Development of a Fluorescent Probe for Measurement of Peroxyl

Article pubs.acs.org/JAFC

Development of a Fluorescent Probe for Measurement of Peroxyl Radical Scavenging Activity in Biological Samples Kubilay Gücļ ü, Gülşah Kıbrıslıoğlu, Mustafa Ö zyürek,* and Reşat Apak Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Turkey ABSTRACT: In antioxidant activity testing, it has been argued that assays capable of measuring the inhibitive action against the biologically relevant peroxyl radicals (ROO•) from a controllable source are preferable in terms of simulating physiological conditions because ROO• is the predominant free radical found in lipid oxidation in foods and biological systems. A new fluorescent probe, p-aminobenzoic acid (PABA), was developed for selective measurement of peroxyl radical scavenging (PRS) activity of biological samples, in view of the fact that the existing PRS assays are quite laborious and require the application of strictly optimized conditions. The earlier probe, β-phycoerythrin, of a similar PRS assay of wide use, oxygen radical absorbance capacity (ORAC), varies from lot to lot of production, undergoes photobleaching, and interacts with polyphenols via non-specific protein binding, while the current probe, fluorescein, undergoes undesired fluorescence (FL) quenching and side reactions. The developed technique is based on the fluorescence decrease of the PABA probe (within an optimal time of 30 min) because of its oxidation by ROO•, generated from the thermal dissociation of 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH). In the absence of the scavenger, ROO• reacted with the probe, generating non-fluorescent products, and caused a decrease in PABA fluorescence, whereas the ROO• scavenger resulted in a fluorescence increase because of the inhibition of the probe oxidation by ROO•. Thus, the fluorescence increment of intact PABA is proportional to the ROO• scavenging activity of samples. The linear range of relative fluorescence intensity versus the PABA concentration was in the interval of 0.5−5.0 μM. Assay precision and accuracy were assessed by analyzing two spiked homogenates of liver and kidney at clinically relevant concentrations with 97−105% recovery and 2.3% interday reproducibility. The proposed method was successfully applied to assay the ROO• scavenging activity of some amino acids, plasma and thiol-type antioxidants, and albumin, with the latter showing the strongest activity with respect to both ORAC and developed PABA methods. On the other hand, the original ORAC method suffers a limitation from protein thiols in total radical-trapping antioxidant parameter (TRAP) calculations, and inconsistent results have been reported by various researchers for ORAC values of thiols, such as vastly differing values for glutathione and zero value for cysteine. KEYWORDS: peroxyl radical scavenging, PABA method, amino acids, thiol-type antioxidants, plasma antioxidants, spectrofluorometric method, classical ORAC method

■

INTRODUCTION There are five major reactive oxygen species (ROS) that may regularly interact and damage biological macromolecules under oxidative stress conditions: superoxide anion (O2• −), hydrogen peroxide (H2O2), peroxyl radical (ROO•), hydroxyl radical (OH•), and singlet oxygen (1O2).1 The peroxyl radical is the analogue of the perhydroxyl radical (HOO•), in which the H atom is replaced by an organic group. ROO• is less reactive than OH• and, thus, possesses an extended half-life of seconds instead of nanoseconds.2 In comparison to other ROS species, ROO• is a stable species capable of diffusing to remote cellular locations. These radicals are commonly found in food and biological samples, where the principal pathway of ROO• formation is autoxidation (e.g., lipid peroxidation). They have harmful effects on human health and are also associated with quality deterioration of foods. The relevance of ROO• in food science and biochemistry fostered the development of methods for determining ROO• scavenging capacity, which measure the ability of an antioxidant to scavenge peroxyl radicals by hydrogen atom transfer (HAT) reactions.3,4 Because the existing HAT-based assays are quite laborious and require the application of strictly optimized conditions, simple and sensitive methods should be devised for measuring the ROO• © 2014 American Chemical Society

scavenging activity of antioxidants. Although certain spectrophotometric procedures have been developed to resolve the shortcomings and limitations of available methods,5−8 there is a need for developing new fluorometric methods having higher sensitivity for ROO• scavenging activity assay. The most frequently applied ROO• generators are watersoluble 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) and 2,2′-azobis(2-amidinopropane) dihydrochloride (ABAP) and lipid-soluble 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN).9 β-Phycoerythrin (β-PE), fluorescein (FL), dichlorofluorescein, crocin, and 4-difluoro-5-(4-phenyl-1,3-butadienyl)4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY 581/591) were the most frequently used spectroscopic probes subjected to oxidation in competitive kinetics with antioxidants.10 In these competitive assays, the presence of antioxidant compounds inhibits or retards the ROO•-induced oxidation of the probe (Figure 1). Therefore, at the beginning of the assay, insignificant spectroscopic changes of the probe Received: Revised: Accepted: Published: 1839

December 5, 2013 February 5, 2014 February 10, 2014 February 10, 2014 dx.doi.org/10.1021/jf405464v | J. Agric. Food Chem. 2014, 62, 1839−1845

Journal of Agricultural and Food Chemistry

Article

a wide range of reduction potentials (E°) ranging between 0.77 and 1.44 V, the E° of alkyl peroxyl radicals derived from polyunsatured fatty acids (PUFA) is 1.00 V at pH 7.0.19 On the other hand, in strongly to weakly acidic solutions, the oxidation peak potentials of the p-aminobenzoic acid (PABA) probe (on a carbon paste electrode) vary in the range of 0.84−0.98 V.20 With other modified electrodes (e.g., with crown ethers), the PABA oxidation potential may rise up to 1.055 V. Thus, it may be envisaged that ROO• can oxidize PABA, whereas O2• − (E° = 0.940 V), 1O2 (E° = 0.650 V), PUFA alkyl radicals (E° = 0.600 V), and H2O2 (E° = 0.320 V)21 may not easily do so. The oxidation potential of the ORAC probe, FL, was reported as 0.78 and 1.21 V versus saturated calomel electrode (SCE) in protonated and deprotonated forms, respectively, corresponding to 1.024 and 1.45 V calculated potentials versus standard hydrogen electrode (SHE),22 and consequently, FL was characterized as “prone to be oxidized by several reactive species into a non-fluorescent product”.23 Thus, the PABA probe is expected to be a right choice for ROO• detection. We report herein the development of a novel fluorescence probe for ROO•, PABA, which can specifically detect ROO• and measure its scavenging activity in terms of an increase of fluorescence in the presence of a scavenger compound. ROO• scavenging activity of several pure compounds (thiol-type antioxidants, glutathione (GSH), cysteine, N-acetyl-cysteine (NAC), homocysteine, 1,4-dithioerythritol (DTE), and cysteamine; amino acids, methionine, serine, and proline; plasma antioxidants, albumin, bilirubin, uric acid, and ascorbic acid) and tissue homogenates (kidney, liver, and heart) was determined by the proposed method in comparison to the classical ORAC method.

Figure 1. Scheme of competitive reactions showing the decomposition of AAPH, generation of peroxyl radicals, and conversion of the PABA probe to non-fluorescent products.

would be observed. As the reaction proceeds, the antioxidants are consumed by the constant flux of ROO• and the oxidation of the probe would progress at a slower rate when compared to the control (i.e., absence of antioxidant compounds). When the antioxidants are depleted, the rate of oxidation of the probe is similar to that of the control.2 The oxygen radical absorbance capacity (ORAC) assay is one of the most common methods for assessing ROO• scavenging activity based on the fluorescence intensity decrease of the ORAC probe with time under a reproducible and constant flux of ROO•, generated from the thermal decomposition of AAPH in aqueous buffer. The area under the fluorescence decay curve (AUC) is the parameter used to analyze the results in conventional ORAC tests. In the presence of chain-breaking antioxidants, the decay of fluorescence is inhibited.11 The protein isolated from Porphyridium cruentum, β-PE, was initially used as the fluorescent probe, which reacts with ROO• to form a non-fluorescent product.12 However, some shortcomings were observed with this probe, such as large lot-to-lot variability of production, photobleaching of β-PE after exposure to excitation light, and interaction with polyphenols by nonspecific protein binding.13 To overcome these limitations, the synthetic non-protein fluorescent probe, FL, has replaced the original β-PE,14 but FL has also been criticized to undergo fluorescence quenching, side reactions, and undesired interactions.15 The conventional ORAC method is limited to the measurement of hydrophilic chain-breaking antioxidants but ignores lipophilic ones. The application to both lipophilic and hydrophilic chain-breaking antioxidants was carried out using a mixture of acetone/water containing 7% randomly methylated β-cyclodextrin (M-β-CD) oligosaccharide as a water solubility enhancer,16 but such high concentrations of oligosaccharides may not release the tested antioxidants to undergo oxidation because of the formation of relatively stable inclusion complexes. The total radical-trapping antioxidant parameter (TRAP) assay was based on the measurement of the time period in which oxygen uptake was inhibited by plasma during a controlled ROO• peroxidation reaction.17 TRAP measures oxygen consumption during controlled lipid oxidation induced by thermal decomposition of AAPH as the radical generator and R-phycoerythrin (PE) as the fluorescent probe and reports results in micromoles of peroxyl radical deactivated per liter of solution.17 In the crocin bleaching assay, which has found limited applications in food and biochemistry, the ability of antioxidant compounds to protect the carotenoid derivative crocin from oxidation by ROO• is measured.18 The selectivity of a probe for ROO• detection mainly depends upon its standard redox potential. Although ROO• has

■

MATERIALS AND METHODS

Reagents and Apparatus. Bilirubin, serine, cysteamine, homocysteine, uric acid, GSH, AAPH, N-acetyl-L-cysteine, and dithioerythritol were purchased from Sigma-Aldrich (St. Louis, MO). Ascorbic acid, albumin from bovine serum, and proline were donated from Merck (Darmstadt, Germany), and PABA, cysteine, methionine, and FL were obtained from Fluka (Buchs, Switzerland). All other reagents used were of analytical grade. The fluorescence spectra were measured in quartz cuvettes using a Varian Cary Eclipse model spectrofluorometer (Mulgrave, Victoria, Australia). A Waters Breeze 2 model high-performance liquid chromatography (HPLC) system (Milford, MA) equipped with a 2998 photodiode array detector (Chelmsford, MA) was used for chromatographic measurements. Preparation of Solutions. For the PABA method, the following solutions were prepared: PABA solution (20 μM) and AAPH (20 mM) were prepared in pure distilled water (Millipore Simpak 1 Synergy 185, Billerica, MA). For the classical ORAC method, FL (78 nM) and AAPH (221 mM) were prepared in phosphate buffer (pH 7.4). This buffer (7.5 × 10−2 M NaH2PO4−Na2HPO4 equimolar mixture) was prepared in distilled water. All scavengers were freshly prepared in distilled water from 1.0 × 10−5 to 5.0 × 10−5 M concentrations, except uric acid and bilirubin, which were initially prepared in 0.5 M NaOH solution and then diluted with distilled water for the PABA method. The original uric acid and bilirubin solutions in 0.5 M aqueous NaOH were diluted with phosphate buffer (pH 7.4) for the classical ORAC assay. Preparation of Tissue Homogenates. Male, 8−10-week-old, Wistar rats (weight range of 290−310 g) were supplied by the animal facility from the Faculty of Veterinary Medicine of Istanbul University. The rats were housed in polycarbonate cages (450 cm2 area per animal), acclimatized for 2 weeks under laboratory conditions (21 ± 2 °C, humidity of 60 ± 5%, and 12 h light/dark cycle), and fed with water and standard rat chow ad libitum. Liver, heart, and kidney tissues 1840

dx.doi.org/10.1021/jf405464v | J. Agric. Food Chem. 2014, 62, 1839−1845

Journal of Agricultural and Food Chemistry

Article

ROO• scavenging activity was calculated using a modified version of eq 1

were isolated after sacrifice by decapitation of the rat. The tissue samples were washed with 0.9% (w/v) NaCl solution, weighed (10%, w/v), and homogenized by adding cold 1.15% (w/v) KCl solution in a glass homogenizer. Homogenates were immediately frozen in liquid nitrogen and kept at −80 °C until analysis.24 Homogenates were centrifugated at 12 000 rpm for 15 min at 4 °C and filtered through a 0.45 μm membrane filter before analysis. PABA Assay (Proposed Method). To a test tube were added 0.5 mL of 20 μM PABA (probe), 0.5 mL of scavenger solution (at different concentrations), 2.0 mL of distilled water, and 2.0 mL of 2.0 × 10−2 M AAPH in this order. The reaction started by adding AAPH solution. The mixture in a total volume of 5.0 mL was incubated for 30 min in a water bath kept at 37 °C. At the end of this period, the fluorescence intensity (λex = 267 nm, and λem = 334 nm) of the reaction mixture was recorded. The half-maximal inhibitory concentration (IC50) values of the scavengers (i.e., defined as the concentration of the scavenger antioxidant that results in 50% inhibition of the fluorescence-decay reaction caused by peroxyl radicals) were determined with the use of PABA by means of a linear plot of inhibition percentage as a function of Cscavenger, where I0 is the initial fluorescence intensity of the original PABA solution and I1 and I2 are the fluorescence intensities of PABA subjected to peroxyl radical action in the absence and presence of a scavenger compound (having a molar concentration, C), respectively. The IC50 values were then compared to those found with the classical ORAC method. The fluorescence intensity of PABA was higher in the presence of ROO• scavengers; therefore, a higher intensity of the reaction mixture was an indication of enhanced ROO• scavenging activity. ROO• scavenging activity was calculated using the following equation:

inhibition ratio (%) = 100[(I2 − I1)/(I0 − I1)]

inhibition ratio (%) = 100[(A 2 − A1)/(A 0 − A1)]

where A1 and A2 are the peak areas of the PABA probe in the absence and presence of the ROO• scavenger, respectively, and A0 is the peak area of the PABA probe at the initial concentration in the reaction mixture. Statistical Analysis. The two-way analysis of variance (ANOVA) was used for data evaluation, and the significance of the difference between means was determined (p = 0.05) using SPSS software for Windows (version 13).26 Results were reported as the mean ± standard deviation (SD) by carrying out measurements for at least three replicates of each scavenger compound or biological sample.

■

RESULTS AND DISCUSSION Optimization of the PABA Assay Parameters. In this study, we developed a rapid and sensitive ROO• scavenging activity assay for pure antioxidant compounds (i.e., amino acids, thiol-type antioxidants, and plasma antioxidants) and biological fluids. PABA has first been used as a specific probe for the determination of ROO• scavenging activity. The PABA assay measures ROO• scavenging potential based on the ability of a compound to protect the oxidative probe PABA from oxidation by ROO•. In the competitive reaction, ROO• generated by thermal dissociation of AAPH attacks both the PABA probe and scavengers to form non-fluorescent products. The attenuation in the fluorescence intensity loss of the PABA probe is dependent upon the ROO• scavenging activity of the tested compound or sample. With the aid of PABA fluorescence values recorded in the presence and absence of scavengers, the ROO• scavenging activity of scavenger compounds can be calculated. Although the non-fluorescent oxidation products were not identified under the same conditions of this assay, photochemical oxidation products were detected by Shaw et al., which may give some idea about the nature of such products.27 Rapid discoloration of the photolyzed sample was observed when PABA was irradiated in aerated solution. Although a number of products were detected under these conditions, the three most abundant stable compounds have been isolated and identified [by nuclear magnetic resonance (NMR) spectroscopy] as 4-amino-3-hydroxybenzoic acid, 4-aminophenol, and 4(4′-hydroxyphenyl)aminobenzoic acid. Likewise, a rapid reaction of singlet oxygen with PABA was reported by Allen et al.,28 but oxidation products were not identified. Under the optimized conditions, the molar fluorescence coefficient for PABA in the proposed method was ε = 1.0 × 108 M−1 cm−1 and the linear range of relative fluorescence intensity versus the concentration of PABA was in the interval of 0.5−5.0 μM, with a correlation coefficient (r) of 0.992 (N = 3), within which the relative standard deviation (RSD) was 3.1%. The limit of detection (LOD) and limit of quantification (LOQ) of the proposed method were calculated using the equations: LOD = 3sbl/m and LOQ = 10sbl/m, respectively, where sbl is the standard deviation of a blank and m is the slope of the calibration line. LOD and LOQ were found to be 0.09 and 0.30 μM, respectively. Assay precision and accuracy were assessed by analyzing two spiked homogenates (i.e., liver and kidney) at clinical relevant concentrations of 1, 2, and 5 μM analyzed (Table 1). The precision data of the proposed assay (expressed as the percent RSD values) was approximately 4.14%. Recovery values from 97.5 to 105.0% were obtained. Reproducibility studies were carried out in five replicates for PABA at 2.0 μM.

(1)

The inhibition percentage (y) can be linearly correlated to the concentration of the scavenger antioxidant (x) y ≈ mx + n

(2)

where m and n are the slope and intercept of this line, respectively. IC50 can then be calculated for 50% inhibition by substituting an ordinate (y) value of 50.

y = 50 = m(IC50) + n

or

IC50 = (50 − n)/m

(4)

(3)

Classical ORAC Assay. The ORAC method, using FL as the fluorescent probe, was carried out as described by Ou et al.14 Briefly, to a test tube were added 2.0 mL of 78 nM FL, 2.0 mL of scavenger solution (at different concentrations), and 1.0 mL of 221 μM AAPH rapidly in this order. The reaction was started by adding AAPH solution. The reaction was performed at 37 °C through the thermal decomposition of AAPH because of the sensitivity of FL to pH.13 The mixture in a total volume of 5.0 mL was incubated for 10 min in a water bath kept at 37 °C. At the end of this period, the fluorescence intensity (λex = 493 nm, and λem = 515 nm) of the reaction mixture was recorded. ROO• scavenging activity was calculated using eqs 1−3. HPLC Assay. The chromatographic analysis was carried out on a Waters Breeze 2 model HPLC system (Milford, MA), equipped with a 2998 model photodiode array detector (Chelmsford, MA). A total of 20 μL of incubation solution was injected into a reverse-phase ACE C18 column (4.6 × 250 mm, 5 μm particle size) (Milford, MA), and the separation was performed by gradient elution. The mobile phase consisting of a mixture of (A) 5% methanol/94% water (v/v) and 1% acetic acid and (B) 55% methanol, 44% water, and 1% acetic acid was delivered to the column at a flow rate of 1.0 mL min−1. The gradient elution program for the analysis of the incubation mixture was 0−3 min, mobile phase A (slope 1.0); 3−8 min, gradient to 100% mobile phase B (slope 1.0); 8−23 min, mobile phase B (slope 1.0); 23−23 min, and linear gradient to 100% mobile phase A (slope 1.0).25 The detection wavelength was set at 286 nm with a total run time of 23 min. Data acquisition and handling were performed by Empower PRO (Waters Associates, Milford, MA). 1841

dx.doi.org/10.1021/jf405464v | J. Agric. Food Chem. 2014, 62, 1839−1845

Journal of Agricultural and Food Chemistry

Article

Table 1. Precision and Recovery of the Proposed Method PABA additions to tissue homogenates liver

kidney

added concentration (μM)

mean (μM)

SDa

RSD (%)b

REC (%)c

1.0 2.0 5.0 1.0 2.0 5.0

1.02 2.08 5.01 1.05 1.95 4.98

0.04 0.05 0.03 0.03 0.03 0.07

4.14 2.16 0.52 3.05 1.32 1.36

101.5 104.3 100.2 105.0 97.5 99.5

a SD = standard deviation. bRSD = relative standard deviation. cREC = recovery (N = 3).

Figure 3. Fluorescence spectra of the remaining PABA in the absence and presence of cysteine: (a) 2 μM PABA, (b) 5 μM cysteine, (c) 4 μM cysteine, (d) 3 μM cysteine, (e) 2 μM cysteine, (f) 1 μM cysteine, and (g) 0 μM cysteine (reference).

The intraday and interday precisions of the developed method, expressed as the RSD for the analyte, were 0.98 and 2.27% (N = 5), respectively. For comparison, time-dependent fluorescence intensity changes were recorded using final mixture solutions (PABA, PABA + AAPH, and PABA + AAPH + cysteine) incubated under identical conditions (Figure 2). There was no note-

the reference (containing no cysteine) because of the competitive scavenging effect of cysteine. The results indicate that PABA is converted into non-fluorescent products with ROO• and also there is a proportional increase of 334 nm peak heights (Figure 3) with an increasing concentration of scavenger (cysteine). With the aid of PABA fluorescence intensity values recorded in the presence and absence of scavengers at varying concentrations, the IC50 values of scavengers and tissue homogenates can be calculated. HPLC Results of the Tested Scavengers. The developed method for measurement of ROO• scavenging activity was validated by quantifying the probe (PABA) and its oxidation product(s) by the HPLC method, followed by statistically comparing the results. Conversion of the PABA probe to products and inhibition of this reaction with a scavenger (i.e., NAC) were followed by the proposed and HPLC methods. As shown in Figure 4, the peak area of the PABA probe (retention time of PABA of 8.8 min) was higher in the presence of a ROO• scavenger, such as NAC, compared to its absence under identical conditions of radical attack (AAPH). The concentrations of PABA (in μg mL−1) remaining after ROO• attack to PABA in the presence of NAC (3.0 × 10−4 M) measured by the proposed method and HPLC method were 6.76 ± 0.03 (N = 3) and 6.62 ± 0.07 (N = 3), respectively, where the initial concentration of PABA was 13.71 ± 0.09 (N = 3). The results obtained using these two methods were quite close, and the proposed method was verified (Figure 4b). As a result of the incubation reaction, the conversion ratio of the original probe to products in the absence of scavenger (reference) were found to be 78.9 and 81.1% with respect to the HPLC and proposed methods, respectively. Evaluation of Results for Synthetic Scavengers. The PABA assay results for ROO• activity measurement were compared to those of the ORAC assay. Albumin had the lowest IC50 value among the studied antioxidants [i.e., highest peroxyl radical scavenging (PRS) activity] with respect to both the developed fluorometric (PABA) and ORAC methods. IC50 values of albumin were found to be 0.6 and 2.8 μM, respectively (Table 2). Zulueta et al. reported that,29 in terms of PRS activity, albumin, a very powerful chain-breaking and transitionmetal-binding extracellular antioxidant,30 was the most effective antioxidant among plasma antioxidants, in accordance with the findings of the proposed method. Inspection of IC50 values of amino acids reveals that the highest scavenging activity was found for methionine (i.e., an amino acid not containing a free thiol group) and the lowest scavenging activity was found for serine with respect to classical ORAC and PABA methods

Figure 2. Fluorescence intensity versus incubation time curves of PABA alone and PABA subjected to peroxyl radical in the absence (reference) and presence (5.0 × 10−5 M cysteine) of a scavenger.

worthy change in intensity of the PABA standard solution within the 0−35 min time interval (Figure 2). It was observed that the fluorescence intensity response in the presence of cysteine decreased with the increase in incubation time from 0 to 30 min and reached a plateau after 30 min. Therefore, 30 min was chosen as the optimal measurement time. Thus, because of the high conversion yield of PABA, IC50 values of the studied scavengers could be relatively rapidly and precisely determined by recording the relative intensities within 30 min. On the other hand, for a typical human serum sample using the ORAC assay system originally used by Cao et al.,12 oxidation of β-PE was reported to reach completion within about 90 min. The fluorescence spectra of PABA, recorded in aqueous solutions at pH 7.0 with varying cysteine concentrations, are shown in Figure 3, where the maximal decrease in fluorescence intensity of PABA was due to ROO• oxidation of the probe without competition and its relative increase from the baseline level was proportional to the scavenging ability of the competitive scavenger. Upon mixing PABA (2.0 μM) with ROO• in the presence of cysteine at various concentrations (1.0−5.0 μM), the fluorescence intensities of PABA (remaining intact after ROO• attack) at 334 nm were greater than that of 1842

dx.doi.org/10.1021/jf405464v | J. Agric. Food Chem. 2014, 62, 1839−1845

Journal of Agricultural and Food Chemistry

Article

Figure 4. HPLC chromatograms for remaining PABA after AAPH reaction in the absence (reference) and presence of NAC (λ = 280 nm): (a) reference (0.5 mL of 1.0 × 10−3 M PABA + 2.5 mL of water + 2.0 mL of 0.4 M AAPH) and (b) 0.5 mL of 1.0 × 10−3 M PABA + 0.5 mL of 3.0 × 10−4 M NAC + 2.0 mL of water + 2.0 mL of 0.4 M AAPH.

Table 2. ROO• Scavenging Activity of Various Scavengers Measured by the PABA Method in Comparison to the ORAC Method (IC50 Values Were Calculated with Respect to Equations 1 and 4; N = 4 or 5 Data Points)a peroxyl scavengers GSH NAC cysteine homocysteine DTE cysteamine methionine serineb prolineb albumin uric acid ascorbic acid bilirubin

IC50 value with respect to the PABA method (μM)

have quite close rate constants for radical disulfide anion formation in pulse radiolysis31 and exhibited very close trolox equivalent antioxidant capacities (TEAC) in the ABTS/ persulfate assay,32 these two thiol compounds may behave differently against lipid peroxidation. Aruoma et al. have shown that, although cysteamine is an excellent scavenger of hydroxyl radicals and hypochlorous acid, it reacts slowly with hydrogen peroxide and does not react at an appreciable rate with superoxide anion radicals.33 As a general drawback for the measurement thiols, the original ORAC method suffers a limitation from protein −SH, because the antioxidant action of plasma proteins was included in the total radical-trapping antioxidant parameter (TRAP) calculation independent of their thiol groups34 and the ORAC values found for GSH with the two probes (β-PE and FL) of the assay greatly differ by a ratio of 1:2.14 Some researchers even found zero ORAC value for cysteine in water or acetone−water solvents using the classical ORAC assay with the FL probe.35 Lussignoli et al. reported that both ascorbic and uric acids exhibited high PRS activities,5 again compatible with the results of the PABA method, because the IC50 values obtained by the developed method for ascorbic acid and uric acid were 5.6 and 11.6 μM, respectively (in a microplate-based crocin bleaching assay of ROO• scavenging, the IC50 values of ascorbic and uric acids were reported as 3.97 and 8.15 μM, respectively).5 Using the classical ORAC method, the IC50 values for ascorbic and uric acids were found to be 10.7 and 9.6 μM, respectively (Table 2). The differences between the types of assays and their conditions (pH, temperature, etc.) may compensate for the different activities reported. Both thiol-type antioxidants and plasma antioxidants with lower IC50 values had higher ROO• scavenging activity than non-thiol amino acids with respect to the PABA method, and this finding is in accordance with the results of other antioxidant activity assays, regardless of the mode of action, i.e., HAT- or electron transfer (ET)-based reactions.33,36,37

IC50 value with respect to the ORAC method (μM)

Thiol-Type Antioxidants 6.5 ± 0.3 8.7 ± 0.4 6.7 ± 0.3 5.7 ± 0.2 12.1 ± 1.4 121 ± 12 Amino Acids 97.9 ± 8.2 136 ± 15 129 ± 9 Plasma Antioxidants 0.6 ± 0.1 11.6 ± 1.1 5.6 ± 0.4 6.2 ± 0.3

16.2 9.3 24.9 14.2 13.0 197

± ± ± ± ± ±

1.0 0.4 1.1 0.4 1.2 19

21.9 ± 2.5 338 ± 39 326 ± 30 2.8 9.6 10.7 10.8

± ± ± ±

0.1 0.3 1.3 1.1

Data presented as the mean ± SD (N = 3). All tested samples: 1.98IC50(PABA) − 6.91 = IC50(ORAC) (r = 0.882). Excluding amino acids: 1.61IC50(PABA) + 1.09 = IC50(ORAC) (r = 0.993). bIC50 values were calculated in millimolar.

a

(Table 2). The IC50 values for NAC (8.7 ± 0.4) and DTE (12.1 ± 1.4) obtained by the proposed method were similar to those of the classical ORAC assay (9.3 ± 0.4 and 13.0 ± 1.2, respectively). It is also noteworthy from Table 2 that cysteamine exhibited a much greater IC50 value (therefore lower peroxyl scavenging activity) than cysteine, having a similar structure. Cysteamine has less electron donation ability through the −SH group than cysteine because the electronegative N atom of the amine group in cysteamine counterbalances the effect of thiol. Although cysteine and cysteamine 1843

dx.doi.org/10.1021/jf405464v | J. Agric. Food Chem. 2014, 62, 1839−1845

Journal of Agricultural and Food Chemistry

Article

The findings for the 13 ROO• scavengers (IC50 values) were statistically compared to those of classical ORAC using the twoway ANOVA test. It is concluded that there was no significant difference between the precisions of these two methods at the 95% confidence level (p = 0.05; Fexp = 2.416; Fcrit = 4.747; and Fexp < Fcrit). For IC50 values of all of the tested samples, PABA correlated linearly with ORAC (r = 0.882); by exclusion of the values for amino acids, this correlation was higher (r = 0.993). Thus, the proposed methodology was validated against the classical ORAC method of wide use. Application of the Proposed Method to Some Tissue Homogenates. The PRS activity of heart homogenate as inhibition percentage versus dilution ratio is shown in Figure 5, where the inhibition percentage was curvilinearly correlated to the homogenate dilution ratio within the interval of 1:50− 1:600 dilution.

to be higher than that of kidney and heart homogenate with respect to both methods. Since the liver was described as the most important organ involved in the regulation of the redox metabolism because of the synthesis of key enzymes responsible for ROS clearance and the production of GSH,38 this result is acceptible. Linear regression analysis of PRS activity data presented in Figure 6 found with the PABA assay showed that this assay correlated well with the classical ORAC assay. The correlation equation between PABA and classical ORAC methods was calculated (y = 0.940x + 12.98; r = 0.991; and N = 3), with the closeness of the slope to unity showing one-to-one correspondence between results. Thus, the proposed methodology was validated for real samples. Because the existing ROO• scavenging assays require much labor and strictly optimized conditions, new assay development using the PABA probe may be useful for more comparable applications in food and biochemical research. ORAC as the most widely used PRS measurement assay has used two probes since it was first launched. The initial probe, β-PE, showed variable performance from one production cycle to another, undesired photobleaching, and non-specific protein binding to polyphenols. Thus, it was replaced with FL, but this latter probe showed rather high ORAC values, some photobleaching and undesired side reactions, and inconsistent findings with thiols. As alternative peroxyl scavenging measurement methods, TRAP uses a lag phase detection of antioxidants, but not all antioxidants possess a lag phase, giving rise to difficulties in comparison of end-point detection. The less frequently used crocin bleaching assay suffers from the initial color of crocin in the presence of other carotenoids and colored compounds, and there are no standard formats for expressing results.39 In regard to redox potential data, the new PABA probe is expected to be more specific for ROO•, because the R-PE and β-PE protein probes of TRAP and ORAC assays, respectively, have been claimed to respond to more than one ROS. Moreover, PABA responds relatively quickly (i.e., within 30 min) to ROO• scavengers, such as cysteine, in reaching a plateau of fluorescence intensity, whereas the classical ORAC assay generally requires longer times for completion of the oxidation reaction for most antioxidants. It is envisaged that future research in HAT-based antioxidant assays (preferentially showing comparable results to those of one or more ETbased assays) should be directed to the design of “turn-on” fluorescence/absorbance probes for selectively detecting a given reactive species [ROS/reactive nitrogen species (RNS)] at a time. It is also preferable to develop probes not affected from side reactions with compounds not having genuine antioxidant status.

Figure 5. Inhibition percentage of tissue homogenates (heart) as a function of the homogenate dilution ratio using the PABA method.

The proposed method was successfully applied to tissue homogenate samples. The ROO• scavenging activity values measured with the proposed PABA and classical ORAC methods are comparatively depicted in a bar diagram (Figure 6), and the percentage inhibitions of identical tissue homogenates found with PABA were very close in relative intensity to those measured with the reference method. The ROO• scavenging activity of liver tissue homogenate was found

■

AUTHOR INFORMATION

Corresponding Author

*Telephone: +90-212-473-7070/17627. Fax: +90-212-4737180. E-mail: [email protected]. Funding

Gülşah Kıbrıslıoğlu thanks the Istanbul University Research Fund, Bilimsel Arastirma Projeleri (BAP) Yurutucu Sekreterligi, for the support given to her M.Sc. Thesis Project 29739 and Istanbul University, Institute of Pure and Applied Sciences (I.U. Fen Bilimleri Enstitüsü), for the support given to her M.Sc. thesis work with the title: “Development of Spectrofluorometric Methods for Measurement of Peroxyl Radical Scavenging Activity in Biological Samples”. The authors express their

Figure 6. Inhibition percentage of some tissue homogenates calculated with the PABA method in comparison to the classical ORAC method (1:50 diluted homogenate). Data are presented as the mean ± SD (error bars) (N = 3) (p = 0.05; Fexp = 0.584; Fcrit(table) = 7.709; and Fexp < Fcrit(table)). 1844

dx.doi.org/10.1021/jf405464v | J. Agric. Food Chem. 2014, 62, 1839−1845

Journal of Agricultural and Food Chemistry

Article

(20) Kotkar, R. M.; Srivastava, A. K. Voltammetric determination of para-aminobenzoic acid using carbon paste electrode modified with macrocyclic compounds. Sens. Actuators, B 2006, 119, 524−530. (21) Buettner, G. R. The pecking order of free radicals and antioxidants: lipid peroxidation, α-tocopherol, and ascorbate. Arch. Biochem. Biophsy. 1993, 300, 535−543. (22) Urano, Y.; Kamiya, M.; Kanda, K.; Ueno, T.; Hirose, K.; Nagano, T. Evolution of fluorescein as a platform for finely tunable fluorescence probes. J. Am. Chem. Soc. 2005, 127, 4888−4894. (23) Gomes, A.; Fernandes, E.; Lima, J. L. F. C. Fluorescence probes used for detection of reactive oxygen species. J. Biochem. Biophys. Methods 2005, 65, 45−80. (24) Alía, M.; Horcajo, C.; Bravo, L.; Goya, L. Effect of grape antioxidant dietary fiber on the total antioxidant capacity and the activity of liver antioxidant enzymes in rats. Nutr. Res. (N. Y., NY, U. S.) 2003, 23, 1251−1267. (25) Szoke, A.; Hayton, W. L.; Schultz, I. R. Quantification of benzocaine and its metabolites in channel catfish tissues and fluids by HPLC. J. Pharm. Biomed. Anal. 1997, 16, 69−75. (26) Miller, J. C.; Miller, J. N. Statistics for Analytical Chemists, 3rd ed.; Ellis Horwood and Prentice Hall: New York, 1993. (27) Shaw, A. A.; Wainschel, L. A.; Shetlar, M. D. The photochemistry of p-aminobenzoic acid. Photochem. Photobiol. 1992, 55, 647−656. (28) Allen, J. M.; Engenolf, S.; Allen, S. K. Rapid reaction of singlet molecular oxygen (1O2) with p-aminobenzoic acid (PABA) in aqueous solution. Biochem. Biophys. Res. Commun. 1995, 212, 1145−1151. (29) Zulueta, A.; Esteve, M. J.; Frigola, A. ORAC and TEAC assays comparison to measure the antioxidant capacity of food products. Food Chem. 2009, 114, 310−316. (30) Halliwell, B. AlbuminAn important extracellular antioxidant? Biochem. Pharmacol. 1988, 37, 569−571. (31) Mezyk, S. P. Direct rate constant measurement of radical disulphide anion formation for cysteine and cysteamine in aqueous solution. Chem. Phys. Lett. 1995, 235, 89−93. (32) Walker, R. B.; Everette, J. D. Comparative reaction rates of various antioxidants with ABTS radical cation. J. Agric. Food Chem. 2009, 57, 1156−1161. (33) Aruoma, O. I.; Halliwell, B.; Hoey, B. M.; Butler, J. The antioxidant action of taurine, hypotaurine and their metabolic precursors. Biochem. J. 1988, 256, 251−255. (34) Ghiselli, A.; Serafini, M.; Maiani, G.; Azzini, E.; Ferro-Luzzi, A. A fluorescence-based method for measuring total plasma antioxidant capability. Free Radical Biol. Med. 1995, 18, 29−36. (35) Pérez-Jiménez, J.; Saura-Calixto, F. Effect of solvent and certain food constituents on different antioxidant capacity assays. Food Res. Int. 2006, 39, 791−800. (36) Suzuki, N.; Kochi, M.; Wada, N.; Mashiko, S.; Nomoto, T.; Yoda, B. Antioxidative activity of amino acids and sulfur-containing compounds to superoxide: measurement by quenching the chemiluminescence of a cypridina luciferin analogue. Biosci., Biotechnol., Biochem. 1992, 56, 409−411. (37) Güngör, N.; Ö zyürek, M.; Gücļ ü, K.; Demirci Cekic, S.; Apak, R. Comparative evaluation of antioxidant capacities of thiol-based antioxidants measured by different in vitro methods. Talanta 2011, 83, 1650−1658. (38) Moreno, I.; Pichardo, S.; Jos, A.; Gomez-Amores, L.; Mate, A.; Vazquez, C. M.; Camean, A. M. Antioxidant enzyme activity and lipid peroxidation in liver and kidney of rats exposed to microcystin-LR administered intraperitoneally. Toxicon 2005, 45, 395−402. (39) 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.

gratitude to T. R. Ministry of Development for the Advanced Research Project of Istanbul University (2011K120320). Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) MacDonald-Wicks, L. K.; Wood, L. G.; Garg, M. L. Methodology for the determination of biological antioxidant capacity in vitro: A review. J. Sci. Food Agric. 2006, 86, 2046−2056. (2) Halliwell, B.; Murcia, M. A.; Chirico, S.; Aruoma, O. I. Free radicals and antioxidants in food and in vivo: What they do and how they work. Crit. Rev. Food Sci. Nutr. 1995, 35, 7−20. (3) Laguerre, M.; Lecomte, J.; Villeneuve, P. Evaluation of the ability of antioxidants to counteract lipid oxidation: Existing methods, new trends and challenges. Prog. Lipid Res. 2007, 46, 244−282. (4) Roginsky, V.; Lissi, E. A. Review of methods to determine chainbreaking antioxidant activity in food. Food Chem. 2005, 92, 235−254. (5) Lussignoli, S.; Fraccaroli, M.; Andrioli, G.; Brocco, G.; Bellavite, P. A microplate-based colorimetric assay of the total peroxyl radical trapping capability of human plasma. Anal. Biochem. 1999, 269, 38−44. (6) Valkonen, M.; Kuusi, T. Spectrophotometric assay for total radical-trapping antioxidant potential in human serum. J. Lipid Res. 1997, 38, 823−833. (7) Cabrini, L.; Barzanti, B.; Cipollone, M.; Fiorentini, D.; Grossi, G.; Tolomelli, B.; Zambonin, L.; Landi, L. Antioxidants and total peroxyl radical-trapping ability of olive and seed oils. J. Agric. Food Chem. 2001, 49, 6026−6032. (8) Handelman, G. J.; Cao, G.; Walter, M. F.; Nightingale, Z. D.; Paul, G. L.; Prior, R. L.; Blumberg, J. B. Antioxidant capacity of oat (Avena sativa L.) extracts. 1. Inhibition of low-density lipoprotein oxidation and oxygen radical absorbance capacity. J. Agric. Food Chem. 1999, 47, 4888−4893. (9) Niki, E. Free radical initiators as source of water- or lipid-soluble peroxyl radicals. Methods Enzymol. 1990, 186, 100−108. (10) Yeum, K.-J.; Russell, R. M.; Krinsky, N. I.; Aldini, G. Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compartments of human plasma. Arch. Biochem. Biophys. 2004, 430, 97−103. (11) Glazer, A. N. Phycoerythrin fluorescence-based assay for reactive oxygen species. Methods Enzymol. 1990, 186, 161−168. (12) Cao, G.; Alessio, H. M.; Cutler, R. G. Oxygen-radical absorbance capacity assay for antioxidants. Free Radical Biol. Med. 1993, 14, 303−311. (13) Huang, D.; Ou, B.; Prior, R. L. The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 2005, 53, 1841−1856. (14) Ou, B.; Hampsch-Woodill, M.; Prior, R. L. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J. Agric. Food Chem. 2001, 49, 4619−4626. (15) Apak, R.; Gorinstein, S.; Böhm, V.; Schaich, K. M.; Ö zyürek, M.; Gücļ ü, K. Methods of measurement and evaluation of natural antioxidant capacity/activity. Pure Appl. Chem. 2013, 85, 957−998. (16) Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J. A.; Deemer, E. K. Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated cyclodextrin as the solubility enhancer. J. Agric. Food Chem. 2002, 50, 1815−1821. (17) Wayner, D. D. M.; Burton, G. W.; Ingold, K. U.; Locke, S. Quantitative measurement of the total peroxyl radical-trapping antioxidant capability of human blood plasma by controlled peroxidation: The important contribution made by plasma proteins. FEBS Lett. 1985, 187, 33−37. (18) Bors, W.; Michel, C.; Saran, M. Inhibition of bleaching of the carotenoid crotin, a rapid test for quantifying antioxidant activity. Biochim. Biophys. Acta 1984, 796, 312−319. (19) Koppenol, W. H. Oxyradical reactions: From bond-dissociation energies to reduction potentials. FEBS Lett. 1990, 264, 165−167. 1845

dx.doi.org/10.1021/jf405464v | J. Agric. Food Chem. 2014, 62, 1839−1845