Identification of a novel biomarker for oxidative stress induced by hydrogen peroxide in primary human hepatocytes using the 2-nitrobenzenesulfenyl chloride isotope labeling method.
OBJECTIVE
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 [(12)C]- and [(13)C]-NBS reagents, respectively. Pairs of peaks with 6-Da differences in which the [(13)C]-NBS labeling was more intense than the [(12)C]-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.


CONCLUSIONS
The NBS labeling technique was useful to identify biomarkers. Serum MnSOD may be a useful biomarker that can distinguish between SS and NASH.
Department of Digestive and Lifestyle-related Disease, Health Research, Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima.
Yoichiro
Y
Takami
Hirofumi
H
Uto
Tsutomu
T
Tamai
Yuko
Y
Sato
Yo-Ichi
Y
Ishida
Hiroyuki
H
Morinaga
Yoichi
Y
Sakakibara
Akihiro
A
Moriuchi
Makoto
M
Oketani
Akio
A
Ido
Tomoaki
T
Nakajima
Takeshi
T
Okanoue
Hirohito
H
Tsubouchi