Division of Clinical Pharmacology, Department of Pharmacology, Vanderbilt University, Nashville, TN, USA.
J Lipid Res. 2010 May;51(5):999-1009. doi: 10.1194/jlr.M001040. Epub 2009 Nov 25.
Levuglandins and their stereo- and regio-isomers (termed isolevuglandins or isoketals) are gamma-ketoaldehydes (IsoK) that rapidly react with lysines to form stable protein adducts. IsoK protein adduct levels increase in several pathological conditions including cardiovascular disease. IsoKs can induce ion channel dysfunction and cell death, potentially by adducting to cellular proteins. However, IsoKs also adduct to phosphatidylethanolamine (PE) in vitro, and whether PE adducts form in cells or contribute to the effects of IsoKs is unknown. When radiolabeled IsoK was added to HEK293 cells, 40% of the radiolabel extracted into the chloroform lower phase suggesting the possible formation of PE adducts. We therefore developed methods to measure IsoK-PE adducts in cells. IsoK-PE was quantified by LC/MS/MS after hydrolysis to IsoK-ethanolamine by Streptomyces chromofuscus phospholipase D. In HEK293 and human umbilical vein endothelial cells (HUVEC), IsoK dose-dependently increased PE adduct concentrations to a greater extent than protein adduct. To test the biological significance of IsoK-PE formation, we treated HUVEC with IsoK-PE. IsoK-PE dose dependently induced cytotoxicity (LC(50) 2.2 muM). These results indicate that cellular PE is a significant target of IsoKs, and that formation of PE adducts may mediate some of the biological effects of IsoKs relevant to disease.
列乌古烷及其立体和区域异构体（称为异列乌古烷或异酮缩醛）是γ-酮醛（IsoK），可迅速与赖氨酸反应形成稳定的蛋白质加合物。几种病理情况下，包括心血管疾病，异酮蛋白加合物水平增加。IsoKs 可诱导离子通道功能障碍和细胞死亡，可能通过与细胞蛋白加合。然而，IsoKs 也在体外与磷脂酰乙醇胺（PE）加合，并且细胞内是否形成 PE 加合物或对 IsoKs 的作用是否有贡献尚不清楚。当放射性标记的 IsoK 被添加到 HEK293 细胞中时，40%的放射性标记物被提取到氯仿下层，这表明可能形成了 PE 加合物。因此，我们开发了在细胞中测量 IsoK-PE 加合物的方法。通过链霉菌 chromofuscus 磷脂酶 D 将 IsoK-PE 水解为 IsoK-乙醇胺后，通过 LC/MS/MS 定量 IsoK-PE。在 HEK293 和人脐静脉内皮细胞（HUVEC）中，IsoK 剂量依赖性地增加 PE 加合物浓度，比蛋白质加合物增加的程度更大。为了测试 IsoK-PE 形成的生物学意义，我们用 IsoK-PE 处理 HUVEC。IsoK-PE 剂量依赖性地诱导细胞毒性（LC（50）2.2 μM）。这些结果表明细胞内的 PE 是 IsoKs 的重要靶标，并且 PE 加合物的形成可能介导 IsoKs 与疾病相关的一些生物学效应。
