Development of Micro analysis of NO and its application

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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.