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Development and validation of a LC/MS-based method for the measurement of intracellular superoxide anion
Analytica Chimica Acta ( IF 5.7 ) Pub Date : 2018-01-01 , DOI: 10.1016/j.aca.2017.11.007
Ze-Yuan Wang , Yi Li , Wen-Qi Chang , Jia-Yi Zheng , Ping Li , Li-Fang Liu , Gui-Zhong Xin

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.

中文翻译:

基于 LC/MS 的细胞内超氧阴离子测量方法的开发和验证

超氧阴离子 (O2.-) 作为第一个产生的活性氧 (ROS),被认为对细胞功能非常有害。细胞内 O2.- 水平的测量对于揭示其在各种氧化损伤疾病中的作用非常重要。基于氢乙锭 (HE) 荧光的方法通过监测 HE/O2.- 反应的独特产物 2-OH-E+ 主导细胞内 O2.- 测定。然而,红色荧光不可避免的交叉干扰限制了其提供关于细胞内 O2.- 形成的可靠信息的能力。通过检测 2-OH-E+,我们在此开发并验证了一种改进的基于 LC/MS 的方法,用于测量细胞内 O2.-。首先,我们证明了HE/O2.-反应的比例。第二,在基于 LC/MS 的 2-OH-E+ 检测中,ungerimine 用作内标以消除日基和基质效应。之后,总蛋白质浓度用于细胞数标准化。因此,进一步提出了一个方程来计算细胞内O2.-的相对丰度(RA)。最后,所开发的方法已成功用于评估天然化合物对 O2.- 生成的抑制作用,其结果通过基于 HE 的荧光测量进行了验证。与荧光测量相比,基于 LC/MS 的细胞内 O2.- 测定方法更灵敏、选择性和准确度更高。进一步提出了一个方程来计算细胞内O2.-的相对丰度(RA)。最后,所开发的方法已成功用于评估天然化合物对 O2.- 生成的抑制作用,其结果通过基于 HE 的荧光测量进行了验证。与荧光测量相比,基于 LC/MS 的细胞内 O2.- 测定方法更灵敏、选择性和准确度更高。进一步提出了一个方程来计算细胞内O2.-的相对丰度(RA)。最后,所开发的方法已成功用于评估天然化合物对 O2.- 生成的抑制作用,其结果通过基于 HE 的荧光测量进行了验证。与荧光测量相比,基于 LC/MS 的细胞内 O2.- 测定方法更灵敏、选择性和准确度更高。
更新日期:2018-01-01
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