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Sassan Azarian, Xue Zhang, Van Nguyen-Tran, Michael Petrassi, Sha-Mei Liao, Bruce D. Jaffee, Satchidananda Panda, Richard Glynne, Thomas Hollenbeck; A Rapid, Sensitive, Medium-Throughput Assay for Quantitation of Ocular A2E And Visual Cycle Retinoids. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3354.
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© ARVO (1962-2015); The Authors (2016-present)
Several therapeutic approaches are being evaluated to reduce the accumulation of ocular lipofuscin or its components, e.g. A2E. Some approaches involve visual cycle inhibition. We report a method for quantitation of A2E and retinoids in the same extracts, for labor-intensive drug efficacy studies. As proof of principle, the method was used to analyze a chronic study of Abca4-/- mice treated with retinylamine, a visual cycle inhibitor. In addition, the A2E level in several mouse models was determined.
Extracts from enucleated undissected eyes were prepared and processed in 96-well format. Half of each extract was analyzed for A2E by LC/MS/MS and the other half for retinoids by HPLC. Signals were quantitated by comparison with synthetic standards. In the chronic study, Abca4-/- mice were orally dosed for 8 weeks with 150 mg/kg retinylamine or vehicle (vegetable oil) by weekly gavage.
Due to batch processing of whole eyes, several hundred samples can be processed per day. The LC/MS/MS assay was linear up to => 300 pmol/eye of A2E (R2 = 0.99). Ocular levels of A2E and retinoids in Abca4-/- mice were in good agreement with published values. Chronic retinylamine treatment of Abca4-/- showed significant reduction of visual cycle activity and ocular A2E level. In addition to Abca4-/-, A2E level was significantly elevated in Sod1-/- and Cfh-/- mice. Abca4-/- Rdh8-/- mice will be analyzed next.
Our method was validated with A2E and retinoid data from untreated Abca4-/- mice. In the proof of principle study the method reported the effects of chronic retinylamine treatment on visual cycle activity and A2E level. The increased A2E levels in retinal models of oxidative stress (Sod1-/-) and complement activation (Cfh-/-) suggest a cross talk between various pathogenic mechanisms, potentially culminating in a vicious cycle. This method should prove useful for characterization of animal models and their use in drug efficacy studies.
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