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Assessing growth quality in preterm infants present challenges, particularly with the use of the standard fat-free mass measurement. We report here a moderate correlation between indirect skeletal muscle mass measurements using the D3-creatine dilution method and fat-free mass measured with air-displacement plethysmography. Skeletal muscle mass could serve as an indicator of growth quality., Competing Interests: Declaration of Competing Interest The Eunice Kennedy Shriver National Institute of Child Health and Human Development (K23HD102554; A.A.S.) and The Academy of Nutrition and Dietetics Dietitians in Nutrition Support Practice Group (M.L.J.). W.J.E. is listed as coinventor on the granted patents for the D3-creatine dilution method, but do not own or derive any income from the intellectual property. The other authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Duchenne muscular dystrophy (DMD) results in a progressive loss of functional skeletal muscle mass (MM) and replacement with fibrofatty tissue. Accurate evaluation of MM in DMD patients has not previously been available. Our objective was to measure MM using the D 3 creatine (D 3 Cr) dilution method and determine its relationship with strength and functional capacity in patients with DMD over a wide range of ages. Subjects were recruited for participation in a 12 month, longitudinal, observational study. Here, we report the baseline data. A 20 mg dose of D 3 Cr dissolved in water was ingested by 92 patients with DMD (ages 4-25 years) followed later with a fasting urine sample. Enrichment of D 3 creatinine was determined by liquid chromatography-mass spectrometry analysis. The North Star Ambulatory Assessment (NSAA) total score was determined for ambulatory participants, and the Performance of Upper Limb (PUL 2.0) total score and grip strength for all participants. We observed a significant age-associated increase in body weight along with a substantial decrease in MM/body weight (%MM). MM and %MM were associated with PUL score (r = 0.517, P < 0.0001 and r = 0.764, P < 0.0001 respectively). The age-associated decrease in MM and %MM was strongly associated with ambulatory status. We observed very little overlap in %MM between ambulant and non-ambulant subjects, suggesting a threshold of 18-22% associated with loss of ambulation. MM is substantially diminished with advancing age and is highly related to clinically meaningful functional status. The D 3 Cr dilution method may provide a biomarker of disease progression and therapeutic efficacy in patients with DMD or other neuromuscular disorders. KEY POINTS: The non-invasive D 3 creatine dilution method provides novel data on whole body functional muscle mass (MM) in a wide range of ages in patients with DMD and reveals profoundly low functional MM in older non-ambulant patients. The difference in %MM between ambulant and non-ambulant subjects suggests a threshold for loss of ambulatory ability between 18 and 22% MM. The data suggest that as functional MM declines with age, maintaining a lower body weight may help to conserve ambulatory ability., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Background: How magnetic resonance (MR) derived thigh muscle volume and deuterated creatine dilution derived muscle mass (D3Cr muscle mass) differentially relate to strength, fitness, and other functions in older adults-and whether associations vary by sex-is not known., Methods: Men (N = 345) and women (N = 482) aged ≥70 years from the Study of Muscle, Mobility, and Aging completed leg extension strength (1-repetition max) and cardiopulmonary exercise testing to assess fitness (VO2peak). Correlations and adjusted regression models stratified by sex were used to assess the association between muscle size measures, study outcomes, and sex interactions., Results: D3Cr muscle mass and MR thigh muscle volume were correlated (men: r = 0.62, women: r = 0.51, p < .001). Each standard deviation (SD) decrement in D3Cr muscle mass was associated with lower 1-repetition max strength (-14 kg men, -4 kg women, p < .001 for both; p-interaction = .003) and lower VO2peak (-79 mL/min men, -30 mL/min women, p < .001 for both, p-interaction: .016). Each SD decrement in MR thigh muscle volume was also associated with lower strength (-32 kg men, -20 kg women, p < .001 for both; p-interaction = .139) and lower VO2peak (-217 mL/min men, -111 mL/min women, p < .001 for both, p-interaction = .010). There were associations, though less consistent, between muscle size or mass with physical performance and function; associations varied by sex., Conclusions: Less muscle-measured by either D3Cr muscle mass or MR thigh muscle volume-was associated with lower strength and fitness. Varied associations by sex and assessment method suggest consideration be given to which measurement to use in future studies., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Given limited experience in applying the creatine-(methyl-D 3 ) (D 3 Cr) dilution method to measure skeletal muscle mass (SMM) in young children, the feasibility of deployment in a fielding setting and performance of the method was assessed in a cohort of 4-year-old children in Dhaka, Bangladesh., Methods: Following D 3 Cr oral dose (10 mg) administration, single fasting urine samples were collected at 2-4 days (n = 100). Twenty-four-hour post-dose collections and serial spot urine samples on days 2, 3 and 4 were obtained in a subset of participants (n = 10). Urinary creatine, creatinine, D 3 Cr and D 3 -creatinine enrichment were analyzed by liquid chromatography-tandem mass spectrometry. Appendicular lean mass (ALM) was measured by dual-energy x-ray absorptiometry and grip strength was measured by a hand-held dynamometer., Results: SMM was measured successfully in 91% of participants, and there were no adverse events. Mean ± SD SMM was greater than ALM (4.5 ± 0.4 and 3.2 ± 0.6 kg, respectively). Precision of SMM was low (intraclass correlation = 0.20; 95% CI: 0.02, 0.75; n = 10). Grip strength was not associated with SMM in multivariable analysis (0.004 kg per 100 g of SMM; 95% CI: -0.031, 0.038; n = 91)., Conclusions: The D 3 Cr dilution method was feasible in a community setting. However, high within-child variability in SMM estimates suggests the need for further optimization of this approach., Impact: The D3-creatine (D3Cr) stable isotope dilution method was considered a feasible method for the estimation of skeletal muscle mass (SMM) in young children in a community setting and was well accepted among participants. SMM was weakly associated with both dual-energy x-ray absorptiometry-derived values of appendicular lean mass and grip strength. High within-child variability in estimated values of SMM suggests that further optimization of the D3Cr stable isotope dilution method is required prior to implementation in community research settings., (© 2023. The Author(s).)

Context: Effects of testosterone on integrated muscle protein metabolism and muscle mass during energy deficit are undetermined., Objective: The objective was to determine the effects of testosterone on mixed-muscle protein synthesis (MPS), proteome-wide fractional synthesis rates (FSR), and skeletal muscle mass during energy deficit., Design: This was a randomized, double-blind, placebo-controlled trial., Setting: The study was conducted at Pennington Biomedical Research Center., Participants: Fifty healthy men., Intervention: The study consisted of 14 days of weight maintenance, followed by a 28-day 55% energy deficit with 200 mg testosterone enanthate (TEST, n = 24) or placebo (PLA, n = 26) weekly, and up to 42 days of ad libitum recovery feeding., Main Outcome Measures: Mixed-MPS and proteome-wide FSR before (Pre), during (Mid), and after (Post) the energy deficit were determined using heavy water (days 1-42) and muscle biopsies. Muscle mass was determined using the D3-creatine dilution method., Results: Mixed-MPS was lower than Pre at Mid and Post (P < 0.0005), with no difference between TEST and PLA. The proportion of individual proteins with numerically higher FSR in TEST than PLA was significant by 2-tailed binomial test at Post (52/67; P < 0.05), but not Mid (32/67; P > 0.05). Muscle mass was unchanged during energy deficit but was greater in TEST than PLA during recovery (P < 0.05)., Conclusions: The high proportion of individual proteins with greater FSR in TEST than PLA at Post suggests exogenous testosterone exerted a delayed but broad stimulatory effect on synthesis rates across the muscle proteome during energy deficit, resulting in muscle mass accretion during subsequent recovery., (Published by Oxford University Press on behalf of the Endocrine Society 2022.)

Boys with Duchenne muscular dystrophy (DMD) experience a progressive loss of functional muscle mass, with fibrosis and lipid accumulation. Accurate evaluation of whole-body functional muscle mass (MM) in DMD patients has not previously been possible and the rate of synthesis of muscle proteins remains unexplored. We used non-invasive, stable isotope-based methods from plasma and urine to measure the fractional rate of muscle protein synthesis (FSR) functional muscle mass (MM), and fat free mass (FFM) in 10 DMD (6-17 years) and 9 age-matched healthy subjects. An oral dose of D 3 creatine in 70% 2 H 2 O was administered to determine MM and FFM followed by daily 70% 2 H 2 O to measure protein FSR. Functional MM was profoundly reduced in DMD subjects compared to controls (17% vs. 41% of body weight, P < 0.0001), particularly in older, non-ambulant patients in whom functional MM was extraordinarily low (<13% body weight). We explored the urine proteome to measure FSR of skeletal muscle-derived proteins. Titin, myosin light chain and gelsolin FSRs were substantially lower in DMD subjects compared to controls (27%, 11% and 40% of control, respectively, P < 0.0001) and were strongly correlated. There were no differences in muscle-derived sarcoplasmic proteins FSRs (creatine kinase M-type and carbonic anhydrase-3) measured in plasma. These data demonstrate that both functional MM, body composition and muscle protein synthesis rates can be quantified non-invasively and are markedly different between DMD and control subjects and suggest that the rate of contractile but not sarcoplasmic protein synthesis is affected by a lack of dystrophin. KEY POINTS: Duchenne muscular dystrophy (DMD) results in a progressive loss of functional skeletal muscle but total body functional muscle mass or rates of muscle protein synthesis have not previously been assessed in these patients. D 3 -creatine dilution was used to measure total functional muscle mass and oral 2 H 2 O was used to examine the rates of muscle protein synthesis non-invasively in boys with DMD and healthy controls using urine samples. Muscle mass was profoundly lower in DMD compared to control subjects, particularly in older, non-ambulant patients. The rates of contractile protein synthesis but not sarcoplasmic proteins were substantially lower in DMD. These results may provide non-invasive biomarkers for disease progression and therapeutic efficacy in DMD and other neuromuscular diseases., (© 2021 The Authors. The Journal of Physiology © 2021 The Physiological Society.)

Background: The D3-creatine (D3Cr) dilution method provides a direct measure of skeletal muscle. The aim of this study was to compare the association of D3Cr muscle mass with lean body mass (LBM) measured by dual-energy x-ray absorptiometry (DXA) and examine its relation with physical function in postmenopausal women., Methods: Seventy-four community-dwelling women (mean age 82.3 ± 5.4) participated in this pilot study from the Buffalo, New York clinical site of the Women's Health Initiative (WHI). Participants attended a clinic visit which included anthropometric measures, blood draw, DXA scan, measures of physical function, and initiated the D3Cr protocol. Physical function was evaluated using hand grip strength, short physical performance battery (SPPB), and RAND-36 physical function scale. Descriptive statistics and logistic regression models were used to examine the associations of D3Cr muscle mass with functional outcomes., Results: D3-creatine muscle mass was moderately correlated with DXA LBM (r = 0.50) and DXA appendicular lean mass (ALM) (r = 0.50). Individuals with high D3Cr muscle mass (%) had higher physical function compared to individuals with low muscle mass (%), indicated by high scores on SPPB (odds ratio [OR] = 5.24; 95% confidence interval [CI]: 1.40, 19.58). We observed stronger relationships between high D3Cr and physical function than either DXA LBM (OR = 3.40; 95% CI: 0.88, 13.11) or DXA ALM (OR = 4.15; 95% CI: 1.10, 15.68) and physical function., Conclusions: Our findings provide strong preliminary data for the associations of D3Cr muscle mass with measures of physical function in older women. These findings support and extend prior work on D3Cr muscle mass in older men., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The rate of accrual of muscle mass in neonates has not been assessed. We describe the D 3 -creatine (D 3 Cr) dilution method, a noninvasive assessment of muscle mass in neonates., Methods: A total of 76 neonates >26-week-old corrected gestational age were enrolled and measured at 2-week intervals while admitted to a neonatal intensive care unit (NICU). Additional measures at 6 and 12-20 months after initial measurement were obtained if available. An enteral dose of 2 mg D 3 Cr in 0.5 mL 20% 2 H 2 O was used to determine muscle mass and total body water (TBW)., Results: Muscle mass by the D 3 Cr method was strongly associated with TBW and body weight (r = 0.9272, p < 0.0001 and r = 0.9435, p < 0.0001 for all time points and r = 0.6661, p < 0.0001 and r = 0.8634, p < 0.0001, respectively, while in the NICU). Change in muscle mass vs. change in body weight, TBW, and length were also strongly correlated., Conclusions: The D 3 Cr dilution method provides a noninvasive assessment of muscle mass accrual in neonates, which has not been previously possible and may be an important new tool for the evaluation of nutritional status and normal growth patterns., Impact: We describe a noninvasive method for the measurement of skeletal muscle mass neonates. At the present time, there is no direct measurement of muscle mass in infants available. The D 3 Cr dilution method is a direct and noninvasive measurement of muscle mass. Using a single enteral dose of D 3 Cr in 2 H 2 O followed by urine and saliva samples, rapid and substantial accrual of muscle mass and TBW is assessed. Assessment of muscle mass accrual in premature infants may be a strong indicator of nutritional status. Change in muscle mass is strongly related to change in weight and TBW.

Background: Direct assessment of skeletal muscle mass in older adults is clinically challenging. Relationships between lean mass and late-life outcomes have been inconsistent. The D3-creatine dilution method provides a direct assessment of muscle mass., Methods: Muscle mass was assessed by D3-creatine (D3Cr) dilution in 1,382 men (mean age, 84.2 years). Participants completed the Short Physical Performance Battery (SPPB); usual walking speed (6 m); and dual x-ray absorptiometry (DXA) lean mass. Men self-reported mobility limitations (difficulty walking 2-3 blocks or climbing 10 steps); recurrent falls (2+); and serious injurious falls in the subsequent year. Across quartiles of D3Cr muscle mass/body mass, multivariate linear models calculated means for SPPB and gait speed; multivariate logistic models calculated odds ratios for incident mobility limitations or falls., Results: Compared to men in the highest quartile, those in the lowest quartile of D3Cr muscle mass/body mass had slower gait speed (Q1: 1.04 vs Q4: 1.17 m/s); lower SPPB (Q1: 8.4 vs Q4: 10.4 points); greater likelihood of incident serious injurious falls (odds ratio [OR] Q1 vs Q4: 2.49, 95% confidence interval [CI]: 1.37, 4.54); prevalent mobility limitation (OR Q1 vs Q4,: 6.1, 95% CI: 3.7, 10.3) and incident mobility limitation (OR Q1 vs Q4: 2.15 95% CI: 1.42, 3.26); p for trend < .001 for all. Results for incident recurrent falls were in the similar direction (p = .156). DXA lean mass had weaker associations with the outcomes., Conclusions: Unlike DXA lean mass, low D3Cr muscle mass/body mass is strongly related to physical performance, mobility, and incident injurious falls in older men., (© The Author(s) 2018. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Key Points: Strategies to enhance the loss of fat while preserving muscle mass during energy restriction are of great importance to prevent sarcopenia in overweight older adults. We show for the first time that the integrated rate of synthesis of numerous individual contractile, cytosolic and mitochondrial skeletal muscle proteins was increased by resistance training (RT) and unaffected by dietary protein intake pattern during energy restriction in free-living, obese older men. We observed a correlation between the synthetic rates of skeletal muscle-derived proteins obtained in serum (creatine kinase M-type, carbonic anhydrase 3) and the synthetic rates of proteins obtained via muscle sampling; and that the synthesis rates of these proteins in serum revealed the stimulatory effects of RT. These results have ramifications for understanding the influence of RT on skeletal muscle and are consistent with the role of RT in maintaining muscle protein synthesis and potentially supporting muscle mass preservation during weight loss., Abstract: We determined how the pattern of protein intake and resistance training (RT) influenced longer-term (2 weeks) integrated myofibrillar protein synthesis (MyoPS) during energy restriction (ER). MyoPS and proteome kinetics were measured during 2 weeks of ER alone and 2 weeks of ER plus RT (ER + RT) in overweight/obese older men. Participants were randomized to consume dietary protein in a balanced (BAL: 25% daily protein per meal × 4 meals) or skewed (SKEW: 7:17:72:4% daily protein per meal) pattern (n = 10 per group). Participants ingested deuterated water during the consecutive 2-week periods, and skeletal muscle biopsies and serum were obtained at the beginning and conclusion of ER and ER + RT. Bulk MyoPS (i.e. synthesis of the myofibrillar protein sub-fraction) and the synthetic rates of numerous individual skeletal muscle proteins were quantified. Bulk MyoPS was not affected by protein distribution during ER or ER + RT (ER: BAL = 1.24 ± 0.31%/day, SKEW = 1.26 ± 0.37%/day; ER + RT: BAL = 1.64 ± 0.48%/day, SKEW = 1.52 ± 0.66%/day) but was ∼26% higher during ER + RT than during ER (P = 0.023). The synthetic rates of 175 of 190 contractile, cytosolic and mitochondrial skeletal muscle proteins, as well as synthesis of muscle-derived proteins measured in serum, creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3), were higher during ER + RT than during ER (P < 0.05). In addition, the synthetic rates of CK-M and CA-3 measured in serum correlated with the synthetic rates of proteins obtained via muscle sampling (P < 0.05). This study provides novel data on the skeletal muscle adaptations to RT and dietary protein distribution., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Background: Muscle mass can be measured directly in vivo by isotope dilution, using Creatine-(methyl-d 3 ) monohydrate (D 3 -Cr) by mouth followed by measurement of the steady-state enrichment of D 3 -creatinine (D 3 -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 D 3 -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 D 3 -Cr dilution to other assessments of fat-free mass., Methods: Subjects (19 males, 23-81 years old; 20 females, 20-77 years old) ingested a single dose of 60 mg D 3 -Cr and urine was collected prior to and daily for 4 days following the dose. Fasting morning urine samples was assessed for D 3 -Cr, total Cr, D 3 -Crn, and total Crn concentrations, as well as isotopic enrichments of D 3 -Crn, by LC/MS. The 24-h urine collections over 3 days after the dose of D 3 -Cr were also performed to determine D 3 -Cr spillage. Total body water, fat mass, and fat-free mass were assessed by bioelectrical impedance spectroscopy (BIS)., Results: Spillage of D 3 -Cr in the urine was greater in women than men. D 3 -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 D 3 -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 D 3 -Cr dose excreted. Muscle mass measured by D 3 -Crn enrichment also correlated (r = 0.8579, P < 0.0001, algorithm corrected) with that measured by 24-h Crn excretion. Muscle mass measured by D 3 -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 D 3 -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)., Conclusions: The D 3 -Cr dilution method is further validated here as a non-invasive, easy-to-use test for measuring muscle mass. The technical issue of D 3 -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., (© 2018 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.)

Biomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM., (Copyright © 2016 the American Physiological Society.)

Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.