Abstract
Abstract: :
Purpose: To determine the primary mechanistic pathway involved in the photooxidation of A2E. Methods: A2E was synthesized as described by Parish, CA et al. (PNAS 1998: 95, 14609). All other chemicals were purchased from Sigma–Aldrich. Irradiations were carried out with either a 200W Xe arc lamp fitted with a 400 nm long pass filter or with Philips "Special Blue" 20W Bilirubin bulb through a ¼ inch sheet of plexiglass to block the small UV output of the lamp. After irradiation, the solutions 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. For the EPR measurements, A2E was dissolved in EtOH and a small aliquot (10 µL) was added to aerated PBS buffer containing DMPO (80 mM). Samples were transferred to a flat aqueous EPR cell and illuminated directly inside the microwave cavity from a distance of 30 cm using a tungsten halogen low voltage lamp with a reflector (model EXR). Spectra were recorded using the following instrumental settings: microwave power 20 mW, modulation amplitude 1 G, receiver gain 2 x 106, time constant 81.92 ms, conversion time 40.96 ms, and scan rate of 80G/41.94 s. Results: It has been reported that A2E can act as a photosensitizer of either free radical (type I) or singlet oxygen (type II) mediated mechanisms. In order to ascertain which is the dominate photochemical process, a series of experiments were performed with the following results: 1) the rate of loss of A2E was decreased in deuterated solvents which is not expected for singlet oxygen. 2) Although azide decreased the rate of photooxidation, it was found to be due to the azide assisted photochemical destruction of A2E and not singlet oxygen quenching. 3) The direct photolysis of A2E gives a series of single oxygen additions. In contrast the reaction of A2E with singlet oxygen (photochemically generated from a porphyrin) resulted in the initial formation of a photoproduct containing two oxygens, most likely the expected endoperoxide. 4) the major species trapped by DMSO detected using EPR is superoxide. This signal was negated in the presence of SOD. Conclusions:The major contributions to the mechanism(s) of photooxidation of A2E are via free radical processes and not through the intermediacy of singlet oxygen.
Keywords: retinal pigment epithelium • radiation damage: light/UV • aging