Article pubs.acs.org/JAFC
Overcoming Reductant Interference in Peroxidase-Based Assays for Hydrogen Peroxide Quantification Shu Jiang and Michael H. Penner* Department of Food Science and Technology, Oregon State University, Corvallis, Oregon 97331-6602, United States ABSTRACT: A problem commonly encountered when using peroxidase-based methods for hydrogen peroxide quantification in biobased matrixes is interference due to the presence of endogenous reductants. Such assays are typically based on the generation of an oxidized reporter molecule in direct proportion to the amount of hydrogen peroxide reduced in the peroxidase-catalyzed reaction. Endogenous reductants confound such assays by reducing the oxidized reporter molecule, thus resulting in underestimates of hydrogen peroxide content. In the present work, we demonstrate how this problem can be circumvented by selectively oxidizing offending compounds by treatment with the oxidized reporter molecule prior to initiating the peroxidase reaction for hydrogen peroxide quantification. The approach is demonstrated using horseradish peroxidase, 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid), as the reporter molecule and a representative garlic paste as the hydrogen peroxidecontaining biobased matrix. The approach is expected to be generally applicable to a wide range of peroxidase-based assays when applied to complex biobased systems. KEYWORDS: hydrogen peroxide, peroxidase, reductants, ABTS, spectrophotometric quantification
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INTRODUCTION Hydrogen peroxide is a reactive oxygen species of general relevance to biochemical systems. It is naturally present in cells at relatively low amounts where, depending on the circumstances, it is associated with either beneficial or detrimental consequences.1,2 It is widely used in industrial biomaterial processing due to its oxidizing,3 bleaching,4 and/or antimicrobial5 properties (e.g., the pulp,6 textile,7 food,8,9 dental,10 cosmetic,11 and forensic12 industries). The quantification of hydrogen peroxide in biobased systems is thus relevant to a wide range of applications, from fundamental questions of cellular metabolism to applied aspects of industrial bioprocessing. It is generally recognized that many of the current methods used to quantify hydrogen peroxide are of questionable applicability when applied to biological matrixes due to the confounding nature of endogenous components.13 Advances in approaches toward hydrogen peroxide quantification in biological systems are thus needed to extend the applicability of existing methods. There are several approaches to the quantification of hydrogen peroxide.14 The “best” method for any particular application will be based on typical factors: sensitivity, detection limit, nature of interfering compounds, equipment required, etc. A common approach to the quantification of hydrogen peroxide in biological systems is based on the use of peroxidase-catalyzed hydrogen peroxide-consuming reactions that change the optical properties of a reporter molecule. Reporter molecules are typically reducing substrates in the peroxidase-catalyzed reaction. Such assays include those based on spectrophotometric,15 fluorimetric,16 and chemiluminescent17 techniques. A situation that often limits the interpretation of such methods is the presence of confounding compounds that alter the stoichiometry of the peroxidasecatalyzed reaction, i.e., alter the number of moles of reporter molecules oxidized per mole of hydrogen peroxide consumed.18 One approach to dealing with this problem is to try to mathematically correct for the aberrant stoichiometry by © XXXX American Chemical Society
accounting for the reactivity of the different confounding compounds, provided such reactivity is known.19 A second approach is to separate the hydrogen peroxide from confounding compounds prior to its quantification, e.g., via the use of solid phase extractions and/or chromatography.20 The former of these separation techniques is typically rather nonspecific, and the latter requires relatively sophisticated equipment, is timeconsuming, and is difficult to incorporate into rapid automated/semiautomated analytical systems. Yet another approach, which is the focus of this paper, is to inactivate the confounding compounds in situ prior to hydrogen peroxide quantification. The objective of this study was to evaluate the potential of using a heretofore unexplored in situ method to deal with sample-containing components capable of interfering with peroxidase-based assays for hydrogen peroxide quantification (i.e., a novel method to deal with compounds that confound peroxidase-based assays). The presented method is based on the selective oxidation of confounding compounds prior to initiating the peroxidase-catalyzed reaction. The term “selective” in this sense indicates that the prequantification oxidative treatment affects only those compounds likely to reduce the oxidized reporter molecule used for hydrogen peroxide quantification. The appropriate oxidizing agent for such a treatment is the oxidized form of the reporter molecule itself, thus ensuring maximum selectivity. This approach specifically inactivates those confounding compounds that react with the oxidized reporter molecule. Confounding compounds that work via this mechanism include a wide range of reductants/antioxidants present in biological systems.21,22 The reporter molecule used in Received: May 13, 2017 Revised: July 28, 2017 Accepted: July 29, 2017
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DOI: 10.1021/acs.jafc.7b02248 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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reduction was then monitored as the decrease in absorbance at 734 nm. At specified times in parallel experiments, 0.08 μmol of supplemental ABTS+• was added to the primary GPE−BTS+• reaction mixtures, while monitoring change in absorbance. The supplemental ABTS+• addition is intended to simulate the generation of ABTS+• that may result from the quantitative hydrogen peroxide/peroxidase reaction. Time Course of Hydrogen Peroxide Degradation in GPE. Hydrogen peroxide-free GPE was spiked with known amounts of hydrogen peroxide to initiate the reaction. At selected times, ABTS+• was added to reaction mixtures to pretreat samples prior to subsequent hydrogen peroxide quantification. Peroxidase was then added to pretreated samples and the assay completed as described for the standard method above. The hydrogen peroxide content of the GPE at the initiation of the experiment was taken as the amount measured for a sample to which a known amount of hydrogen peroxide was added to the ABTS+•-pretreated GPE sample and immediately assayed.
this study was 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). When ABTS is oxidized by hydrogen peroxide via the peroxidase-catalyzed reaction, it yields the corresponding cation radical (ABTS+•). ABTS+• is the molecular form of the reporter molecule monitored in the assay due to it having a relatively high molar absorptivity in the visible region.23 The selective oxidizing agent used in the prequantification treatment phase of the assay is thus ABTS+•. The biobased hydrogen peroxide-containing matrix used throughout the work was a garlic (Allium sativum) paste representative of that used in the food processing industry. The primary outcome of the study is substantiation of the concept of using selective oxidations to extend the applicability of peroxidase-based methods for the quantification of hydrogen peroxide in biobased materials.
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MATERIALS AND METHODS
RESULTS AND DISCUSSION A common problem associated with peroxidase-based assays for hydrogen peroxide quantification is illustrated in Figure 1. The
Reagents. Hydrogen peroxide (30 wt %, ACS reagent grade), horseradish peroxidase (HRP, type II), ABTS [2,2′-azino-bis(3ethylbenzo thiazoline-6-sulfonic acid) diammonium salt], monobasic sodium phosphate, and potassium persulfate were purchased from Sigma-Aldrich, USA. Analyte-Containing Sample. A food-grade commercial garlic paste preparation was used as the biobased sample matrix for all analyses. According to the manufacturer, the paste was a blend of dehydrated garlic, palm oil, soy lecithin, tocopherol, and ascorbyl palmitate. In processing, the paste is treated with hydrogen peroxide and heated. This representative analyte-containing sample is herein referred to as “garlic paste”. Sample Extract. A 5 g portion of garlic paste was mixed with 15 mL of 0.1 M sodium phosphate (pH 6.0) in a 20 mL scintillation vial and vortexed for approximately 3 min. Two mL aliquots of the resulting suspension were added to a series of microcentrifuge tubes and subsequently centrifuged at 10,000 rpm (Jouan A14 centrifuge) for 10 min. Sample-containing tubes were then incubated in an ice-bath for approximately 10 min to solidify lipid components. The resulting tubes contained a pellet at the bottom, an aqueous liquid phase, and a solidified-lipid phase at the top. A needle/syringe was then used to quantitatively remove the liquid phase from each centrifuge tube. The liquid phase was then extracted with hexane (1:1, v/v) and filtered using Milipore Swinnex syringe glass fiber filters. The resulting clear solution is herein referred to as “garlic paste extract” (GPE). Preparation of ABTS+• Solution. ABTS cation radical (ABTS+•) containing solutions were prepared by incubating an aqueous 7 mM ABTS, 2.45 mM potassium persulfate, solution overnight (12−16 h) in the dark, at room temperature.24 The solution was then made 100 mM (sodium) phosphate, pH 6.0, prior to being used in subsequent experiments. Standard Method for Hydrogen Peroxide Quantification (Peroxidase−ABTS Assay). In a typical assay for samples containing hydrogen peroxide in noninterfering buffer systems, 0.2 mL of colorforming reagent (0.86 mM ABTS, 0.1 M sodium phosphate, pH 6.0) was added to 2 mL of H2O2-containing sample. The signal producing reaction was then initiated by adding 100 μL of HRP (100 μg/mL); the absorbance was read after mixing (∼30 s, after which color development was stable) at 734 nm. Enzyme solutions were kept on ice until initiation of the reaction. All reactions were done at ambient temperature. Modified Peroxidase−ABTS Assay Incorporating ABTS+• Pretreatment. In a typical assay, to a given amount of hydrogen peroxide-containing sample (typically from 20 to 200 μL) is added sufficient aqueous ABTS+• solution (prepared as described above) such that, following the pretreatment period, the absorbance is in the range 0.1−0.3 (the extent of the pretreatment period is dependent on the sample; see the Results and Discussion). Peroxidase was then added to the pretreated samples for hydrogen peroxide quantification, as described for the standard method above. Time Course of ABTS+• Reduction in GPE. Time courses were initiated by adding 20 μL of GPE to 4 mL of ABTS+• solutions with initial absorbance readings at 734 nm in the range 0.9−1.0. ABTS+•
Figure 1. Calibration curves for hydrogen peroxide determination in a model buffer system (◆) and in GPE (■) using the HRP/ABTS system. Final values are means ± standard deviation from triplicate measurements. Points without visible error bars have standard deviations smaller than the data points.
figure shows a representative standard curve for the quantification of hydrogen peroxide using the HRP/ABTS assay in a model buffer system. The depicted sensitivity and linearity are typical of many such assays when applied to model systems. The second curve of Figure 1, which reflects the complete absence of signal, covers the same amounts of hydrogen peroxide within a biobased matrix (a garlic paste extract, GPE). The latter assay was initiated by the addition of hydrogen peroxide to GPE containing all of the relevant HRP/ABTS assay components. A plausible explanation for the absence of signal when applying the assay to the GPE matrix is that endogenous reductants in the GPE confound the assay by reacting with the product/reporter molecule (ABTS+•) generated in the peroxidase-catalyzed reaction. This rationale is consistent with HRP-catalyzed ABTS oxidation by hydrogen peroxide being rapid relative to the reactivity of hydrogen peroxide with other components typical of biological systems.2 It is also consistent with the documented reactivity of ABTS+• in biological matrixes, including garlic preparations.25 The likelihood of product modification was verified in the present case in a series of experiments in which ABTS+• was prepared in a buffer system and subsequently added to GPE; in all cases, there was a time-dependent decrease in the absorbance attributed to ABTS+•. In further experiments, fleeting color development was observed in the initial seconds following the addition of relatively high amounts of hydrogen peroxide to GPE samples B
DOI: 10.1021/acs.jafc.7b02248 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry containing ample quantities of ABTS and HRP, thus indicating HRP-catalyzed ABTS+• production had occurred but that the presence of ABTS+• was fleeting. A plausible mechanism for assay interference that is consistent with these observations is presented in Figure 2.
Figure 3. Pictorial description of assay to quantify hydrogen peroxide in reductant-containing matrix. Initially, ABTS+• is added to the hydrogen peroxide-containing matrix (i.e., to the sample). This initiates the oxidation of confounding endogenous reductants as a result of their reaction with ABTS+• to generate ABTS. This phase of the assay corresponds to the decrease in absorbance starting at time zero. Horseradish peroxidase (HRP) is then added to the reaction mixture (vertical dashed line) to initiate hydrogen peroxide quantification based on the increase in absorbance due to the peroxidase-catalyzed hydrogen peroxide-dependent oxidation of ABTS to ABTS+• (steep increase in absorbance starting at the vertical dashed line). Hydrogen peroxide is quantified on the basis of the change in absorbance immediately before and after the addition of HRP.
Figure 2. Plausible mechanism for assay interference. The determination of the concentration of hydrogen peroxide in a sample is based on the amount of ABTS+• formed following initiation of the peroxidasecatalyzed reaction. If hydrogen peroxide/peroxidase-generated ABTS+• is reduced as a consequence of endogenous reductants, then the amount of hydrogen peroxide will be underestimated.
In this scenario, ABTS is oxidized to ABTS+• in the process of peroxidase-catalyzed hydrogen peroxide reduction. This reaction alone should result in color/signal formation. However, as depicted in Figure 2, ABTS+• is reduced back to ABTS by confounding compounds. The net result is diminished ABTS+• accumulation and corresponding underestimates of hydrogen peroxide concentration. This type of interference is expected from compounds that show antioxidant activity.22,23 Indeed, a decrease in signal due to ABTS+• reduction is the basis of the Trolox equivalent antioxidant capacity (TEAC) assay, a widely used assay for quantifying antioxidant activity in foods.26,27 The above interference scenario is not limited to peroxidase-based assays which use ABTS as the reducing substrate/reporter molecule. The problem of endogenous components confounding peroxidase-based assays that incorporate a range of different chromophores, fluorophores, and chemiluminescent compounds as reporter molecules is known.28,29 In most instances, this complication is considered an inherent limitation of directly applying peroxidase-based assays to biological samples without prior analyte separation.20,30 In the present work, the cyclic nature of the ABTS/ABTS+• redox reaction was used to circumvent the problem of reporter molecule reduction by endogenous reductants. This is possible because ABTS+• is a relatively stable radical with sufficient lifetime to allow it to be used as a selective oxidizing agent prior to ABTS being used as the reporter molecule for hydrogen peroxide quantification. The pertinent reactions along with a schematic illustrating the nature of the overall assay, including the prequantification ABTS+• treatment, are shown in Figure 3. The schematic depicts the decrease in signal as the sample is first treated with ABTS+•, during which time endogenous interfering compounds are oxidized as they reduce the added ABTS+• to ABTS. Once this treatment has subsided, HRP is added to the sample mixture to initiate the hydrogen peroxide specific conversion of ABTS back to ABTS+•. The analytical signal attributed to hydrogen peroxide is thus the difference in the absorbance at the end of the prequantification ABTS+• treatment and the absorbance obtained following HRP addition (which corresponds to newly generated ABTS+•). Potassium persulfate was found to be a convenient reagent for preparing appropriate ABTS+• solutions for the prequantification treatment.31 Appropriate solutions must contain sufficient ABTS+• such that it is in excess relative to the confounding compounds present in the sample being treated. This can be assured by verifying that the relevant absorbance of the
pretreated sample does not decrease to the point that would be observed in the absence of ABTS+• (i.e., at least some excess ABTS+• should be detectable). Having excess ABTS+• present during the HRP phase of the assay is not a problem in this analytical scheme, since the analyte signal is taken as the difference in absorbance before and after the addition of HRP (extremely high ABTS+• levels should be avoided in order to keep the baseline absorbance within a reasonable range). A second consideration with respect to ABTS/ABTS+• concentrations is that the concentration of ABTS, during the HRPreaction phase, must be sufficient to account for all of the hydrogen peroxide in the system. This is confirmed by verifying that supplemental ABTS does not result in an increase in absorbance. Lastly, it is imperative that excess ABTS be included during the preparation of the ABTS+• solution, since the presence of potassium persulfate in the analyte-containing reaction mixture would be problematic. The stoichiometric ratio for the reaction of ABTS with persulfate is 2:1 (ABTS:persulfate).32,33 The inclusion of an ABTS+• treatment to deal with confounding compounds in peroxidase-based assays is appealing due to its simplicity and because it specifically targets those compounds likely to pose a problem in subsequent assays. Importantly, as used herein, ABTS and ABTS+• do not react with hydrogen peroxide in the absence of HRP and, at least with respect to the systems dealt with here, neither do the ABTS+•oxidized confounding compounds generated during the ABTS+• treatment. Hence, the ABTS+• treatment per se does not interfere with the subsequent HRP-based hydrogen peroxide quantification; it simply quenches compounds likely to confound the assay. The analytical approach described herein is applicable when using reporter molecules which when oxidized have sufficient lifetime to be used in the prequantification treatment phase of the assay. ABTS is particularly well suited for this approach due to the relative stability of its cation radical. Many of the traditional reducing substrates used for monitoring peroxidatic reactions, such as guaiacol34 and the benzidine derivatives,35 are not C
DOI: 10.1021/acs.jafc.7b02248 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry appropriate for this approach due to the instability of the radical formed in the peroxidase-catalyzed reaction. Assays based on the latter types of reporter molecules are typically described as follows:36 HRP
H 2O2 + 2AH 2 ⎯⎯⎯⎯→ 2H 2O + 2AH•
(1)
2AH• → polymerized products (AHHA)
(2)
The problem with such reporter molecules is that the polymerized product is stable and, thus, it cannot be used in a treatment step to quench confounding compounds. Keep in mind that confounders are capable of reducing the initially formed radicals prior to their polymerization, thereby decreasing the analytical signal, just as is observed with the ABTS substrate. The key point with respect to doing a prequantification treatment is that ABTS is rather unique in its suitability for this assay approach. The time course of ABTS+• reduction during the prequantification treatment step is expected to be sample-specific. It is preferable that the rate of ABTS+• reduction be negligible by the end of the treatment step, such that changes in absorbance due to ABTS+• reduction are insignificant relative to the amount of ABTS+• generated during peroxidase-dependent hydrogen peroxide reduction. The HRP-catalyzed reaction upon which hydrogen peroxide quantification is based is relatively fast. In the present experiments, the HRP-catalyzed reaction was essentially completed during the time required to prepare samples for absorbance readings ( 0.05) when quantifying hydrogen peroxide in the model matrix versus the ABTS+•-treated GPE matrix. These results demon-
Figure 4. Calibration curves for hydrogen peroxide determination in ABTS+•-treated GPE using different concentrations of ABTS (◆, 0.06 mM; ■, 0.12 mM) in the reaction mixture. Values are means ± standard deviation from triplicate assays. Points without visible error bars have standard deviations smaller than the data points.
strate the effectiveness of selectively oxidizing endogenous confounding compounds prior to performing the HRP-based quantification assay. The data shown in Figures 1 and 4 also address the extent to which ABTS serves as the sole reducing substrate in the hydrogen peroxide-consuming reaction. This is relevant because of the known nonspecificity of the peroxidase enzyme. It is possible that endogenous components may compete with ABTS as a substrate for this reaction, thus lowering the molar absorbance change for the assay. The similarity of the molar absorbance changes in the model and GPE matrixes, as indicated by the similarity in slopes of the calibration curves in the two systems, suggests that competing substrates in GPE are not an issue in the current experimental design. A further experiment comparing molar absorbance yields at different ABTS concentrations supports this interpretation (see overlapping calibration curves of Figure 4); this rationale is based on the assumption that increasing the concentration of ABTS in the reaction mixture would increase molar absorbance yields if there were significant competition with a fixed amount of alternative substrates. Experiments evaluating substrate competition should be conducted when applying this assay to new matrixes, since significant competition by alternative substrates, such as may be the case with some phenolic-rich foods, would result in underestimates of hydrogen peroxide content. The previous two paragraphs discuss data obtained using ABTS+•-treated GPE as the peroxide-containing matrix. Figure 5 shows a representative time course of ABTS+• reduction during the initial preparation of the ABTS+•-treated GPE. This type of data provides important information when considering prequantification ABTS+•-treatment times. The kinetics of ABTS+• reduction in any given matrix will be dependent on the reactivity and concentration of endogenous constituents.37,38 The relatively slow reduction of ABTS+• depicted in Figure 5 is consistent with data from studies using ABTS+• to assess the antioxidant activity of garlic-based products.25 The inset to Figure 5 shows that the rate of decrease in absorbance, attributable to continued ABTS+• reduction, following ABTS+• treatments of 15, 30, 60, 120, and 180 min, was 0.006, 0.004, 0.003, 0.002, and 0.002 absorbance units/min, respectively. These values provide a guide for calculating the extent to which confounding compounds remaining in samples following ABTS+• treatments are likely to affect absorbance changes D
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rapidly changing biological systems. To this point, solvent extraction techniques have been evaluated as a means of stabilizing hydrogen peroxide levels in living tissues prior to subsequent colorimetric analyses.40 A potential complication of using solvent-based enzyme inactivation methods during sample preparation is that the resulting sample may not be compatible with subsequent peroxidase-based assays.) The extent to which the ABTS+• treatment terminates hydrogen peroxide degradation in the GPE matrix used in this study is illustrated in Figure 6
Figure 5. Time course of ABTS+• reduction during treatment of GPE, depicted as a decrease in absorbance attributable to ABTS+•. Initial conditions were 20 μL of GPE in 4 mL of ABTS+• solution. (The time course also reflects the inactivation of GPE components capable of confounding hydrogen peroxide quantification.) The inset depicts initial rates of ABTS+• reduction following the addition of 0.08 μmol of supplemental ABTS+• to GPE samples already treated with ABTS+• for 15 (◆), 30 (■), 60 (▲), 120 (×), and 180 (●) min (the “already treated” GPE corresponds to the depicted 180 min time course). Rates of supplemental ABTS+• reduction (ΔAbs/min), as depicted in the inset, were 0.006, 0.004, 0.003, 0.002, and 0.002 in GPE initially treated with ABTS+• for 15, 30, 60, 120, and 180 min, respectively. These initial rates of ABTS+• reduction provide insight into the fate of ABTS+• likely to be generated as a result of the hydrogen peroxide quantification reaction in ABTS+•-treated GPE. Data points represent means ± standard deviation from triplicate samples. Points without visible error bars have standard deviations smaller than the data points.
Figure 6. Time course of hydrogen peroxide decay in GPE, depicted as a decrease in absorbance. Curve “a” was obtained by simultaneously adding hydrogen peroxide and ABTS+• to GPE and allowing the mixture to react for the specified times prior to initiating the quantification of hydrogen peroxide by the addition of peroxidase. Curve “b” was obtained by adding hydrogen peroxide to GPE and allowing it to react for the specified times prior to adding ABTS+• to terminate reaction and eliminate confounding compounds; peroxidase was subsequently added to the ABTS +• -treated sample to initiate hydrogen peroxide quantification. The inset depicts a first order plot of the data of curve “b”. Data points are means ± standard deviation from triplicate measurements. Points without visible error bars have standard deviations smaller than the data points.
during the hydrogen peroxide quantification reaction. The values are thus to be considered in the context of the HRP-catalyzed hydrogen peroxide-quantifying reaction, which is complete in approximately 30 s. The relatively small corrections occurring during this time frame can be taken into account, if necessary, when calculating hydrogen peroxide levels. Incorporating the ABTS+• pretreatment into peroxidase-based assays is straightforward for those applications where hydrogen peroxide levels are relatively static, i.e., systems in which the concentration of hydrogen peroxide remains essentially constant over the assay period. This is expected to be the case with most processed foods where the relevant enzyme systems have been inactivated. The incorporation of the ABTS+• pretreatment is more complicated when working with dynamic systems, for example, short time frame kinetic studies following changes in hydrogen peroxide levels in nonthermally treated samples. The primary concern when working with unstable systems is that hydrogen peroxide concentrations may change during the prequantification ABTS+• treatment. The extent to which this may be a factor is dependent on the kinetics of the system, including (a) rates of hydrogen peroxide production and degradation, (b) the relative reactivity of hydrogen peroxide and ABTS+• with endogenous reductants, and (c) the time required for the ABTS+• treatment. It is sometimes informative to view the prequantification ABTS+• treatment as a termination step when working with unstable systems. An optimum termination step would stop all hydrogen peroxide consuming/producing reactions instantaneously while simultaneously quenching those compounds likely to inhibit the subsequent peroxidase-based assay. (In this work, we have focused on the latter, but the former may be important when dealing with
(time course “a”). The time course depicts hydrogen peroxide loss in fresh (untreated) GPE to which ABTS+• and hydrogen peroxide were added simultaneously. The data thus depicts the maximum amount of hydrogen peroxide loss that may occur in the GPE matrix during the ABTS+• treatment. It is a maximum because the reagents were added to fresh GPE (“fresh” meaning the GPE had no previous exposure to hydrogen peroxide or ABTS+•). The relatively small decrease in hydrogen peroxide content in the initial phase of the time course can be rationalized as representing the amount of hydrogen peroxide that reacts with GPE components prior to those components reacting with ABTS+• (the assumption is that peroxide and ABTS+• compete for reaction with at least some of the endogenous reductants; those reductants that preferentially react with hydrogen peroxide in the initial phase of the experiment are hereafter referred to as “fast reacting”). In the optimal case, hydrogen peroxide concentrations would have remained constant following the simultaneous addition of hydrogen peroxide and ABTS+• to GPE (indicating that all endogenous reductants were quenched by reaction with ABTS+• prior to their having a chance to react with hydrogen peroxide). In the present case, hydrogen peroxide concentrations remained constant following an initial small but significant decline. Complementary experiments showed the absolute amount of hydrogen peroxide consumed by reaction with fast reacting components remained constant as the E
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assay due to the presence of confounding compounds. Thus, the data of Figure 6 provides a representative example of how the modified assay, as presented here, expands the applicability of quantitative peroxidase-based assays for hydrogen peroxide. The aim of the presented study was to develop a method that allows the direct use of peroxidase-based assays to quantify hydrogen peroxide in biological matrixes. Such applications of peroxidase-based assays are often not feasible due to the presence of endogenous compounds that reduce the oxidized reporter molecule generated in the peroxidase reaction. Here, we show that this can be circumvented by treatment of the biological matrix with the oxidized reporter molecule itself prior to initiating the peroxidase reaction. This approach requires the use of an amenable reporter molecule, such as ABTS/ABTS+•. The advantages and limitations of this approach have been presented through a series of experiments evaluating the stability of hydrogen peroxide in a garlic-based product representative of those in the food industry. The primary advantage of the assay is that it allows one to directly quantify hydrogen peroxide in confounding compound-containing matrixes. A potential limitation of the method is that the treatment used to inactivate confounding compounds may take on the order of minutes, depending on the sample matrix, and this may limit the extent to which the method can be applied to rapidly reacting systems. The concept behind the assay, of selectively oxidizing confounding compounds with ABTS+• prior to using ABTS/peroxidase-based methods for hydrogen peroxide quantification, is expected to be generally applicable. This includes many widely used peroxidasebased coupled enzyme assays for analytes other than hydrogen peroxide per se (e.g., glucose determination via the glucose oxidase/peroxidase reaction).
concentration of added hydrogen peroxide was increased. This behavior is reflected in the calibration curves of Figure 7, which
Figure 7. Comparison of calibration curves obtained following the spiking of known amounts of hydrogen peroxide into ABTS+•-treated and untreated GPE samples. Curve “a” was obtained by measuring hydrogen peroxide levels following its addition to ABTS+•-treated GPE (as in Figure 4); curve “b” was obtained by measuring hydrogen peroxide levels following the addition of hydrogen peroxide and ABTS+• simultaneously to untreated GPE, waiting 15 min, then adding HRP for hydrogen peroxide quantification. Data points are means ± standard deviation from triplicates.
are parallel to one another but offset to the extent that hydrogen peroxide was consumed during the ABTS+•-treatment period. The parallel nature of the curves is consistent with a constant amount of hydrogen peroxide being consumed in each case. These results are consistent with the existence of a limited amount of endogenous components in fresh GPE that rapidly react with hydrogen peroxide, i.e., react with hydrogen peroxide prior to their reaction with ABTS+• when added simultaneously. The notion of ABTS+• and hydrogen peroxide competing for reaction with endogenous components is expected on the basis of them both being relatively strong oxidizing agents. The competition between hydrogen peroxide and ABTS+• for reaction with certain endogenous components is supported by data from experiments showing that prolonged treatment of fresh GPE with ABTS+• results in an ABTS+•-treated GPE in which hydrogen peroxide was stable (Figure 7, curve “a”). The data discussed in this paragraph serve to illustrate the importance of considering the relative reactivity of hydrogen peroxide and ABTS+• when working with dynamic systems. Time course “b” of Figure 6 illustrates the application of the presented method for the determination of hydrogen peroxide decay in GPE. For reasons discussed in the previous paragraph, the curve does not capture the very rapid decay which occurs immediately following addition of hydrogen peroxide, i.e., decay due to the reaction of hydrogen peroxide with GPE’s “fast reacting” components. Rather, the time course depicts the decay of hydrogen peroxide remaining in the product following the initial fast phase reaction. Data of this type is useful to food processors employing hydrogen peroxide because it allows an estimation of the time required for residual hydrogen peroxide to reach specified levels. The inset to Figure 6 shows that hydrogen peroxide decay in GPE approaches first order behavior; the slight concave-up nature of the curve is expected for systems containing multiple components of varying reactivity. It is important to note that the depicted time course for hydrogen peroxide decay in GPE is not obtainable using the traditional peroxidase-based
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AUTHOR INFORMATION
Corresponding Author
*Phone: 541-737-6513. E-mail:
[email protected]. ORCID
Michael H. Penner: 0000-0002-4995-6885 Funding
This study was supported by a grant from the Oregon Agricultural Research Foundation (ARF8296A). Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Segundo, M. A.; Castro, C.; Magalhães, L. M.; Reis, S. Determination of scavenging capacity against hydrogen peroxide: recent trends on chemical methods. In Hydrogen Peroxide: Detection, Applications and Health Implications, 1st ed.; Aguilar, G., Guzman, R. A., Eds.; Nova Science Pub Inc.: Hauppauge, NY, 2012; pp 203−218. (2) Winterbourn, C. C. The biological chemistry of hydrogen peroxide. Methods Enzymol. 2013, 528, 3−25. (3) Martin, B.; Sedelmeier, J.; Bouisseau, A.; Fernandez-Rodriguez, P.; Haber, J.; Kleinbeck, F.; Kamptmann, S.; Susanne, F.; Hoehn, P.; Lanz, M.; Pellegatti, L.; Venturoni, F.; Robertson, J.; Willis, M. C.; Schenkel, B. Toolbox study for application of hydrogen peroxide as a versatile, safe and industrially-relevant green oxidant in continuous flow mode. Green Chem. 2017, 19, 1439−1448. (4) Campbell, R. E.; Drake, M. A. Cold enzymatic bleaching of fluid whey. J. Dairy Sci. 2013, 96, 7404−7413. (5) Linley, E.; Denyer, S. P.; McDonnell, G.; Simons, C.; Maillard, J.-Y. Use of hydrogen peroxide as a biocide: new consideration of its mechanisms of biocidal action. J. Antimicrob. Chemother. 2012, 67, 1589−1596. F
DOI: 10.1021/acs.jafc.7b02248 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry (6) Pan, G. Improving hydrogen peroxide bleaching of ASPEN CTMP by using aqueous alcohol media. BioResources 2011, 6, 4005−4011, DOI: 10.15376/biores.6.4.40005-4011. (7) Yu, J.; Shao, D.; Sun, C.; Xu, C.; Hinks, D. Pilot-plant investigation on low-temperature bleaching of cotton fabric with TBCC-activated peroxide system. Cellulose 2017, 24, 2647. (8) Jervis, S. M.; Drake, M. The impact of iron on the bleaching efficacy of hydrogen peroxide in liquid whey systems. J. Food Sci. 2013, 78, R129−R137. (9) Ukuku, D. O.; Bari, L.; Kawamoto, S. Hydrogen peroxide. In Decontamination of Fresh and Minimally Processed Produce, 1st ed.; Gómez-López, V. M., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2012; pp 197−214. (10) AL-Omiri, M. K.; Abul Hassan, R. S.; AlZarea, B. K.; Lynch, E. Improved tooth bleaching combining ozone and hydrogen peroxideA blinded study. J. Dent. 2016, 46, 30−35. (11) Monakhova, Y. B.; Diehl, B. W. K. Rapid 1H NMR determination of hydrogen peroxide in cosmetic products and chemical reagents. Anal. Methods 2016, 8, 4632−4639. (12) Barni, F.; Lewis, S. W.; Berti, A.; Miskelly, G. M.; Lago, G. Forensic application of the luminol reaction as a presumptive test for latent blood detection. Talanta 2007, 72, 896−913. (13) Murphy, M. P.; Holmgren, A.; Larsson, N.-G.; Halliwell, B.; Chang, C. J.; Kalyanaraman, B.; Rhee, S. G.; Thornalley, P. J.; Partridge, L.; Gems, D.; Nyströ m, T.; Belousov, V.; Schumacker, P. T.; Winterbourn, C. C. Unraveling the biological roles of reactive oxygen species. Cell Metab. 2011, 13, 361−366. (14) Olenin, A. Y. Methods of nonenzymatic determination of hydrogen peroxide and related reactive oxygen species. J. Anal. Chem. 2017, 72, 243−255. (15) Rhee, S. G.; Chang, T.-S.; Jeong, W.; Kang, D. Methods for detection and measurement of hydrogen peroxide inside and outside of cells. Mol. Cells 2010, 29, 539−549. (16) Winterbourn, C. C. The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells. Biochim. Biophys. Acta, Gen. Subj. 2014, 1840, 730−738. (17) Marquette, C. A.; Blum, L. J. Applications of the luminol chemiluminescent reaction in analytical chemistry. Anal. Bioanal. Chem. 2006, 385, 546−554. (18) Wardman, P. Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: Progress, pitfalls, and prospects. Free Radical Biol. Med. 2007, 43, 995−1022. (19) Wang, N.; Miller, C. J.; Wang, P.; Waite, T. D. Quantitative determination of trace hydrogen peroxide in the presence of sulfide using the Amplex Red/horseradish peroxidase assay. Anal. Chim. Acta 2017, 963, 61−67. (20) Tarvin, M.; McCord, B.; Mount, K.; Sherlach, K.; Miller, M. L. Optimization of two methods for the analysis of hydrogen peroxide: High performance liquid chromatography with fluorescence detection and high performance liquid chromatography with electrochemical detection in direct current mode. J. Chromatogr. A 2010, 1217, 7564− 7572. (21) Tarpey, M. M.; Wink, D. A.; Grisham, M. B. Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations. Am. J. Physiol. Regul. Integr. Comp. Physiol 2004, 286, R431−R444. (22) Kalili, K. M.; De Smet, S.; van Hoeylandt, T.; Lynen, F.; de Villiers, A. Comprehensive two-dimensional liquid chromatography coupled to the ABTS radical scavenging assay: a powerful method for the analysis of phenolic antioxidants. Anal. Bioanal. Chem. 2014, 406, 4233−4242. (23) Prior, R. L.; Wu, X.; Schaich, K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 2005, 53, 4290−4302. (24) Huang, D.; Ou, B.; Prior, R. L. The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 2005, 53, 1841−1856. (25) Leelarungrayub, N.; Rattanapanone, V.; Chanarat, N.; Gebicki, J. M. Quantitative evaluation of the antioxidant properties of garlic and shallot preparations. Nutrition 2006, 22, 266−274.
(26) Zulueta, A.; Esteve, M. J.; Frígola, A. ORAC and TEAC assays comparison to measure the antioxidant capacity of food products. Food Chem. 2009, 114, 310−316. (27) Pellegrini, P.; Serafini, M.; Colombi, B.; Del Rio, D.; Salvatore, S.; Bianchi, M.; Brighenti, F. Total antioxidant capacity of plant foods, beverages and oils consumed in Italy assessed by three different in vitro assays. J. Nutr. 2003, 133, 2812−2819. (28) Staniek, K.; Nohl, H. H2O2 detection from intact mitochondria as a measure for one-electron reduction of dioxygen requires a noninvasive assay system. Biochim. Biophys. Acta, Bioenerg. 1999, 1413, 70− 80. (29) Rodrigues, J. V.; Gomes, C. M. Enhanced superoxide and hydrogen peroxide detection in biological assays. Free Radical Biol. Med. 2010, 49, 61−66. (30) Yue, H.; Bu, X.; Huang, M.-H.; Young, J.; Raglione, T. Quantitative determination of trace levels of hydrogen peroxide in crospovidone and a pharmaceutical product using high performance liquid chromatography with coulometric detection. Int. J. Pharm. 2009, 375, 33−40. (31) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; RiceEvans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med. 1999, 26, 1231−1237. (32) Venkatasubramanian, L.; Maruthamuthu, P. Kinetics and mechanism of formation and decay of 2,2’-azinobis- (3-ethylbenzothiazole-6-sulphonate) radical cation in aqueous solution by inorganic peroxides. Int. J. Chem. Kinet. 1989, 21, 399−421. (33) Henriquez, C.; Aliaga, C.; Lissi, E. Formation and decay of the ABTS derived radical cation: a comparison of different preparation procedures. Int. J. Chem. Kinet. 2002, 34, 659−665. (34) Doerge, D. R.; Divi, R. L.; Churchwell, M. I. Identification of the colored guaiacol oxidation product produced by peroxidases. Anal. Biochem. 1997, 250, 10−17. (35) Josephy, P. D.; Eling, T.; Mason, R. P. The horseradish peroxidase-catalyzed oxidation of 3,5,3′,5′-tetramethylbenzidine. J. Biol. Chem. 1982, 257, 3669−3675. (36) Adak, S.; Mazumder, A.; Banerjee, R. K. Probing the active site residues in aromatic donor oxidation in horseradish peroxidase: involvement of an arginine and a tyrosine residue in aromatic donor binding. Biochem. J. 1996, 314, 985−991. (37) Walker, R. B.; Everette, J. D. Comparative reaction rates of various antioxidants with ABTS radical cation. J. Agric. Food Chem. 2009, 57, 1156−1161. (38) Tian, X.; Schaich, K. M. Effects of molecular structure on kinetics and dynamics of the Trolox equivalent antioxidant capacity assay with ABTS+•. J. Agric. Food Chem. 2013, 61, 5511−5519. (39) Childs, R. E.; Bardsley, W. G. The steady-state kinetics of peroxidase with 2,2’-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as chromogen. Biochem. J. 1975, 145, 93−103. (40) Mátai, A.; Hideg, É. A comparison of colorimetric assays detecting hydrogen peroxide in leaf extracts. Anal. Methods 2017, 9, 2357−2360.
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DOI: 10.1021/acs.jafc.7b02248 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
