Muscle Mass Measured by D3‐Creatine Dilution Method Correlates with Assessments by Bio‐Impendance Spectroscopy and 24‐hour Urinary Creatinine
Authors: Hellerstein Marc 
Published: November 2023

Accurate understanding of changes in muscle mass is useful for monitoring the effectiveness of interventions in healthy adults and children, ranging from wellness and lifestyle changes to the efficacy of medical therapies. The total‐body creatine pool size reflects muscle mass as >95% of creatine is present in muscle, and can be assessed directly in vivo by an isotope dilution method. D3‐creatine (D3‐Cr) is administered by mouth and the steady‐state enrichment of D3‐creatinine is measured in urine (creatinine is converted from muscle creatine by a non‐enzymatic step and is excreted in urine in large quantities). Isotope dilution methods have few assumptions but do require knowledge of the amount of tracer delivered to the pool of interest. The objectives of this study were to determine muscle mass in healthy human subjects across different age‐groups of men and women using the D3‐Cr test, and to compare to assessments of fat free mass (FFM). In addition, we have observed that a subset of human subjects directly spill some of the administered D3‐Cr in urine; accordingly, a second objective of this study was to develop a method to correct for spillage of administered D3‐Cr. Subjects (19 males, 23–81 years old; 20 females, 20–77 years old) ingested a single, oral dose of 60 mg D3‐Cr and urine samples were collected prior to and for 4 days following the dose. Urine samples were used for measuring D3‐Cr, total creatine, D3‐creatinine (D3‐Crn) and total creatinine (Crn). Isotopic enrichments of D3‐Crn were measured in fasting morning urine samples by LC/MS and expressed as mole percent excess (MPE). D3‐Crn MPE and the ratio of Cr/Crn were used to calculate creatine pool size and muscle mass. 24‐hour urine collections over 3 days after the oral dose of D3‐Cr were also performed to determine D3‐Cr spillage in the urine. Total body water, extracellular fluid, intracellular fluid (ICW), fat mass and fat‐free mass (FFM) were also assessed by bioimpedence spectroscopy with an Impedimed SFB7 device. Spillage of D3‐Cr in the urine was observed in a subset of predominantly female subjects. An algorithm was developed for the estimation of spillage based on the relationship between the fasting Cr/Crn ratio and the cumulative proportion of D3‐Cr dose excreted over 3 days from 24‐hr urine collections. There were significant correlations between muscle mass measured by D3‐Cr dilution method versus ICW, with spillage corrected either by the algorithm (r=0.9130, p<0.0001) or measured 3 day D3‐Cr losses (r2=0.9173, p <0.0001) or with FFM (r=0.8959 and 0.9022, respectively, p<0.0001). Muscle mass measured by D3‐Crn enrichment also correlated (r=0.8568, p < 0.0001, algorithm corrected) with that measured by 24‐hour creatinine excretion. These results demonstrate that the D3‐Cr dilution method is a validated, non‐invasive, easy to use test for measuring muscle mass in a wide range of subjects and that the technical issue of D3‐Cr spillage can be corrected for. Muscle mass measurement by creatine dilution potentially has broad applications in clinical and non‐clinical settings.Support or Funding InformationKineMed, Inc.