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 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.
Morrow
J D
JD
Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232-6602, USA.
Awad
J A
JA
Wu
A
A
Zackert
W E
WE
Daniel
V C
VC
Roberts
L J
LJ
D000818:Animals; D004951:Esterification; D005609:Free Radicals / Q000737:chemistry; D006865:Hydrogenation; D007536:Isomerism; D015227:Lipid Peroxidation; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000096:biosynthesis; D051381:Rats; D013929:Thromboxane B2 / Q000737:chemistry* / Q000378:metabolism*
D005609:Free Radicals; D010743:Phospholipids; D013929:Thromboxane B2