Measurement of human mixed muscle protein fractional synthesis rate depends on the choice of amino acid tracer

Abstract

Smith GI, Villareal DT, Mittendorfer B. Measurement of human mixed muscle protein fractional synthesis rate depends on the choice of amino acid tracer. Am J Physiol Endocrinol Metab 293: E666-E671, 2007. First published May 29, 2007; doi:10.1152/ajpendo.00185.2007.-The goal of this study was to discover whether using different tracers affects the measured rate of muscle protein synthesis in human muscle. We therefore measured the mixed muscle protein fractional synthesis rate (FSR) in the quadriceps of older adults during basal, postabsorptive conditions and mixed meal feeding (70 mg protein ⅐ kg fat-free mass Ϫ1 ⅐ h Ϫ1 ϫ 2.5 h) by simultaneous intravenous infusions of [5,5,5-2 H3]leucine and either [ring-13 C6]phenylalanine or [ring-2 H5]phenylalanine and analysis of muscle tissue samples by gas chromatography-mass spectrometry. Both the basal FSR and the FSR during feeding were ϳ20% greater (P Ͻ 0.001) when calculated from the leucine labeling in muscle tissue fluid and proteins (fasted: 0.063 Ϯ 0.005%/h; fed: 0.080 Ϯ 0.007%/h) than when calculated from the phenylalanine enrichment data (0.051 Ϯ 0.004 and 0.066 Ϯ 0.005%/h, respectively). The feeding-induced increase in the FSR (ϳ20%; P ϭ 0.011) was not different with leucine and phenylalanine tracers (P ϭ 0.69). Furthermore, the difference between the leucine-and phenylalaninederived FSRs was independent of the phenylalanine isotopomer used (P ϭ 0.92). We conclude that when using stable isotope-labeled tracers and the classic precursor product model to measure the rate of muscle protein synthesis, absolute rates of muscle protein FSR differ significantly depending on the tracer amino acid used; however, the anabolic response to feeding is independent of the tracer used. Thus different precursor amino acid tracers cannot be used interchangeably for the evaluation of muscle protein synthesis, and data from studies using different tracer amino acids can be compared qualitatively but not quantitatively. muscle protein turnover MUSCLE MASS IS MAINTAINED by a balance between the rate of muscle protein synthesis and breakdown (29), which can be measured in vivo by using a variety of tracer methods (4,27,28,32,40,42). The most commonly used approach to measure the rate of muscle protein synthesis is based on the incorporation of a stable isotope-labeled amino acid (usually phenylalanine or leucine) during continuous intravenous infusion of the tracer to determine the muscle protein fractional synthesis rate (FSR) (5,22,31,35,37,41). The reported rates of mixed muscle protein synthesis from these studies varies substantially even during standard overnight fasted conditions in young, healthy individuals (from ϳ0.04 to 0.08%/h) (11-13, 16, 23, 24, 36, 37, 41). The reason(s) for this variability in results is not clear.One potential explanation for the disparity in results may be the use of different tracers and/or surrogate precursor pools for the calculation of the FSR. Although the FSR is known to be influenced, to some degree, by the precursor pool used for calculat...

A new method to measure muscle protein synthesis in humans by endogenously introduced d9-leucine and using blood for precursor enrichment determination

“…Because the amino acid enrichment in this later report (Smith et al. 2007) was measured using GC-MS, comparable responses in the calculated FSR values when using the leucine and the phenylalanine tracers between that report and the present study suggest that the leucine tracer itself, as well as the type of mass spectrometer employed to analyze d 9 -leucine enrichment (i.e., LC-MS/MS vs. GC-MS), do not have any effects on the evaluation of muscle protein synthesis. Such lack of analytical (i.e., sample processing/mass spectrometry-related) effects in the present study was also expected because the deuteriums in the d 9 -leucine are all bonded to carbon atoms that do not exchange (Whitelegge 2009; Rudowska et al.…”

Isotopic and modeling investigation of long-term protein turnover in rat tissues

“…However, FSR values in the literature varied considerably, depending on the studies, by up to five-or threefold for muscle or kidney, respectively. Given the singularity of our approach compared with classic AA tracer studies and the fact that FSR estimations in such studies appear to be strongly dependent on the particular method and specific experimental conditions (20,26,49,52), comparisons between our results and the literature should remain qualitative only and cannot be used as an external validation of our approach. However, the ranking of tissues according to their FSR values was globally the same when comparing the results of the present method and mean data in the literature (Table 2).…”

“…In particular, their FSR estimates may be biased because of nonphysiological conditions that artificially increase the P turnover when the flooding-dose method is used and because of the large uncertainty regarding the isotopic enrichment of the true precursor pool when the constant-infusion method is used (12,20). The FSR values reported in the literature, therefore, differ markedly, depending on the tracer method, the particular AA tracer, and the surrogate precursor pool for P synthesis that are used (20,26,49,52).Precursor-product AA tracer studies have established that the different body P have distinct FSR [e.g., much more rapid in gut and liver than in muscle (10, 52)] that are also differently stimulated by particular nutritional, physiological, and pathophysiological conditions (9,19,36,52). Because of the necessarily limited duration of such AA tracer studies, most FSR modulations have been measured acutely over short time periods and have been reported to differ according to the particular metabolic state of the individual (i.e., fed or fasted), but it is difficult to extrapolate conclusions from such results regarding average FSR values over longer periods of time.…”

“…In particular, their FSR estimates may be biased because of nonphysiological conditions that artificially increase the P turnover when the flooding-dose method is used and because of the large uncertainty regarding the isotopic enrichment of the true precursor pool when the constant-infusion method is used (12,20). The FSR values reported in the literature, therefore, differ markedly, depending on the tracer method, the particular AA tracer, and the surrogate precursor pool for P synthesis that are used (20,26,49,52).…”

Exercise and Regulation of Protein Metabolism

“…Typically, in the postabsorptive (fasted) state, rates of MPB exceed those of MPS (i.e., 0.08-0.1% h À1 for MPB and 0.03-0.07% h À1 for MPS [1][2][3], engendering negative protein balance (À0.04% to 0.05% h À1 ), and hence, an overall loss of muscle proteins. However, this catabolic scenario is reversed following intake of dietary protein, upon which rates of MPS exceed that of MPB, and muscle protein balance becomes positive again through an approximate doubling of MPS.…”