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X-band and S-band EPR detection of nitric oxide in murine endotoxaemia using spin trapping by ferro-di(N-(dithiocarboxy)sarcosine).

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Plonka P. M., Wisniewsk M., Chlopicki S., Elas M., Rosen G. M.
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2003
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vol. 50
|
issue 3
799-806
EN
Ammonium salt of N-(dithiocarboxy)sarcosine (DTCS) chelated to ferrous salt was tested as an NO-metric spin trap at room temperature for ex vivo measurement of g·NO production in murine endotoxaemia. In a chemically defined in vitro model system EPR triplet signals of NO-Fe(DTCS)g2 were observed for as long as 3 hours, only if samples were reduced with sodium dithionite. This procedure was not necessary for the ex vivo detection of ·NO in endotoxaemic liver homogenates at X-band or in the whole intact organs at S-band, whereas only a weak signal was observed in endotoxaemic lung. These results suggest that in endotoxaemia not only high level of ·NO, but also the redox properties of liver and lung might determine the formation of complexes of ·NO with a spin trap. Nevertheless, both S- and X-band EPR spectroscopy is suitable for ·NO-metry at room temperature using Fe(DTCS)2 as the spin trapping agent. In particular, S-band EPR spectroscopy enables the detection of ·NO production in a whole organ, such as murine liver.
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Kinetics of increased generation of ·NO in endotoxaemic rats as measured by EPR.

100%
Plonka P. M., Chlopicki S., Wisniewska M., Plonka B. K.
|
2003
|
vol. 50
|
issue 3
807-813
EN
Ferrous-diethyldithiocarbamate (Fe(DETC)2) chelate is a lipophilic spin trap developed for g·NO detection by electron paramagnetic resonance (EPR) spectroscopy. Using this spin trap we investigated the kinetics of ·NO production in endotoxaemia in rats induced by lipopolysaccharide (Escherichia coli, 10 mg/kg). The NO-Fe(DETC)2 complex was found to give a characteristic EPR signal, and the amplitude of the 3rd (high-field) component of its hyperfine splitting was used to monitor the level of ·NO. We found that in blood, kindey, liver, heart and lung ·NO production starts to increase as early as 2 h after LPS injection, reaches the maximum 6 h after LPS injection and then returns to basal level within further 12-18 h. Interestingly, in the eye bulb the maximum of ·NO production was detected 12 h after LPS, and the signal was still pronounced 24 h after LPS. In brief, the highly lipophilic exogenous spin trap, Fe(DETC)2 is well suited for assessment of ·NO production in endotoxaemia. We demonstrated that the kinetics of increased production of ·NO in endotoxaemic organs, with the notable exception of the eye, do not follow the known pattern of NOS-2 induction under those conditions. Accordingly, only in early endotoxaemia a high level of ·NO is detected, while in late endotoxaemia ·NO detectability is diminished most probably due to concomitant oxidant stress.
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