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E. Gaillard, L. Avalle, J. Dillon; The autooxidation of A2E, a component of human retinal lipofuscin . Invest. Ophthalmol. Vis. Sci. 2004;45(13):755.
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Purpose: To determine the stability of A2E in air. Methods: A2E was synthesized as described by Parish, CA et al. (PNAS 1998: 95, 14609). All other chemicals were purchased from Sigma–Aldrich. Water was purified with a Millipore Milli–Q Plus filtration system. Synthetic melanin was prepared by the exhaustive oxidation of L–DOPA. After solutions of A2E were kept in the dark for 10 to 16 days, they were extracted with CH3OH:CHCl3 and the extract was analyzed by LC–MS (Thermo Finnigan, LCQ Advantage, Surveyor). The instrument consists of a Surveyor LC with PDA detector and a quadrupole ion trap mass analyzer with an electrospray ion source. Results: Upon standing in water in the dark, A2E (m/z = 592) forms a series of compounds with the sequential addition of single oxygen atoms (i.e., m/z = 608, 624 etc). In addition there is also the formation of M–2 associated with each molecular ion. This series of oxidation products is similar to that found in the direct photooxidation of A2E (although much slower; e.g., days vs. minutes) with the MS/MS data for the autooxidation products being comparable to the MS/MS data for the photoproducts. For example, the 608 amu product gives fragments of M–140, M–150, M–166, M–174 and M–190 which is the same as that found for the 608 amu product generated from the photooxidation of A2E. The appearance of the M–140 fragment suggests that it is a furanoid oxide as has been observed in the oxidation of beta–carotene (Baldas, J. and Porter, Q.N. 1966: Chem. Comm. 23, 852). In addition to these higher molecular weight products, smaller compounds were also formed. Again, in analogy with beta–carotene, these appear to arise from oxidative cleavage of the side chain with the subsequent formation of aldehydes. These oxidations are attenuated in organic solvents, with the addition of EDTA and in the presence of synthetic melanin. Conclusions:1) These oxidations are, out of necessity, free radical processes. Because the autooxidation products are similar to the products of photolysis, the photoproducts are most likely formed from free radical processes. 2) The formation of highly reactive aldehydes in RPE cells would have damaging consequences to the tissue. 3) Care must be taken in the storage and handling of A2E.
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