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D.C. Paik, T.F. Hu, J.C. Merriam, J.P. Dillon; Photonitration Induced Protein Cross–Linking in Age–Related Cataract . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1686.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Covalent lens protein cross–linking occurs during cataract development. Photochemical damage is a well known mechanism involved in cataract formation. Nitrite ion, whose sources include smoking and inflammation, is elevated in the cataractous lens and can function as a photosensitizer. Thus, the present study was undertaken in order to investigate "photonitration" induced protein cross–linking as a possible mechanism for age–related lens cataract formation. Methods: Lens calf protein (2mg/ml) with 10 mM NaNO2 (pH 7.4) was photolyzed at constant temperature using a Xenon lamp (350 nm high pass filter). The reaction was stopped by addition of equimolar ammonium sulfamate. The samples were then acid hydrolyzed using isodesmosine as an internal standard. Cross–link enrichment was performed using the cellulose mini–column method of Skinner (1982). The water fraction was collected and analyzed by RP–HPLC with diode array and fluorescence (ex350/em440) detectors. Gradient conditions using 2–20% ACN over 60 min and 20 mM heptafluorobutyric acid (ion pairing) were employed. Pump speed was 1 ml/min. Cross–links analysis from photonitrated lens protein was then compared to human cataract samples. Results: Three photonitration products were identified and designated PN–1, –2, and –3. The compounds absorb maximally ca. 350 nm and exhibit strong blue fluorescence (ca. ex 350/em 440). Based on similar Rt and UV/fluorescence, all 3 compounds were detected in 7 human cataract samples and not in 4 age–matched controls (p<0.05). Conclusion: These results suggest that photonitration induced protein cross–linking may contribute to the formation of age–related cataract. This mechanism provides a potential explanation for the association between smoking/inflammation and cataract.
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