Identification of a novel biomarker for oxidative stress induced by hydrogen peroxide in primary human hepatocytes using the 2‐nitrobenzenesulfenyl chloride isotope labeling method

Aim:  Oxidative stress is involved in the progression of non-alcoholic steatohepatitis (NASH). However, there are few biomarkers that are easily measured and accurately reflect the disease states. The aim of this study was to identify novel oxidative stress markers using the 2-nitrobenzenesulfenyl (NBS) stable isotope labeling method and to examine the clinical utility of these diagnostic markers for NASH. Methods:  Proteins extracted from phosphate buffered saline- and hydrogen peroxide-loaded human primary hepatocyte were labeled with the [12C]- and [13C]-NBS reagents, respectively. Pairs of peaks with 6-Da differences in which the [13C]-NBS labeling was more intense than the [12C]-NBS labeling were detected by MALDI-TOF/MS and identified by MS/MS ion searching. Results:  Four pairs of peaks, m/z 1705–1711, m/z 1783–1789, m/z 1902–1908 and m/z 2790–2796, were identified as cytochrome c oxidase VIb (COX6B), liver carboxylesterase 1 (CES1), carbamoyl-phosphate synthase 1 (CPS1) and superoxide dismutase (MnSOD), respectively. Furthermore, serum MnSOD protein levels were significantly higher in NASH patients than in simple steatosis (SS) patients. The serum MnSOD levels tended to increase in parallel with the stage of fibrosis. Conclusion:  The NBS labeling technique was useful to identify biomarkers. Serum MnSOD may be a useful biomarker that can distinguish between SS and NASH.