MECHANISM OF FRUCTOSAMINE ASSAY - EVIDENCE AGAINST ROLE OF SUPEROXIDEAS INTERMEDIATE IN NITROBLUE TETRAZOLIUM REDUCTION

We studied the chemistry of the fructosamine assay for glycated serum proteins by using the model Amadori compound N-alpha-formyl-N-epsilon- fructoselysine (fFL), an analog of glycated lysine residues in protein . Free lysine was formed at similar to 70% yield during a standard 20- min incubation of fFL with alkaline nitroblue tetrazolium (NBT) at 37 degrees C. Although superoxide dismutase (SOD; EC 1.15.1.1) and catala se (EC 1.11.1.6) decreased the yield of the product, monoformazan dye (MF(+)), the yield of MF(+) was slightly greater under anaerobic than aerobic conditions, excluding a role for superoxide as an intermediate in the reduction of NBT during the fructosamine assay. SOD added to d iabetic patients' sera at physiological concentrations also caused a s ignificant (similar to 50%) inhibition of MF(+) formation. This inhibi tion was reduced by addition of nonionic detergents, which contain org anic peroxide inhibitors of SOD, to the fructosamine reagent. Overall, these data indicate that the Amadori compound is the direct reductant of NBT in the fructosamine assay and that superoxide is not an interm ediate in the reaction. The inhibitory effects of SOD and catalase are most likely the result of oxygen regeneration in the assay mixture.