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

Abstract

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.

Atomic spectrometry update. Atomic mass spectrometry

“…An on-line GC-combustion-SIRMS method for the determination of d 15 N in urea in plasma and urine samples in the abundance range 0.37-0.52 atom% 15 N was based on the preparation of the dimethylaminomethylene (DAM) derivative prior to injection on to the GC column. 316 The two derivative peaks identified contained either one or two DAM groups. The latter peak was chosen for mass analysis even though introduction of two derivative DAM groups introduced extra N, which changed the d 15 N value by 20.54%.…”

An improved analytical method for the determination of urea nitrogen isotopomers in biological samples utilizing continuous flow isotope ratio mass spectrometry

“…Different urea tracers ( 13 C‐, 18 O‐, 13 C 18 O‐ and 15 N 15 N‐urea) have been infused into humans and animals to determine urea production rate by in vivo isotopic dilution 1, 2, 4–6. The analytical determination of the resulting isotopic enrichment has been conducted by gas chromatography/mass spectrometry (GC/MS)7 or gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) 8, 9. The advantages of GC/MS are the small sample size required and the capability to determine multiple isotopomers in the same run; however, relatively high enrichments (0.50 mol percent excess, MPE) are needed due to the limited accuracy and precision of this technique 10.…”

“…The advantages of GC/MS are the small sample size required and the capability to determine multiple isotopomers in the same run; however, relatively high enrichments (0.50 mol percent excess, MPE) are needed due to the limited accuracy and precision of this technique 10. In contrast, GC/C/IRMS can measure isotopic composition at lower enrichments and natural abundance levels 8, 10…”