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

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仪(IRMS)来 测量极低的浓缩物(示踪剂；示踪比率为0.005 0.10%)。我们开发了一种方法来测量低浓缩物 常规气相色谱/质谱仪(GC/MS)。这个 方法包括三个要素：1)使用高度替代的 含有三个或更多标记原子的示踪剂，用于测量浓缩度 在非常低的自然丰度之上，有高度取代的同位异构体； 2)使用高取代度的天然丰度同分异构体作为碱 用于比较的离子而不是最丰富的m+0同位素异构体 缩小同位素比率测量的动态范围；以及3)a) 灵敏的质谱分析，测量天然的 用作示踪剂的同种异构体的丰度 信噪比(&gt；100：1)。这种方法被用来测量肌肉 连续注入预充剂后的蛋白质合成速度 禁食犬中L-[13C6]-苯丙氨酸8只，L-[-2-H3]-亮氨酸5只 禁食的人类。[13C6]-苯丙氨酸的GC/MS富集值 利率几乎与传统的 高效液相色谱分离苯丙氨酸、燃烧成二氧化碳和 用红外光谱仪测量~(13)CO_2的富集度(红外光谱仪富集度=0.9988× GC/MS富集度，R2=0.891)，使肌肉的值相同 分数合成速率(平均(扫描电子显微镜：2.7)(0.2和2.5(0.2%/天 GC/MS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 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).