Development and Application of a Stable Isotope Dilution Analysis for the Quantitation of Advanced Glycation End Products of Creatinine in Biofluids of Type 2 Diabetic Patients and Healthy Volunteers

Abstract

<i>N</i>-(1-Methyl-4-oxoimidazolidin-2-ylidene) α-amino acids were recently identified in roasted meat as so far unknown advanced glycation end products (AGEs) of creatinine. For the first time, this paper reports on the preparation of ¹³C-labeled twin molecules of six <i>N</i>-(1-methyl-4-oxoimidazolidin-2-ylidene) α-amino acids and the development of a stable isotope dilution analysis (SIDA) for their simultaneous quantitation in meat, plasma, and urine samples by means of HPLC-MS/MS. Method validation demonstrated good precision (<14% RSD) and accuracy (97–118%) for all analytes and a lower limit of quantitation of 1 pg injected onto the column. The SIDA was applied to monitor plasma appearance and urinary excretion of these AGEs in type 2 diabetes mellitus patients (DM, <i>n</i> = 7) and healthy controls (<i>n</i> = 10) prior to and after ingestion of a bolus of processed beef meat. Interestingly, the basal concentration of <i>N</i>-(1-methyl-4-oxoimidazolidin-2-ylidene) aminopropionic acid was elevated in plasma and urine of DM patients compared to healthy individuals. Further, ingestion of processed meat led to a significantly higher concentration of this AGE in biofluids from DM patients when compared to healthy controls. These findings suggest a favored in vivo formation, as demonstrated by physiological model incubations of creatinine and carbohydrates (37 °C, pH 7.4), or a more efficient dietary up-take of <i>N</i>-(1-methyl-4-oxoimidazolidin-2-ylidene) α-amino acids in hyperglycemic diabetes patients