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Photo-induced oxygenation generates biologically active, oxidatively truncated lipids in the retina. Previously, doubly allylic dihydroperoxides, 9,12- dihydroperoxyoctadeca-10,13-dienoic acid (9,12-diHPODE) and 10,13- dihydroperoxyoctadeca-8,11-dienoic acid (10,13-diHPODE), were postulated as key intermediates in the free radical-promoted oxidative fragmentation of linoleate that generates aldehydes, such as the cytotoxic ã-hydroxyalkenal 4-hydroxy-2-nonenal (HNE), in vivo. We developed an efficient preparation of regioisomerically pure 9,12- and 10,13-diHPODE, devised to enable studies of their fragmentation reactions. Free radical-induced oxygenation of linoleate initially generates conjugated monohydroperoxy octadecadienoates (HPODEs) that are then converted into diHPODEs. In contrast, we found that singlet oxygenation of conjugated HPODEs does not produce diHPODEs. Unconjugated HPODEs are unique products of singlet oxygenation of linoleate that are coproduced with conjugated HPODEs. Preparative separation of the mixture of regioisomeric mono and diHPODEs generated by singlet oxygenation of linloeate is impractical. However, a simple tactic circumvented the problem. Thus, selective conversion of the undesired conjugated HPODEs into Diels-Alder adducts could be accomplished under mild conditions by reaction with N-phenyltriazolinedione. These adducts were readily removed and the two remaining unconjugated HPODEs could then be easily isolated as regioisomerically pure compounds. Each of these was subsequently converted into a different, regioisomerically pure, diHPODE through further singlet oxygenation.A study of the fragmentation of authentic linoleic dihydroperoxides provided for the first time, direct experimental evidence for their validity as immediate precursors for 9-keto-12-oxo-10-dodecenoic acid (KODA) and 9-hydroxy-12-oxo-10(E)-dodecenoic acid (HODA). Treating dihydroperoxides with vitamin E gave an unexpected result. Vitamin E, instead of preventing, promoted their oxidative fragmentation into truncated bifunctional electrophiles. The reaction was inhibited by the addition of the metal chelator, DTPA, indicating metal ion dependence. These findings may help to explain accumulating paradoxical evidence that vitamin E supplements often provide little or no protection against oxidative injury. The observation that UV light can promote decomposition of diHPODEs is especially important regarding the oxidative damage occurring in retina. Subsequent formation and breakdown of PUFA-derived hydroperoxides into truncated small molecules may occur through photo-induced singlet oxygenation even under conditions that block free radical-induced oxidation.