Balagopal P, Ford G C, Ebenstein D B, Nadeau D A, Nair K S
Endocrine Research Unit, Mayo Clinic and Foundation, 200 First Street S.W., Rochester, Minnesota, 55905, USA.
Anal Biochem. 1996 Jul 15;239(1):77-85. doi: 10.1006/abio.1996.0293.
Two modified GC-based isotope ratio MS (IRMS) techniques, for measurement of [13C]leucine enrichment in muscle protein, were compared with a conventional dual inlet technique. Of these three, two involved HPLC purification of leucine and liberation of carbon dioxide (CO2) using the ninhydrin reaction. In the conventional technique the CO2 was introduced into the MS by a dual inlet system following cryogenic concentration. In the second method (ninhydrin/GC/IRMS) the CO2 was purified by on-line GC. In the third technique, GC/combustion/IRMS, derivatized amino acids are separated by GC and analyzed after combustion. A primed continuous infusion of L-[1-13C]leucine was given intravenously to human subjects and needle quadriceps muscle biopsies were taken to measure [13C]leucine enrichment in muscle protein. All three methods demonstrated excellent correlation (r > 0. 999). Differences in the measurement of [13C]leucine enrichment in muscle protein were <6%. The ninhydrin techniques require microgram quantities of leucine, with the conventional technique requiring twice the amount as the ninhydrin/GC/IRMS method. The GC/combustion/IRMS technique requires only nanogram quantities of leucine with similar precision enabling measurement of synthesis rates of individual proteins from biopsy samples. We have measured the isotopic enrichment of myosin heavy chain and mixed muscle protein in human subjects using the GC/combustion/IRMS technique.
将两种基于气相色谱（GC）的改良同位素比率质谱（IRMS）技术用于测量肌肉蛋白质中[¹³C]亮氨酸的富集情况，并与传统的双进样技术进行比较。在这三种技术中，有两种涉及通过高效液相色谱（HPLC）纯化亮氨酸，并使用茚三酮反应释放二氧化碳（CO₂）。在传统技术中，CO₂在低温浓缩后通过双进样系统引入质谱仪。在第二种方法（茚三酮/GC/IRMS）中，CO₂通过在线气相色谱进行纯化。在第三种技术GC/燃烧/IRMS中，衍生化的氨基酸通过气相色谱分离并在燃烧后进行分析。对人类受试者静脉内给予一次负荷剂量的L-[¹-¹³C]亮氨酸，并采集股四头肌活检针吸组织样本以测量肌肉蛋白质中[¹³C]亮氨酸的富集情况。所有三种方法均显示出极好的相关性（r>0.999）。肌肉蛋白质中[¹³C]亮氨酸富集量的测量差异<6%。茚三酮技术需要微克量的亮氨酸，而传统技术所需量是茚三酮/GC/IRMS方法的两倍。GC/燃烧/IRMS技术仅需要纳克量的亮氨酸，且具有相似的精密度，能够测量活检样本中单个蛋白质的合成速率。我们已使用GC/燃烧/IRMS技术测量了人类受试者中肌球蛋白重链和混合肌肉蛋白质的同位素富集情况。
