Using Deuteromics to Measure Protein Turnover
Muscle Protein Turnover
Traditional approaches for measuring muscle protein turnover use the precursor-product method with stable isotope analysis by gas chromatography-mass spectrometry. This technique involves administration of 13C-, 15N-, or 2H-labeled amino acids and measuring protein enrichment. The isotopes are either continuously infused for 4 to 12 hours or a flooding dose is used to achieve a steady state. Both approaches have many serious drawbacks:
- Administration of a flooding amino acid can alter protein turnover by itself
- Continuous infusions require special facilities
- IV infusions restrict subject’s real world activities
- Infusion protocols are over short time periods
- True precursor enrichment within muscle cells difficult to measure – must use proxies (enrichment in plasma, KIC, or urinary hippurate)
- Experiments are costly due to sterile IV solutions
- Difficult to test many subjects at once
Labeling with deuterated water is a more convenient and less costly alternative for measuring protein turnover. Only a single oral dose of deuterium oxide is required (see Wilkinson and MacDonald papers below). The basis of the technique is that deuterium oxide rapidly equilibrates with total body water. Free amino acids are labeled with deuterium through various enzymatic reactions. Alanine has 4 possible sites of labeling. Belluoto et al. (Am J Physiol E1340-47, 2007) has shown that labeling of plasma free alanine rapidly equilibrates with total body water within 20 minutes. The precursor enrichment of total body water is easily measured with plasma, saliva or urine.
Gas chromatography combustion isotope ratio mass spectrometry (GCC-IRMS) is used to measure very low levels of deuterium in muscle proteins. We hydrolyze mixed muscle or muscle subfractions. The GCC-IRMS separates derivatized alanine by gas chromatography. The alanine peak is then combusted to hydrogen gas at 1450°C, which is analyzed by isotope ratio mass spectrometry. We can detect enrichment levels of 0.001% of deuterium with accuracy and precision. The fractional protein synthesis rate is calculated from the stable isotope analysis of deuterium in alanine.
The following papers are good references for these techniques:
- Gaiser, Fluckey and Previs (2010) Application of 2H2O to measure skeletal muscle protein synthesis
- MacDonald et al. (2013) A novel oral tracer procedure for measurement of myofibrillar protein synthesis
- Wilkinson et al. (2013) A validation of the application of D2O stable isotope tracer techniques for monitoring day-to-day changes in muscle protein subfraction synthesis in humans
Plasma Protein Turnover
Similar deuterium labeling studies can be used to measure synthesis rates of mixed plasma proteins or specific plasma proteins with the stable isotope analysis of alanine in these proteins. Using a single deuterium administration, it is possible to measure simultaneously both plasma and muscle protein turnover.
Please contact us with your questions about Deuteromic tracer studies.