Bw. Patterson et al., MEASUREMENT OF VERY-LOW STABLE-ISOTOPE ENRICHMENTS BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY - APPLICATION TO MEASUREMENT OF MUSCLE PROTEIN-SYNTHESIS, Metabolism, clinical and experimental, 46(8), 1997, pp. 943-948
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.