Development and validation of a LC/MS-based method for the measurement of intracellular superoxide anion.
Development and validation of a LC/MS-based method for the measurement of intracellular superoxide anion.
复制标题
DOI：
10.1016/j.aca.2017.11.007
复制
发表时间：
2018-01
影响因子：
6.2
通讯作者：
Ze Wang;Yi Li;Wen‐Qi Chang;Jia-Yi Zheng;Ping Li;Li-Fang Liu;G. Xin
中科院分区：
文献类型：
--
作者：
Ze Wang;Yi Li;Wen‐Qi Chang;Jia-Yi Zheng;Ping Li;Li-Fang Liu;G. Xin
超氧阴离子（O2）-)作为第一个产生的活性氧（ROS），一直被认为对细胞功能有很大的危害。细胞内氧的测量对揭示其在多种氧化损伤疾病中的作用具有重要意义。氢乙二酸（HE）荧光法是主导细胞内氧。通过监测HE/ o2 -反应的独特产物2-OH-E+。然而，红色荧光的不可避免的交叉干扰限制了它提供细胞内o2形成可靠信息的能力。通过检测2-OH-E+，我们开发并验证了一种改进的基于LC/ ms的细胞内O2.-测定方法。首先，我们证明了HE/ o2 -反应的比例性。其次，在LC/ ms检测2-OH-E+时，采用不洁利胺作为内标，消除日基效应和基质效应。之后，利用总蛋白浓度进行细胞数归一化。因此，我们进一步提出了计算细胞内O2.-相对丰度（RA）的公式。最后，该方法已成功用于评价天然化合物对O2的抑制作用。-代，其结果通过基于he的荧光测量验证。与荧光法比较，LC/ ms法测定细胞内氧含量。-测定法灵敏度高，选择性好，准确度高。
Superoxide anion (O2.-), as the first generated reactive oxygen species (ROS), has been considered to be highly deleterious to cell functions. The measurement of intracellular O2.-level is of great importance to uncover its roles in a variety of oxidative damage diseases. Hydroethidium (HE) fluorescence-based method is dominating intracellular O2.-assay by monitoring the unique product 2-OH-E+of HE/O2.-reaction. However, the avoid-less cross-interference of red fluorescence limited its ability to provide trustworthy information on intracellular O2.-formation. By the detection of 2-OH-E+, we herein developed and validated an improved LC/MS-based method for the measurement of intracellular O2.-. Firstly, we demonstrated the proportionality of HE/O2.-reaction. Secondly, ungerimine was used as internal standard to eliminate daily basis and matrix effect in the LC/MS-based detection of 2-OH-E+. Afterward, the total protein concentration was utilized for cell number normalization. Accordingly, an equation was further proposed to calculate the relative abundance (RA) of intracellular O2.-. Finally, the developed method has been successfully utilized to evaluate the inhibitory effects of natural compounds on O2.-generation, the result of which was validated by the HE-based fluorescent measurement. Compared with the fluorescent measurement, the LC/MS-based intracellular O2.-assay method is more sensitive, selective and accurate.