Dilution of oral D₃‐Creatine to measure creatine pool size and estimate skeletal muscle mass: development of a correction algorithm

Abstract

BackgroundMuscle mass can be measured directly in vivo by isotope dilution, using Creatine‐(methyl‐d3) monohydrate (D3‐Cr) by mouth followed by measurement of the steady‐state enrichment of D3‐creatinine (D3‐Crn) in urine. Isotope dilution methods require knowledge of the amount of tracer delivered to the pool of interest. In a subset of human subjects, a small amount of orally administered D3‐Cr ‘spills’ into urine after absorption and prior to transport into skeletal muscle cells. The objectives were to develop a method to correct for spillage to compare the estimate of muscle mass by D3‐Cr dilution to other assessments of fat‐free mass.MethodsSubjects (19 males, 23–81 years old; 20 females, 20–77 years old) ingested a single dose of 60 mg D3‐Cr and urine was collected prior to and daily for 4 days following the dose. Fasting morning urine samples was assessed for D3‐Cr, total Cr, D3‐Crn, and total Crn concentrations, as well as isotopic enrichments of D3‐Crn, by LC/MS. The 24‐h urine collections over 3 days after the dose of D3‐Cr were also performed to determine D3‐Cr spillage. Total body water, fat mass, and fat‐free mass were assessed by bioelectrical impedance spectroscopy (BIS).ResultsSpillage of D3‐Cr in the urine was greater in women than men. D3‐Crn enrichment and the ratio of Cr/Crn were used in an algorithm to calculate Cr pool size and muscle mass. Specifically, an algorithm was developed for the estimation of spillage based on the relationship between the fasting Cr/Crn ratio and the cumulative proportion of the D3‐Cr dose excreted over 3 days based on 24‐h urine collections. Muscle mass corrected using the algorithm based on fasting urine levels correlated (r = 0.9967, P < 0.0001) with that corrected by measuring D3‐Cr dose excreted. Muscle mass measured by D3‐Crn enrichment also correlated (r = 0.8579, P < 0.0001, algorithm corrected) with that measured by 24‐h Crn excretion. Muscle mass measured by D3‐Cr dilution method correlated with intracellular water by BIS, whether using spillage corrected by the algorithm (r = 0.9041, P < 0.0001) or measured by 3 day D3‐Cr losses (r = 0.91, P < 0.0001) and similarly correlated with fat‐free mass by BIA (r = 0.8857 and 0.8929, P < 0.0001, respectively).ConclusionsThe D3‐Cr dilution method is further validated here as a non‐invasive, easy‐to‐use test for measuring muscle mass. The technical issue of D3‐Cr spillage can be corrected for with a simple algorithm based on fasting spot urine samples. Muscle mass by Cr dilution potentially has broad applications in clinical and research settings.

Association of change in muscle mass assessed by D3‐creatine dilution with changes in grip strength and walking speed

“…Muscle mass was determined by the D 3 Cr dilution method as previously described . Briefly, total body creatinine pool size, and subsequently total body muscle mass, was assessed using a single oral dose of deuterated creatine (D 3 ‐creatine), which is absorbed and diluted by entry into the endogenous pool of creatine in skeletal muscle.…”

“…Labelled creatinine (D 3 ‐creatinine) and unlabelled creatinine are then measured in a fasting, single‐void urine sample 3–6 days after ingestion of the dose. These measures are then included in an algorithm to determine total body creatine pool size and skeletal muscle mass (kilograms) . To account for body size differences across participants, the primary analyses utilized the ratio of muscle mass (kilograms) to body mass (kilograms), that is, D 3 Cr muscle mass/weight.…”

“…The D 3 ‐creatine (D 3 Cr) dilution method is a direct assessment of muscle mass . We found that low D 3 Cr muscle mass is strongly related to poor physical performance and subsequent falls and mobility problems in older men, but these relationships were not observed using DXA measures of lean body mass .…”

D3‐Creatine dilution and the importance of accuracy in the assessment of skeletal muscle mass

“…In some subjects, a small amount of the oral tracer dose is ‘spilled’ into urine and not transported into muscle. However, the amount of spillage can be estimated and corrected with an algorithm based on urine levels of creatine and creatinine . The excretion of Crn and measurement of D 3 ‐enrichment in urine Crn provides an opportunity to ‘sample’ the intra‐myocellular enrichment of D 3 ‐Cr and thus determines the dilution of the oral label in the whole body creatine pool in skeletal muscle.…”

“…A particular value of D 3 ‐creatine dilution is that it can be assessed in large number of subjects with very little subject burden. A subject simply swallows a capsule and produces a fasting urine sample later . This ease of use may allow the D 3 ‐creatine dilution method to be used in large subject and patient populations including many where CT or MRI cannot be used, such as paediatric or international studies in malnourished populations.…”