Spectrophotometric determination of browning products of glycation of protein amino groups based on their reactivity with nitro blue tetrazolium salts

A simple spectrophotometric method has been devised for determining the advanced products of glycation of lysine and albumin (browned compounds) based on their reactivity with nitro blue tetrazolium (NBT) salts. Aminoiminopropene derivatives of lysine and malondialdehyde, which share the same optical and fluorescence characteristics of browned compounds, were unreactive in the assay. Of the early products of glycation of lysine and albumin, the Schiff base adduct was unreactive in the assay, whereas the Amadori product (1-deoxy-1-morpholinofructose), when determined with NBT, gave a positive reaction that was slightly less marked than that of browned derivatives of albumin. It is concluded that the NBT assay is specific for Amadori and browned derivatives produced by glycation of amino groups and can be used for detecting such compounds in proteins (circulating and structural) exposed to high levels of carbohydrates.