977Reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 Ϫ ), hydroxyl radical ( · OH), nitric oxide (NO), peroxynitrite (ONOO Ϫ ), and peroxide (R-OOH) affect the living body and cause various types of diseases such as cancer, 1) cardiovascular disorders, 2) and neurodegenerative diseases. 3,4) On the other hand, it is accepted that some ROS control disinfection in cells and work as a biofactor for signal transmission of insulin. 5) In addition, H 2 O 2 is a major by-product of ROS in living organisms and a common marker for oxidative stress in the field of clinical inspection.6-8) The development of methods for the determination of these substances is significant in the fields of clinical and biological studies. Several methods for the determination of the spectrophotometry, 9-11) fluorophotometry, [12][13][14][15][16][17][18] and chemiluminescence 19,20) of H 2 O 2 and other ROS have already been reported. These methods focus on the determination of individual ROS. However, it appears that most of ROS have only minute life times under biological environments. Therefore, we considered that it is more practicable to measure the total rather than individual ROS for a clinical purpose. Hence, we developed a handy and highly sensitivity method for the determination of ROS, including H 2 O 2 . We have already developed methods for the fluorometric determination of cobalt(II) and H 2 O 2 with fluorescein hydrazide (FH), 21) however, the method adopted for the determination of H 2 O 2 exhibited remarkably poor reproducibility. In this study, method to obtain a more reproducible and sensitive spectrofluorimetric determination of H 2 O 2 and other ROS were examined. Furthermore, the applicability of the method for measuring H 2 O 2 in urine samples was examined. Moreover, because an excellent crystal of FH was obtained, structural analysis by X-ray diffractometry was performed. As a result, this FH crystal was confirmed to have a five-membered spirolactam structure. The relationship between fluorescence and the chemical structure was studied; the fluorescein, which was the strong fluorescent substance that caused the spirolactam formation by acting as the hydrazine conductor, became a colorless, non-fluorescent substance.
Experimental Synthesis of Fluorescein Hydrazide (FH)A modification of the procedure of Baeyer and Akita. 22,23) for the synthesis of fluoescein and FH was employed. Resorcinol 2 mol and phthalic anhydride 1 mol were combined with methanesulfonic acid. The mixture was heated and dissolved in 5% sodium hydroxide solution. The solution was poured into 30% acetic acid; the resulting fluorescein, a yellow precipitate, was collected with a yield of 92.5%. Subsequently, fluorescein was added to a solution of excess 98% anhydrous hydrazine and heated at 80°C in a warm water bath for 3 h. Upon cooling to room temperature and with the addition of strong hydrochloric acid into the solution, the light yellow substance was deposited. Then, the substance obtained was dissolved in ethan...
