| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2002: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
In order to measure quantitatively the amount of hydrogen peroxide (H_2O_2) generated by phagocytes or irradiation of ionizing radiation in whole body, a cation radical formed by the reaction of H_2O_2 with 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) in the presence of peroxidase (HRP) was measured using L-band ESR with surface-coil-type resonator. The amount of H_2O_2 may be related to signal intensity of the ESR spectrum of ABTS cation radical, where H_2O_2 reacts with HRP, resulting in the production of compound I, and then the compound I oxidized ABTS to yield ABTS cation radical.
Liposomes (1.24 mol ABTS/mol phospholipid (DMPC : cholesterol : stearylamine=1:1:0.1)) containing ABTS and HRP (ABTS-HRP-liposome) were prepared. However, the liposomes were not used for further analysis.
A solution of pre-formed ABTS cation radical solution produced by mixing an ABTS-HRP solution with various concentrations of H_2O_2,was intraperitoneally injected into mouse and measured using a L-band ESR spectrometer to elucidate sensitivity of the L-band ESR. The L-band ESR signal of ABTS cation radical was observed in the presence of H_2O_2 more than 2 ? mol, suggesting that the lowest limiting concentration of H_2O_2 is probably 2 ? mol.
On the other hand, the ABTS-HRP solution was intraperitoneally injected into mouse, followed by the injection of a large amount of H_2O_2 into mouse. As a result, the L-band ESR spectrum of ABTS cation radical was obtained. On the contrary, the injection of LPS, which should cause the generation accumulation of phagocytes in the peritoneal cavity, gave no ESR spectrum.
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