MEASURE VERY LOW STABLE ISOTOPE ENRICHMENTS BY GC & MS MUSCLE PROTEIN SYNTHESIS

用稳定同位素测量肌肉蛋白质合成

Measurement of muscle protein synthesis using stable isotope tracers usually requires isotope ratio mass spectrometry (IRMS) to measure very low enrichments (tracer; tracee ratios of 0.005% to 0.10%). We developed an approach to measure low enrichments by conventional gas chromatography/mass spectrometry (GC/MS). The approach includes three elements: 1) use of a highly substituted tracer containing three or more labeled atoms to measure enrichment above a very low natural abundance a highly substituted isotopomers; 2) use of a highly substituted natural abundance isotopomer as a base ion for comparison rather than the most abundant m+0 isotopomer to reduce the dynamic range of the isotopomer ratio measurement; and 3) a sensitive mass spectrometric analysis which measures the natural abundance content of the isotopomer used as a tracer with a high signal/noise ratio (>100:1). The approach was used to measure muscle protein synthesis rates following a primed continuous infus ion of L-[13C6]-phenylalanine in 8 fasted dogs and L-[-2-H3]-leucine in 5 fasted humans. Values of [13C6]-phenylalanine enrichment by GC/MS rates were virtually identical to those obtained by a conventional approach using HPLC to isolate phenylalanine, combustion to CO2, and measurement of 13CO2 enrichment by IRMS (IRMS enrichment = 0.9988 x GC/MS enrichment, r2=0.891) resulting in identical values for muscle fractional synthesis rates (mean(SEM:2.7(0.2 and 2.5(0.2%/day for GC/MS and IRMS values, respectively).