The D3 -creatine dilution method non-invasively measures muscle mass in mice

Abstract

Developing accurate methods to quantify age-related muscle loss (sarcopenia) could greatly accelerate development of therapies to treat muscle loss in the elderly, as current methods are inaccurate or expensive. The current gold standard method for quantifying sarcopenia is dual-energy X-ray absorptiometry (DXA) but does not measure muscle directly-it is a composite measure quantifying "lean mass" (muscle) excluding fat and bone. In humans, DXA overestimates muscle mass, which has led to erroneous conclusions about the importance of skeletal muscle in human health and disease. In animal models, DXA is a popular method for measuring lean mass. However, instrumentation is expensive and is potentially limited by anesthesia concerns. Recently, the D₃ -creatine (D₃ Cr) dilution method for quantifying muscle mass was developed in humans and rats. This method is faster, cheaper, and more accurate than DXA. Here, we demonstrate that the D₃ Cr method is a specific assay for muscle mass in mice, and we test associations with DXA and body weight. We evaluated the D₃ Cr method compared to DXA-determined lean body mass (LBM) in aged mice and reported that DXA consistently overestimates muscle mass with age. Overall, we provide evidence that the D₃ Cr dilution method directly measures muscle mass in mice. Combined with its ease of use, accessibility, and non-invasive nature, the method may prove to more quickly advance development of preclinical therapies targeting sarcopenia.

Keywords: aging; mice; sarcopenia; skeletal muscle.

FIGURE 1

Creatine pool size and muscle mass can be calculated from mouse urine by D3‐creatinine enrichment following an IP injection of D3‐labeled creatine. (a) D₃‐creatinine enrichment values at varying timepoints from individual urine samples of young mice following an IP injection of D₃‐labeled creatine. (b) Creatine pool size (mg) values of individual mice calculated using their respective D₃‐creatinine enrichment at 24 h. Bars represent creatine pool sizes either uncorrected or corrected with spillage effects observed at 0, 2, 6, and, 12 h post‐IP. (c) Muscle mass (g) values of individual mice calculated using their respective D3‐creatinine enrichment at 24 h. Bars represent creatine pool sizes either uncorrected or corrected with spillage effects observed at 0, 2, 6, and, 12 h post‐IP. “M#” individual male mice, “F#” individual female mice.

FIGURE 2

Cross‐sectional analysis of aged mice revealed lower DXA‐determined lean mass and increased fat mass independent of D₃Cr determined skeletal muscle mass. (a) Body weights of mice, grouped by age. (b) DXA‐determined fat body mass (FBM) percentages, grouped by age. (c) DXA‐determined lean body mass (LBM) percentages, grouped by age. (d) D₃Cr dilution method calculated muscle mass (D₃Cr MM) percentages, grouped by age. (e) Both DXA LBM and D3‐Crn MM correlate significantly with total body weight. (f) Decreasing skeletal muscle mass determined by D3‐Crn contrasted with DEXA in mice of varying ages. BM, bone mass, FBM, fat body mass, LBM, lean body mass, D3‐Crn MM, skeletal muscle mass as determined by D₃Cr dilution method. Significance – ns p > 0.05, * p < 0.05, **<0.005, ***<0.0005).