MEASUREMENT OF VERY-LOW STABLE-ISOTOPE ENRICHMENTS BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY - APPLICATION TO MEASUREMENT OF MUSCLE PROTEIN-SYNTHESIS

Measurement of muscle protein synthesis using stable isotopically labe led tracers usually requires isotope ratio mass spectrometry (IRMS) be cause of the need to measure very low enrichments of stable isotopical ly labeled tracers (tracer to tracee ratio [TTR], 0.005% to 0.10%). Th is approach is laborious, requiring purification of the metabolite of interest and combustion to a gas for IRMS analysis, and is best suited for use with C-13 tracers. We have developed an approach whereby low enrichments can be conveniently measured by a conventional gas chromat ography/mass spectrometry (GC/MS) instrument. The approach includes th ree critical elements: (1) use of a highly substituted tracer containi ng three or more labeled atoms, to measure enrichment above a very low natural abundance of highly substituted isotopomers; (2) use of a hig hly substituted natural abundance isotopomer as a base ion for compari son rather than the most abundant m + 0 isotopomer, to reduce the dyna mic range of the isotopomer ratio measurement; and (3) a sensitive mas s spectrometric analysis that measures the natural abundance of the is otopomer used as a tracer with a high signal to noise ratio (> 100:1). This approach was used to measure the rate of synthesis of muscle pro tein following a primed continuous infusion of L-[C-13(6)]-phenylalani ne (PHE) in eight fasted dogs and L-[H-2(3)]-leucine in five fasted hu man subjects. Values for [C-13(6)]-PHE enrichment by GC/MS rates were virtually identicalthose obtained by a conventional approach using hig h-performance liquid chromatography (HPLC) to isolate PHE, combustion to CO2, and measurement of (CO2)-C-13 enrichment by IRMS (IRMS enrichm ent = 0.9988 x GC/MS enrichment, R-2 = .891), resulting in identical v alues for muscle fractional synthesis rates ([FSRs] mean +/- SEM: 2.7 +/- 0.2 and 2.5 +/- 0.2%/d for GC/MS and IRMS, respectively). Human mu scle synthesis rates measured by GC/MS analysis of [H-2(3)]-leucine en richment (1.90 +/- 0.17%/d) were similar to published values based on IRMS analysis using a 1-C-13-leucine tracer. We conclude that compared with the IRMS approach, the GC/MS approach offers faster throughput, has a lower sample requirement, and is suitable for a wider variety of tracers such as H-2. The principles outlined here should be applicabl e to the measurement of low enrichments by GC/MS in a wide variety of stable isotope tracer applications. Copyright (C) 1997 by W.B. Saunder s Company.