Free radical-induced generation of isoprostanes in vivo: evidence for the formation of D-ring and E-ring isoprostanes

We recently reported the discovery that a series of novel prostaglandin (PG)F₂-like compounds (F₂-isoprostanes) are produced in vivo independent of the cyclooxygenase as products of free radical-catalyzed lipid peroxidation. F₂-isoprostanes are initially formed in situ from arachidonic acid esterified to phospholipids and then released preformed. We have now investigated whether PGD₂/E₂-like isoprostanes are also produced by rearrangement of the PGG₂-like intermediates involved in isoprostane formation. Using a variety of approaches utilizing mass spectrometry, compelling evidence was obtained for the presence of D₂/E₂-isoprostane-containing phosphospholipids in the liver (85 ± 33 ng/g of liver) and free D₂/E₂-isoprostanes in the circulation (215 ± 90 pg/ml) of rats treated with CCl₄ to induce lipid peroxidation. In untreated rats, levels of D₂/E₂-isoprostanes esterified in liver phospholipids were much lower (0.90 ± 0.10 ng/g), and free compounds could not be detected in the circulation (2-isoprostanes that would be expected to be formed in abundance, 8-epi-PGE₂, was found to be a potent renal vasoconstrictor, and these effects could be abrogated by SQ29548, a thromboxane receptor antagonist. Further understanding of the biological consequences of the formation of these novel compounds and factors that influence their formation may provide valuable insights into the pathophysiology of oxidant injury.