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 (Ra meal ) and endogenous phenylalanine and the rate of phenylalanine disposal (Rd ). This was compared with the "traditional" dual-tracer method, using one (1-CM)- and two (2-CM)-compartment models. For both methods, [13 C6 ]phenylalanine was given orally, and [15 N]phenylalanine was constantly infused; the triple-tracer method added [2 H5 ]phenylalanine, infused at rates to mimic meal [13 C6 ]phenylalanine appearance. Additionally, incorporation of meal-derived phenylalanine into specific proteins was measured after purification by two-dimensional electrophoresis. The triple-tracer approach reduced modeling errors, allowing improved reconstruction of Ra meal 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 2-CM 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.