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.
Metges
Cornelia C
CC
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
Daenzer
Maren
M
Petzke
Klaus J
KJ
Elsner
Angelika
A
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