Application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the analysis of stable isotope enrichments of phenylalanine and tyrosine

Abstract

Whole-body protein synthesis and breakdown are measured by a combined tracer infusion protocol with the stable isotope amino acids L-[ring-(2)H(5)]-phenylalanine, L-[ring-(2)H(2)]-tyrosine and L-[ring-(2)H(4)]-tyrosine that enable the measurement of the phenylalanine to tyrosine conversion rate. We describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of very low tracer-tracee ratios (TTR) of the amino acids L-phenylalanine and L-tyrosine in human plasma. TTR calibration curves of the tracers L-[ring-(2)H(5)]-phenylalanine, L-[ring-(2)H(2)]-tyrosine and L-[ring-(2)H(4)]-tyrosine were linear (r(2)>0.99) in the range between 0.01% and 5.0% TTR and lowest measurable TTR for the tracers was 0.01% at a physiological concentration of 60 microM. The method was applied successfully to plasma samples from a clinical study reaching a steady state enrichment plateau (mean+/-SD) of 3.33+/-0.19% for L-[ring-(2)H(5)]-phenylalanine, 2.40+/-0.43% for L-[ring-(2)H(2)]-tyrosine and 0.29+/-0.07% for L-[ring-(2)H(4)]-tyrosine, respectively. The LC-MS/MS method can be applied for measurement of very low plasma enrichments of phenylalanine and tyrosine for the determination of whole-body protein synthesis and breakdown rates in humans.