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Introduction/Objective. Age-related cataract is a significant cause of visual impairment worldwide. Oxidative damage and the effects of free radical species are considered important in the etiopathogenesis of cataracts. The aim of this study was to evaluate the antioxidative capacity and oxidative stress in the aqueous humor (AH) according to age and cataracts maturity. Methods. Clinical and biochemical researches were carried out in 55 patients with age-related cataract. According to the cataract maturity, patients were classified into incipient (cortical – group C, 18 patients; nuclear – group N, 20 patients; mature – group M, 17 patients). In order to evaluate the impact of age patients within each group were divided into Group I (65–69 years) and Group II (70 ≥ years). The antioxidant activity of AH was measured by the reduction power method and the activity of glutathione peroxidase (GPx) spectrophotometrically. Changes in the concentrations of hydroxyl and ascorbyl radicals were detected by electron spin resonance spectroscopy. Results. Both reduction power and GPx activity were significantly (p < 0.001) reduced in group N compared to group C and in group M compared to group N. Concentrations of hydroxyl (29.45 ± 1.01% in group C, 38.12 ± 1.29% in group N, and 74.14 ± 2.52% in group M) and ascorbyl radicals (26.12 ± 0.89% in group C, 41.15 ± 1.39% in group N, and 83.56 ± 2.84% in group M) increased significantly (p < 0.05) with progression of age-related cataract. Significant negative correlation (r = -0.759, p < 0.05) was determined between concentrations of HO• and content of GPx. Conclusion. Our research proved that the level of oxidative stress in the AH is significantly affected during aging and cataract progression, The obtained data support the hypothesis that during aging, depending on the maturity of the cataract, the antioxidant capacity in the AH decreases due to an increase in the concentration of reactive HO•.

Ascorbyl radical is often used as a biomarker of oxidative stress in human organism. It is a product of the antioxidant activity of ascorbic acid and it is not expected to react further with biologically important molecules. For the first time, the reactivity of catecholamines and their precursors was investigated theoretically and experimentally towards ascorbyl radical and the main structural parameters governing activity were analyzed. It was shown that catechol moiety plays an important role, which classifies norepinephrine and 3,4-dihydroxyphenylacetic acid as the most reactive when compared to homovanillic acid, vanillylmandelic acid, and octopamine. DFT methods have been employed to determine the most probable mechanism of the reaction. Based on the change in reaction enthalpy it was concluded that Hydrogen Atom Transfer (HAT) is a preferred mechanism both in water and pentyl ethanoate. The stabilization interactions in ascorbic acid, its anion and radical are carefully analyzed in order to understand the preferability of the mentioned mechanism. The stability of the ascorbyl radical is explained in detail. The results prove that ascorbyl radical is not just a product of antioxidant activity, but also a potentially harmful species in an organism. [ABSTRACT FROM AUTHOR]

Paenibacillus larvae , the causal agent of American foulbrood disease (AFB), affects Apis mellifera larvae and can induce oxidative stress by overproduction of radical oxygen species (ROS). This study aimed to assess the oxidative stress levels in larvae exposed to three different strains of P. larvae through their diet by examining the ascorbyl radical (A) to ascorbate anion (AH¯) ratio. The results revealed that larvae inoculated with P. larvae exhibited a lower value of this index compared to uninoculated ones. Interestingly, the level of A remained constant, while the concentration of AH¯ increased. Said increase correlated with the virulence of the specific P. larvae strain used in the inoculation. These findings suggest a potential link between AH¯ molecules and a defense response in A. mellifera larvae against infection, consistent with their resistance to P. larvae (LD 50). [Display omitted] • Paenibacillus larvae can induce oxidative stress of Apis mellifera larvae. • Oxidative stress was analyzed by ascorbyl radical (A)/ascorbate anion (AH¯) ratio. • The ratio A /AH¯ of larvae inoculated reflected less oxidative stress that uninoculated ones. • The level of A remained unchanged despite AH¯ increased. • The intensity of AH¯ increased was according to larvae resistance (LD 50). [ABSTRACT FROM AUTHOR]

Selenocysteine (Sec) is the 21st amino acid in the genetic code and it is present in a small number of proteins where it replaces the much more commonly used amino acid cysteine (Cys). It is both more complicated and bioenergetically costly to insert Sec into a protein in comparison to Cys, and this cost is most likely compensated by a gain of function to the enzyme/protein in which it is incorporated. Here we investigate one such gain of function, the enhancement of one‐electron transfer catalysis. We compared the ability of Sec‐containing mouse mitochondrial thioredoxin reductase (mTrxR2) to catalyze the reduction of bovine cytochrome c, ascorbyl radical, and dehydroascorbate in comparison to Cys‐containing thioredoxin reductases from D. melanogaster (DmTrxR) and P. falciparum (PfTrxR). The Sec‐containing mTrxR2 was able to reduce all three substrates, while the Cys‐containing enzymes had little or no activity. In addition, we constructed Cys➔Sec mutants of DmTrxR and PfTrxR and found that this substitution resulted in a gain of function, as these mutant enzymes were now able to catalyze the reduction of these substrates. We also found that in the case of PfTrxR, reduction of cytochrome c was enhanced five‐fold in a truncated PfTrxR in which the C‐terminal redox center was removed. This shows that some of the ability of thioredoxin reductase to reduce this substrate comes from the flavin coenzyme. We also discuss a possible mechanism by which Sec‐containing thioredoxin reductase reduces dehydroascorbate to ascorbate by two sequential, one‐electron reductions, in part catalyzed by Sec. [ABSTRACT FROM AUTHOR]

Ascorbyl radical was generated by the pulse radiolysis method and observed with the fast kinetic spectrophotometry within reverse micelles stabilized by AOT in n-heptane or by Igepal CO-520 in cyclohexane at different water to surfactant molar ratio, w 0 . Rate constants for the disproportionation of the ascorbyl radicals were smaller than those for intermicellar exchange for both type of reverse micelles and slower than those in homogeneous aqueous solutions. However, they increased with increasing w 0 for AOT/n-heptane system, while they decreased for Igepal CO-520 system. The absorption spectra of ascorbic acid AOT/n-heptane reverse micellar system showed that the “pH” sensed by this molecule is lower than that in respective homogeneous aqueous solutions. The obtained results were rationalized taking into account three main factors (i) preferential location of ascorbic acid molecules in the interfacial region of the both types of reverse micelles; (ii) postulate that the pH of the interface is lower than that of the water pool of reverse micelles and (iii) different structure of the interface of the reverse micelles made by AOT in n-heptane and those formed by Igepal CO-520 I cyclohexane. Some possible consequences of these findings are discussed. [ABSTRACT FROM AUTHOR]

A detailed kinetic study of the reduction of cis-dichloridobispicolinatoruthenate(III) by l-ascorbic acid that leads to the formation of the corresponding RuII complex, was carried out spectrophotometrically using the stopped-flow technique. The reaction was studied as a function of [AscH2]T, [RuIII], and pH. The observed kinetic traces could only be fitted to a three-exponential function, characteristic for three parallel reaction paths. However, an earlier study clearly showed that under the selected experimental conditions only a single complex species is present in solution. The observed complication is suggested to arise from the low driving force of the reaction during which the re-oxidation of RuII by the semi-oxidized l-ascorbic acid, i.e. ascorbyl radical Asc ·-, accounts for the apparent three-exponential behavior of the reaction. A detailed account of different modeling efforts and a comparison with related systems is presented. [ABSTRACT FROM AUTHOR]

The effect of oxygen on the two separate one-electron reactions involved in the oxidation of ascorbic acid was investigated. The rate of ascorbate radical ( Asc − ) formation (and stability) was strongly dependent on the presence of oxygen. A product of ascorbic acid oxidation was measurable levels of hydrogen peroxide, as high as 32.5 μM from 100 μM ascorbic acid. Evidence for a feedback mechanism where hydrogen peroxide generated during the oxidation of ascorbic acid accelerates further oxidation of ascorbic acid is also presented. The second one-electron oxidation reaction of ascorbic acid leading to the disappearance of Asc − was also strongly inhibited in samples flushed with argon. In the range of 0.05–1.2 mM ascorbic acid, maximum levels of measurable hydrogen peroxide were achieved with an initial concentration of 0.2 mM ascorbic acid. Hydrogen peroxide generation was greatly diminished at ascorbic acid levels of 0.8 mM or above. [ABSTRACT FROM AUTHOR]

The aim of this work was to study the oxidative profile of gills of two limpet species (Nacella (Patinigera) magellanica and Nacella (Patinigera) deaurata) (Gmelin, 1971) exposed to different environmental conditions. Due to the tidal characteristics of the Beagle Channel, N. magellanica are exposed to air twice daily for 3 to 5 hours each time, whereas N. deaurata are exposed to air for 3 hours only during spring tides. The different regime of exposure includes extreme temperatures under 0ºC during winter and more than 20°C in summer for N. magellanica, whereas N. deaurata are usually covered by more than 0.3 m of water at 4°C in winter and 11°C in summer. No significant differences were found between the two molluscs regarding the oxygen uptake, the content of ?-tocopherol and ?-carotene and the activities of the antioxidant enzymes catalase and superoxide dismutase. Lipid peroxidation in gills was estimated as the content of lipid radicals, assessed by electron paramagnetic resonance (EPR). Lipid radical content and total iron content were respectively 80.6 and 62% lower in N. magellanica than in N. deaurata. A typical EPR spectrum of ascorbyl radical in gills from both limpets was observed. Both the ascorbyl radical content and the ascorbyl radical content/ascorbate content ratio were significantly lower in N. magellanica than in N. deaurata. In the Antarctic Nacella concinna inhabits all levels of the littoral zone. Limpets at the highest level in the intertidal showed significantly increased activities of both catalase and superoxide dismutase as compared to their intertidal and subtidal relatives. Thus, it seems that Antarctic high intertidal conditions, involving regular exposure to air and presumably also thermal stress on sunny days during the Antarctic summer, cause a necessity for N. concinna to ward off higher oxygen radical species production by increasing its antioxidant defence. Taken as a whole, the data presented here indicate that coping with environmentally demanding conditions requires a complex adjustment of the physiological metabolic pathways to ensure survival by minimising intracellular damage.

Abstract: Electron paramagnetic resonance (EPR) spectroscopy detects the presence of radicals of biological interest, such as ascorbyl radical (A•) and lipid radicals. A• is easily detectable by EPR even in aqueous solution at room-temperature. Under oxidative conditions leading to changes in total ascorbate (AH−) content, the A•/AH− ratio could be used to estimate early oxidative stress in the hydrophilic milieu. This methodology was applied to a wide range of aquatic systems including algae, sea urchin, limpets, bivalves and fish, under physiological and oxidative stress conditions as well. The A•/AH− ratio reflected the state of one part of the oxidative defense system and provided an early and simple diagnosis of environmental stressing conditions. Oxidative damage to lipids was assessed by the EPR-sensitive adduct formation that correlates well with cell membrane damage with no interference from other biological compounds. Probe instability, tissue metabolism, and lack of spin specificity are drawback factors for employing EPR for in vivo determination of free radicals. However, the dependability of this technique, mostly by combining it with other biochemical strategies, enhances the value of these procedures as contributors to the knowledge of oxidative condition in aquatic organisms. [Copyright &y& Elsevier]

Context: Oxidative stress may contribute to lymphedema and subsequent tissue damage. However, the causal role of oxidative stress in lymphedema remains unclear. Objective: We attempted to detect and identify the free radicals formed in lymphedema fluid and assessed the protective mechanisms and effects of specific enzyme inhibitors and natural antioxidants. Materials and methods: To study the level of postsurgical oxidative stress with lymphedema in a mouse tail model, we used an electron spin resonance (ESR) method and an ascorbyl radical's ESR spectrum as an oxidative stress biomarker. The drug-treatment group received an i.p. injection with indomethacin (2 mg/kg), baicalein (15 mg/kg), MK-886 (3 mg/kg), zileuton (6.25 mg/kg), diphenyleneiodonium (DPI; 1 mg/kg), sulforaphane (30 mg/kg), oryzanol (30 mg/kg) or sesamol (30 mg/kg) once daily for 14 d from the day of operation. All animals were sacrificed on day 14. Results: Administration of indomethacin, sulforaphane, oryzanol and sesamol significantly suppressed both the tail volume (56.9%, 77.8%, 72.2% and 38.1% inhibition, respectively, p < 0.01) and ascorbyl radical signals (31.4%, 54.5%, 79.3% and 57.1% inhibition, respectively, p < 0.01), compared with the control mice. No significant differences were found between any of the baicalein, MK-886, or zileuton groups compared with the control. DPI suppressed the tail volume (25.9% inhibition, p < 0.01) but not the ascorbyl radical signals. Conclusion: This study showed that COX-derived oxidative stress plays a major role in the pathological mechanisms of surgically induced lymphedema. Indomethacin, sulforaphane, oryzanol and sesamol exhibit potent protective properties against surgically induced lymphedema. [ABSTRACT FROM AUTHOR]

Electron paramagnetic resonance uses the resonant microwave radiation absorption of paramagnetic substances to detect highly reactive and, therefore, short-lived oxygen and nitrogen centered radicals. Previously, steady state concentrations of nitric oxide, ascorbyl radical (A·) and the labile iron pool (LIP) were determined in digestive gland of freshly collected animals from the North Sea bivalve Mya arenaria. The application of a simple kinetic analysis of these data based on elemental reactions allowed us to estimate the steady state concentrations of superoxide anion, the rate of A· disappearance and the content of unsaturated lipids. This analysis applied to a marine invertebrate opens the possibility of a mechanistic understanding of the complexity of free radical and LIP interactions in a metabolically slow, cold water organism under unstressed conditions. This data can be further used as a basis to assess the cellular response to stress in a simple system as the bivalve M. arenaria that can then be compared to cells of higher organisms. [ABSTRACT FROM AUTHOR]

The delicate balance between the advantageous and detrimental effects of free radicals is one of the important aspects of human (patho)physiology. The controlled production of reactive oxygen and nitrogen species has an essential role in the regulation of various signaling switches. On the other hand, imbalanced generation of radicals is highly correlated with the pathogenesis of many diseases which require the application of selected antioxidants to regain the homeostasis. In the era of growing interest for redox processes, electron paramagnetic resonance (EPR) spectroscopy is arguably the best-suited technique for such research due to its ability to provide a unique insight into the world of free radicals and antioxidants. Herein, I present the principles of EPR spectroscopy and the applications of this method in assessing: (i) the oxidative status of biological systems, using endogenous long-lived free radicals (ascorbyl radical (Asc•), tocopheroxyl radical (TO•), melanin) as markers; (ii) the production of short-lived radicals (hydroxyl radical (OH•), superoxide radical anion (O2•−), sulfur- and carbon-centered radicals), which are implicated in both, oxidative stress and redox signaling; (iii) the metabolism of nitric oxide (NO•); (iv) the antioxidative properties of various drugs, compounds, and natural products; (v) other redox-relevant parameter. Besides giving a comprehensive survey of up-to-date literature, I also provide illustrative examples in sufficient detail to provide a means to exploit the potential of EPR in biochemical/physiological/medical research. The emphasis is on the features and characteristics (both positive and negative) relevant for EPR application in clinical sciences. My aim is to encourage fellow colleagues interested in free radicals and antioxidants to expand their base knowledge or methods used in their laboratories with data acquired by EPR or some of the EPR techniques outlined in this review, in order to boost up the exciting area of redox science. [ABSTRACT FROM AUTHOR]

Abstract: The role of fullerene as a pro-oxidant or anti-oxidant in Ultraviolet B ray (UV-B)-induced disorders in mouse skin was investigated. Fullerene gave no photo-toxic effect to UV-B-irradiated mouse skin. Since erythema was concentrated at the pore circumference in a UV-B irradiation experiment in mouse skin, the sebaceous gland pairs was strongly implicated as a site for the generation of reactive oxygen species (ROS). In a histological evaluation of the skin stained with CH3MDFDA (ROS index) and YO-Pro-1 (apoptosis index), the fluorescence intensity of a sebaceous gland significantly increased with UV-B irradiation. With the application of fullerene to UV-irradiated mouse skin, no toxicity was recognized in comparison with the control, and erythema, the ROS index, and the apoptosis index decrease with the application of fullerene. Ascorbyl radical (AAed with the application of ascorbate (AA) to UV-B-irradiated mouse skin, and AAd with the application of fullerene. The co-application of AA and fullerene, which suppressed AA, significantly suppressed erythema, and also suppressed both the ROS index and apoptosis index in mouse skin after UV-B irradiation. In both mouse skin at 48h after UV-B irradiation and in an attempt to reproduce this phenomenon artificially in vitro, a similar high AAph name="rad" /> >4) was observed in electron spin resonance (ESR) charts. The binding of fullerene with AA impairs the Fenton reaction between AA and Fe-protein based on the observation of ascorbate-specific UV absorption and a linear equation for the calibration curve. Therefore, fullerene may impair the intercalation of AA to a heme pocket by binding with AA. These results suggest that the co-application of AA and fullerene is effective against oxidative skin damage caused by UV-B irradiation, and the development of an AAr such as fullerene should be useful for reducing organ damage associated with Fe-protein oxidation. [Copyright &y& Elsevier]

Abstract: The hypothesis of this study was that mammalian ferritin (FER) has the ability of releasing Fe in the tissue to catalyze the generation of free radicals, such as ascorbyl (Aroxyl radical ( might lead to the damage of FER itself. The rat liver homogenates exhibited an electron paramagnetic resonance (EPR) signal with the spectral features (a H =1.88G, g =2.0054) of Aition to the reaction medium of isolated rat liver FER increased by 3-fold the EPR signal, as compared to the recorded value in its absence. Isolated microsomes from rat liver incubated during 10min showed a signal with the spectral features (a H =15G, g =2.0062) of ddition of FER in the presence of either ethylenediamine-tetraacetic acid (EDTA) or adenosine-5′-triphosphate (ATP) significantly increased the recorded spectra. The labile Fe pool (LIP) in the homogenate was assessed by EPR. The rat liver homogenates exhibited an EPR signal with the spectral features (g =4.3) of the Fe2+ and was significantly increased by the addition of FER (3-fold). The oxidation profile of the isolated FER from rat liver was analyzed after incubation with 10mM ascorbate (AH−). A drastic increase in the width of the band suggested alterations to the protein structure. The FER content of tryptophan (Trp) and thiols was significantly lower when the incubation was performed in the presence of AH− as compared to the recorded effect in its absence. The data in tissue homogenates presented here showed that radical generation is associated to FER Fe release, and moreover that the FER protein itself was affected during this process. [Copyright &y& Elsevier]

The potential of free radical formation in serum of β-thalassemia/Hb E patients receiving a single oral dose of 25 mg/kg body weight of deferiprone, a bidentate orally active iron chelator, was evaluated using EPR/spin trapping technique. In the presence of ascorbic acid and tert-butylhydroperoxide, EPR signals of ascorbyl radical (aH=0.18 mT) and DMPO-carbon centred adduct (aH=2.37 mT, aN=1.65 mT) were detected. Shortly after deferiprone administration, EPR signal intensities decreased concomitant with an increase in serum levels of deferiprone. Unfortunately, enhanced EPR signal intensities were observed at 300 min after dosing in patients with serum molar ratio of deferiprone to iron less than 3, suggesting the formation of incomplete iron-deferiprone complexes and consequently free radical formation. To avoid adverse effects of deferiprone, a dosage regimen should be designed according to iron status of the patients and aimed at maintaining an adequate ratio of serum chelator-to-iron concentration. [ABSTRACT FROM AUTHOR]

Objective: To study the effects of cold ischemia and reperfusion after transplantation of the sheep uterus and to compare the preservation solution Perfadex (Vitrolife, Mölndal, Sweden) with Ringer''s acetate. Design: Experimental animal study. Setting: University hospital. Animal(s): Adult, female sheep. Intervention(s): One uterine horn with the common uterine cavity and cervix of sexually mature ewes was auto-transplanted after 1 hour of cold ischemic storage in either Perfadex (n = 5) or Ringer''s acetate (n = 5). During 3 hours of reperfusion, uterine venous blood was collected and analyzed for several parameters that were indicative of oxidative stress and reperfusion injury. A biopsy was taken for histological analysis at the end of the experiment. Main Outcome Measure(s): Lipid peroxidation and ascorbyl radicals in uterine venous blood during reperfusion. Light microscopy and quantification of neutrophils in tissue after 3 hours of reperfusion. Result(s): A decline in pH and a rise in lactate and pCO2-pO2, as well as an elevation of antioxidant capacity, lipid peroxidation, and intensity of ascorbyl radical electron spin resonance signal, was found that was more prominent after storage in Ringer''s acetate. The histological analysis revealed mild inflammation in both study groups. Conclusion(s): Short-time cold ischemic storage of the sheep uterus does not induce any severe reperfusion damage, but the use of the protective buffer Perfadex decreases oxidative stress and inflammation when compared with a more simple solution. [Copyright &y& Elsevier]

The objective of this work was to analyze oxidative metabolism in Mya arenaria. Total Fe content in M. arenaria collected in the German Wadden Sea was 1.9 ± 0.7, 0.7 ± 0.1 and 0.17 ± 0.01 nmol/mg fresh weight (FW), in digestive glands (DG), mantle and gills, respectively. Labile Fe pool, assessed by electronic paramagnetic resonance (EPR), was 146 ± 10 pmol/mg FW, and by the fluorescence method employing calcein it was 118 ± 9 pmol/mg FW. The lipid radical content in the DG, assessed by EPR, was 27 ± 7 pmol/mg FW, and the thiobarbituric reactive substances content amounted to 57 ± 8 pmol/mg FW. Ascorbyl radical (A•) content, assessed by quantification of EPR signals, was 0.04 ± 0.01 pmol/mg FW, and the ascorbate content (AH−) was 478 ± 12 pmol/mg FW. The ratio A•/AH− was (8 ± 1) × 10−5 AU, suggesting a minimum oxidative stress even under physiological conditions, presumably depending on basal metabolic functions. The content of nitric oxide (NO), assessed by EPR, was 99 ± 3 pmol/mg FW. The generation rate of NO by nitric oxide synthase-like activity (NOS-like) was assayed as NO production detected by EPR in the presence of l-arginine and NADPH, and was 3.16 ± 0.06 pmol/(mg FW min). The data presented here document the detectable presence of highly reactive species in M. arenaria. [Copyright &y& Elsevier]

Abstract: As an index of oxidative status, we analyzed ascorbyl radical generation during and after kainate-induced seizures in mouse hippocampus, using an ESR spectrometer equipped with a special tissue-type quartz cell. A specific doublet ESR spectrum was observed after seizures, and the g value and the hyperfine coupling constant (hfcc) of the spectrum were identical with those of ascorbyl radical itself. Antiepileptic zonisamide inhibited the generation of ascorbyl radical accompanying the seizures. [Copyright &y& Elsevier]

Abstract: Background: Ascorbate is the most effective water-soluble antioxidant and its plasma concentration is usually measured by different methods including colorimetric assays, HPLC or capillary electrophoresis. Plasma antioxidant capacity is determined by indexes such as total reactive antioxidant potential, total antioxidant reactivity, oxygen radical absorbance capacity, etc. We developed an alternative method for the evaluation of the plasma antioxidant status due to ascorbate. Methods: TEMPO kinetics scavenging analyzed by ESR spectroscopy was performed on plasma samples in different antioxidant situations. Plasma ascorbate concentrations were determined by capillary electrophoresis. Ascorbyl radical levels were measured by ESR. Results: Plasma reactivity with TEMPO (PR-T) reflected plasma ascorbate levels. Average PR-T for normal plasmas resulted 85±27 μmol/l (n =43). PR-T during ascorbic acid intake (1 g/day) increased to an average value of 130±20 μmol/l (p &#60;0.001, n =20). PR-T correlated with the plasmatic ascorbate levels determined by capillary electrophoresis (r =0.92), presenting as an advantage the avoiding of the deproteination step. Plasma ascorbyl radical levels increase from 16±2 to 24±3 nmol/l (p &#60;0.005, n =14) after ascorbate intake. Conclusions: PR-T could be considered as a measure of the plasmatic antioxidant capacity due to the plasma ascorbate levels and could be useful to investigate different antioxidant situations. [Copyright &y& Elsevier]

Damage to DNA and disruption of membrane integrity by lipid peroxidation processes are two of the proposed causes of UV-B-induced growth inhibition in plants. However, the relative significance of these different types of molecular damage has not been established in experiments carried out under realistic physiological conditions. Plants ofGunnera magellanica(a native herb from southern Patagonia) were exposed to a gradient of biologically effective UV-B doses (from 0 to 6.5 kJ m−2 d−1 of UV-Bbe) in a greenhouse study. Leaf expansion was measured and sensitive techniques were used to detect damage to DNA (in the form of cyclobutane pyrimidine dimers; CPDs) and lipid peroxidation (via electronic-paramagnetic resonance; EPR). Leaf expansion decreased and the CPD density increased with increasing UV-B doses, but the degree of lipid peroxidation remained unaffected. The highest UV-B dose induced a transient oxidative stress situation (as evaluated using the ratio of ascorbyl radical to ascorbate, A·/AH–), which was rapidly controlled by an increase in the ascorbate pool. The present results suggest that under a range of UV-Bbe doses that overlaps the range of doses thatG. magellanicaplants experience in their natural environment, growth inhibition is better explained by DNA damage than by increased lipid peroxidation. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function. [Copyright &y& Elsevier]

Previous work from our laboratory demonstrated that pyridoxal isonicotinoyl hydrazone (PIH) has in vitro antioxidant activity against iron plus ascorbate-induced 2-deoxyribose degradation due to its ability to chelate iron; the resulting Fe(III)–PIH2 complex is supposedly unable to catalyze oxyradical formation. A putative step in the antioxidant action of PIH is the inhibition of Fe(III)-mediated ascorbate oxidation, which yields the Fenton reagent Fe(II) [Biochim. Biophys. Acta 1523 (2000) 154]. In this work, we demonstrate that PIH inhibits Fe(III)–EDTA-mediated ascorbate oxidation (measured at 265 nm) and the formation of ascorbyl radical (in electron paramagnetic resonance (EPR) studies). The efficiency of PIH against ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation was dose dependent and directly proportional to the period of preincubation of PIH with Fe(III)–EDTA. The efficiency of PIH in inhibiting ascorbate oxidation and ascorbyl radical formation was also inversely proportional to the Fe(III)–EDTA concentration in the media. When EDTA was replaced by the weaker iron ligand nitrilotriacetic acid (NTA), PIH was much more effective in preventing ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation. Moreover, the replacement of EDTA with citrate, a physiological chelator with a low affinity for iron, also resulted in PIH having a higher efficiency in inhibiting iron-mediated ascorbate oxidation and 2-deoxyribose degradation. These results demonstrate that PIH removes iron from EDTA (or from either NTA or citrate), forming an iron–PIH complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. These results are of pharmacological relevance because PIH has been considered for experimental chelating therapy in iron-overload diseases. [Copyright &y& Elsevier]

The biological activity of phenolic compounds ingested by caterpillars is commonly believed to result from their oxidation, although the products of oxidation have been well-characterized in only a few cases. The initial oxidation products of phenols (semiquinone or phenoxyl radicals) can be measured with electron paramagnetic resonance (EPR) spectrometry. In this study semiquinone radicals formed from tannic acid and gallic acid in the gut fluids of two species of caterpillars were measured. In Orgyia leucostigma, in which ingested phenols are not oxidized, semiquinone radicals were absent or at very low intensities. By contrast, in Malacosoma disstria, in which ingested phenols are oxidized, high semiquinone radical intensities were measured. In the absence of detectable levels of semiquinone radicals, ascorbyl radicals were detected in the EPR spectra instead. High molar ratios of ascorbate to phenols in an artificial diet produced ascorbyl radicals in the midgut fluids of both species, while diets containing low molar ratios produced semiquinone radicals. Similar results were obtained in M. disstria fed the leaves of red oak or sugar maple. The results of this study provide further evidence that ascorbate is an essential antioxidant that prevents the oxidation of phenols in the gut fluids of caterpillars, and demonstrate that EPR spectrometry is a valuable method for determining the degree of oxidative activation of phenols ingested by herbivorous insects. [Copyright &y& Elsevier]

Oxidative stress has been developed using dietary carbonyl-iron and iron-dextran parenteral administration as models of in vivo iron overload in rats. Carbonyl-iron led to a 2-fold increase in plasma iron content, a significant decrease (34%) in ascorbate plasma content and non-significant changes in plasma ascorbyl radical content. Iron-dextran produced a dramatic increase (6.7-fold) in plasma iron content, overwhelming the plasma total iron binding capacity. The ascorbyl radical content increased significantly in iron-dextran treatment (2.6-fold) and plasma ascorbate level was not affected. Ascorbyl radical/ascorbate ratio was significantly higher in both iron treated groups as compared with the control group (4×10−4±1×10−4). Data reported here indicate that the ascorbyl radical/ascorbate ratio is an appropriate in vivo indicator of oxidative stress under conditions of iron overload. The overall mechanism that describes the ascorbate status in plasma seems to be strongly dependent on the way the excess of iron is stored and thus, to the availability of the catalytically active iron for interacting with the plasma components. On this regard, evaluation of A⋅/AH− ratio did not help to discriminate between the possible involved mechanisms. [Copyright &y& Elsevier]

Incubation of MC-1010 cells with the spin-trapping agent 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) followed by brief treatment with the solid oxidant lead dioxide (PbO) yielded, after filtration, a cell-free solution that contained two nitroxyl adducts. The first was the hydroxyl radical adduct, 5,5-dimethyl-2-hydroxypyrrolidine-1-oxyl (DMPO-OH), which formed immediately upon PbO oxidation. The second had a 6-line EPR spectrum typical of a carbon-centered radical (A=15.9 G; A=22.4 G) and formed more slowly. No radical signals were detected in the absence of either cells or PbO treatment. The 6-line spectrum could be duplicated in model systems that contained ascorbate, DMPO and DMPO-OH, where the latter was formed from hydroxyl radicals generated by sonolysis or the cleavage of hydrogen peroxide with Fe (Fenton reaction). In addition, enrichment of MC-1010 cells with ascorbate prior to spin trapping yielded the 6-line EPR spectrum as the principal adduct following PbO oxidation and filtration. These results suggest that ascorbate reacted with DMPO-OH to form a carbon-centered ascorbyl radical that was subsequently trapped by DMPO. The requirement for mild oxidation to detect the hydroxyl radical adduct suggests that DMPO-OH formed in the cells was reduced to an EPR-silent form (i.e., the hydroxylamine derivative). Alternatively, the hydroxylamine derivative was the species initially formed. The evidence for endogenous hydroxyl radical formation in unstimulated leukocytes may be relevant to the leukemic nature of the MC-1010 cell line. The spin trapping of the ascorbyl radical is the first report of formation of the carbon-centered ascorbyl radical by means other than pulse radiolysis. Unless it is spin trapped, the carbon-centered ascorbyl radical immediately rearranges to the more stable oxygen-centered species that is passive to spin trapping and characterized by the well-known EPR doublet of A=1.8 G. [ABSTRACT FROM AUTHOR]

Ascorbic acid (AscH2) is one of the most important vitamins found in the human diet, with many biological functions including antioxidant, chelating, and coenzyme activities. Ascorbic acid is also widely used in medical practice especially for increasing iron absorption and as an adjuvant therapeutic in iron chelation therapy, but its mode of action and implications in iron metabolism and toxicity are not yet clear. In this study, we used UV–Vis spectrophotometry, NMR spectroscopy, and EPR spin trapping spectroscopy to investigate the antioxidant/pro-oxidant effects of ascorbic acid in reactions involving iron and the iron chelator deferiprone (L1). The experiments were carried out in a weak acidic (pH from 3 to 5) and neutral (pH 7.4) medium. Ascorbic acid exhibits predominantly pro-oxidant activity by reducing Fe3+ to Fe2+, followed by the formation of dehydroascorbic acid. As a result, ascorbic acid accelerates the redox cycle Fe3+ ↔ Fe2+ in the Fenton reaction, which leads to a significant increase in the yield of toxic hydroxyl radicals. The analysis of the experimental data suggests that despite a much lower stability constant of the iron–ascorbate complex compared to the FeL13 complex, ascorbic acid at high concentrations is able to substitute L1 in the FeL13 chelate complex resulting in the formation of mixed L12AscFe complex. This mixed chelate complex is redox stable at neutral pH = 7.4, but decomposes at pH = 4–5 during several minutes at sub-millimolar concentrations of ascorbic acid. The proposed mechanisms play a significant role in understanding the mechanism of action, pharmacological, therapeutic, and toxic effects of the interaction of ascorbic acid, iron, and L1. [ABSTRACT FROM AUTHOR]

Selenocysteine (Sec) is the 21st amino acid in the genetic code and it is present in a small number of proteins where it replaces the much more commonly used amino acid cysteine (Cys). It is both more complicated and bioenergetically costly to insert Sec into a protein in comparison to Cys, and this cost is most likely compensated by a gain of function to the enzyme/protein in which it is incorporated. Here we investigate one such gain of function, the enhancement of one-electron transfer catalysis. We compared the ability of Sec-containing mouse mitochondrial thioredoxin reductase (mTrxR2) to catalyze the reduction of bovine cytochrome c, ascorbyl radical, and dehydroascorbate in comparison to Cys-containing thioredoxin reductases from D. melanogaster (DmTrxR) and P. falciparum (PfTrxR). The Sec-containing mTrxR2 was able to reduce all three substrates, while the Cys-containing enzymes had little or no activity. In addition, we constructed Cys➔Sec mutants of DmTrxR and PfTrxR and found that this substitution resulted in a gain of function, as these mutant enzymes were now able to catalyze the reduction of these substrates. We also found that in the case of PfTrxR, reduction of cytochrome c was enhanced five-fold in a truncated PfTrxR in which the C-terminal redox center was removed. This shows that some of the ability of thioredoxin reductase to reduce this substrate comes from the flavin coenzyme. We also discuss a possible mechanism by which Sec-containing thioredoxin reductase reduces dehydroascorbate to ascorbate by two sequential, one-electron reductions, in part catalyzed by Sec., (© 2018 The Protein Society.)