Kool Jeroen, Van Liempd Sebastiaan M, Harmsen Stefan, Schenk Tim, Irth Hubertus, Commandeur Jan N M, Vermeulen Nico P E
LACDR-Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, Amsterdam, The Netherlands.
Anal Bioanal Chem. 2007 Jun;388(4):871-9. doi: 10.1007/s00216-007-1296-x. Epub 2007 Apr 28.
Reactive oxygen species (ROS) can damage proteins, cause lipid peroxidation, and react with DNA, ultimately resulting in harmful effects. Antioxidants constitute one of the defense systems used to neutralize pro-oxidants. Since pro-oxidants and antioxidants are found ubiquitously in nature, pro-and antioxidant effects of individual compounds and of mixtures receive much attention in scientific research. A major bottleneck in these studies, however, is the identification of the individual pro-oxidants and antioxidants in mixtures. Here, we describe the development and validation of an on-line post-column biochemical detection system for ROS-producing compounds and antioxidants in mixtures. Inclusion of cytochrome P450s and cytochrome P450 reductase also permitted the screening of compounds that need bioactivation to exert their ROS-producing properties. This pro-oxidant and antioxidant detection system was integrated on-line with gradient HPLC. The resulting high-resolution screening technology was able to separate mixtures of ROS-producing compounds and antioxidants, allowing each species to be characterized rapidly and sensitively.
活性氧（ROS）可损伤蛋白质、引发脂质过氧化并与DNA发生反应，最终产生有害影响。抗氧化剂是用于中和促氧化剂的防御系统之一。由于促氧化剂和抗氧化剂在自然界中普遍存在，因此单个化合物以及混合物的促氧化和抗氧化作用在科研中备受关注。然而，这些研究中的一个主要瓶颈是鉴定混合物中的单个促氧化剂和抗氧化剂。在此，我们描述了一种用于混合物中ROS生成化合物和抗氧化剂的在线柱后生化检测系统的开发与验证。加入细胞色素P450和细胞色素P450还原酶还可筛选需要生物活化才能发挥其ROS生成特性的化合物。该促氧化剂和抗氧化剂检测系统与梯度高效液相色谱（HPLC）在线集成。由此产生的高分辨率筛选技术能够分离ROS生成化合物和抗氧化剂的混合物，从而快速灵敏地对每种物质进行表征。
