Tretter L, Ambrus A
Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary; MTA-SE Laboratory for Neurobiochemistry, Budapest, Hungary.
Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary; MTA-SE Laboratory for Neurobiochemistry, Budapest, Hungary.
Methods Enzymol. 2014;547:199-223. doi: 10.1016/B978-0-12-801415-8.00012-6.
In this chapter, we describe the currently most advanced methods applied for the quantitative assessment of ROS homeostasis inside the mitochondrion. These techniques are of particular interest in the field of oxidative stress. After discussing the importance of quantifying mitochondrial ROS homeostasis, three major aspects of this phenomenon and the pertinent methodologies for detection are delineated in detail. First the most important methods, based on fluorimetric or spectrophotometric approaches, for the detection of mitochondrial ROS are described. Elimination of ROS generated inside the mitochondrion is another crucial mechanism that also needs to be quantified accurately to estimate the antioxidant capacity of mitochondria under specific conditions. Since ROS generation and elimination manifest in concert, there needs to exist independent methods for the estimation of the net effect. Such a sensitive biochemical marker in the mitochondrion is aconitase, a citric acid cycle enzyme which is greatly sensitive to ROS. We describe two procedures for the precise determination of aconitase activity. A few auxiliary techniques and good practices having relevance in the successful accomplishment of the more delicate approaches are also mentioned. All other relevant technical considerations including advantages/disadvantages of the various methods and the most common artifacts are also discussed.
在本章中，我们描述了目前用于定量评估线粒体内活性氧（ROS）稳态的最先进方法。这些技术在氧化应激领域尤为重要。在讨论了量化线粒体ROS稳态的重要性之后，详细阐述了这一现象的三个主要方面以及相关的检测方法。首先描述了基于荧光或分光光度法检测线粒体ROS的最重要方法。线粒体内产生的ROS的消除是另一个关键机制，为了评估特定条件下线粒体的抗氧化能力，也需要对其进行准确量化。由于ROS的产生和消除是协同表现的，因此需要有独立的方法来估计净效应。线粒体中的一种敏感生化标志物是乌头酸酶，它是一种对ROS非常敏感的柠檬酸循环酶。我们描述了两种精确测定乌头酸酶活性的方法。还提到了一些有助于成功完成更精细方法的辅助技术和良好实践。此外，还讨论了所有其他相关技术考量，包括各种方法的优缺点以及最常见的假象。
