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

Biochim Biophys Acta. 2006 May;1760(5):730-44. doi: 10.1016/j.bbagen.2005.12.023. Epub 2006 Jan 24.


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 (2H2O) labeling can safely maintain stable body water 2H enrichments for weeks or months. 2H is metabolically incorporated into C-H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so 2H incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of 2H2O-labeled rodent tissue proteins that metabolic 2H flux into C-H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By 2H2O 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 t1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, 2H2O 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / metabolism*
  • Animals
  • Blood Proteins / analysis
  • Blood Proteins / metabolism
  • Brain / metabolism
  • Brain Chemistry
  • Carbon Tetrachloride / toxicity
  • Collagen / analysis
  • Collagen / metabolism
  • Deuterium Oxide / administration & dosage*
  • Deuterium Oxide / pharmacokinetics
  • Female
  • Fibrosis
  • Gas Chromatography-Mass Spectrometry
  • Humans
  • Isotope Labeling / methods*
  • Kinetics
  • Liver / drug effects
  • Liver / metabolism
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Muscles / chemistry
  • Muscles / metabolism
  • Protein Biosynthesis*
  • Proteins / analysis*
  • Proteins / metabolism
  • Rats
  • Rats, Sprague-Dawley


  • Amino Acids
  • Blood Proteins
  • Proteins
  • Collagen
  • Carbon Tetrachloride
  • Deuterium Oxide