Abstract
Methods::
A2E was synthesized as described by Parrish et al. (1995). A2E was separated from the mixture by HPLC using a Phenomenex Synergi Max RP C12 4 µm semi-preparative column (250 x 10 mm) and an isocratic mobile phase of 97% methanol: 3% water with 0.1% TFA for 1 hour at 1 mL/min. A2E was dissolved into 100µL of methanol and 2mL of water with 0.1% formic acid. The mixture was divided into 4 samples. The first sample was irradiated under blue light (two Philips "Special Blue" 20W Bilirubin bulbs) for 60 minutes. The second and third 200µL aliquots were also irradiated for 60 minutes as above but GSH was added either before or after irradiation. The fourth sample was mixed with glutathione and placed in dark for 24 hours. The samples were then directly analyzed by ESI-MS ((ThermoFinnegan Advantage LC/Surveyor ion trap MS).
Results::
: There are numerous oxidation products of A2E that result from irradiation with blue light such as the furanoid oxides and other oxygen addition products with m/z 608, 624, 640 etc. and a series of aldehydes with m/z 488, 472, 448, 432, 422, 406, 382, and 366. These are observed for both samples with and without glutathione after 60 minutes irradiation. Moreover, the irradiation mixture in the presence of glutathione (m/z = 308) showed the formation of GSSG (m/z 616 peak). Two reactions were observed between GSH and A2E oxidation products. The first is the reduction of the higher molecular weight A2E oxidation product (eg, shift in m/z 640 to 642) with the concomitant oxidation of GSH to GSSG. The second is the formation A2E aldehyde adducts with GSH presumably via Michael addition. For example, the products with m/z 778.7 (m/z = 472 + m/z 308 - 2) yields the parent aldehyde ion (m/z = 472) and GSH ion (m/z = 308) in the MS/MS fragmentation. Conclusion : A2E oxidation can lead to the disruption of the redox stasis of the RPE by leading to the oxidation of GSH to GSSG and by forming GSH/A2E adducts. If these reactions occur in vivo, they could severely compromise RPE cell viability.
Keywords: oxidation/oxidative or free radical damage • retina • aging