|Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1995: ¥1,800,000 (Direct Cost: ¥1,800,000)
The condition of NO trapping with N-methylglucamine dithiocarbamate-iron complex (MGD-Fe) was optimized, and NO derived from mouse macrophage and human leukocytes was determined by electron paramagnetic resonance (EPR) spectrosopy. In situ determination of NO was made by cultureing cells in EPR cuvettes. Cell culture using ordinary plastic culture plates was also performed and the results were compared because culture in EPR cuvette has a limit in oxygen supply.
MGD-Fe can trap NO only when the iron is in ferrous state. The stability of ferrous iron in solutions depends on pH,ions and MGD/Fe ratio. In RPMI,ferrous iron is easily oxidized, and hepes inhibits the oxidation. When the ratio of MGD/Fe is more than 10, ferrous iron in RPMI/hepes is sufficiently stable. The spin adduct, MGD-Fe-NO is unstable when the adduct is made in the abscence of oxygen followed by oxygen exposure. However, when the MGD-Fe complex is made in nitrogen atmospher, and then oxygenated, the adduct of MGD-Fe-NO
was stable for hours.
EPR signal composed of 3 lines, assigned as MGD-Fe-NO was obtained when peritoneal macrophages from BULB/c mice elicited with thioglycolate broth were cultured in EPR cuvettes and perfused with RPMI media containing gamma-IF and LPS for 6 hours followed by the replacement with RPMI/hepes containg MGD-Fe spin trap. The intensity of the signal grew for minutes and then reached plateau. The supply of fresh trap media resulted in a growth of the same signal. When comparable experiments were carried out using ordinary plastic culture plates, the signal growth continued longer, suggesting that termination of the trapping in the in situ system is due to the limitation of oxygen supply. NO generation was started 3 hr after gamma-IF/LPS stimulaton and then the generating rate increased to reached plateau at 6 hrs.
The same signal could be obtained from human peripheral neutrophils or monocytes after stimulation with LPS.However, the reproducibility is not enough for quantitative study. Less