ANALYSIS OF ENDOGENOUSLY PRODUCED NITRIC OXIDE (NO) EMPLOYING IRON COMPLEXES AS AN NO TRAPPING REAGENT AND PHYSIOLOGICAL STUDY ON NO

一氧化氮（NO）是一种双原子分子，是一种无机自由基。它被认为是一种大气污染物和潜在的危害。然而，它现在被广泛认为是一种内源性分子，在血管平滑肌松弛、神经元传递和巨噬细胞诱导的细胞毒性等生物学过程中具有重要的生理作用。为了阐明内源性NO的各种作用，有关NO在细胞、组织和器官中的数量和分布的信息是必不可少的。然而，由于NO在体内形成的浓度很低，并且在生命系统中的半衰期很短，因此很难确定其数量和分布。作为克服这些困难的分析方法之一，自旋捕集技术结合电子顺磁共振（EPR）技术已被用于体内外不稳定自由基的测定，铁与二硫代氨基甲酸酯衍生物的配合物是其中值得注意的一种。 关于我们 NO的自旋捕获试剂，因为NO对铁络合物具有高亲和力，并且所得的NO结合的铁络合物在室温下在体外EPR测量中表现出强烈的三线信号。这样的二硫代氨基甲酸酯衍生物包括N，N-二乙基二硫代氨基甲酸酯（DETC）、N-甲基-D-葡糖胺二硫代氨基甲酸酯（MGD）和N-（二硫代羧基）肌氨酸（DTCS）。铁与DTCS的络合物（Fe-DTCS）及其NO络合物（NO-Fe-DTCS）在水介质中溶解度高，稳定性好。因此，Fe-DTCS和NO-Fe-DTCS配合物分别具有作为生物良性的、有效的NO捕获试剂和作为水溶性自旋标记试剂（或自旋探针）的潜力。此外，如果来自内源性产生或外源性供应的NO-Fe-DTCS复合物的EPR信号能够在活的小动物的整个身体中测量，则可以进行内源性和外源性NO的体内EPR成像。为了获得EPR谱和图像，我们使用了自制的700-MHz-microwave EPR成像系统，该系统由一个连接有一对场梯度线圈的电磁铁、一个数据处理计算机和一个700-MHz-microwave EPR单元组成。此外，我们报道了应用NO捕获试剂的内源性产生的NO的检测和成像。首先，我们尝试从猪主动脉内皮细胞的NO生成的检测。其次，我们证明了在体内EPR成像内源性产生的NO，捕获的Fe-DTCS复合物，在一个活的小鼠的腹部。本实验在败血症休克模型中通过经腹腔途径向小鼠施用脂多糖来进行。由此获得的图像的轮廓对应于肝脏，提示NO-Fe-DTCS complex.We的代谢途径，在这项研究中，我们可以证明，铁二硫代氨基甲酸盐作为一个NO捕获试剂是适合在体内真实的时间检测的生理和病理条件下产生的NO。少

Diatomic molecule, nitric oxide (NO) is an inorganic free radical. It has been known as an atomospheric pollutant and a potential hazard. However, it is now widely known as an endogenous molecule with critical physiological roles in the biological processes of vascular smooth muscle relazation, neuronal transmission and macrophage-induced cytotoxicity. To elucidate a variety of actions of endogenous NO,information concerning the quantities and distributions of NO in cells, tissues, and organs is essential. However, it is rather difficult to determine quantities and distribution due to the very small concentration of NO formed in vivo and the very short half-life of NO in living system. As one of the analytical methods to overcome these difficulties, spin-trapping technique combined with electron paramagnetic resonance (EPR) spectroscopy has been used for the determination of unstable free radicals in vitro and in vivo.Iron complexes with dithiocarbamate derivatives are noted among the … More spin trapping reagents for NO because NO has a high affinity for the iron complexes and resultant NO-bound iron complexes exhibit an intense three-line signal at room temperature on in vitro EPR measurement. Such dithiocarbamate derivatives include N,N-diethyldithiocarbamate (DETC), N-methyl-D-glucamine dithiocarbamate (MGD), and N-(dithiocarboxy) sarcosine (DTCS). Iron complex with DTCS (Fe-DTCS) and its NO complex (NO-Fe-DTCS) are fairy soluble and stable in aqueous media. Therefore, Fe-DTCS and NO-Fe-DTCS complexes have a potential as a biologically benign, effective NO-trapping reagent and as a water-soluble spin-labeling reagent (or spin probe), respectively. Further, if the EPR signal from endogenously produced or exogenously supplied NO-Fe-DTCS complex is able to measure in the whole body of a live small animal, in vivo EPR imaging of endogenous and exogenous NO would be performed. Such EPR image would permit noninvasive observation of in vivo spatial distribution of NO.To obtain EPR spectra and image, we used a home built 700-MHz-microwave EPR imaging system, which is composed of an electromagnet attached with a pair of field gradient coils, a data-processing computer, and a 700-MHz-microwace EPR unit.In this study, we demonstrated the usefulness of iron dithiocarbamates as an NO trapping reagent. Further, we reported the applications of the NO trapping reagent to the detection and imaging of endogenously produced NO.First, we tried the detection of NO generation from porcine aorta endothelial cells. Second, we demonstrated in vivo EPR imaging of endogenously produced NO,trapped by an Fe-DTCS complex, in the abdomen of a live mouse. This experiment has been cariied out in a septic-shock model by administering lipopolysaccharide to mice via intraperitoneal route. The outline of the image thus obtained corresponds to the liver, suggesting the metabolic pathway of NO-Fe-DTCS complex.We could demonstrate in this study that iron dithiocarbamates as an NO trapping reagent are suitable for in vivo real time detection of NO produced under physiological and pathological conditions. Less