Dual-calibration coefficient:a more accurate protocol for simultaneous determination of superoxide a

• 作 者: Hongmin Li Lu Li Xu Wang Qingling Li Miao Du Bo Tang
• 机 构: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of
• 出 处: Science China(Chemistry)
• 发表日期: 2015年05期
• 关键词: calibration superoxide extracts xanthine peroxide simultaneous providing probes

We present a more accurate method for the quantification of superoxide anion(O2??) and hydrogen peroxide(H2O2) simultaneously in human Hep G2 cell extracts. After the xanthine/xanthine oxidase system was added into cell extract which was devoid of O2?? and H2O2, steady-state and in-situ produced O2?? and H2O2 by xanthine/xanthine oxidase system was labeled by fluorescent probes and subsequently separated by microchip electrophoresis. Based on this method, two differential equations with the calibration coefficients were established for O2?? and H2O2, respectively. Using the established dual-calibration coefficients, we obtained the calibrated concentrations of O2?? and H2O2 that produced in human Hep G2 cells, which were lower(0.66±0.03 and 0.82±0.04 ?mol/L for O2?? and H2O2, respectively) than that(0.85±0.03 and 0.96±0.03 ?mol/L for O?? and H2O2, respectively) obtained from statutory working curve. The proposed dual-calibration coefficient protocol takes into account both the complex matrix effect of the biological system and real time decaying of O2?? and H2O2, providing a method with higher accuracy.

We present a more accurate method for the quantification of superoxide anion (O2 ??) and hydrogen peroxide (H2O2) simultaneously in human Hep G2 cell extracts. After the xanthine / xanthine oxidase system was added into cell extract which was devoid of O2 and H2O2, steady-state and in-situ produced O2- and H2O2 by xanthine / xanthine oxidase system was labeled by fluorescent probes and subsequently separated by microchip electrophoresis. Based on this method, two differential equations with the calibration coefficients were established for O2 ?? and H2O2, respectively. Using the established dual-calibration coefficients, we obtained the calibrated concentrations of O2 ?? and H2O2 that produced in human Hep G2 cells, which were lower (0.66 ± 0.03 and 0.82 ± 0.04? mol / L for O2 and H2O2, respectively) than that (0.85 ± 0.03 and 0.96 ± 0.03? Mol / L for O and H2O2, respectively) obtained from statutory working curve. The proposed dual-calibration coefficient protocol takes into account both the comp lex matrix effect of the biological system and real time decaying of O2- and H2O2, providing a method with higher accuracy.