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ABSTRACT: Membrane-permeable, phenanthrene-derivedo-quinodimethanes of type 1 react with nitric oxide in a cheletropic fashion to produce fluorescent nitroxide radicals 2 and hydroxylamines 3. This method allows the sensitive and quantitative detection of nitric oxide production in biological samples by means of fluorescence microscopy, as is demonstrated by the monitoring of NO production from alveolar macrophages.
Chemistry 05/1999; 5(6):1738 - 1747. · 5.93 Impact Factor
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ABSTRACT: Organic amine-based buffer compounds such as HEPES (Good’s buffers) are commonly applied in experimental systems, including
those where the biological effects of peroxynitrite are studied. In such studies 3-morpholinosydnonimineN-ethylcarbamide (SIN-1), a compound that simultaneously releases nitric oxide (⋅NO) and superoxide (O⨪2), is often used as a source for peroxynitrite. Whereas in mere phosphate buffer H2O2 formation from 1.5 mmSIN-1 was low (∼15 μm), incubation of SIN-1 with Good’s buffer compounds resulted in continuous H2O2 formation. After 2 h of incubation of 1.5 mm SIN-1 with 20 mm HEPES about 190 μm H2O2 were formed. The same amount of H2O2 could be achieved from 1.5 mm SIN-1 by action of superoxide dismutase in the absence of HEPES. The increased H2O2 level, however, could not be related to a superoxide dismutase or to a NO scavenger activity of HEPES. On the other hand,
SIN-1-mediated oxidation of both dihydrorhodamine 123 and deoxyribose as well as peroxynitrite-dependent nitration ofp-hydroxyphenylacetic acid were strongly inhibited by 20 mm HEPES. Furthermore, the peroxynitrite scavenger tryptophan significantly reduced H2O2 formation from SIN-1-HEPES interactions. These observations suggest that peroxynitrite is the initiator for the enhanced
formation of H2O2. Likewise, authentic peroxynitrite (1 mm) also induced the formation of both O⨪2 and H2O2 upon addition to HEPES (400 mm)-containing solutions in a pH (4.5–7.5)-dependent manner. In accordance with previous reports it was found that at pH ≥5
oxygen is released in the decay of peroxynitrite. As a consequence, peroxynitrite(1 mm)-induced H2O2 formation (∼80 μm at pH 7.5) also occurred under hypoxic conditions. In the presence of bicarbonate/carbon dioxide (20 mm/5%) the production of H2O2 from the reaction of HEPES with peroxynitrite was even further stimulated. Addition of SIN-1 or authentic peroxynitrite to
solutions of Good’s buffers resulted in the formation of piperazine-derived radical cations as detected by ESR spectroscopy.
These findings suggest a mechanism for H2O2 formation in which peroxynitrite (or any strong oxidant derived from it) initially oxidizes the tertiary amine buffer compounds
in a one-electron step. Subsequent deprotonation and reaction of the intermediate α-amino alkyl radicals with molecular oxygen
leads to the formation of O⨪2, from which H2O2 is produced by dismutation. Hence, HEPES and similar organic buffers should be avoided in studies of oxidative compounds.
Furthermore, this mechanism of H2O2formation must be regarded to be a rather general one for biological systems where sufficiently strong oxidants may interact
with various biologically relevant amino-type molecules, such as ATP, creatine, or nucleic acids.
Journal of Biological Chemistry 05/1998; 273(21):12716-12724. · 4.77 Impact Factor
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ABSTRACT: We have previously demonstrated an energy-dependent injury to cultured liver endothelial cells during cold incubation in University of Wisconsin (UW) solution. In the present study, we report experimental evidence for the involvement of reactive oxygen species in this injury: LDH release during 48 h of cold incubation in UW solution was decreased from 40–55% under aerobic conditions to less than 20% under hypoxic conditions or by the presence of KCN (1 mM). Similar protection was achieved by the addition of the spin trap 5,5-dimethyl-1-pyrroline N-oxide, the hydroxyl radical scavenger dimethyl sulfoxide, or the flavonoid silibinin to UW solution under aerobic conditions. Preincubating the cells with the iron chelator deferoxamine even decreased the injury to less than 5%. The residual injury (as observed after longer incubation times) under hypoxic conditions or in cells preincubated with deferoxamine was no longer energy dependent. The amount of thiobarbituric acid-reactive substances markedly increased during cold incubation of the cells in UW solution. This increase was not observed in UW solution to which KCN had been added, i.e., under the conditions of energy depletion. These results suggest that an iron-dependent generation of reactive oxygen species with subsequent lipid peroxidation is involved in the pathogenesis of the injury to cultured liver endothelial cells in cold UW solution. Copyright © 1996 Elsevier Science Inc.
Free Radical Biology and Medicine 02/1997; · 5.42 Impact Factor
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ABSTRACT: In order to elucidate the potential of substituted o-quinodimethanes as reagents for the trapping of nitric oxide (NO) in biological systems, the reaction of alkoxyl- and alkyl-substituted 7,8-diphenyl- and 7,7,8-triphenyl-o-quinodimethanes with nitric oxide in solution was investigated by ESR spectroscopic and UV/vis stopped-flow techniques. Photolytic decarbonylation of 1,3-diphenyl- and 1,1,3-triphenylindan-2-ones gave the corresponding phenyl-substituted benzocyclobutenes as the major products and low photostationary concentrations of o-quinodimethanes. During 266-nm laser flash photolysis (LFP) of 1,3-dimethoxy-1,3-diphenylindan-2-one and 1-methoxy-1,3,3-triphenylindan-2-one in acetonitrile, species absorbing in the 400-600 nm range were produced, which were attributed to configurational isomers of the corresponding 7,7,8,8-substituted o-quinodimethanes. The isomeric o-quinodimethanes decayed at significantly different rates, indicating a strong influence of the relative orientation of the terminal substituents on their stability. Reaction of the raw photolysates of the 2-indanones with NO produced strong ESR spectra of the corresponding cyclic nitroxide radicals, isoindolin-2-oxyls. The nitroxide radicals were generated in a two-phase process, the first, rapid phase being attributed to the reaction of NO with the photolytically formed o-quinodimethanes and the second, slow phase reflecting the reaction with small amounts of o-quinodimethanes, generated by thermal ring opening of the phenyl-substituted benzocyclobutenes and probably a direct reaction of NO with the benzocyclobutenes. The kinetics of both steps, as evaluated by stopped-flow UV/vis and ESR spectroscopy, revealed a strong dependence of the rate constants of the o-quinodimethane + NO reaction on the substitution pattern of the o-quinodimethanes, with rate constants spanning a range of 10-4000 M(-)(1) s(-)(1). The rate constants ((0.4-7.5) x 10(-)(4) s(-)(1)) for the reaction of NO with the 7,7,8,8-tetrasubstituted benzocyclobutenes are much less influenced by the substitution pattern. The utility of phenyl-substituted benzocyclobutenes as "reservoirs" for o-quinodimethane-type nitric oxide traps is discussed.
The Journal of Organic Chemistry 11/1996; 61(20):6835-6848. · 4.45 Impact Factor
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ABSTRACT: Nitrogen dioxide (*NO2) is an oxidizing free radical which can initiate a variety of destructive pathways in living systems, and several diseases are suspected to be connected with both exogenously and endogenously formed *NO2. Peroxynitrite (ONOO-/ONOOH) is believed to be an important endogenous source of *NO2 radicals, but other sources, among them enzymatically ones, have been identified recently. It also became clear during the last few years that in vivo formation of 3-nitrotyrosine strictly depends on the availability of *NO2 radicals. Since nitrogen dioxide is a very toxic compound an arsenal of antioxidants (e.g. vitamin C, glutathione, vitamin E, and beta-carotene) must eliminate this harmful radical in vivo. Here the recently identified superoxide (O2*-)-dependent formation of peroxynitrate (O2NOO-) and the central role of vitamin C are of special importance.
Biological Chemistry 383(3-4):389-99. · 2.96 Impact Factor
