Nonenzymatic Free Radical-catalyzed Generation of Thromboxane-like Compounds (Isothromboxanes) in Vivo

Abstract

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.

Formation of F-ring Isoprostane-like Compounds (F3-Isoprostanes) in Vivo from Eicosapentaenoic Acid

“…This extended area of the TLC plate was determined to contain F 3 -IsoPs by analyzing small 5-mm cuts using approaches for their identification described below. The M-⅐CH 2 C 6 F 5 ions were monitored for quantification (m/z 567 for F 3 total amount of F 3 -IsoPs was determined by integrating the peak area of material in the m/z 567 channel in comparison with the m/z 573 channel. GC/NICI/MS was carried out using an Agilent Technologies 6890N Network GC/MS system.…”

“…Formation of these compounds proceeds through the generation of bicyclic endoperoxide PGH 2 -like intermediates that are reduced to PGF 2 -like compounds termed F 2 -IsoPs (1) or undergo rearrangement to PGD 2 and PGE 2 -like compounds (D 2 /E 2 -IsoPs) (2) and thromboxane-like compounds (isothromboxanes) (3). Unlike cyclooxygenase-derived PGs, IsoPs are formed in situ esterified to phospholipids and are subsequently released, presumably by the action of a phospholipase A 2 (4).…”

Thromboxane A2 Signalling in Humans: a ‘Tail’ of Two Receptors

“…The isoprostanes were originally discovered in 1990 by Morrow et al, [64] and are now believed to act as potent mediators of oxidative injury [64][65][66][67][68]. As such, the isoprostanes are mainly generated from arachidonic acid non-enzymatically and are synthesised in situ in phospholipids and then released through the actions of phospholipases, such as phospholipase A 2 [64,65].…”

“…A wide range of prostanoid-like isoprostanes is generated including the PGF 2α -, the PGD 2 -, and PGE 2 -series [64,69]. The F 2 series are the most common and one of these compounds 8-epi PGF 2α , also recently termed iPF 2α -III [69], is the most abundant isoprostane generated in situations of oxidative injury [64][65][66][67][68][69]. More recently, similar free-radical derived isomers of other polyunsaturated fatty acids have been described [69,70].…”

Formation of Highly Reactive γ-Ketoaldehydes (Neuroketals) as Products of the Neuroprostane Pathway

“…Their formation proceeds via PGH 2 -like bicyclic endoperoxide intermediates, which are reduced to form F-ring IsoPs (F 2 -IsoPs) (1) or undergo rearrangement to form D-ring and E-ring IsoPs (2) and isothromboxanes (3). Recently, we reported that IsoP endoperoxide intermediates also undergo rearrangement to form highly reactive ␥-ketoaldehyde levuglandin-like compounds (4).…”