Longitudinal changes in total body creatine pool size and skeletal muscle mass using the D₃‐creatine dilution method

Background We recently validated in cross‐sectional studies a new method to determine total body creatine pool size and skeletal muscle mass based on D 3 ‐creatine dilution from an oral dose and detection of urinary creatinine enrichment by isotope ratio mass spectrometry (IRMS). Routine clinical use of the method in aging and disease will require repeated application of the method, with a more widely available technology than IRMS, to enable determination of change in skeletal muscle mass in longitudinal studies. We therefore adapted the method to liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) technology, and sought to establish proof of concept for the repeated application of the method in a longitudinal study. Because the turnover of creatine is slow, it was also critical to determine the impact of background enrichment from an initial dose of oral D 3 ‐creatine on subsequent, longitudinal measurements of change in muscle mass. Methods Rats were given an oral tracer dose of D 3 ‐creatine (1.0 mg/kg body weight) at 10 and 17 weeks of age. LC‐MS/MS was used to determine urinary D 3 ‐creatine, and urinary D 3 ‐creatinine enrichment, at time intervals after D 3 ‐creatine administration. Total body creatine pool size was calculated from urinary D 3 ‐creatinine enrichment at isotopic steady state 72 h after administration of D 3 ‐creatine tracer. Results At 10 weeks of age, rat lean body mass (LBM) measured by quantitative magnetic resonance correlated with creatine pool size ( r = 0.92, P = 0.0002). Over the next 7 weeks, the decline in urinary D 3 ‐creatinine enrichment was slow and linear, with a rate constant of 2.73 ± 0.06 %/day. Subtracting background urinary D 3 ‐creatinine enrichment from the elevated enrichment following a second dose of D 3 ‐creatine at 17 weeks permitted repeat calculations of creatine pool size. As at 10 weeks, 17‐week LBM correlated with creatine pool size ( r = 0.98, P <0.0001). In addition, the change in creatine pool size was correlated with the change in LBM during the 7 weeks of rat growth between measurements ( r = 0.96, P <0.0001). Conclusion The LC‐MS/MS‐based D 3 ‐creatine dilution method can be applied repeatedly to measure total body creatine skeletal muscle mass change in longitudinal study.