Determination of low isotopic enrichment ofL-[1-13C]valine by gas chromatography/combustion/isotope ratio mass spectrometry: a robust method for measuring protein fractional synthetic ratesin vivo
✍ Scribed by Reijngoud, Dirk-Jan; Hellstern, Gerald; Elzinga, Henk; de Sain-van der Velden, Monique G.; Okken, Albert; Stellaard, Frans
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 225 KB
- Volume
- 33
- Category
- Article
- ISSN
- 1076-5174
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✦ Synopsis
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 lineair relationship was observed between the increment of the 13C/12C isotope ratio in gas derived from the combustion of deriva-CO 2 tized 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 0.000 236 » 0.000 038 (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.