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Stable isotope approaches to study muscle mass outcomes in clinical populations

  • Lee-anne S. Chapple
    Correspondence
    Corresponding author. Dr Lee-anne Chapple Mailing address: 4G751, ICU Research, Royal Adelaide Hospital, Port Road, Adelaide, South Australia 5000, Australia. Tel.: +61 8 7074 1763.
    Affiliations
    Intensive Care Unit, Royal Adelaide Hospital, Adelaide, Australia

    Discipline of Acute Care Medicine, The University of Adelaide, Adelaide, Australia

    Centre of Research Excellence in Translating Nutritional Science to Good Health, The University of Adelaide, Adelaide, Australia
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  • Marlou L. Dirks
    Affiliations
    Department of Sport and Health Sciences, College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom
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  • Imre W.K. Kouw
    Affiliations
    Intensive Care Unit, Royal Adelaide Hospital, Adelaide, Australia

    Discipline of Acute Care Medicine, The University of Adelaide, Adelaide, Australia

    Centre of Research Excellence in Translating Nutritional Science to Good Health, The University of Adelaide, Adelaide, Australia
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Open AccessPublished:February 28, 2021DOI:https://doi.org/10.1016/j.nutos.2021.01.004

      Abstract

      Both low muscle mass and muscle loss are associated with reduced physical function, mobility, independence, and quality of life, and are characteristic of a number of clinical conditions including diabetes, cardiovascular disease (CVD), chronic obstructive pulmonary disease (COPD), and critical illness. The accurate measurement of muscle mass is critical to assess the efficacy of an intervention or therapy. Stable isotope amino acid approaches can be used to quantify specific aspects of whole-body and muscle protein turnover, including synthesis and breakdown, which play distinctive roles in muscle mass maintenance in direct response to therapies. This review aims to elucidate whether acute responses measured using stable isotope amino acid tracers relate to changes in muscle mass in vulnerable clinical populations. Experimental studies quantifying whole-body protein synthesis and breakdown rates in clinical populations have been conducted to determine the response to nutritional interventions or to compare disease with health; however, these studies show limited potential to translate to expected muscle mass outcomes. In addition, clinical studies that have assessed both muscle mass and acute changes in whole-body or muscle protein turnover are lacking. We argue that the assessment of both muscle protein synthesis and breakdown rates, or simply limb net balance, obtains the most complete picture in relation to muscle-specific outcomes. While stable isotope amino acid tracer experiments provide meaningful mechanistic insight into the acute response to clinical interventions, they should be combined with, and/or followed-up by, longer-term studies incorporating measurements of muscle mass to ascertain the impact of an intervention on muscle mass maintenance in clinical populations.

      Keywords

      1. The relevance of muscle mass to clinical outcomes

      Skeletal muscle is one of the largest groups of tissues in the human body, and is highly adaptive to external stimuli such as nutrition and physical (in)activity [
      • Parry S.M.
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      ]. Low muscle mass, irrespective of body size, is prevalent in a number of metabolic conditions including type two diabetes mellitus [
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      Patients with type 2 diabetes show a greater decline in muscle mass, muscle strength, and functional capacity with aging.
      ], cancer [
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      ], and COPD [
      • Vestbo J.
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      • et al.
      Body mass, fat-free body mass, and prognosis in patients with chronic obstructive pulmonary disease from a random population sample: findings from the Copenhagen City Heart Study.
      ], and is associated with poor clinical outcomes in hospitalised patients, including longer hospital length of stay (LOS), more postoperative complications, worsened disease prognosis, and increased mortality [
      • Welch C.
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      The impact of sarcopenia on survival and complications in surgical oncology: a review of the current literature.
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      Sarcopenia adversely impacts postoperative complications following resection or transplantation in patients with primary liver tumors.
      ,
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      Association of diabetes, comorbidities, and A1C with functional disability in older adults: results from the National Health and Nutrition Examination Survey (NHANES), 1999-2006.
      ,
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      • Bosmans J.
      • Tijssen F.
      • Stoot J.
      Functional compromise cohort study (FCCS): sarcopenia is a strong predictor of mortality in the intensive care unit.
      ]. Similarly, acute muscle loss, such as that observed during hospitalisation for critical illness [
      • Puthucheary Z.A.
      • Rawal J.
      • McPhail M.
      • Connolly B.
      • Ratnayake G.
      • Chan P.
      • et al.
      Acute skeletal muscle wasting in critical illness.
      ,
      • Parry S.M.
      • El-Ansary D.
      • Cartwright M.S.
      • Sarwal A.
      • Berney S.
      • Koopman R.
      • et al.
      Ultrasonography in the intensive care setting can be used to detect changes in the quality and quantity of muscle and is related to muscle strength and function.
      ,
      • Dirks M.L.
      • Hansen D.
      • Van Assche A.
      • Dendale P.
      • Van Loon L.J.
      Neuromuscular electrical stimulation prevents muscle wasting in critically ill comatose patients.
      ], is also associated with poor outcomes including increased intensive care unit (ICU) LOS and reduced physical function that persists after hospital discharge [
      • Gruther W.
      • Benesch T.
      • Zorn C.
      • Paternostro-Sluga T.
      • Quittan M.
      • Fialka-Moser V.
      • et al.
      Muscle wasting in intensive care patients: ultrasound observation of the M. quadriceps femoris muscle layer.
      ]. Correspondingly, the maintenance of muscle mass in hospitalised patients is associated with improved survival [
      • de van der Schueren M.A.E.
      • de Smoker M.
      • Leistra E.
      • Kruizenga H.M.
      The association of weight loss with one-year mortality in hospital patients, stratified by BMI and FFMI subgroups.
      ]. Given the importance of muscle mass for clinical outcomes, the ability to quantify the response to interventions aimed to attenuate muscle loss or increase muscle mass over time is critical, both for the patient and the healthcare system.
      A number of techniques exist that enable the quantification of muscle mass in a clinical setting. Magnetic resonance imaging (MRI) and computed tomography (CT; including of the 3rd lumbar region) are considered the gold standard imaging methods and, along with dual energy x-ray absorptiometry (DXA), are typically used in clinical research to measure skeletal muscle (or lean) mass [
      • Mitsiopoulos N.
      • Baumgartner R.N.
      • Heymsfield S.B.
      • Lyons W.
      • Gallagher D.
      • Ross R.
      Cadaver validation of skeletal muscle measurement by magnetic resonance imaging and computerized tomography.
      ]. Ultrasonography and bio-electrical impedance (BIA) have gained momentum as cost-effective bedside alternatives to determine muscle thickness/cross-sectional area (CSA) or fat free mass, respectively [
      • Puthucheary Z.A.
      • Rawal J.
      • McPhail M.
      • Connolly B.
      • Ratnayake G.
      • Chan P.
      • et al.
      Acute skeletal muscle wasting in critical illness.
      ,
      • Tillquist M.
      • Kutsogiannis D.J.
      • Wischmeyer P.E.
      • Kummerlen C.
      • Leung R.
      • Stollery D.
      • et al.
      Bedside ultrasound is a practical and reliable measurement tool for assessing quadriceps muscle layer thickness.
      ,
      • Teigen L.M.
      • Kuchnia A.J.
      • Mourtzakis M.
      • Earthman C.P.
      The use of technology for estimating body composition: strengths and weaknesses of common modalities in a clinical setting [formula: see text].
      ]. However, techniques used to measure muscle mass in patients often rely on estimations, and can be influenced by differences in methodology [
      • Ishida H.
      • Watanabe S.
      Influence of inward pressure of the transducer on lateral abdominal muscle thickness during ultrasound imaging.
      ] and patient variations (‘noise’) that are difficult to control in a clinical setting, such as physical activity level, nutritional intake, and fluid status [
      • Earthman C.P.
      Body composition tools for assessment of adult malnutrition at the bedside: a tutorial on research considerations and clinical applications.
      ]. Furthermore, studies comparing different techniques have often reported poor levels of agreement for measuring muscle mass [
      • Puthucheary Z.A.
      • Rawal J.
      • McPhail M.
      • Connolly B.
      • Ratnayake G.
      • Chan P.
      • et al.
      Acute skeletal muscle wasting in critical illness.
      ,
      • Jones D.J.
      • Lal S.
      • Strauss B.J.
      • Todd C.
      • Pilling M.
      • Burden S.T.
      Measurement of muscle mass and sarcopenia using anthropometry, bioelectrical impedance, and computed tomography in surgical patients with colorectal malignancy: comparison of agreement between methods.
      ]. For example, over seven days of ICU admission Puthucheary et al. showed greater degrees of atrophy when measured using the ratio of protein to DNA (30% loss) when compared to muscle fibre CSA (18% loss) and ultrasound-derived m. rectus femoris CSA (10% loss) [
      • Puthucheary Z.A.
      • Rawal J.
      • McPhail M.
      • Connolly B.
      • Ratnayake G.
      • Chan P.
      • et al.
      Acute skeletal muscle wasting in critical illness.
      ]. Therefore, it is important to recognise that the observed degree of muscle loss depends on the quantification technique used. While changes in muscle mass are related to clinical outcomes and provide insight into the longer-term response to interventions, acute responses may be used to test the efficacy and response to an intervention and gain understanding of the underlying mechanisms as well as the expected magnitude of change in muscle mass. Our review aims to elucidate whether acute responses measured using stable isotope amino acid tracers relate to changes in muscle mass in vulnerable clinical populations. It is envisaged that this will support researchers and clinicians in their interpretation of findings from these acute tracer studies and expected impact on clinical outcomes.

      2. Stable isotope tracer techniques to measure protein turnover and net balance

      The continuous turnover of muscle proteins is required to maintain muscle quality, and the net balance between synthesis and breakdown determines whether muscle protein is lost, maintained, or gained. In clinical populations experiencing muscle loss, muscle protein breakdown (MPB) must exceed muscle protein synthesis (MPS). This may be due to an increase in breakdown, a reduction in synthesis, or a combination of both, thereby resulting in a negative net balance. Decades of research and continuous advances in the field of muscle protein metabolism have resulted in various techniques to measure MPS and MPB in humans. Although acute measurements of MPS or MPB rates provide insight into the mechanism and physiological response to external stimuli such as nutrition and exercise, it has been argued that the determination of net balance on a whole-body or skeletal muscle level provides a more complete account of protein turnover and, as such, relates to muscle mass outcomes [
      • Millward D.J.
      • Smith K.
      The application of stable-isotope tracers to study human musculoskeletal protein turnover: a tale of bag filling and bag enlargement.
      ].
      There are several approaches to quantify net balance on a whole-body or skeletal muscle tissue level, with the most commonly used methods included in Table 1. Although there are other approaches that can measure MPS or MPB alone (e.g. 3-methylhistidine, with or without stable isotope tracers, for MPB [
      • Vesali R.F.
      • Klaude M.
      • Thunblad L.
      • Rooyackers O.E.
      • Wernerman J.
      Contractile protein breakdown in human leg skeletal muscle as estimated by [2H3]-3-methylhistidine: a new method.
      ]), for conciseness we have only included methods that allow the quantification of net balance. A commonly used approach, due to its applicability in a clinical setting, is the nitrogen balance technique. There are limitations to this technique in that factors such as losses via sweat and respiration affect the accuracy of absolute daily nitrogen balance (i.e. this technique under-estimates losses and over-estimates intake) [
      • Rand W.M.
      • Pellett P.L.
      • Young V.R.
      Meta-analysis of nitrogen balance studies for estimating protein requirements in healthy adults.
      ] and muscle-specific nitrogen balance is difficult to obtain (Table 1), yet this technique can provide useful insight into whole-body nitrogen balance patterns and can assess the response to an intervention over multiple days to weeks. In a more detailed, yet also more complex and costly way, intravenous infusion of stable isotope amino acids combined with repeated blood samples can be used to provide insight into the turnover of specific amino acids on a whole-body level. However, factors such as inflammation or disease state may influence organ protein turnover. As muscle protein turnover represents only ~25–30% of whole-body protein turnover [
      • Nair K.S.
      • Halliday D.
      • Griggs R.C.
      Leucine incorporation into mixed skeletal muscle protein in humans.
      ], whole-body protein turnover does not accurately reflect the response of skeletal muscle [
      • Barle H.
      • Hammarqvist F.
      • Westman B.
      • Klaude M.
      • Rooyackers O.
      • Garlick P.J.
      • et al.
      Synthesis rates of total liver protein and albumin are both increased in patients with an acute inflammatory response.
      ]. Therefore, muscle-specific measurements are required to determine the effect of specific interventions on muscle. The most commonly used approach to measure net balance in vivo in humans is the arteriovenous balance (AV) approach across an arm or leg, which uses simultaneous sampling from an artery (or arterialised hand vein) and a vein (e.g. femoral or antecubital), combined with arterial blood flow measurements of the arm or leg (e.g. via Doppler ultrasound or indocyanine green infusion). This relatively simple approach results in the net balance of amino acids, as well as other metabolites if required [
      • Dirks M.L.
      • Wall B.T.
      • Otten B.
      • Cruz A.M.
      • Dunlop M.V.
      • Barker A.R.
      • et al.
      High-fat overfeeding does not exacerbate rapid changes in forearm glucose and fatty acid balance during immobilization.
      ], which is the most direct method to determine changes in net balance, and may be used to predict the effect of a longer-term intervention on muscle mass. An advantage of this method is that only amino acid concentrations and blood flow measurements are required for the calculation of net balance. However, when combined with a stable isotope amino acid tracer infusion this approach provides a more complete description of protein turnover [
      • Wolfe R.R.C.
      D.L.: Isotope Tracers in Metabolic Research: Principles and Practice of Kinetic Analysis.
      ], especially when muscle biopsy collection is also included [
      • Biolo G.
      • Fleming R.Y.
      • Maggi S.P.
      • Wolfe R.R.
      Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle.
      ]. While it can be assumed that with correct cannula placement AV balance represents net balance of muscle tissue (and not other tissues such as skin and adipose tissue [
      • van Hall G.
      • Gonzalez-Alonso J.
      • Sacchetti M.
      • Saltin B.
      Skeletal muscle substrate metabolism during exercise: methodological considerations.
      ,
      • Guo Z.
      • Jensen M.D.
      Arterio-venous balance studies of skeletal muscle fatty acid metabolism: what can we believe?.
      ]), an alternative approach is to calculate muscle net balance directly. Specifically, this can be achieved via the combination of two different stable isotope amino acid tracers (using the same amino acid with a different label, e.g. 13C-Phe for fractional synthesis rate; FSR and 15N-Phe for fractional breakdown rate; FBR) with repeated blood and muscle tissue sampling, thereby measuring the FSR via the direct incorporation of labelled amino acids in muscle tissue [
      • Rennie M.J.
      • Edwards R.H.
      • Halliday D.
      • Matthews D.E.
      • Wolman S.L.
      • Millward D.J.
      Muscle protein synthesis measured by stable isotope techniques in man: the effects of feeding and fasting.
      ] as well as the FBR via the dilution of the muscle intracellular free pool [
      • Zhang X.J.
      • Chinkes D.L.
      • Sakurai Y.
      • Wolfe R.R.
      An isotopic method for measurement of muscle protein fractional breakdown rate in vivo.
      ]. Given the relative burden of repeated muscle biopsies, the limited evidence available applying the assessment of postprandial responses with the dilution technique [
      • Tuvdendorj D.
      • Chinkes D.L.
      • Herndon D.N.
      • Zhang X.J.
      • Wolfe R.R.
      A novel stable isotope tracer method to measure muscle protein fractional breakdown rate during a physiological non-steady-state condition.
      ], and the technical challenges associated with the FBR calculations, there is a paucity of data applying this method in human intervention studies [
      • Phillips S.M.
      • Tipton K.D.
      • Aarsland A.
      • Wolf S.E.
      • Wolfe R.R.
      Mixed muscle protein synthesis and breakdown after resistance exercise in humans.
      ,
      • Symons T.B.
      • Sheffield-Moore M.
      • Chinkes D.L.
      • Ferrando A.A.
      • Paddon-Jones D.
      Artificial gravity maintains skeletal muscle protein synthesis during 21 days of simulated microgravity.
      ]. The invasiveness of this approach also limits its feasibility in clinical populations. Moreover, a downside of this method is that to date it cannot provide net balance of specific muscle fractions such as the myofibrillar (i.e. contractile) fractions, as FBR is measured from mixed muscle protein. An increasingly used, logistically feasible approach that can provide crucial insight in the integrative FSR and FBR of specific proteins of interest is oral ingestion of deuterium oxide (2H2O/D2O). Unfortunately, there is a difference in required body water and muscle protein bound enrichments to measure MPS and MPB using deuterium oxide [
      • Wilkinson D.J.
      • Franchi M.V.
      • Brook M.S.
      • Narici M.V.
      • Williams J.P.
      • Mitchell W.K.
      • et al.
      A validation of the application of D(2)O stable isotope tracer techniques for monitoring day-to-day changes in muscle protein subfraction synthesis in humans.
      ]. As a result, the timeframes for performing these measurements do not align, which precludes FSR and FBR to be measured in parallel. Of note, it is possible to use D2O-derived MPS rates and a measurement of muscle mass to estimate MPB (e.g. Ref. [
      • Brook M.S.
      • Wilkinson D.J.
      • Mitchell W.K.
      • Lund J.N.
      • Phillips B.E.
      • Szewczyk N.J.
      • et al.
      Synchronous deficits in cumulative muscle protein synthesis and ribosomal biogenesis underlie age-related anabolic resistance to exercise in humans.
      ]), which might provide information on the impact of a nutritional intervention in clinical practice. Altogether, although oral deuterium oxide consumption is a powerful method that will further increase our insight into human muscle protein turnover under free-living conditions, this method cannot be used to calculate net balance in the way that other discussed methods (Table 1) can. In the next section we will further discuss the feasibility of these acute stable isotope techniques in clinical populations and explore studies that have used these techniques in relation to measures of muscle mass.
      Table 1Overview of methods for the quantification of whole-body and skeletal muscle protein balance, with the rows in grey highlighting the recommended approaches to quantify net balance in humans.
      LevelMethodOutcome and calculationStrengthsLimitationsReferences
      Whole-bodyNitrogen balanceWhole-body nitrogen balance = nitrogen ingested - nitrogen excreted
      • Simple
      • Non-invasive
      • Allows for longer-term measurements (including multiple meals)
      • Cannot measure muscle-specific changes
      • Overestimates intake and underestimates excretion
      [
      • Kopple J.D.
      Uses and limitations of the balance technique.
      ]
      Stable isotope tracer kineticsWhole-body protein net balance = whole-body protein synthesis – endogenous Ra
      • Relatively non-invasive
      • Cannot measure muscle-specific changes
      • Validity of non-steady state postprandial measurements is subject to discussion [
        • Wolfe R.R.
        • Park S.
        • Kim I.Y.
        • Starck C.
        • Marquis B.J.
        • Ferrando A.A.
        • et al.
        Quantifying the contribution of dietary protein to whole body protein kinetics: examination of the intrinsically labeled proteins method.
        ]
      [
      • Wolfe R.R.C.
      D.L.: Isotope Tracers in Metabolic Research: Principles and Practice of Kinetic Analysis.
      ]
      Skeletal muscleAV balanceLimb net balance = (CA – CV) ∗ BF
      • Direct measurement of limb net balance
      • Relatively non-invasive. Can be performed by using arterialised instead of arterial samples.
      • Can be combined with stable isotope tracer infusion for 2-pool calculations, and muscle biopsies for 3-pool calculations
      • Measurement is potentially influenced by non-muscle tissues, although this can be minimized by correct cannulate placement [
        • van Hall G.
        • Gonzalez-Alonso J.
        • Sacchetti M.
        • Saltin B.
        Skeletal muscle substrate metabolism during exercise: methodological considerations.
        ]
      [
      • Biolo G.
      • Fleming R.Y.
      • Maggi S.P.
      • Wolfe R.R.
      Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle.
      ]
      Stable isotope tracer kineticsMuscle net balance = FSR - FBR
      • Direct measurement of skeletal muscle protein net balance
      • Sensitive
      • Can assess changes over short time periods
      • Relatively invasive due to requirement for repeated muscle biopsies and intravenous infusions.
      • Can only measure NB of mixed muscle protein
      • Limited time frame for FBR measurement prevents NB measurement over entire postprandial period
      [
      • Rennie M.J.
      • Edwards R.H.
      • Halliday D.
      • Matthews D.E.
      • Wolman S.L.
      • Millward D.J.
      Muscle protein synthesis measured by stable isotope techniques in man: the effects of feeding and fasting.
      ,
      • Zhang X.J.
      • Chinkes D.L.
      • Sakurai Y.
      • Wolfe R.R.
      An isotopic method for measurement of muscle protein fractional breakdown rate in vivo.
      ]
      AV: arteriovenous; BF: blood flow; CA: arterial(ised) concentration; CV: venous concentration; FBR: fractional breakdown rate; FSR: fractional synthesis rate; NB: net balance; Ra: rate of appearance.

      3. Studies applying stable isotope techniques in clinical populations to assess the potential relationship with muscle mass

      3.1 Acute tracer studies and changes in muscle mass in healthy populations

      In healthy populations, acute MPS rates following a single bout of resistance exercise increase by 50–100% and align with more modest MPS responses observed over a prolonged period (i.e. increases of ~20–35% in daily MPS rates during several days using deuterium oxide) [
      • Wilkinson D.J.
      • Franchi M.V.
      • Brook M.S.
      • Narici M.V.
      • Williams J.P.
      • Mitchell W.K.
      • et al.
      A validation of the application of D(2)O stable isotope tracer techniques for monitoring day-to-day changes in muscle protein subfraction synthesis in humans.
      ,
      • Fuchs C.J.
      • Kouw I.W.K.
      • Churchward-Venne T.A.
      • Smeets J.S.J.
      • Senden J.M.
      • Lichtenbelt W.
      • et al.
      Postexercise cooling impairs muscle protein synthesis rates in recreational athletes.
      ,
      • Holwerda A.M.
      • Paulussen K.J.M.
      • Overkamp M.
      • Smeets J.S.J.
      • Gijsen A.P.
      • Goessens J.P.B.
      • et al.
      Daily resistance-type exercise stimulates muscle protein synthesis in vivo in young men.
      ,
      • Devries M.C.
      • McGlory C.
      • Bolster D.R.
      • Kamil A.
      • Rahn M.
      • Harkness L.
      • et al.
      Leucine, not total protein, content of a supplement is the primary determinant of muscle protein anabolic responses in healthy older women.
      ]. While these increases in MPS often occur in parallel with muscle hypertrophy following weeks to months of exercise training in young and older individuals, the acute increase in MPS may not quantitatively predict the degree of associated changes in muscle mass [
      • Mitchell C.J.
      • Churchward-Venne T.A.
      • Parise G.
      • Bellamy L.
      • Baker S.K.
      • Smith K.
      • et al.
      Acute post-exercise myofibrillar protein synthesis is not correlated with resistance training-induced muscle hypertrophy in young men.
      ,
      • Mayhew D.L.
      • Kim J.S.
      • Cross J.M.
      • Ferrando A.A.
      • Bamman M.M.
      Translational signaling responses preceding resistance training-mediated myofiber hypertrophy in young and old humans.
      ]. In line, the acute MPS response to protein ingestion does not always translate to protein supplementation-induced changes in muscle mass over the longer term [
      • Tieland M.
      • Franssen R.
      • Dullemeijer C.
      • van Dronkelaar C.
      • Kyung Kim H.
      • Ispoglou T.
      • et al.
      The impact of dietary protein or amino acid supplementation on muscle mass and strength in elderly people: individual participant data and meta-analysis of RCT's.
      ,
      • Cruz-Jentoft A.J.
      • Landi F.
      • Schneider S.M.
      • Zuniga C.
      • Arai H.
      • Boirie Y.
      • et al.
      Prevalence of and interventions for sarcopenia in ageing adults: a systematic review. Report of the International Sarcopenia Initiative (EWGSOP and IWGS).
      ,
      • Xu Z.R.
      • Tan Z.J.
      • Zhang Q.
      • Gui Q.F.
      • Yang Y.M.
      Clinical effectiveness of protein and amino acid supplementation on building muscle mass in elderly people: a meta-analysis.
      ,
      • Ten Haaf D.S.M.
      • Nuijten M.A.H.
      • Maessen M.F.H.
      • Horstman A.M.H.
      • Eijsvogels T.M.H.
      • Hopman M.T.E.
      Effects of protein supplementation on lean body mass, muscle strength, and physical performance in nonfrail community-dwelling older adults: a systematic review and meta-analysis.
      ]. While these acute studies provide relevant data on the magnitude and impact of nutrition and exercise interventions on muscle mass changes in health when conducted in a fairly controlled setting, there is a paucity of studies that have applied acute stable isotope tracer measurements in clinical populations and assessed their relation to changes in muscle mass.

      3.2 Whole-body protein net balance studies

      Acute stable isotope tracer studies have assessed whole-body protein turnover rates (i.e. protein synthesis and breakdown, and the resulting net balance) in a broad variety of clinical populations. This includes studies in patients following surgery [
      • Hatzakorzian R.
      • Carvalho G.
      • Bui H.
      • Sato T.
      • Wykes L.
      • Shum-Tim D.
      • et al.
      High-dose insulin administration is associated with hypoaminoacidemia during cardiac surgery.
      ,
      • Hatzakorzian R.
      • Shum-Tim D.
      • Wykes L.
      • Hulshoff A.
      • Bui H.
      • Nitschmann E.
      • et al.
      Glucose and insulin administration while maintaining normoglycemia inhibits whole body protein breakdown and synthesis after cardiac surgery.
      ], undergoing dialysis [
      • Veeneman J.M.
      • Kingma H.A.
      • Boer T.S.
      • Stellaard F.
      • De Jong P.E.
      • Reijngoud D.J.
      • et al.
      Protein intake during hemodialysis maintains a positive whole body protein balance in chronic hemodialysis patients.
      ,
      • Tjiong H.L.
      • Fieren M.W.
      • Rietveld T.
      • Wattimena J.L.
      • Schierbeek H.
      • Huijmans J.G.
      • et al.
      Albumin and whole-body protein synthesis respond differently to intraperitoneal and oral amino acids.
      ], with COPD [
      • Jonker R.
      • Deutz N.E.P.
      • Ligthart-Melis G.C.
      • Zachria A.J.
      • Veley E.A.
      • Harrykissoon R.
      • et al.
      Preserved anabolic threshold and capacity as estimated by a novel stable tracer approach suggests no anabolic resistance or increased requirements in weight stable COPD patients.
      ,
      • Engelen M.P.
      • Deutz N.E.
      • Wouters E.F.
      • Schols A.M.
      Enhanced levels of whole-body protein turnover in patients with chronic obstructive pulmonary disease.
      ], liver cirrhosis [
      • Tessari P.
      • Barazzoni R.
      • Kiwanuka E.
      • Davanzo G.
      • De Pergola G.
      • Orlando R.
      • et al.
      Impairment of albumin and whole body postprandial protein synthesis in compensated liver cirrhosis.
      ], or cancer cachexia [
      • Winter A.
      • MacAdams J.
      • Chevalier S.
      Normal protein anabolic response to hyperaminoacidemia in insulin-resistant patients with lung cancer cachexia.
      ,
      • van Dijk D.P.
      • van de Poll M.C.
      • Moses A.G.
      • Preston T.
      • Olde Damink S.W.
      • Rensen S.S.
      • et al.
      Effects of oral meal feeding on whole body protein breakdown and protein synthesis in cachectic pancreatic cancer patients.
      ], and critically ill and burns patients [
      • Rooyackers O.
      • Kouchek-Zadeh R.
      • Tjader I.
      • Norberg A.
      • Klaude M.
      • Wernerman J.
      Whole body protein turnover in critically ill patients with multiple organ failure.
      ,
      • Berg A.
      • Rooyackers O.
      • Bellander B.M.
      • Wernerman J.
      Whole body protein kinetics during hypocaloric and normocaloric feeding in critically ill patients.
      ,
      • Biolo G.
      • Fleming R.Y.
      • Maggi S.P.
      • Nguyen T.T.
      • Herndon D.N.
      • Wolfe R.R.
      Inverse regulation of protein turnover and amino acid transport in skeletal muscle of hypercatabolic patients.
      ]. These studies report an overall negative whole-body protein net balance, with rates of breakdown exceeding synthesis in patients when compared to healthy controls, which reflects the catabolic state observed in these clinical conditions. In response to nutritional interventions, such as hyperaminoacidaemia [
      • Hatzakorzian R.
      • Carvalho G.
      • Bui H.
      • Sato T.
      • Wykes L.
      • Shum-Tim D.
      • et al.
      High-dose insulin administration is associated with hypoaminoacidemia during cardiac surgery.
      ,
      • Tjiong H.L.
      • Fieren M.W.
      • Rietveld T.
      • Wattimena J.L.
      • Schierbeek H.
      • Huijmans J.G.
      • et al.
      Albumin and whole-body protein synthesis respond differently to intraperitoneal and oral amino acids.
      ,
      • Winter A.
      • MacAdams J.
      • Chevalier S.
      Normal protein anabolic response to hyperaminoacidemia in insulin-resistant patients with lung cancer cachexia.
      ,
      • Berg A.
      • Rooyackers O.
      • Bellander B.M.
      • Wernerman J.
      Whole body protein kinetics during hypocaloric and normocaloric feeding in critically ill patients.
      ] or protein supplementation [
      • Veeneman J.M.
      • Kingma H.A.
      • Boer T.S.
      • Stellaard F.
      • De Jong P.E.
      • Reijngoud D.J.
      • et al.
      Protein intake during hemodialysis maintains a positive whole body protein balance in chronic hemodialysis patients.
      ,
      • van Dijk D.P.
      • van de Poll M.C.
      • Moses A.G.
      • Preston T.
      • Olde Damink S.W.
      • Rensen S.S.
      • et al.
      Effects of oral meal feeding on whole body protein breakdown and protein synthesis in cachectic pancreatic cancer patients.
      ,
      • Jonker R.
      • Deutz N.E.
      • Erbland M.L.
      • Anderson P.J.
      • Engelen M.P.
      Hydrolyzed casein and whey protein meals comparably stimulate net whole-body protein synthesis in COPD patients with nutritional depletion without an additional effect of leucine co-ingestion.
      ,
      • Liebau F.
      • Wernerman J.
      • van Loon L.J.
      • Rooyackers O.
      Effect of initiating enteral protein feeding on whole-body protein turnover in critically ill patients.
      ], whole-body protein balance becomes positive, primarily due to increases in whole-body protein synthesis of up to 50%, while whole-body protein breakdown remains unchanged or slightly decreased. This reflects a comparable anabolic response to a healthy population, and as such, provides insight into the efficacy of interventional strategies that over time may result in the attenuation of muscle mass loss in clinical populations. In line, two studies in ICU patients have assessed the effect of both enteral and parenteral nutrition on whole-body protein kinetics (via primed intravenous infusions of L-[ring-2H5]-phenylalanine and L-[3,3-2H2]-tyrosine and the uptake of amino acids via L-[1-13C]-phenylalanine added to ongoing nutrition) in the acute phase (24 h [
      • Sundstrom Rehal M.
      • Liebau F.
      • Tjader I.
      • Norberg A.
      • Rooyackers O.
      • Wernerman J.
      A supplemental intravenous amino acid infusion sustains a positive protein balance for 24 hours in critically ill patients.
      ]) and over several days of ICU admission [
      • Liebau F.
      • Sundstrom M.
      • van Loon L.J.
      • Wernerman J.
      • Rooyackers O.
      Short-term amino acid infusion improves protein balance in critically ill patients.
      ]. These studies show that amino acid administration increases whole-body protein synthesis rates to levels above rates of breakdown (which remained unchanged), leading to improved whole-body protein net balance and suggesting a potential anabolic effect of amino acid administration during critical illness. However, whole-body protein turnover does not necessarily align with the response in skeletal muscle protein turnover and skeletal muscle protein turnover has been shown to be much slower than the turnover of splanchnic tissues [
      • Moreau K.
      • Walrand S.
      • Boirie Y.
      Protein redistribution from skeletal muscle to splanchnic tissue on fasting and refeeding in young and older healthy individuals.
      ], so that whole-body protein turnover may overestimate the muscle protein anabolic response. In addition, as the impact of organ turnover and disease state likely affects whole-body protein turnover rates in clinical populations [
      • Barle H.
      • Hammarqvist F.
      • Westman B.
      • Klaude M.
      • Rooyackers O.
      • Garlick P.J.
      • et al.
      Synthesis rates of total liver protein and albumin are both increased in patients with an acute inflammatory response.
      ,
      • van Dijk D.P.J.
      • Horstman A.M.H.
      • Smeets J.S.J.
      • den Dulk M.
      • Grabsch H.I.
      • Dejong C.H.C.
      • et al.
      Tumour-specific and organ-specific protein synthesis rates in patients with pancreatic cancer.
      ], acute measurements of whole-body protein net balance are less accurate to predict changes in muscle. As such, although whole-body protein turnover can provide valuable insight into the whole-body response to an intervention it does not necessarily reflect changes in muscle mass, and muscle-specific measurements are required to assess the effect on skeletal muscle tissue.

      3.3 Muscle net balance studies

      Both the AV-balance methodology (e.g. forearm or leg net balance), as well as combined FSR and FBR measurements, provide greater insight into skeletal muscle amino acid kinetics than whole-body measurements, and have been applied in clinical populations to assess alterations in muscle protein metabolism [
      • Biolo G.
      • Fleming R.Y.
      • Maggi S.P.
      • Nguyen T.T.
      • Herndon D.N.
      • Wolfe R.R.
      Inverse regulation of protein turnover and amino acid transport in skeletal muscle of hypercatabolic patients.
      ,
      • Luzi L.
      • Piceni Sereni L.
      • Spessot M.
      • Dodesini R.
      • Pastore M.R.
      • Bianchi E.
      • et al.
      Postabsorptive muscle protein metabolism in type 1 diabetic patients after pancreas transplantation.
      ,
      • Luzi L.
      • Regalia E.
      • Pulvirenti A.
      • Piceni Sereni L.
      • Spessot M.
      • Romito R.
      • et al.
      Post-absorptive and insulin-mediated muscle protein metabolism in liver-transplanted patients.
      ,
      • Murton A.
      • Bohanon F.J.
      • Ogunbileje J.O.
      • Capek K.D.
      • Tran E.A.
      • Chao T.
      • et al.
      Sepsis increases muscle proteolysis in severely burned adults, but does not impact whole-body lipid or carbohydrate kinetics.
      ,
      • Ferrando A.A.
      • Chinkes D.L.
      • Wolf S.E.
      • Matin S.
      • Herndon D.N.
      • Wolfe R.R.
      A submaximal dose of insulin promotes net skeletal muscle protein synthesis in patients with severe burns.
      ,
      • Klaude M.
      • Mori M.
      • Tjader I.
      • Gustafsson T.
      • Wernerman J.
      • Rooyackers O.
      Protein metabolism and gene expression in skeletal muscle of critically ill patients with sepsis.
      ,
      • Gamrin-Gripenberg L.
      • Sundstrom-Rehal M.
      • Olsson D.
      • Grip J.
      • Wernerman J.
      • Rooyackers O.
      An attenuated rate of leg muscle protein depletion and leg free amino acid efflux over time is seen in ICU long-stayers.
      ]. For example, basal whole-body and leg protein synthesis and breakdown rates have been shown to be higher in severely burned patients when compared to healthy controls [
      • Biolo G.
      • Fleming R.Y.
      • Maggi S.P.
      • Nguyen T.T.
      • Herndon D.N.
      • Wolfe R.R.
      Inverse regulation of protein turnover and amino acid transport in skeletal muscle of hypercatabolic patients.
      ]; however, since absolute breakdown rates were higher (+80% versus controls) than synthesis rates (+50% versus controls), this resulted in a more negative net protein balance in burns patients. In critically ill patients, leg protein breakdown has been shown to be significantly elevated compared with leg protein synthesis, resulting in negative muscle protein net balance over the first 1–3 weeks of the ICU stay [
      • Puthucheary Z.A.
      • Rawal J.
      • McPhail M.
      • Connolly B.
      • Ratnayake G.
      • Chan P.
      • et al.
      Acute skeletal muscle wasting in critical illness.
      ,
      • Klaude M.
      • Mori M.
      • Tjader I.
      • Gustafsson T.
      • Wernerman J.
      • Rooyackers O.
      Protein metabolism and gene expression in skeletal muscle of critically ill patients with sepsis.
      ,
      • Gamrin-Gripenberg L.
      • Sundstrom-Rehal M.
      • Olsson D.
      • Grip J.
      • Wernerman J.
      • Rooyackers O.
      An attenuated rate of leg muscle protein depletion and leg free amino acid efflux over time is seen in ICU long-stayers.
      ]. Similarly, a study that assessed FSR and FBR of mixed-muscle proteins in septic patients following burn injury observed a reduced muscle protein net balance as a result of a 2.4 fold increased FBR and no effect on FSR when compared with non-septic burned patients [
      • Murton A.
      • Bohanon F.J.
      • Ogunbileje J.O.
      • Capek K.D.
      • Tran E.A.
      • Chao T.
      • et al.
      Sepsis increases muscle proteolysis in severely burned adults, but does not impact whole-body lipid or carbohydrate kinetics.
      ]. Thus, the negative muscle protein net balance is reflective of the significant muscle wasting observed during critical illness, which is predominantly driven by accelerated muscle protein breakdown. The anabolic response to a nutritional intervention assessed by muscle protein net balance has been studied acutely [
      • Luzi L.
      • Regalia E.
      • Pulvirenti A.
      • Piceni Sereni L.
      • Spessot M.
      • Romito R.
      • et al.
      Post-absorptive and insulin-mediated muscle protein metabolism in liver-transplanted patients.
      ,
      • Ferrando A.A.
      • Chinkes D.L.
      • Wolf S.E.
      • Matin S.
      • Herndon D.N.
      • Wolfe R.R.
      A submaximal dose of insulin promotes net skeletal muscle protein synthesis in patients with severe burns.
      ] and over more prolonged periods [
      • Gamrin-Gripenberg L.
      • Sundstrom-Rehal M.
      • Olsson D.
      • Grip J.
      • Wernerman J.
      • Rooyackers O.
      An attenuated rate of leg muscle protein depletion and leg free amino acid efflux over time is seen in ICU long-stayers.
      ,
      • Garibotto G.
      • Sofia A.
      • Parodi E.L.
      • Ansaldo F.
      • Bonanni A.
      • Picciotto D.
      • et al.
      Effects of low-protein, and supplemented very low-protein diets, on muscle protein turnover in patients with CKD.
      ] in patient populations. As an example, a study in chronic kidney disease (CKD) patients assessed the anabolic response to different protein diets over 6 weeks using repeated measurements of forearm perfusion combined with infusions of L-[ring-2H5]-phenylalanine. It was demonstrated that the diet higher in protein did not alter forearm phenylalanine rate of disappearance (i.e. a proxy for MPS) but lowered whole-body and forearm phenylalanine rate of appearance (i.e. MPB), resulting in a 40% higher (yet still negative) forearm protein net balance [
      • Garibotto G.
      • Sofia A.
      • Parodi E.L.
      • Ansaldo F.
      • Bonanni A.
      • Picciotto D.
      • et al.
      Effects of low-protein, and supplemented very low-protein diets, on muscle protein turnover in patients with CKD.
      ]. However, the lack of muscle mass measurements preclude any conclusions on the relationship between improved (forearm and whole-body) net balance and changes in absolute muscle mass. A recent study by Davies et al., shows a reduced acute forearm branched chain amino acid (BCAA) net balance following ingestion of a mixed meal in patients with Crohn's disease when compared with healthy controls [
      • Davies A.
      • Nixon A.
      • Muhammed R.
      • Tsintzas K.
      • Kirkham S.
      • Stephens F.B.
      • et al.
      Reduced skeletal muscle protein balance in paediatric Crohn's disease.
      ]. Interestingly, the lower postprandial BCAA response (suggestive of anabolic resistance, i.e. a reduced anabolic response to protein ingestion) was associated with a lower baseline appendicular lean mass in the patient group. Although no stable isotope tracers were applied to gain insight into amino acid kinetics, this study illustrates that forearm net balance can be used to measure protein net balance following a nutritional intervention and corresponds with absolute muscle mass. In support, a continuous net release of phenylalanine and total amino acids from the leg has been observed in the first 2 weeks of ICU stay (despite continuous enteral and parenteral feeding) [
      • Vesali R.F.
      • Klaude M.
      • Rooyackers O.E.
      • Tjäder I.
      • Barle H.
      • Wernerman J.
      Longitudinal pattern of glutamine/glutamate balance across the leg in long-stay intensive care unit patients.
      ], which corresponds with the substantial muscle loss generally observed in these patients. Consequently, in a catabolic state, protein net balance being negative over a substantial timeframe can likely, for a large part, be attributed to an increase in the breakdown of muscle proteins. Therefore, the assessment of protein net balance arguably provides a more valid estimation of long-term muscle mass changes in clinical populations with acute muscle wasting than isolated measures of MPS alone.

      3.4 Predictive changes in muscle mass in clinical conditions

      With data available on FSR and FBR in healthy conditions, and; therefore, net balance, we can calculate theoretically expected changes in muscle mass in clinical populations. As an example, a change of 10% in basal (i.e. 0.003–0.007%/h) or postprandial (i.e. 0.004–0.005%/h) MPS or MPB rates would result in a ~0.5–1% decrease in muscle protein turnover over 1 week (assuming 12 h in basal or postprandial state). Such a 1% decrease in turnover represents 450 g of lean mass (assuming a body weight of 75 kg and 60% lean mass), or 250 g of appendicular lean mass. If both the postprandial rise in MPS and suppression of MPB were affected, changes in postprandial protein handling as small as 5% could result in a ~1 kg loss of muscle mass per week. While such values of muscle protein turnover are realistic, they do not always align with the degree of muscle mass loss observed. A reason for this might be that muscle loss is heavily influenced by other factors such as disease state and medication use, dietary intake, and physical (in)activity levels. Moreover, the sensitivity of both mass spectrometry analyses for stable isotope enrichments and muscle mass measurements affect the potential to predict the impact of an intervention on long-term muscle mass from acute tracer data. This might imply that although an intervention strategy is able to stimulate net balance acutely, this may not translate into a sufficiently large increase in muscle mass to exceed the ‘noise’ of the measurement (i.e. timeframe, sensitivity, and method used). Additionally, any acute increase in protein net balance in response to interventional strategies will need to be of a significant magnitude, and applied repeatedly over an extended timeframe, to eventually result in muscle protein accretion.

      3.5 Studies combining muscle-specifc measurements of protein turnover and muscle mass assessments

      The inclusion of both measurements of muscle protein turnover rates using acute stable isotope tracer methodology combined with the assessment of muscle mass in clinical populations has only been assessed in a handful of studies [
      • Puthucheary Z.A.
      • Rawal J.
      • McPhail M.
      • Connolly B.
      • Ratnayake G.
      • Chan P.
      • et al.
      Acute skeletal muscle wasting in critical illness.
      ,
      • Biolo G.
      • Fleming R.Y.
      • Maggi S.P.
      • Nguyen T.T.
      • Herndon D.N.
      • Wolfe R.R.
      Inverse regulation of protein turnover and amino acid transport in skeletal muscle of hypercatabolic patients.
      ,
      • Gamrin-Gripenberg L.
      • Sundstrom-Rehal M.
      • Olsson D.
      • Grip J.
      • Wernerman J.
      • Rooyackers O.
      An attenuated rate of leg muscle protein depletion and leg free amino acid efflux over time is seen in ICU long-stayers.
      ,
      • Williams J.P.
      • Phillips B.E.
      • Smith K.
      • Atherton P.J.
      • Rankin D.
      • Selby A.L.
      • et al.
      Effect of tumor burden and subsequent surgical resection on skeletal muscle mass and protein turnover in colorectal cancer patients.
      ]. Firstly, Biolo et al. combined the AV-balance technique, skeletal muscle biopsies, and stable isotope amino acid tracers [
      • Biolo G.
      • Fleming R.Y.
      • Maggi S.P.
      • Nguyen T.T.
      • Herndon D.N.
      • Wolfe R.R.
      Inverse regulation of protein turnover and amino acid transport in skeletal muscle of hypercatabolic patients.
      ] to assess whole-body and leg protein synthesis and breakdown, and MPS rates in patients with burns compared to healthy controls. Both whole-body and leg protein synthesis and breakdown rates were increased when compared with healthy controls, with breakdown rates exceeding protein synthesis rates, resulting in a negative protein net balance. Fractional MPS rates were 50% higher in patients than in healthy controls; however, no measures of MPB were included and with the absence of muscle mass measurements it can only be assumed that the more negative protein net balance on a whole-body and leg level is representative of the muscle wasting observed in burns patients. Gamrin-Gripenberg et al. [
      • Gamrin-Gripenberg L.
      • Sundstrom-Rehal M.
      • Olsson D.
      • Grip J.
      • Wernerman J.
      • Rooyackers O.
      An attenuated rate of leg muscle protein depletion and leg free amino acid efflux over time is seen in ICU long-stayers.
      ] assessed both whole-body and muscle protein net balance (applying the 2- and 3-pool AV model, with 3-methylhistidine for myofibrillar breakdown) in 20 ICU patients, and observed an improved (yet still negative) protein net balance over the course of ICU stay from both the leg (2-pool AV) and muscle (3-pool AV) data, primarily due to an increase in rates of MPS without any change in MPB. These data align with the greater degree of muscle loss that occurs during the early days of ICU stay [
      • Puthucheary Z.A.
      • Rawal J.
      • McPhail M.
      • Connolly B.
      • Ratnayake G.
      • Chan P.
      • et al.
      Acute skeletal muscle wasting in critical illness.
      ,
      • Chapple L.S.
      • Deane A.M.
      • Williams L.T.
      • Strickland R.
      • Schultz C.
      • Lange K.
      • et al.
      Longitudinal changes in anthropometrics and impact on self-reported physical function after traumatic brain injury.
      ], and as such, the assessments of muscle protein net balance using either AV-balance methods with or without muscle biopsies can be an informative indicator of changes in muscle mass during critical illness. To the best of our knowledge, only two studies have directly combined acute measurements of muscle protein turnover with the assessment of muscle mass changes over time in a clinical cohort. A study conducted in colorectal cancer patients planned for surgery showed a significant inverse correlation between the loss of lean leg mass after surgery (−27%) and postprandial FSR [
      • Williams J.P.
      • Phillips B.E.
      • Smith K.
      • Atherton P.J.
      • Rankin D.
      • Selby A.L.
      • et al.
      Effect of tumor burden and subsequent surgical resection on skeletal muscle mass and protein turnover in colorectal cancer patients.
      ]. Moreover, they demonstrated that the evident recovery in postprandial MPS rates following the provision of amino acids is accompanied by a decrease in leg protein breakdown (measured as phenylalanine rate of appearance) post-surgery. Although this is suggestive of an increase in muscle protein net balance post-surgery, these data were unfortunately not included. This study does however, demonstrate that changes in muscle protein turnover align with absolute changes in muscle mass. Similarly, Puthucheary et al. used leg AV-balance, primed constant stable isotope amino acid infusions, and muscle biopsies for the measurement of whole-body and muscle protein kinetics in critically ill patients. Muscle mass was assessed over 7 days via ultrasound measurements of the m. rectus femoris CSA [
      • Puthucheary Z.A.
      • Rawal J.
      • McPhail M.
      • Connolly B.
      • Ratnayake G.
      • Chan P.
      • et al.
      Acute skeletal muscle wasting in critical illness.
      ]. Acute FSR increased by >100% over the study period, which, if this had been the only measurement made, would suggest a highly anabolic state. However, although both leg protein synthesis and breakdown rates increased (with nutrition provision as per standard care), overall leg protein net balance remained negative due to higher absolute breakdown rates (29% and 14% higher than leg protein synthesis on days 1 and 7, respectively), these acute changes were accompanied by a severe decline in muscle mass (−13% in m. rectus femoris CSA and −18% in muscle fibre CSA) [
      • Puthucheary Z.A.
      • Rawal J.
      • McPhail M.
      • Connolly B.
      • Ratnayake G.
      • Chan P.
      • et al.
      Acute skeletal muscle wasting in critical illness.
      ]. This demonstrates again that while the increase in leg protein synthesis and FSR provides insightful data on muscle protein turnover in clinical conditions, the overall net balance provides a more complete picture of muscle mass changes. The fact that net balance was similarly negative on days 1 and 7, and muscle loss was linear, suggests that net balance represents a valuable indication of the changes in skeletal muscle mass in a clinical setting.

      4. Conclusion

      The development of effective (nutritional) interventions to preserve muscle mass in clinical populations is important to improve patient outcomes. Stable isotope amino acid approaches can be conducted in smaller cohorts of patients to obtain insight into the dynamic and mechanistic responses to an intervention that are not directly possible from the quantification of muscle mass alone. These approaches provide insight into acute responses that occur prior to detectable changes in muscle mass and may be used prior to embarking on a large-scale clinical trial focussed on improving muscle mass as the primary outcome. However, in this review we have provided evidence that the standard practice of solely quantifying MPS is insufficient to predict the longer-term effect on muscle mass in clinical populations. Since increases in MPB are often accelerated in illness when compared to healthy individuals, we argue that measurements of protein net balance provide greater insight into predicted changes in muscle mass in a clinical setting. While whole-body measures of protein synthesis, breakdown, and net balance are fairly common in clinical research, measures across a limb or muscle-specific measurements are preferred since they are less likely to be influenced by changes in organ protein turnover. We argue that the preferred methods are 2- or 3-pool AV-balance, or the calculation of net protein balance from measurements of both MPS and MPB. While these acute measurements of net protein balance may not quantitatively predict the change in muscle mass within a long-term interventional trial, they are more likely to reflect the potential of a (nutritional) intervention to result in longer-term benefits on muscle mass than isolated measurements of MPS or MPB alone. In addition, acute changes in net balance may not directly align with the associated change in absolute muscle mass, due to a variety of anabolic and catabolic factors in clinical practice including disease state, medical therapy, inflammation, nutritional intake, physical activity, sleep, hormonal fluctuations, or fluid status that contribute to muscle mass regulation. We conclude that stable amino acid tracers provide valuable mechanistic insight into the acute change in net balance in response to clinical interventions yet these must be combined with, or followed by, a measurement of muscle mass to determine the true influence of an intervention on clinically-relevant outcomes prior to implementation into clinical practice.

      Financial disclosure

      Nil funding to disclose.

      Author contribution

      LSC, MLD, and IWKK were responsible for conceptualisation, data curation, and original draft writing and review and editing of the final manuscript.

      Declaration of interests

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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