Search

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. Fil: González, Paula Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Aguiar, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Malanga, Gabriela Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Puntarulo, Susana Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina

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 x 10(-4)+/-1 x 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.

Even though the aminopeptidase and amylase are the major liquefying factors in the liquefaction process of the ejaculated semen-coagulum, the exact nature of "switching on" phenomena has not been elucidated yet. Monitoring the rate of production of the asborbyl radical, an indirect method for oxyradical-stress measurement, this prima facie study reports the involvement of oxygen radical in the liquefaction process of coagulated semen.

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)x10(-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.

Catalytic transition metals are an absolute requirement for the aerobic oxidation of ascorbate monoanion. Thus, for example, the concentration of iron can be determined by the metal-dependent rate of ascorbate oxidation in near-neutral solutions. The lower limit of detection of iron, as Fe(III)EDTA, by monitoring the decrease in absorbance at 265 nm of ascorbate is about 200 nM. However, by measuring the concentration of the ascorbyl radical by ESR spectroscopy the lower limit is about 10 nM. Using these assays, I have shown that the typical microliter laboratory syringe can introduce significant iron into solutions. Thus, for studies involving iron, these two tests can be used to determine the amount of contaminating iron in reagents as well as iron from other sources such as laboratory equipment.

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.

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. D 2002 Elsevier Science B.V. All rights reserved.

We have detected and analyzed a free radical in human cerebrospinal fluid (CSF) of acute lymphoblastic leukemia (ALL) for the first time using electron paramagnetic resonance (EPR) at ambient temperature. We have also introduced an alternative capillary method to measure the radical. EPR spectra of the radical show a characteristic doublet with hyperfine coupling value of 1.8 G and g = 2.005. Based on EPR measurements, computer simulation, and literature values, we have determined that the species is ascorbyl radical (AsR). The radical has been investigated in CSF samples from ALL patients having no therapy, undergoing chemotherapy, and following therapy. Determination of the ascorbyl radical concentrations in CSF and serum was attempted using known concentrations of a nitroxyl radical. In addition, comparison in CSF and serum for ALL has been made along with statistical analyses of the data obtained. We found that AsR in CSF and serum has a strong correlation in patients undergoing chemotherapy (n = 57, r = 0.57, P0.0001). Ascorbate in CSF and serum show good correlation in patients having therapy but not for patients after therapy.

The interaction of ascorbate with different heme iron redox states of myoglobin (ferrylmyoglobin, FeIV=O; metmyoglobin, FeIII; and oxymyoglobin FeIIO2) was examined by e.s.r. and absorption spectroseopy. The reaction of ascorbate with ferryl- or met-myoglobin resulted in ascorbyl radical production. The interaction of ascorbate with oxymyoglobin proceeded with formation of ascorbyl radical, hydrogen peroxide, and an overall oxidation of oxymyoglobin to metmyoglobin. The latter reaction proceeded via an oxoferryl complex intermediate - corresponding to ferrylmyoglobin and identified by treatment of the reaction mixture with Na2S. These observations are consistent with a concerted electron transfer mechanism, whereby the two electrons required for the reduction of oxygen to hydrogen peroxide are donated by ascorbic acid and the heme iron. The antioxidant and prooxidant aspects of these redox transitions are discussed in terms of their kinetic properties.

The main contribution to ESR spectra of lyophilized tissues of plant or animal origin usually is given by an anisotropic singlet, which is known to be caused at least in the majority of cases by the adsorption of an ascorbyl radical. Model systems have been in vestigated in order to discuss experimental parameters, which in fluence these spectra qualitatively and quantitatively.