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L. Avalle, J. Dillon, E.R. Gaillard; Action Spectrum for Singlet Oxygen Production by Human Retinal Lipofuscin . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1623.
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Purpose: To measure the action spectrum for singlet oxygen production by the organic soluble portion of human retinal lipofuscin. Methods: Human retinal lipofuscin granules were isolated according to the method of Feeney-Burns (Trans. Ophthal. Soc. U.K., 1983) and the organic soluble portion was obtained by extraction with 1:1:1 CHCl3:CH3OH:H2O. Samples were prepared in air saturated CH3OH and excitation was provided with the output of a Continuum Powerlite 9010 Nd:YAG pumped Sunlite OPO. Singlet oxygen emission was monitored at right angles to the excitation beam with a Ge diode. The excitation wavelength was tuned in 5 nm steps from 300 nm to 600 nm while maintaining constant beam energy. The output of the Ge diode was amplified, stopped at a 500 MHz digitizing oscilloscope (Tektronix TDS 620B) and passed to a PC for data storage and analysis. Results: The system was calibrated by measuring the action spectrum for singlet oxygen sensitization by tetraphenylporphyrin (H2TPP) where there was excellent agreement between the action spectrum and the ground state absorption spectrum of H2TPP. The action spectrum for singlet oxygen sensitization by the organic soluble fraction of lipofuscin had an absorption maximum at 380 nm and absorbs well above 400 nm. This spectrum corresponds to a component observed in the HPLC chromatogram of lipofuscin which has been isolated. Conclusions: In our original work on the photochemical properties of human retinal lipofuscin (Photochem. Photobiol., 1995), we observed that there was a very photoactive chromophore present in the mixture that did not correspond to A2E. This component has been identified by measuring the action spectrum for singlet oxygen production by lipofuscin. These data may further our understanding of the molecular mechanisms involved in light damage to the human retina.
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