AGEs (advanced glycation end products): an advanced method for an advanced problem

Advanced glycation end products (AGEs) are heterogeneous, sugar-derived protein modifications [Fig.1] that have received attention in various research fields, such as food preparation, the normal aging process and pathophysiological conditions in the human body. [1]

Several in vitro experiments are reported to identify AGEs inhibitors, for instance: measuring glycation of bovine serum albumin, determination of fructosamine adducts and measuring α-dicarbonyl compounds. [2] However, the latter methods suffer from various drawbacks like a complex formation between test products and the protein can result in quenching of the fluorescence signal and misinterpretation of the data. Therefore, it was of utmost importance to develop and validate a reliable and universal method to circumvent these problems. Subsequently, several classes of natural products will be tested in the new method for their AGEs inhibition properties.

In this research, the precise quantification of N(6)-carboxymethyllysine (CML), one of the most abundant AGEs [3], was achieved through HILIC Ultra-Performance Liquid Chromatography coupled to a Xevo G2-XS QTof MS system, using d₂-CML as an internal standard. CML was generated by incubation of GK-peptide and ribose. The sample preparation included a hydrolysis step with hydrochloric acid and a clean up step with P3 (Protein Precipitation Plate) cartridges. The method was validated following the FDA guidelines of Bioanalytical Method Validation [4] regarding linearity, LLOQ, precision and quality controls.

Overall, advanced glycation is a leading cause for the progression and pathogenesis of many chronic diseases (e.g. diabetes mellitus). Specifically, this validated method can contribute to the unambiguous discovery of new potential anti-AGEs agents.

Fig. 1 General scheme of AGEs formation.

• References

• 1 Rahbar S, Figarola B. Inhibitors and breakers of advanced glycation endproducts (AGEs): A review. Curr Med Chem Endocr Metab Agents 2002; 2: 135-161
• 2 Hodge J. Chemistry of Browning Reactions in Model Systems. 1953: 928-943 .
• 3 Nguyen H, Fels-Klerx H. N(6)-(carboxymethyl)lysine: A review on analytical methods, formation, and occurrence in processed food, and health impact. Food Rev Int 2014; 30: 36-52.
• 4 U.S. Department of Health and Human Services. Food and Drug Administration (FDA), Guidance for Industry: Bioanalytical Method Validation,. 2018