title: Photo-products of retinal pigment epithelial bisretinoids react with cellular thiols.
abstract: OBJECTIVE
  Bisretinoids such as A2E that accumulate as components of the lipofuscin of retinal pigment epithelial cells are implicated in some retinal disease processes. These compounds undergo light-induced oxidation and cleavage with the latter releasing of a mixture of aldehyde-bearing fragments, including dicarbonyl methylglyoxal. We tested for the reactivity of photooxidation and photodegradation products of A2E with thiol-containing glutathione (GSH).
  
  
  METHODS
  In cell-free assays, we measured the ability of photooxo-A2E to competitively inhibit the GSH-mediated reduction of the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid). Cellular GSH was assayed colorimetrically. Products of GSH reduction and GSH-adducts were detected by electrospray ionization mass spectrometry (ESI-MS) and GSH and oxidized GSH (glutathione disulfide [GSSG]) were quantified from chromatographic peak areas.
  
  
  RESULTS
  We found that GSH can donate hydrogen atoms to, and form conjugates with, photooxidized forms of the bisretinoid A2E and with its photocleavage products. Reaction with non-photooxidized A2E was not observed. Chemical reduction by GSH involved the donation of a hydrogen atom from each of two GSHs. The ratio of GSH consumed to GSSG formed was consistent with GSH being used for both reduction and adduct formation. With the aid of synthesized standards, methylglyoxal-GSH adducts were identified within mixtures of GSH and photooxidized A2E; the adducts formed noncatalytically and by glutathione-S-transferase mediation.
  
  
  CONCLUSIONS
  Reduction and adduct formation by GSH likely limits the reactivity of bisretinoid photoproducts and may aid their elimination from the cells. These findings are significant to forms of macular degeneration associated with bisretinoid formation and maculopathy stemming from GSH synthase deficiency.
authors.affiliation: Department of Ophthalmology, Columbia University, New York, NY, USA.
authors.forename: Kee Dong
authors.initials: KD
authors.lastname: Yoon
authors.forename: Kazunori
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Jilin
authors.initials: J
authors.lastname: Zhou
authors.forename: Janet R
authors.initials: JR
authors.lastname: Sparrow
mesh_terms: D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002474:Cell-Free System / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008027:Light; D008062:Lipofuscin / Q000737:chemistry / Q000378:metabolism*; D008268:Macular Degeneration / Q000378:metabolism* / Q000473:pathology; D010084:Oxidation-Reduction; D055668:Photochemical Processes / Q000528:radiation effects; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D012168:Retinal Pigments / Q000378:metabolism*; D012176:Retinoids / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D000447:Aldehydes; D008062:Lipofuscin; D012168:Retinal Pigments; D012176:Retinoids; D013438:Sulfhydryl Compounds; D005982:Glutathione Transferase; D005978:Glutathione; D019803:Glutathione Disulfide