Low-abundance plasma and urinary [(15)N]urea enrichments analyzed by gas chromatography/combustion/isotope ratio mass spectrometry.
We report a method for determining plasma und urinary [(15)N]urea enrichments in an abundance range between 0.37 and 0.52 (15)N atom% (0-0.15 atom% excess (APE) (15)N) using a dimethylaminomethylene derivative. Compared with conventional off-line preparation and (15)N analysis of urea, this method requires only small sample volumes (0.5 ml of plasma and 25 microl of urine). The (15)N/(14)N 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 per thousand delta(15)N) 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 [(15)N]glycine (95 AP (15)N) or 0.4 mg of [(15)N]-alpha-lysine (95 AP (15)N), respectively. Urine and plasma were collected at hourly intervals over 7 h after the meal intake. After (15)N glycine intake, maximum urinary urea (15)N enrichments were 330 and 430 per thousand delta(15)N (0.12 and 0.16 APE (15)N) measured by GC/C/IRMS, whereas plasma [(15)N]glycine enrichments were 2.5 and 3.3 APE (15)N in the two human subjects 2 h after the meal. (15)N enrichments of total urine and urine samples devoid of ammonia were higher enriched than urinary [(15)N]urea measured by GC/C/IRMS, reflecting the presence of other urinary N-containing substances (e.g. creatinine). In rats plasma urea (15)N enrichments were 15-20 times higher than those in urinary urea (10-20 per thousand delta(15)N). The different [(15)N]urea enrichments observed after ingestion of [(15)N]-labeled glycine and lysine confirm known differences in the metabolism of these amino acids.
Research Unit 'Nutritional Physiology,' Forschungsinstitut für die Biologie landwirtschaftlicher Nutztiere (Research Institute for the Biology of Farm Animals), D-18196 Dummerstorf, Germany. metges@fbn-dummerstorf.de
Cornelia C
CC
Metges
Maren
M
Daenzer
Klaus J
KJ
Petzke
Angelika
A
Elsner
D000328:Adult; D000818:Animals; D002138:Calibration; D005240:Feasibility Studies; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D005998:Glycine / Q000493:pharmacokinetics; D006801:Humans; D008239:Lysine / Q000493:pharmacokinetics; D008297:Male; D009587:Nitrogen Isotopes / Q000097:blood / Q000493:pharmacokinetics* / Q000652:urine; D051381:Rats; D017208:Rats, Wistar; D013045:Species Specificity; D014508:Urea / Q000097:blood* / Q000652:urine*
D009587:Nitrogen Isotopes; D014508:Urea; D008239:Lysine; D005998:Glycine