Resorcinol as a Spectrofluorometric Probe for the Hypochlorous Acid

Oct 9, 2012 - Roberto Biondi , Stefano Brancorsini , Giulia Poli , Maria Giulia Egidi , Enrico Capodicasa , Livio Bottiglieri , Sandro Gerli , Eleonor...
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Resorcinol as a Spectrofluorometric Probe for the Hypochlorous Acid Scavenging Activity Assay of Biological Samples Mustafa Ö zyürek, Burcu Bekdeşer, Kubilay Gücļ ü, and Reşat Apak* Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Turkey S Supporting Information *

ABSTRACT: A novel spectrofluorometric method was developed and validated for hypochlorous acid (HOCl) scavenging activity estimation using resorcinol, which is a highly sensitive and chemically stable fluorogenic probe. This assay is based on the chlorination of resorcinol to its nonfluorescent products in the presence of HOCl. HOCl reacts with both resorcinol and HOCl scavengers incubated in solution for 10 min, where scavengers compete with resorcinol for the HOCl. Thus, the relative increase in fluorescence intensity of intact resorcinol is proportional to the antioxidative activity of HOCl scavengers. Using this reaction, a kinetic approach was adopted to assess the HOCl scavenging activity of amino acids, vitamins, and plasma and thiol antioxidants. This assay, which is applicable to small molecule antioxidants and tissue homogenates, proved to be efficient for thiol-type antioxidants for which the widely used 5-thio-2-nitrobenzoic acid (TNB) test is not accurately responsive. Thus, conventional problems of the TNB assay arising from the reactivity of thiol-type scavengers to produce extra TNB by direct reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were overcome. Moreover, unlike enzymatic assays (e.g., elastase), there is no confusion as to whether the putative scavenger actually reacts with HOCl or inhibits the enzyme.

H

HOCl reacts with many biologically important molecules, including thiols, amino acids, and amine.4 The reaction of thiols (RSH) with HOCl initially yields a sulfenyl chloride intermediate, which reacts with another thiol to form a disulfide (RSSR).5 The rate constants for the reaction of HOCl with lowmolecular-weight thiols are >107 M−1 s−1.6

ypochlorous acid (HOCl) is a powerful oxidant generated in neutrophils via the reaction of the Cl− ion with H2O2 catalyzed by myeloperoxidase (MPO). Its reaction with the superoxide anion radical (O2• −) which occurs at a rate constant close to 107 M−1 s−1 yields the most reactive oxygen species (ROS), hydroxyl radical (•OH) thus causing tissue damage.1 MPO

H 2O2 + Cl− + H+ ⎯⎯⎯⎯→ HOCl + H 2O

HOCl + O2

•−

•

−

→ OH + Cl + O2

(1.1) (1.2)

(1.3)

RSCl + RSH → RSSR + H+ + Cl−

(1.4)

Several methods have been developed to detect HOCl, all employing a spectrophotometric, fluorogenic, or chemiluminogenic “probe”, i.e., a molecule that reacts with HOCl producing a detectable product. In a widely used TNB (5-thio-2-nitrobenzoic acid) method (λmax = 412 nm) based on the generation of 5,5′dithiobis(2-nitrobenzoic acid) (DTNB) upon the oxidation of TNB by HOCl, scavengers containing free thiol groupswhich are expected to compete with TNB for oxidation by HOCl actually react with DTNB; therefore, this method can neither be accurately applied to −SH-containing scavenger compounds (glutathione, cysteine, lipoic acid, etc.) nor to real samples containing these scavengers, because such thiols yield an excess of TNB when present.7,8 Yan et al.9 developed a protein carbonyl assay based on the inhibition of the formation of carbonyl groups in bovine serum albumin (BSA) in the presence of HOCl

A potent microbicidal agent, HOCl plays an important role in bacterial cell killing as a critical component of host defenses against invading bacteria, fungi, and viruses; however, abnormal levels of HOCl or insufficient cellular antioxidant defense can stimulate free-radical chain reactions by interacting with a multitude of biological molecules that may eventually give rise to various health disorders, such as cancer and atherosclerosis.2 The involvement of myeloperoxidase-derived HOCl in the pathogenesis of inflammatory diseases, in atherosclerosis through oxidation of low-density lipoprotein (LDL) contained in the artery wall, and in cell damages at the bronchial epithelium in respiratory diseases, may arise from the adverse reactions between HOCl and sulfhydryl groups of proteins/peptides of amino groups of amino acids.3 Therefore, it is important to eliminate excessive HOCl in vivo to prevent HOCl-originated disease, using antioxidant defense systems consisting of low- and high-molecular-weight constituents to defend against ROS attack. © 2012 American Chemical Society

RSH + HOCl → RSCl + H 2O

Received: August 16, 2012 Accepted: October 9, 2012 Published: October 9, 2012 9529

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calculated, in comparison with the reference (KI/taurine) method.

scavengers (i.e., thiols, uric acid, ascorbic acid), but a variety of other oxidation mechanisms also lead to the formation of carbonyls, so the carbonyl assay, as a biomarker of protein damage,10 cannot be taken as specific evidence of HOCl. In enzyme-based methods criticized for being laborious and timeconsuming, enzymes such as α1-antiproteinase (i.e., a major target of HOCl attack) or elastase,11 glutathione peroxidase and catalase (i.e., inactivated by relatively higher concentrations of HOCl)12 may be inhibited by potential scavengers existing in the medium, and this may cause errors in HOCl scavenging activity measurement, because of the confusion as to whether the putative scavenger actually reacts with HOCl or inhibits the enzyme. In addition, the fact that both the detector molecule (probe) and the HOCl scavenger analyte may simultaneously absorb light at the selected analytical wavelength is a frequently encountered interference in these spectrophotometric determinations, e.g., the KI/taurine chlorination assay involving ultraviolet-visible light (UV−vis) spectrophotometry could not be used for the HOCl scavenging ability measurement of oxicam group nonsteroidal anti-inflammatory drugs, because of the strong absorbance of the studied molecules in the range of 200− 420 nm.3 Through the use of chemiluminigenic probes based on the luminol-elicited CL, the HOCl scavenging activity of antioxidants was determined by measuring the amount of light produced by the oxidation of luminol,13 where results also must be expressed as IC50 values, since no linear relationship exists between the concentration of the scavenging molecule and the amount of light.3 A fluorogenic probe, para-aminobenzoic acid, acting as a nonselective target for HOCl (because of its peroxyl radical scavenging ability), was used to determine the HOCl scavenging activity of nonsteroidal anti-inflammatory drugs where interference removal leading to specificity increase required LC separation.3 For all these reasons, novel methods for the measurement of HOCl scavenging activity applicable to biological samples need to be devised to eliminate the aforementioned constraints and fill this literature gap. For the novel method to be devised, resorcinol (1,3-dihydroxy benzene), which is a highly fluorescent compound (excitation 276 nm, emission 304 nm), was selected as the HOCl probe molecule for the first time.14 Heasley et al. determined, through the identification of the intermediates, that successive electrophilic chlorination of resorcinol from monochlororesorcinol to dichlororesorcinol and then to 2,4,6-trichlororesorcinol occurs before ring opening.15 The standard one-electron redox potentials (E°) for ROO• (H+/ROOH) and polyunsaturated fatty acid (PUFA)-alkyl radicals (PUFA•) (H+/PUFA) were reported as 1000 and 600 mV, respectively.16 Resorcinol (E° = 1179 mV), on the other hand, has only negligible activity as a potential scavenger of peroxyl radical, singlet oxygen (E° = 650 mV) and hydrogen peroxide (E° = 320 mV) due to its higher redox potential.16,17 The fluorescence intensity of resorcinol is attenuated as a result of its reaction with HOCl. Resorcinol is fluorescent, but its chlorination products are not. The attenuated intensity of resorcinol fluorescence is dependent upon the HOCl scavenging activity of the tested compound, as measured by competitive reaction kinetics. With the aid of resorcinol fluorescence values recorded in the presence and absence of biological samples (i.e., thiol-type antioxidants (e.g., glutathione, cysteine), amino acids (e.g., L-serine, L-valine), plasma antioxidants (e.g., albumin, bilirubin), the HOCl scavenging rate constants and IC50 (50% inhibitive concentration) values of standards and tissue homogenates (kidney and liver) were

■

EXPERIMENTAL SECTION Reagents and Apparatus. The following chemical substances of analytical reagent grade were supplied from the corresponding sources: L-serine, L-valine, alanine, cysteamine, homocysteine, lipoic acid, taurine, glycine, dithioerythritol, methanol (MeOH), and ethanol (EtOH) were obtained from Sigma (Steinheim, Germany); uric acid, 4-chlororesorcinol, and 4,6-dichlororesorcinol were obtained from Aldrich (Steinheim, Germany); L-glutathione (reduced), L-ascorbic acid, proline, BSA, and sodium hypochlorite (NaOCl) were obtained from Merck (Darmstadt, Germany); acetic acid and potassium iodide were obtained from Riedel-de Haen (Steinheim, Germany); resorcinol, L-cysteine, L-methionine, lipoic acid (reduced), and N-acetyl-L-cysteine were obtained from Fluka (Buchs, Switzerland). The emission spectra and intensity measurements were recorded in a quartz cuvette, using a VARIAN Cary Eclipse spectrofluorometer (Mulgrave, Victoria, Australia). The ultraviolet-visible light (UV−vis) spectra and absorption measurements were recorded in matched quartz cuvettes using a Varian CARY 100 UV−vis spectrophotometer (Mulgrave, Victoria, Australia). The chromatograph was from a Model Waters Breeze 2 HPLC system (Waters Associates, Milford, MA, USA) equipped with a Model 2998 photodiode array detector (Waters, Chelmsford, MA, USA). Data acquisition was accomplished using Empower PRO (Waters Associates, Milford, MA). Preparation of Solutions. The resorcinol solution (50 μM), KI (2.0 M), and taurine (2-aminoethanesulfonic acid) (150 mM), were all prepared in pure distilled water (Millipore Simpak1 Synergy 185, USA). The HOCl stock solution was prepared by diluting 0.5% (v/v) solution of NaOCl from commercial solution immediately before use and adjusting a solution of NaOCl to pH 6.2 with diluted H2SO4. A working solution of HOCl was prepared by 10-fold dilution of the stock solution with distilled water. The concentration of HOCl was further determined spectrophotometrically at 235 nm using the molar absorption coefficient of ε = 100 M−1 cm−1.18 All antioxidants were freshly prepared in distilled water with 0.5−1.0 × 10−4 M uric acid in 0.5 M NaOH solution prior to measurement. The mobile phase constituents for HPLC analysis with gradient elution were pure methanol and 1% acetic acid. Animal Treatments and Preparation of Tissue Homogenate. Wistar rats (8 weeks old) were obtained from the animal facility from the Faculty of Veterinary Medicine of Istanbul University. The rats were housed in polycarbonate cages (450 cm2 area per animal) and acclimatized under laboratory conditions (23 ± 2 °C, humidity 50%−60%, 12 light/dark cycles). Liver and kidney tissues were isolated after sacrifice by decapitation from the rats. The tissue samples were washed with 0.9% NaCl solution, weighed (10%, w/v), and homogenized by adding cold 1.15% KCl solution in a glass homogenizer. Homogenates were immediately frozen in liquid nitrogen and kept at −80 °C until analysis.19 Homogenates were filtered through a 0.45-μm membrane filter before analysis. Resorcinol Assay. To a test tube were added 1.0 mL of 50 μM resorcinol (probe material), x mL of scavenger solution at a suitable concentration, (3.5 − x) mL of distilled water, and 0.5 mL of 0.8 mM HOCl rapidly in this order. The reaction started by adding HOCl solution. 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 9530

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Figure 1. Scheme of competitive reactions of the resorcinol probe and AOX; chlorinated probe products (Resorcinol-(Cl)x are nonfluorescent (namely, monochlororesorcinols, dichlororesorcinols, and trichlororesorcinols).

end of this period, the fluorescence intensity (λex = 276 nm, λem = 304 nm) of the reaction mixture was recorded. The effect of incubation time on the final mixture solution in the presence and absence of ascorbic acid (0.2 mM) were compared, with respect to their stabilities, the corresponding fluorescence intensities being recorded between 0 and 35 min. The IC50 (50% inhibitive concentration) values of the scavengers were determined using a resorcinol probe, by means of a linear plot of the inhibition percentage as a function of Cscavenger, where Io is the initial fluorecence intensity of the original resorcinol probe solution, I1, and I2 of the resorcinol probe subjected to HOCl action in the absence and presence of a HOCl scavenger, respectively, and C is the molar concentration of relevant scavenger (see Figure S-1 in the Supporting Information). The IC50 values were then compared with those found with the KI-taurine method.20 The fluorescence intensity of the resorcinol probe was higher in the presence of HOCl scavengers (due to less conversion to chlorinated resorcinol products); therefore, an increase in the intensity of the reaction mixture indicated an increase in HOCl scavenging activity. The HOCl scavenging activity was calculated using the following equation: ⎛ I − I1 ⎞ Inhibition ratio (%) = 100⎜ 2 ⎟ ⎝ I0 − I1 ⎠

with HOCl, where the only constituent giving rise to a fluorescence intensity in the system is the resorcinol probe itself (see Figure S-2 in the Supporting Information). KI/Taurine Assay. The spectrophotometric KI/taurine reference method20 of determining HOCl is based on the oxidation of I− to I2 by taurine chloramine. The I2 concentration was determined spectrophotometrically at 350 nm (ε = 2.29 × 104 M−1 cm−1).21 To a test tube were added 1.0 mL of phosphate saline buffer (pH 7.4), (2.0 − x) mL of distilled water, 0.5 mL of taurine, x mL of scavenger solution (x = 0−2.0 mL) at a suitable concentration, and 0.2 mL of HOCl, in this order. The solution was mixed and 0.5 mL of KI was added. A yellow coloration developed, and the absorbance was read at 350 nm. The concentration of tissue homogenate (1:20 diluted) was selected so as not to give an initial absorbance at 350 nm. The spectrophotometric scavenging assay was applied with incubation for 20 min at 37 °C to less-effective scavengers (proline, serine, valine, and alanine). The inhibition ratio of scavengers (%) was calculated using the following formula: ⎛A − A⎞ inhibition ratio (%) = 100⎜ 0 ⎟ ⎝ A0 ⎠

where A0 and A are the absorbances of the system in the absence and presence of a scavenger, respectively. HPLC Assay. The analyses were carried out using a reversephase Model C18 column (4.6 mm × 250 mm, 5 μm particle size) (ACE, Milford, MA, USA). The mobile phase consisted of two solvents, i.e., methanol (A) and 1% acetic acid (v/v) (B). The following parameters and gradient were used for the analysis of incubation mixture: Vsample = 20 μL; flow rate = 1.0 mL min−1; λResorcinol = 280 nm); 1. min 50% A−50% B (slope 1.0); 4. min 60% A−40% B (slope 1.0); 7. min 70% A−30% B (slope 1.0); 10. min 80% A−20% B (slope 1.0); 13. min 90% A−10% B (slope 1.0). The capability of scavenging hypochlorite was calculated using a modified version of eq 1:

(1)

The inhibition percentage (y) can be correlated to the concentration of inhibitor antioxidant (x): y = mx + n

(2)

where m and n are the slope and intercept of this linear correlation, respectively. IC50 can then be calculated for 50% inhibition (y = 50), such that y = 50 = m(IC50) + n

or

IC50 =

50 − n m

(4)

(3)

⎛ A − A1 ⎞ inhibition ratio (%) = 100⎜ 2 ⎟ ⎝ A 0 − A1 ⎠

Possible interferences from biological samples are overcome by taking sufficiently low amounts of these samples for competitive analysis in the course of chlorination of resorcinol. In that case, the HOCl scavengers contained therein would not show an initial fluorescence intensity, and yet, they would react

(5)

where A1 and A2 are the peak areas of the resorcinol probe in the absence and presence of the HOCl scavenger, respectively, A0 is 9531

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the peak area of the resorcinol probe at the initial concentration in the reaction mixture. Statistical Analysis. Descriptive statistical analyses were performed using Excel software (Microsoft Office 2002) for calculating the means and the standard error of the mean. Results were expressed as the mean ± standard deviation (SD). Using SPSS software for Windows (version 13), the data were evaluated by two-way ANalysis Of VAriance (ANOVA).22

■

RESULTS AND DISCUSSION Optimization of the Resorcinol Assay. A novel method for the assessment of HOCl scavenging activity of amino acids, thiol-type antioxidants, and plasma antioxidants is described, based on the fluorometric determination of unoxidized resorcinol (after HOCl chlorination) in the presence and absence of the tested scavengers. HOCl, generated from NaOCl at pH 6.2, attacks both the resorcinol probe and potential scavengers in 37 °C-incubated solutions for 10 min. At the end of this period, resorcinol gives rise to basically nonfluorescent chlorination products (i.e., monochlororesorcinols, dichlororesorcinols, trichlororesorcinols), resulting in a decrease in the fluorescence of resorcinol.23 A scheme of the conversion of resorcinol to chlorinated products (resorcinol(Cl)x) is shown in Figure 1. It has been reported in the literature that resorcinol is a highly fluorescent molecule but its chlorination products are not.14 The chlorination products had a negligible fluorescence intensity. The molar extinction coefficient for resorcinol in the proposed method was given as ε = 1.59 × 107 M−1 cm−1, and the linear concentration range was given as 1.60 × 10−6−5.0 × 10−5 M (correlation coefficient of r = 0.9999), within which the relative standard deviation (RSD) was 2.4%. The limit of detection (LOD) and limit of quantification (LOQ) were calculated using the equations LOD =

Figure 2. Fluorescence intensity versus incubation time curves of resorcinol alone and resorcinol subjected to HOCl in the absence (reference) and the presence (2.0 × 10−5 M AA) of a scavenger.

intensity of resorcinol standard solutions within the 0−30 min time interval (Figure 2). The fluorescence spectra of resorcinol, recorded in aqueous solutions at pH 6.2 with varying GSH concentrations, are shown in Figure 3, where the maximal decrease in fluorescence intensity

3s bl m

and

LOQ =

10s bl m Figure 3. Fluorescence spectra of the remaining resorcinol in the absence and presence of GSH ((a) 10 μM resorsinol, (b) 20 μM GSH, (c) 16 μM GSH, (d) 12 μM GSH, (e) 8.0 μM GSH, (f) 4.0 μM GSH, and (g) 0 μM GSH (reference)).

where sbl is the standard deviation of a blank and m is the slope of the calibration line. The LOD and LOQ values for resorcinol were determined to be 0.48 μM and 1.60 μM, respectively. The repeatability and reproducibility of the method for IC50 values of glutathione (as intra-day and inter-day (n = 3) RSD) were 0.9 and 1.3%, respectively. Conversion of the resorcinol probe to resorcinol-(Cl)x and inhibition of this reaction with a HOCl scavenger (i.e., AA) were followed by measuring the fluorescence intensity of the mixture, as a function of time (Figure 2). Inhibition of this reaction proceeded slowly in the presence of AA, and an optimal measurement time of 10 min was chosen when a fluorescence intensity plateau was reached. This optimal period was sufficient to achieve an intensity difference between the presence and absence of a scavenger, yielding a satisfactory inhibition ratio. Thus, because of the high conversion yield of resorcinol, IC50 values of the studied scavengers could be rapidly and precisely determined by recording the relative intensities within 10 min. For comparison, time-dependent fluorescence intensity changes were recorded using resorcinol standard solutions incubated under identical conditions. There was no noteworthy change in

of resorcinol was due to HOCl oxidation without competition, and its relative increase from baseline level was proportional to the scavenging ability of the competitive scavenger. Upon mixing 10 μM resorcinol with HOCl in the presence of GSH at concentrations of 4.0−20 μM, the fluorescence intensities of resorcinol at 304 nm increased, indicating that the reaction of resorcinol with HOCl results in its oxidation to nonfluorescent resorcinol-(Cl)x and that there is a proportional increase of 304nm peak heights (Figure 3) with increasing concentration of scavenger (GSH). With the aid of resorcinol 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. Precision and accuracy were evaluated by spiking two matrices (1:500 diluted liver and kidney homogenates) with known amounts of resorcinol at three different levels in the 5.0−10.0 μM 9532

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Figure 4. HPLC chromatograms for remaining resorcinol and its chlorination products after HOCl reaction in the presence and absence (reference) of GSH (λ = 280 nm): (a) reference (1.0 mL of 1.0 × 10−2 M resorcinol + 3.5 mL of water + 0.5 mL of 8.0 × 10−2 M HOCl) and (b) 1.0 mL of 1.0 × 10−2 M resorcinol + 0.3 mL of 1.0 × 10−2 M GSH + 3.2 mL of water + 0.5 mL of 8.0 × 10−2 M HOCl.

the conversion efficiency of resorcinol probe to resorcinol-(Cl)x calculated using the linear equation

concentration interval (see Table S-1 in the Supporting Information). The precision, which is expressed as the relative standard deviation (RSD, %) in fluorescence intensity measurement within the tested concentration range, was ∼2.1%. The recovery of the method varied from 102% to 105%. Comparison of the Findings of Resorcinol and HPLC Assays. Conversion of resorcinol probe to chlorination products and inhibition of this reaction with a scavenger (i.e., GSH) were followed by the proposed and HPLC methods. In the chromatograms of this work (Figure 4), the retention times for resorcinol, 4-chlororesorcinol, and 2,4,6-trichlororesorcinol (detected at 280 nm) were 4.18, 7.60, and 12.70 min, respectively. The amounts of resorcinol were found with the aid of the calibration curve drawn as peak area versus concentration. Concentrations of resorcinol (in μg mL−1) remaining after HOCl reaction in the presence of a scavenger (0.3 mL GSH), using the proposed and HPLC methods, were 57.0 ± 0.04 and 57.4 ± 0.06, respectively (the initial concentration of resorcinol was 220.0 ± 0.14), and both methods correctly reflected the relative decrease in probe (resorcinol) conversion into chlorination products (resorcinol(Cl)x) in the presence of GSH (Figure 4b). On the other hand,

y = 2.2 × 109C + 6.2 × 104

(r = 0.9999)

was found to be 73.9% without any scavenger (reference), compared to that of the proposed method (74.1%). Comparison of the Findings of the Resorcinol and KI/ Taurine Assays. HOCl can react with taurine to form the stable chloramine. Although taurine chloramine can be quantified directly using its absorbance at 250 nm, the system is not sufficiently sensitive for neutrophil studies, because of the low extinction coefficient of chloramine (∼398 M−1 cm−1). Its concentration can be sensitively determined through its oxidative ability to convert I− to I2. The I2 concentration was determined spectrophotometrically in the presence of excess I− as I3−.24 HOCl + TauNH 2 → TauNHCl + H 2O

(1.5)

TauNHCl + 2I− + H+ → TauNH 2 + I 2 + Cl−

(1.6)

On the other hand, HOCl and chloramines react readily with thiols, although chloramines differ from HOCl in discriminating between low-molecular-weight thiols, based on their pKa.25 9533

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RNHCl + 2RSH → RNH 2 + HCl + RSSR

respectively).29 These observations are in compliance with our findings (Table 1). Halliwell et al. concluded that ascorbic acid, at physiologically relevant concentrations, is able to protect α1antiprotease against inactivation by HOCl, and this protective effect of ascorbic acid is greatly enhanced by preincubating it with HOCl before adding the α1-antiprotease, suggesting that ascorbic acid is acting by scavenging HOCl.30 When 27 μM ascorbic acid was incubated with 25 μM HOCl at pH 7.4 and 37 °C for 20 min, the ascorbic acid concentration decreased to 17 μM (shown by HPLC); ascorbic acid concentration decreased to 5 μM and 1.5 μM upon incubation with 50 μM and 100 μM HOC1, respectively, where oxidation products of ascorbic acid were not observable with the UV detector of the HPLC system.30 The reaction of HOCl with amino compounds produces chloroamines (as in reaction 1.5), which retain the oxidizing ability of HOCl with less reactivity.25 An α-amino acid such as serine is finally converted to the corresponding aldehyde (e.g., glycolaldehyde), NH4Cl, and CO2 by HOCl oxidation through the formation of chloramine.31 BSA has a much lower IC50 value, found by the proposed method, compared to that by KI/taurine (see Table 1), in agreement with the observation that HOCl damage to α1-antiprotease was almost completely (95%) inhibited by 10 mg/mL albumin acting as the HOCl scavenger, whereas inhibition was 81% at 3.3 mg/mL albumin.32 In this work, Halliwell concluded that concentrations of albumin less than those normally present in plasma are able to completely prevent inactivation of α1-antiprotease by HOCl via preferential scavenging of this molecule.32 KI/taurine method was applied with incubation for 20 min in a water bath kept at 37 °C for albumin, valine, proline, serine, and alanine. HOCl scavenging activity of these scavengers could not be measured by KI/taurine without incubation (for glycine, the KI/taurine test did not work at the studied concentration level, even with incubation). The two-way ANalysis Of VAriance (ANOVA) comparison, via the aid of F-test of the mean-squares of “between-treatments” (i.e., IC50 values of different samples, with respect to the fluorometric and KI/taurine methods depicted in Table 1) and of residuals22 for several real samples (consisting of 12 HOCl scavengers) enabled us to conclude that there was no significant difference between treatments. In other words, the experimentally found resorcinol results and KI/taurine results were statistically alike at the 95% confidence level (Fexp = 0.7019, Fcrit = 4.543, Fexp < Fcrit at P = 0.05) (By exclusion of the values for compounds with the highest IC50 variability, i.e., BSA, and ascorbic acid; Fexp = 3.1856, Fcrit = 4.667, Fexp < Fcrit at P = 0.05). Thus, the proposed methodology was validated against the KI/ taurine method. Application of the Resorcinol Method to Some Tissue Homogenates. The proposed method can be effectively used to assay the HOCl scavenging activity of biological fluids on the condition that sufficiently low amounts of samples are taken for analysis. The HOCl scavenging activity of kidney and liver homogenates, plotted as percentage inhibition versus homogenate volume, is shown in Figure 5, where the inhibition percentage was almost linearly correlated to homogenate volume within the interval of 0.5−2.5 mL. The HOCl scavenging activity values measured with the proposed resorcinol method and the reference KI/taurine method are comparatively depicted in a bar diagram (Figure 6); the percentage inhibitions of identical tissue homogenates found with resorcinol were almost equal in relative intensity to those measured with the reference method. The HOCl scavenging activity of kidney tissue homogenate was

(1.7)

The reaction of methionine with HOCl is accepted to initially produce a sulfonium chloride that subsequently hydrolyzes, giving the sulfoxide as the final product. Peskin and Winterbourn hypothesized that the sulfenyl chloride formed from chloramine and HOCl condenses with the thiol to a disulfide more rapidly than it hydrolyzes.25 Magalhaes et al. reported13 HOCl scavenging activity of lipoic acid and cysteine as IC50 values of 26.3 and 9.1 μM, respectively, in accordance with the values found with the fluorometric method (see Table 1). The activity Table 1. HOCl Scavenging Activity of Various Scavengers Measured by the Resorcinol Method in Comparison to the KI/Taurine Methoda IC50 value HOCl scavenger glutathione (GSH) N-acetyl-cysteine (NAC) cysteine homocysteine 1,4-dithioerythritol methionine lipoic acid dihydrolipoic acid cysteamine serineb glycine prolineb alanineb valineb albuminb uric acid ascorbic acid

via the resorcinol method Thiol-Type Antioxidants 11.32 ± 0.76 μM 15.76 ± 0.07 μM 9.27 ± 0.88 μM 25.51 ± 1.60 μM 11.71 ± 0.31 μM 15.59 ± 0.65 μM 22.25 ± 1.05 μM 12.16 ± 0.70 μM 13.51 ± 0.23 μM Amino Acids 30.33 ± 2.00 μM 34.97 ± 1.89 μM 29.75 ± 0.73 μM 25.60 ± 0.36 μM 33.00 ± 1.45 μM Plasma Antioxidants 0.41 ± 0.012 μM 13.27 ± 0.90 μM 46.41 ± 2.48 μM

via the KI/taurine method 8.95 ± 0.47 μM 7.46 ± 0.48 μM 14.50 ± 1.47 μM 7.47 ± 0.58 μM 20.43 ± 1.24 μM 36.00 ± 3.20 μM 23.11 ± 2.30 μM 18.06 ± 1.61 μM 33.30 ± 1.24 μM 60.00 ± 6.41 mM NDc 18.49 ± 1.67 mM 52.43 ± 6.41 mM 58.00 ± 5.70 mM 18.38 ± 0.68 μM 16.95 ± 0.97 μM 16.00 ± 0.37 μM

a

P = 0.05, Fexp = 0.7019, Fcrit (table) = 4.543, Fexp < Fcrit (table). Data in table presented in the form of “mean ± SD”, N = 3. IC50 values were calculated with respect to eqs 1 and 4 (N = 4 or 5 data points). bThe KI/taurine method was applied with incubation. c“ND” indicates that the HOCl scavenging activity at the studied concentration level could not be detected.

of lipoic acid is likely due to its strained five-membered ring, which is well-known to react rapidly with electrophilic reagents.26,27 Pattison and Davies found the second-order rate constant for the reaction of cysteine with HOCl to be 3.0 × 107 M−1 s−1,28 in accordance with the value found in the present study (3.39 × 107 M−1 s−1) (see Table S-2 and Figure S-3 in the Supporting Information). The relative HOCl scavenging activity of compounds can be proposed in some cases, based on their functional groups, e.g., the stronger scavenging activities of dihydrolipoic acid, glutathione, and cysteine can be attributed to their thiol groups.9 Yan et al. reported that −SH groups are more effective than −OH groups in scavenging HOCl. They observed that ascorbic acid (with two −OH groups) showed lower scavenging ability, compared to thiol compounds where BSA carbonyl assay was used.9 The HOCl scavenging activity of (thiols-rich) raw garlic extract was reported to be greater than that of ascorbic acid (i.e., IC50 values were 0.339 ± 0.028 and 0.500 ± 0.049 mg mL−1, 9534

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used TNB method arising from the extra capability of thiols to directly reduce DTNB to TNB without involvement of HOCl scavenging action were overcome. In addition, the major problem inherent in the widely used enzymatic (e.g., elastase) assays is addressed: Does the putative scavenger actually react with HOCl or does it inhibit the enzyme? The resorcinol assay results were close or comparable to those found by the conventional KI/taurine and HPLC methods. This alternative cost-effective method has emerged as a promising tool for studies of HOCl biochemistry.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +90 212 473 7180. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

Figure 5. Percentage inhibition of tissue homogenates, as a function of homogenate volume using the resorcinol method ((●) 1:500 diluted liver and (▲) 1:500 diluted kidney homogenate).

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ACKNOWLEDGMENTS One of the authors (B.B.) would like to thank Istanbul University Research Fund, Bilimsel Arastirma Projeleri (BAP) Yurutucu Sekreterligi, for the support given to her Ph.D. Thesis Project No. T-5761 and to Istanbul University, Institute of Pure and Applied Sciences (I.U. Fen Bilimleri Enstitüsü), for the support given to her Ph.D. thesis work with the title “Development of Spectrophotometric Methods for Measurement of Reactive Oxygen Species Scavenging Activity in Biological Samples”. The authors also extend their gratitude to TUBITAK (Turkish Scientific and Technical Research Council) for the Research Project No. 112T372, and to T.R. Ministry of Development for the Advanced Research Project of Istanbul University (No. 2011K120320).

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Figure 6. Percentage inhibition of some tissue homogenates calculated with the resorcinol method (1:500 diluted homogenate), in comparison to that calculated using the KI/taurine method (1:20 diluted homogenate).

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CONCLUSIONS HOCl is believed to be the key reactive oxygen species (ROS) responsible for oxidative damage to various biological molecules, and the deleterious effects of excessive HOCl formation can be attenuated using antioxidants. Despite the great importance of HOCl detection, the existing literature methods for meeting this demand are quite few and lack selectivity. The proposed method for the detection of HOCl is simple, sensitive, and easy to operate (only a conventional spectrofluorometer is needed). This method renders the relatively specific detection of HOCl (thanks to the high oxidation potential of the probe), and is applied to HOCl scavenging activity estimation of a rich variety of biologically active compounds (i.e., amino acids, thiols, plasma antioxidants) and tissue homogenates over a relatively wide concentration range. Thus, conventional problems of the widely 9535

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Analytical Chemistry

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