Development of Micro analysis of NO and its application

本文研究了用羧基对苯二酚测定一氧化氮(NO)的电子自旋共振(ESR)法，该方法的检出限(S/N=3)为100nM，比润滑法和荧光法具有更高的灵敏度和特异性。我们建立了高效液相/电子捕获/电子自旋共振体系，能够分离多种自由基，用反相高效液相色谱在10分钟内测定每个自由基。由于羧基-PTIO很容易被生物体中的抗坏血酸、生育酚、尿酸等还原剂氧化，所以不能很好地测定NO。因此，用卵磷脂制备的脂质体-PTIO考察了测定生物体系中NO的稳定性。这说明脂质体-PTIO可以通过脂双层的屏障来保护其免受多种还原剂的伤害。在10μM~100μM范围内线性关系良好，检出限(S/N=3)为5μM，ESR为50 nM。结果表明，在大鼠泪液中可识别产生0.4μM的NO。大鼠胸主动脉内有NO生成，引起血管松弛。

Electron spin resonance (ESR) method was examined for determination of nitric oxide (NO) using Carboxy-PTIO.the Proposed method was 100 nM as detection limit (S/N=3) of NO, and more high sensitivety and saecificitv than these of Grease method and fluorescence method. We developed the HPLC/ECD/ESR system, which be able to separate for the many free radicals, determining each radical within 10 min by using the reverse-phase HPLC.Scince Carboxy-PTIO is easy to oxidize by reductants such as ascorbic acid, tocopherol and uric acid in the organism, good determination for NO could not obtained. Therefore, Liposome- PTIO which was prepared bv using the lecithin, examined stability for determination of NO in the biological systems. It is elucidate that Liposome-PTIO could be protected from many reductants by barrier of lipid bilayer. The good linearity is obtained between 10μM to 100μM, and detection limit (S/N=3) is 5 μM by the ECD detector and 50 nM by ESR.We applied proposed method to determination of NO of lacrimal fluid in rat suffered from conjunctivitis. As the results, generation of 0.4 μM for NO was recognized in the lacrimal fluid of rat. Formation of NO was confirmed in the aorta thoracica of the rat which caused the blood vessel relaxation.