Assessment of Muscle Protein Synthesis

1. Assessment of Muscle Protein Synthesis 637 tutorial…

2. Definitions… • Fractional Synthesis Rates (FSR): the amount of labeled to total enrichment of a specific precursor. Frequently used precursors are: Leucine, Valine, Phenylalanine, Methionine and Alanine. Always expressed in percent. • Rates of muscle protein synthesis (RPS or MPS): the amount of label incorporated into skeletal muscle over a defined period of time. Typically, nmolsincorp. / g tissue / unit of time

3. Definitions… • Mixed muscle: muscle protein synthesis occurring in the total muscle homogenate (consisting of a mixture of proteins). • Myofibrillar FSR: muscle protein synthesis occurring in the myofibrillar rich protein fraction • Cytosolic FSR: muscle protein synthesis occurring in the cytosolic rich fraction • Mitochondrial FSR: protein synthesis occurring in the mitochondrial rich fraction

4. Definitions… • Tracer: The molecule chosen to assess protein synthesis. The selection of this molecule (typically an amino acid) is very important. This molecule is typically a stable or radioactive isotope of the molecule of choice. • Precursor pool: the availability of label and unlabeled tracer (typically an amino acid). The ratio of labeled to unlabeled molecules should remain steady and must be accurately assessed/measured to have any interpretable data for calculations of protein synthesis. • The stability of the precursor pool should be measured at the tRNA level, but this is rarely done. Most use the blood values or cytosolic values to determine the “enrichment” of the tRNA, which is not always appropriate…

5. Amino Acid Availability Vessel Interstitial Space Protein Intracellular Space

6. Amino Acid Availability Vessel Interstitial Space Protein Intracellular Space

7. Amino Acid Availability Vessel Interstitial Space Protein Intracellular Space

8. Method of delivery for precursor molecules 2H2O 2H2O 2H2O

9. Flooding Dose Calculations • RPS by Vary • (DPM ∙ g wet wt-1) ∙ (SĀ)-1∙ (t)-1 • where DPM (disintegrations per minute) ∙ g wet wt-1 is the quantity of L-[2,3,4,5,6-3H]F incorporated into the available pool (mg protein per g of tissue), SĀ is the mean specific activity in plasma (DPM · nmol F-1) over 12 minutes of flooding and t is incorporation time in hours. • Fractional rates of protein synthesis (FSR) with F were calculated using the equation described by Garlick et al. (19): • SP ∙ (SĀ x t)-1 x 100

10. Primed-constant infusion calculations

11. FSR by deuterium oxide by Gasier:

12. Assumptions for synthesis rates that may or may not have been validated: Rates of label incorporation are linear over time. (This does not mean that protein synthesis is linear over time; which often confuses people in the field. The intent of the statement is to reflect that for a given rate of synthesis, label incorporation for that rate will be linear over time.) Recycling of label is constant/negligible during periods of assessment. Specific activities of the cytosol and tRNA are similar. Detectable enrichments are indicative of the protein pool being measured.

13. The flooding dose technique includes the following assumptions which may or may not have been validated: • Rates of label incorporation are linear over time. • There is an immediate equilibration between the extracellular and intracellular phenylalanine compartments. • The massive dose of the tracer does not contribute to calculated synthesis rates. • Rates of protein degradation during this short period (10-15 min) are negligible. • SA pools are not concentrated or diluted during the period of assessment.

14. The primed-constant infusion method assumptions that may or may have not been validated: Rates of label incorporation are linear over time. SA pools are relatively constant over the measuring period, and that these pools are equilibrated with tRNA pools (equal access for label with low concentrations). Rates of protein degradation are constant or negligible during the period of measure (no dilution of pools or re-using labeled substrates). There are no changes in protein pool during period of measure. Labels are not ‘swapped’ or if they are, measured during the period.

15. Deuterium oxide methodologies also have assumptions: • Rates of label incorporation are linear over time. • There is rapid equilibration between body water labeling and amino acid tracer. • Labels are not transferred once amino acids have been incorporated into skeletal muscle protein • Fast turnover proteins are not saturated with label during period of measure.