Assessment of threonine metabolism in vivo by gas chromatography/mass spectrometry and stable isotope infusion.
The fractional contributions (FC) of threonine to glycine and 2-ketobutyrate (KB) fluxes in fed pigs have been assessed by the constant infusion of L-[1-13C]-threonine. The analysis of the enantiomeric purity of labeled threonine by gas chromatography/mass spectrometric (GC/MS) analysis is reported as the N-TFA isopropyl ester derivative. The commercially available [1-13C]threonine comprised 98.7% of the L-enantiomer, enriched at 99 atom percentage excess (APE), and 1.3% of L-allo-threonine contaminant, also enriched at 99 APE. The enantiomeric purity of threonine in plasma of pigs infused for 10 h with [1-13C]threonine showed that the L-allo contaminant did not accumulate. The t-butyl dimethylsilyl derivatives of threonine, glycine, and 2-aminobutyrate (ABA) were used to measure the enrichment of these compounds in plasma and liver samples by GC/MS/selected ion monitoring analysis. Analyses were performed on between 1 and 5 nmol of each amino acid extracted from biological fluids and a 1:10 split injection. GC/MS parameters were assessed with standards at similar quantities and found to be satisfactory; e.g., injection of 1-10 nmol of glycine did not significantly alter the slope and the precision of the standard curve. The coefficient of variation of enrichment determination was less than 10% for standards enriched at 0.4 APE or more and biological samples enriched at 0.6 APE or greater. Within-animal coefficients of variation for four plasma samples obtained at equal intervals between 8 and 10 h of [1-13C]threonine infusion were 4, 21, and 24% for threonine, ABA, and glycine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Ballèvre
O
O
Station de Recherches Porcines, INRA St Gilles, L'Hermitage, France.
Prugnaud
J
J
Houlier
M L
ML
Arnal
M
M
D000613:Aminobutyrates / Q000032:analysis* / Q000097:blood; D000818:Animals; D002247:Carbon Isotopes; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D005998:Glycine / Q000032:analysis* / Q000097:blood; D008099:Liver / Q000737:chemistry; D017646:Organosilicon Compounds; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012825:Silicon; D013552:Swine; D013912:Threonine / Q000032:analysis* / Q000097:blood
D000613:Aminobutyrates; D002247:Carbon Isotopes; D017646:Organosilicon Compounds; C009660:t-butyldimethylsilyl compounds; D013912:Threonine; D005998:Glycine; D012825:Silicon