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Abstract
Rationale: Dietary protein may attenuate the muscle atrophy experienced by patients in the ICU, yet protein handling is poorly understood.
Objectives: To quantify protein digestion and amino acid absorption and fasting and postprandial myofibrillar protein synthesis during critical illness.
Methods: Fifteen mechanically ventilated adults (12 male; aged 50 ± 17 yr; body mass index, 27 ± 5 kg⋅m−2) and 10 healthy control subjects (6 male; 54 ± 23 yr; body mass index, 27 ± 4 kg⋅m−2) received a primed intravenous L-[ring-2H5]-phenylalanine, L-[3,5-2H2]-tyrosine, and L-[1-13C]-leucine infusion over 9.5 hours and a duodenal bolus of intrinsically labeled (L-[1-13C]-phenylalanine and L-[1-13C]-leucine) intact milk protein (20 g protein) over 60 minutes. Arterial blood and muscle samples were taken at baseline (fasting) and for 6 hours following duodenal protein administration. Data are mean ± SD, analyzed with two-way repeated measures ANOVA and independent samples t test.
Measurements and Main Results: Fasting myofibrillar protein synthesis rates did not differ between ICU patients and healthy control subjects (0.023 ± 0.013% h−1 vs. 0.034 ± 0.016% h−1; P = 0.077). After protein administration, plasma amino acid availability did not differ between groups (ICU patients, 54.2 ± 9.1%, vs. healthy control subjects, 61.8 ± 13.1%; P = 0.12), and myofibrillar protein synthesis rates increased in both groups (0.028 ± 0.010% h−1 vs. 0.043 ± 0.018% h−1; main time effect P = 0.046; P-interaction = 0.584) with lower rates in ICU patients than in healthy control subjects (main group effect P = 0.001). Incorporation of protein-derived phenylalanine into myofibrillar protein was ∼60% lower in ICU patients (0.007 ± 0.007 mol percent excess vs. 0.017 ± 0.009 mol percent excess; P = 0.007).
Conclusions: The capacity for critically ill patients to use ingested protein for muscle protein synthesis is markedly blunted despite relatively normal protein digestion and amino acid absorption.
Using contemporary stable isotope amino acid methodology, this study quantified fasting and postprandial myofibrillar protein synthesis rates, as well as the amount of enteral protein digested and absorbed and used for de novo myofibrillar protein accretion in skeletal muscle. This study shows that protein digestion and amino acid absorption are relatively normal in critically ill patients when compared to healthy control subjects, yet postprandial muscle protein synthesis is markedly reduced in critical illness, reflecting an anabolic resistance to ingested protein.
This study demonstrates that overcoming anabolic resistance to dietary protein administration could provide a key mechanistic target for clinical interventions aimed at attenuating muscle wasting in critically ill patients.
Marked skeletal muscle atrophy (1) is a common sequela of critical illness and is associated with prolonged ICU and hospital stay, increased mortality (2), and considerable long-term disability (3–6). In health, muscle mass is maintained by precisely regulated muscle protein synthesis and breakdown rates. Protein ingestion stimulates muscle protein synthesis and inhibits breakdown, making it a major determinant for muscle mass maintenance (7). To improve patient recovery, strategies to attenuate muscle loss are recommended, and augmented protein doses provide a promising target. International guidelines suggest greater protein doses for critically ill patients than those recommended in health (e.g., 1.2–2.0 g · kg−1 · day−1) (8, 9), and a number of recent small or phase 2 randomized trials have studied the effect of augmented protein doses on patient outcomes, including muscle strength, with conflicting results (10–12). However, there are no data to show whether enterally administered protein is effectively digested and absorbed and used for muscle protein synthesis during critical illness, and without this, clinical studies are difficult to interpret.
Given that the absorption of other macronutrients (carbohydrate and lipid) is diminished during critical illness (13–15), it is plausible that protein malabsorption and subsequent compromised amino acid availability could contribute to muscle wasting. In a small exploratory study, the systemic availability of amino acids after continuous gastric protein delivery was reduced, potentially because of abnormal gastrointestinal motility, impaired amino acid absorption, and/or increased splanchnic amino acid extraction (16). However, protein digestion and amino acid absorption after protein administration directly into the small intestine (thereby bypassing the variable influence of gastric emptying rate) has not been quantified, and we hypothesized that protein digestion and amino acid absorption are impaired after duodenal protein administration in critically ill patients.
Another potential cause of muscle wasting in critical illness could be a reduced capacity to stimulate muscle protein synthesis in response to the presence of amino acids in the plasma. This phenomenon, coined anabolic resistance (17, 18), has been demonstrated in aging (19), muscle disuse (20, 21), and other clinical populations (22–24), but limited data exist in critical illness. Previous work has shown that ICU patients have increased rates of whole-body protein synthesis in response to feeding (25–27); yet skeletal muscle protein turnover represents as little as ∼25% of whole-body protein turnover, and hence whole-body protein assessment does not reflect skeletal muscle responses (28, 29). Few studies to our knowledge have directly assessed muscle protein synthesis rates in critical illness (1, 30), and no study has assessed the impact of protein administration on rates of muscle protein synthesis in these patients compared with healthy control subjects. We hypothesized that critically ill patients have a blunted anabolic response to administered protein.
To test our hypotheses, contemporary stable isotope methodology combined with the enteral administration of an intrinsically labeled intact milk protein was applied in critically ill patients and healthy control subjects. This methodology allowed the assessment of fasting and postprandial myofibrillar protein synthesis rates, as well as the precise quantification of the amount of enteral protein-derived phenylalanine released into the circulation and used for de novo myofibrillar protein accretion in skeletal muscle.
This prospective single-center study was approved by the Central Adelaide Local Health Network Human Research Ethics Committee (HREC/15/RAH/153) and performed according to the National Health and Medical Research Council National Statement on Ethical Conduct in Human Research. The trial was registered prospectively at the Australia New Zealand Clinical Trials Registry (ACTRN12616001652460).
Patients admitted to the Royal Adelaide Hospital ICU from October 2017 to May 2019 were eligible if they were 1) expected to remain mechanically ventilated for at least 48 hours; 2) 18 years of age or older; and 3) receiving, or suitable for, enteral nutrition. Exclusion criteria are reported in the online supplement. Written informed consent was obtained from the legal authorized guardian.
Healthy control subjects were eligible if they were 18 years of age or older and met none of the exclusion criteria (see the online supplement).
An overview of the experimental protocol is presented in Figure 1, and further details are provided in the online supplement, including pretesting and patient data extraction. In brief, a nasoduodenal catheter was placed using the Cortrak device (Avanos Medical) in patients on the preceding day confirmed by X-ray and in control subjects before the first muscle biopsy. Following a 4-hour fast in patients (from 20:00 the night before in control subjects), the plasma and muscle intracellular pools were primed with a single intravenous dose of L-[ring-2H5]-phenylalanine (2.0 μmol · kg−1), L-[ring-3,5-2H2]-tyrosine (0.6 μmol · kg−1), and L-[1-13C]-leucine (4.0 μmol · kg−1) delivered via an intravenous pump (Module VP 8100; Alaris, Care Fusion), followed by a continuous infusion of L-[ring-2H5]-phenylalanine (0.050 μmol · kg−1 · min−1), L-[ring-3,5-2H2]-tyrosine (0.015 μmol · kg−1 · min−1), and L-[1-13C]-leucine (at 0.100 μmol · kg−1 · min−1) over 9.5 hours. At t = 0 minutes, a duodenal infusion of intrinsically labeled L-[1-13C]-phenylalanine and L-[1-13C]-leucine intact milk protein (20 g protein, 7.1 kJ · min−1), dissolved in water to 240 ml was delivered over 60 minutes via an enteral feeding pump (Flocare; Nutricia). Protein was delivered directly into the duodenum to exclude any influence of gastric emptying rate on the rate of protein absorption, as abnormal gastric motility occurs frequently and variably in critically ill patients.
Figure 1. Study day procedures. i.d. = Intraduodenal administration of L-[1-13C]-phenylalanine and L-[1-13C]-leucine; i.v. = Intravenous continuous dose of L-[ring-2H5]-phenylalanine, L-[ring-3,5-2H2]-tyrosine and L-[1-13C]-leucine; BS = Arterial blood sample; MB = Muscle biopsy; P = i.v. priming dose of L-[ring-2H5]-phenylalanine, L-[ring-3,5-2H2]-tyrosine and L-[1-13C]-leucine.
[More] [Minimize]L-[ring-2H5]-phenylalanine was present in the intravenous infusion only and was used to assess whole-body protein metabolism as well as fasting and postprandial muscle protein synthesis rates. L-[1-13C]-leucine was present in both the intravenous infusion and administered milk protein and allowed the assessment of fasting and postprandial muscle protein synthesis rates (providing a correction for precursor pool dilution). L-[1-13C]-phenylalanine was present in the milk protein only and was used to measure digestion and absorption of administered protein and to quantify amino acid incorporation (from the provided protein) into skeletal muscle protein that was used for muscle protein synthesis. L-[ring-3,5-2H2]-tyrosine was present in the intravenous infusion only (in combination with L-[ring-2H5]-phenylalanine) and was used for the assessment of whole-body protein metabolism (i.e., rates of protein breakdown and amino acid oxidation).
Whole-blood glucose was assessed at the bedside using a portable glucometer (Freestyle Optium Neo H; Abbott, Abbott Diabetes Care Ltd.) and serum insulin, plasma amino acid concentrations (total phenylalanine, leucine, and tyrosine, labeled and unlabeled), and plasma amino acid enrichments (the percentage of labeled phenylalanine, leucine, and tyrosine/total [labeled and unlabeled] amino acids) were determined from arterial blood samples.
Skeletal muscle tissue biopsies were obtained at t = −120, 0, and 360 minutes relative to protein administration from the middle region of the M. vastus lateralis using percutaneous needle biopsy technique (Bergstrom [31]) after administration of local anesthetic (Xylocaine 2% with 1:200,000 adrenaline), alternated between legs (first leg randomized) using separate incisions. Tissue preparation and analysis is described in the online supplement.
Whole-body amino acid kinetics and fractional synthesis rates (FSR in %⋅h−1) were calculated as per previous methodologies (32, 33) as detailed in the online supplement. Area under the curve (AUC) was calculated as AUC divided by total minutes.
On the basis of previous data (34, 35) and an independent samples t test at a significance level of 0.05, 15 ICU patients and 10 healthy control subjects would provide 80% power to detect a difference between groups in postprandial myofibrillar fractional synthesis rates (primary outcome) of 0.044% · h−1 (effect size of 1.2 on the basis of an assumed 60% upper confidence limit of SD = 0.037% · h−1). An upper 60% confidence limit of the SD was used to account for the uncertainty in the observed values (36). Baseline characteristics and myofibrillar-protein bound L-[1-13C]-phenylalanine enrichments between ICU patients and healthy control subjects were compared using independent samples t tests. Whole-blood glucose, serum insulin, plasma amino acid concentrations and enrichments, whole-body protein kinetics, and fractional synthesis rates over time for the fasting (t = −120 to 0 min) and postprandial (t = 0 to 360 min) periods were analyzed by two-factor repeated-measures ANOVA with time as the within-subjects factor and group as the between-subjects factor. The ANOVA included time as a categorical variable within the model (with one category per time point), such that the model estimates the mean outcome at each time point and does not make any assumptions about the form of the pattern of responses over time (linear or otherwise). In case of significant time by group interactions, separate analyses were performed to determine time effects for each group (one-factor repeated measures ANOVA with time points as a categorical factor) with a Bonferroni post hoc test applied to the pairwise comparisons of time points, and between-group tests at each time point (independent samples t test). Peak values, time to peak, and AUC were presented for plasma time curves and whole-body protein turnover rates (synthesis, breakdown, oxidation, and protein net balance) during the fasting (t = −120 to 0 min) and postprandial (t = 0 to 360 min) periods and differences were determined using independent samples t tests. All data are presented as mean ± SD. All analyses were conducted as two-sided tests and statistical significance was set at P < 0.05. All calculations were performed using SPSS (IBM Statistics, version 26.0; IBM Corp.).
Consent was obtained for 21 patients, but the study was aborted in 6 before commencement owing to inability to place nasoduodenal tube (n = 3), consent withdrawal (n = 2), and medical instability (n = 1). Demographic and clinical data for the 15 ICU patients and 10 healthy control subjects are provided in Table 1.
| ICU Patients (n = 15) | Healthy Control Subjects (n = 10) | P Value | |
|---|---|---|---|
| Age, yr | 49.7 ± 16.5 | 53.9 ± 22.9 | 0.601 |
| Male, n (%) | 12 (80) | 6 (60) | |
| Weight, kg | 81.5 ± 16.5 | 81.4 ± 14.9 | 0.982 |
| Body mass index, kg · m−2 | 26.5 ± 5.2 | 26.7 ± 4.4 | 0.938 |
| 72-h prestudy caloric intake, kJ · 24 h−1 | 6,349 ± 2,506 | 7,730 ± 1,498 | 0.132 |
| Prestudy protein intake | |||
| g · 24 h−1 | 74.6 ± 29.6 | 80.5 ± 20.9 | 0.589 |
| g · kg·24 h−1 | 0.94 ± 0.43 | 1.00 ± 0.21 | 0.701 |
| APACHE II score | |||
| On admission | 18.7 ± 5.0 | — | — |
| On study day | 16.1 ± 6.4 | — | — |
| Days in ICU before study | 4.6 ± 2.9 | — | — |
| Length of ICU stay, d | 12.7 ± 9.0 | — | — |
| Duration of mechanical ventilation, d | 11.7 ± 8.9 | — | — |
| Diagnostic category, n | — | — | |
| Trauma | 5 | ||
| Neurological | 6 | ||
| Respiratory | 4 | ||
| SOFA score on study day | 5.5 ± 2.1 | — | — |
| RIFLE criteria on study day, n | — | — | |
| No AKI | 12 | ||
| Injury | 1 | ||
| Failure | 2 | ||
| Creatinine on study day, μmol · L−1 | 91.0 ± 57.8 | — | — |
| Urea:creatinine on study day, mmol · L−1 | 104.0 ± 56.9 | ||
| Vasopressors/catecholamine, μg · kg−1 · min−1 (n = 3) | |||
| Peak dose administered during study | 4.3 ± 3.2 | — | — |
| Opiates, mc · h−1 (n = 9) | |||
| Peak dose administered during study | 48 ± 26 | — | — |
| Propofol administered, mg | |||
| During study (n = 10) | 1840 ± 481 | — | — |
| 3 d prior to fasting (n = 13) | 8242 ± 4043 | — | — |
| Insulin before study fasting period, IU · 72h−1 (n = 4) | 133.0 ± 66.8 | — | — |
Plasma amino acid concentrations are presented in Figure 2, whole-blood glucose and serum insulin concentrations are presented in Figures E1A and E1B, and plasma amino acid enrichments are presented in Figure E2.
Figure 2. (A–C) Total (labeled and unlabeled) arterial plasma phenylalanine (A), leucine (B), and tyrosine (C) concentrations (μmol·L−1) before, during, and after intraduodenal protein administration indicative of the amino acid concentrations present in the circulation over the study period. The gray section represents the period of duodenal protein administration. Phenylalanine, fasting: P-interaction = 0.326; main time effect P = 0.869; main group effect P < 0.001. Postprandial: P-interaction = 0.224; main time effect P < 0.001; main group effect P < 0.05. Leucine, fasting: P-interaction = 0.867; main time effect P = 0.586; main group effect P = 0.046. Postprandial: P-interaction = 0.005; time effect, both P < 0.001. Peak: 121.6 ± 27.5 μmol⋅L−1 versus 97.8 ± 17.7 μmol⋅L−1; P = 0.045. Tyrosine, fasting: P-interaction < 0.05; time effect, controls, P < 0.05; patients:, P = 0.378. Postprandial: P-interaction < 0.05; time effect, both P < 0.001. *Significant difference between groups.
[More] [Minimize]Plasma arterial L-[1-13C]-phenylalanine enrichments, reflective of the absolute amount of labeled phenylalanine in plasma from the duodenally administered protein (proof of methodology), increased rapidly after duodenal protein administration in both groups (P < 0.001), reaching peak values of 12.1 ± 2.6 mol percent excess (MPE) at 86 ± 25 minutes in ICU patients and 17.4 ± 1.6 MPE at 63 ± 21 min in healthy control subjects (P < 0.001), remaining lower in ICU patients from t = 30–90 minutes when compared with healthy control subjects (P < 0.05) (Figure E2C). Rates of protein-derived amino acid appearance in the plasma from the labeled duodenal protein (i.e., a direct measure of protein digestion and amino acid absorption) increased after duodenal protein administration in both groups (Figure 3A) (P-interaction = 0.004, time effect, both P < 0.001), with lower rates observed in ICU patients than in healthy control subjects at t = 30 and 45 minutes (P < 0.05), and then higher rates in ICU patients from t = 120 to 360 minutes (P < 0.05). By calculating the area under the curve of exogenous amino acid rate of appearance, the total amount of dietary protein-derived phenylalanine appearing in the circulation over the 6-hour postprandial period was determined (Phe plasma; expressed as a fraction of the amount of phenylalanine in the duodenally administered protein), which did not differ between ICU patients and healthy control subjects (61.8 ± 13.1 vs. 54.2 ± 9.1%; P = 0.12) (Figure 3B).
Figure 3. Exogenous protein-derived phenylalanine appearance rates (μmol phenylalanine·kg−1·min−1 [A] indicative of the rate of protein digestion and amino acid absorption) and total amount of protein-derived phenylalanine that appeared in the circulation throughout the 6-hour postprandial period, expressed as a percentage of the total amount of exogenous protein-derived phenylalanine that was administered intraduodenally (Phe plasma, %; [B] indicative of protein digestion and amino acid absorption over the total 6-hour postprandial period). The gray section represents the period of intraduodenal protein administration. Exogenous protein-derived phenylalanine rates of appearance: P-interaction = 0.004; time effect, both P < 0.001. *Significant difference between groups.
[More] [Minimize]Endogenous phenylalanine rates of appearance (endogenous Ra; reflective of tissue protein breakdown), total phenylalanine rates of appearance (total Ra), total phenylalanine rates of disappearance (total Rd), and phenylalanine oxidation rates, used for the calculation of whole-body protein metabolism, are presented in Figure E3. Whole-body protein turnover (i.e., protein synthesis, breakdown, and amino acid oxidation rates and protein net balance, calculated from the rates of amino acid appearance in and disappearance from the circulation) are presented in Figure 4. Fasting and postprandial whole-body protein synthesis rates were higher in ICU patients than in healthy control subjects (fasting, 0.85 ± 0.17 μmol phenylalanine · kg−1 · min−1 vs. 0.49 ± 0.09 μmol phenylalanine · kg−1 · min−1; P < 0.001; postprandial, 0.87 ± 0.19 μmol phenylalanine · kg−1 · min−1 vs. 0.53 ± 0.08 μmol phenylalanine · kg−1 · min−1, respectively; P < 0.001), with a blunted postprandial increase in ICU patients (P-interaction < 0.001). Similarly, whole-body protein breakdown rates were higher in ICU patients than in healthy control subjects in both the fasting (0.85 ± 0.18 μmol phenylalanine · kg−1 · min−1 versus 0.48 ± 0.09 μmol phenylalanine · kg−1 · min−1; P < 0.001) and postprandial (0.75 ± 0.18 μmol phenylalanine · kg−1 · min−1 vs. 0.42 ± 0.08 μmol phenylalanine · kg−1 · min−1; P < 0.001) periods and reduced to a greater extent following protein administration in ICU patients (P-interaction < 0.001). Whole-body protein oxidation rates did not differ between groups in the fasting (P = 0.210) and postprandial (P-interaction = 0.279; main group effect P = 0.209) period. Overall, protein net balance did not differ between the two groups in the fasting period (P = 0.089) and became positive in the postprandial state in both groups (main time effect, P < 0.001) with no differences between ICU patients and healthy control subjects (0.11 ± 0.04 μmol phenylalanine · kg−1 · min−1 vs. 0.10 ± 0.02 μmol phenylalanine · kg−1 · min−1; P-interaction = 0.382; main group effect = 0.752).
Figure 4. Whole-body protein synthesis, breakdown, and oxidation rates and whole-body protein net balance (area under the curve per minute expressed as μmol phenylalanine·kg−1 · min−1) for the fasting and postprandial period. Synthesis: P-interaction < 0.001; time effect, both P < 0.001. Breakdown: P-interaction < 0.001; time effect, both P < 0.001. Oxidation: P-interaction = 0.279; main time effect, P < 0.001; main group effect, P = 0.209. Net balance: P-interaction = 0.382; main time effect, P < 0.001; main group effect, P = 0.752. *Significant difference between groups.
[More] [Minimize]Myofibrillar protein fractional synthesis rates (FSR: % · h−1) are presented in Figures 5A and 5B. Fasting myofibrillar protein synthesis rates based upon the L-[ring-2H5]-phenylalanine tracer (the rate at which amino acids are synthesized into skeletal muscle protein in the fasted state) (Figure 5A) did not differ between groups (patients, 0.023 ± 0.013% · h−1, and controls, 0.034 ± 0.016% · h−1; P = 0.077). After intraduodenal protein administration, myofibrillar protein FSR (the rate at which amino acids are synthesized into skeletal muscle protein in response to dietary protein administration) increased to 0.028 ± 0.010% · h−1 in ICU patients compared with 0.043 ± 0.008% · h−1 in healthy control subjects (P-interaction = 0.584; main time effect, P = 0.046), with lower myofibrillar protein synthesis rates in patients than in control subjects (main group effect, P = 0.001). Fasting myofibrillar protein fractional synthesis rates, calculated from the L-[1-13C]-leucine tracer (Figure 5B), did not differ between groups (P = 0.789), and no significant increase in postprandial myofibrillar protein synthesis was observed after protein administration (P-interaction = 0.311; main time effect, P = 0.781). Myofibrillar protein-bound L-[1-13C]-phenylalanine enrichments were significantly lower in ICU patients than in healthy control subjects, representative of 60% less protein-derived phenylalanine incorporated into myofibrillar protein in ICU patients than in healthy control subjects (0.007 ± 0.007 MPE vs. 0.017 ± 0.009 MPE; P = 0.007) (Figure 6).
Figure 5. (A and B) Fasting and postprandial myofibrillar protein fractional synthesis rates (FSR, %·h−1) assessed using either intravenous L-[ring-2H5]-phenylalanine infusions (A) or intravenous and dietary protein-derived muscle protein-bound L-[1-13C]-leucine administration (B), indicative of the rate of skeletal muscle protein synthesis within the fasted state and following dietary protein administration. FSR based upon L-[ring-2H5]-phenylalanine: P-interaction = 0.584; main time effect, P = 0.046; main group effect, P = 0.001. FSR based upon L-[1-13C]-leucine: P-interaction = 0.311; main time effect, P = 0.781; main group effect, P = 0.100. **Significant difference from fasting rates.
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Figure 6. Myofibrillar protein-bound L-[1-13C]-phenylalanine enrichments (MPE; %) 6 hours after intraduodenal administration of intrinsically L-[1-13C]-phenylalanine labeled protein, as a direct measure of dietary protein incorporation into contractile skeletal muscle protein. MPE = mole % excess. *Significant difference between groups.
[More] [Minimize]Our study shows that the extent of protein digestion and amino acid absorption after duodenal protein administration in critically ill patients is essentially normal. Despite this, the incorporation of dietary-derived amino acids into skeletal muscle protein is markedly impaired.
Macronutrient malabsorption is common in critical illness. Protein absorption has previously been reported to be impaired following continuous intragastric delivery (16), presumably attributed to slower gastric emptying, a problem frequently encountered in critical illness (37). To avoid the variable and unpredictable influence of gastric emptying, we delivered protein directly into the duodenum. After intraduodenal delivery, the rate of protein digestion and amino acid absorption appeared initially slightly delayed in ICU patients compared with healthy control subjects. However, the extent, as measured by the total amount of protein-derived amino acids in the systemic circulation over the 6-hour study period, did not differ between the cohorts (e.g., ∼55–60%), suggesting that protein digestion and amino acid absorption after intraduodenal protein administration is not compromised in critical illness.
In agreement with previous literature (25–27), we observed higher fasting and postprandial whole-body protein synthesis and breakdown rates in critically ill patients than in healthy control subjects. Protein net balance increased in both groups after protein administration, yet there were no differences in fasting or postprandial protein net balance between the two groups. Three other studies have previously examined whole-body protein turnover in response to nutrient administration in critically ill patients. Two studies showed an increase in whole-body protein balance following intravenous amino acid supplementation of enteral nutrition (26, 27), whereas the third study showed a higher whole-body protein balance following 100% compared to 50% goal of total enteral nutrition delivery (38). Although whole-body protein net balance increases and becomes positive after protein administration in ICU patients, which may be thought to equate to an attenuation of skeletal muscle loss, whole-body protein kinetics do not accurately reflect the response of skeletal muscle to protein delivery (29, 39), as muscle protein turnover represents only 25–30% of whole-body protein turnover (28). Therefore, it is essential to quantify changes in muscle protein synthesis rather than whole-body protein turnover in response to dietary protein.
Skeletal muscle mass comprises around 40% of total body weight and is in a constant state of amino acid flux (40), with fasting and fed rates of muscle protein synthesis being involved in muscle mass regulation. In our study, the measurement of myofibrillar protein synthesis allowed assessment of structural protein synthesis as opposed to acute phase proteins that are rapidly synthesized in response to inflammation in critical illness. Our data showed that fasting myofibrillar protein synthesis rates do not differ in a critically ill cohort from those in a healthy cohort, suggesting that fasting myofibrillar protein synthesis is not impaired in critical illness. To our knowledge, this is a novel finding.
Our data also show that postprandial myofibrillar protein synthesis rates increase after duodenal protein administration in both ICU patients and healthy control subjects. However, the deposition of dietary protein-derived amino acids into myofibrillar protein was ∼60% lower 6 hours after protein administration in ICU patients, showing, for the first time an impaired muscle protein synthetic response to dietary protein (i.e., anabolic resistance). Only two studies to our knowledge have quantified rates of muscle protein synthesis in critical illness, both of which aimed to assess change in muscle protein synthesis over the ICU stay (30). Puthucheary and colleagues compared muscle protein synthesis rates from 11 patients on Days 1 and 7 of ICU stay with fasted and fed healthy control subjects. They reported that muscle protein synthesis rates did not differ between ICU patients on Day 1 and fasted healthy control subjects (patients, 0.035% · h−1, vs. control, 0.039% · h−1; P = 0.57) and between patients on Day 7 (variable doses of enteral nutrition provided) and fed controls (patients, 0.076% · h−1, vs. control, 0.065% · h−1; P = 0.30) (1). Gamrin-Gripenberg and colleagues combined arteriovenous balance methodology with a single skeletal muscle biopsy in critically ill adults receiving continuous enteral nutrition and observed an increase in rates of muscle protein synthesis over the ICU stay (Days 10–20 vs. 30–40) (30). However, the methodology in these studies differs substantially from ours, making direct comparisons impossible. Our trial is the first to assess the physiological response to a protein dose delivered directly into the duodenum in critically ill patients, from digestion, absorption, whole-body protein turnover, through to myofibrillar protein synthesis and the direct incorporation of the administered protein dose into skeletal muscle tissue. Our results show that although ICU patients respond to dietary protein with increased postprandial rates of muscle protein synthesis, they are less able to use the protein provided to incorporate into muscle than healthy control subjects, representing a blunted muscle protein synthetic response.
The demonstrated anabolic resistance to dietary protein is likely to contribute to the skeletal muscle wasting observed during critical illness. A number of conditions are known to be associated with anabolic resistance, including older age, obesity, acute inflammation, insulin resistance, and physical inactivity or muscle disuse, all of which are prevalent in ICU patients (37, 38). Older age has been shown to impair muscle protein synthesis by 10–20% (19), and older obese adults have lower rates of muscle protein synthesis after protein ingestion compared with young lean individuals (41). However, our two cohorts were well matched for age (mean age, 49.7 yr vs. 53.9 yr; P = 0.601) and body mass index (27 kg · m−2 in both cohorts), so these factors are likely not responsible for the observed differences. Physical inactivity reduces the muscle protein synthetic response to protein ingestion (42). The 60% reduction in incorporation of ingested amino acids into muscle is higher than the amount previously observed in enforced bedrest in healthy young men (20). To minimize the anticipated difference in physical activity between our cohorts, the healthy individuals were instructed to reduce physical activity before the study day and were confined to bed throughout the study period. Nevertheless, the extended and enforced bedrest of the ICU patients may have a marked effect on muscle protein turnover and may be an important cause of the observed differences in postprandial muscle protein synthesis. Furthermore, both insulin resistance (43) and an exaggerated inflammation-induced acute phase response are characteristic of critical illness (44) and could be relevant in this cohort. Future studies should explore mechanisms underlying anabolic resistance to dietary protein to guide therapeutic strategies.
Our trial has a number of strengths, including delivery of the protein dose intraduodenally and recruiting an age- and body mass index–matched healthy cohort. Contemporary stable isotope methodology was combined with administration of a highly enriched intrinsically labeled intact protein source to allow a direct assessment of protein digestion and amino acid absorption kinetics, fasting, and postprandial muscle protein synthesis rates, and assessment of the incorporation of enterally administered intact dietary protein into skeletal muscle tissue. Although continuous enteral feeding is frequently used in practice, we were able to apply a single bolus dose of protein to determine the physiological response to dietary protein. Limitations include that patients were studied relatively early during their ICU admission, yet anabolic resistance may vary over time. Also, by necessity, owing to the complexity of study procedures, the patient cohort is a convenience sample. Nevertheless, the ICU cohort was representative of typical high-acuity critically ill patients (Table 1). Given the more frequent plasma sampling conducted, calculations of fractional myofibrillar protein synthesis rates were based on plasma, rather than muscle-free (i.e., intracellular) enrichments, to allow for a more accurate assessment of the precursor pool. In addition, muscle protein breakdown may contribute to changes in muscle mass, but breakdown rates were not measured in this study. Finally, using state-of-the-art methodology, this study provides novel data on the physiological response to dietary protein in a highly challenging and understudied population, compared with a healthy cohort, and hence contributes significantly to our understanding of muscle wasting and its potential management.
A number of potential strategies have been proposed to overcome anabolic resistance in health and other clinical populations. Augmented protein delivery is a strategy recommended by international critical care nutrition guidelines (8, 9), yet so far, only two small clinical randomized controlled trials support this strategy with attenuation of ultrasound-derived muscle loss (10, 11). In health, ingestion of greater protein doses has been shown to increase postprandial plasma availability of amino acids (45) and to further stimulate muscle protein synthesis (35, 46, 47). On the basis of the results of this study, it is plausible that increased doses of enterally delivered protein may be effectively digested and absorbed; however, it is uncertain whether additional amounts of protein can overcome the observed anabolic resistance to dietary protein and further stimulate myofibrillar protein synthesis in critically ill patients. Similarly, although not examined in this study, these data suggest that the provision of hydrolyzed or intravenous free amino acids may not offer benefit in terms of muscle protein synthesis (48), as digestion and absorption appear relatively intact and the defect appears to be at the myocyte level.
This study demonstrates for the first time that, although the extent of digestion and amino acid absorption of intact dietary protein administered directly into the intestine is not impaired in critical illness, the incorporation of dietary protein-derived amino acids into skeletal muscle protein is markedly blunted, representing anabolic resistance to dietary protein. Although the capacity for effective usage of additional protein in critical illness should be explored, these data caution against the widespread use of augmented enteral protein doses in this population without further evaluation. The mechanisms underlying the impaired anabolic response to protein administration are unclear but may contribute to muscle atrophy and poor functional recovery in critically ill patients.
The authors thank the following medical doctors for their assistance with conducting muscle biopsies and inserting arterial lines on study days: Michael Davies, Anneleen Neuts, Karel Heytens, Misha Yadav, and Tejaswini Arunachala Murthy. They also thank the clinical trials pharmacists at the Royal Adelaide Hospital for preparation of the intravenous tracer (Steven Duong, Yee Chai, Huilin Zhou), technical staff at Maastricht University Medical Centre for the stable isotope analysis (Annemie Gijsen, Stefan Gorissen, Joan Senden, Janneau Kranenburg, and Joy Goessens), and biostatistician Kylie Lange at the University of Adelaide for her statistical advice.
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Data sharing agreement: Nonidentifiable data that underlie the results reported in this trial will be made available 3 years after publication and ending 5 years after publication of the main manuscript. Availability will only be made to independent researchers who provide a written proposal for data evaluation that is judged to be methodologically sound by an independent committee approved by the sponsor. Proposals should be directed to [email protected]. If the proposal is approved, applicants will be required to sign a data access agreement and will remain responsible for all costs incurred.
Supported by the National Health and Medical Research Council project grant APP1144496. L.-a.S.C. has been supported by a European Society for Clinical Nutrition and Metabolism Research Fellowship (2018) and a National Health and Medical Research Council Early Career Fellowship (2019–2022). The Royal Adelaide Hospital provided infrastructure and administrative support.
Author Contributions: L.-a.S.C., I.W.K.K., S.S., A.M.D., L.J.C.v.L., and M.J.C. made substantial contributions to the conception or design of the work. L.-a.S.C., I.W.K.K., M.J.S., L.M.W., S.G., E.R., P.S., A.M.D., L.J.C.v.L., and M.J.C. were responsible for the acquisition, analysis, or interpretation of data for the work. L.-a.S.C., I.W.K.K., L.J.C.v.L., and M.J.C. drafted the work, and all other authors revised it critically for important intellectual content. All authors provided final approval of the version submitted for publication and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
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Originally Published in Press as DOI: 10.1164/rccm.202112-2780OC on May 18, 2022
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