biochem. 239 (1996) 70–76. [5] L.H. Long, M.V. Clement, B. Halliwell, Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of ( )-epigallocatechin, ( )-epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media, Biochem. Biophys. Res. Commun. 273 (2000) 50–53. [6] N.C. Veich, Structural determinants of plant peroxidases function, Phytochem. Rev. 3 (2004) 3–18. [7] D. Job, H.B. Dunford, Substituent effect on the oxidation of phenols and aromatic amines by horseradish peroxidase compound I, Eur. J. Biochem. 66 (1976) 607–614. [8] E. Monzani, A.L. Gatti, A. Profumo, L. Casella, M. Gullotti, Oxidation of phenolic compounds by lactoperoxidase: evidence for the presence of a low-potential compound II during catalytic turnover, Biochemistry 36 (1997) 1918–1926.
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