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It has heretofore been assumed that the cyclooxygenases (COXs) are solely responsible for peostaglandin (PG) synthesis in vivo. An important structural feature of PGH2 formed by COX is the trans-configuration of side chains relative to the prostane ring. Previously, we reported that a series of PG-like compounds termed isoprostanes (IsoPs) are formed in vivo in humans from the free radical-catalyzed peroxidation of arachidonate independent of COX. A major difference between these compounds and PGs is that IsoPs are formed from endoperoxide intermediates, the vast majority of which contain side chains that are cis relative to the prostane ring. In addition, unlike the formation of eicosanoids from COX, IsoPs are formed as racemic mixtures because they are generated nonenzymatically. IsoPs containing E- and D-type prostane rings (E2/D2-IsoPs) are one class of IsoPs formed, and we have reported previously that one of the major IsoPs generated is 15-E2t-IsoP (8-iso-PGE2). Unlike PGE2, 15-E2t-IsoP is significantly more unstable in buffered solutions in vitro and undergoes epimerization to PGE2. Analogously, the D-ring IsoP (15-D2c-IsoP) would be predicted to rearrange to PGD2. We now report that compounds identical in all respects to PGE2 and PGD2 and their respective enantiomers are generated in vivo via the IsoP pathway, presumably by epimerization of racemic 15-E2t-IsoP and 15-D2c-IsoP, respectively. Racemic PGE2 and PGD2 were present esterified in phospholipids derived from liver tissue from rats exposed to oxidant stress at levels of 24 +/- 16 and 37 +/- 12 ng/g of tissue, respectively. In addition, racemic PGs, particularly PGD2, were present unesterified in urine from normal animals and humans and represented up to 10% of the total PG detected. Levels of racemic PGD2 increased 35-fold after treatment of rats with carbon tetrachloride to induce oxidant stress. In this setting, PGD2 and its enantiomer generated by the IsoP pathway represented approximately 30% of the total PGD2 present in urine. These findings strongly support the contention that a second pathway exists for the formation of bioactive PGs in vivo that is independent of COX.