The retinoid (visual) cycle is a complex enzymatic pathway that operates in the retina for the regeneration of 11-cis-retinal (11-cis-Ral), the inherent visual chromophore indispensable for vision. Deficiencies in the retinoid metabolism are involved in pathologic mechanisms of several forms of retinal diseases including age-related macular degeneration, Stargardt's disease, and Leber's congenital amaurosis, for which no effective cures presently exist. Nevertheless, the interference of abnormal retinoid metabolism with chemicals has been considered to be a promising strategy aimed at alleviating these retinal dysfunctions. Moreover, since gene therapy is gaining increasing importance in clinical practice, the modulation of key enzymes implicated with the retinoid cycle at a genetic level will hold great promise for the treatment of patients with degenerative diseases of the retina.
Toxic lipofuscin in the RPE (retinal pigment epithelium) is implicated in blindness in AMD (age-related macular degeneration) or recessive Stargardt's disease patients. In the present study, we identified a novel fluorescent lipofuscin component in human and bovine RPEs. Using 1D and 2D NMR and MS, we confirmed the structure of this pigment and called it pdA2E. It exhibits absorbance maxima at 492 and 342 nm, and is susceptible to photocatalytic isomerization and oxidation. This fluorophore was also detected in the eyecup extracts of Abca4(-/-)Rdh8(-/-) (Abca4 encodes ATP-binding cassette transporter 4 and Rdh8 encodes retinol dehydrogenase 8) mice, an AMD/recessive Stargardt's disease model. Excess amassing of pdA2E within RPE cells caused significant cell viability loss and membrane damage. The formation of pdA2E occurred when atRAL (all-trans-retinal) reacted with excess ethanolamine in the absence of acetic acid, and the process is likely to involve the participation of three atRAL molecules. Our findings suggest that endogenous pdA2E may serve as a sensitizer for yielding singlet oxygen and a singlet oxygen quencher, as well as a by-product of retinal metabolism, and its complete characterization facilitates the understanding of biosynthetic pathways by which adverse RPE lipofuscin constituents form.
Gene and drug therapies are being developed to alleviate vision loss in patients with Stargardt's disease and age-related macular degeneration (AMD). To evaluate the therapeutic effects of these treatments, organic solvents are routinely used to extract and quantify bisretinoid lipofuscin constituents, such as N-retinylidene-N-retinylethanolamine (A2E) and all-trans-retinal dimer (ATR-dimer). By high-performance liquid chromatography (HPLC), we found that A2E and ATR-dimer were both altered by tetrahydrofuran (THF) and chloroform, but were stable in dimethyl sulfoxide (DMSO) or methanol (MeOH). In addition, cyclohexane and ethanol (EtOH) did not alter ATR-dimer, whereas an alteration of A2E occurred in EtOH. On the basis of these findings, we designed processes II-IV, generated by modifications of process I, a routine method to measure bisretinoid compounds in vivo. Extra amounts of either ATR-dimer or A2E in mouse eyecups were released by processes II-IV versus process I. Efforts to clarify the effects of organic solvents on lipofuscin pigments are important because such studies can guide the handling of these fluorophores in related experiments.
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