MEASUREMENT OF VERY LOW STABLE ISOTOPE ENRICHMENTS BY GAS CHROMATOGRAPHY

利用稳定同位素测定肌肉蛋白质合成 示踪剂通常需要同位素比质谱（IRMS）， 测量非常低的富集度（示踪剂;示踪比为0.005%至 0.10%）。 我们开发了一种方法来测量低浓缩， 常规气相色谱/质谱法（GC/MS）。 的 该方法包括三个要素：1）使用高度取代的示踪剂 含有三个或更多个标记原子，以测量浓度高于 非常低的天然丰度高度取代的同位素异构体; 2）使用 高度取代的天然丰度同位素异构体作为基离子， 比较，而不是最丰富的m+0同位素异构体，以减少 同位素异构体比率测量的动态范围;以及3）灵敏的 质谱分析，测量天然丰度 同位素异构体的含量用作具有高信噪比的示踪剂 比值（>1001）。 该方法被用于测量肌肉蛋白质 合成速率， 对8只禁食狗输注L-[13 C6]-苯丙氨酸， 5名禁食人体中的L-[-2-H3]-亮氨酸。 [13 C6]-苯丙氨酸的值 通过GC/MS的富集率几乎与获得的那些相同 通过使用HPLC分离苯丙氨酸的常规方法， 燃烧生成CO2，并通过IRMS（IRMS）测量13 CO2富集度 富集度= 0.9988 x GC/MS富集度，r2=0.891）， 肌肉部分合成速率的相同值 （平均值（SEM）：2.7（0.2和2.5（GC/MS为0.2%/天 和IRMS值）。

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).