Detection of 3-Nitro-Tyrosine in the Cataractous Lens

Abstract

Abstract: : Purpose: We have previously shown that non-enzymatic nitration of alpha crystallin produces effects that could lead to the formation of lens cataracts. The present study was undertaken in order to identify and quantitate levels of 3-nitro-tyrosine (a biomarker of nitrosative stress) in the cataractous lens. Methods: The phacoemulsion of 4 patients undergoing placement of an intraocular prosthetic lens for mixed type cataract was compared to 4 non-cataractous eye bank lenses. The phacoemulsion was dialysed to remove salts and then digested to free amino acids with protease (Strep Griseus). The controls were from persons age 42 (2 lenses) and 71 (2 lenses). HPLC analysis with diode array detection was performed with the following conditions. Pump speed was 0.7 ml/min using an isocratic method of 20mM NaHPO₄ (pH 3.2)/5% MeOH on a C₁₈ reverse phase column. 3-nitro-tyrosine detection was confirmed by spiking with standard 3-nitro-tyrosine. Results: 3-nitro-tyrosine was detectable in all 4 cataract specimens examined and was not detectable in the control lenses. The range of 3-nitro-tyrosine detected was 0.415 to 1.312 umol/mg lens protein with an average of 0.707 umol/mg (p=0.015). A methods control experiment using non-cataractous lens confirmed that 3-nitro-tyrosine was not produced through the process of phacoemulsion. Conclusions: 3-nitro-tyrosine, a biomarker of nitrosative stress is present in the cataractous lens and not in control lenses. These findings support the hypothesis that nitration plays a role in the development of senile lens cataract. CR: None Support NIH grant K08 AG00863  

Keywords: nitric oxide • cataract • oxidation/oxidative or free radical damage