Tetrahydrobiopterin (H4B) in the absence of l-arginine has been shown to be an important factor in promoting the direct formation of hydrogen peroxide (H2O2) at the expense of superoxide (O2−) by neuronal nitric oxide synthase (NOS1) [Rosen GM, Tsai P, Weaver J, Porasuphatana S, Roman LJ, Starkov AA, et al. Role of tetrahydrobiopterin in the regulation of neuronal nitric-oxide synthase-generated superoxide. J Biol Chem 2002;277:40275–80]. Based on these findings, it is hypothesized that l-arginine also shifts the equilibrium between O2− and H2O2. Experiments were designed to test this theory. As the concentration of l-arginine and Nω-hydroxyl-l-arginine increases, the rate of NADPH consumption for H4B-bound NOS1 decreased resulting in lower rates of both O2− and H2O2 generation, while increasing the rate of nitric oxide (NO) production. At saturating concentrations of l-arginine or Nω-hydroxyl-l-arginine (50μM), NOS1 still produced O2− and H2O2. Both l-arginine and Nω-hydroxyl-l-arginine have greater impact on the rate of generation of O2− than on H2O2.

[reaction: see text] Ester-containing nitrones, including 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide 5, have been reported to be robust spin traps for superoxide (O2*-). Using a chiral column, we have been able to isolate the two enantiomers of nitrone 5. With enantiomerically pure nitrone 5a and 5b we explored whether one of these isomers was solely responsible for the EPR spectrum of aminoxyl 6. Data obtained demonstrate that the spin trapping of O2*- by nitrone 5a and nitrone 5b affords the identical EPR spectra and lifetimes in homogeneous aqueous solution and exhibits the same ratio of cis and trans isomers. Quantum chemical modeling in vacuo also finds no difference, aside from the expected optical activity, arising from the difference in stereochemistry.

Nitric oxide synthases (NOS) independent of the isozyme, produce nitric oxide (.NO), superoxide (O2.-), and hydrogen peroxide (H2O2). Since .NO has been implicated in many physiological processes, the importance of O2.- and H2O2 in regulating cell signaling by .NO cannot be overlooked. Before addressing these questions, we investigated the production of .NO, O2.-, and H2O2 by purified NOS. NOS 1 and NOS 2 were chosen, as the flux of .NO from each isozyme supports differential biological activity. We found that the initial rate and sustained production of .NO was considerably greater for NOS 2 as compared to NOS 1. In the absence of L-arginine, however, NOS 1 generation of O2.- and H2O2 was found to be substantially greater than that measured for NOS 2. Differences between NOS 1 and NOS 2 production of .NO, O2.-, and H2O2 may define the specific physiologic function of each isozyme.

Spin trapping, a technique used to characterize short-lived free radicals, consists of using a nitrone or nitroso compound to "trap" an unstable free radical as a long-lived aminoxyl that can be characterized by EPR spectroscopy. The resultant aminoxyl exhibits hyperfine splitting constants that are dependent on the spin trap and the free radical. Such is the case with 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO-OH) and 2,2-dimethyl-5-hydroperoxy-1-pyrrodinyloxyl (DMPO-OOH) whose hyperfine splitting constants, A(N) = A(H) = 14.9 G and A(N) = 14.3 G, A(H)(beta) = 11.7 G, and A(H)(gamma) = 1.25 G, respectively, have been used to demonstrate the generation of HO(*) and O(2)(*)(-). However, to date, the source of the apparent A(H)(gamma) hyperfine splitting in DMPO-OOH is not known. We consider three possible explanations to account for the unique EPR spectrum of DMPO-OOH. The first is that the gamma-splitting arises from one of the hydrogen atoms at either carbon 3 or carbon 4 of DMPO-OOH. The second is that the gamma-splitting originates from the hydrogen atom of DMPO-OOH. The third is that the conformational properties of DMPO-R change upon going from DMPO-OH to DMPO-OOH. Experimental and theoretical chemical approaches as well as EPR spectral modeling were used to investigate which of these hypotheses may explain the asymmetric EPR spectrum of DMPO-OOH. From these studies it is shown that the 12-line EPR spectrum of DMPO-OOH results not from any proximal hydrogen, but from additional conformers of DMPO-OOH. Thus, the 1.25 G hyperfine splitting, which has been assigned as a gamma-splitting, is actually from two individual EPR spectra associated with different conformers of DMPO-OOH.

Apparent rate constants, at acidic pH and neutral pH for the reaction of a family of ester-containing 5-carboxyl-5-methyl-1-pyrroline N-oxides with superoxide (O-2(.-)) were estimated, using ferricytochrome c as a competitive inhibitor. It was of interest to note that the rate constants were similar among the different nitrones and not that significantly different from that found for 5-(diethoxyphosphoryl)-5-dimethyl-1-pyrroline N-oxide. At acidic pH, the rate constant for spin trapping O-2(.-) was 3-fold greater than that at physiological pH. Subsequent experiments determined the half-life of aminoxyls, derived from the reaction of these nitrones with O-2(.-). The EPR spectra were modeled by using a global analysis method. The results clearly demonstrated that EPR spectra of all the aminoxyls were inconsistent with a model that included a single gamma-hydrogen splitting. A better interpretation modeled them as two diastereomers with identical nitrogen splittings and slightly different beta-hydrogen splittings. Detailed line width analyses slightly favored an equal line width-unequal population ratio for the two diastereomers.

Tetrahydrobiopterin (H4B) is a critical element in the nitric-oxide synthase (NOS) metabolism Of L-arginine to L-citrulline and NO.. It has been hypothesized that in the absence of or under nonsaturating levels Of L-arginine where O-2 reduction is the primary outcome of NOS activation, H4B promotes the generation of H2O2 at the expense of O-2(.-). The experiments were designed to test this hypothesis. To test this theory, two different enzyme preparations, H4B-bound NOS I and H4B-free NOS I, were used. Initial rates of NADPH turnover and O-2 utilization were found to be considerably greater in the H4B-bound NOS I preparation than in the H4B-free NOS I preparation. In contrast, the initial generation of O-2(.-) from the H4B-free NOS I preparation was found to be substantially greater than that measured using the H4B-bound NOS I preparation. Finally, by spin trapping nearly all of the NOS I produced O-2(.-) we found that the initial rate of H2O2 production by H4B-bound NOS I was considerably greater than that for H4B-free NOS I.

Neuronal nitric oxide synthase (NOS I) has been shown to generate nitric oxide (NO⋅) and superoxide (O2⋅−) during enzymatic cycling, the ratio of each free radical is dependent upon the concentration of l-arginine. Using spin trapping and electron paramagnetic resonance (EPR) spectroscopy, we recently reported that NOS I can oxidize ethanol (EtOH) to α-hydroxyethyl radical (CH3⋅CHOH). We speculated that the perferryl complex of NOS, (NOS–[Fe5+O]3+) was responsible for the generation of CH3⋅CHOH. Using potassium monopersulfate (KHSO5) to oxidize the heme of NOS I to NOS–[Fe5+O]3+, we were able to demonstrate that this perferryl complex can oxidize l-arginine to l-citrulline and NO⋅. Even in the absence of l-arginine, EtOH was oxidized to CH3⋅CHOH by NOS–[Fe5+O]3+. Sodium cyanide (NaCN), a heme blocker, inhibited the formation of CH3⋅CHOH by NOS.

Four conclusions derive from the studies exploring NOS-generated O⨪2 and the kinetic data outlined here. 1) When sufficientl-arginine and H4B are present, NOS dimers secrete small amounts of O⨪2 or H2O2and instead couple their heme and O2 reduction to NO⋅ synthesis. 2) Significant O⨪2 production may occur when concentrations of H4B (≤2μm) orl-arginine (≤100μm) fall below levels required to saturate the enzyme. In these circumstances, O⨪2forms by heme-catalyzed O2 reduction. 3) NOS reductase domain flavins are protected from autooxidation and do not secrete large amounts of O⨪2 unless certain redox-active xenobiotics are present. These cause O⨪2 production by catalyzing electron transfer from the NOS reductase domain to O2. One recent example suggests that NOS-derived O⨪2 participates in tissue injury associated with the xenobiotic (54Day B.J. Patel M. Calavetta L. Chang L.-Y. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12760-12765Crossref PubMed Scopus (169) Google Scholar). 4) NOS may generate both NO⋅ and O⨪2 when concentrations ofl-arginine or H4B are low (41Xia Y. Roman L.J. Masters B.S.S. Zweier J.L. J. Biol. Chem. 1998; 273: 22635-22639Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 42Vásquez-Vivar J. Kalyanaraman B. Martasek P. Hogg N. Masters B.S. Karoui H. Tordo P. Pritchard K.A.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1242) Google Scholar, 43Xia Y. Tsai A.-L. Berka V. Zweier J.L. J. Biol. Chem. 1998; 273: 25804-25808Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 44Pou S. Keaton L. Surichamorn W. Rosen G.M. J. Biol. Chem. 1999; 274: 9573-9580Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 45Vásquez-Vivar J. Hogg N. Martásek P. Karoui H. Pritchard Jr., K.A. Kalyanaraman B. J. Biol. Chem. 1999; 274: 26736-26742Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 51Klatt P. Heinzel B. John M. Kastner M. Bohme E. Mayer B. J. Biol. Chem. 1992; 267: 11374-11378Abstract Full Text PDF PubMed Google Scholar, 59Culcasi M. Lafon-Cazal M. Pietri S. Bockaert J. J. Biol. Chem. 1994; 269: 12589-12593Abstract Full Text PDF PubMed Google Scholar). When the steady-state flux of O⨪2 was high there was evidence for ONOO− formation (57Xia Y. Dawson V.L. Dawson T.M. Snyder S.H. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6770-6774Crossref PubMed Scopus (657) Google Scholar, 58Xia Y. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6954-6958Crossref PubMed Scopus (610) Google Scholar). Indeed, certain pathologic states might promote formation of ONOO− such as ischemia/reperfusion injury (67Grisham M.B. Granger D.N. Lefer D.J. Free Radic. Biol. Med. 1998; 25: 404-433Crossref PubMed Scopus (251) Google Scholar). Formation of HO⋅, either through metal ion-catalyzed H2O2 decomposition (68Haber F. Weiss J. Proc. R. Soc. Lond. A. 1934; 147: 332-351Crossref Google Scholar) or from decomposition of ONOO− (69van der Vliet A. O'Neill C.H. Halliwell B. Cross C.E. Kaur H. FEBS Lett. 1994; 339: 89-92Crossref PubMed Scopus (361) Google Scholar, 70Pou S. Nguyen S.Y. Gladwell T. Rosen G.M. Biochim. Biophys. Acta. 1995; 1244: 62-68Crossref PubMed Scopus (84) Google Scholar, 71Richeson C.E. Mulder P. Bowry V.W. Ingold K.U. J. Am. Chem. Soc. 1998; 120: 7211-7219Crossref Scopus (152) Google Scholar), at sensitive cellular sites may also contribute to cytotoxicity. Finally, it is worth noting that sequential formation of NO⋅ and O⨪2can result in differing cell signaling pathways (15Lane P. Gross S.S. Semin. Nephrol. 1999; 19: 215-229PubMed Google Scholar, 16Finkel T. Curr. Opin. Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (1019) Google Scholar), few of which have been well defined. Therefore, under different conditions a variety of oxidants may derive from NOS that can impact cell function in ways that are significant.

One of the difficult tasks confronting the study of free radicals in biology is the inability to measure “on line” injury to a biological target, while characterizing the reactive species responsible for the toxic event. This is particularly relevant in light of the fact that specific free radicals play a critical role in host immune response. An approach towards addressing this important issue draws upon the unique EPR spectral properties of 14N/15N-labeled compounds. In particular, Neisseria gonorrhoeae has been covalently labeled with 15N-deuterium17-containing 4-maleimido-2,2,6,6-tetramethylpiperidin-1-yloxyl (15N-D17-4-MAL-TEMPO). The EPR spectrum from bacteria so labeled exhibited two low-field peaks: (a) a broad, strongly immobilized species classified as “S”; (b) a more narrow, weakly immobilized component termed “W”. The W/S ratio is an indicator of changes in membrane organization. In the presence of the superoxide-generating system, hypoxanthine/xanthine oxidase, an increase in the W/S ratio from 3.3 for control to 6.4 was observed, which was only partially inhibited by superoxide dismutase (W/S ratio of 4.4). When the spin trap 5,5-dimethyl-1-pyrroline 14N-oxide (DMPO) was included in the above reaction mixture, an EPR spectrum was recorded, which was a composite of 2,2,-dimethyl-5-hydroperoxypyrrolidin-1-yl-14N-oxyl (DMPO-OOH) and 15N-D17-4-MAL-TEMPO-labeled Neisseria gonorrhoeae. With the use of computer subtraction procedures, the W/S ratio was found to be 6.4. The experiments demonstrate the utility of 14N/15N-labeled aminoxyls as a valuable tool in accessing the effects of specific free radicals on the fluidity of cell membranes.

The ideal spin trap for the in vivo in situ detection of HO˙ is one that reacts specifically with this free radical at near diffusion controlled rates. Further, the corresponding spin trapped adducts must be sufficiently long-lived to allow the acquisition of EPR spectra for prolonged periods of time. Herein, we report on our studies in which we explored the ability of various imidazole N-oxides, an isoquinoline N-oxide and pyrroline N-oxides to specifically react with HO˙ at the expense of O2˙–. Further, we estimated the rate constant for spin trapping HO˙. Finally, we measured the stability of the corresponding spin trapped adducts. Our data suggest that imidazole N-oxides, in particular 2,2-dimethyl-4-methoxycarbonyl-2H-imidazole 1-oxide (3), appear to be the best spin traps for the in vivo in situ detection of HO˙ in real time.

Biologically generated nitric oxide appears to play a pivotal role in the control of a diverse series of physiologic functions. Iron-chelates and low-frequency EPR spectroscopy have been used to verify in vivo production of nitric oxide. The interpretation of in vivo identification of nitric oxide localized at the site of evolution in real time is complicated by the varied kinetics of secretion. The quantitative efficiency of the spectroscopic measurement, so important in understanding the physiology of nitric oxide, remains elusive. The development of a more stable iron-chelate will help better define nitric oxide physiology. In this report, we present data comparing the commonly used ferro-di(N-methyl-d-glucamine-dithiocarbamate) (Fe2+(MGD)2) and the novel chelate ferro-di(N-(dithiocarboxy)sarcosine) (Fe2+(DTCS)2) quantifying the in vitro and in vivo stability of the corresponding spin trapped adducts, NO-Fe(MGD)2 and NO-Fe(DTCS)2. Finally, very low frequency EPR spectroscopy has been used to evaluate the pharmacokinetics of NO–Fe(MGD)2 and NO–Fe(DTCS)2 in mice in real time.

Neuronal nitric-oxide synthase (NOS I) in the absence of L-arginine has previously been shown to generate superoxide (O-2(radicalanion)) (Pou, S., Pou, W. S., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992) J. Biol. Chem. 267, 24173-24176). In the presence of L-arginine, NOS I produces nitric oxide (NO.). Yet the competition between O-2 and L-arginine for electrons, and by implication formation of O-2(radicalanion), has until recently remained undefined. Herein, we investigated this relationship, observing O-2(radicalanion) generation even at saturating levels of L-arginine. Of interest was the finding that the frequently used NOS inhibitor N-G-monomethyl L-arginine enhanced O-2(radicalanion) production in the presence of L-arginine because this antagonist attenuated NO. formation. Whereas diphenyliodonium chloride inhibited O-2(radicalanion), blockers of heme such as NaCN, 1-phenylimidazole, and imidazole likewise prevented the formation of O-2(radicalanion) at concentrations that inhibited NO. formation from L-arginine, Taken together these data demonstrate that NOS I generates O-2(radicalanion) and the formation of this free radical occurs at the heme domain.

The free radical intermediates formed during the nitrosation of thiols including glutathione (GSH), L-cysteine (CystSH), captopril (CapSH) and N-acetylpenicillamine (NAPenSH) by NO˙ at physiological pH have been investigated using EPR spectroscopy combined with the spin trap DMPO. We have found that NO˙ in the presence of oxygen reacted with GSH, CystSH and CapSH to generate the corresponding thiyl radicals, which are spin trapped by DMPO to give the spin trapped adducts DMPO-SG, DMPO-SCyst and DMPO-SCap respectively. In the case of NAPenSH no spin trapped adduct is detected. Desferrioxamine, an inhibitor of peroxynitrite (ONOO–), has no effect on the formation of either DMPO-SG or DMPO-SCyst, suggesting that ONOO– is not involved in the formation of GS˙ and Cyst˙. In contrast, desferrioxamine inhibits the detection of DMPO-SCap, pointing to a role for ONOO– in the formation of CapS˙. Our results demonstrate that ˙NO2 is the most likely reactive species in the nitrosation of GSH and CystSH by oxygenated NO˙, thereby generating the corresponding thiyl radicals. Furthermore, our data indicate that the scavenging of NO˙ by thiols may lead to the production of superoxide (O2˙–) via the formation of thiyl radicals.

During his studies on the properties of oxygen, Priestley () noted that this gas, an essential ingredient for life processes, appears to "burn out the candle of life too quickly." More than two centuries would elapse, however, before this observation would be associated with Grubbé's () accounts of redness and irritation on the hands of his workers testing X-ray tubes. By 1954, Gerschman et al. () suggested that free radicals were the common element linking the observed toxicity of oxygen to the harmful effects of ionizing radiation. The implication of this hypothesis seemed remote at that time. However, within a decade, the search for biologically generated free radicals would lead to the discovery of superoxide and an enzyme that attenuated cellular levels of this free radical (,). In the intervening years, free radicals have been recognized as common intermediates in cellular metabolism (,), found to play an essential role in host immune response () and demonstrated to regulate many essential physiologic functions ().

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIssues Pertinent to the in Vivo in Situ Spin Trapping of Free Radicals§Sovitj Pou, Howard J. Halpern, Pei Tsai, and Gerald M. RosenView Author Information Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, and Department of Radiation and Cellular Oncology, The University of Chicago, Chicago, Illinois 60637 Cite this: Acc. Chem. Res. 1999, 32, 2, 155–161Publication Date (Web):November 4, 1998Publication History Received26 February 1998Published online4 November 1998Published inissue 1 February 1999https://pubs.acs.org/doi/10.1021/ar970251lhttps://doi.org/10.1021/ar970251lresearch-articleACS PublicationsCopyright © 1999 American Chemical SocietyRequest reuse permissionsArticle Views281Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Electron paramagnetic resonance spectroscopy,Free radicals,Nitrogen compounds,Oxides,Quantum mechanics Get e-Alerts

Neutrophils release elastase, which is known secondarily to cause tissue damage. However, it is rapidly inactivated by the endogenous alpha(1)-proteinase inhibitor (alpha(1)Pi). Nevertheless, under pathological conditions, alpha(1)Pi is inactivated by oxidants released from neutrophils, resulting in an excess of elastase at the site of inflammation, This elastase/alpha(1)Pi imbalance has been implicated as a pathogenic factor in cystic fibrosis, acute respiratory distress syndrome, and emphysema, Elastase inhibitors, which do not interfere with the microbicidal activity of neutrophils and are resistant to neutrophil-released oxidants, would undoubtedly represent an important advance in the management of neutrophil-mediated tissue injury. We report that a new family of elastase inhibitors ICI200355 and ZD0892 Tvas found to be resistant toward superoxide, hypochlorous acid, hydrogen peroxide, hydroxyl radical, and peroxynitrite mediated degradation as well as having no effect on the formation of these oxidants by activated neutrophils, More importantly, we found that these inhibitors did not interfere with the ability of hunan neutrophils to phagocytose and to kill Staphylococcus aureus. In conclusion, a new potent class of elastase inhibitors, while blocking the effects of neutrophil elastase, was found not to impede various physiological functions of human neutrophils, in particular the ability of these phagocytic cells to phagocytose and kill bacteria.

Phagocytes mediate their innate immunological response by releasing products that damage invading microorganisms. These products include proteins such as lysozyme, peroxidases, and elastase as well as reactive oxygen species such as superoxide, hydrogen peroxide, hypohalous acid, and hydroxyl radical. Although it is clear that many phagocytic secretory products have direct cytotoxic potential, understanding is limited of how multiple products interact to generate and modulate the cytotoxic response. This review focuses on recent findings that elucidate the biochemical nature of secretory product interaction in the formation of free radicals, particularly the highly reactive hydroxyl radical. The possible role of these reactions in phagocyte microbicidal activity and inflammatory tissue injury is discussed.

Central to the study of free radical processes is the ability to identify and localize their cellular site of formation. Under the best of experimental conditions, spin trapping/ESR spectroscopy can only characterize intracellular production of specific free radicals and confocal microscopy can only localize the site of their formation. In this article, we report on the development of a fluorophore-containing nitrone, alpha-[4-[5-((2-carboxy)phenyl)-5-hydroxy-4-oxo-3-phenyl)-2-pyrrolin-1-yl]phenyl]-N-(tert-butyl)nitrone sodium salt (4).(3) This nitrone (4) reacts with alpha-hydroxyethyl radical with a second order rate constant of 1.7 x 10(5) M(-1) s(-1) to give a characteristic ESR spectrum. However, we were unable to decrease the fluorescence emission, due in part to the small concentration of nitroxide generated from the reaction of alpha-hydroxyethyl radical with nitrone (4). Using the fluorophore-containing nitroxide (7) as a model, we found that only 12% of the nitroxide needs to be reduced to give an almost 400% increase in the fluorescent emission of (7). Our findings suggest new approaches to the development of various fluorophore-containing nitrones that can both characterize specific free radicals and localize their site of intracellular formation.