Analysis of peroxynitrite reactions with guanine, xanthine, and adenine nucleosides by high-pressure liquid chromatography with electrochemical detection: C8-nitration and -oxidation

Peroxynitrite, the reaction product of nitric oxide and superoxide anion, a nd a powerful oxidant, was found to nitrate as well as oxidize adenine, gua nine, and xanthine nucleosides. A highly sensitive reverse-phase HPLC metho d with a dual-mode electrochemical detector, which reduces the nitro produc t at the first electrode and detects the reduced product by oxidation at th e second electrode, was applied to detect femtomole levels of 8-nitroguanin e and 8-nitroxanthine. This method was used to separate and identify the pr oducts of nitration and oxidation from the reactions of nucleosides with pe roxynitrite. Peroxynitrite nitrates deoxyguanosine at neutral pH to give th e very unstable 8-nitrodeoxyguanosine, in addition to 8-nitroguanine. 8-Nit rodeoxyguanosine, with a half-life of similar to 10 min at room temperature and less than or equal to3 min at 37 degreesC, hydrolyzes at pH 7 to 8-nit roguanine. A decrease in the reaction pH resulted in a decrease in the leve l of CS-nitration. Peroxynitrite also oxidizes deoxyguanosine in a pH-depen dent manner, to give 8-oxodeoxyguanosine with a maximum yield (0.5-0.7%) at pH 5. Guanosine and xanthosine exhibit reactivity similar to that of deoxy guanosine toward peroxynitrite at neutral pH, producing only the correspond ing 8-nitronucleosides as well as 8-nitroguanine and 8-nitroxanthine, respe ctively. 8-Nitroguanosine at pH 7, with a half-life of several weeks at 5 d egreesC and 5 h at 37 degreesC, was much more stable than 8-nitrodeoxyguano sine. C8-nitration was confirmed by dithionite reduction to the correspondi ng amino nucleosides, which cochromatographed with synthesized 8-amino nucl eoside standards. In contrast to guanine nucleosides, adenine nucleosides u ndergo peroxynitrite-mediated C8 oxidation even at neutral pH to give the c orresponding 8-oxoadenine nucleosides in similar to0.3% yield. Adenine nitr ation, though minor compared to C8-oxidation, appears to occur at both C2 a nd C8 positions of the adenine ring. Lowering the reaction pH from 7 to 5 r esults in 2.4- and 2.2-fold increases in the yields of 8-oxo-dA and 8-oxo-A do, respectively, but the level of nitration is not altered.