Determination of low isotopic enrichment of L-[1-13C]valine by gas chromatography/combustion/isotope ratio mass spectrometry: a robust method for measuring protein fractional synthetic rates in vivo.
A method was developed for measuring protein fractional synthetic rates using the N-methoxycarbonylmethyl ester (MCM) derivative of L-[1-13C]valine and on-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The derivatization procedure can be performed rapidly and GC separation of valine from the other branched-chain amino acids, leucine and isoleucine, is easily obtained. A good linear relationship was observed between the increment of the 13C/12C isotope ratio in CO2 gas derived from the combustion of derivatized valine and the tracer mole ratio of L-[1-13C]valine to unlabelled valine. The limit of quantitation was at an L-[1-13C]valine tracer mole ratio of 0.0002. The method was used to measure the isotopic enrichment of L-[1-13C]valine in standard mixtures and in skeletal muscle of six growing piglets infused with L-[1-13C]valine (2 mg kg-1 h-1 for 6 h). After infusion of L-[1-13C]valine the mean tracer mole ratio in plasma of L-[1-13C]valine at the isotopic steady state was 0.0740 +/- 0.0056 (GC/MS, mean +/- SEM) and the mean tracer mole ratio of valine in muscle protein fraction at 6 h was 0.000236 +/- 0.000038 (GC/C/IRMS). The resulting mean protein fractional synthetic rate in piglet skeletal muscle was 0.052 +/- 0.007% h-1, which is in good agreement with literature data obtained with alternative, more elaborate techniques. By this method protein fractional synthetic rates can be measured at low isotopic enrichment levels using L-[1-13C]valine, the MCM derivative and on-line GC/C/IRMS.
Laboratory of Metabolic Disorders, Groningen University Hospital, The Netherlands. d.j.reijngoud@med.rug.nl
D J
DJ
Reijngoud
G
G
Hellstern
H
H
Elzinga
M G
MG
de Sain-van der Velden
A
A
Okken
F
F
Stellaard
D000818:Animals; D002247:Carbon Isotopes; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006868:Hydrolysis; D007553:Isotope Labeling; D007700:Kinetics; D024510:Muscle Development; D009124:Muscle Proteins / Q000096:biosynthesis / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry / Q000254:growth & development; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D013552:Swine; D014633:Valine / Q000032:analysis*
D002247:Carbon Isotopes; D009124:Muscle Proteins; D011506:Proteins; D014633:Valine