Research article Received: 17 December 2014,
Accepted: 13 January 2015
Published online in Wiley Online Library: 8 March 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2879
Reactivity of pyruvic acid and its derivatives towards reactive oxygen species Aleksandra Kładna,a Mariola Marchlewicz,b Teresa Piechowska,c Irena Krukc and Hassan Y. Aboul-Eneind* ABSTRACT: Pyruvic acid and its derivatives occurring in most biological systems are known to exhibit several pharmacological properties, such as anti-inflammatory, neuroprotective or anticancer, many of which are suggested to originate from their antioxidant and free radical scavenger activity. The therapeutic potential of these compounds is a matter of particular interest, due to their mechanisms of action, particularly their possible antioxidant behaviour. Here, we report the results of a study of the effect of pyruvic acid (PA), ethyl pyruvate (EP) and sodium pyruvate (SP) on reactions generating reactive oxygen species (ROS), such as superoxide anion radicals, hydroxyl radicals and singlet oxygen, and their total antioxidant capacity. Chemiluminescence (CL) and spectrophotometry techniques were employed. The pyruvate analogues studied were found to inhibit the CL signal arising from superoxide anion radicals in a dose-dependent manner with IC50 = 0.0197 ± 0.002 mM for EP and IC50 = 69.2 ± 5.2 mM for PA. These compounds exhibited a dose-dependent decrease in the CL signal of the luminol + H2O2 system over the range 0.5–10 mM with IC50 values of 1.71 ± 0.12 mM for PA, 3.85 ± 0.21 mM for EP and 22.91 ± 1.21 mM for SP. Furthermore, these compounds also inhibited hydroxyl radical-dependent deoxyribose degradation in a dose-dependent manner over the range 0.5–200 mM, with IC50 values of 33.2 ± 0.3 mM for SP, 116.1 ± 6.2 mM for EP and 168.2 ± 6.2 mM for PA. All the examined compounds also showed antioxidant capacity when estimated using the ferric–ferrozine assay. The results suggest that the antioxidant activities of pyruvate derivatives may reflect a direct effect on scavenging ROS and, in part, be responsible for their pharmacological actions. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: pyruvic acid; ethyl pyruvate; sodium pyruvate; reactive oxygen species; chemiluminescence; spectrophotometry
Introduction
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* Correspondence to: H. Y. Aboul-Enein, Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Giza 12622, Egypt. E-mail: [email protected] a
Department of History of Medicine and Medical Ethics, Pomeranian Medical University, Szczecin, Poland
b
Department of Aesthetic Dermatology, Pomeranian Medical University, Szczecin, Poland
c
Institute of Physics, Faculty of Mechanical Engineering and Mechatronics, West Pomeranian University of Technology in Szczecin, Szczecin, Poland
d
Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Giza 12622, Egypt
Copyright © 2015 John Wiley & Sons, Ltd.
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Pyruvic acid is an α-keto acid being a key intermediate of glucose metabolism; its carboxylate anion is known as pyruvate. This compound enters into Krebs cycle to generate the ATP. Several authors have reported that pyruvate and its derivatives possess anti-inflammatory activities: suppressing the generation of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and inhibiting the expression of pro-inflammatory agents such as inducible nitric oxidase synthase (iNOS), cyclooxygenase-2 (COX-2), and other pro-inflammatory mediators (1–8). Biological mechanisms of the anti-inflammatory action are not well recognized, and are in part ascribed to their antioxidant activity (2). Over the past two decades, growing research has revealed that oxidative stress can lead to chronic inflammation that is associated with the development of most chronic diseases including cardiovascular and neurological diseases, diabetes and cancer (9–13). Oxidative stress may be defined as an imbalance between the generation of pro-oxidants [reactive oxygen species (ROS), reactive nitrogen species (RNS) and their metabolites] and their elimination by antioxidants. ROS and RNS playing the most important roles in biological systems and include the superoxide anion radical (O ¯2• ), hydroxyl radical (HO°), hydrogen peroxide (H2O2), peroxynitrite (ONOO–) and singlet oxygen (1O2) (13–15). Among these reactive species, HO° and 1O2 are the most potent oxidants for organic and inorganic compounds. With regard to anti-inflammatory properties and the ability to scavenger ROS, confirmed in numerous studies of pyruvate and its analogues in animal models of human disease (8,16), it is suggested that these compounds have anti-
cancer, anti-ischemic, cardio- and neuroprotective activities, and are promising for use as therapeutic agents for cancer and inflammatory diseases. However, the possibility that pyruvate and its analogues might be used clinically requires, among other things, quantitative evaluation of their ability to scavenge particular reactive species. To address this, a series of in vitro experiments designed to evaluate the reactivity of pyruvic acid (PA), ethyl pyruvate (EP) and sodium pyruvate (SP) towards superoxide radical, hydroxyl radical, hydrogen peroxide and singlet oxygen were carried out. The chemical structures of the tested compounds are shown in Fig. 1.
A. Kładna et al.
Figure 1. Chemical structures of pyruvic acid and its derivatives.
The IC50 value was measured from a linear regression curve of the scavenging ratios (Q) against the logarithmic concentrations of each the tested compound.
Experimental Reagents All reagents were of analytical grade. PA, EP, SP, luminol, trolox (6-hydroxy-2,5,7,8-tetramethyl-2-carboxyl acid), 18-crown-6-ether (1,4,7,10,13,16)hexaoxacyclooctadecane, ascorbic acid, sodium trifluoroacetate, imidazole (A) and p-mitrosodimethylaniline (RNO) were from Sigma-Aldrich GmbH (Sternheim, Germany). Anhydrous dimethylsulfoxide (DMSO) was from Aldrich (St. Louis, MO, USA). Ammonium ferrous sulfate (FeSO4(NH4)2SO4) and potassium superoxide (KO2) were from Fluka (Buchs, Switzerland). Tiron (1,2-dihydroxybenzene-3,5-disulfonic acid) and other reagents were from Merck (Darmstadt, Germany). For water solution preparation, double distilled water was used. Antioxidant activity determination Chemiluminescence measurements Superoxide radical scavenging activity. The superoxide anion radical was produced according to the method described by Valentine et al. (17) as follows: a 60-mg aliquot of 18-crown-6-ether was dissolved in 10 mL of dry DMSO and 7 mg of KO2 was quickly added to the flash using a syringe to avoid contact with the air. This mixture was stirred for 60 min to give a pale yellow solution of 10 mM superoxide anion radical stable at room temperature. In the measurements, the radical was used as a 1 mM solution in DMSO. The tested compounds (PA, EP, SP and Tiron) were dissolved in DMSO. Tiron, a compound known to be an effective antioxidant was used as a positive control. O ¯2• scavenging activity was measured using a previously reported chemiluminescence (CL) technique (18). Briefly, reagents were mixed in a thermostated glass cuvette placed in a light-tight chamber before an EMI 9553Q photomultiplier with a S20 cathode sensitive over the range 200–800 nm, interfaced with a computer for data acquisition and handling. The reactor cell was washed using a B-169 vacuum system (Büchi, Flawill, Switzerland). All measurements were performed at 293 ± 1 K. A personal computer was equipped with a home-written software program which provides the opportunity to calculate of the light sum (∑I), i.e. an area under the kinetic curve, I = f(t), for any chosen time interval t
∑I ¼ ∫ IðtÞdt t0
where I(t) is the CL intensity, t0 is the measurement at the start time (after mixing reagents) and t is the measurement at the finish. Scavenging activity Q(%) was calculated from the equation Qð%Þ ¼ ½1 – ∑I=∑Io 100
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where ∑Io and ∑I are the relative integrals of the control reaction and sample solutions over 70 s, respectively. The results were expressed as a sample concentration giving 50% inhibition of the O¯2• -induced light emission, as a measure of antioxidant activity.
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In order to evaluate the scavenger activity of the tested pyruvate derivatives against hydrogen peroxide, a previously described CL method based on H2O2-induced oxidation of luminol was used (19). The reaction mixture contained different concentrations of the examined compound, 100 μM luminol, phosphate buffer pH 7.0 and 5 mM H2O2. The reaction was initiated by addition of H2O2. The scavenging activity was defined as percentage (%) CL quenching Q (%) = [1 – ∑I/∑Io] × 100, where ∑Io is the relative integral of a control reaction (luminol + pH + H2O2) and ∑I is the sum of the CL detected from the control reaction in the presence of the tested compound (PA,EP or SA). Scavenging potentials were calculated from linear regression curves of the scavenging ratios against the logarithmic concentrations of each the tested compound as IC50 values. Hydrogen peroxide scavenging activity.
Hydroxyl radical scavenging activity. The Fenton reaction (Fe(II) + H2O2) was used as a source of hydroxyl radicals (20,21). The influence of tested compounds on the system producing HO radicals was detected using deoxyribose degradation caused by the radicals according to the method described by Halliwell and Gutteridge (22,23). In this method, degradation of deoxyribose by hydroxyl radical results in malonaldehyde formation. When reacted with thiobarbituric acid, this forms a coloured thiobarbituric acid–malonaldehyde adduct (2: 1), absorbing strongly at 532 nm in acid solution. The reaction mixture consisted of deoxyribose (2 mM), sodium trifluoroacetate buffer (10 mM, pH 6.15), ferrous sulfate (62.5 μM) and H2O2 (1 mM) (control). The tested compounds were added to the reaction mixture before starting the reaction by adding H2O2. The reaction mixture was incubated for 30 min at 310 K. The extent of deoxyribose oxidation by the hydroxyl radical was measured after mixing 3 mL of sample with 1 mL trichloroacetic acid (2.8%) and 1 mL thiobarbituric acid in 50 mM NaOH. The prepared mixture was heated at 373 K for 15 min and cooled to room temperature. Individual samples were placed in a Zeiss M-40 UV/Vis spectrophotometer with Win-Aspect software ( Jena, Germany) to measure the thiobarbituric acid–malonaldehyde adduct absorption at 532 nm. The resulting absorbance was read against appropriate blanks. The radical scavenging activity of PA, EP, SP and the positive control trolox was defined as the percentage deviations of deoxyribose degradation with respect to the tested compounds-free control Q(%) = [1 – A/Ao] × 100%, where A is the absorbance detected at 532 nm in the presence of the tested pyruvate compound or trolox dissolved in water, and Ao is the absorbance without the compound, after addition of the same amount of water. Similarly, IC50 values were calculated from linear regression curves of Q(%) against logarithmic concentrations for each the examined compound. Ferric reducing activity measurements. The reducing activities of the tested compounds were measured using an antioxidant assay developed by Berker et al. (24). In this method, reduction of Fe
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 1153–1158
Pyruvic acid and its derivatives towards reactive oxygen species
(III)–ferrozine agent by a compound having an antioxidant capacity leads to stable Fe(II)–ferrozine complex formation that exhibits a single sharp absorbance peak at 562 nm. The ferric–ferrozine complex was prepared according to the following procedure. An aqueous solution containing 0.024 g of NH4Fe(SO4)2 · 12H2O in 1 mL of HCl (1 M) was mixed with a solution of 0.123 g ferrozine dissolved in water. After mixing, the reaction mixture was diluted to 25 mL with distilled water to obtain a final Fe(III) ion concentration of 2 mM, and ferrozine concentration of 0.1 mM. The formed Fe(III)–ferrozine complex was kept in a stoppered dark-coloured bottle. Working solutions were prepared from this stock solution as follows 1.5 mL of the complex was added to 1 mL of antioxidant solution (1 mM) and mixed with 2 mL of buffer solution (0.2 M/L acetic acid/sodium acetate, pH 5.5). The increase in absorbance was determined at 562 nm after standing for 1.5 h at room temperature. The effect of the tested compounds on the formation of singlet oxygen was performed according to the method developed by Kraljic and El-Mohsni (25). The method is based on the bleaching of RNO by the intermediate product a transannular peroxide (AO2) arising in the reaction of 1O2 with imidazole Effect of the tested compounds on singlet oxygen production.
1
O2 þ A → AO2
AO2 þ RNO → -RNO þ products The bleaching of RNO was measured as the decrease in absorbance at 440 nm using a Zeiss M-40 spectrometer. All experiments were conducted at least three times. Linear regression analyses were computed using the statistical package STATISTICA 6.0 2002 (Stat Soft Polska, Kraków, Poland).
Results and discussion Use of the 18-crown-6-ether/KO2/DMSO reaction as a source of superoxide anion radical and the sensitive CL method allowed us to measure the radical-scavenging activity of PA, EP and SP. The reaction is accompanied by the emission of electromagnetic radiation (CL) with emission bands with maxima at 480, 580, 640 and 700 nm, corresponding to the simultaneous transition in the singlet oxygen dimoles (1O2)2 during their radiative deactivation to the ground state (3O2) (26,27). The spectra and the reactions responsible for the 1O2 production have been presented previously (18). According to the reported mechanism, 1O2 is generated in the following reactions (28)
Figure 2. (A) Representative scavenging effects of various concentrations of EP on the chemiluminescence (CL) intensity recorded from 1 mM superoxide anion radical produced in DMSO (control). Curves: 1, control; 2, 0.005 mM; 3, 0.01 mM; 4, 0.03 mM; 5, 0.05 mM; 6, 0.2 mM. (B) A dose-dependent inhibition of the light sum recorded from the superoxide anion radical/DMSO solution measured at various concentrations of EP, under the same conditions as in (A). (C) Dose-dependent inhibition of the light sum recorded from the superoxide anion radical/DMSO solution in the presence of PA and SP, under the same conditions as in (B). Denotations of the tested compounds are given in Fig. 1.
Table 1. Linear range and IC50 (mean ± SD) value for the examined pyruvate analogues against superoxide anion radical, hydrogen peroxide, and hydroxyl radical Species
2 O2¯• þ 2ðCH3 Þ2 SO→1 O2 þ ðCH3 Þ2 SO2 þ CH3 SOðCH2 Þ
O 2¯
þOH- O2¯• →1 O2 þ electron
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H2O2
HO°
PA EP SP Tiron PA EP SP Ascorbic acid PA EP SP Trolox
Range (mM) 20–250 0.005–0.2 0.2–2.0 0.1–2.0 0.5–10.0 0.5–10.0 0.5–10.0 0.0005–0.02 0.5–200 0.5–200 0.5–200 0.01–3.0
IC50 (mM) 69.2 ± 5.2 0.0197 ± 0.002 40% (2 mM)* 0.57 ± 0.06 1.71 ± 0.12 3.83 ± 0.21 22.91 ± 1.21 0.00069 ± 0.00002 168.2 ± 6.2 116.2 ± 6.2 33.2 ± 0.3 0.052 ± 0.03
* Scavenging effect observed at the highest examined concentration.
Copyright © 2015 John Wiley & Sons, Ltd.
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Figure 2(A) demonstrates the typical CL kinetic curves obtained from the superoxide anion radical/DMSO system alone (curve 1) and in the presence of 0.005–0.2 mM EP, as representative behaviour. This plot shows that CL intensity from the light-emitting system decreased in the presence of EP, in a concentrationdependent manner (curves 2–6). A positive linear relationship was observed between antioxidant activity and EP concentration (Fig. 2B). The compounds PA and SP demonstrated similar inhibitory behaviour (Fig. 2C). Table 1 gives the calculated IC50 values for these pyruvates. SP reached a 40% effect at the maximum tested concentration (2 mM). The IC50 values of PA and EP were compared with that calculated for a powerful inhibitor of
Compound
A. Kładna et al.
superoxide anion radical such as Tiron under the same experimental conditions. The IC50 value for the superoxide anion radical scavenging activity by EP was significantly lower than that of Tiron. This behaviour indicates that EP is more efficient inhibitor of superoxide anion radical than Tiron, for which the rate constant of the reaction with this species was reported to be 5 × 108 M/s in aqueous solution (29). The radical scavenging activities of the examined pyruvates seem to depend particularly on the presence of the hydrogen atom in the carboxylic acid group (PA) and the hydrogen atoms of the ethyl group of the aliphatic ester (EP). Findings from the superoxide anion radical scavenging method demonstrate that the examined pyruvates might act as reducing agents, by donating a hydrogen atom to superoxide anion (30): O2•¯ þH→HO 2 Under our experimental conditions, the HO 2 species may decompose to OHˉ in the reaction with DMSO (28): HO 2 þ ðCH3 Þ2 SO→HO þ ðCH3 Þ2 SO2
To confirm that the tested pyruvates are able to react with H2O2, luminol-amplified CL was applied. All the pyruvate analogues tested reduced the CL signal in terms of both light intensity and the CL sum in a concentration-dependent manner (Fig. 3A). The percentage of light intensity quenching was similar to that of the CL sum quenching (not shown). A linear relationship between
A PA EP SP
100 80 60 40 20 0 0
20
40
60
80
100
Concentration (mM)
H2O2 scavenging potency and the concentration of particular pyruvates was observed (Fig. 3B). The IC50 values calculated from these plots are given in Table 1. They vary from 1.71 to 22.91 mM and are significantly higher than the IC50 value calculated for the standard control, ascorbic acid (0.69 ± 0.02 μM). Luminol was the first compound used as a CL probe for the detection of H2O2 (31) and the mechanism of electronic excitation of luminol has been intensively investigated. CL accompanying oxidation of luminol satisfies criteria for its use in bioanalysis, due to its ultrasensitivity and nontoxicity (19,32–34). The literature has shown that the CL mechanism accompanying oxidation of luminol may be summarized briefly as follows. In the first step luminol is dehydrogenated to the diazoquinone, which is oxygenated with H2O2 to give a cyclic peroxide. The second step involves decomposition of the cyclic peroxide to a 3-aminophthalate dianion in an electronically excited state. In the last step, the dianion returns to the ground state with the emission of light (λmax = 425 nm) (31,35,36). Thus, the luminolamplified CL, used to quantify the H2O2 scavenging potency of pyruvate analogues demonstrates that the tested compounds can react directly with H2O2, irreversibly removing this oxidant from the reaction medium. Nath et al. (37) and Kang et al. (38) reported that pyruvate undergoes decarboxylation in the presence of H2O2, and acetate, carbon dioxide and water were identified as the reaction products. Thus, the protective effects of pyruvate derivatives observed is cells and tissues (5–8) may be due, in part, to their antioxidant action and are in accordance with our CL results. All the tested pyruvate analogues were also able to scavenge hydroxyl radicals in a concentration-dependent manner (Fig. 4). It is well recognized that deoxyribose degradation depends on the presence of ROS, mainly hydroxyl radical (39). Deoxyribose degradation was efficiently prevented by PA, EP and SP, with SP being the most effective pyruvate derivative, giving an IC50 value of 33.2 ± 0.3 mM (Table 1), although it demonstrated very weak potency in comparison with trolox (IC50 = 0.052 ± 0.03 mM). These results demonstrate, once again, an important ability of pyruvate analogues to scavenge hydroxyl radicals directly. It is worth noting that this species is the most important radical involved in cell damage, due to its high reactivity with biomolecules, usually exceeding 109 M–1 s–1 (14). The high level of radical activity may be important in the oxidative damage of cells, because these hydroxyl radicals can be involved in several reactions, such as adding to a double bond, abstraction of a hydrogen atom and oxidation via electron donation.
B 100
PA
50
80 40
R
= 0,9869
R
= 0,9915
R
= 0,9869
SP
EP
PA
EP
60
30
40
20
Q
SP
20
10 0
0
1
1
10
Concentration (mM)
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Figure 3. (A) Suppression effect of the tested compounds on the luminol-based CL. (B) Dose-dependent inhibition of the light sum; calculation of EC50 values of the examined compounds towards H2O2. For reaction conditions, see Experimental.
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10
100
Concentration (mM) Figure 4. Dose-dependent effect of the tested compounds on the deoxyribose oxidation by hydroxyl radicals generated by the Fenton reaction. Experimental conditions are given in Experimental.
Copyright © 2015 John Wiley & Sons, Ltd.
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Because antioxidants can exert their antioxidant activity via donation of hydrogen atoms or single-electron transfer to a radical (40), we further studied the antioxidant potential of pyruvate analogues using a ferric–ferrozine assay, troxol antioxidant capacity (TAC). In this assay, the examined compounds were able, in the presence of ferrozine, to reduce the ferric ion in the Fe(III)– ferrozine complex to the Fe(II) ion. The Fe(II)–ferrozine complex formed exhibits an absorbance maximum at 562 nm, and the band intensity depends on the reducing power of an antioxidant. The latter complex absorbance is positively correlated with the antioxidant reducing potential (an electron transfer way). Figure 5(A) illustrates the ferric reducing powers of the tested antioxidants and a comparison with the reduction power of trolox, as the positive control. The reducing activities of the pyruvate analogues at 1 mM and the standard compound trolox at 0.0133 mM were observed to be in the following order: trolox > PA > SP > EP. The reducing ability of trolox was ~ 100× that of PA. The most important advantage of the ferric–ferrozine method of measuring antioxidant measurement is a high sensitivity for antioxidants that have even weak reducing ability, due to the very high molar extinction coefficient (Ɛ = 2.8 × 104 M-1 cm-1) of the Fe(II)–ferrozine complex (24). By contrast to their beneficial effects, some efficient antioxidants, e.g. flavonoids, have been reported to exert pro-oxidant
A
1.4 PA
1.2
Absorbance
1.0
O2•¯ þ H2 O2 → O2 þ HO° þ HOˉ This reaction is slow (k = 3.0 ± 0.6 M-1 s-1) but is strongly catalysed by transition metals ions, such as iron(II) and copper(II), and also ions whose presence in cells is due to environmental pollution (13,43), such as cobalt(II), chromium(III) and nickel(II) (the rate constant k ≥ 100 M-1 s-1). The experimental findings and the literature data (44) allow us to suggest that the base-catalysed disproportionation of H2O2 is responsible for 1O2 formation under our experimental conditions:
H2 O2 þ HOOˉ →1 O2 þ H2 O þ HOˉ
EP
0.4 0.2 450
500
550
600
650
700
Wavelength (nm)
B EP
0.3 PA
0.2
SP
0.1
Control
0
The toxicity of H2O2 in the cell is mainly due to its decomposition to HO• in a reaction with transition metal ions or is based on the Haber–Weiss reaction (42)
H2 O2 þ HOˉ → HOOˉ þ H2 O
SP
0.6
0.0
FeðIIÞ þ H2 O2 → HOo þ HOˉ þ FeðIIIÞ
Trolox
0.8
0.0 400
activity in vitro (41). The possibility that the pyruvate compounds would exert pro-oxidant activity under our experimental conditions and their effect on the generation of 1O2 in the Fenton reaction was evaluated (Fig. 5B). As shown in Fig. 5, the bleaching of RNO in the RNO + imidazole + Fenton reagents + pyruvate compound system was significantly increased in comparison with the RNO + imidazole + Fenton reagent reaction (control) (P < 0.05). The efficacy of RNO bleaching yields a ranking order of EP > PA > SP, suggesting the generation of increased amounts of 1 O2 in the presence of these pyruvate derivatives. Such behaviour of these compounds is in accordance with their reducing ability observed in the TAC assay; the compounds can reduce the free Fe(III) ion to Fe(II), thereby supporting the Fenton reaction:
5
10
15
20
25
30
35
40
Time (min)
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Figure 5. (A) Comparison of total antioxidant activity of the examined compounds and trolox. (B) Time course of the bleaching of RNO in the presence of the examined compounds (80 μM RNO + 60 mM imidazole + 10 mM sodium trifluoroacetate + 0.1 mM FeSO4(NH4)2SO4 + 1 mM tested compound + 1 mM H2O2), and in their absence (control) (80 μM RNO + 60 mM imidazole + 10 mM sodium trifluoroacetate + 0.1 mM FeSO4(NH4)2SO4 + 1 mM H2O2). ΔA = difference in absorbance of the reaction mixture with a tested compound and the control reaction. Bleaching of RNO was followed at 440 nm, an absorption maximum of RNO.
Because of the relatively high concentration of H2O2 in the cell (~10-2 μM) (42) due to its accumulation, and also its ability to diffuse across cell membranes, the observed enhancing effect of pyruvate derivatives on 1O2 generation seen in Fig. 5(B), demonstrates that the compounds tested may be involved in the redox regulation of cellular stress. Our findings demonstrate that the preventive role of pyruvate derivatives against oxidative stress, as reported for EP (32), is mainly due to scavenging of H2O2 and superoxide anion radicals. The high IC50 values of PA and SA suggest that these compounds are not efficient hydroxyl radical scavengers (Table 1) under our experimental conditions, regardless of their high rate constants for the reactions with pyruvic acid and pyruvate in aqueous solution – (1.2 ± 0.4) × 108 and (7 ± 2) × 108 M-1 s-1, respectively (45). This seems to confirm the high potency of pyruvate derivatives to decompose H2O2, leading to suppression of hydroxyl radical formation in the Fenton reaction. In conclusion, the above findings indicate that pyruvate analogues possess good scavenging potential towards superoxide anion radical, weaker scavenging potential towards hydroxyl radical and efficiently decompose H2O2. Moreover, these compounds show relatively high reducing power. One question arises, whether the examined compounds are better scavengers of oxygen free radicals then the standard antioxidant Tiron. We suggest that EP is a much better inhibitor of superoxide radicals then Tiron in a hydrophobic medium. To the best our knowledge, comparison of the scavenging activity for ROS and the reducing potential of PA, EP and SP is reported for the first time. For this reason, it is difficult
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to compare IC50 values measured in our study with those obtained previously. Only one study (32) has investigated the antioxidant activity of pyruvate and EP towards H2O2. The authors used luminol-amplified CL and found IC50 values for these compounds of ~ 47 and 12 mM, respectively. These values are about 10 times higher than ours. In the same study, the IC50 value for the reaction of EP with superoxide anion radical generated in aqueous solution using a xanthine–xanthine oxidase system as a source of the radical was estimated to be 80 mM. This inconsistency between the previous findings and ours for a value of IC50 for EP may be due to methodological differences in study design. Our study provides further evidence that pyruvic acid and its derivatives are important endogenous scavengers of ROS, especially superoxide radical and H2O2, and thereby have potential for controlling inflammation and preventing diseases with an oxidative stress aetiology, such as neurodegenerative, disorders diabetes, cardiovascular disease or cancer.
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19. Tao X, Wang W, Wang Z, Cao X, Zhu J, Niu L, et al. Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate. Luminescence 2014;29:301–6. 20. Goldstein S, Meyerstein D, Czapski G. The Fenton reagents. Free Radical Biol Med 1993;15:435–45. 21. Walling C. Fenton’s reagents revisited. Acc Chem Res 1975;8:125–31. 22. Halliwell B, Gutteridge JMC. Formation of a thiobarbituric acid – reactive substance from deoxyribose in the presence of iron salts: the role of superoxide and hydroxyl radicals. FEBS Lett 1981;128:347–52. 23. Laughton MJ, Halliwell B, Evans PJ, Hoult JRS. Antioxidant and prooxidant actions of the plant phenolics quercetin, gossypol and myricetin: effects on lipid peroxidation, hydroxyl radical generation and bleomycin-dependent damage to DNA. Biochem Pharmacol 1989;38:2859–65. 24. Berker KI, Güçlü K, Demirata B, Apak R. A novel antioxidant assay of ferric reducing capacity measurement using ferrozine as the colour forming complexation reagent. Anal Methods 2010;2:1770–8. 25. Kralijć I, Mohsini SEI. A new method for the detection of singlet oxygen in aqueous solutions. Photochem Photobiol 1978;28:577–81. 26. Khan AU, Kasha M. Chemiluminescence arising from simultaneous transition in pairs of singlet oxygen molecules. J Am Chem Soc 1970;92:3293–300. 27. Khan AU. Activated oxygen: singlet molecular oxygen and superoxide anion. Photochem Photobiol 1978;28:615–27. 28. Gampp H, Lippard SJ. Reinvestigation of 18-crown-6 ether/potassium superoxide solutions in Me2SO. Inorg Chem 1983;22:357–8. 29. Greenstock CL, Miller RW. The oxidation of Tiron by superoxide anion. Kinetics of the reaction in aqueous solution and in chloroplasts. Biochim Biophys Acta 1975;396:11–16. 30. Seyoum A, Asres K, El-Fiky FK. Structure–radical scavenging activity relationships of flavonoids. Phytochemistry 2006;67:2058–70. 31. Allen RC. Biochemiexcitation: chemiluminescence and the study of biological oxygenation reactions. In: Chemical and biological generation of excited states. New York: Academic Press, 1982:327–33. 32. Fedeli D, Falcioni G, Olek RA, Massi M, Cifani C, Polidori C, et al. Protective effect of ethyl pyruvate on msP rat leukocytes damaged by alcohol intake. J Appl Toxicol 2007;27:561–70. 33. Aitken RJ, Baker MA, O’Bryan M. Shedding light on chemiluminescence: the application of chemiluminescence in diagnostic andrology. J Androl 2004;25:455–65. 34. Khan MN, Jan MR, Shah J, Lee SH. A validated silver-nanoparticleenhanced chemiluminescence method for the determination of citalopram in pharmaceutical preparations and human plasma. Luminescence 2014;29:266–74. 35. Marguette CA, Blum LJ. Chemiluminescent enzyme immunoassays a review of bioanalytical applications. Bioanalysis 2009;7:1259–69. 36. Lind J, Mernyi G, Eriksen TE. Chemiluminescence mechanism of cyclic hydrazides such as luminol in aqueous solutions. J Am Chem Soc 1983;105:7655–61. 37. Nath KA, Ngo EO, Hebbel RP, Croatt AJ, Zhou B, Nutter LM. α-Ketoacids scavenge H2O2 in vitro and in vivo and reduce menadione-induced DNA injury and cytotoxicity. Am J Physiol 1995;268:C227–36. 38. Kang Y-H, Park S-H, Lee Y-J, Kang J-S, Kang I-J, Shin H-K, et al. Antioxidant α-keto-carboxylate pyruvate protects low-density lipoprotein and atherogenic macrophages. Free Radical Res 2002;36:905–14. 39. Gutteridge JMC. Lipid peroxidation and possible hydroxyl radical formation stimulated by the self-reduction of a doxorubicin–iron(III) complex. Biochem Pharmac 1984;33:1725–8. 40. Quideau S, Deffieux D, Douat-Casassus C, Pouységu L. Plant polyphenols: chemical properties, biological activities, and synthesis. Angew Chem Int Ed 2011;50:586–621. 41. Cao G, Sofic E, Prior RL. Antioxidant and pro-oxidant behavior of flavonoids: structure–activity relationships. Free Radical Biol Med 1997;22:749–60. 42. Kehrer JP. The Haber–Weiss reaction and mechanisms of toxicity. Toxicol 2009;149:32–46. 43. Gupte A, Mumper RJ. Elevated copper and oxidative stress in cancer cells as a target for cancer treatment. Cancer Treat Rev 2009;35:32–46. 44. Smith LL, Kulig MJ. Sterol metabolism. XXXIX. Singlet molecular oxygen from hydrogen peroxide disproportionation. J Am Chem Soc 1976;98:1027–9. 45. Ervens B, Gligorowski S, Hermann H. Temperature-dependent rate constants for hydroxyl radical reactions with organic compounds in aqueous solutions. Phys Chem Chem Phys 2003;5:1811–24.
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 1153–1158
Accepted: 13 January 2015
Published online in Wiley Online Library: 8 March 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2879
Reactivity of pyruvic acid and its derivatives towards reactive oxygen species Aleksandra Kładna,a Mariola Marchlewicz,b Teresa Piechowska,c Irena Krukc and Hassan Y. Aboul-Eneind* ABSTRACT: Pyruvic acid and its derivatives occurring in most biological systems are known to exhibit several pharmacological properties, such as anti-inflammatory, neuroprotective or anticancer, many of which are suggested to originate from their antioxidant and free radical scavenger activity. The therapeutic potential of these compounds is a matter of particular interest, due to their mechanisms of action, particularly their possible antioxidant behaviour. Here, we report the results of a study of the effect of pyruvic acid (PA), ethyl pyruvate (EP) and sodium pyruvate (SP) on reactions generating reactive oxygen species (ROS), such as superoxide anion radicals, hydroxyl radicals and singlet oxygen, and their total antioxidant capacity. Chemiluminescence (CL) and spectrophotometry techniques were employed. The pyruvate analogues studied were found to inhibit the CL signal arising from superoxide anion radicals in a dose-dependent manner with IC50 = 0.0197 ± 0.002 mM for EP and IC50 = 69.2 ± 5.2 mM for PA. These compounds exhibited a dose-dependent decrease in the CL signal of the luminol + H2O2 system over the range 0.5–10 mM with IC50 values of 1.71 ± 0.12 mM for PA, 3.85 ± 0.21 mM for EP and 22.91 ± 1.21 mM for SP. Furthermore, these compounds also inhibited hydroxyl radical-dependent deoxyribose degradation in a dose-dependent manner over the range 0.5–200 mM, with IC50 values of 33.2 ± 0.3 mM for SP, 116.1 ± 6.2 mM for EP and 168.2 ± 6.2 mM for PA. All the examined compounds also showed antioxidant capacity when estimated using the ferric–ferrozine assay. The results suggest that the antioxidant activities of pyruvate derivatives may reflect a direct effect on scavenging ROS and, in part, be responsible for their pharmacological actions. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: pyruvic acid; ethyl pyruvate; sodium pyruvate; reactive oxygen species; chemiluminescence; spectrophotometry
Introduction
Luminescence 2015; 30: 1153–1158
* Correspondence to: H. Y. Aboul-Enein, Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Giza 12622, Egypt. E-mail: [email protected] a
Department of History of Medicine and Medical Ethics, Pomeranian Medical University, Szczecin, Poland
b
Department of Aesthetic Dermatology, Pomeranian Medical University, Szczecin, Poland
c
Institute of Physics, Faculty of Mechanical Engineering and Mechatronics, West Pomeranian University of Technology in Szczecin, Szczecin, Poland
d
Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Giza 12622, Egypt
Copyright © 2015 John Wiley & Sons, Ltd.
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Pyruvic acid is an α-keto acid being a key intermediate of glucose metabolism; its carboxylate anion is known as pyruvate. This compound enters into Krebs cycle to generate the ATP. Several authors have reported that pyruvate and its derivatives possess anti-inflammatory activities: suppressing the generation of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and inhibiting the expression of pro-inflammatory agents such as inducible nitric oxidase synthase (iNOS), cyclooxygenase-2 (COX-2), and other pro-inflammatory mediators (1–8). Biological mechanisms of the anti-inflammatory action are not well recognized, and are in part ascribed to their antioxidant activity (2). Over the past two decades, growing research has revealed that oxidative stress can lead to chronic inflammation that is associated with the development of most chronic diseases including cardiovascular and neurological diseases, diabetes and cancer (9–13). Oxidative stress may be defined as an imbalance between the generation of pro-oxidants [reactive oxygen species (ROS), reactive nitrogen species (RNS) and their metabolites] and their elimination by antioxidants. ROS and RNS playing the most important roles in biological systems and include the superoxide anion radical (O ¯2• ), hydroxyl radical (HO°), hydrogen peroxide (H2O2), peroxynitrite (ONOO–) and singlet oxygen (1O2) (13–15). Among these reactive species, HO° and 1O2 are the most potent oxidants for organic and inorganic compounds. With regard to anti-inflammatory properties and the ability to scavenger ROS, confirmed in numerous studies of pyruvate and its analogues in animal models of human disease (8,16), it is suggested that these compounds have anti-
cancer, anti-ischemic, cardio- and neuroprotective activities, and are promising for use as therapeutic agents for cancer and inflammatory diseases. However, the possibility that pyruvate and its analogues might be used clinically requires, among other things, quantitative evaluation of their ability to scavenge particular reactive species. To address this, a series of in vitro experiments designed to evaluate the reactivity of pyruvic acid (PA), ethyl pyruvate (EP) and sodium pyruvate (SP) towards superoxide radical, hydroxyl radical, hydrogen peroxide and singlet oxygen were carried out. The chemical structures of the tested compounds are shown in Fig. 1.
A. Kładna et al.
Figure 1. Chemical structures of pyruvic acid and its derivatives.
The IC50 value was measured from a linear regression curve of the scavenging ratios (Q) against the logarithmic concentrations of each the tested compound.
Experimental Reagents All reagents were of analytical grade. PA, EP, SP, luminol, trolox (6-hydroxy-2,5,7,8-tetramethyl-2-carboxyl acid), 18-crown-6-ether (1,4,7,10,13,16)hexaoxacyclooctadecane, ascorbic acid, sodium trifluoroacetate, imidazole (A) and p-mitrosodimethylaniline (RNO) were from Sigma-Aldrich GmbH (Sternheim, Germany). Anhydrous dimethylsulfoxide (DMSO) was from Aldrich (St. Louis, MO, USA). Ammonium ferrous sulfate (FeSO4(NH4)2SO4) and potassium superoxide (KO2) were from Fluka (Buchs, Switzerland). Tiron (1,2-dihydroxybenzene-3,5-disulfonic acid) and other reagents were from Merck (Darmstadt, Germany). For water solution preparation, double distilled water was used. Antioxidant activity determination Chemiluminescence measurements Superoxide radical scavenging activity. The superoxide anion radical was produced according to the method described by Valentine et al. (17) as follows: a 60-mg aliquot of 18-crown-6-ether was dissolved in 10 mL of dry DMSO and 7 mg of KO2 was quickly added to the flash using a syringe to avoid contact with the air. This mixture was stirred for 60 min to give a pale yellow solution of 10 mM superoxide anion radical stable at room temperature. In the measurements, the radical was used as a 1 mM solution in DMSO. The tested compounds (PA, EP, SP and Tiron) were dissolved in DMSO. Tiron, a compound known to be an effective antioxidant was used as a positive control. O ¯2• scavenging activity was measured using a previously reported chemiluminescence (CL) technique (18). Briefly, reagents were mixed in a thermostated glass cuvette placed in a light-tight chamber before an EMI 9553Q photomultiplier with a S20 cathode sensitive over the range 200–800 nm, interfaced with a computer for data acquisition and handling. The reactor cell was washed using a B-169 vacuum system (Büchi, Flawill, Switzerland). All measurements were performed at 293 ± 1 K. A personal computer was equipped with a home-written software program which provides the opportunity to calculate of the light sum (∑I), i.e. an area under the kinetic curve, I = f(t), for any chosen time interval t
∑I ¼ ∫ IðtÞdt t0
where I(t) is the CL intensity, t0 is the measurement at the start time (after mixing reagents) and t is the measurement at the finish. Scavenging activity Q(%) was calculated from the equation Qð%Þ ¼ ½1 – ∑I=∑Io 100
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where ∑Io and ∑I are the relative integrals of the control reaction and sample solutions over 70 s, respectively. The results were expressed as a sample concentration giving 50% inhibition of the O¯2• -induced light emission, as a measure of antioxidant activity.
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In order to evaluate the scavenger activity of the tested pyruvate derivatives against hydrogen peroxide, a previously described CL method based on H2O2-induced oxidation of luminol was used (19). The reaction mixture contained different concentrations of the examined compound, 100 μM luminol, phosphate buffer pH 7.0 and 5 mM H2O2. The reaction was initiated by addition of H2O2. The scavenging activity was defined as percentage (%) CL quenching Q (%) = [1 – ∑I/∑Io] × 100, where ∑Io is the relative integral of a control reaction (luminol + pH + H2O2) and ∑I is the sum of the CL detected from the control reaction in the presence of the tested compound (PA,EP or SA). Scavenging potentials were calculated from linear regression curves of the scavenging ratios against the logarithmic concentrations of each the tested compound as IC50 values. Hydrogen peroxide scavenging activity.
Hydroxyl radical scavenging activity. The Fenton reaction (Fe(II) + H2O2) was used as a source of hydroxyl radicals (20,21). The influence of tested compounds on the system producing HO radicals was detected using deoxyribose degradation caused by the radicals according to the method described by Halliwell and Gutteridge (22,23). In this method, degradation of deoxyribose by hydroxyl radical results in malonaldehyde formation. When reacted with thiobarbituric acid, this forms a coloured thiobarbituric acid–malonaldehyde adduct (2: 1), absorbing strongly at 532 nm in acid solution. The reaction mixture consisted of deoxyribose (2 mM), sodium trifluoroacetate buffer (10 mM, pH 6.15), ferrous sulfate (62.5 μM) and H2O2 (1 mM) (control). The tested compounds were added to the reaction mixture before starting the reaction by adding H2O2. The reaction mixture was incubated for 30 min at 310 K. The extent of deoxyribose oxidation by the hydroxyl radical was measured after mixing 3 mL of sample with 1 mL trichloroacetic acid (2.8%) and 1 mL thiobarbituric acid in 50 mM NaOH. The prepared mixture was heated at 373 K for 15 min and cooled to room temperature. Individual samples were placed in a Zeiss M-40 UV/Vis spectrophotometer with Win-Aspect software ( Jena, Germany) to measure the thiobarbituric acid–malonaldehyde adduct absorption at 532 nm. The resulting absorbance was read against appropriate blanks. The radical scavenging activity of PA, EP, SP and the positive control trolox was defined as the percentage deviations of deoxyribose degradation with respect to the tested compounds-free control Q(%) = [1 – A/Ao] × 100%, where A is the absorbance detected at 532 nm in the presence of the tested pyruvate compound or trolox dissolved in water, and Ao is the absorbance without the compound, after addition of the same amount of water. Similarly, IC50 values were calculated from linear regression curves of Q(%) against logarithmic concentrations for each the examined compound. Ferric reducing activity measurements. The reducing activities of the tested compounds were measured using an antioxidant assay developed by Berker et al. (24). In this method, reduction of Fe
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 1153–1158
Pyruvic acid and its derivatives towards reactive oxygen species
(III)–ferrozine agent by a compound having an antioxidant capacity leads to stable Fe(II)–ferrozine complex formation that exhibits a single sharp absorbance peak at 562 nm. The ferric–ferrozine complex was prepared according to the following procedure. An aqueous solution containing 0.024 g of NH4Fe(SO4)2 · 12H2O in 1 mL of HCl (1 M) was mixed with a solution of 0.123 g ferrozine dissolved in water. After mixing, the reaction mixture was diluted to 25 mL with distilled water to obtain a final Fe(III) ion concentration of 2 mM, and ferrozine concentration of 0.1 mM. The formed Fe(III)–ferrozine complex was kept in a stoppered dark-coloured bottle. Working solutions were prepared from this stock solution as follows 1.5 mL of the complex was added to 1 mL of antioxidant solution (1 mM) and mixed with 2 mL of buffer solution (0.2 M/L acetic acid/sodium acetate, pH 5.5). The increase in absorbance was determined at 562 nm after standing for 1.5 h at room temperature. The effect of the tested compounds on the formation of singlet oxygen was performed according to the method developed by Kraljic and El-Mohsni (25). The method is based on the bleaching of RNO by the intermediate product a transannular peroxide (AO2) arising in the reaction of 1O2 with imidazole Effect of the tested compounds on singlet oxygen production.
1
O2 þ A → AO2
AO2 þ RNO → -RNO þ products The bleaching of RNO was measured as the decrease in absorbance at 440 nm using a Zeiss M-40 spectrometer. All experiments were conducted at least three times. Linear regression analyses were computed using the statistical package STATISTICA 6.0 2002 (Stat Soft Polska, Kraków, Poland).
Results and discussion Use of the 18-crown-6-ether/KO2/DMSO reaction as a source of superoxide anion radical and the sensitive CL method allowed us to measure the radical-scavenging activity of PA, EP and SP. The reaction is accompanied by the emission of electromagnetic radiation (CL) with emission bands with maxima at 480, 580, 640 and 700 nm, corresponding to the simultaneous transition in the singlet oxygen dimoles (1O2)2 during their radiative deactivation to the ground state (3O2) (26,27). The spectra and the reactions responsible for the 1O2 production have been presented previously (18). According to the reported mechanism, 1O2 is generated in the following reactions (28)
Figure 2. (A) Representative scavenging effects of various concentrations of EP on the chemiluminescence (CL) intensity recorded from 1 mM superoxide anion radical produced in DMSO (control). Curves: 1, control; 2, 0.005 mM; 3, 0.01 mM; 4, 0.03 mM; 5, 0.05 mM; 6, 0.2 mM. (B) A dose-dependent inhibition of the light sum recorded from the superoxide anion radical/DMSO solution measured at various concentrations of EP, under the same conditions as in (A). (C) Dose-dependent inhibition of the light sum recorded from the superoxide anion radical/DMSO solution in the presence of PA and SP, under the same conditions as in (B). Denotations of the tested compounds are given in Fig. 1.
Table 1. Linear range and IC50 (mean ± SD) value for the examined pyruvate analogues against superoxide anion radical, hydrogen peroxide, and hydroxyl radical Species
2 O2¯• þ 2ðCH3 Þ2 SO→1 O2 þ ðCH3 Þ2 SO2 þ CH3 SOðCH2 Þ
O 2¯
þOH- O2¯• →1 O2 þ electron
Luminescence 2015; 30: 1153–1158
H2O2
HO°
PA EP SP Tiron PA EP SP Ascorbic acid PA EP SP Trolox
Range (mM) 20–250 0.005–0.2 0.2–2.0 0.1–2.0 0.5–10.0 0.5–10.0 0.5–10.0 0.0005–0.02 0.5–200 0.5–200 0.5–200 0.01–3.0
IC50 (mM) 69.2 ± 5.2 0.0197 ± 0.002 40% (2 mM)* 0.57 ± 0.06 1.71 ± 0.12 3.83 ± 0.21 22.91 ± 1.21 0.00069 ± 0.00002 168.2 ± 6.2 116.2 ± 6.2 33.2 ± 0.3 0.052 ± 0.03
* Scavenging effect observed at the highest examined concentration.
Copyright © 2015 John Wiley & Sons, Ltd.
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Figure 2(A) demonstrates the typical CL kinetic curves obtained from the superoxide anion radical/DMSO system alone (curve 1) and in the presence of 0.005–0.2 mM EP, as representative behaviour. This plot shows that CL intensity from the light-emitting system decreased in the presence of EP, in a concentrationdependent manner (curves 2–6). A positive linear relationship was observed between antioxidant activity and EP concentration (Fig. 2B). The compounds PA and SP demonstrated similar inhibitory behaviour (Fig. 2C). Table 1 gives the calculated IC50 values for these pyruvates. SP reached a 40% effect at the maximum tested concentration (2 mM). The IC50 values of PA and EP were compared with that calculated for a powerful inhibitor of
Compound
A. Kładna et al.
superoxide anion radical such as Tiron under the same experimental conditions. The IC50 value for the superoxide anion radical scavenging activity by EP was significantly lower than that of Tiron. This behaviour indicates that EP is more efficient inhibitor of superoxide anion radical than Tiron, for which the rate constant of the reaction with this species was reported to be 5 × 108 M/s in aqueous solution (29). The radical scavenging activities of the examined pyruvates seem to depend particularly on the presence of the hydrogen atom in the carboxylic acid group (PA) and the hydrogen atoms of the ethyl group of the aliphatic ester (EP). Findings from the superoxide anion radical scavenging method demonstrate that the examined pyruvates might act as reducing agents, by donating a hydrogen atom to superoxide anion (30): O2•¯ þH→HO 2 Under our experimental conditions, the HO 2 species may decompose to OHˉ in the reaction with DMSO (28): HO 2 þ ðCH3 Þ2 SO→HO þ ðCH3 Þ2 SO2
To confirm that the tested pyruvates are able to react with H2O2, luminol-amplified CL was applied. All the pyruvate analogues tested reduced the CL signal in terms of both light intensity and the CL sum in a concentration-dependent manner (Fig. 3A). The percentage of light intensity quenching was similar to that of the CL sum quenching (not shown). A linear relationship between
A PA EP SP
100 80 60 40 20 0 0
20
40
60
80
100
Concentration (mM)
H2O2 scavenging potency and the concentration of particular pyruvates was observed (Fig. 3B). The IC50 values calculated from these plots are given in Table 1. They vary from 1.71 to 22.91 mM and are significantly higher than the IC50 value calculated for the standard control, ascorbic acid (0.69 ± 0.02 μM). Luminol was the first compound used as a CL probe for the detection of H2O2 (31) and the mechanism of electronic excitation of luminol has been intensively investigated. CL accompanying oxidation of luminol satisfies criteria for its use in bioanalysis, due to its ultrasensitivity and nontoxicity (19,32–34). The literature has shown that the CL mechanism accompanying oxidation of luminol may be summarized briefly as follows. In the first step luminol is dehydrogenated to the diazoquinone, which is oxygenated with H2O2 to give a cyclic peroxide. The second step involves decomposition of the cyclic peroxide to a 3-aminophthalate dianion in an electronically excited state. In the last step, the dianion returns to the ground state with the emission of light (λmax = 425 nm) (31,35,36). Thus, the luminolamplified CL, used to quantify the H2O2 scavenging potency of pyruvate analogues demonstrates that the tested compounds can react directly with H2O2, irreversibly removing this oxidant from the reaction medium. Nath et al. (37) and Kang et al. (38) reported that pyruvate undergoes decarboxylation in the presence of H2O2, and acetate, carbon dioxide and water were identified as the reaction products. Thus, the protective effects of pyruvate derivatives observed is cells and tissues (5–8) may be due, in part, to their antioxidant action and are in accordance with our CL results. All the tested pyruvate analogues were also able to scavenge hydroxyl radicals in a concentration-dependent manner (Fig. 4). It is well recognized that deoxyribose degradation depends on the presence of ROS, mainly hydroxyl radical (39). Deoxyribose degradation was efficiently prevented by PA, EP and SP, with SP being the most effective pyruvate derivative, giving an IC50 value of 33.2 ± 0.3 mM (Table 1), although it demonstrated very weak potency in comparison with trolox (IC50 = 0.052 ± 0.03 mM). These results demonstrate, once again, an important ability of pyruvate analogues to scavenge hydroxyl radicals directly. It is worth noting that this species is the most important radical involved in cell damage, due to its high reactivity with biomolecules, usually exceeding 109 M–1 s–1 (14). The high level of radical activity may be important in the oxidative damage of cells, because these hydroxyl radicals can be involved in several reactions, such as adding to a double bond, abstraction of a hydrogen atom and oxidation via electron donation.
B 100
PA
50
80 40
R
= 0,9869
R
= 0,9915
R
= 0,9869
SP
EP
PA
EP
60
30
40
20
Q
SP
20
10 0
0
1
1
10
Concentration (mM)
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Figure 3. (A) Suppression effect of the tested compounds on the luminol-based CL. (B) Dose-dependent inhibition of the light sum; calculation of EC50 values of the examined compounds towards H2O2. For reaction conditions, see Experimental.
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10
100
Concentration (mM) Figure 4. Dose-dependent effect of the tested compounds on the deoxyribose oxidation by hydroxyl radicals generated by the Fenton reaction. Experimental conditions are given in Experimental.
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 1153–1158
Pyruvic acid and its derivatives towards reactive oxygen species
Because antioxidants can exert their antioxidant activity via donation of hydrogen atoms or single-electron transfer to a radical (40), we further studied the antioxidant potential of pyruvate analogues using a ferric–ferrozine assay, troxol antioxidant capacity (TAC). In this assay, the examined compounds were able, in the presence of ferrozine, to reduce the ferric ion in the Fe(III)– ferrozine complex to the Fe(II) ion. The Fe(II)–ferrozine complex formed exhibits an absorbance maximum at 562 nm, and the band intensity depends on the reducing power of an antioxidant. The latter complex absorbance is positively correlated with the antioxidant reducing potential (an electron transfer way). Figure 5(A) illustrates the ferric reducing powers of the tested antioxidants and a comparison with the reduction power of trolox, as the positive control. The reducing activities of the pyruvate analogues at 1 mM and the standard compound trolox at 0.0133 mM were observed to be in the following order: trolox > PA > SP > EP. The reducing ability of trolox was ~ 100× that of PA. The most important advantage of the ferric–ferrozine method of measuring antioxidant measurement is a high sensitivity for antioxidants that have even weak reducing ability, due to the very high molar extinction coefficient (Ɛ = 2.8 × 104 M-1 cm-1) of the Fe(II)–ferrozine complex (24). By contrast to their beneficial effects, some efficient antioxidants, e.g. flavonoids, have been reported to exert pro-oxidant
A
1.4 PA
1.2
Absorbance
1.0
O2•¯ þ H2 O2 → O2 þ HO° þ HOˉ This reaction is slow (k = 3.0 ± 0.6 M-1 s-1) but is strongly catalysed by transition metals ions, such as iron(II) and copper(II), and also ions whose presence in cells is due to environmental pollution (13,43), such as cobalt(II), chromium(III) and nickel(II) (the rate constant k ≥ 100 M-1 s-1). The experimental findings and the literature data (44) allow us to suggest that the base-catalysed disproportionation of H2O2 is responsible for 1O2 formation under our experimental conditions:
H2 O2 þ HOOˉ →1 O2 þ H2 O þ HOˉ
EP
0.4 0.2 450
500
550
600
650
700
Wavelength (nm)
B EP
0.3 PA
0.2
SP
0.1
Control
0
The toxicity of H2O2 in the cell is mainly due to its decomposition to HO• in a reaction with transition metal ions or is based on the Haber–Weiss reaction (42)
H2 O2 þ HOˉ → HOOˉ þ H2 O
SP
0.6
0.0
FeðIIÞ þ H2 O2 → HOo þ HOˉ þ FeðIIIÞ
Trolox
0.8
0.0 400
activity in vitro (41). The possibility that the pyruvate compounds would exert pro-oxidant activity under our experimental conditions and their effect on the generation of 1O2 in the Fenton reaction was evaluated (Fig. 5B). As shown in Fig. 5, the bleaching of RNO in the RNO + imidazole + Fenton reagents + pyruvate compound system was significantly increased in comparison with the RNO + imidazole + Fenton reagent reaction (control) (P < 0.05). The efficacy of RNO bleaching yields a ranking order of EP > PA > SP, suggesting the generation of increased amounts of 1 O2 in the presence of these pyruvate derivatives. Such behaviour of these compounds is in accordance with their reducing ability observed in the TAC assay; the compounds can reduce the free Fe(III) ion to Fe(II), thereby supporting the Fenton reaction:
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Figure 5. (A) Comparison of total antioxidant activity of the examined compounds and trolox. (B) Time course of the bleaching of RNO in the presence of the examined compounds (80 μM RNO + 60 mM imidazole + 10 mM sodium trifluoroacetate + 0.1 mM FeSO4(NH4)2SO4 + 1 mM tested compound + 1 mM H2O2), and in their absence (control) (80 μM RNO + 60 mM imidazole + 10 mM sodium trifluoroacetate + 0.1 mM FeSO4(NH4)2SO4 + 1 mM H2O2). ΔA = difference in absorbance of the reaction mixture with a tested compound and the control reaction. Bleaching of RNO was followed at 440 nm, an absorption maximum of RNO.
Because of the relatively high concentration of H2O2 in the cell (~10-2 μM) (42) due to its accumulation, and also its ability to diffuse across cell membranes, the observed enhancing effect of pyruvate derivatives on 1O2 generation seen in Fig. 5(B), demonstrates that the compounds tested may be involved in the redox regulation of cellular stress. Our findings demonstrate that the preventive role of pyruvate derivatives against oxidative stress, as reported for EP (32), is mainly due to scavenging of H2O2 and superoxide anion radicals. The high IC50 values of PA and SA suggest that these compounds are not efficient hydroxyl radical scavengers (Table 1) under our experimental conditions, regardless of their high rate constants for the reactions with pyruvic acid and pyruvate in aqueous solution – (1.2 ± 0.4) × 108 and (7 ± 2) × 108 M-1 s-1, respectively (45). This seems to confirm the high potency of pyruvate derivatives to decompose H2O2, leading to suppression of hydroxyl radical formation in the Fenton reaction. In conclusion, the above findings indicate that pyruvate analogues possess good scavenging potential towards superoxide anion radical, weaker scavenging potential towards hydroxyl radical and efficiently decompose H2O2. Moreover, these compounds show relatively high reducing power. One question arises, whether the examined compounds are better scavengers of oxygen free radicals then the standard antioxidant Tiron. We suggest that EP is a much better inhibitor of superoxide radicals then Tiron in a hydrophobic medium. To the best our knowledge, comparison of the scavenging activity for ROS and the reducing potential of PA, EP and SP is reported for the first time. For this reason, it is difficult
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to compare IC50 values measured in our study with those obtained previously. Only one study (32) has investigated the antioxidant activity of pyruvate and EP towards H2O2. The authors used luminol-amplified CL and found IC50 values for these compounds of ~ 47 and 12 mM, respectively. These values are about 10 times higher than ours. In the same study, the IC50 value for the reaction of EP with superoxide anion radical generated in aqueous solution using a xanthine–xanthine oxidase system as a source of the radical was estimated to be 80 mM. This inconsistency between the previous findings and ours for a value of IC50 for EP may be due to methodological differences in study design. Our study provides further evidence that pyruvic acid and its derivatives are important endogenous scavengers of ROS, especially superoxide radical and H2O2, and thereby have potential for controlling inflammation and preventing diseases with an oxidative stress aetiology, such as neurodegenerative, disorders diabetes, cardiovascular disease or cancer.
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Luminescence 2015; 30: 1153–1158
