OXIDATIVE ALTERATIONS IN THE EXPERIMENTAL GLYCATION MODEL OF DIABETES-MELLITUS ARE DUE TO PROTEIN GLUCOSE ADDUCT OXIDATION - SOME FUNDAMENTAL DIFFERENCES IN PROPOSED MECHANISMS OF GLUCOSE-OXIDATION AND OXIDANTPRODUCTION

Modification of human serum albumin (HSA) with formaldehyde resulted i n a loss of 75 % of available lysine residues, but there was no change in histidine content or susceptibility to free-radical-mediated fragm entation. The modified HSA appeared resistant to glycation and glucose -mediated fragmentation. Native HSA inhibited oxidant production by fr ee glucose, as assessed by the hydroxylation of benzoic acid, but modi fied HSA had little effect. Thus the oxidation of free glucose appeare d to be inhibited by glycatable protein, but not by unglycatable prote in. Also, a close proximity of glucose to protein (decreased in the ca se of modified HSA) would seem to be a prerequisite for glucose-mediat ed protein fragmentation. This latter observation, in particular, led us to examine the role of oxidation of glucose attached to HSA in the production of reactive oxidants and subsequent molecular damage. Glyca ted HSA, washed free of unbound glucose, became fragmented and generat ed oxidants capable of hydroxylating benzoic acid and oxidizing choles teryl linoleate-HSA complexes. Significant levels of benzoate hydroxyl ation and HSA fragmentation occurred with HSA (10 mg/ml) containing 3. 3 mol of glucose bound/mol of HSA. This is equivalent to incubation of 10 mg/ml native HSA with 0.66 mM glucose, conditions which lead to li ttle fragmentation or oxidant formation. The oxidative activity of gly cated HSA was dependent on transition-metal concentration. The level o f protein-bound glucose appeared to decrease during the oxidant produc tion and protein fragmentation. Thus glucose can oxidize and generate reactive oxidants, whether in solution or attached to protein. We disc uss which is the more likely mechanism of glucose oxidation under the near-physiological conditions used to study the effects of protein exp osure to glucose in vitro.