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E.R. Gaillard, Z. Wang, J. Dillon; Identification Of Reactive Intermediate Species Formed During The Autooxidation And Photooxidation Of A2E . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2084.
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To characterize the autooxidation and photooxidation products of A2E. We have previously characterized two groups of oxidation products, oxides and smaller molecular weight aldehydes, and here we report on the characterization of the third group of products, ketones. These species are more reactive than A2E itself and thus constitute a group of potential cytotoxins to the retinal pigment epithelium. They are observed in extracts of human retinal lipofuscin.
A2E was kept in the dark for 3 weeks (autooxidation) or irradiated for one hour (photooxidation) with Phillips "special blue" Bilirubin bulbs througha ¼ inch sheet of plexiglass. Auto–oxidized or photo–oxidized A2E was dissolved in methanol and the 2, 4–dinitrophenylhydrazine solution was added. A 5:1 molar ratio of phenylhydrazine and the starting material A2E was used. The solution was kept at room temperature for 30 mins and followed by MS analysis (ThermoElectron LCQ Advantage).
The oxidation of A2E results in a complex mixture of products consisting of 1) lower molecular weight aldehydes, 2) a series of oxygen additions up to nine oxygen atoms and 3) those oxidations minus 2 amu (M–2). As reported previously, the initial oxidations give primarily furanoid oxides in the 5–8 positions after a reaarangement from the 5,6 epoxide. Up to the first four oxygens appear to be within the cyclohexenyl rings. This is based upon the appearance of a loss of m/z 106 which can only result from an unmodified acyclic moiety. A series of oxidation products also reacted with nitrophenylhydrazine (MW= 180 amu) suggesting the formation of aldehydes or ketones. Based on the molecular weights of 2,4–dinitrophenyldrazones, the molecular weights of those ketones or aldehydes are calculated to be 606 amu, 622 amu, or 638 amu, etc. Therefore, they belong to the second group (M–2 group) of the oxidation products. Since the formation of aldehydes does not result in the loss of two hydrogens simultaneously, we deduced that the M–2 series in the second group of oxidation products have ketone groups. MS/MS spectra indicated the carbonyl groups are on the cyclohexenyl ring.
the oxidation of A2E primarily results in the oxidation of the cyclohexenyl ring. This includes the formation of carbonyl compounds. These latter may react further and contribute to the modification of proteins or other important cellular components.
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