Mass Spectrometric Methods for Determination of [13C]Leucine Enrichment in Human Muscle Protein

Abstract

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 CO2was introduced into the MS by a dual inlet system following cryogenic concentration. In the second method (ninhydrin/GC/IRMS) the CO2was 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 ofL-[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.