Development of Micro analysis of NO and its application

研究了电子自旋共振（ESR）法测定一氧化氮（NO）的方法。该方法对NO的检测限为100 nM (S/N=3)，比油脂法和荧光法具有更高的灵敏度和特异性。我们开发了HPLC/ECD/ESR系统，该系统能够对多种自由基进行分离，利用反相HPLC在10 min内测定每个自由基。由于羧基ptio易被机体内抗坏血酸、生育酚、尿酸等还原剂氧化，故无法得到对NO的良好测定。因此，利用卵磷脂制备的脂质体- PTIO，考察了生物系统中NO测定的稳定性。说明脂质体- ptio可以通过脂质双分子层的屏障对多种还原剂进行保护。在10μM ~ 100μM范围内线性良好，ECD检测器的检出限（S/N=3）为5 μM， ESR检测器的检出限为50 nM。将该方法应用于结膜炎大鼠泪液中一氧化氮的测定。结果显示，大鼠泪液中一氧化氮生成0.4 μM。证实大鼠胸主动脉有一氧化氮形成，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.