The aim of this work was to determine by ion-exchange liquid
chromatography and isotope ratio mass spectrometry the specific
15N enrichment of
amino acids in casein and plasma proteins in cows receiving three successive daily
oral doses (300, 150 and 150 g) of
(15NH4)2SO4 (10 atom per cent
isotopic enrichment) and to examine the
15N enrichments obtained with regard to nitrogen transport and
metabolism in the lactating cow. To investigate the
15N distribution in amino acids
in casein and in plasma proteins, samples of
15N-labelled casein and plasma proteins
were extracted either from a pool of several milkings (36–96 h
after starting to
administer the tracer) or from pooled venous blood (removed on the fourth day after
the start of administration) from the four lactating cows.
15N enrichments of the
proteins studied, expressed as atoms percent excess, were
0·2509 for casein and
0·0577 for plasma protein. Chromatographic fractionation
of the amino acid mixture
(protein hydrolysates) resulted in nine groups containing between one and four
amino acids: Asp, Ser and Thr; Glu; Pro; Gly, Ala, Val and Met; Ileu and Leu; Tyr;
Phe; His and Lys; and Arg. High
15N incorporation was demonstrated in all
individual or groups of amino acids studied. In both proteins, Glu
appeared to be the
most enriched amino acid, Phe and Arg the least enriched. Most aliphatic molecules
with a single amino group were highly enriched. The much lower
(3·5–7·7-fold)
enrichments in plasma protein compared with casein suggest considerable
intracellular dilution at the site of liver protein synthesis.
Finally, the amino acid
separation methods are discussed and suggestions for improving them considered.