title: Preparative and biosynthetic insights into pdA2E and isopdA2E, retinal-derived fluorophores of retinal pigment epithelial lipofuscin.
abstract: OBJECTIVE
  Retinal-derived fluorophores that accumulate as RPE lipofuscin are implicated in pathological mechanisms of AMD. One component of RPE lipofuscin has been characterized as pdA2E, a pyridinium adduct derived from all-trans-retinal and excess ethanolamine. One-step preparation and biosynthetic studies of pdA2E and its novel isomer called isopdA2E are reported.
  
  
  METHODS
  Biosynthetic reaction mixtures, RPE/choroids and neural retinas dissected from bovines, eyes harvested from Abca4(-/-)Rdh8(-/-) mice, irradiated samples, and enzyme-treated solutions were analyzed by HPLC, mass spectrometry, nuclear magnetic resonance spectroscopy, fluorescence spectrophotometry, and density functional theory (DFT).
  
  
  RESULTS
  Optimization of the in vitro synthesis of pdA2E resulted in a biomimetic preparation of this pigment in a yield of 15%; this protocol also allowed the identification of isopdA2E, a double-bond isomer of pdA2E at the C13C14 position in bovine RPE lipofuscin. Interconversion between these two molecules occurs when either pdA2E or isopdA2E is exposed to light. A phospholipase D-based assay demonstrated the possibility of pdA2-PE being formed in neural retina and served as a precursor of pdA2E in the biosynthetic pathway. DFT calculations revealed that the 492-nm absorbance was assigned to the long arm of pdA2E/isopdA2E and the 340/342-nm absorbance to the short arm. Fluorescence efficiency of pdA2E and isopdA2E is very similar, but is much weaker in comparison with A2E, isoA2E, and iisoA2E.
  
  
  CONCLUSIONS
  Our results facilitate the understanding of compositions and biosynthetic pathways of adverse RPE lipofuscin.
authors.affiliation: College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
authors.forename: Junli
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Eye Center of the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Yao
authors.affiliation: College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
authors.forename: Qiuxia
authors.initials: Q
authors.lastname: Jin
authors.affiliation: Key Laboratory of Organosilicon Chemistry and Material Technology, Hangzhou Normal University, Hangzhou, China.
authors.forename: Kezhi
authors.initials: K
authors.lastname: Jiang
authors.affiliation: School of Medicine, Zhejiang University City College, Hangzhou, China.
authors.forename: Jingmeng
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Ophthalmology, Zhejiang Provincial People's Hospital, Hangzhou, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Liu
authors.affiliation: College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China Eye Center of the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
authors.forename: Yalin
authors.initials: Y
authors.lastname: Wu
mesh_terms: D000818:Animals; D032701:Biomimetics; D002417:Cattle; D002829:Choroid / Q000378:metabolism*; D004195:Disease Models, Animal; D005451:Fluorescein Angiography; D007536:Isomerism; D008062:Lipofuscin / Q000737:chemistry*; D051379:Mice; D018345:Mice, Knockout; D011726:Pyridinium Compounds / Q000737:chemistry; D012162:Retinal Degeneration / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D012176:Retinoids / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D008062:Lipofuscin; D011726:Pyridinium Compounds; D012176:Retinoids; C000590172:pdA2E compound
keywords: biomarker; biomimetic synthesis; biosynthetic pathway; electronic transition; lipofuscin; retinal pigment epithelium