Validation of a single biopsy approach and bolus protein feeding to determine myofibrillar protein synthesis in stable isotope tracer studies in humans


Journal Title (Medline/Pubmed accepted abbreviation): Nutr.&Metab.
Year: 2011
Volume: 8
Number:1
Page numbers: 15
Website http://www.nutritionandmetabolism.com/content/8/1/15

Summary of Background and Research Design

Background:In order to measure the rate of protein synthesis in response to a nutritional component, subjects are often administered the nutrient that is enriched with a rare isotope. The isotopes are safe and have exactly the same properties, but the fates of the rare isotopes are able to be followed through cellular uptake and metabolism. For example, one can observe isotopically-labeled amino acids being incorporated into proteins. Carbon-13 and hydrogen-2 (deuterium) are commonly employed rare isotopes. Typically, the participant is primed with an initial dose of the labeled nutrient (ex. 2 µmol/kg body weight) and then is provided with a constant infusion of the labeled nutrient (ex. 0.05 µmol/kg/min). A muscle biopsy is obtained about 90 min after the priming dose and then a second biopsy after a longer period of time, approximately 3 hrs. The muscle biopsy entails 3.0-4.0 mm diameter needles, which can be painful for the subject. Therefore, there is an ethical drive to minimize the number of biopsies required to answer the science question.
       Another commonly employed experimental technique is to add isotopically-labeled amino acids to a protein drink in order to study the rates of muscle protein synthesis and breakdown after exercise. However, there is concern that the physiological fates of the amino acids are different than the intact protein, therefore providing false information about whole body protein synthesis.

Hypothesis:1) The proposed mathematical model with a single muscle biopsy can determine the rate of muscle synthesis as accurately as the common two biopsy method. 2) After exercise, 4% of isotopically-labeled tracer added to a protein drink will be able to accurately report rates of muscle protein synthesis and breakdown.

Subjects:Sixteen active males were randomly assigned to perform study 1 (8 subjects, 21 ± 4 yrs old) or study 2 (8 subjects, 22 ± 3 yrs old).

Treatments and protocol:
  • Study 1- Participants arrived at the laboratory in the morning after an overnight fast. A baseline blood sample was drawn and blood samples were drawn once every hour. The subject was primed intravenously with 2 µmol/kg body weight of L-[ring-13C6]phenylalanine and then received a constant infusion at 0.05 µmol/kg/min. Muscle biopsies were obtained at 90 and 240 min after the priming dose. The rate of myofibrillar protein synthesis was calculated using the traditional method, using both time points. The individual biopsies were also used in two different proposed mathematical models, one for the “short” incubation time and one for the “long” incubation time to calculate the rate of protein synthesis (see paper for details).
  • Study 2- Participants reported to the laboratory after an overnight fast and the priming and infusion were administered as in Study 1. The subjects then performed unilateral leg press exercises with one leg while the other leg served as a control. After exercise, subjects consumed 25 g whey protein isolate enriched with 4% of the 13C-labeled phenylalanine tracer (35.6 mg). Muscle biopsies were obtained from both legs at 60, 180, and 300 min after exercise. Arterialized blood samples were acquired every hour before the whey beverage and every 10 min after the beverage.

Summary of research findings:
Study 1)
  • Muscle intracellular concentration enrichment was not significantly different between the “short” incubation time (biopsy 1) and the “long” incubation time (biopsy 2).
  • The calculation of the rate of myofibrillar protein synthesis was the same for the “long” one biopsy method and the two biopsy method. However, the “short” one biopsy method yielded an apparent overestimate of the rate of protein synthesis (statistically different result).
Study 2)
  • Plasma enrichment levels of isotopes peaked 20 min after drinking the enriched whey beverage. By measuring the ratio of tracer to tracee, the plasma isotope concentrations appeared to have reached steady state over the duration of the infusion (ideal for protein synthesis rate measurements).

Interpretation of findings/Key practice applications:

Their one biopsy model appears to be an accurate alternative from the 2 biopsy method of calculating the rate of myofibrillar protein synthesis when the incorporation time is about 3 hrs. They also showed that 4% enrichment of a protein beverage after exercise is not too much to overload the amino acid equilibrium, thus providing an accurate way to measure protein synthesis and breakdown.
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