Nonenzymatic free radical-catalyzed generation of thromboxane-like compounds (isothromboxanes) in vivo

The isoprostanes (IsoPs) are novel bioactive prostaglandin-like compounds produced in vivo by free radical catalyzed peroxidation of arachidonyl-containing lip ids, 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 B-2 (TxB(2))-like compounds, termed B-2-isothromboxanes (B-2-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 TxB(2). Incubations of arachidonic acid with Fe/ADP/ascorbate for 30 min in vitro generated a series of peaks representing putative B-2-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 B-2-IsoTx. Analysis of the compounds by electron ionization mass spectrometry yielded multiple mass spectra similar to those of TxB(2). B-2-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 B-2-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 B-2-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.