The accumulation of hydrogen peroxide (H2O2) during incubations of protein with glucose (experimental glycation) has previously been too low for direct measurement although it is suggested to be the precursor of protein-damaging hydroxylating agents. We have thus developed a simple H2O2-measuring technique which relies upon the rapid peroxide-mediated oxidation of Fe2+ to Fe3+ (catalysed by sorbitol) under acidic conditions followed by reaction of the latter cation with the dye, xylenol orange. We have used the method to demonstrate that incubation mixtures of protein and glucose generates nanomolar levels of hydrogen peroxide in the presence of protein under physiological conditions of pH and temperature.
