Scavenging of reactive oxygen species by chlorophyllin: An ESR study

The antioxidant effects of chlorophyllin (CHL), a water-soluble analog of the green plant pigment chlorophyll, on different reactive oxygen species (ROS) were investigated by electron spin resonance (ESR) spectroscopy. As a standard, we have used the ability of CHL to scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. CHL inhibits the formation of 5,5-dimethyl-1-pyrroline-N-oxide adduct with hydroxyl radical (DMPO-OH adduct) generated by γ-radiation in a dose-dependent manner. At a concentration of 1 mM, CHL caused more than 90% inhibition of ESR signal intensity of this adduct. However, the results obtained with the Fenton reaction were different. We also found evidence for the inhibition of ¹O₂-dependent formation of the 2,2,6,6-tetramethyl-piperidine oxide (TEMPO) radical during photosensitization of methylene blue with visible light. CHL was also able to inhibit hydrogen peroxide induced oxidation of phenol red. The rate constant of the reaction of CHL with H₂O₂ was found to be 2.7×10⁶ M^-1s^-1. In conclusion, CHL has potent antioxidant ability involving scavenging of various physiologically important ROS.     

Kinetics of Parallel Electron Transfer from β-Carotene to Phenoxyl Radical and Adduct Formation Between Phenoxyl Radical and β-Carotene

Phenoxyl radicals generated by laser flash photolysis were found to react with β-carotene with concomitant β-carotene bleaching in two parallel reactions with similar rates: (i) formation of a β-carotene adduct with a (pseudo) first order rate constant of 1-1.5 ± 10⁴ s^-1 with absorption maximum around 800 nm, and (ii) formation of a β-carotene radical cation with a (pseudo) first order rate constant of 2-3 ± 10⁴ s^-1 with absorption maximum around 920 nm. Both β-carotene radicals decay on a similar time scale and have virtually disappeared after 100 ms, the β-carotene adduct by a second order process. Oxygen had no effect on β-carotene bleaching or radical formation and decay. The reduction of phenoxyl radicals by β-carotene may prove important for an understanding of how β-carotene acts as an antioxidant.     

Hydroxyl Radical-Induced Reactions in Polyadenylic Acid as Studied by Pulse Radiolysis: Part I. Transformation Reactions of Two Isomeric OH-Adducts

The absorption spectra of polyadenylic acid (polyA) radicals in N₂0 saturated aqueous solution have been measured as a function of time (up to 15 s) following an 0.4μS electron pulse. The spectra and their changes were analysed by comparison with those from monomeric adenine derivatives (nucleosides and nucleotides) which had been studied by Steenken.¹

The reaction of OH^· radicals with the adenine moiety in poly A results in the formation of two hvdroxvl adducts at the positions C-4 [polyA40H^·] and C-8 [polyA80H^·]. Each OH-adduct undergoes a unimol-ecular transformation reaction before any bimolecular or other unimolecular decay occurs. These reactions are characterized by different rate constants and _pH dependencies. The polyA40H^· adduct undergoes a dehydration reaction to yield a neutral N⁶ centered radical (rate constant K_deh= 1.4 × 10⁴s^-1 at ^pH7.3). This reaction is strongly inhibited by H⁺. In comparison with the analogous reactions in adenosine phosphates, the kinetic _pK value for its inhibition is two ^pH units higher. This shift is the result of the counter ion condensation or double-strand formation. The polyA80H^· adduct undergoes an imidazole ring opening reaction to yield an enol type of formamidopyrimidine radical with the resulting base damage (k^r.o. = 3.5 × 10⁴ s ^-1 at pH7.3). This reaction in contrast is strongly catalysed by H⁺and OH^-, similar as for adenosine but different compared to the nucleotides.     

Role of reactive oxygen species in cardiac preconditioning: study with photoactivated Rose Bengal in isolated rat hearts

Oxygen radical scavengers have been shown to prevent the development of ischemic preconditioning, suggesting that reactive oxygen species (ROS) might be involved in this phenomenon. In the present study, we have investigated whether direct exposure to ROS produced by photoactivated Rose Bengal (RB) could mimic the protective effects of ischemic preconditioning.

Methods In vitro generation of ROS from photoactivated RB in a physiological buffer was first characterised by ESR spectroscopy in the presence of 2,2,6,6-tetramethyl-1-piperidone (oxoTEMP) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In a second part of the study, isolated rat hearts were exposed for 2.5 min to photoactivated RB. After 5 min washout, hearts underwent 30 min no-flow normothermic ischemia followed by 30 min of reperfusion.

Results and Conclusions The production of singlet oxygen (¹O₂) by photoactivated RB in the perfusion medium was evidenced by the ESR detection of the nitroxyl radical oxoTEMPO. Histidine completely inhibited oxoTEMPO formation. In addition, the use of DMPO has indicated that (i) superoxide anions (O^·-₂) are produced directly and (ii) hydroxyl radicals (HO^·) are formed indirectly from the successive O^·-₂ dismutation and the Fenton reaction. In the perfusion experiments, myocardial post-ischemic recovery was dramatically impaired in hearts previously exposed to the ROS produced by RB photoactivation (¹O₂, O^·-₂, H₂O₂ and HO^·) as well as when ¹O₂ was removed by histidine (50 mM) addition. However, functional recovery was significantly improved when hearts were exposed to photoactivated RB in presence of superoxide dismutase (10⁵ IU/L) and catalase (10⁶ IU/L).

Further studies are now required to determine whether the cardioprotective effects of Rose Bengal in presence of O^·-₂ and H₂O₂ scavengers are due to singlet oxygen or to other species produced by Rose Bengal degradation.     

Scavenging of reactive oxygen species by a novel glucurinated flavonoid antioxidant isolated and purified from spinach

NAO is a natural water soluble antioxidant that was isolated and purified from spinach leaves. Using HPLC, NMR, and CMR spectroscopy, the main components were identified as flavonoids and p-coumaric acid derivatives. The NAO was found to be a very effective antioxidant in several in vivo and in vitro biological systems. In the present study, the antioxidant activity of the novel antioxidant glucurinated flavonoid (GF) isolated and characterized from NAO, is compared to well-known antioxidants. In addition, the direct free radical scavenging properties of the purified component GF were studied using the electron spin resonance (ESR) technique. GF and NAO were found to be superior to EGCG and NAC and to the Vitamin E homologue Trolox in inhibiting reactive oxygen species (ROS) formation in the autooxidation system of linoleic acid and in fibroblasts exposed to metal oxidation. GF and NAO were found to inhibit the ESR signal intensity of DMPO-O(2) radical formation during the riboflavin photodynamic reaction. 10 mM GF caused approximately 90% inhibition in the intensity of the ESR signal, while NAO at a concentration of 60 microg/ml caused an inhibition of about 50%. Using the Fenton reaction, GF and NAO were found to inhibit DMPO-OH radical formation. A concentration of 2 mM GF caused a 70% inhibition in the intensity of the DMPO-OH radical ESR signal, while propyl gallate at the same concentration caused only 50% inhibition. Furthermore, both GF and NAO also inhibited the (1)O(2) dependent TEMPO radical generated in the photoradiation TPPS4 system. About 80% inhibition was obtained by 4 mM GF. The results obtained indicate that the natural antioxidants derived from spinach may directly affect the scavenging of ROS and, as a consequence, may be considered as effective sources for combating oxidative damage.     

A Kinetic and ESR Investigation of Iron(II) Oxalate Oxidation by Hydrogen Peroxide and Dioxygen as a Source of Hydroxyl Radicals

The reaction of Fe^II oxalate with hydrogen peroxide and dioxygen was studed for oxalate concentrations up to 20 mM and pH 2-5, under which conditions mono- and bis-oxalate comlexes (Fe^II(ox) and Fe^II(ox)₂^2-) and uncomplexed Fe²⁺ must be considered. The reaction of Fe^II oxalate with hydrogen peroxide (Fe²⁺ + H₂O₂ → Fe³⁺ + *OH + OH^-) was monitored in continuous flow by ESR with t-butanol as a radical trap. The reaction is much faster than for uncomplexed Fe²⁺ and a rate constant, k = 1 × 10⁴ M-¹ s^-1 is deduced for Fe^II(ox). The reaction of Fe^II oxalate with dioxygen is strongly pH dependent in a manner which indicates that the reactive species is Fe^II(ox)₂^2-, for which an apparent second order rate constant, k = 3.6 M^-1 s^-1, is deduced. Taken together, these results provide a mechanism for hydroxyl radical production in aqueous systems containing Fe^II complexed by oxalate. Further ESR studies with DMPO as spin trap reveal that reaction of Fe^II oxalate with hydrogen peroxide can also lead to formation of the carboxylate radical anion (CO₂^*-), an assignment confirmed by photolysis of Fe^III oxalate in the presence of DMPO.     

Chlorophyllin as an effective antioxidant against membrane damage in vitro and ex vivo

Chlorophyllin (CHL), the sodium-copper salt and the water-soluble analogue of the ubiquitous green pigment chlorophyll, has been attributed to have several beneficial properties. Its antioxidant ability, however, has not been examined in detail. Using rat liver mitochondria as model system and various sources for the generation of reactive oxygen species (ROS) we have examined the membrane-protective properties of CHL both under in vitro and ex vivo conditions. Oxidative damage to proteins was assessed as inactivation of the enzymes, cytochrome c oxidase and succinic dehydrogenase besides formation of protein carbonyls. Damage to membrane lipids was measured by formation of lipid hydroperoxides and thiobarbituric acid reactive substances. The effect of this compound on the antioxidant defense system was studied by estimating the level of glutathione and superoxide dismutase. ROS were generated by gamma-radiation, photosensitization, ascorbate-Fe(2+), NADPH-ADP-Fe(3+) and the peroxyl radical generating agent, azobis-amidopropane hydrochloride. Our results show that CHL is highly effective in protecting mitochondria, even at a low concentration of 10 microM. The antioxidant ability, at equimolar concentration, was more than that observed with ascorbic acid, glutathione, mannitol and tert-butanol. When CHL was fed to mice at a dose of 1% in drinking water, there was a significant reduction in the potential for oxidative damage in cell suspensions from liver, brain and testis. To examine the possible mechanisms responsible for the observed antioxidant ability we have studied the reaction of CHL with the potent ROS in the form of hydroxyl radical and singlet oxygen. The compound shows a fairly high rate constant with singlet oxygen, in the order of 1.3x10(8) M(-1) s(-1). In conclusion, our studies showed that CHL is a highly effective antioxidant, capable of protecting mitochondria against oxidative damage induced by various ROS.     

Esr Spin Trapping Studies Into of Nature of the Oxidizing Species Formed in the Fenton Reaction: Pitfalls Associated With of Use Of 5,5-Dimethyl-1-Pyrroline-n-Oxide in the Detection of the Hydroxyl Radical

Several investigators have challenged the widely held view that the hydroxyl radical is the primary oxidant formed in the reaction between the ferrous ion and hydrogen peroxide. In recent studies, using the ESR spin trapping technique, Yamazaki and Piette found that the stoichiometry of oxidant formation in the reaction between Fe²⁺ and H₂O₂ often shows a marked deviation from the expected value of 1:1 (I. Yamazaki and L. H. Piette (1990) J. Am. Chem. Soc. 113, 7588-7593). In order to account for these observations, it was suggested that additional oxidizing species are formed, such as the ferryl ion (FeO²⁺), particularly when iron is present at high concentration and chelated to EDTA.

In this paper it is shown that secondary reactions, involving the redox cycling of iron and the oxidation of the hydroxyl radical adduct of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide(DMPO) by iron, operate under the reaction conditions employed by Yamazaki and Piette. Consequently, the stoichiometry of oxidant formation can be rationalized without the need to envisage the formation of oxidizing species other than the hydroxyl radical. It is also demonstrated that the iron(III) complex of DETAPAC can react directly with DMPO to form the DMPO hydroxyl radical adduct (DMPO/OH) in the absence of hydrogen peroxide. Therefore, to avoid the formation of (DMPO/OH) as an artefact, it is suggested that DETAPAC should not be used as a reagent to inactivate containating adventitious iron in experiments using DMPO.     

Ozone exposure generates free radicals in the blood samples in vitro. Detection by the ESR spin-trapping technique

Generation of free radicals in the reaction of ozone with blood samples and related salt solutions was investigated in vitro by using ESR spin-trapping technique with DMPO. In the reactions of low levels of ozone, a carbon-centered radical was spin-trapped with DMPO, giving rise to the 6-line ESR signal in both whole blood and blood plasma. In the blood plasma, DMPO-spin adduct of hydroxyl radical (DMPO-OH) was detected together with the spin adduct of carbon-centered radical. The present spin-trapping study demonstrates that, when exposed to ozone, 0.9% NaCl solution in the presence of DMPO gives rise to the formation of DMPO-OH. The addition effects of ethanol, which is a ^·OH scavenger, into the NaCl solution reveal that DMPO-OH is produced by the reaction of DMPO with both ^·OH and unidentified oxidants originated from the reaction of Cl^- and ozone. Based on these observations, we consider that ^·OH is generated similarly in the blood plasma exposed to ozone. The ESR study of DMPO-spin adducts in the ozone-exposed aqueous solution of NaOCl indicates that Cl^- reacts with ozone to give ClO^-. Presumably, further oxidation of ClO^- by ozone leads to the formation of ^·OH and the unidentified oxidants.     

Clarification of the Relationship Between Free Radical Spin Trapping and Carbon Tetrachloride Metabolism in Microsomal Systems

It has been proposed that the C-phenyl-N-tert-butylnitrone/trichloromethyl radical adduct (PBN/^•CCl₃) is metabolized to either the C-phenyl-N-tert-butylnitrone/carbon dioxide anion radical adduct (PBN/^•CO₂⁻) or the glutathione (GSH) and CCl₄-dependent PBN radical adduct (PBN/[GSH-^•CCl₃]). Inclusion of PBN/^•CCl₃ in microsomal incubations containing GSH, nicotinamide adenine dinucleotide phosphate (NADPH), or GSH plus NADPH produced no electron spin resonance (ESR) spectral data indicative of the formation of either the PBN/[GSH-^•CCl₃] or PBN/^•CO₂⁻ radical adducts. Microsomes alone or with GSH had no effect on the PBN/^•CCl₃ radical adduct. Addition of NADPH to a microsomal system containing PBN/^•CCl₃ presumably reduced the radical adduct to its ESR-silent hydroxylamine because no ESR signal was observed. The Folch extract of this system produced an ESR spectrum that was a composite of two radicals, one of which had hyperfine coupling constants identical to those of PBN/^•CCl₃. We conclude that PBN/^•CCl₃ is not metabolized into either PBN/[GSH-^•CCl₃] or PBN/^•CO₂⁻ in microsomal systems.     

Rapid scavenging of peroxynitrous acid by monohydroascorbate

The reaction of peroxynitrous acid with monohydroascorbate, over the concentration range of 250 μM to 50 mM of monohydroascorbate at pH 5.8 and at 25°C, was reinvestigated and the rate constant of the reaction found to be much higher than reported earlier (Bartlett, D.; Church, D. F.; Bounds, P. L.; Koppenol, W. H. The kinetics of oxidation of L-ascorbic acid by peroxynitrite. Free Radic. Biol. Med. 18:85–92; 1995; Squadrito, G. L.; Jin, X.; Pryor, W. A. Stopped-flow kinetics of the reaction of ascorbic acid with peroxynitrite. Arch. Biochem. Biophys. 322:53–59; 1995). The new rate constants at pH 5.8 are k₁ = 1 × 10⁶ M⁻¹ s⁻¹ and k₋₁ = 500 s⁻¹ for 25°C and k₁ = 1.5 × 10⁶ M⁻¹ s⁻¹ and k₋₁ = 1 × 10³ s⁻¹ for 37°C. These values indicate that even at low monohydroascorbate concentrations most of peroxynitrous acid forms an adduct with this antioxidant. The mechanism of the reaction involves formation of an intermediate, which decays to a second intermediate with an absorption maximum at 345 nm. At low monohydroascorbate concentrations, the second intermediate decays to nitrate and monohydroascorbate, while at monohydroascorbate concentrations greater than 4 mM, this second intermediate reacts with a second monohydroascorbate to form nitrite, dehydroascorbate, and monohydroascorbate. EPR experiments indicate that the yield of the ascorbyl radical is 0.24% relative to the initial peroxynitrous acid concentration, and that this small amount of ascorbyl radicals is formed concomitantly with the decrease of the absorption at 345 nm. Thus, the ascorbyl radical is not a primary reaction product. Under the conditions of these experiments, no homolysis of peroxynitrous acid to nitrogen dioxide and hydroxyl radical was observed. Aside from monohydroascorbate's ability to “repair” oxidatively modified biomolecules, it may play a role as scavenger of peroxynitrous acid.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

A novel method of measuring hydroxyl radical-scavenging activity of antioxidants using γ-irradiation

A new method using ESR spin trapping was proposed for measuring the scavenging activity of antioxidants for the hydroxyl (OH) radical. (-)-Epigallocatechin gallate (EGCg) and 5,5-dimethyl-1-pyrrolline N-oxide (DMPO) were used as the antioxidant and spin trapping agent, respectively. The conventional method using a Fenton reaction had problems associated with the estimation of activity, because the antioxidant disturbs the system for generating OH radical by coordinating on Fe²⁺ and by consuming H₂O₂, besides scavenging the spin adduct (DMPO-OH). Intense γ-irradiation was therefore used to generate OH radicals, and the intensity decrease in DMPO-OH after irradiation was followed to obtain the rate constant for the scavenging of DMPO-OH by EGCg. The intensities were extrapolated to zero time to estimate the quantity of DMPO-OH formed during γ-irradiation. By using these values, the reaction rate constant between OH radical and EGCg was calculated as a ratio to that of DMPO. It was shown that this method is useful for comparing the OH radical-scavenging activity of various antioxidants.     

Free Radical Transients in Photobleaching of Xanthophylls and Carotenes

Carotenoicls in chloroform and carbon tetrachloriclc photobleach upon nanosecond laser flash photolysis in two steps: instantaneously and in a second-order reaction. The rate constant for second-order reaction (first-order in a solvent derived radical and first-order in (excess) ccirotenoid) is largest for carotenes (9.8·10⁸ M^-1 s^-1 for β-carotene), intermediate for hydroxylated carotenoids, and smallest for carbonyl containing carotenoids (1.0·10⁸ M^-1 s^-1 for astaxanthin) in chloroform at 20°C. Near infrared, ibsorbing transients are formed concomitant with pliotohleaching in chloroform (not detected in cxbon tetrachloride). A species formed instantaneously is tentatively identified as either a carotenoid/solvent adduct or an ion-pair. A second species is formed by decay of the instantaneously formed species and is identified as the carotenoid radical cation. This species is formed in a first-order reaction with a rate constant of approx. 5·10⁴ s^-1 and absorbing at longer wavelength than the precursor. The lifetime (second-order decay) of the interniediates appears to be longest for the carotenoids with the longest conjugated system. The results indicate that carotenes are better antioxidants than xantliophylls as the carotenes, at least in the present lipophilic solvents, react faster with free radicals.     

The Horseradish Peroxidase Catalysed Oxidation of Deoxyribose Sugars

The ability of horseradish peroxidase (E.C. 1.11.1.7. Donor: H₂O₂ oxidoreductase) to catalytically oxidize 2-deoxyribose sugars to a free radical species was investigated. The ESR spin-trapping technique was used to denionstrate that free radical species were formed. Results with the spin trap 3.5-dibronio-4-nitrosoben-zene sulphonic acid showed that horseradish peroxidase can catalyse the oxidation of 2-deoxyribose to produce an ESR spectrum characteristic of a nitroxide radical spectrum. This spectrum was shown to be a composite of spin adducts resulting from two carbon-centered species, one spin adduct being characterized by the hyperfine coupling constants a^N = 13.6Ganda^H_β = 11.0G, and the other by a^N = 13.4G and a^H = 5.8 G. When 2-deoxyribose-5-phosphate was used as the substrate, the spectrum produced was found to be primarily one species characterized by the hyperfine coupling constants a^N = 13.4G and a^H= 5.2. All the radical species produced were carbon-centered spin adducts with a β hydrogen, suggesting that oxidation occurred at the C(2) or C(5) moiety of the sugar. Interestingly, it was found that under the same experimental conditions, horseradish peroxidase apparently did not catalyze the oxidation of either 3-deoxyribose or D-ribose to a free radical since no spin adducts were found in these cases.

It can be readily seen that 2-deoxyribose and 2-deoxyribose-5-phosphate can be oxidized by HRP/H₂O₂ to form a free radical species that can be detected with the ESR spin-trapping technique. There are two probable sites for the formation of a CH type radical on the 2-deoxyribose sugar, these being the C(2) and the C(5) carbons. The fact that there is a species produced from 2-deoxy-ribose, but not 2-deoxy-ribose-5-phosphate, suggests that there is an involvement of the C(5) carbon in the species with the 1 1.0G β hydrogen. In the spectra formed from 2-deoxy-ribose, there is a big difference in the hyperfine splitting of the β hydrogens, suggesting that the radicals are formed at different carbon centers, while the addition of a phosphate group to the C(5) carbon seems to inhibit radical formation at one site. In related work, the chemiluminescence of monosaccharides in the presence of horseradish peroxidase was proposed to be the consequence of carbon-centered free radical formation (10).     

Essential Oil Phenyl Propanoids. Useful as OH Scavengers?

In order to search for radical scavengers which could be used as raw materials for cosmetics, phenyl propanoids (eugenol, isoeugenol, dehydrodieugenol, dehydrodieugenol B and coniferyl aldehyde) were examined for their hydroxyl radical (· OH) scavenging ability. A Fenton system was used to produce -OH. In order to see scavenging by these phenyl propanoids, competition reactions between a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and these phenyl propanoids for -OH were studied. The relative yield of the spin adduct of -OH (DMPO-OH) was measured by electron spin resonance spectroscopy. The approximate rate constants of the reactions between these phenyl propanoids and -OH estimated by measuring the reduced height of the ESR signals of DMPO-OH were found to be at least in the order of 10⁹ M^-1 s^-1 (diffusion-controlled). Also, using the TBA tests, the reactions between ·OH and several compounds reactive with ·OH were investigated in the presence of the phenyl propanoids and it was found that the phenyl propanoids compete with such reactive compounds for ·OH. These results indicate that these phenyl propanoids can be used as antioxidants for skin damage perhaps caused by -OH generated by UV-light.     

Mechanism of reaction of nitrogen dioxide radical with hydroxycinnamic acid derivatives: A pulse radiolysis study

Nitrogen dioxide radical (NO^·₂) is known as a toxic agent produced in the metabolism of nitrates and nitrites. By the use of the pulse radiolysis technique, the mechanism of the reaction of NO^·₂ radical with hydroxycinnamic acid derivatives (HCA) was studied and the rate constants have been measured. The rate constants were found to be 7.4 × 10⁸, 7.2 × 10⁸, 8.6 × 10⁸ dm³ mol^-1s^-1 for ferulic acid, sinapic acid and caffeic acid, respectively. The reactions produce the corresponding phenoxyl radical.     

Oxidation of deseferrioxamine to nitroxide free radical by activated human neutrophils

Human neutrophils activatd by PMA were found to induced the formation of a nitroxide radical from DFO. The presence of SOD was necessary to permit the formation of the DFO radical. The inactive phorbol ester did not induce DFO radical, and _sphinganine suppressed the radical produced by the active phorbol ester. Other cell stimuli (Zymocel and the chemotactic peptide) also induced the formation of the DFO radical, although radical concentration was very much lower than with PMA. Participation of ^.NO, ^,OH or ¹O₂ was ruled out by the inability of N^G-methyl-L-arginine, N^G-nitro-L-arginine, DMSO, mannitol, histidine, and methionine to inhibit the formation of DFO radical produced by PMA-activated cells. Furthermore, PMA-activated cells dod not produce detectable levels of NO₂⁻, as a stable oxidation product of ^.NO, and D₂, which enhances the lifetime of singlet oxygen, did not modify the intensity or the lifetime of DFO radical. The involvement of cell MPO was suggested by the inhibition of the DFO radical observed after treatment with catalase or with antihuman MPO antibodies. Also, HOCI was found to induce the DFO radical in cell-free reactions, but our data indicate that the reaction leading to DFO radical formation by neutrophils involves the reduction of MPO compound II back to active enzyme (ferric-MPO). Anti-inflammatory drugs strongly increased the DFO radical produced by activated neutrophils. On the contrary, none of these drugs was able to increase the DFO radical produced by HOCl. Histidine and methionine that inhibited the DFO radical intensity in cell-free reactions, were shown to act directly onm HOCl. Experiments with MPO-H₂O₂ in SOD- and Cl⁻-free conditions showed the formation of DFO radical and confirmed the hypothesis of the involvement of compound II. The conversion of compound II to ferric MPO by DFO optimized the enzymatic activity of neurophils, and in the presence of monochlorodimedon (compound II promoting agent) we measured an increased HOCl production. When DFO was modified by conjugation with hydroxyethyl starch, it lost the ability to produce the radical either by neutrophils or by MPO-H₂O₂ and did not increase HOCl production. The inability of these DFO derivatives to produce potentially toxic species migh explain their reported lower toxicity in vivo.     

Redox reactions of the urate radical/urate couple with the superoxide radical anion, the tryptophan neutral radical and selected flavonoids in neutral aqueous solutions

The kinetics of several processes involving the potential antioxidant role of urate in physiological systems have been investigated by pulse radiolysis. While the monoanionic urate radical, ^·UH^-, can be produced directly by oxidation with ^·Br^-₂ or ^·OH, it can also be generated by oxidation with the neutral tryptophan radical, ^·Trp, with a rate constant of 2 × 10⁷ M^-1s^-1. This radical, ^·UH^-, reacts with ^·O^-₂ with a rate constant of 8 × 10⁸ M^-1s^-1. Also, ^·UH^- is reduced by flavonoids, quercetin and rutin in CTAB micelles at rate constants of 6 × 10⁶ M^-1s^-1 and 1 × 10⁶ M^-1s^-1, respectively. These results can be of value by providing reference data useful in further investigation of the antioxidant character of urate in more complex biological systems.     

The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid

N-acetylcysteine has been widely used as an antioxidant in vivo and in vitro. Its reaction with four oxidant species has therefore been examined. N-acetylcysteine is a powerful scavenger of hypochlorous acid (H---OCl); low concentrations are able to protect ₁-antiproteinase against inactivation by HOCl. N-acetylcysteine also reacts with hydroxyl radical with a rate constant of 1.36 × 10¹⁰ M⁻¹s⁻¹, as determined by pulse radiolysis. It also reacts slowly with H₂O₂, but no reaction of N-acetylcysteine with superoxide (O₂⁻) could be detected within the limits of our assay procedures.