The determination of low d₅‐phenylalanine enrichment (0.002–0.09 atom percent excess), after conversion to phenylethylamine, in relation to protein turnover studies by gass chromatography/electron ionization mass spectrometry

Abstract

A gas chromatography/mass spectrometry (GC/MS) method for measuring very low levels of enrichment of d5-phenylalanine (0.002-0.09 atom percent excess) is described. This method makes it possible to determine the enrichment of amino acid incorporated into tissue protein during studies of protein synthesis in man. Phenylalanine is enzymatically converted to phenylethylamine and the d5-enrichment is measured in the heptafluorobutyryl derivative by selective-ion recording under electron ionization conditions. The coefficients of variation for muscle-protein hydrolysate samples enriched with d5-phenylalanine at the 0.005 and 0.05 atom percent excess levels were 6.0 and 1.2%, respectively. This precision at low enrichment and the small amount of protein needed (about 1 mg) provide real advantages for clinical studies of tissue protein synthesis. Moreover, in contrast to the conventional approach which uses GC/MS for plasma amino acids (typically 2-20 atom percent excess) but gas isotope-ratio mass spectrometry for protein-bound amino acids, the enrichment of both plasma-free and protein-bound d5-phenylalanine can be measured with a single instrument.

The Response of Muscle Protein Anabolism to Combined Hyperaminoacidemia and Glucose-Induced Hyperinsulinemia Is Impaired in the Elderly

“…The intracellular concentration of phenylalanine was then calculated from the tissue value, accounting for the ratio of intracellular to extracellular water (21). Mixed muscle protein-bound phenylalanine enrichment was measured by GCMS (GC 8000 series, MD 800, Fisons Instruments, Manchester, UK) using the standard curve approach previously described (22).…”

Short-term insulin and nutritional energy provision do not stimulate muscle protein synthesis if blood amino acid availability decreases

“…standard (43). Mixed-muscle protein-bound phenylalanine enrichment was analyzed by GC-MS after protein hydrolysis and amino acid extraction (43), using the external standard curve approach (7).…”

Tracers to investigate protein and amino acid metabolism in human subjects

“…The tracer laboratories and suppliers of tracer MS, therefore, should focus on the development of improved equipment and methodology that either allows the measurement of lower enrichments with GC-MS equipment or improves the sensitivity of the existing GC-combustion-IRMS equipment. An elegant solution of the first type has been presented by Calder et al (1992). In the case of tracers with multiple heavy atoms (e.g.…”