Abstract
Abstract: :
Purpose: To elucidate the effects of melanin on A2E photooxidation in RPE cells. Methods: A2E was synthesized as described by Parish, CA et al. (PNAS 1998: 95, 14609). Passage 5–6 calf RPE cells were fed calf melanin and old human melanin. All human melanin was from the donor eyes aged from 60–90. Cells were incubated at 37oC fed melanin over a period of six days. Then fresh media or media containing 25uM A2E was added to cultures. Cells were allowed to take up A2E for 5 hours. The media was removed and cells were washed with PBSA. Fresh PBSA was added and the cells were irradiated (Philips "Special Blue" Bilirubin bulb, 20W) through a ¼ inch sheet of plexiglass to block the small UV output of the lamp. The samples were irradiated for either 20 or 30 minute intervals. After irradiation, cells were subjected to trypsin digestion, collected in PBSA and were homogenized. Suspensions were extracted using the Folch chloroform–methanol extraction method. The organic layer was collected and analyzed by LC–MS (Thermo Finnigan, LCQ Advantage, Surveyor; Surveyor LC with PDA detector, quadrupole ion trap mass analyzer, electrospray ion source). Results: The direct irradiation of A2E leads to a mixture of photoproducts. The first group consists of a series of single additions of oxygen resulting in M+16 (i.e., 608 amu, 624 amu etc.) The second group results from two hydrogen atom reduction or oxidation giving M+/– 2 amu. Calf melanin dramatically inhibits A2E oxidation. Over a total irradiation period of 90 minutes, calf melanin consistently reduces the rate of formation of the 608 amu and 624 amu products by 50%. In addition to the formation of the 608 and 624 amu products, dehydrogenation products of m/z 606 and 622 amu were also detected. Calf melanin was also effective in inhibiting the formation of these products. Finally, old human melanin did not inhibit the photooxidation of A2E. In fact, there was a small, but consistent, increase in A2E photooxidation in the presence of old human RPE melanin comparable to that observed for irradiation of depigmented cells. Conclusions:Calf and presumably young human RPE melanin is an effective antioxidant against photooxidation in RPE cells but this ability apparently deteriorates with age. In order to fully understand how RPE cells respond to either oxidative or photooxidative stress it is important to examine pigmented cells.
Keywords: retinal pigment epithelium • antioxidants • aging