The overall objective of this proposal is to define the biochemical basis and mechanisms of crystallin pigmentation and crosslinking in lens aging and cataractogenesis. Progress achieved in the past 2 1/2 years in the applicant's laboratory unraveled a high correlation between lens pigmentation and pentosidine, an ascorbate-derived protein crosslink formed in a Maillard-type reaction. This and other observations strongly implicate toxicity of ascorbate oxidation products in lens senescence and cataractogenesis. We propose to investigate the mechanism(s) by which ascorbate turns into a Maillard reactant and the extent to which the ascorbate mediated Maillard reaction explains human lens crystallin pigmentation and crosslinking in aging and cataractogenesis. Specifically, we hypothesize that several lines of defense must be weakened before ascorbate mediates lens crystallin pigmentation and crosslinking by the Maillard reaction. In order to clarify the nature of the biochemical defect the following experiments will be performed: 1. We will first identify differences in patterns of ascorbate degradation products in young, old and cataractous human lenses and elucidate the structure of these products. 2. We will identify specific human lens fractions that contain inhibitory activity against the ascorbate mediated Maillard reaction and characterize the biochemical nature of the ascorbate degradation inhibitory activity(ies). 3. We will elucidate the structure of the advanced Maillard products specific for ascorbate, focusing especially on the structure of long-wave fluorescent molecules. 4. Finally, we will systematically correlate specific defects in ascorbate metabolism uncovered in specific aims I-III with extent of crystallin pigmentation and crosslinking and the presence of specific advanced Maillard reaction products as a function of age and presence of cataract. The successful completion of this project may result in generating clues for a novel hypothesis of cataractogenesis based on genetic defects in ascorbate metabolism in the lens.
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