A Novel Triple Tracer Approach to Assess Postprandial Protein Turnover

Insulin and nutrients have profound effects on proteome homeostasis. Currently no reliable methods are available to measure postprandial protein turnover. A triple tracer method was developed using phenylalanine stable isotope tracers to estimate appearance rates of ingested (Rameal) and endogenous (Raend) phenylalanine, and the rate of phenylalanine disposal (Rd). This was compared to the "traditional" dual tracer method, using one (1-CM) and two (2-CM) compartment models. For both methods, [13C6]phenylalanine was given orally and [15N]phenylalanine constantly infused; the triple tracer method added [2H5]phenylalanine, infused at rates to mimic meal [13C6]phenylalanine appearance. Additionally, incorporation of meal-derived phenylalanine into specific proteins was measured after purification by 2-dimentional electrophoresis. The triple tracer approach reduced modeling errors, allowing improved reconstruction of Rameal with a tracer-to-tracee ratio that was more constant and better estimated Rd. The 2-CM better described phenylalanine kinetics and Rd than 1-CM. Thus, the triple tracer approach using the 2-compartment model is superior for measuring non-steady state, postprandial protein turnover. This novel approach also allows measurement of postprandial synthesis rates of specific plasma proteins. We offer a valid non-steady state model to measure postprandial protein turnover and synthesis of plasma proteins that can safely be applied in adults, children and pregnant women.