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ABSTRACT: Nitric oxide (NO) generation in murine macrophages was determined in real time using the electron paramagnetic resonance (EPR) spin trapping method. An iron complex of N-methyl D-glucamine dithiocarbamate was utilized as the spin trap. This spin trapping compound reacts with NO in solution to form a specific room-temperature stable, mononitrosyl complex which is readily detected and identified by EPR spectroscopy. Mouse peritoneal macrophages were placed in an EPR sample-cell and activated by lipopolysaccharide and gamma-interferon at 37 degrees C, followed by an additional incubation in oxygenated medium without these activation agents. After various incubation periods, spin trap solution was infused to replace the medium in the sample-cell, and the time-evolution of the EPR signal of the spin adduct (NO-complex) was recorded. Rates of NO generation were calculated based upon the initial slopes of the increase in the EPR intensity with time. In comparison to the NO (or NO2-) generation rate obtained under similar experimental conditions using the Griess reaction assay, the spin trapping method was found to be more sensitive, with a lowest limit of the detection of 3 pmol/min. In addition, by using the spin trapping method, NO generation from the same cells could be measured consecutively during various stages of activation, because infusion of the spin trap solution did not affect the viability of macrophages.
Biochimica et Biophysica Acta 05/1996; 1289(3):362-8. · 4.66 Impact Factor
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ABSTRACT: The unique capabilities of EPR spin trapping of nitric oxide based on a ferrous-dithiocarbamate spin trap have been demonstrated in a study verifying the source of the nitrogen and oxygen atoms in nitric oxide produced from activated macrophages. Spin trapping experiments were performed during nitric oxide generation from activated mouse peritoneal macrophages using the ferrous complex of N-methyl D-glucamine dithiocarbamate as a spin trap. When 15N-substituted arginine was given to the activated macrophages in the presence of the spin trap, a characteristic EPR spectrum of the nitric oxide spin adduct was obtained, which indicates the presence of the 15N atom in the nitric oxide molecule. The hyperfine splitting (hfs) constant of the 15N nucleus was 17.6 gauss. When 17O-containing dioxygen (55%) was supplied to the medium, an EPR spectrum consistent with the 17O-substituted nitric oxide spin adduct was observed in the composite spectrum. The hfs of 17O was estimated to be 2.5 gauss. The 14NO spin adduct observed after prolonged incubation in the medium which contains [15N]L-arginine as the only extracellular source of arginine demonstrates that arginine is recycled through its metabolite in activated macrophages.
Free Radical Research 10/1995; 23(3):287-95. · 2.88 Impact Factor
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ABSTRACT: Polymorphonuclear leukocytes (PMNs) have been suggested to be damaged by superoxide radical generated on their own. The protective capacity of a spin trapping compound, phenyl-N-tert-butyl nitrone (PBN) was evaluated for this damage which occurs after the induction of superoxide generation. The life span of PMNs after superoxide generation was measured in the presence of PBN using the cell counting method, and effects of PBN on the amount of superoxide generated were quantitated using both cytochrome c reduction and spin trapping with DMPO. Results indicated significant extension of life span when PBN was present, and the extension was dose dependent. However, the magnitude of life span extension was not as large as expected from the decrease of superoxide generation. Possible mechanisms for the protection of PMNs by PBN are discussed.
Free Radical Research 08/1995; 23(1):73-80. · 2.88 Impact Factor
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ABSTRACT: Superoxide generation by polymorphonuclear leukocytes (PMNs) in suspension, or adherent to glass or plastic, after stimulation with N-formylmethionyl-leucyl-phenylalanine or phorbol myristate acetate was measured by cytochrome c reduction and spin trapping. Amounts of superoxide generated by adherent PMNs were inversely related to cell density. The generation of hydrogen peroxide was also inhibited at higher cell densities. In contrast to adherent cells, superoxide released by PMNs in suspension linearly increased with respect to cell number over a wider range. Microscopic observation indicated that the number of cells in mutual contact increased rapidly at cell densities higher than 4 x 10(4) cells/cm2, and inhibition of superoxide became apparent at higher cell densities. Mediators which could be released by PMNs, such as NO and adenosine, were not the cause of inhibition. These data suggest that mutual contact of PMNs suppresses their generation of superoxide. Survival rates of PMNs after stimulation increased at higher densities, indicating that the mutual contact-induced inhibition of superoxide generation by PMNs may be physiologically relevant at sites of inflammation.
Free Radical Research 05/1995; 22(4):361-73. · 2.88 Impact Factor
