Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence
Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence
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DOI：
10.1073/pnas.0501719102
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发表时间：
2005-04-19
影响因子：
11.1
通讯作者：
Kalyanaraman, B
中科院分区：
文献类型：
--
作者：
Zhao, HT;Joseph, J;Kalyanaraman, B
在这里，我们报告的产物的结构表征之间的反应形成的氢乙啶（HE）和超氧化物（O-2（中心点-））。通过质谱和核磁共振技术，确定该产物的化学结构为2-羟基乙硫铵（2-OH-E+）。通过使用一个真实的标准，我们开发了一种高效液相色谱法检测和定量的反应产物的HE和O-2（中心点-）后，在牛主动脉内皮细胞处理甲萘醌或抗霉素A诱导细胞内活性氧。同时，我们使用了自旋陷阱，5-叔丁氧羰基-5-甲基-1-吡咯啉N-氧化物（BMPO），以检测和确定所形成的活性氧物种的结构。BMPO捕获了在细胞外形成的O-2（中心点-），并在EPR技术的使用过程中作为BMPO-OH加合物被检测到。BMPO具有细胞渗透性，抑制细胞内2-OH-E+的形成。然而，未检测到细胞内BMPO自旋加合物。此处描述的O-2（中心点-）与HE反应产物的确定性表征构成了用于O-2（中心点-）细胞内检测和定量的明确测定的基础。对乙锭（E+）和2-OH-E+的荧光特性的分析强烈表明，目前可用的荧光方法不适用于定量细胞内O-2（中心点-）。我们得出的结论是，使用HE作为探针的HPLC/荧光测定法更适合检测细胞内活性氧（中心点-）。
Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (O-2(center dot-)). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E+). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and O-2(center dot-) formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the O-2(center dot-) that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E+. However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of O-2(center dot-) with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of O-2(center dot-). Analysis of the fluorescence characteristics of ethidium (E+) and 2-OH-E+ strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular O-2(center dot-). We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable reactive oxygen species for detecting intracellular O-2(center dot-).