of ZNS significantly (77% of control: p < 0.05) scavenged ·OH (Figure 1).
ZNS was treated with H2O2-without UV (Exp. A), or H2O2 with UV, generating ·OH in Exp. B; each reactant was analyzed by LC/MS. In these mass spectrograms, the following fragment peaks were observed except for m/z: 213 (ZNS itself): m/z: 62, 106, 118, 132, 214, 215, 230, 235 and 251. These fragments were analyzed by SIM, and it was found that m/z: 213 (ZNS itself) was markedly decreased and m/z: 118 was detected specifically in the conditions with H2O2 and UV irradiation for 30 sec (Exp. B). No significant difference was found between other fragments compairing Exp. A and Exp. B (Table 1). Meanwhile a fragment peak was not detected by these analyses that would correspond to the hydroxyl radical-adducted molecule.
Mori et al. , using ESR spectrometry, first reported that ZNS scavenged •OH. This experiment confirms that this scavenging activity occurs in a clinically relevant concentration of ZNS. Using experimental animals, it has been shown that ZNS is metabolized by direct acetylation, by glucronyl conjugation, and via hydroxylation
Table 1. Analysis of ZNS and its decomposed fragments by LC/MS.
followed by oxidation of the methylen carbon of the sulfamoyl group, finally resulting loss of the sulfamoyl group. In addition, there is N-O bound cleavage of the isoxazole ring to produce hydroxylation in the benzene ring. Formed ring-cleft metabolites are followed by conjugation with sulfuric acid or glucronic acid. The 1,2-benzisoxazole N-O cleavage product of ZNS, 2-sulphamolacetylphenol (SMAP; m/z: 215) is one of the major metabolites [6,7]. Reduction of ZNS to SMAP is mediated by cytochrome P450 [7,8] and by mammalian intestinal bacteria in vivo .
Yoshida and Masuda, from the Research Laboratory of Dainippon Pharmaceutical Co., Ltd. (Yoshida & Masuda, 1997, on the reaction of ZNS with •OH radical. Personal communication to A.M.) treated ZNS with •OH radicals induced by Fenton’s reaction, and using HPLC, observed decreased level of ZNS. Using GC/MS, they analyzed the possible decomposed fragments from ZNS, but did not identify the specific fragments that originated from ZNS. In this LC/MS study, UV-H2O2 system, but not the Fenton’s reaction, was used for •OH generation. Moreover, UV irradiation was used only for 30 sec, and the decomposing reaction for ZNS was already in progress with the peak height of m/z for ZNS slightly but significantly decreased.
In the analysis by SIM mode, many signals of m/z were observed both in the Exp. A (H2O2 without UV) and B (H2O2 with UV). The common signals may be due to oxidative reaction by H2O2 itself. Some higher m/z signals, e.g., m/z: 230, 235, 251, may be due to secondary reactions among fragments by the oxidation reaction by H2O2. Meanwhile, m/z: 118 in the Exp. B was significantly increased comparing with in Exp. A, which could
Figure 1. Dose-dependent of •OH scavenging activity by ZNS (39 µM - 5 mM). Values are expressed as mean ± SEM (n=3 - 4). *p < 0.05 compared with control (ZNS = 0).
indicate induction by •OH. This fragment was estimated as decomposed products by cleavage of ZNS from the side chain, methane sulfonamide, and may be related to opening of the ring at -O-Nposition. We compared our mass spectral data of decomposed fragments of ZNS by UV-H2O2 system with the data of ZNS metabolites in the rat, and found no common mass fragmental datum existed including SMAP. On the other hand, m/z: 118, we found, was not observed in metabolites of ZNS in the urine using in vivo experiments , we suspect these metabolites may be unstable in vivo. These data suggested that ZNS may react directly with free radicals.