Optimization forSimultaneous Determination of a Panelof Advanced Glycation End Products as Biomarkers for Metabolic Diseases

Both dietary and endogenous reactive carbonyl species, such as methylglyoxal (MGO) and glyoxal (GO), react with proteins to generate advanced glycation end products (AGEs), which contribute to metabolic diseases. However, accurately determining individual AGEs in biological samples remains challenging due to the lack of standardized methods. In this study, we optimized and detailed procedures for AGE digestion using enzyme cocktails and separation and detection via high-resolution LC–MS/MS. For the first time, we observed that enzyme backgrounds contained higher levels of methylglyoxal-derived hydroimidazolone 1 (MG-H1) and glucosepane than mouse plasma by 1.4–3 times (e.g., 1512.55 ± 18.89 nM in enzymes vs 496.95 ± 90.91 nM in plasma for MG-H1). Using this optimized method, we quantified fructosyl-lysine and nine AGEs in the plasma, kidneys, and urine of mice. MGO-derived AGEs increased significantly in the plasma and kidneys after MGO treatment. Additionally, both MGO- and GO-derived AGEs were elevated in high-fat-diet (HF)-fed mice compared to low-fat-diet (LF)-fed controls, with further increases in HF-fed mice supplemented with MGO (HFM). This optimized method provides accurate AGE quantification, enabling insights into their role as biomarkers for metabolic syndrome and advancing the understanding of dietary and metabolic contributions to AGE formation.