ASSESSMENT OF THREONINE METABOLISM INVIVO 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. The FC (×100) of threonine to glycine and KB fluxes were, in fed pigs, 4.6 ± 0.7 and 7.1 ± 0.6, respectively (mean ± SE, n =3). Comparisons are drawn between the variabilities associated with the use of the stable isotope procedure and those observed with [U14C]threonine in a previous experiment. © 1991.