scholarly journals 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

Amino Acids ◽  
2021 ◽  
Author(s):  
Svetlana Baskal ◽  
Alexander Bollenbach ◽  
Catharina Mels ◽  
Ruan Kruger ◽  
Dimitrios Tsikas
Keyword(s):  
Amino Acids ◽  
Human Urine ◽  
Simultaneous Measurement ◽  
De Novo ◽  
Time Window ◽  
Negative Ion ◽  
Gas Chromatography Mass Spectrometry ◽  
Post Translational Modification ◽  
Relevant Concentration ◽  
Relative Standard

AbstractA 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).

scholarly journals Sensitive Gas Chromatography-Mass Spectrometry Method for Simultaneous Measurement of MDEA, MDMA, and Metabolites HMA, MDA, and HMMA in Human Urine

2006 ◽  
Vol 52 (9) ◽  
pp. 1728-1734 ◽  
Author(s):  
Stephane O Pirnay ◽  
Tsadik T Abraham ◽  
Marilyn A Huestis
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Human Urine ◽  
Simultaneous Measurement ◽  
Dynamic Range ◽  
Solid Phase ◽  
Gas Chromatography Mass Spectrometry ◽  
Spectrometry Method ◽  
Mass Spectrometry Method ◽  
Chromatography Mass Spectrometry

Abstract Background: A sensitive gas chromatography-mass spectrometry method was developed and validated for the simultaneous measurement of MDEA, MDMA, and its metabolites, 3,4-methylenedioxy-N-ethylamphetamine (MDEA), 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy), and its metabolites, 4-hydroxy-3-methoxyamphetamine (HMA), 3,4-methylenedioxyamphetamine (MDA), and 4-hydroxy-3-methoxyamphetamine (HMMA) in human urine. Methods: We hydrolyzed 1 mL urine, fortified with MDMA-d5, MDA-d5, and MDEA-d6, with 100 μL of concentrated hydrochloric acid at 120 °C for 40 min, then added 100 μL 10 N sodium hydroxide and 3 mL phosphate buffer 0.1 N (pH 6.0) were added to hydrolyzed urine specimens before solid-phase extraction. After elution and evaporation, we derivatized extracts with heptafluorobutyric acid anhydride and analyzed with gas chromatography-mass spectrometry operated in EI-selected ion-monitoring mode. Results: Limits of quantification were 25 μg/L for MDEA, MDMA, and its metabolites. Calibration curves were linear to 5000 μg/L for MDEA, MDMA, HMA, MDA, and HMMA, with a minimum r2 &gt; 0.99. At 3 concentrations spanning the linear dynamic range of the assay, mean overall extraction efficiencies from urine were &gt;85.5% for all compounds of interest. Intra- and interassay imprecisions, produced as CV, were &lt;15% for all drugs at 30, 300, and 3000 μg/L. Conclusions: This gas chromatography-mass spectrometry assay provides adequate sensitivity and performance characteristics for the simultaneous quantification of MDEA, MDMA, and its metabolites HMMA, MDA, and HMA in human urine. The method meets and exceeds the requirements of the proposed Substance Abuse and Mental Health Services Administration’s guidelines for federal workplace drug testing of MDEA and MDMA in urine.

scholarly journals Analysis of Protein Amino Acids in Tobacco Using Microwave Digestion of Plant Material

2005 ◽  
Vol 21 (8) ◽  
pp. 451-465 ◽  
Author(s):  
SC Moldoveanu
Keyword(s):  
Amino Acids ◽  
Free Amino Acids ◽  
Free Amino ◽  
High Performance ◽  
Microwave Digestion ◽  
Gas Chromatography Mass Spectrometry ◽  
Protein Levels ◽  
Relative Standard ◽  
Protein Amino Acids ◽  
The Difference

AbstractThis paper describes a technique using microwave digestion and gas chromatography-mass spectrometry (GC-MS), which makes possible the analysis of protein amino acids in tobacco. The technique involves first the measurement of free amino acids, a hydrolysis using microwave digestion, and a measurement of total resulting amino acids. The content of protein amino acids is determined from the difference of total and free amino acids. The digestion is performed with aqueous 6 N HCl (with 1% phenol) for two hours in a microwave at 120°C in sealed vials. The GC-MS analysis is performed after the amino acids are derivatized with N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA). The technique provides reliable results with less than 10% relative standard deviation (RSD) for most amino acids. Only the determination of very low level amino acids is affected by larger errors. The method provides results for free amino acids that are in very good agreement with those obtained by high performance liquid chromatography (HPLC), and also results for protein levels in tobacco in agreement with data previously reported in the literature. Results are given for several single grade tobaccos and for tobacco blends from four Kentucky reference cigarettes.

scholarly journals An enzymatic activation of formaldehyde for nucleotide methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charles Bou-Nader ◽  
Frederick W. Stull ◽  
Ludovic Pecqueur ◽  
Philippe Simon ◽  
Vincent Guérineau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Crystallographic Structure ◽  
De Novo Synthesis ◽  
Active Site Residues ◽  
Enzymatic Activation ◽  
Enzyme Cofactors ◽  
Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

scholarly journals Diverse metabolic response of cancer cells treated with a 213Bi-anti-EGFR-immunoconjugate

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benedikt Feuerecker ◽  
Philipp Biechl ◽  
Christof Seidl ◽  
Frank Bruchertseifer ◽  
Alfred Morgenstern ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cancer Cells ◽  
Treatment Response ◽  
Cell Lines ◽  
Egf Receptor ◽  
Metabolic Response ◽  
Gas Chromatography Mass Spectrometry ◽  
Alpha Emitter ◽  
Early Treatment Response ◽  
Bladder Cancer Cells

AbstractEvaluation of treatment response is among the major challenges in modern oncology. We herein used a monoclonal antibody targeting the EGF receptor (EGFR) labelled with the alpha emitter 213Bi (213Bi-anti-EGFR-MAb). EJ28Luc (bladder) and LN18 (glioma) cancer cells, both overexpressing EGFR, were incubated for 3 h with the radioimmunoconjugate. To assess the responses in the core carbon metabolism upon this treatment, these cancer cell lines were subsequently cultivated for 18 h in the presence of [U-13C6]glucose. 13C-enrichment and isotopologue profiles of key amino acids were monitored by gas chromatography–mass spectrometry (GC/MS), in order to monitor the impacts of the radionuclide-treatment upon glucose metabolism. In comparison to untreated controls, treatment of EJ28Luc cells with 213Bi-anti-EGFR-MAb resulted in a significantly decreased incorporation of 13C from [U-13C6]glucose into alanine, aspartate, glutamate, glycine, proline and serine. In sharp contrast, the same amino acids did not display less 13C-enrichments during treatment of the LN18 cells. The data indicate early treatment response of the bladder cancer cells, but not of the glioma cells though cell lines were killed following 213Bi-anti-EGFR-MAb treatment. The pilot study shows that the 13C-labelling approach is a valid tool to assess the responsiveness of cancer cells upon radionuclide-treatment in considerable metabolic detail.

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