Interaction and reactivity of urocanic acid towards peroxyl radicals

The capacity of urocanic acid to interact with peroxyl radicals has been evaluated in several systems: oxidation in the presence of a free radical source (2,2'-azobis(2-amidinopropane; AAPH), protection of phycocyanin bleaching elicited by peroxyl radicals, and Cu(II)- and AAPH-promoted LDL oxidation. The results indicate that both isomers (cis and trans) are mild peroxyl radical scavengers. For example, trans-urocanic acid is nearly 400 times less efficient than Trolox in the protection of the peroxyl radical promoted bleaching of phycocyanin. Regarding the removal of urocanic acid by peroxyl radicals, nearly 100 muM trans-urocanic acid is required to trap half of the produced radicals under the employed conditions (10 mM AAPH, 37 degrees C). Competitive experiments show that the cis-isomer traps peroxyl radicals 30% less efficiently than the trans-isomer. Given the high concentrations that trans-urocanic acid reaches in skin, its capacity to trap peroxyl radicals could contribute to the protection of the tissue towards ROS-mediated processes. Furthermore, both isomers, and particularly the cis-isomer, protect LDL from Cu(II)-induced oxidation.     

Interaction and reactivity of urocanic acid towards peroxyl radicals

Abstract

The capacity of urocanic acid to interact with peroxyl radicals has been evaluated in several systems: oxidation in the presence of a free radical source (2,2′-azobis(2-amidinopropane; AAPH), protection of phycocyanin bleaching elicited by peroxyl radicals, and Cu(II)- and AAPH-promoted LDL oxidation. The results indicate that both isomers (cis and trans) are mild peroxyl radical scavengers. For example, trans-urocanic acid is nearly 400 times less efficient than Trolox in the protection of the peroxyl radical promoted bleaching of phycocyanin. Regarding the removal of urocanic acid by peroxyl radicals, nearly 100 μM trans-urocanic acid is required to trap half of the produced radicals under the employed conditions (10 mM AAPH, 37°C). Competitive experiments show that the cis-isomer traps peroxyl radicals ~30% less efficiently than the trans-isomer. Given the high concentrations that trans-urocanic acid reaches in skin, its capacity to trap peroxyl radicals could contribute to the protection of the tissue towards ROS-mediated processes. Furthermore, both isomers, and particularly the cis-isomer, protect LDL from Cu(II)-induced oxidation.     

Inhibition of the metmyoglobin-induced peroxidation of linoleic acid by dietary antioxidants: Action in the aqueous vs. lipid phase

The gastric digestion of food containing oxidizable lipids and iron catalysts for peroxide decomposition such as (met)myoglobin from muscle meat can be accompanied by an extensive formation of potentially toxic lipid hydroperoxides. An early protective action by dietary antioxidants in the gastro-intestinal tract is plausible, especially for poorly bioavailable antioxidants such as polyphenols. Hence, the ability of antioxidants to inhibit lipid peroxidation initiated by dietary iron in mildly acidic emulsions is a valuable and general model. In this work, the ability of some ubiquitous dietary antioxidants representative of the main antioxidant classes (alpha-tocopherol, the flavonol quercetin, beta-carotene) to inhibit the metmyoglobin-induced peroxidation of linoleic acid is investigated by UV-visible spectroscopy and HPLC in mildly acidic emulsions. The phenolic antioxidants quercetin and alpha-tocopherol come up as the most efficient peroxidation inhibitors. Inhibition by quercetin essentially proceeds in the aqueous phase via a fast reduction of an unidentified activated iron species (with a partially degraded heme) produced by reaction of metmyoglobin with the lipid hydroperoxides. This reaction is faster by, at least, a factor 40 than the reduction of ferrylmyoglobin (independently prepared by reacting metmyoglobin with hydrogen peroxide) by quercetin. By contrast, alpha-tocopherol mainly acts in the lipid phase by reducing the propagating lipid peroxyl radicals. The poorer inhibition afforded by beta-carotene may be related to both its slower reaction with the lipid peroxyl radicals and its competitive degradation by autoxidation and/or photo-oxidation.     

A fluorometric method for measurement of oxygen radical-scavenging activity of water-soluble antioxidants

The relative activities of the antioxidants Trolox, ascorbic acid, uric acid, quercetin, and rutin, and the activities of total antioxidants in serum samples were determined using a fluorometric assay based on the dye 6-carboxyfluoroscein (6C-Fl) as a fluorescent indicator; 2,2'-azobis-2-amidinopropane hydrochloride (AAPH) as a peroxyl radical generator; 6-hydroxy-2,5,7, 8-tetramethyl-1-chroman-2-carboxylic acid (Trolox) as a calibrator; and phosphate buffer (pH 7.0) as a solvent. Incubation of 6C-Fl in 0. 075 M phosphate buffer, in the presence of AAPH at 37 degrees C, resulted in loss of its fluorescence signal at 520 nm with excitation at 495 nm. The antioxidants Trolox, ascorbic acid, and uric acid provided protection of the fluorescence of 6C-Fl, and the relative antioxidant activities, determined by the net protection area under curve technique, were found to be 1:0.4:1, respectively. Trolox and ascorbic acid were used to validate this assay. A linear correlation of the net protection value with the concentration of serum, Trolox, ascorbic acid, and uric acid was demonstrated. Quercetin and rutin were shown to have strong antioxidant activities, nearly 10 times those of vitamin C. This assay is simple, reliable, and suitable for automation to handle many samples and requires few microliters of serum samples.     

Enhancement of peroxidase-induced lipid peroxidation by indole-3-acetic acid: effect of antioxidants

Summary

Indole-3-acetic acid (IAA) enhanced the peroxidase-induced lipid peroxidation in phosphatidylcholine liposomes, as measured by loss of fluorescence of cis-parinaric acid. α-Tocopherol or β-carotene in the lipid phase or ascorbate or Trolox in the aqueous phase inhibited the loss of fluorescence induced by the peroxidase + IAA system, but glutathione had only a small inhibitory effect. The peroxyl radical formed by one-electron oxidation of IAA, followed by decarboxylation and reaction with oxygen, is suggested to act as the initiator of lipid peroxidation. The protection by ascorbate or Trolox is explained by the reactivity of these compounds with the IAA indolyl radical, as shown by pulse radiolysis experiments, whereas the weak effect of glutathione agrees with its low reactivity towards the IAA-derived peroxyl radical and its precursors.     

In vitro antioxidant activity of 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), a vitamin E metabolite

2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC) has been identified as a major water-soluble metabolite of vitamin E, which circulates in the blood and is excreted with the urine. The aim of this study was to assess the antioxidant activity of alpha-CEHC using several methods with different prooxidant challenges. In the Oxygen Radical Absorbance Capacity assay, a fluorescent protein acts as a marker for oxidative damage induced by peroxyl radicals. In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, a stable free radical, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS.+) is reduced directly by antioxidants. Scavenging properties vs. reactive nitrogen species were studied measuring the effects on tyrosine nitration after reaction with peroxynitrite. Trolox, alpha-tocopherol, ascorbic acid, and (-)-epicatechin were simultaneously tested in order to compare their antioxidant activities. In all mentioned systems, alpha-CEHC exhibited antioxidant properties similar to those of Trolox. We conclude that alpha-CEHC is a molecule with good antioxidant activity, having the advantage over Trolox of being a naturally occurring compound. These properties might be useful for research or industrial purposes.     

Reactions of linoleic acid peroxyl radicals with phenolic antioxidants: a pulse radiolysis study

Linoleic acid peroxyl radicals (LOO.) can be viewed as model intermediates occurring during lipid peroxidation processes. Formation and reactions of these species were investigated in aqueous alkaline solution using the technique of pulse radiolysis combined with kinetic spectroscopy. Irradiation of linoleic acid in N2O/O2-saturated solutions leads to a mixture of peroxyl radical isomers, whereas reaction of 13-hydroperoxylinoleic acid (13-LOOH) with azide radicals in N2O-saturated solution produces 13-LOO. radicals specifically. These peroxyl radicals cannot be observed directly, but their reactions with the two flavonols, kaempferol and quercetin, acting as radical-scavenging antioxidants, produced strongly absorbing aroxyl radicals (ArO.). The same aroxyl radicals were generated by .OH and N3. with rate constants exceeding 10(9) dm3 mol-1 s-1. Applying a reaction scheme that includes competing generation and decay reactions of both LOO. and ArO. radicals, we derived individual rate constants for LOO. reactions with the phenols (greater than 10(7) dm3 mol-1 s-1), with the aroxyl radicals to form covalent adducts (greater than 10(8) dm3 mol-1 s-1), as well as for their bimilecular decay (3.0 X 10(8) dm3 mol-1 s-1). These results demonstrate the high reactivity of both fatty acid peroxyl radicals and the flavone antioxidants in aqueous solution.     

A novel and simple ORAC methodology based on the interaction of Pyrogallol Red with peroxyl radicals

Oxygen radicals absorbance capacities (ORAC) indexes are frequently employed to characterize the radical trapping capacity of pure compounds and their complex mixtures. A drawback of ORAC values obtained using phycoerythrin, fluorescein (FL) or c-phycocyanin as targets, makes it possible to conclude that for very reactive compounds they are much more related to stoichiometric factors than to the reactivity of the tested compound. In the present paper, we propose a simple methodology, based on the bleaching of Pyrogallol Red (PGR) absorbance that provides ORAC indexes that are almost exclusively determined by the reactivity of the tested compounds. This difference is due to the high reactivity of PGR and the high concentrations of this compound employed in the experiments.     

Improved peroxyl radical scavenging TOSC assay to quantify antioxidant capacity using SIFT-MS

Abstract

We report a new, fast, sensitive variation of the total oxyradical scavenging capacity (TOSC) assay for measuring the antioxidant capacity of pure compounds, plant extracts and biological fluids using selected ion flow tube mass spectrometry (SIFT-MS). The TOSC assay examines the partial inhibition of ethene formation in the presence of antioxidants that compete with α-keto-γ-methiolbutyric acid (KMBA) for reactive oxygen species. The SIFT-MS-TOSC assay takes 15 s for each ethene analysis and the time interval between consecutive analyses is 20 s. We demonstrate the method by monitoring the antioxidant capacity of several standard radical scavengers of peroxyl radicals. For peroxyl radicals the measured SIFT-MS-TOSC concentrations necessary to produce 50% inhibition of radical reaction with KMBA are 6.1 ± 0.3 μM for Trolox, 5.7 ± 0.3 μM for ascorbic acid, 8.4 ± 0.4 μM for uric acid and 38 ± 2 μM for reduced glutathione.     

Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.     

A method to evaluate capacity and efficiency of water soluble antioxidants as peroxyl radical scavengers

In this paper, we report on a method to evaluate the activity of water soluble and H-atom donor antioxidants as peroxyl radical scavengers in a micelle system reproducing the conditions occurring in the upper small intestine in humans, during digestion and absorption of lipids. This method, which overcomes some of the problems of the total radical trapping antioxidant parameter (TRAP) assays, measures the peroxyl radical trapping capacity (n) and the peroxyl radical trapping efficiency IC50(-1) of antioxidants, that is the number "n" of peroxyl radicals trapped by one molecule of the studied antioxidant and the reciprocal of the antioxidant concentration that halves the steady-state concentration of peroxyl radicals, respectively. These two fundamental parameters characterizing the radical chain breaking of many water soluble antioxidants, among which dietary polyphenols, can be obtained with relatively good precision from a single experiment, on the basis of a rigorous treatment of the kinetic data.     

Melatonin and structurally-related,endogenous indoles act as potent electron donors and radical scavengers in vitro

Summary

The radical scavenging properties of melatonin, structurally-related indoles and known antioxidants were investigated in kinetic competition studies using the specific radical trapping reagent 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS). In the presence of highly reactive radicals, ABTS is oxidized to the stable thiazoline cation radical, ABTS*⁺ which, due to its intense green color, can be measured photometrically at 420 nm absorbance. The indoles melatonin, 5-methoxytryptophol, 5-methoxyindole acetic acid and 5-methoxytryptamine as well as the phenolic and thiolic antioxidants ascorbic acid, Trolox, and glutathione inhibited ABTS cation radical formation and catalyzed ABTS radical cation reduction. Melatonin was the most potent radical scavenger and electron donor when compared with the methoxylated indole analogs and the other antioxidants tested. Melatonin, the methoxylated indole analogs and the other antioxidants tested acted as potent electron donors which scavenged initiating and propagating radicals and repaired oxidative damage due to electrophile intermediates.     

Inhibition of free radical-induced erythrocyte hemolysis by 2-O-substituted ascorbic acid derivatives

Inhibitory effects of 2-O-substituted ascorbic acid derivatives, ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S), on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of ascorbic acid (AA) and other antioxidants. The order of the inhibition efficiency was AA-2S> or =Trolox=uric acid> or =AA-2P> or =AA-2G=AA>glutathione. Although the reactivity of the AA derivatives against AAPH-derived peroxyl radical (ROO(*)) was much lower than that of AA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis and membrane protein oxidation. In addition, the AA derivatives were found to react per se with ROO(*), not via AA as an intermediate. These findings suggest that secondary reactions between the AA derivative radical and ROO(*) play a part in hemolysis inhibition. Delayed addition of the AA derivatives after AAPH-induced oxidation of erythrocytes had already proceeded showed weaker inhibition of hemolysis compared to that of AA. These results suggest that the AA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AA in vivo.     

Structural effects on the reactivity 1,4-dihydropyridines with alkylperoxyl radicals and ABTS radical cation

A series of eight commercial C-4 substituted 1,4-dihydropyridines and other synthesized related compounds were tested for direct potential scavenger effect towards alkylperoxyl radicals and ABTS radical cation in aqueous Britton-Robinson buffer pH7.4. A direct quenching radical species was established. The tested 1,4-dihydropyridines were 8.3-fold more reactive towards alkylperoxyl radicals than ABTS cation radical, expressed by their corresponding kinetic rate constants. Furthermore, NPD a photolyte of nifedipine and the C-4 unsubstituted 1,4-DHP were the most reactive derivatives towards alkylperoxyl radicals. The pyridine derivative was confirmed by GC/MS technique as the final product of reaction. In consequence, the reduction of alkylperoxyl and ABTS radicals by 1,4-dihydropyridines involved an electron transfer process. Also, the participation of the hydrogen of the 1-position appears as relevant on the reactivity. Results of reactivity were compared with Trolox.     

Total antioxidant potential of resinous exudates from Heliotropium species, and a comparison of the ABTS and DPPH methods

Total reactive antioxidant potential (TRAP) of resinous exudates from Heliotropium species was evaluated by measuring the bleaching of stable free radicals. The antioxidant capacity of the resinous exudates in Trolox equivalents, evaluated from the bleaching of ABTS derived radical cations, ranged from 2.0M (H. huascoense) to 5.2 M (H. stenophyllum), indicating a very high concentration of phenolic compounds. Considerably smaller values were obtained by measuring the bleaching of DPPH radicals. The ratio between the values obtained employing ABTS derived radicals and DPPH, ranged from 37 (H. megalanthum) to 4.5 (H. chenopodiaceum variety typica). The magnitude of the difference can be considered as an indication of the relative reactivity of the antioxidants present in the exudates. Similar ratios were observed when stoichiometric coefficients were evaluated for representative purified flavonoids obtained from the resinous exudates.     

The antioxidant effect of hydroxyl-substituent Schiff bases on the free-radical-induced hemolysis of human erythrocytes

The major objectives of the present work were focused on assessing the antioxidant capacities of two hydroxyl-substituent Schiff bases, 2-((o-hydroxylphenylimino)methyl)phenol (OSAP) and 2-((p-hydroxylphenylimino)methyl)phenol (PSAP) either used alone or in combination with some familiar water-soluble antioxidants i.e. 6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and L-ascorbic acid (VC), and lipophilic ones i.e. alpha-tocopherol (TOH) and L-ascorbyl-6-laurate (VC-12). 2,2'-Azobis(2-amidinopropane hydrochloride) (AAPH). Induced hemolysis of human erythrocytes functioned as the evaluation experimental system in this research. The present findings showed that either OSAP or PSAP not only was an antioxidant with high activity in protecting erythrocytes against AAPH-induced hemolysis concentration-dependently, but can also protect erythrocytes by acting with Trolox, TOH, VC and VC-12 synergistically. Based on chemical kinetic deduction, the number of trapping peroxyl radicals, n, of the above-mentioned antioxidants can be calculated in relation to Trolox that traps two peroxyl radicals; thus, TOH can trap 3.83 peroxyl radicals, VC-12 traps 2.87 and VC can only trap 1.08. As for OSAP and PSAP, 8.71 and 13.7 peroxyl radicals can be trapped, respectively, indicating that they were the most efficient inhibitors against AAPH-induced hemolysis. Moreover, the total number of peroxyl radicals trapped by OSAP+Trolox, OSAP+TOH, OSAP+VC and PSAP+VC were higher than the sum of the above individual antioxidant used alone, demonstrating that a mutual promotive effect existed in the above mixed antioxidants. In contrast, owing to the fact that the total number of peroxyl radicals trapped by OSAP+VC-12, PSAP+Trolox, PSAP+TOH and PSAP+VC-12 were less than the sum of the above individual antioxidant used alone, a mutual antagonistic effect was suggested in these combinative usages. This information may be helpful in the pharmaceutical application of two Schiff bases.     

The cytoprotective effect of Trolox demonstrated with three types of human cells

Trolox, a hydrophilic analogue of alpha-tocopherol, was reported to scavenge peroxyl radicals better than vitamin E in sodium dodecyl sulfate micelles and in liposomes. However, it was not known if Trolox protects human cells against oxyradical damage or if it acts as an antioxidant there. Here we demonstrate that Trolox prolonged substantially the survival of human ventricular myocytes and hepatocyte against oxyradicals generated with xanthine oxidase plus hypoxanthine, and prevented lysis of red cells exposed to an azo-initiator (2,2'-azo-bis(2-amidinopropane) HCl). Note that Trolox did not inhibit xanthine oxidase. In each cell type, the protection by Trolox was dose dependent and surpassed those given by such water-soluble antioxidants as ascorbic acid, superoxide dismutase, and (or) catalase, each examined at or near its optimal level in the same system. Using hepatocytes as a model, we further observed that Trolox reduced markedly the quantity of phospholipid conjugated dienes (a chemical imprint of oxyradical damage) in cells despite their exposure to oxyradicals. These data suggested that Trolox behaves as an antioxidant in cells as illustrated in hepatocytes.     

Total antioxidant potential of resinous exudates from Heliotropium species,and a comparison of the ABTS and DPPH methods

Total reactive antioxidant potential (TRAP) of resinous exudates from Heliotropium species was evaluated by measuring the bleaching of stable free radicals. The antioxidant capacity of the resinous exudates in Trolox equivalents, evaluated from the bleaching of ABTS derived radical cations, ranged from 2.0M (H. huascoense) to 5.2 M (H. stenophyllum), indicating a very high concentration of phenolic compounds. Considerably smaller values were obtained by measuring the bleaching of DPPH radicals. The ratio between the values obtained employing ABTS derived radicals and DPPH, ranged from 37 (H. megalanthum) to 4.5 (H. chenopodiaceum variety typica). The magnitude of the difference can be considered as an indication of the relative reactivity of the antioxidants present in the exudates. Similar ratios were observed when stoichiometric coefficients were evaluated for representative purified flavonoids obtained from the resinous exudates.     

In Vitro Antioxidant Activity of 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman (α-CEHC), a Vitamin E Metabolite

2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman ( &#102 -CEHC) has been identified as a major water-soluble metabolite of vitamin E, which circulates in the blood and is excreted with the urine. The aim of this study was to assess the antioxidant activity of &#102 -CEHC using several methods with different prooxidant challenges. In the Oxygen Radical Absorbance Capacity assay, a fluorescent protein acts as a marker for oxidative damage induced by peroxyl radicals. In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, a stable free radical, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS &#148 + ) is reduced directly by antioxidants. Scavenging properties vs. reactive nitrogen species were studied measuring the effects on tyrosine nitration after reaction with peroxynitrite. Trolox, &#102 -tocopherol, ascorbic acid, and ( &#109 )-epicatechin were simultaneously tested in order to compare their antioxidant activities. In all mentioned systems, &#102 -CEHC exhibited antioxidant properties similar to those of Trolox. We conclude that &#102 -CEHC is a molecule with good antioxidant activity, having the advantage over Trolox of being a naturally occurring compound. These properties might be useful for research or industrial purposes.     

A pyranine based procedure for evaluation of the total antioxidant potential (TRAP) of polyphenols. A comparison with closely related methodologies

A novel procedure for the evaluation of total reactive antioxidant potentials (TRAP) is described. The method is based on the measurement of the bleaching of pyranine by peroxyl radicals. The addition of the antioxidants produces a clear induction time whose magnitude is directly related to the antioxidant concentration. A comparison of the values obtained with those reported employing closely related methodologies shows that the results are significantly affected by the substrate employed to monitor the steady state free radical concentration. Possible sources of this dependence are discussed.