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 oxidant production.

Modification of human serum albumin (HSA) with formaldehyde resulted in a loss of 75% of available lysine residues, but there was no change in histidine content or susceptibility to free-radical-mediated fragmentation. The modified HSA appeared resistant to glycation and glucose-mediated fragmentation. Native HSA inhibited oxidant production by free glucose, as assessed by the hydroxylation of benzoic acid, but modified HSA had little effect. Thus the oxidation of free glucose appeared to be inhibited by glycatable protein, but not by unglycatable protein. Also, a close proximity of glucose to protein (decreased in the case of modified HSA) would seem to be a prerequisite for glucose-mediated 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. Glycated HSA, washed free of unbound glucose, became fragmented and generated oxidants capable of hydroxylating benzoic acid and oxidizing cholesteryl linoleate-HSA complexes. Significant levels of benzoate hydroxylation 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 little fragmentation or oxidant formation. The oxidative activity of glycated HSA was dependent on transition-metal concentration. The level of protein-bound glucose appeared to decrease during the oxidant production and protein fragmentation. Thus glucose can oxidize and generate reactive oxidants, whether in solution or attached to protein. We discuss which is the more likely mechanism of glucose oxidation under the near-physiological conditions used to study the effects of protein exposure to glucose in vitro.