Figure 8.

Identification and characterization of metabolite IX as a benzothiazine compound. A. Presence of metabolite IX in the urine of wild-type mice treated with 400 mg/kg APAP, [acetyl-²H₃]-APAP and 2,3,5,6-[²H₄]-APAP. Ion chromatograms were prepared by extracting ions in the 20 ppm mass range of 281.005⁺ (APAP), 281.005⁺ ([acetyl-²H₃]-APAP) and 284.025⁺ (2,3,5,6-[²H₄]-APAP), respectively. B. Presence of metabolite IX in the urine of wild-type mice treated with 400 mg/kg p-aminophenol (PAP). Peaks corresponding to metabolite IX were labeled in the extracted ion chromatograms of PAP (lower panel) and APAP (upper panel) treatments. C. MS² spectrum of metabolite IX. Major fragment ions were interpreted in the inlaid structural diagrams. D. LC-MS analysis of the products from the HRP-mediated reaction between Cys-APAP and H₂O₂. Peaks corresponding to Cys-APAP (271.045⁺) and N-acetylamino-1,4-benzothiazine (207.059⁺) were labeled in the ion chromatogram of the reaction. E. Relative abundances of metabolite IX in the 24-h urine of wild-type and Cyp2e1-null mice treated with 200 and 400 mg/kg APAP (n=8). F. Relative abundances of metabolite IX in the 1-h, 2-h, 4-h and 24-h urine of wild-type and Cyp2e1-null mice treated with 400 mg/kg APAP. Relative abundances of metabolite IX (mean ± SD in parts per ten thousand) were determined by normalizing the single ion counts of metabolite IX versus the total ion counts of each urine sample (n=4 for 1-h, 2-h, 4-h urine, * for P < 0.05 and ** for P < 0.01).