Abstract
Ribose produces advanced glycation end-products (AGEs) more rapidly than glucose. It has also been reported that Nε-(carboxymethyl)arginine (CMA) is formed in collagen, which is the most abundant protein in organisms. In this study, AGEs produced by the reaction of ribose with gelatin, a soluble collagen, were evaluated by immunochemical methods using anti-AGEs antibodies, mass spectrometry, and fluorescence intensity measurements. Measurement by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that Nε-(carboxymethyl)lysine (CML), Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1) and CMA were significantly increased in the ribose-gelatin sample compared to the gelatin alone sample after a 7-day incubation at 37°C. The fluorescence intensity at excitation and emission of 370 nm and 440 nm, respectively, was also significantly increased, whereas the AGE structure detected was unknown. In addition, the pericarp hot water extract ofTrapa bispinosa (TBE) and/or lutein were added to this reaction system to evaluate the inhibitory effect of AGE formation. As a result, inhibition of CML, MG-H1, and CMA formation was observed at ≥ 5 μg/mL of TBE in ELISA and LC-MS/MS. However, no similar inhibitory effect was detected in fluorescence measurements, suggesting that TBE does not inhibit the formation of fluorescent AGEs. Since the inhibition of AGE formation by lutein was not confirmed, it may be a difficult compound to evaluate by in vitro studies. This study showed that ELISA and LC-MS/MS measurement can detect three types of AGEs formation in the ribose-gelatin system and that they are useful for screening compounds, e.g., TBE, that inhibit AGE formation.
