Volume 91, Issue 2, February (2004), pp. 253-262 © The Author 2004
doi:10.1079/BJN20031040

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Use of stable isotopes to measure de novo synthesis and turnover of amino acid-C and -N in mixed micro-organisms from the sheep rumen in vitro

C. Atasoglu†¹, A. Y. Guliye¹, R. J. Wallace^1
1Rowett Research Institute, Bucksburn, Aberdeen, AB21 9SB, UK

 (Received 17 June 2003–Revised 9 October 2003–Accepted 9 October 2003)

Protein synthesis and turnover in ruminal micro-organisms were assessed by stable-isotope methods in order to follow independently the fate of amino acid (AA)-C and -N in different AA. Rumen fluid taken from sheep receiving a grass hay–concentrate diet were strained and incubated in vitro with starch–cellobiose–xylose in the presence of NH₃ and 5 g algal protein hydrolysate (APH)/l, in incubations where the labels were ¹⁵NH₃, [¹⁵N]APH or [¹³C]APH. Total ¹⁵N incorporation was calculated from separate incubations with ¹⁵NH₃ and [¹⁵N]APH, and net N synthesis from the increase in AA in protein-bound material. The large difference between total and net AA synthesis indicated that substantial turnover of microbial protein occurred, averaging 3·5 %/h. Soluble AA-N was incorporated on average more extensively than soluble AA-C (70 v. 50 % respectively, P=0·001); however, incorporation of individual AA varied. Ninety percent of phenylalanine-C was derived from the C-skeleton of soluble AA, whereas the incorporation of phenylalanine-N was 72 %. In contrast, only 15 % aspartate-C+asparagine-C was incorporated, while 45 % aspartate-N+asparagine-N was incorporated. Deconvolution analysis of mass spectra indicated substantial exchange of carboxyl groups in several AA before incorporation and a condensation of unidentified C₂ and C₄ intermediates during isoleucine metabolism. The present results demonstrate that differential labelling with stable isotopes is a way in which fluxes of AA synthesis and degradation, their biosynthetic routes, and separate fates of AA-C and -N can be determined in a mixed microbial population.

Keywords:
Amino acids, Carbon skeletons, Rumen, Sheep

Abbreviations:
AA, amino acid, ape, atom percent excess, APH, algal protein hydrolysate, I, isotopomer correction factor