A validation of the application of D(2)O stable isotope tracer techniques for monitoring day-to-day changes in muscle protein subfraction synthesis in humans

Abstract

Quantification of muscle protein synthesis (MPS) remains a cornerstone for understanding the control of muscle mass. Traditional [(13)C]amino acid tracer methodologies necessitate sustained bed rest and intravenous cannulation(s), restricting studies to ~12 h, and thus cannot holistically inform on diurnal MPS. This limits insight into the regulation of habitual muscle metabolism in health, aging, and disease while querying the utility of tracer techniques to predict the long-term efficacy of anabolic/anticatabolic interventions. We tested the efficacy of the D2O tracer for quantifying MPS over a period not feasible with (13)C tracers and too short to quantify changes in mass. Eight men (22 ± 3.5 yr) undertook one-legged resistance exercise over an 8-day period (4 × 8-10 repetitions, 80% 1RM every 2nd day, to yield "nonexercised" vs. "exercise" leg comparisons), with vastus lateralis biopsies taken bilaterally at 0, 2, 4, and 8 days. After day 0 biopsies, participants consumed a D2O bolus (150 ml, 70 atom%); saliva was collected daily. Fractional synthetic rates (FSRs) of myofibrillar (MyoPS), sarcoplasmic (SPS), and collagen (CPS) protein fractions were measured by GC-pyrolysis-IRMS and TC/EA-IRMS. Body water initially enriched at 0.16-0.24 APE decayed at ~0.009%/day. In the nonexercised leg, MyoPS was 1.45 ± 0.10, 1.47 ± 0.06, and 1.35 ± 0.07%/day at 0-2, 0-4, and 0-8 days, respectively (~0.05-0.06%/h). MyoPS was greater in the exercised leg (0-2 days: 1.97 ± 0.13%/day; 0-4 days: 1.96 ± 0.15%/day, P < 0.01; 0-8 days: 1.79 ± 0.12%/day, P < 0.05). CPS was slower than MyoPS but followed a similar pattern, with the exercised leg tending to yield greater FSRs (0-2 days: 1.14 ± 0.13 vs. 1.45 ± 0.15%/day; 0-4 days: 1.13 ± 0.07%/day vs. 1.47 ± 0.18%/day; 0-8 days: 1.03 ± 0.09%/day vs. 1.40 ± 0.11%/day). SPS remained unchanged. Therefore, D2O has unrivaled utility to quantify day-to-day MPS in humans and inform on short-term changes in anabolism and presumably catabolism alike.

Keywords: deuterium oxide; protein synthesis; skeletal muscle.

Fig. 1.

Schematic of resistance exercise (Ex) study protocol. RET, resistance exercise training; 1RM, 1-repetition maximum.

Fig. 2.

A: exponential time course of body water enrichment over 8 days following oral bolus of 150 ml of deuterium oxide (D₂O; 70 atom%). B: natural logarithm transformed body water enrichment for calculation of decay constant and elimination half-life. C: correlation of 2 different body water analytical techniques using high-temperature conversion elemental analyzer (TC-EA) direct liquid injection and gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-Pyr-IRMS). D: comparison of plasma alanine enrichment and saliva enrichment on day 8. *Significantly different from initial day 1 time point, P < 0.01. APE, atom% excess.

Fig. 3.

A: linear incorporation of deuterium into protein bound alanine over the 1st 4 days. B: illustration of nonlinear incorporation of deuterium into protein-bound alanine beyond 4 days. C: myofibrillar protein synthesis rates in exercised (solid line) and nonexercised (dashed line) legs over 8-day training period. D: temporal pattern of myofibrillar protein synthesis in exercised and nonexercised legs. ***Significantly different from rested leg at the same time point, P < 0.001; **P < 0.01; *P < 0.05; §significantly different from previous time point in same leg P < 0.05. FSR, fractional synthetic rate.

Fig. 4.

Muscle collagen (A) and sarcoplasmic protein synthesis (B) in exercised (solid line) and nonexercised (dashed line) legs over 8-day training period.