title: Toward an understanding of bisretinoid autofluorescence bleaching and recovery.
abstract: OBJECTIVE
  To understand molecular mechanisms underlying photobleaching of the RPE fluorophores responsible for fundus autofluorescence.
  
  
  METHODS
  ARPE-19 cells were allowed to accumulate the bisretinoid, A2E, and were irradiated at 430 nm. For some experiments, the cells were pretreated with vitamin E or sulforaphane and N-acetylcysteine; samples included A2E-free cells. The cells were analyzed by fluorescence microscopy and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis. A2E free cells were also irradiated and analyzed. Cell death was quantified by double labeling with a membrane impermeable dye and 4',6'-diamino-2-phenylindole (DAPI).
  
  
  RESULTS
  A2E that had accumulated in ARPE-19 cells exhibited irradiation-associated autofluorescence bleaching despite the absence of appreciable cell death. Chromatographic analysis with absorbance, fluorescence, and mass spectrometry detection revealed that irradiation of A2E was associated with A2E photoisomerization, photooxidation, and photodegradation. Pretreatment with vitamin E favored fluorescence recovery; this finding was consistent with a process involving photooxidation. A2E that was not cell-associated underwent irradiation-induced bleaching, but fluorescence recovery was not observed.
  
  
  CONCLUSIONS
  Using cell-associated A2E as a model of RPE bisretinoid behavior, photobleaching and autofluorescence recovery was observed; these changes were similar to RPE autofluorescence reduction in vivo. The potential for autofluorescence recovery is dependent on light dose and antioxidant status. Fluorescence bleaching of bisretinoid involves photooxidative and photodegradative processes.
authors.affiliation: Department of Ophthalmology, Columbia University, New York, New York 10032, USA.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Jilin
authors.initials: J
authors.lastname: Zhou
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Hunter
authors.forename: David R
authors.initials: DR
authors.lastname: Williams
authors.forename: Janet R
authors.initials: JR
authors.lastname: Sparrow
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D017209:Apoptosis / Q000528:radiation effects; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005453:Fluorescence; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D017879:Isothiocyanates; D008027:Light; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D011726:Pyridinium Compounds / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects*; D012176:Retinoids / Q000378:metabolism*; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology; D014810:Vitamin E / Q000494:pharmacology
chemical_list: C112040:A2-E (N-retinylidene-N-retinylethanolamine); D017879:Isothiocyanates; D011726:Pyridinium Compounds; D012176:Retinoids; D013454:Sulfoxides; D013861:Thiocyanates; D014810:Vitamin E; C016766:sulforaphane; D005978:Glutathione; D000111:Acetylcysteine