河野 雅弘 日本電子(株), 分析機器技術本部・応用研, 研究室長
LEE Chang-il Kanagawa Dental College, Pharmacology, Instructor, 歯学部, 助手 (60220795)
TAKAHASHI Shun-suke Kanagawa Dental College, Pharmacology, Instructor, 歯学部, 助手 (60206810)
TODOKI Kazuo Kanagawa Dental College, Pharmacology, Assistant Professor, 歯学部, 講師 (90139577)
KOHNO Masahiro JEOL Ltd., Application and Research Center, Director of ESR Laboratory
|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1997 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1996 : ¥1,000,000 (Direct Cost : ¥1,000,000)
In recent years, reactive oxygen species have been implicated in the pathogenesis of a wide variety of disorders. Although the existence of reactive oxygen intermediates in drug metabolism can be inferred from end product analysis or from the effects of antioxidants or enzymes such as superoxide dismutase, only the technique of electron spin resonance (ESR) allows the direct detection of these highly reactive species. However, some free radical species cannot be detected by ESR due to their extremely short half-lives, which result in low steady-state concentrations of the radicals or to short radical relaxation times, which lead to a very broad line. These facts made recent development of spin-trapping and chemiluminescence techniques are widely used to detect free radicals. The goal of this project is to develop the various assays available for measurement of reactive oxygen species in biological models. In this research project, we wish to establish (1) the spin-trapping/ESR techniqu
e in vitro and vivo and (2) the chemiluminescence-optical biosensor application of this technique, a very sensitive method that has the advantage of being able to provide continuous, online, nondestructive monitoring of reactive oxygen species.
1.Desferioxamine (DFX), a transition metal ion chelator, has been used to detect H_2O_2 and peroxidase enzyme. We demonstrated that DFX's one-electron oxidation product, DFX nitroxide free radical, is an intermediate of H_2O_2-peroxidase reaction ; thus, H_2O_2 and peroxidase enzyme are detected (-muM) with ESR spectroscopy (by detecting DFX radical).
2.ESR spectrometry method using NO-trapping reagent, Fe (DTCS) _2 complex, may be useful for detecting NO in vitro system ; however, the conditions used for NO detection in vevo are not optimal due to Fe (DTCS) _2 toxicity or to low L-band ESR sensitivity.
3.Generation of singlet oxygen (^1O_2) from photoactivation of rose bengal was studied by ESR spectroscopy by use of the spin-trap 2,2,6,6-tetramethylpiperidine (TEMP). The result of ESR study showed that ^1O_2 was produced by photoactivation of rose bengal and was detected as ^1O_2-TEMP product (TEMPO ; 2,2,6,6-tetramethylpiperidine-N-oxyl).
4.Optical biosensor was developed for the batch analysis system of hypoxanthine in the concentration range between 10^<-8> and 10^<-4> M.With this optical biosensor, it should be possible to measure oxygen-derived free radical generation in a variety of conditions and with multiple interventions.
5.Oxygen-derived free radicals, possibly hydroxyl radical, directly produces vasoconstriction in the dog dental pulp of canine teeth. This could be explained by the destruction of basally released EDRF and/or endothelial dysfunction.
(1)口腔付近の酸化還元状態:L-バンド帯域でin vivo ESR計測を行い,安定ニトロキシドラジカルをプローブとしてマウス腹部および口腔付近の酸化還元状態を評価した。その結果、口腔付近では酸化還元状態が低いことが示唆された。