Development for measuring the concentration of hydrogen peroxide in vivo using ESR equipment

为了定量测定吞噬细胞或电离辐射照射全身产生的过氧化氢（H_2O_2）的量，用表面线圈型共振器L-带ESR测量了H_2O_2与2，2-连氮基-双（3-乙基苯并噻唑啉）-6-磺酸（ABTS）在过氧化物酶（HRP）存在下反应生成的阳离子自由基。H_2O_2的量可能与ABTS阳离子自由基的ESR谱信号强度有关，H_2O_2与HRP反应生成化合物I，化合物I再氧化ABTS生成ABTS阳离子自由基，制备了ABTS-HRP-脂质体（1.24 mol ABTS/mol磷脂（DMPC：胆固醇：硬脂胺=1：1：0.1））。将ABTS-HRP溶液与不同浓度的H_2O_2混合，制成ABTS阳离子自由基溶液，经腹腔注射给小鼠，用L带ESR谱仪测定，以阐明L带ESR的灵敏度。当H_2O_2浓度大于2？mol，表明H_2O_2的最低极限浓度可能为2？另一方面，将ABTS-HRP溶液腹腔注射小鼠，然后注射大量H_2O_2。结果得到了ABTS阳离子自由基的L-带ESR谱。而腹腔注射内毒素（LPS）则不能产生ESR谱，因为内毒素可引起腹腔吞噬细胞的生成和聚集。

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.