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M Satake, J Madaj, B Dmochovska, Y Nishikawa, PL Rinaldi, VM Monnier; Formation of Vitamin C Degradation Products in Diabetic Rat and Human Lens Revealed with 6-deoxy-6-fluoro Ascorbic Acid (F-ASA) and NMR Spectroscopy . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2385.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Ascorbic acid oxidation products are thought to play a major role in the postsynthetic modification of crystallins in aging and diabetes. We previously reported (ARVO 2001) that F-ASA oxidation in HLE B-3 lens epithelial cells was surprisingly resistant to high glucose and galactose stress but not to GSH depletion by BSO. We have now investigated F-ASA degradation in diabetic rat and human lens. Methods: S.D. rats with 5 months of STZ-diabetes were administered i.p. F-ASA (4 mg/rat) every 12 hours for 5 days. Human lenses obtained fresh at autopsy were incubated with F-ASA (1mM) and F-dehydroascorbic acid (F-DHA,1mM) for 12 hours. From each lens a protein-free extract was prepared and analyzed by 19F-NMR using a 750 MHz Varian instrument with proton decoupling device. Results:F-ASA in control rat lens was 4.66 nmol/lens and was decreased by 50.1% in diabetic cataracts (P<0.05). Preliminary results indicate the presence of F-DHA and F-unknown at -212.4 ppm. Surprisingly, these were the only two detectable F-ASA degradation products compared to the ten to fifteen peaks that were detectable during in vitro F-ASA degradation. The diabetic rat lens behaved similarly to GSH depleted HLE B-3 cells in terms of ascorbic acid oxidation. In fresh human lenses (ages 44-63yrs) incubated with F-ASA (1mM) for 12 hours, F-ASA, F-unknown at -212.4 ppm and F-2,3-diketogluconic acid (F-DKG) were the main products. In several human lenses incubated with F-DHA (1mM) for 12 hours, no F-ASA but F-DKG as the major degradation product and several unknown products were detectable that were not present in the culture medium. Conclusion:These data for the first time demonstrate that cataract formation and the human lens are associated with the selective presence of ascorbic acid degradation products that may participate in aging of lens crystallins.
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