A supplemental intravenous amino acid infusion sustains a positive protein balance for 24 hours in critically ill patients
Sundström Rehal et al. Critical Care
A supplemental intravenous amino acid infusion sustains a positive protein balance for 24 hours in critically ill patients
Martin Sundström Rehal 0 1
Felix Liebau 0 1
Inga Tjäder 0 1
Åke Norberg 0 1
Olav Rooyackers 0 1
Jan Wernerman 0 1
0 Department of Perioperative Medicine and Intensive Care (PMI), Karolinska University Hospital Huddinge , Stockholm , Sweden
1 Department of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institute , Stockholm , Sweden
Background: Providing supplemental amino acids to ICU patients during a 3-h period results in improved wholebody net protein balance, without an increase in amino acid oxidation. The primary objective was to investigate if a 24-h intravenous amino acid infusion in critically ill patients has a sustained effect on whole-body protein balance as was seen after 3 h. Secondary objectives were monitoring of amino acid oxidation rate, urea and free amino acid plasma concentrations. Methods: An infusion of [1-13C]-phenylalanine was added to ongoing enteral nutrition to quantify the enteral uptake of amino acids. Primed intravenous infusions of [ring-2H5]-phenylalanine and [3,3-2H2]-tyrosine were used to assess whole-body protein synthesis and breakdown, to calculate net protein balance and to assess amino acid oxidation at baseline and at 3 and 24 hours. An intravenous amino acid infusion was added to nutrition at a rate of 1 g/kg/day and continued for 24 h. Results: Eight patients were studied. The amino acid infusion resulted in improved net protein balance over time, from -1.6 ± 7.9 μmol phe/kg/h at 0 h to 6.0 ± 8.8 at 3 h and 7.5 ± 5.1 at 24 h (p = 0.0016). The sum of free amino acids in plasma increased from 3.1 ± 0.6 mmol/L at 0 h to 3.2 ± 0.3 at 3 h and 3.6 ± 0.5 at 24 h (p = 0.038). Amino acid oxidation and plasma urea were not altered significantly. Conclusion: We demonstrated that the improvement in whole-body net protein balance from a supplemental intravenous amino acid infusion seen after 3 h was sustained after 24 h in critically ill patients. Trial registration: This trial was prospectively registered at Australian New Zealand Clinical Trials Registry. ACTRN, 12615001314516. Registered on 1 December 2015.
Critical illness; Protein balance; Intensive care; Nutrition; Amino acid supplementation; Stable isotope tracers
Several observational studies have demonstrated correlation
between a low protein intake in the ICU and poor
outcomes, making protein supplementation an appealing
]. Current guidelines recommend between 1.2
and 2.5 g/kg/day for critically ill patients [
]. There are
no high-quality randomized controlled trials (RCTs)
demonstrating improved patient outcomes in support of
these recommendations. A number of studies with
surrogate endpoints have failed to demonstrate any
advantage of protein deliveries above 1.5 g/kg/day [
employing short-term nitrogen balance also report
improved nitrogen balance at intakes above 1.5 g/kg/day [
There is a paucity of evidence to inform clinical
practice. Clinical trials are urgently needed, but these in turn
require physiological studies to elucidate the biological
effects of protein supplementation during critical illness.
The design of these RCTs should rest upon knowledge
of physiology and safety of the suggested feeding
regimens in subjects with compromised organ function.
Stable isotope tracer techniques provide a method
of quantifying protein synthesis, breakdown and
oxidation in vivo [
]. Our research group has previously
demonstrated that an intravenous infusion of amino
acids in addition to enteral feeding improves net
whole-body protein balance for up to 3 h during the
initial week of ICU stay, and that a similar response
is seen when this therapy is repeated 2–4 days later
]. In addition, safety in terms of unaltered amino
acid oxidation and urea levels was demonstrated. We
therefore wanted to investigate if a prolonged
intravenous amino acid infusion could sustain improved
protein balance for up to 24 h (primary outcome).
Changes in amino acid oxidation rate, serum urea
and plasma free amino acid concentrations were
monitored as secondary outcomes.
All patients in a 12-bed mixed surgical-medical ICU
were screened for participation. Exclusion criteria
were (1) < 18 years of age, (2) severe hemodynamic
instability requiring resuscitation, (3) renal
replacement therapy (RRT), (4) expectations that a patient
would not complete the protocol (planned
interruptions of enteral nutrition, transfer etc. within 24 h),
(5) hospital stay > 2 weeks prior to screening for
participation, (6) no arterial line for sampling in situ,
and (7) lack of informed consent. This study was
prospectively registered at Australian New Zealand
Clinical Trials Registry (ANZCTR) (trial ID
All aspects of care were determined by the attending
physician and nursing team. Nutritional therapy adhered
to a local protocol emphasizing early enteral feeding
with the addition of parenteral nutrition after 5 days
when the target was not reached using enteral nutrition.
Caloric targets were determined by indirect calorimetry
when possible or as 20 kcal/kg ideal bodyweight. The
feeding rates were kept unaltered during the study
period. In the event of intravenous glutamine
supplementation this therapy was interrupted during the study
At time (T)1 = 0 on day 1, an infusion of
[1-13C]-phenylalanine was added to ongoing enteral nutrition to
quantify the uptake of enteral amino acids, at a rate
calculated to obtain 30% enrichment of enteral
phenylalanine content. At T2 = 120 min, a priming intravenous
bolus of 0.5 mg/kg [ring-2H5]-phenylalanine, 0.15 mg/kg
[2H4]-tyrosine and 0.3 mg/kg [2H2]-tyrosine was
administered, after which a continuous infusion of
0.5 mg/kg/h [ring-2H5]-phenylalanine and 0.3 mg/kg/h
[3,3-2H2]-tyrosine was started and continued for
360 min. After expected isotopic equilibrium at T3 =
285–300 min, four blood samples were drawn from the
arterial line at 5-min intervals. At T4 = 300 min, an
intravenous amino acid infusion (Glavamin, Fresenius
Kabi) was started at a rate of 0.3 ml/kg/h (equivalent to
1 g/kg/day) to be continued for 24 h. Blood sampling
was repeated at T5 = 465–480 min, after which the
tracer infusions were discontinued for the remainder
of day 1. The protocol is illustrated in Fig. 1.
On day 2, enteral and parenteral tracers were
administered in identical order and dose to day 1, 24 h
after T1. A third set of blood samples were drawn
between T8 = 285–300 min after the start of the enteral
tracer on day 2.
Plasma samples were obtained from blood by
centrifugation and immediately frozen to -80 °C. Labeled amino
acids were analyzed by gas chromatography-mass
spectrometry as previously described [
plasma sample from each sampling point of T3, T5 and
T8, was also analyzed for urea (Urea kit on Indiko
analyser, Thermo Fisher Scientific) and free amino acid
concentrations. Plasma free amino acid concentration is
presented as the sum of all free amino acids except
proline, which was not a part of our assay.
Gastric residual volumes were checked before starting
the enteral tracer. Energy expenditure was quantified in
mechanically ventilated patients by indirect calorimetry
(Quark RMR, Cosmed, Rome, Italy) during sampling at
T3 and T8 in the absence of contraindications to gas
exchange measurements (fraction of inspired oxygen
(FiO2) >0.6, gas leaks).
Whole-body protein kinetics were calculated using a
single-pool model as previously described (Fig. 2) [
At steady state conditions, the phenylalanine rate of
appearance (RaPhe) = the phenylalanine rate of
disappearance (RdPhe). RaPhe is calculated from the mean value of
isotopic enrichment (molar percentage excess (MPE)) in
the four samples. Synthesis and breakdown are then
derived as follows:
Breakdown ¼ RaPhe – ðEnteralPhe þ ParenteralPheÞ
Synthesis ¼ RaPhe − OxidationPhe
Phenylalanine oxidation rate was estimated from the
hydroxylation of [ring-2H5]-phenylalanine to
[2H4]-tyrosine. Appearance of enteral phenylalanine was corrected
for splanchnic extraction of amino acids [
weight adjustments for tracer data are by actual body
weight. Descriptive data corrected for body weight are
½Length ðcmÞ – 100 þ
Actual body weight on admission
– ðLength ðcmÞ ‐ 100Þ
the formula used for dosing nutrition according to
Descriptive statistics are provided as median and range.
Normality was assessed using the Shapiro-Wilk
normality test. Parametric (two-tailed Student’s t test for paired
samples) and non-parametric (Wilcoxon signed-rank
test) methods were used as appropriate for paired
samples. One-way analysis of variance (ANOVA) with
repeated measures was used for comparisons of multiple
paired samples. Sphericity was assessed with Mauchly’s
test of sphericity, and a Greenhouse-Geisser correction
applied as appropriate. The Bonferroni correction was
used for post-hoc testing. Based on a previous study
from our group a recruitment target of n = 10 was
determined sufficient to assess the primary endpoint with
80% power [
]. An α value ≤0.05 was regarded as
statistically significant. Tests were performed in SPSS
Statistics version 24 (IBM, Armonk, NY, USA) and Prism
version 7 (GraphPad Software Inc, La Jolla, CA, USA).
A total of 12 patients in the ICU of Karolinska
University Hospital Huddinge were recruited between January
and December of 2016. Baseline protein balance was
assessed in all patients, with eight completing the entire
protocol. In four cases protein balance could not be
assessed at 3 h (n = 1) or 24 h (n = 3) due to protocol
violations or clinical circumstances (Fig. 3). One patient
died during the study period. The proportion of males to
females was 3:1, median age was 65 (47–74) years, and
median body mass index (BMI) 27.9 (22–38.9) kg/m2.
Other patient characteristics and nutritional therapy are
summarized in Table 1 and 2.
In the eight patients in whom protein kinetics were
analyzed at all three time points, the amino acid infusion
resulted in improved net protein balance over time
(p = 0.0016). Protein balance increased between 0 and
3 h (p = 0.01) and remained unaltered between 3 and
24 h (p = 1.00). A similar pattern was seen in the four
patients in whom change in protein balance could
only be measured at a single time point. The
temporal changes in individual patients are illustrated in
Fig. 4. The relative contributions from changes in
synthesis and breakdown towards the improvement in
net protein balance were not statistically significant
(p = 0.500 and p = 0.292, respectively). Protein kinetics
are summarized in Table 3.
The amino acid supplementation did not result in
altered amino acid oxidation (p = 0.147) or plasma urea
concentrations (p = 0.053). Plasma total free amino acid
concentrations increased over time (p = 0.038, Fig. 5).
In this study we investigated the effects on whole-body
protein kinetics of adding a supplemental intravenous
amino acid infusion to enteral feeding for 24 h in
critically ill patients. We found that intravenous amino acid
supplementation resulted in improved net protein
balance that was sustained unaltered from 3 h up to 24 h.
This response was not associated with increases in
protein oxidation or plasma urea concentrations, but
plasma total free amino acid concentrations increased
over time. The results after 3 h confirmed our earlier
reported results [
Our results reinforce the physiological rationale for
protein supplementation during critical illness. However,
several gaps in the knowledge remain to be clarified.
The optimal dosing of protein delivery is uncertain. Our
study employed a pragmatic protocol, meaning that
nutrition was not protocolized beyond the nutritional
guidelines of the unit and the supplementation studied.
This resulted in median amino acid delivery of 1.11 and
2.07 g/kg/day prior to and during therapy, respectively.
It is uncertain if higher or lower delivery within the
recommended intervals of different guidelines (1.2–2.5 g
protein/kg/day) would result in a similar or improved
protein balance, although pooled results from tracer
studies indicate linear dose-response correlation [
The potential patient-centered benefits of protein
supplementation and the optimal dose and timing of the
intervention require further exploration in the context
The finding that the additional intravenous dose in
our study resulted in improved net protein balance
without increasing amino acid oxidation supports its
adequacy. Our results are only partially in agreement with
those of Shaw et al., who examined protein kinetics in
patients with sepsis during total parenteral nutrition
with varying protein content [
]. They observed an
optimal protein-sparing effect at 1.5 g/kg/day, with no
improvement at higher doses. The mean amino acid dose
in our study was similar to that delivered in the largest
current RCT of supplemental intravenous amino acids
]. Although this trial was designed to study the
possible protective effect of extra protein supplementation
on kidney function, there was no difference in 90-day
mortality between groups. This may be indicative of
safety, but it must be emphasized that inclusion criteria
make the study population selective, and the limited
statistical power in this secondary outcome parameter
SOFA sequential organ failure assessment, SAPS III simplified acute physiology score III, LoS length of stay, GI gastrointestinal
*LoS on first study day
Kg denotes calculated body weight according to the formula provided in “Methods”
AA amino acid, IV intravenous
aN = 4
bN = 6
calls for caution in interpretations. Furthermore, there
was a significant increase in serum urea concentration
in the intervention group, but this did not translate to
increased RRT requirements. The numeric increase in
mean serum urea at 24 h in our study was not
statistically significant, but as a safety parameter it needs
to be explored further in future research. In another
recent study, 1.5 g/kg/day of protein as compared to
0.5 g/kg/day resulted in marginally improved protein
balance but no difference in outcomes. Both serum urea
concentration and urinary urea excretion were increased
]. This study may also be regarded as an indication of
safety, but again a specific selected patient group was
eligible for inclusion.
Beyond assessing the effects on protein balance,
physiological and kinetic studies are essential to
investigating safety aspects of therapeutic regimens. Although
the prevalent opinion in critical care nutrition is leaning
towards high protein delivery early in the course of
illness, there are limited data supporting this position [
The increased load of non-volatile acids and azotemia
associated with protein feeding are potentially harmful
in patients in the ICU, who have altered metabolism and
compromised organ function. The REDOXS trial by
All patients (n = 12)
Heyland et al. is the largest RCT of amino acid
(glutamine + alanine + glycine) supplementation to date,
indicating a trend towards greater mortality in the intervention
]. It should be noted that 13% of patients in
the treatment group had urea levels >50 mmol/L,
compared to 4% in the control arm. This result warrants
caution when administering high doses of amino acids,
and especially as an unbalanced amino acid composition,
to vulnerable patients [
]. We propose that indices of
amino acid oxidation, plasma urea and free amino acid
concentrations should be routinely monitored as an
adjunct to other measures of outcomes in future clinical
trials or observational studies on protein delivery. This
would provide important information regarding safety
and a physiological framework to explain treatment
effects. In our study we did not observe any significant
increases in amino acid oxidation or ureagenesis. This
may be due to the small sample size, but our data
provide some basis for power calculations to determine the
appropriate recruitment targets for monitoring these
parameters in future studies.
It is important to stress that the single-pool model we
used provides no information on where the changes in
synthesis and breakdown take place and which proteins
are synthesized and broken down. Studying the protein
balance of individual organs or the immune system in
the critically ill is technically demanding. In a study of
15 patients in the ICU, Essen et al. found that protein
synthesis rate was upregulated in peripheral lymphocytes
and liver (albumin synthesis), but on average was
unaffected in muscle, as compared to healthy subjects [
Several investigators have also used the arteriovenous
balance technique to demonstrate negative protein
balance in the leg muscle in patients with sepsis or burns
]. These findings support the theory that skeletal
muscle acts as a protein reservoir during critical illness,
providing indispensable host functions with necessary
]. The effects of protein supplementation
on regional synthesis and breakdown are yet to be
characterized. The mean phenylalanine balance observed at
Table 3 Protein kinetics (phenylalanine)
BL–3 h–24 h (n = 8)
3 h and 24 h in our study corresponds to net synthesis
of approximately 45–55 g protein/day (containing 4%
phenylalanine), in a patient weighing 75 kg. Assuming a
protein content of 20%, this would equate to 250 g of
lean body mass. Given the biological implausibility of
patients gaining muscle mass during critical illness, any
increases in net synthesis from protein feeding must
occur mainly elsewhere. On the other hand, the loss of
muscle protein during critical illness is mainly the result
of increased breakdown, and inhibition of breakdown
due to protein feeding might prevent larger losses.
Simultaneous quantification of regional protein balance in
skeletal muscle and whole-body kinetics in response to
amino acid supplementation would be a logical next step
towards elucidating the response to protein feeding in
critically ill patients.
Our study has several strengths. Measuring protein
kinetics during enteral feeding in adult patients in the
ICU has, to our knowledge, so far only been performed
by our research group. As we used both enteral and
parenteral tracers to determine protein kinetics, no changes
were made to any ongoing nutritional therapy. Patients
were primarily fed by the enteral route in accordance
with international guidelines. The repeated
quantification of protein balance in patients in the ICU is also
novel. Although the stable isotope tracer method only
provides a snapshot of whole-body protein metabolism,
the repeatability of measurements between 3 h and 24 h
demonstrates that this technique can be used to assess the
physiological response to therapeutic interventions by
repeated measures. Related work exploring the effects of
increased protein intake as part of parenteral nutrition in
an ICU setting with stable isotope tracer methodology has
been performed in preterm neonates, infants undergoing
cardiac surgery, infants with bronchiolitis and adolescents
with sepsis [
]. The fundamental differences in the
metabolism of children preclude any extrapolations of
these results to an adult population.
Limitations of our study include the small sample size
and heterogeneity in age, time course of the ICU stay
and anthropometric characteristics of patients. Despite
these differences the trend in the change in protein
balance over time was similar in all patients, regardless of
baseline conditions (Fig. 4). The non-randomized
design, non-standardized nutrition and the fact that not
all patients were eligible for analysis at three time
points due to protocol violations also limit
generalizability of the results. All 12 patients were
analyzed on a minimum of two occasions, and the results
of partial analyses were in agreement with those of
patients who completed the full protocol. There are also
inherent limitations in the stable isotope tracer
technique and single-pool model used to determine
wholebody protein kinetics. It is possible that synthesis and
oxidation rates are underestimated because the
technique only allows measurements from plasma
precursors of protein synthesis, neglecting intracellular amino
acid recycling or accumulation. The choice of tracer
may influence the measured rates of synthesis,
breakdown and oxidation depending on the metabolism of
different amino acids. As demonstrated in earlier work
by our group [
], simultaneous quantification of
protein balance with leucine and phenylalanine tracers
in critically ill patients results in different rates of
synthesis and breakdown. Absolute values in the
magnitude of change in protein balance should therefore be
interpreted with caution. However, as net changes with
different tracers are clearly correlated, we believe the
trend towards improved protein balance seen in this
study is independent of the choice of tracer. Also, the
fact that we measure whole-body protein kinetics does
not allow us to determine which proteins/tissues
represent the more positive net balance. Despite these
limitations we consider the methodology used to be the
best available technique to quantify protein kinetics under
relevant clinical circumstances. Another limitation is that
our study only concerns the effects of intravenous amino
acid supplementation. As the route of protein delivery
may have importance for the physiological and therapeutic
effects during critical illness, future studies should
consider investigating protein kinetics during exclusively
We demonstrated that a supplemental intravenous
infusion of amino acids in addition to routine nutrition,
mainly by the enteral route, sustained improved protein
balance from 3 h up to 24 h in critically ill patients.
These findings reinforce the potential importance of
protein supplementation during critical illness. Given
the unique properties of protein kinetic studies to
investigate physiological effects and safety endpoints of
protein delivery during critical illness, we advocate this
methodology to guide the design of future research.
AA: Amino acid; ANOVA: Analysis of variance; BL: Baseline; ICU: Intensive care
unit; IV: Intravenous; Phe: Phenylalanine; Ra: Rate of appearance; RCT: Randomized
controlled trial; Rd: Rate of disappearance; RRT: Renal replacement therapy; SAPS
III: Simplified acute physiology score; SD: Standard deviation; SOFA: Sequential
assessment of organ failure
We would like to express our deepest gratitude to the following co-workers:
Kristina Kilsand and Sara Rydén for performing the clinical studies and Eva
Nejman-Skoog, Towe Jakobsson and Christina Hebert for performing the
This study was funded by a grant from the Stockholm County Council and
the Swedish Research Council.
Availability of data and materials
The datasets used and analyzed during the current study are available from
the corresponding author on request.
MSR, FL, IT, OR and JW designed the study. MSR and IT participated in the
clinical studies. OR was responsible for laboratory analyses and data handling.
MSR, OR, JW and ÅN analyzed the data. MSR, ÅN and OR performed the statistical
analysis. MSR drafted the manuscript. FL, IT, OR, ÅN and JW contributed with
critical revisions to the manuscript. All authors have read and approve of the final
version of the manuscript.
Ethics approval and consent to participate
Informed consent was obtained directly from the patients or in the case where
a patient could not communicate her or his intentions, the closest relative.
Information was provided both in writing and orally, underscoring that the
patient could withdraw from the study at any time. The study was approved by
the regional ethics committee in Stockholm county (Dnr 2015/0048-32).
JW and OR are giving paid lectures on the subject of protein nutrition in the
ICU for Nestlé, Nutricia and Fresenius Kabi. The remaining authors declare
that they have no competing interests.
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