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

Abstract

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 NH 3 and 5 g algal protein hydrolysate (APH)/l, in incubations where the labels were 15 NH 3 , [ 15 N]APH or [ 13 C]APH. Total 15 N incorporation was calculated from separate incubations with 15 NH 3 and [ 15 N]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 C 2 and C 4 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.