Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical.

It has been shown that BH(4) ameliorates endothelial dysfunction associated with conditions such as hypertension, cigarette smoking, and diabetes. This effect has been proposed to be due to a superoxide scavenging activity of BH(4). To examine this possibility we determined the rate constant for the reaction between BH(4) and superoxide using electron paramagnetic resonance (EPR) spin trapping competition experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). We calculated a rate constant for the reaction between BH(4) and superoxide of 3.9 +/- 0.2 x 10(5) M(-1)s(-1) at pH 7.4 and room temperature. This result suggests that superoxide scavenging by BH(4) is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH(2) and pterin are the stable products generated from the reaction. The formation of BH(4) cation radical (BH(4)(*+)) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH(4)(*+) is mainly delocalized on the pyrazine ring of BH(4). In parallel experiments, we investigated the effect of ascorbate on 7,8-BH(2) reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH(2) to BH(4), nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH(2). In conclusion, it is likely that BH(4)-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH(4) in the vasculature.

Mitochondrial aconitase is a source of hydroxyl radical. An electron spin resonance investigation.

Mitochondrial aconitase (m-aconitase) contains a [4Fe-4S](2+) cluster in its active site that catalyzes the stereospecific dehydration-rehydration of citrate to isocitrate in the Krebs cycle. It has been proposed that the [4Fe-4S](2+) aconitase is oxidized by superoxide, generating the inactive [3Fe-4S](1+) aconitase. In this reaction, the likely products are iron(II) and hydrogen peroxide. Consequently, the inactivation of m-aconitase by superoxide may increase the formation of hydroxyl radical ((*)OH) through the Fenton reaction in mitochondria. In this work, evidence for the generation of (*)OH from the reaction of m-aconitase with superoxide is provided using ESR spin trapping experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide and alpha-phenyl-N-tert-butylnitrone. Formation of free ( small middle dot)OH was verified with the (*)OH scavenger Me(2)SO, which forms methyl radical upon reacting with (*)OH. The addition of Me(2)SO to incubation mixtures containing m-aconitase and xanthine/xanthine oxidase yielded methyl radical, which was detected by ESR spin trapping. Methyl radical formation was further confirmed using [(13)C]Me(2)SO. Parallel low temperature ESR experiments demonstrated that the generation of the [3Fe-4S](1+) cluster increased with increasing additions of superoxide to m-aconitase. This reaction was reversible, as >90% of the initial aconitase activity was recovered upon treatment with glutathione and iron(II). This mechanism presents a scenario in which (*)OH may be continuously generated in the mitochondria.

Detection of superoxide anion using an isotopically labeled nitrone spin trap: potential biological applications.

We describe the synthesis and biological applications of a novel nitrogen-15-labeled nitrone spin trap, 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide ([(15)N]EMPO) for detecting superoxide anion. Superoxide anion generated in xanthine/xanthine oxidase (100 nM min(-1)) and NADPH/calcium-calmodulin/nitric oxide synthase systems was readily detected using EMPO, a nitrone analog of 5,5'-dimethyl-1-pyrroline N-oxide (DMPO). Unlike DMPO-superoxide adduct (DMPO-OOH), the superoxide adduct of EMPO (EMPO-OOH) does not spontaneously decay to the corresponding hydroxyl adduct, making spectral interpretation less confounding. Although the superoxide adduct of 5-(diethoxyphosphoryl)-5-methyl-pyrroline N-oxide is more persistent than EMPO-OOH, the electron spin resonance spectra of [(14)N]EMPO-OOH and [(15)N]EMPO-OOH are less complex and easier to interpret. Potential uses of [(15)N]EMPO in elucidating the mechanism of superoxide formation from nitric oxide synthases, and in ischemia/reperfusion injury are discussed.

Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase.

The binding of calcium/calmodulin stimulates electron transfer between the reductase and oxygenase domains of neuronal nitric oxide synthase (nNOS). Here, we demonstrate using electron spin resonance spin-trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide that pterin-free nNOS generates superoxide from the reductase and the oxygenase domain by a calcium/calmodulin-dependent mechanism. Tetrahydrobiopterin (BH(4)) diminishes the formation of superoxide by a mechanism that does not cause inhibition of NADPH consumption. In contrast, BH(4) analogs 7,8-dihydrobiopterin and sepiapterin do not affect superoxide yields. L-Arginine alone inhibits the generation of superoxide by nNOS but not by C331A-nNOS mutant that has a low affinity for L-arginine. A greater decrease in superoxide yields is observed when nNOS is preincubated with L-arginine. This effect is in accordance with the slow binding rates of L-arginine to NOS in the absence of BH(4). L-Arginine alone or in combination with BH(4) decreases the rates of NADPH consumption. The effect of L-arginine on superoxide yields, however, was less dramatic than that caused by BH(4) as much higher concentrations of L-arginine are necessary to attain the same inhibition. In combination, L-arginine and BH(4) inhibit the formation of superoxide generation and stimulate the formation of L-citrulline. We conclude that, in contrast to L-arginine, BH(4) does not inhibit the generation of superoxide by controlling electron transfer through the enzyme but by stimulating the formation of the heme-peroxo species.

Electron spin resonance spin-trapping detection of superoxide generated by neuronal nitric oxide synthase.

NOS is a ubiquitous enzyme that has an oxygenase and reductase activity. NOS reduces electron acceptors, at the reductase domain, by a one-electron mechanism that is not inhibited by SOD. One example of this activity is the direct reduction of ferricytochrome c by nNOS. Redox cycling electron acceptors (EA in Scheme 1), such as lucigenin and NBT, are reduced by NOS to generate an intermediate radical (EAred). This radical can then be reoxidized to the parent compound by oxygen, and in the process generate superoxide. Consequently, both NBT and lucigenin will enhance NADPH-dependent superoxide generation in the presence of flavoprotein reductases such as NOS. The artificial generation of superoxide from lucigenin and NBT is a major pitfall in the use of these compounds as superoxide probes. We conclude that the use of ESR spin-trapping techniques, although not free of problems, is a viable technique for the detection and quantification of superoxide in systems containing nNOS.

Effect of redox-active drugs on superoxide generation from nitric oxide synthases: biological and toxicological implications.

In this article, we address the mechanism of superoxide formation from constitutive nitric oxide synthases (NOS). Merits and drawbacks of the various superoxide detection assays are reviewed. One of the most viable techniques for measuring superoxide from NOS is electron spin resonance (ESR) spin-trapping using a novel phosphorylated spin trap. Implications of superoxide and peroxynitrite formation from NOS enzymes in cardiovascular and cerebrovascular disorders are discussed.

Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors.

The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of L-arginine, NG-hydroxy-L-arginine, L-thiocitrulline, and L-NG-monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.

Formation of spin trap adducts during the decomposition of peroxynitrite.

Peroxynitrite-mediated one-electron oxidations may be an important event in its cytotoxic mechanisms, and yet, free radical formation in the presence of peroxynitrite is difficult to study by EPR-spin trapping because adducts from most spin traps are destroyed by the oxidant. This led to some controversy with regard to the interpretation of experiments in the presence of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), an adequate spin trap to study most of free radicals. In this report we reexamined peroxynitrite-mediate formation of spin-trap adducts. Kinetic studies and EPR experiments with water labeled with 17O are in agreement with the reaction of DMPO with a highly reactive intermediate derived from peroxynitrite to produce the DMPO-hydroxyl radical adduct by a mechanism not involving the oxidation of DMPO to a cation radical followed by water addition. The results cannot discriminate between two mechanisms of DMPO-hydroxyl radical formation, either spontaneous peroxynitrite homolysis to the hydroxyl radical or DMPO-assisted peroxynitrite homolysis. The formation of DMPO adducts during peroxynitrite-mediated oxidation of dimethyl sulfoxide, ethanol, and formate occurs through free radical mechanisms as confirmed by studies of oxygen consumption and product formation. Accordingly, spin-trapping experiments in the presence of 3,5-dibromo-4-nitrosobenzenesulfonic acid, a spin trap that is more resistant to nitrogen dioxide, led to the detection of the methyl and the beta-hydroxyethyl radical during peroxynitrite-mediated oxidation of dimethyl sulfoxide and ethanol, respectively. Oxidation of these hydroxyl radical scavengers to detectable radicals favors the hypothesis that the hydroxyl radical is produced during peroxynitrite homolysis. Bicarbonate was able to modulate peroxynitrite-mediated one-electron oxidations.

Endothelial nitric oxide synthase-dependent superoxide generation from adriamycin.

Adriamycin (or doxorubicin) is an active and broad spectrum chemotherapeutic agent. Unfortunately, its clinical use is severely restricted by a dose-limiting cardiotoxicity which has been linked to the formation of superoxide. Enzymatic one-electron reduction of adriamycin forms adriamycin semiquinone radical, which rapidly reacts with oxygen to form superoxide and adriamycin. In this way, adriamycin provides a kinetic mechanism for the one-electron reduction of oxygen by flavoenzymes such as NADPH-cytochrome P450 reductase and mitochondrial NADH dehydrogenase. We demonstrate here that the endothelial isoform of nitric oxide synthase (eNOS) reduces adriamycin to the semiquinone radical. As a consequence, superoxide formation is enhanced and nitric oxide production is decreased. Adriamycin binds to eNOS with a Km of approximately 5 microM, as calculated from both eNOS-dependent NADPH consumption and superoxide generation. Adriamycin stimulated superoxide formation is not affected by calcium/calmodulin and is abolished by the flavoenzyme inhibitor, diphenyleneiodonium. This strongly suggests that adriamycin undergoes reduction at the reductase domain of eNOS. A consequence of eNOS-mediated reductive activation of adriamycin is the disruption of the balance between nitric oxide and superoxide. This may lead eNOS to generate peroxynitrite and hydrogen peroxide, potent oxidants implicated in several vascular pathologies.

Peroxynitrite-mediated decarboxylation of pyruvate to both carbon dioxide and carbon dioxide radical anion.

There has been a recent renewal of interest in the antioxidant properties of pyruvate which are usually attributed to its capacity to undergo oxidative decarboxylation in the presence of hydrogen peroxide. The interaction of pyruvate with other oxidizing biological intermediates, however, has been scarcely considered in the literature. Here we report that peroxynitrite, the oxidant produced by the reaction between superoxide anion and nitric oxide, reacts with pyruvate with an apparent second-order rate constant of 88 +/- 7 M-1 s-1 at pH 7.4 and 37 degrees C. Kinetic studies indicated that pyruvate reacts with peroxynitrite anion (k = 100 +/- 7 M-1 s-1, peroxynitrous acid (k = 49 +/- 7 M-1 s-1, and a highly oxidizing species derived from peroxynitrous acid. Pyruvate decarboxylation was proved by anion exchange chromatography detection of acetate in incubations of peroxynitrite and pyruvate at pH 7.4 and 5.5. Formation of carbon dioxide radical anion was ascertained by EPR spin-trapping studies in the presence of GSH and the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The use of pyruvate labeled with 13C at the 1-position led to the detection of the labeled DMPO carbon dioxide radical anion adduct. In the absence of GSH, oxygen consumption studies confirmed that peroxynitrite mediates the decarboxylation of pyruvate to free radical intermediates. Comparing the yields of acetate and free radicals estimated from the oxygen uptake studies, it is concluded that pyruvate is oxidized by both one- and two-electron oxidation pathways, the latter being preponderant. Hydrogen peroxide-mediated pyruvate oxidation does not produce detectable levels of carbon dioxide radical anion except in the presence of iron(II)-ethylenediamine-N,N,N',N'-tetraacetate (EDTA). The apparent second-order rate constant of the reaction between pyruvate and hydrogen peroxide was determined to be 1 order of magnitude lower than that of the reaction between pyruvate and peroxynitrite. The latter process may contribute to the antioxidant properties of pyruvate.

Superoxide anion formation from lucigenin: an electron spin resonance spin-trapping study.

Lucigenin (LC2+) is frequently used as a superoxide probe. To detect superoxide, lucigenin must be reduced to the lucigenin cation radical (LC.+). We show, using the phosphorylated spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), that lucigenin stimulates NADPH-dependent superoxide production by endothelial nitric oxide synthase (eNOS). The formation of the DEPMPO-superoxide adduct is calcium/calmodulin independent. DEPMPO-superoxide adduct formation is inhibited by diphenyleneiodonium and is abolished by superoxide dismutase. It is likely that eNOS/NADPH can reduce lucigenin to LC.+ which reduces oxygen to superoxide. Consequently, lucigenin cannot be used to measure superoxide formation.

Peroxynitrite-mediated formation of free radicals in human plasma: EPR detection of ascorbyl, albumin-thiyl and uric acid-derived free radicals.

Formation of peroxynitrite by the fast reaction between nitric oxide and superoxide anion may represent a critical control point in cells producing both species, leading to either down-regulation of the physiological effects of superoxide anion and nitric oxide by forming an inert product, nitrate, or to potentiation of their toxic effects by oxidation of nearby molecules by peroxynitrite. (The term peroxynitrite is used to refer to the sum of all possible forms of peroxynitrite anion and peroxynitrous acid unless otherwise specified.) In this report we demonstrate that, in spite of all the antioxidant defences present in human plasma, its interaction with peroxynitrite leads to generation of free radical intermediates such as (i) the ascorbyl radical, detected by direct EPR, (ii) the albumin-thiyl radical, detected by spin-trapping experiments with both N-tert-butyl-alpha-phenylnitrone and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and (iii) a uric acid-derived free radical, detected as the DMPO radical adduct in plasma whose thiol groups were previously blocked with 5,5-dithiobis-(2-nitrobenzoic acid). The identity of the latter adduct was confirmed by parallel experiments demonstrating that it is not detectable in plasma pretreated with uricase, whereas it is formed in incubations of peroxynitrite with uric acid. Peroxynitrite-mediated oxidations were also followed by oxygen consumption and ascorbate and plasma-thiol depletion. Our results support the view that peroxynitrite-mediated one-electron oxidation of biomolecules may be an important event in its cytotoxic mechanism. In addition, the data have methodological implications by providing support for the use of EPR methodologies for monitoring both free radical reactions and ascorbate concentrations in biological fluids.

Oxidative activity of primaquine metabolites on rat erythrocytes in vitro and in vivo.

The oxidative activities of primaquine [6-methoxy-8-(4-amino-1-methylbutylamino)quinoline] and its metabolites, the quinone-imine derivatives of 5-hydroxyprimaquine [5-hydroxy-6-methoxy-8-(4-amino-1-methylbutylamino)quinoline] and 5-hydroxydemethylprimaquine [5-hydroxy-6-demethyl-8-(4-amino-1-methylbutylamino)quinoline], 6-methoxy-8-amino quinoline and hydrogen peroxide, were studied on rat erythrocytes in vitro and in vivo. In both cases, the most effective metabolites in oxidizing hemoglobin and depleting non-protein sulfhydryl groups from erythrocytes were the quinone-imine derivatives of the ring-hydroxylated metabolites, 5-hydroxyprimaquine and 5-hydroxydemethyl-primaquine. The latter quinone-imines were shown by light absorption spectroscopy and oxygen consumption studies to be able to oxidize purified rat hemoglobin to methemoglobin but to be unable to react directly with reduced glutathione. In agreement with these results, no radical adduct was detected by electron paramagnetic resonance spectroscopy in incubations of rat erythrocytes with the quinone-imines and the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide; metabolite-derived free radicals were detected instead. Taken together, the results suggest that 5-hydroxyprimaquine and 5-hydroxydemethylprimaquine are important metabolites in the expression of primaquine hemotoxicity, in contrast to 6-methoxy-8-aminoquinoline. Additionally, the results indicate that hydrogen peroxide is the ultimate oxidant formed from the ring-hydroxylated metabolites by redox-cycling of the corresponding quinone-imine derivatives both in vitro and in vivo.

Free radicals and excited species in the metabolism of indole-3-acetic acid and its ethyl ester by horseradish peroxidase and by neutrophils.

The peroxidative metabolization of indole-3-acetic acid, a biologically important process, has been followed by EPR spectroscopy with the aim of obtaining information on the mechanism of generation of electronically excited species. The skatole-3-methylene radical detected during oxidation by horseradish peroxidase, does not appear to be involved in a major oxygen consuming process or in the generation of singlet oxygen. The chemiluminescence spectrum exhibits several maxima, which are also observed when the ethyl ester of indole-3-acetic acid is metabolized by horseradish peroxidase or by myeloperoxidase in neutrophils. When the ester is metabolically activated in either of these systems, the EPR spectrum indicates a tertiary carbon-centered radical. This radical centered on the carbon in the 3-position participates in a chemiexcitation/emissive route. Within the cell, this emissive process is responsible for a large part of the oxygen consumed. Some of the emitters originate in the cleavage of the 2,3 double bond. The ester, which is capable of penetrating into the cells, also emits with other myeloperoxidase-containing cells. This compound may have useful applications as an intracellular chemiluminescent probe for the presence of myeloperoxidase.

Hydroxylated metabolites of the antimalarial drug primaquine. Oxidation and redox cycling.

Oxidation and redox cycling of the hydroxylated metabolites of the antimalarial drug primaquine (i.e. 5-hydroxyprimaquine, 5-hydroxydemethylprimaquine, and 5,6-dihydroxy-8-aminoquinoline) were studied. The three metabolites readily oxidized under physiological conditions, forming hydrogen peroxide and the corresponding quinone-imine derivatives as the main products. The latter compounds were characterized by visible, NMR, and infrared spectroscopy. Concomitant formation of drug-derived radicals and hydroxyl radicals was attested by direct and spin-trapping EPR experiments, respectively. The use of the spin stabilization method indicated that the radicals derived from 5-hydroxydemethylprimaquine and 5,6-dihydroxy-8-aminoquinoline are of the o-semiquinone type. Tentative structures are proposed for the radicals based on product identification and computer simulation of the experimental EPR spectra. The quinone-imines obtained from the reduced metabolites did not react at appreciable rates with NADPH but underwent redox cycling upon addition of ferredoxin:NADP+ oxidoreductase, forming hydrogen peroxide and hydroxyl radicals. The effect of antioxidant enzymes on hydroxyl radical yield obtained during oxidation and redox cycling indicates that the main route for hydroxyl radical formation is the metal ion-catalyzed reaction between the drug-derived radicals and hydrogen peroxide. Taken together, the results indicate that hydrogen peroxide is the potential toxic product formed from the primaquine metabolites.

ESR detection of free radical intermediates during autoxidation of 5-hydroxyprimaquine.

Autoxidation of 5-hydroxyprimaquine, a putative metabolite of the antimalarial primaquine, was studied by oxygen consumption and ESR spectroscopy. 5-Hydroxyprimaquine underwent fast autoxidation under mild conditions (pH 7.4-8.5, 25 degrees C, and presence of 1 mM diethylenetriamine pentaacetic acid); each mol of the drug consumed 0.75 mol of oxygen and formed 0.5 mol of hydrogen peroxide. Direct-ESR experiments demonstrated that 5-hydroxyprimaquine autoxidation was accompanied by generation of a drug-derived free radical that is oxygen sensitive. Generation of hydroxyl radical was also established by spin-trapping experiments in the presence of 5,5-dimethyl-1-pyrroline N-oxide. The effect of antioxidant enzymes on hydroxyl radical adduct yield and analysis of autoxidation stoichiometry suggest that the main route for hydroxyl radical generation is the iron-catalyzed reaction between the drug-derived free radical and hydrogen peroxide.