Low-abundance plasma and urinary [15N]urea enrichments analyzed by gas chromatography/combustion/isotope ratio mass spectrometry

• Published in: Journal of mass spectrometry. 2002-05, Vol.37 (5), p.489-494
• Main Authors: Metges, Cornelia C., Daenzer, Maren, Petzke, Klaus J., Elsner, Angelika
• Format: Article
• Language: English
• Subjects: [15N]glycine; [15N]lysine; Adult; Animals; Biological and medical sciences; Calibration; dimethylaminomethylene; Feasibility Studies; Female; Fundamental and applied biological sciences. Psychology; Gas Chromatography-Mass Spectrometry - methods; gas chromatography/combustion/isotope ratio mass spectrometry; Glycine - pharmacokinetics; Humans; Lysine - pharmacokinetics; Male; Metabolisms and neurohumoral controls; Nitrogen Isotopes - blood; Nitrogen Isotopes - pharmacokinetics; Nitrogen Isotopes - urine; Nitrogen metabolism. Proteins. Glycoproteins. Nucleic acids. Collagen; protein metabolism; Rats; Rats, Wistar; Species Specificity; stable isotopes; urea; Urea - blood; Urea - urine; Vertebrates: anatomy and physiology, studies on body, several organs or systems
• ISSN: 1076-5174; 1096-9888
• DOI: 10.1002/jms.306

We report a method for determining plasma und urinary [15N]urea enrichments in an abundance range between 0.37 and 0.52 15N atom% (0–0.15 atom% excess (APE) 15N) using a dimethylaminomethylene derivative. Compared with conventional off‐line preparation and 15N analysis of urea, this method requires only small sample volumes (0.5 ml of plasma and 25 µl of urine). The 15N/14N ratio of urea derivatives was measured by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Two peaks were separated; one was identified by gas chromatography/mass spectrometry (GC/MS) as the complete derivatized urea. Calibration of the complete urea derivative was performed by linear regression of enrichment values of known standard mixtures. Replicate standard (6–465‰ δ15N) derivatizations showed a relative standard deviation ranging from 0.1 to 7%. In order to test the feasibility of the method, human subjects and rats ingested a single meal containing either 200 mg of [15N]glycine (95 AP 15N) or 0.4 mg of [15N]‐α‐lysine (95 AP 15N), respectively. Urine and plasma were collected at hourly intervals over 7 h after the meal intake. After 15N glycine intake, maximum urinary urea 15N enrichments were 330 and 430‰ δ15N (0.12 and 0.16 APE 15N) measured by GC/C/IRMS, whereas plasma [15N]glycine enrichments were 2.5 and 3.3 APE 15N in the two human subjects 2 h after the meal. 15N enrichments of total urine and urine samples devoid of ammonia were higher enriched than urinary [15N]urea measured by GC/C/IRMS, reflecting the presence of other urinary N‐containing substances (e.g. creatinine). In rats plasma urea 15N enrichments were 15–20 times higher than those in urinary urea (10–20‰ δ15N). The different [15N]urea enrichments observed after ingestion of [15N]‐labeled glycine and lysine confirm known differences in the metabolism of these amino acids. Copyright © 2002 John Wiley & Sons, Ltd.