THE PYRIDOINDOLE ANTIOXIDANT STOBADINE INHIBITED GLYCATION-INDUCED ABSORBENCY AND FLUORESCENCE CHANGES IN ALBUMIN

We studied the effect of the pyridoindole antioxidant stobadine on gly cation-induced absorbance and fluorescence changes in bovine serum alb umin (BSA), used as a model protein. Incubation of BSA (4 mg/ml) with glucose (100-400 mM) in 0.12 M phosphate buffer, pH 7.4, in the presen ce of 100 mu M Cu2+ at 37 degrees C resulted in a time-dependent incre ase of absorbance (320 nm) and fluorescence (excitation 350 nm, emissi on 415 nm). The process was found to be dependent on the presence of o xygen and transition metal ions, but equimolar iron could not fully su bstitute for the activity of copper. The glucose-induced chromo- and f luorophore formation was reduced significantly by stobadine. For 200 m M glucose, in 7- and 14-day incubations, 51%-60% inhibition was obtain ed at a stobadine concentration of 0.1 mM, and the effect leveled off at higher concentrations of the drug. No inhibition was observed with N-acetyl stobadine, a derivative with restricted antioxidant activity. Since stobadine did not affect the Amadori product formation determin ed by the thiobarbituric acid (TEA) method as 5-hydroxymethyl furfural (5-HMF) released in boiling oxalic acid, the inhibitory action of sto badine may be explained by its interference with metal-catalyzed oxida tion reactions following after the glycation step. The results obtaine d suggest that antioxidant therapy could be used to limit the damage f rom adverse glycation-induced processes in diabetes mellitus.