Hyperglycemic conditions of diabetes accelerate
protein modifications by
glucose leading to the accumulation of
advanced glycation end-products (AGEs). We have investigated the conversion of
protein-Amadori intermediate to
protein-AGE and the mechanism of its inhibition by
pyridoxamine (PM), a potent AGE inhibitor that has been shown to prevent
diabetic complications in animal models. During incubation of
proteins with physiological diabetic concentrations of
glucose, PM prevented the degradation of the protein glycation intermediate identified as
fructosyllysine (Amadori) by 13C NMR using [2-13C]-enriched
glucose. Subsequent removal of
glucose and PM led to conversion of
protein-Amadori to AGE Nepsilon-
carboxymethyllysine (CML). We utilized this inhibition of post-Amadori reactions by PM to isolate
protein-Amadori intermediate and to study the inhibitory effect of PM on its degradation to
protein-CML. We first tested the hypothesis that PM blocks Amadori-to-CML conversion by interfering with the catalytic role of redox
metal ions that are required for this glycoxidative reaction. Support for this hypothesis was obtained by examining structural analogs of PM in which its known bidentate
metal ion binding sites were modified and by determining the effect of endogenous
metal ions on PM inhibition. We also tested the alternative hypothesis that the inhibitory mechanism involves formation of covalent adducts between PM and
protein-Amadori. However, our 13C NMR studies demonstrated that PM does not react with the Amadori. Because the mechanism of interference with redox
metal catalysis is operative under the conditions closely mimicking the diabetic state, it may contribute significantly to PM efficacy in preventing
diabetic complications in vivo. Inhibition of
protein-Amadori degradation by PM also provides a simple procedure for the isolation of
protein-Amadori intermediate, prepared at physiological levels of
glucose for relevancy, to study both the
biological effects and the chemistry of post-Amadori pathways of AGE formation.