Davies Sean S, Amarnath Venkataraman, Brame Cynthia J, Boutaud Olivier, Roberts L Jackson
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 27232-6602, USA.
Nat Protoc. 2007;2(9):2079-91. doi: 10.1038/nprot.2007.298.
Measurement of F(2)-isoprostanes (F(2)-IsoPs) has been independently verified as one of the most reliable approaches to assess oxidative stress in vivo. However, the rapid clearance of F(2)-IsoPs makes the timing of sample collection critical for short-lived oxidative insults. Isoketals (IsoKs) are gamma-ketoaldehydes formed via the IsoP pathway of lipid peroxidation that rapidly react with lysyl residues of proteins to form stable protein adducts. Oxidative stress can also activate cyclooxygenases to produce prostaglandin H(2), which can form two specific isomers of IsoK-levuglandin (LG) D(2) and E(2). Because adducted proteins are not rapidly cleared, IsoK/LG protein adduct levels can serve as a dosimeter of oxidative and inflammatory damage over prolonged periods of time as well as brief episodes of injury. Quantification of IsoK/LG protein adducts begins with liquid-phase extraction to separate proteins from lipid membranes, allowing measurement of both IsoK/LG protein adducts and F(2)-IsoP from the same sample if desired. IsoK/LG-lysyl-lactam adducts are measured by liquid chromatography tandem mass spectrometry after proteolytic digestion of extracted proteins, solid-phase extraction and preparative HPLC.
F(2)-异前列腺素（F(2)-IsoPs）的测量已被独立验证为评估体内氧化应激最可靠的方法之一。然而，F(2)-IsoPs的快速清除使得样本采集时间对于短暂的氧化损伤至关重要。异酮（IsoKs）是通过脂质过氧化的异前列腺素途径形成的γ-酮醛，它能迅速与蛋白质的赖氨酰残基反应形成稳定的蛋白质加合物。氧化应激还可激活环氧化酶产生前列腺素H(2)，其可形成异酮-列腺素（LG）D(2)和E(2)的两种特定异构体。由于加合的蛋白质不会被快速清除，异酮/列腺素蛋白质加合物水平可作为长时间以及短暂损伤期氧化和炎症损伤的剂量计。异酮/列腺素蛋白质加合物的定量分析首先通过液相萃取将蛋白质与脂质膜分离，如果需要，可从同一样本中同时测量异酮/列腺素蛋白质加合物和F(2)-IsoP。在对提取的蛋白质进行蛋白水解消化、固相萃取和制备型高效液相色谱后，通过液相色谱串联质谱法测量异酮/列腺素-赖氨酰内酰胺加合物。
