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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 lipids. Previously, we have identified IsoPs containing F-type and D- and E-type prostane rings that are formed by reduction and rearrangement o...

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Bibliographic Details
Published in:The Journal of biological chemistry 1996-09, Vol.271 (38), p.23185-23190
Main Authors: Morrow, J D, Awad, J A, Wu, A, Zackert, W E, Daniel, V C, Roberts, 2nd, L J
Format: Article
Language:English
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Summary: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 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 CCl 4 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 (
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.271.38.23185