Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems HIGH-THROUGHPUT REAL-TIME ANALYSES
Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems HIGH-THROUGHPUT REAL-TIME ANALYSES
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DOI：
10.1074/jbc.m111.309062
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发表时间：
2012-01-27
影响因子：
4.8
通讯作者：
Kalyanaraman, Balaraman
中科院分区：
文献类型：
--
作者：
Zielonka, Jacek;Zielonka, Monika;Kalyanaraman, Balaraman
在这里，我们描述了一个高通量的荧光和高效液相色谱为基础的方法，在生物系统中的活性氧和氮物种（ROS/RNS）的全球概况。HPLC和荧光检测的结合使用是成功实施和验证该方法的关键。本文包括特异性检测和定量ROS/RNS物质与荧光探针之间相互作用形成的产物的方法，如下所示：使用氢乙啶的超氧化物，使用硼酸盐基探针的过氧亚硝酸盐，使用4，5-二氨基荧光素的一氧化氮衍生的亚硝化物质，和使用10-乙酰基-3，3-二氨基荧光素的过氧化氢和其它氧化剂，7-二羟基吩恶嗪（Amplex（R）Red）分别与辣根过氧化物酶和不与辣根过氧化物酶反应。在这项研究中，我们展示了实时监测活性氧/RNS在活化的巨噬细胞使用高通量荧光和HPLC方法。这种全局分析方法，同时检测多个ROS/RNS产品的荧光探针，在这项研究中开发的将是有用的，在解开复杂的作用ROS/RNS在氧化还原调节，细胞信号，细胞氧化过程和高通量筛选抗炎抗氧化剂。
Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex (R) Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.