MEASUREMENT OF VERY LOW STABLE ISOTOPE ENRICHMENTS BY GAS CHROMATOGRAPHY

通过气相色谱法测量极低稳定同位素富集度

• 批准号: 6486672
• 负责人: BRUCE W PATTERSON
• 金额: $ 15.75万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6486672
• 关键词: biological products; biomedical resource; neoplasm /cancer; nucleic acids; spectrometry

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).

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 该方法包括三个要素：1)使用高度取代的示踪剂 含有三个或更多标记原子的，用于测量 天然丰度极低的高度取代的同位异构体；2)使用 一种高度取代的天然丰度同分异构体，用作 而不是最丰富的m+0同位异构体，以减少 同分异构体比率测量的动态范围；以及3)灵敏 测量自然丰度的质谱分析 用作高信号噪声示踪剂的同种异构体含量 比率(&gt；1001)。这种方法被用来测量肌肉蛋白质 素数连续后的合成速率 L-[13C6]-苯丙氨酸在8只禁食犬体内的输注 L-[-2-H3]-亮氨酸在5名禁食人群中的分布。[13C6]-苯丙氨酸的值 GC/MS的富集率与获得的结果基本相同 通过使用高效液相分离苯丙氨酸的传统方法， 燃烧为CO_2，并用红外光谱(IRMS)测量13CO_2的浓缩 浓缩=0.9988 x GC/MS浓缩，R2=0.891) 肌肉部分合成率的等效值 (平均(扫描电子显微镜：2.7(0.2和2.5，GC/MS为0.2%/天 和IRMS值)。