Oxidative Lipidomics of Apoptosis: Quantitative Assessment of Phospholipid Hydroperoxides in Cells and Tissues
Oxidative Lipidomics of Apoptosis: Quantitative Assessment of Phospholipid Hydroperoxides in Cells and Tissues
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DOI：
10.1007/978-1-60327-029-8_21
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发表时间：
2010-01-01
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影响因子：
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通讯作者：
Kagan, Valerian E.
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文献类型：
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作者：
Tyurin, Vladimir A.;Tyurina, Yulia Y.;Kagan, Valerian E.
氧化磷脂在线粒体凋亡阶段的执行和凋亡细胞的清除中起重要作用。氧化脂质组学可以成功地鉴定和定量细胞凋亡过程中产生的氧化磷脂。利用这种方法，可以通过电喷雾电离（ESI）等软电离质谱技术鉴定和表征磷脂及其氢过氧化物的多种分子种类。脂质氢过氧化物的定量评估是通过基于荧光高效液相色谱的方案进行的。该方案是基于磷脂的油分离使用二维高效薄层色谱（2-D-HPTLC）。磷脂通过磷脂酶A(2)水解。用Amplex红色试剂和微过氧化物酶-11 （MP-11）荧光法定量测定脂肪酸氢过氧化物（FA-OOH）的释放。本方案的检测限为1- 2pmol脂质氢过氧化物。脂质阵列与氧化脂质阵列通过比较磷脂的丰度与氧化磷脂的丰度来进行。利用氧化脂质组学方法，我们发现细胞凋亡过程中磷脂氧化的模式是非随机的，并且不遵循它们在几种细胞凋亡和各种疾病状态中的丰度。这对体内细胞凋亡的评估具有重要意义。阴离子磷脂，心磷脂（CL）和磷脂酰丝氨酸（PS），是首选的过氧化底物。
Oxidized phospholipids play essential roles in execution of mitochondrial stage of apoptosis and clearance of apoptotic cells. The identification and quantification of oxidized phospholipids generated during apoptosis can be successfully achieved by oxidative lipidomics. With this approach, diverse molecular species of phospholipids and their hydroperoxides arc identified and characterized by soft-ionization mass-spectrometry techniques Such as electrospray ionization (ESI). Quantitative assessment of lipid hydroperoxides is performed by fluorescence HPLC-based protocol. The protocol is based oil separation of phospholipids using two-dimensional-high-performance thin-layer chromatography (2-D-HPTLC). Phospholipids are hydrolyzed using phospholipase A(2). The fatty acid hydroperoxides (FA-OOH) released is quantified by a fluorometric assay using Amplex red reagent and microperoxidase-11 (MP-11). Detection limit of this protocol is 1-2 pmol of lipid hydroperoxides. Lipid arrays vs. oxidized lipid arrays call be performed by comparing the abundance of phospholipids with the abundance of oxidized phospholipids. Using oxidative lipidomics approach we show that the pattern of phospholipid oxidation during apoptosis is nonrandom and does not follow their abundance in several types of cells undergoing apoptosis and a variety of disease states. This has important implications for evaluation of apoptosis in vivo. The anionic phospholipids, cardiolipin (CL) and phosphatidylserine (PS), arc the preferred peroxidation substrates.