Measurement of protein turnover rates by heavy water labeling of nonessential amino acids

In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water ((H2O)-H-2) labeling can safely maintain stable body water H-2 enrichments for weeks or months. H-2 is metabolically incorporated into C-H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, So H-2 incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of (HO)-H-2-O-2-labeled rodent tissue proteins that metabolic H-2 flux into C-H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By (H2O)-H-2 labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl4 was detected. Kinetics of several human serum proteins were also measured, reproducing published t(1/2) estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, (H2O)-H-2 labeling permits sensitive, quantitative, operationally simple measurements of protein turnover in vivo by the rise-to-plateau approach, especially for proteins with slow constitutive turnover. (c) 2006 Elsevier B.V. All rights reserved.