Abstract
Skeletal muscle mass is determined by the balance between rates of protein synthesis and degradation. Protein synthesis rates can be measured in vivo by administering an amino acid as a tracer that is labeled with an isotope (radioactive or stable) of C, H, or N. The rate at which the labeled amino acid is incorporated into muscle protein, as a function of the amount of labeled amino acid in the precursor pool at the site of translation, reflects the rate of protein synthesis. There are a number of approaches for performing this measurement depending on the question being addressed and the experimental system being studied. In this chapter, we describe the “flooding dose” approach using l-[3H]-phenylalanine as the tracer and that is suitable for determining the rate of skeletal muscle protein synthesis (total and myofibrillar proteins) over an acute period (ideally less than 30 min) in any size animal; details for working with mice are presented. The method describes how to administer the tracer without anesthesia, the tissue collection, and the preparation of muscle and blood samples for analysis of the tracer and tracee amino acids in the precursor pool and in muscle proteins.
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References
Davis TA, Fiorotto ML (2009) Regulation of muscle growth in neonates. Curr Opin Clin Nutr Metab Care 12:78–85
Davis TA, Fiorotto ML (2005) Regulation of skeletal muscle protein metabolism in growing animals. In: Burrin DG, Mersmann HJ (eds). Biology of Metabolism of Growing Animals. Elsevier, The Netherlands, pp. 37–68
Rooyackers OE, Nair KS (1997) Hormonal regulation of human muscle protein metabolism. Annu Rev Nutr 17:457–485
Davis TA, Fiorotto ML, Burrin DG, et al (1999) Protein synthesis in organs and tissues: quantitative methods in laboratory animals. In: El-Khoury AE (ed), Methods for Investigation of Amino Acid and Protein Metabolism. CRC Press, Boca Raton, 49–68
Reeds PJ, Davis TA (1999) Of flux and flooding: the advantages and problems of different isotopic methods for quantifying protein turnover in vivo: I. Methods based on the dilution of a tracer. Curr Opin Clin Nutr Metab Care 2:23–28
Davis TA, Reeds PJ (2001) Of flux and flooding: the advantages and problems of different isotopic methods for quantifying protein turnover in vivo: II. Methods based on the incorporation of a tracer. Curr Opin Clin Nutr Metab Care 4:51–56
Garlick PJ, McNurlan MA, Essen P, et al (1994) Measurement of tissue protein synthesis rates in vivo: a critical analysis of contrasting methods. Am J Physiol Endocrinol Metab 266:E287–E297
Wagenmakers AJ (1999) Tracers to investigate protein and amino acid metabolism in human subjects. Proc Nutr Soc 58:987–1000
Chinkes DL (2005) Methods for measuring tissue protein breakdown rate in vivo. Curr Opin Clin Nutr Metab Care 8:534–537
Bergen WG (2008) Measuring in vivo intracellular protein degradation rates in animal systems. J Anim Sci 86:E3–12
Vissers YL, von Meyenfeldt MF, Braulio VB, et al (2003) Measuring whole-body actin/myosin protein breakdown in mice using a primed constant stable isotope-infusion protocol. Clin Sci (Lond) 104:585–590
Emery PW, Preedy VR (2003) Measuring muscle protein turnover in vivo: what can 3-methylhistidine production tell us? Clin Sci (Lond) 104:557–558
Johnson JD, Dunham T, Skipper BJ, et al (1986) Protein turnover in tissues of the rat fetus following maternal starvation. Pediatr Res 20:1252–1257
Fiorotto ML, Davis TA, Reeds PJ (2000) Regulation of myofibrillar protein turnover during maturation in normal and undernourished rat pups. Am J Physiol Regul Integr Comp Physiol 278:R845–R854
Garlick PJ, McNurlan MA, Preedy VR (1980) A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H]phenylalanine. Biochem J 192:719–723
Davis TA, Fiorotto ML, Nguyen HV, et al (1999) Aminoacyl-tRNA and tissue free amino acid pools are equilibrated after a flooding dose of phenylalanine. Am J Physiol Am J Physiol Endocrinol Metab 277:E103–E109
Louis M, Poortmans JR, Francaux M, et al (2003) No effect of creatine supplementation on human myofibrillar and sarcoplasmic protein synthesis after resistance exercise. Am J Physiol Endocrinol Metab 266:E287–E297
Balagopal P, Ljungqvist O, Nair KS (1997) Skeletal muscle myosin heavy-chain synthesis rate in healthy humans. Am J Physiol Endocrinol Metab 272:E45–E50
Rooyackers O, Adey D, Ades P, et al (1996) Effect of age on in vivo rates of mitochodrial protein synthesis in human skeletal muscle. Proc Nat Acad Sci USA 93:15364–15369
Welle S, Thornton C, Jozefowicz R, et al (1993) Myofibrillar protein synthesis in young and old men. Am J Physiol Endocrinol Metab 264:E693–E698
Rennie MJ, Smith K, Watt PW (1994) Measurement of human tissue protein synthesis: an optimal approach. Am J Physiol Endocrinol Metab 266:E298–E307
Smith PK, Krohn RI, Hermanson GT, et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85
Davis TA, Fiorotto ML, Nguyen HV, et al (1991) Response of muscle protein synthesis to fasting in suckling and weaned rats. Am J Physiol Regul Integr Comp Physiol 261:R1373–R1380
Heys SD, Norton AC, Dundas CR, et al (1989) Anaesthetic agents and their effect on tissue protein synthesis in the rat. Clin Sci (Lond) 77:651–655
Jepson MM, Pell JM, Bates PC, et al (1986) The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats. Biochem J 235:329–336
Bregendahl K, Liu L, Cant JP, et al (2004) Fractional protein synthesis rates measured by an intraperitoneal injection of a flooding dose of L-[ring-2H5]phenylalanine in pigs. J Nutr 134:2722–2728
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Fiorotto, M.L., Sosa, H.A., Davis, T.A. (2012). In Vivo Measurement of Muscle Protein Synthesis Rate Using the Flooding Dose Technique. In: DiMario, J. (eds) Myogenesis. Methods in Molecular Biology, vol 798. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-343-1_14
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DOI: https://doi.org/10.1007/978-1-61779-343-1_14
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