Morrow J D, Awad J A, Wu A, Zackert W E, Daniel V C, Roberts L J
Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232-6602, USA.
J Biol Chem. 1996 Sep 20;271(38):23185-90. doi: 10.1074/jbc.271.38.23185.
The isoprostanes (IsoPs) are novel bioactive prostaglandin-like compounds produced in vivo by free radical-catalyzed peroxidation of arachidonyl-containing lipids. Previously, we have identified IsoPs containing F-type and D- and E-type prostane rings that are formed by reduction and rearrangement of IsoP endoperoxide intermediates, respectively. We now explore whether thromboxane B2 (TxB2)-like compounds, termed B2-isothromboxanes (B2-IsoTxs), are formed by rearrangement of IsoP endoperoxides. Detection of these compounds was carried out using a stable isotope dilution mass spectrometric assay originally developed for quantification of cyclooxygenase-derived TxB2. Incubations of arachidonic acid with Fe/ADP/ascorbate for 30 min in vitro generated a series of peaks representing putative B2-IsoTx at levels of 62.4 +/- 21.0 ng/mg arachidonate. Using various chemical modification and derivatization approaches, it was determined that these compounds contained hemiacetal ring structures and two double bonds, as would be expected for B2-IsoTx. Analysis of the compounds by electron ionization mass spectrometry yielded multiple mass spectra similar to those of TxB2. B2-IsoTxs are also formed esterified to phospholipids; oxidation of arachidonyl-containing phosphatidylcholine in vitro followed by hydrolysis resulted in the release of large amounts of these compounds. To explore whether B2-IsoTxs are also formed in vivo, a well characterized animal model of lipid peroxidation consisting of orogastric administration of CCl4 to rats was used. Levels of B2-IsoTx esterified in lipids in the liver increased 41-fold from 2.5 +/- 0.5 to 102 +/- 30 ng/g of liver. In addition, circulating levels of free compounds increased from undetectable (<5 pg/ml) to 185 +/- 30 pg/ml after CCl4, a 37-fold increase. Thus, we have provided evidence that IsoTxs constitute another novel class of eicosanoids produced in vivo nonenzymatically by free radical-catalyzed lipid peroxidation. These studies thus expand our understanding of products of lipid peroxidation formed in vivo from the free radical-catalyzed peroxidation of arachidonic acid.
异前列腺素（IsoPs）是一类新型的生物活性前列腺素样化合物，在体内由含花生四烯酸的脂质经自由基催化的过氧化反应产生。此前，我们已鉴定出含有F型以及分别由IsoP内过氧化物中间体还原和重排形成的D型和E型前列腺环的异前列腺素。我们现在探究类似血栓素B2（TxB2）的化合物，即B2 - 异血栓素（B2 - IsoTxs），是否由IsoP内过氧化物重排形成。使用最初开发用于定量环氧化酶衍生的TxB2的稳定同位素稀释质谱分析法对这些化合物进行检测。在体外将花生四烯酸与铁/二磷酸腺苷/抗坏血酸孵育30分钟，产生了一系列代表假定B2 - IsoTx的峰，其水平为62.4±21.0 ng/mg花生四烯酸。使用各种化学修饰和衍生化方法确定，这些化合物含有半缩醛环结构和两个双键，这与B2 - IsoTx预期的结构相符。通过电子电离质谱法对这些化合物进行分析，得到了多个与TxB2相似的质谱图。B2 - IsoTxs也以酯化形式与磷脂结合存在；体外对含花生四烯酸的磷脂酰胆碱进行氧化然后水解，导致大量此类化合物的释放。为了探究B2 - IsoTxs是否也在体内形成，使用了一种特征明确的脂质过氧化动物模型，即对大鼠经口胃给予四氯化碳。肝脏中脂质酯化的B2 - IsoTx水平从2.5±0.5 ng/g肝脏增加到102±30 ng/g肝脏，增加了41倍。此外，游离化合物的循环水平在给予四氯化碳后从不可检测（<5 pg/ml）增加到185±30 pg/ml，增加了37倍。因此，我们提供了证据表明异血栓素构成了另一类由自由基催化的脂质过氧化在体内非酶促产生的新型类二十烷酸。这些研究从而扩展了我们对体内由花生四烯酸自由基催化过氧化形成的脂质过氧化产物的理解。
