MEASUREMENT OF VERY LOW STABLE ISOTOPE ENRICHMENTS BY GAS CHROMATOGRAPHY

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 signalnoise ratio (>1001). The approach was used to measure muscle protein synthesis rates following a primed continuous infusi on 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 andIRMS values, respectively).