Improved Methods for the Rapid Formation and Prevention of Advanced Glycation End Products (AGEs) In Vitro by Coupling to the Hypoxanthine/Xanthine Oxidase Assay System

Abstract

Advanced glycation end products (AGEs) represent a set of molecules that contribute directly to the initiation and aggravation of diseases associated with ageing. AGEs are produced by the reaction between reducing sugars (or α-dicarbonyl compounds), proteins, and amino acid residues. Previous in vitro methods using non-enzymatic procedures described in the literature require an incubation period of 1–3 weeks to generate AGEs. In this study, the reaction time for the formation of AGEs (48 and 3 h) was significantly reduced by adaptation of methods previously described in the literature and coupling them to the free radical generation system termed hypoxanthine/xanthine oxidase assay. The incorporation of this assay into the experimental system accelerated the production of AGEs as a result of the formation of reactive oxygen species (ROS), as shown by increased fluorescence. The capacity of different classes of chemical compounds (aminoguanidine, chlorogenic acid, rutin, and methanol extracts of Hancornia speciosa Gomes) to inhibit protein glycation by acting as scavenging agents of α-dicarbonyl species was evaluated. Aminoguanidine and, especially, rutin identified in the leaf extracts of H. speciosa Gomes showed a high capacity to act as scavengers of reactive carbonyl species RCS-trapping, resulting in the inhibition of AGEs formation.

Keywords

• advanced glycation end products
• glyoxal
• hypoxanthine/xanthine oxidase
• methyl glyoxal
• RCS-trapping
• rutin
• xanthine oxidase