The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane

The autofluorescent lipofuscin that accumulates in retinal pigment epitheli al cells with age may contribute to an age-related decline in cell function . The major lipofuscin fluorophore, A2E, is a pyridinium bisretinoid. We pr eviously proposed that the biogenesis of A2E involves the following: (i) fo rmation of the Schiff base, N-retinylidene phosphatidylethanolamine from al l-trans-retinal and phosphatidylethanolamine in the photoreceptor outer seg ment membrane; (ii) further reaction of N-retinylidene phosphatidylethanola mine with retinal to yield phosphatidylethanolamine-bisretinoid, A2-PE; (ii i) hydrolysis of A2-PE to generate A2E. To provide evidence for this biogen ic scheme, all-trans-retinal was reacted with dipalmitoyl-L-alpha-phosphati dylethanolamine to yield DP-A2-PE (A2-PE), as confirmed by UV, with mass sp ectrometry revealing the molecular ion at m/z 1222.9 (C77H124O8PN) accompan ied by product ion at m/z 672.8, representing the phosphoryl-A2E fragment o f A2-PE. In reaction mixtures of retinal and outer segments and in samples of Royal College of Surgeons rat retina containing outer segment membranous debris, A2-PE was detected as a series of high performance liquid chromato graphy peaks, each with UV similar to reference A2-PE. By mass spectrometry , A2-PE consisted of multiple peaks, representing fatty acids with differen t chain lengths, and the phosphoryl-A2E moiety, m/z 673. Incubation of the retinal/outer segment reaction mixture with phospholipase D generated A2E, as detected by high performance liquid chromatography, thus confirming A2-P E as the A2E precursor.