Determining 15N to 14N ratios in biofluids by single-pulse 1H nuclear magnetic resonance.

In the 1H NMR spectra of acidified biofluids from our studies of nitrogen metabolism the 1:1 doublet signal (1J-1H-15N = 73.2 Hz) from the protons of 15N-enriched ammonia is clearly resolved from the 1:1:1 triplet signal (1J-1H-14N = 52.3 Hz) from (naturally abundant) 14N ammonia. The five spectral lines, infinitely broad at neutral pH, produce a "pseudo-pentet" at 7.1 ppm from which the fractional 15N-enrichment is easily calculated. 1H NMR spectra of plasma and urine from rats infused with 15N-enriched ammonium acetate illustrate the technique. The greater sensitivity of 1H NMR over 15N and 14N NMR spectroscopy suggests 15N-ammonia determinations by this method will compare favorably with other methods for determining nitrogen labeling.