O APAP-induced liver injury in miceAfter urine profiling, an increased abundance in protein peaks was observed for mice treated with 275 and 350 mg/kg APAP compared to control and AMAP (Figure 2A). In total, 66 protein peaks in the WCX beads spectra, and 75 protein peaks in the C8 beads spectra were detected as significantly different between all APAP treatments and control. These proteins presented with increasing peak I-BRD9 web intensities in urine of mice with elevated plasma ALT values (Figure 2B). Most protein peaks were only detectable in urine of mice with relatively severe APAP-induced liver injury. However, two proteins of 15.9 and 16.8 kDa, later identified as SOD1 and CaM, respectively, were observed in the C8 beads spectra at low plasma ALT levels. The eleven differentiating proteins that were identified using vMALDI LTQ are depicted in Table 2. LC-MS/MS analysis confirmed the presence of these proteins and additionally retrieved the identity of the 16.8 kDa protein, which was not found using vMALDI-LTQ (Table 3). Besides SOD1 and CaM, also peak intensities of fragments of CA3 correlated closely with plasma ALT values (Figure 2C ), and therefore these 3 proteins were investigated further. To confirm the presence of CA3 and SOD1 in urine by a specific antibody, we used Western blot analysis, as shown in Figure 3A. Whereas CA3 could be detected only in urine of mice with high plasma ALT (.3500 U/L) values, SOD1 was associated with minor elevations in plasma ALT (.100 U/L) and it gradually amplified with increasing plasma ALT values. After measuring the intensities of the SOD1 signal in the Western blot, linear MedChemExpress 4EGI-1 regression analysis showed a significant correlation between urinary SOD1 and plasma ALT levels (Figure 3B). The third potential biomarker, CaM, was confirmed with an immunocapUrinary Biomarkers of Acetaminophen HepatotoxicityFigure 3. Identification of CA3, SOD1 and CaM in mouse urine. Western blots show the relation between urinary SOD1 and CA3, and plasma ALT levels in individual mice (n = 13; panel A), of which urinary SOD1 intensity on Western blot was analyzed by linear regression analysis (B). Immunoprecipitation demonstrated the specific protein profile of CaM, i.e. the mass peak for CaM at 16.8 kDa (CaM+H) and its double and triple charged form (CaM+2H 24272870 and CaM+3H), in mouse urine after APAP treatment (C). ALT: alanine aminotransferase; APAP: acetaminophen; CA3: carbonic anhydrase 3; CaM: calmodulin; SOD1: superoxide distmutase 1. doi:10.1371/journal.pone.0049524.gDiscussionThe present study was designed to identify novel biomarkers in urine for acute DILI by using APAP as model compound. Applying multiple proteomics techniques allowed us to identify twelve proteins related to APAP-induced liver injury. 15857111 For the firsttime, we report the presence of CA3, SOD1 and CaM in urine to be related to APAP-induced liver injury, of which CaM had never been linked to liver injury before. Of these proteins, principally SOD1 and CaM closely associated with plasma ALT, as observed by proteomic profiling and antibody-based methods. CA3 fragments showed a good correlation with plasma ALT withUrinary Biomarkers of Acetaminophen HepatotoxicityFigure 4. Detection of SOD1, CA3 and CaM in human urine samples. Presence of CA3 and SOD1 was assessed by Western blot in urine samples of masterpool control (I), severe APAP intoxication sample 1 (II) and 2 (III) and a positive control (IV) (panel A). Using an ELISA assay, the urinary concentration of C.O APAP-induced liver injury in miceAfter urine profiling, an increased abundance in protein peaks was observed for mice treated with 275 and 350 mg/kg APAP compared to control and AMAP (Figure 2A). In total, 66 protein peaks in the WCX beads spectra, and 75 protein peaks in the C8 beads spectra were detected as significantly different between all APAP treatments and control. These proteins presented with increasing peak intensities in urine of mice with elevated plasma ALT values (Figure 2B). Most protein peaks were only detectable in urine of mice with relatively severe APAP-induced liver injury. However, two proteins of 15.9 and 16.8 kDa, later identified as SOD1 and CaM, respectively, were observed in the C8 beads spectra at low plasma ALT levels. The eleven differentiating proteins that were identified using vMALDI LTQ are depicted in Table 2. LC-MS/MS analysis confirmed the presence of these proteins and additionally retrieved the identity of the 16.8 kDa protein, which was not found using vMALDI-LTQ (Table 3). Besides SOD1 and CaM, also peak intensities of fragments of CA3 correlated closely with plasma ALT values (Figure 2C ), and therefore these 3 proteins were investigated further. To confirm the presence of CA3 and SOD1 in urine by a specific antibody, we used Western blot analysis, as shown in Figure 3A. Whereas CA3 could be detected only in urine of mice with high plasma ALT (.3500 U/L) values, SOD1 was associated with minor elevations in plasma ALT (.100 U/L) and it gradually amplified with increasing plasma ALT values. After measuring the intensities of the SOD1 signal in the Western blot, linear regression analysis showed a significant correlation between urinary SOD1 and plasma ALT levels (Figure 3B). The third potential biomarker, CaM, was confirmed with an immunocapUrinary Biomarkers of Acetaminophen HepatotoxicityFigure 3. Identification of CA3, SOD1 and CaM in mouse urine. Western blots show the relation between urinary SOD1 and CA3, and plasma ALT levels in individual mice (n = 13; panel A), of which urinary SOD1 intensity on Western blot was analyzed by linear regression analysis (B). Immunoprecipitation demonstrated the specific protein profile of CaM, i.e. the mass peak for CaM at 16.8 kDa (CaM+H) and its double and triple charged form (CaM+2H 24272870 and CaM+3H), in mouse urine after APAP treatment (C). ALT: alanine aminotransferase; APAP: acetaminophen; CA3: carbonic anhydrase 3; CaM: calmodulin; SOD1: superoxide distmutase 1. doi:10.1371/journal.pone.0049524.gDiscussionThe present study was designed to identify novel biomarkers in urine for acute DILI by using APAP as model compound. Applying multiple proteomics techniques allowed us to identify twelve proteins related to APAP-induced liver injury. 15857111 For the firsttime, we report the presence of CA3, SOD1 and CaM in urine to be related to APAP-induced liver injury, of which CaM had never been linked to liver injury before. Of these proteins, principally SOD1 and CaM closely associated with plasma ALT, as observed by proteomic profiling and antibody-based methods. CA3 fragments showed a good correlation with plasma ALT withUrinary Biomarkers of Acetaminophen HepatotoxicityFigure 4. Detection of SOD1, CA3 and CaM in human urine samples. Presence of CA3 and SOD1 was assessed by Western blot in urine samples of masterpool control (I), severe APAP intoxication sample 1 (II) and 2 (III) and a positive control (IV) (panel A). Using an ELISA assay, the urinary concentration of C.