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V. M. Monnier, X. Fan, C. Strauch; In vitro Model for Screening Inhibitors of Lens Crystallin Modification by Ascorbic Acid Oxidation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1463.
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Human lens crystallins become progressively pigmented and crosslinked with age, in part due to glycation reactions. We hypothesized this process might be in part mediated by ascorbic acid oxidation products, as unequivocally confirmed by our Δen-αA-hSVCT2 mouse model. This animal model condenses the lens discoloration phenomenon into 12 months instead of several decades as in the human. In previous studies we identified several low molecular weight compounds that can inhibit this process. To understand their mechanism of action, we have here modeled this reaction in vitro.
Three different type of in vitro incubation conditions were tested either in anaerobic and aerobic conditions by using calf lens protein extract incubated with ascorbic acid or dehydroascorbic acid (DHA), an oxidized product of ascorbic acid, with or without inhibitors. The inhibition efficiency was monitored by analysis of known advanced glycation end products (AGEs), including carboxymethyllysine (CML), carboxyethyllysine (CEL), pentosidine and autofluorescence. The candidate inhibitors were also previously tested in the Δen-αA-hSVCT2 mice.
Five compounds were found to inhibit ascorbylation in all three in vitro incubation conditions. Penicillamine gave the best inhibition during in vitro incubations. Pyridoxamine, aminoguanidine, NC-1 and NC-2 also were found significantly prevent ascorbylation. Incubation using DHA in anaerobic and metal free condition proved to be a powerful way to test the inhibitors whereby pentosidine was the most sensitive marker of inhibition efficiency. However, some disparate results were observed between in vitro and in vivo. Penicillamine worked very well in vitro, but had no effect in vivo at all, possibly because of poor bioavailability
Using an in vitro incubation approach, pentosidine was identified as useful probe for testing of ascorbylation inhibitors using HTS. Disparate results between in vitro and in vivo implicate bioavailability as an issue for candidate inhibitor selection. The strong correlation between lens fluorescence and progression of senile cataracts suggest that nucleophilic inhibitors of ascorbylation might be one day useful as inhibitors of cataractogenesis.
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