DETERMINATION OF LOW ISOTOPIC ENRICHMENT OF L-[1-C-13]VALINE BY GAS-CHROMATOGRAPHY COMBUSTION ISOTOPE RATIO MASS-SPECTROMETRY - A ROBUST METHOD FOR MEASURING PROTEIN FRACTIONAL SYNTHETIC RATES IN-VIVO

A method was developed for measuring protein fractional synthetic rate s using the N-methoxycarbonylmethyl ester (MCM) derivative of L-[1-C-1 3]valine and on-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The derivatization procedure can be performe d rapidly and GC separation of valine from the other branched-chain am ino acids, leucine and isoleucine, is easily obtained. A good lineair relationship was observed between the increment of the C-13/C-12 isoto pe ratio in CO, gas derived from the combustion-of derivatized valine and the tracer mole ratio of L-[1-C-13]valine to unlabelled valine. Th e limit of quantitation was at an L-[1-C-13]valine tracer mole ratio o f 0.0002. The method was used to measure the isotopic enrichment of L- [1-C-13]valine-in standard mixtures and in skeletal muscle of six grow ing piglets infused with L-[1-C-13]valine (2 mg kg(-1) h(-1) for 6 h). After infusion of L-[1-C-13]valine the mean tracer mole ratio in plas ma of L-[1-C-13]valine at the isotopic steady state was 0.0740 +/- 0.0 056 (GC/MS, mean +/- SEM) and the mean tracer mole ratio of valine in muscle protein fraction at 6 h was 0.000236 +/- 0.000038 (GC/C/IRMS). The resulting mean protein fractional synthetic rate in piglet skeleta l muscle was 0.052 +/- 0.007% h(-1), which is in good agreement with l iterature data obtained with alternative, more elaborate techniques. B y this method protein fractional synthetic rates Can be measured at lo w isotopic enrichment levels using L-[1-C-13]valine, the MCM derivativ e and on-line GC/C/IRMS. (C) 1998 John Whey & Sons, Ltd.