Development, validation of a GC–MS method for the simultaneous measurement of amino acids, their PTM metabolites and AGEs in human urine, and application to the bi-ethnic ASOS study with special emphasis to lysine

Abstract

A gas chromatography-mass spectrometry (GC–MS) method was developed and validated in relevant concentration ranges for the simultaneous measurement of l-lysine (Lys, L) and its Nε- and Nα-methylated (M), Nε- and Nα-acetylated (Ac), Nε-carboxymethylated (CM) and Nε-carboxyethylated (CE) metabolites in human urine. Analyzed Lys metabolites were the post-translational modification (PTM) products Nε-mono-, di- and trimethyllsine, Nε-MML, Nε-DML, Nε-TML, respectively, Nα-ML, Nε-AcL, Nα-AcL, and its advanced glycation end-products (AGEs) Nε-CML, Nε-CM-[2,4,4-2H3]Lys (d3-CML), Nε-CEL and furosine. AGEs of arginine (Arg) and cysteine (Cys) were also analyzed. De novo synthesized trideutero-methyl esters (R-COOCD3) from unlabelled amino acids and derivatives were used as internal standards. Native urine samples (10 µL aliquots) were evaporated to dryness under a stream of nitrogen. Analytes were esterified using 2 M HCl in methanol (60 min, 80 °C) and subsequently amidated by pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C). The generated methyl ester-pentafluoropropionyl (Me-PFP) derivatives were reconstituted in borate buffer and extracted immediately with toluene. GC–MS analyses were performed by split-less injection of 1-µL aliquots, oven-programmed separation and negative-ion chemical ionization (NICI). Mass spectra were generated in the scan mode (range, m/z 50–1000). Quantification was performed in the selected-ion monitoring (SIM) mode using a dwell time of 50 or 100 ms for each ion. The GC–MS method was suitable for the measurement of Lys and all of its metabolites, except for the quaternary ammonium cation Nε-TML. The Me-PFP derivatives of Lys, Arg and Cys and its metabolites eluted in the retention time window of 9 to 14 min. The derivatization of Nε-CML, d3-CML and Nε-CEL was accompanied by partial Nε-decarboxylation and formation of the Me-PFP Lys derivative. The lowest derivatization yield was observed for Nε-DML, indicating a major role of the Nε-DML group in Lys derivatization. The GC–MS method enables precise (relative standard deviation, RSD < 20%) and accurate (bias, < ± 20%) simultaneous measurement of 33 analytes in human urine in relevant concentration ranges. We used the method to measure the urinary excretion rates of Lys and its PTM metabolites and AGEs in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). No remarkable differences were found indicating no ethnic-related differences in PTM metabolites and AGEs except for Nε-monomethyllysine and S-(2-carboxymethylcysteine).

Urinary excretion of amino acids and their advanced glycation end-products (AGEs) in adult kidney transplant recipients with emphasis on lysine: furosine excretion is associated with cardiovascular and all-cause mortality

“…In our study, after each urination the container was put immediately into a refrigerator and kept at approximately 4 °C until the next urination. Under such collection and storage conditions, the AGEs measured in the present study are stable for at least 3 days in human and rat urine (Baskal et al 2021a(Baskal et al , 2021b. To the best of our knowledge, there is no need for stabilization of AGEs in urine or even in blood as demonstrated for CML and CEL (Hull et al 2013).…”

“…The urine samples were transferred from UMCG frozen on dry ice to the Institute of Toxicology at Hannover Medical School and stored there (at − 20 °C) until analyses. Lys and AGEs were measured by GC-MS in 10-µL urine aliquots after solvent evaporation to dryness and a two-step derivatization first with methanolic 2 M HCl and then with pentafluoropropionic anhydride in ethyl acetate as described elsewhere in detail (Baskal et al 2021a). This method is based on a GC-MS method previously reported for free amino acids in plasma and urine (Hanff et al 2019b).…”

“…This method is based on a GC-MS method previously reported for free amino acids in plasma and urine (Hanff et al 2019b). GC-MS analyses were performed on the model ISQ from ThermoFisher (Dreieich, Germany) in the selected-ion monitoring mode (SIM) as reported in detail elsewhere (Baskal et al 2021a).…”

“…The aim of present study was to measure urinary concentrations of several AGEs, including CML, CEL and furosine, and their common precursor Lys, in urine samples of KTR and healthy donors, and to investigate potential associations between AGEs, transplantation outcome and donation of a kidney. To reach this goal, we newly analyzed urine samples for AGEs and amino acids by means of a validated gas chromatography-mass spectrometry (GC-MS) method that uses deuterium-labeled AGEs as internal standards (Baskal et al 2021a).…”

Advanced Glycation End-Products (AGEs) of Lysine and Effects of Anti-TCR/Anti-TNF-α Antibody-Based Therapy in the LEW.1AR1-iddm Rat, an Animal Model of Human Type 1 Diabetes

“…Known AGEs of Lys include N ε -carboxymethyl lysine (CML) (Figure 1, right panel), N ε -carboxyethyl lysine (CEL), furosine (i.e., [N ε -(2-furoylmethyl)-L-lysine]) and pentosidine, an AGE composed of Lys and Arg (not shown). LMM N ε -and N G -glycated AGEs circulate in the blood and are excreted in the urine of humans and animals [5,6]. The vast majority of Lys methylation occurs in non-histone proteins [2].…”