Comprehensive metabolic amino acid flux analysis in critically ill patients

Abstract

Amino acid (AA) metabolism is severely disturbed in critically ill ICU patients. To be able to make a more scientifically based decision of when and in which protein and AA composition to deliver in ICU, comprehensive AA phenotyping with measurements of plasma concentrations and whole body production (WBP) is needed. Therefore, we studied ICU patients and matched control subjects using a novel pulse isotope method to obtain in-depth metabolic analysis.

In 51 critically ill ICU patients (SOFA~6.6) and 49 healthy controls, we measured REE and body composition/phase-angle using BIA. In the postabsorptive state, we collected arterial(ized) blood for CRP and AA. Then, we administered an 8 ml solution containing 18 stable AA tracers as a pulse and calculated WBP. Enrichments: LC-MS/MS and statistics: t-test, ANCOVA.

Critically ill ICU patients had lower phase-angle (p<0.00001), and higher CRP (p<0.0001). Most AA concentrations were lower in ICU patients (p<0.0001), except tau-methylhistidine and phenylalanine. WBP of most AA were significantly (p<0.0001) higher with increases in glutamate (160%), glutamine (46%), and essential AA. Remarkably, net protein breakdown was lower. There were only weak relationships between AA concentrations and WBP.

Critically ill ICU patients (SOFA 8–16) had lower values for phase angle (p=0.0005) and small reductions of most plasma AA concentrations, but higher tau-methylhistidine (p=0.0223) and hydroxyproline (p=0.0028). Remarkably, the WBP of glutamate and glutamine were lower (p<0.05), as was theirclearance, but WBP of tau-methylhistidine (p=0.0215) and hydroxyproline (p=0.0028) were higher. Our study in critically ill ICU patients shows that comprehensive metabolic phenotyping was able to reveal severe disturbances in specific AA pathways, in a disease severity dependent way. This information may guide improving nutritional compositions to improve the health of the critically ill patient.

Introduction

Many studies have reported metabolic perturbations in critically ill patients, (1) mostly focussing on amino acid metabolism as muscle protein degradation increases the release of amino acids from muscle. After initially mainly measuring changes in the plasma concentration (2–5), we (6–10) and others (11–16) have used stable tracer technology to study the production of specific amino acids in ICU patients, as it became apparent that plasma concentration changes do not fully represent alterations in actual production and disposal of amino acids (1). This research subsequently has been used to develop scientifically-based nutritional approaches. For instance, the observation that plasma glutamine concentrations are low in ICU patients has stimulated research that studied the effects of glutamine enriched parenteral nutrition (17–19) or glutamine pharmacological intervention (20). However, the lack of more in-depth information about the specific alterations in the glutamine-related pathways could potentially explain why this nutritional intervention has not improved clinical outcomes evenly in all trials (21), despite having shown a significant impact on infectious complications and length of mechanical ventilation (22).

The loss of body and muscle mass has been related to increased protein breakdown and reduced protein synthesis (23, 24). Breakdown of body protein to amino acids will affect the disposal and production of almost all amino acids as most of the amino acids are part of body protein or are metabolites of those amino acids. However, this has never been measured in individual subjects, as well as the calculation of the clearance of individual amino acids without the need to parenterally infuse large amounts of amino acids (4, 25, 26).

To achieve an integrative and comprehensive view of amino acid metabolism in critically ill patients, we applied our metabolic phenotyping approach (27, 28), using a pulse of a composition of many stable isotope tracers of amino acids, in a group of 51 ICU patients as compared to 49 healthy-matched subjects. In addition, we stratified the ICU group into moderately and severely ill patients to examine the effects of disease severity. The ultimate aim is to identify changes in amino acid metabolism that might help to design improved clinical nutrition approaches in critically ill patients.

Material and methods

Subject inclusion

Fifty-one critically ill ICU patients and 49 healthy controls matched for age, gender, and BMI participated in the study (Table 1a). Critically ill patients were admitted to the ICU between September 2018 and October 2019. Two ICU sites were used for enrollment. Thirty-seven subjects were recruited from CHUV (Lausanne University Hospital,Service of Intensive Care Medicine & Burns, Lausanne, Switzerland) and 14 patients from RMC (Tel Aviv University -Rabin Medical Center, Israel) (Table 1b). ICU patients were studied 4 days (Lausanne) and 14 days (Tel Aviv) after admission.

Table 1a-.

General and clinical characteristics of the healthy and critically ill populations

	Healthy (n=49)	ICU (n=51)	Pvalue
Age (years)	53.4 [47.6, 59.8]	60.1 [54.7, 66.1]	0.107
Sex (male/female)	27/22	36/15	0.147
Body Weight (kg)	87.8 [81.8, 94.2]	82.7 [78.1, 87.6]	0.191
BMI	30.5 [28.6, 32.4]	28.9 [27.4, 30.5]	0.193
Fat-free Mass (kg)	55.7 [51.7, 59.9]	61.8 [58.1, 65.8]	0.03039
Fat Mass (kg)	29.2 [25.0, 34.2]	14.2 [11.2, 18.2]	<0.00001
Intracellular H2O (L)	20.9 [19.2, 22.8]	27.5 [25.7, 29.4]	0.00001
Extracellular H2O (L)	19.1 [17.8, 20.5]	19.2 [17.8, 20.8]	0.921
Phase Angle	5.99 [5.67, 6.32]	3.74 [3.46, 4.04]]	<0.00001
(hs)CRP (mg/L)	1.17 [0.82, 1.67]	148.7 [122.9, 180.0]	<0.00001
Glucose (mM)	5.27 [4.94, 5.62]	7.45 [7.00, 7.92]	<0.00001
REE (kcal/day)	1922 [1697, 2147]	1802 [1679, 1924]	0.406
Blood Pressure Systole (mm Hg)	131.2 [126.6, 135.9]	116.8 [111.4, 122.4]	0.00017
Blood Pressure Diastole (mm Hg)	78.7 [75.8, 81.6]	57.7 [55.0, 60.4]	<0.00001
Heart Rate (beats/min)	67.5 [64.6, 70.6]	87.1 [81.7, 92.8]	<0.00001
SOFA Score	-	6.58 [5.64, 7.67]	-
APACHE 2 Score	-	20.42 [18.48, 22.56]	-
SAPS2 Score	-	46.0 [42.1, 50.3]	-

Values are geometric mean [95% CI] except for Sex. CRP: C-reactive protein. Statistics are done on logtransformed data by unpaired t-test (except sex: Mann-Whitney), bold is p<0.05 and red is after log transformation.

Table 1b -.

General and clinical characteristics of the two centers that contributed to the critically ill populations

	CHUV	RMC	P-value
# of subjects	37	14	-
Age	62.9 [57.1, 69.4]	53.4 [41.9, 68.0]	0.122
Sex (male/female)	28/9	8/6	0.301
Body Weight (kg)	83.1 [77.9, 88.7]	81.7 [71.6, 93.2]]	0.786
BMI (kg/m2)	29.3 [27.6, 31.0]	27.9 [24.3, 32.1]	0.451
APACHE 2 Score	21.6 [19.4, 24.0]	17.7 [14.0, 22.4]	0.075
On Mechanical ventilation	37/37	12/14	0.0714-
SOFA score on study day	7.66 [6.58, 8.93]	4.39 [3.15, 6.11]	0.00071
Time of study measurement after admission to the ICU	4	14

Values are geometric mean [95% CI] except for Sex. CHUV = Lausanne University Hospital (Service of Intensive Care Medicine & Burns, Lausanne, Switzerland), RMC = Tel Aviv University - Rabin Medical Center, Israel). Statistics are when lognormal done on logtransformed data by unpaired t-test (except sex and “on Mechanical Ventilation”: Mann-Whitney test), bold is p<0.05 and red is after log transformation.

The healthy subjects were recruited via advertisements in the local community. Medical history and medication use were assessed as part of the screening process. Exclusion criteria for healthy controls were metabolic diseases including hepatic and renal disorders; presence of acute illness or metabolically unstable chronic illness like CHF, CRF, COPD or diabetes mellitus; an established diagnosis of malignancy; the presence of fever within the last 3 days prior to the study day; pregnancy; use of oral corticosteroids (<4 weeks); use of protein or amino acid containing nutritional supplements; and participation in a weight-management program within 6 months prior to study day. Written informed consent was obtained from all participants or legal representatives before performing any study-related procedures. The study was approved by the local Institutional Review Boards. We report here data from baseline measurements of 2 intervention trials (NCT02770092, NCT03628365) and from the RMC study.

Study design

The healthy subjects were studied at the Clinical Research Unit of the Center for Translational Research in Aging and Longevity, at Texas A&M University. The ICU patients were studied at their respective locations. The severity of the illness on admission was quantified by the APACHE 2 and SAPS2 scores, while the Sequential Organ Failure Assessment (SOFA) score was used to assess organ failure on study days (29). As the SOFA score is an indicator of the level of organ failure in critically ill patients (29, 30), the ICU patients were also stratified on the basis of this score (SOFA-moderate 1–7 and SOFA-high 8–16) to observe whether the metabolic changes are related to the severity of the illness.

All healthy subjects were studied in the postabsorptive condition (after overnight fast) and the critically ill patients were studied when enteral nutrition was stopped for 6 h. No subjects have received parenteral nutrition.

Anthropometrics and body composition

In the ICU patients, body weight was measured using the Hill-Rom and Linet Bed scales and height was predicted from ulna length. Body composition was determined by multi frequency bioelectrical impedance analyzer (InBody S10®, InBody Corp., Seoul, South Korea, (31) or BodyStat, UK (32)).

In the healthy subjects, body weight and height were measured on a Health-O-Meter Professional 500KL (Pelstar LLC, Alsip, IL, USA) self-calibrating digital scale (±0.02 kg) and stadiometer, respectively and body composition using the model SFB7 multi frequency bioelectrical impedance analyzer (ImpediMed, San Diego, CA, USA).

Phase angle was calculated as the arctangent of the reactance to resistance ratio measured at 50kHz (33).

Resting Energy Expenditure (REE)

REE was measured in the critically ill ICU patients using indirect calorimetry measurements (34) only if patients were in steady state, including hemodynamic and respiratory stability without changing any therapy (vasopressors dosage or ventilator setting) one hour before and during the measurement. Duration of the indirect calorimetry measurement was between 20 and 30 min, during which a steady state condition, defined as coefficient of variation of VO2 and VCO2 was less than 5% for a 5 min measurement or less than 10% for a 25 min measurement. In mechanically ventilated ICU patients, REE was measured with the Q-NRG (Cosmed, Rome, Italy).

In the healthy group, ParvoMedics TrueMax 2400 Metabolic Measurement System (Parvomedics Inc, Sandy, UT, USA) was used after calibration with a series 5530 three-liter syringe (Hans Rudolph Inc., Kansas City, MO, USA). Participants were in a supine position and instructed to stay awake for at least 20 minutes. After 10 minutes, the last consecutive time points were used to calculate the average value.

Protein metabolism by stable isotope IV pulse

In the ICU patients, blood was collected from an arterial catheter. In healthy subjects, after an overnight fast, one peripheral line was placed in a superficial dorsal vein of the non-dominant hand for blood sampling. This hand was placed in a thermostatically controlled hot box (internal temperature: 60°C), a technique to mimic direct arterial sampling (35). After collecting a blood sample for amino acid concentration and baseline enrichment analysis (t=0 min), the amino acid mixture (bolus) of stable isotopes (Cambridge Isotope Laboratories (Woburn, MA, USA) was given within 10 seconds. The composition of the pulse tracer solution was identical in both groups (Table 2). Arterialized-venous or arterial blood was sampled at t= 10, 20, 30, 60, 120 minutes for analysis of tracer enrichments.

Table 2 -.

Composition of the infused stable tracers

	Amount of tracer (mg)
L-Arginine [guanidino-15N2]	22.73
L-Citrulline [5-13C;4,4,5,5-2H2]	2.30
L-Glutamate [1,2,-13C2]	9.17
L-Glutamine [15N2]	7.47
Glycine [2,2-2H2]	12.02
L-Histidine [15N3]	1.04
L-Hydroxyproline [2,5,5-2H3]	0.75
L-Isoleucine [1-13C]	4.96
L-Leucine [13C6]	5.04
L-Methionine [1-13C]	2.14
L-Methionine [methyl-D3]	2.17
L-Ornithine [13C5]	1.43
L-Phenylalanine [ring-13C6]	13.10
tau-Methyl-L-Histidine [methyl-2H3]	0.49
L-Taurine [1,2,-13C2]	3.43
L-Tryptophan [indole-2H5]	4.87
L-Tyrosine [ring-D4]	1.19
L-Valine [13C5]	3.96

Volume is about 8 mL, made isotonic with NaCl.

Biochemical analysis and calculations of metabolic parameters

All blood samples were put in Li-heparinized or EDTA tubes, immediately put on ice, and centrifuged (4°C, 3120 × g for 5 min) to obtain plasma. Plasma was aliquoted with 0.1 vol of 33% (w/w) trichloroacetic acid and stored at −80°C. Tracer enrichments [tracer:tracee ratio (TTR)] and amino acid concentrations were analyzed batch-wise by LC-MS/MS by isotope dilution (27).

The sum of branched-chain amino acids (BCAA) is the sum of valine, leucine, and isoleucine. The sum of the essential amino acids (EAA) represents the sum of the concentration of all essential amino acids (isoleucine, leucine, valine, histidine, lysine, methionine, phenylalanine, threonine, and tryptophan). The sum of the non-essential amino acids (NEAA) represents the sum of the concentration of all non-essential amino acids measured (aspartate, glutamate, asparagine, glutamine, serine, glycine, arginine, alanine, proline, and tyrosine). The sum of the neutral amino acids (LNAA) represents the sum of the concentration of the neutral amino acids (isoleucine, leucine, valine, tyrosine, and phenylalanine). Sum of amino acids (sumAA) represents the sum of the concentration of all measured amino acids.

The area under the curve (AUC) of the change in the TTR after the pulse was calculated from t=10 to t=120 min (28) and, as the first 10–60 min contribute most to the AUC, the TTR in that period is relatively high and thus is measured with the lowest variation. WBP was calculated as TracerDOSE/AUC. The conversion of an amino acid into another metabolite was calculated by using the WBP of the product amino acid and the ratio between the AUC of the TTR of the product and the TTR of the substrate (36). The conversion of phenylalanine to tyrosine equals net protein breakdown.

Whole body clearance is the WBP, corrected for the plasma concentration.

Plasma glucose concentrations of baseline samples were measured using a COBAS c111 semi-automatic analyzer (Gluc2 Kit; Roche Diagnostics®). High-sensitivity C-reactive protein of baseline samples (hsCRP) was measured by a COBAS c111 (CRP HS Kit; Roche Diagnostics®) in the healthy subjects and by routine plasma C-reactive protein (CRP) in the critically ill patients.

Statistical analysis

Results are expressed as mean [95% Confidence Interval ([95% CI]). All data were checked for normality and if necessary converted logarithmically and statistical tests conducted on these data. We did not remove any possible outliers from the logarithmically converted data. Population characteristics and baseline measurements were compared using an unpaired Student’s t-test. Categorical values were compared using Mann-Whitney test. The comparison between ICU patients with a SOFA score of 1 to 7 (SOFA-moderate) and ICU patients with a SOFA score of 8 to 16 (SOFA-high) was done with the unpaired Student’s t-test and ANCOVA with sex, age, and BMI as confounders.

For TRP- and TYR-to-LNAA ratios, ANCOVA was performed with plasma concentration of TRP or TYR as the dependent and the LNAA plasma concentration as the independent variable. For calculation of clearance, ANCOVA was done with WBP as dependent and plasma concentration as independent variables and sex, BMI, and age as confounders.

For figure 6, we calculated that a correlation coefficient larger than 0.27 with 51 observations, has a p-value less than 0.05 (37).

Figure 6.

The correlation between the plasma amino acids concentration, depicted as [amino acid], and the whole body production (WBP) of the measured amino acids. The color bar on the right shows a gradual color change from dark blue (correlation=1) to white (correlation=0) to dark red (correlation= −1). A correlation coefficient larger than 0.27 indicates a p-value less than 0.05 (37).

Graphpad Prism (Version 9.0.0) was used for data analysis. The level of significance (α) was set at p<0.05 and tested two-sided.

Results

General Characterization of the subjects

We were able to match the ICU patients with healthy control subjects in relation to age, sex, body weight, and BMI (Table 1). We also compared the patients from the two locations that recruited the ICU patients. The patients from Rabin Medical Center had a lower SOFA score (4.4 vs 7.7; p=0.00071), but only showed statistically higher plasma concentrations for taurine and glutamate, and lower alanine (not shown).

Comparison of the healthy subjects with the ICU patients shows that, although there were no differences in the REE, ICU patients had lower fat-free and fat mass, intracellular water content, phase angle, and blood pressure; and higher plasma CRP, glucose concentrations, and heart rate.

The elevated APACHE 2 and SAPS2 scores reflect admission severity with a predicted mortality rate of around 20% (29, 38). The SOFA scores on study day reflect persistent major organ failures.

Comparing ICU patients and healthy controls

Plasma amino acid concentrations

The plasma concentration (Table 3) of most of the amino acids was lower in ICU patients with significant reductions in plasma glutamate (Figure 1), hydroxyproline, glutamine (Figure 2), citrulline (Figure 3), serine, glycine, arginine (Figure 4), alanine, proline, tryptophan, histidine, and lysine that were related to the reduction of the summation of all amino acids. Although concentrations of some amino acids, including leucine, valine and isoleucine, were unchanged, increased concentrations were observed for tau-methylhistidine, methionine, and phenylalanine. The ratio between tryptophan or tyrosine and the neutral amino acids was reduced.

Table 3 -.

Plasma Amino Acid concentrations and ratios of the healthy and critically ill populations

	Healthy (n=49)	ICU (n=51)	P value
Aspartate	2.0 [1.7, 2.4]	1.6 [1.4, 1.9]	0.051
Glutamate	60.5 [51.8, 70.8]	37.3 [31.6, 44.0]	0.00004
HydroxyProline	10.8 [9.0, 12.9]	6.2 [5.2, 7.3]	0.00002
Asparagine	57.6 [53.7, 61.7]	60.4 [53.7, 67.9]	0.494
Glutamine	612 [577, 648]	394 [364, 427]	<0.00001
Citrulline	30.1 [27.3, 33.1]	17.9 [15.5, 20.7]	<0.00001
Serine	74.5 [69.5, 79.9]	58.3 [52.8, 64.4]	0.00017
Glycine	232 [210, 256]	131 [118, 146]	<0.00001
Arginine	67.7 [62.7, 73.2]	47.2 [41.4, 53.9]	0.00001
Threonine	100 [92.4, 109]	96.5 [109, 85.2]]	0.622
Alanine	303 [283, 324]	151 [132, 172]	<0.00001
Taurine	37.1 [34.3, 40.1]	30.8 [25.5, 37.1]	0.069
Proline	158 [145, 172]	120 [106, 137]	0.00072
tau-Methylhistidine	3.85 [3.44, 4.31]	6.57 [5.32, 8.10]	0.00003
Valine	166 [156, 177]	155 [139, 174]	0.307
Methionine	18.9 [17.8, 20.1]	21.1 [17.7, 25.2]	0.245
Isoleucine	56.5 [52.1, 61.1]	58.9 [51.4, 67.6]	0.595
Leucine	106 [98.0, 115]	102 [89.4, 116]	0.587
Tryptophan	37.2 [34.5, 40.1]	22.4 [19.7, 25.5]	<0.00001
Phenylalanine	47.2 [44.6, 49.9]	63.2 [56.4, 70.8]	0.00002
Ornithine	49.1 [45.1, 53.5]	50.4 [43.3, 58.6]	0.770
Histidine	69.7 [65.3, 74.5]	45.0 [41.6, 48.6]	<0.00001
Lysine	166 [155, 178]	128 [115, 142]	0.00011
Tyrosine	53.7 [50.4, 57.3]	48.2 [41.7, 55.8]	0.185
BCAA	330 [308, 353]	319 [284, 358]	0.616
EAA	771 [729, 816]	710 [649, 777]	0.125
NEAA	1653 [1572, 1737]	1222 [1129, 1322]	<0.00001
LNAA	432 [406, 460]	461 [414, 512]	0.312
SumAA	2509 [2377, 2648]	1794 [1667, 1932]	<0.00001
	ICU (n=51) estimated difference with healthy group	P value	% change
TRP, LNAA corrected	−16.87 [−20.47, −13.27]	<0.0001	−44.0%
TYR, LNAA correction	−11.14 [−17.47, −4.819]	0.0007	−20.3%

Values are μM; geometric mean [95% CI] or mean (estimated differences). Statistics are done on logtransformed datae by unpaired t-test, bold is p<0.05 and red is after log transformation. For TRP and TYR/LNAA ratio, ANCOVA with TRP or TYR as dependent and LNAA plasma concentration as independent. Values (μM) are estimated difference with healthy group, mean [95% CI]; bold is p<0.05. % change is estimated difference mean difference divided by TRP or TYR LNAA of healthy group.

Figure 1.

Glutamate plasma concentration (left panel) and whole body production (right panel) in healthy subjects and critically ill patients with a moderate or high SOFA score. Individual data points and geometric mean with 95% CI are shown. Statistics are between healthy subjects and critically ill patients (plasma concentration: p=0.00004, whole body production: p<0.00001) and between critically ill patients with a moderate or high SOFA score (plasma concentration: p=0.0067, whole body production: p=0.0501).

Figure 2.

Glutamine plasma concentration (left panel) and whole body production (right panel) in healthy subjects and critically ill patients with a moderate or high SOFA score. Individual data points and geometric mean with 95% CI are shown. Statistics are between healthy subjects and critically ill patients (plasma concentration: p<0.00001, whole body production: p<0.00001) and between critically ill patients with a moderate or high SOFA score (plasma concentration: p=0.0679, whole body production: p=0.032).

Figure 3.

Citrulline plasma concentration (left panel), whole-body production (middle panel), and arginine de novo production (right panel) in healthy subjects and critically ill patients with a moderate or high SOFA score. Individual data points and geometric mean with 95% CI are shown. Statistics are between healthy subjects and critically ill patients (plasma concentration: p<0.00001, whole body production: p=0.00001, arginine de novo production: p<0.00001) and between critically ill patients with a moderate or high SOFA score (plasma concentration: p=0.0869, whole body production: p=0.0275, arginine de novo production: p=0.0001).

Figure 4.

Arginine plasma concentration (left panel) and whole-body production (right panel) in healthy subjects and critically ill patients with a moderate or high SOFA score. Individual data points and geometric mean with 95% CI are shown. Statistics are between healthy subjects and critically ill patients (plasma concentration: p=0.00001, whole body production: p=0.0083) and between critically ill patients with a moderate or high SOFA score (plasma concentration: p=0.065, whole body production: p=0.46).

Whole-body amino acid production and conversions

We have summarized the changes in the different WBP and conversions in Figure 5. In the ICU group, the whole body production (Table 4) of several amino acids were higher (arginine (Figure 4), glycine, hydroxyproline, phenylalanine, tau-methylhistidine, methionine, tyrosine, tryptophan, glutamate (Figure 1), isoleucine, glutamine (Figure 2), taurine, valine, leucine, and ornithine) while for many of these amino acids the plasma concentration was either reduced or increased or unchanged, indicating that the relationship between whole-body productions and plasma concentrations of amino acids is not strong (Figure 6). Remarkably, the whole body production was significantly lower for citrulline (Figure 3). The whole body production of the sum of leucine, valine, and isoleucine and of essential amino acids is increased by 50–60%, indicative of a massive increase of the whole-body protein breakdown rate. The largest absolute increase of the whole body production was for glutamate (96 mmol/hour; 260%).

Figure 5.

Summary of the whole body production and conversions of the healthy subjects and the critically ill patients in % difference from healthy subjects. Data are geometric mean with 95% CI (see Table 4).

Table 4 -.

Whole Body Production of the healthy and critically ill populations

	Healthy (n=49)	ICU (n=51)	P value
Arginine	9709 [8926, 10561]	11501 [10473, 12630]	0.00830
Glycine	17485 [15858, 19278]	21405 [19344, 23465]	0.02377
Hydroxyproline	508 [424, 609]	357 [306, 417]	0.00373
Citrulline	1157 [1067, 1255]	795 [695, 910]	0.00001
Phenylalanine	4195 [3814, 4614]	5994 [5573, 6446]	<0.00001
tau-methylhistidine	78.37 [70.20, 87.48]	123.5 [100.7, 151.6]	0.00018
Methionine	2100 [1917, 2301]	2654 [2325, 3028]	0.00439
Tyrosine	3995 [3588, 4449]	5134 [4714, 5591]	0.00036
Tryptophan	913 [791, 1054]	1957 [1813, 2114]	<0.00001
Glutamate	58982 [51406, 67675]	155375 [133442, 180913]	<0.00001
Isoleucine	3034 [2600, 3540]	5429 [4825, 6110]	<0.00001
Histidine	4815 [4434, 5229]	4394 [4003, 4824]	0.144
Glutamine	37845 [34470, 41551]	55241 [50908, 59943]	<0.00001
Taurine	2171 [1877, 2510]	3328 [2949, 3757]	0.000038
Valine	10507 [9474, 11652]	16922 [15780, 18123]	<0.00001
Leucine	10604 [9622, 11685]	17148 [16010, 18367]	<0.00001
Ornithine	2004 [1833, 2191]	3016 [2698, 3371]	<0.00001
Essential Amino Acids	36631 [33534, 40013]	55754 [52199, 59550]	<0.00001
Branched-Chain Amino Acids	24575 [22410, 26949]	39925 [37399, 42621]	<0.00001
Glutamate>>Glutamine	16965 [13666, 21061]	52915 [44302, 63202]	<0.00001
Citrulline ➨ Arginine	1160 [1034, 1302]	609 [507, 732]]	<0.00001
Arginine ➨ Citrulline	198 [120, 325]	88.1 [71.7, 108]	0.00258
Arginine ➨ Ornithine	1376 [959, 1975]	3765 [3295, 4301]	<0.00001
Ornithine ➨ Citrulline	124 [83.1, 184]	83.0 [65.8, 105]	0.120
Citrulline ➨ Ornithine	164 [116, 233]	203 [165, 250]	0.295
Phenylalanine ➨ Tyrosine	369 [318, 427]	200 [161, 247]	<0.00001

Values are μmol/hour; geometric mean [95% CI]. Statistics are on logtransformed data by unpaired t-test, bold is p<0.05.

By observing the conversion of the pulsed stable tracers of amino acids into their products of several pathways, we were able to calculate these conversions (Table 4). We found a 311% increase in the conversion of glutamate to glutamine. The conversions in the arginine, ornithine, and citrulline pathways show a substantial reduction in the citrulline to arginine conversion (arginine de novo production; Figure 3), the arginine to citrulline conversion (NO production), and the ornithine to citrulline conversion. In contrast, the arginine to ornithine conversion was elevated, indicating that the urea production increased substantially. The 33% reduction of the phenylalanine to tyrosine conversion (Table 4), a direct estimation of net whole-body protein breakdown, was observed in relation to the large increase of the appearance of amino acids, including phenylalanine and tyrosine. Whole-body clearance The calculation of the clearance of amino acids (Table 5) can be done when both the plasma concentration (Table 3) and the whole body production (Table 4) data are available. We found that the clearance of several amino acids was increased but not for hydroxyproline, citrulline, tau-methylhistidine, and histidine.

Table 5 -.

Whole Body Clearance estimates critically ill population, compared to the healthy controls

	ICU (n=51) estimated difference from healthy group	P value	% change
Arginine	3029 [1699, 4360]	<0.0001	34.0%
Glycine	5353 [3107, 7598]	<0.0001	32.4%
Hydroxyproline	92.8 [−33.1, 218.6]	0.1467	-
Citrulline	1.167 [−130, 132]	0.9859	-
Phenylalanine	1736 [1268, 2204]	<0.0001	44.2%
tau-methylhistidine	1.75 [−6.11, 9.60]	0.6599	-
Methionine	771.7 [470.1, 1073]	<0.0001	38.9%
Tyrosine	1320 [889, 1750]	<0.0001	34.1%
Tryptophan	1392 [1182, 1601]	<0.0001	137.6%
Glutamate	120867 [98184, 143551]	<0.0001	102.9%
Isoleucine	2334 [1447, 3222]	<0.0001	72.1%
Histidine	264 [−336, 864]	0.3843	-
Glutamine	21018 [15811, 26224]	<0.0001	60.0%
Taurine	1571 [807, 2335]	0.0001	75.2%
Valine	6944 [5911, 7978]	<0.0001	68.2%
Leucine	6956 [5865, 8047]	<0.0001	69.3%
Ornithine	1034 [770, 1298]	<0.0001	55.5%

Statistics are on logtransformed data. ANCOVA with WBP as dependent and plasma concentration as independent and confounders sex, bmi and age. Values (μmol/hour) are estimated difference with healthy group, mean [95% CI]; bold is p<0.05. % change is estimated difference mean divided by WBP mean of healthy group (Table 4).

Comparison of whole-body productions and plasma concentrations of amino acids in ICU patients

We compared the correlation between the plasma concentrations of the amino acids and their whole body productions in the ICU patients (Figure 6). The concentrations of plasma amino acids of the different pathways correlated to each other (e.g. leucine, isoleucine, valine), while concentrations of plasma amino acids from different pathways correlated less well. For instance, the plasma taurine and tau-methylhistidine concentrations correlated with only a few amino acids. We calculated that a correlation coefficient larger than 0.27 with 51 observations, has a p-value less than 0.05 (37). The correlation between the whole body productions of the different amino acids was diverse, indicating that several amino acids have a clearly different response in severe illness. In contrast, the relationships between the plasma amino acids and their whole body productions were very weak (right upper quadrant). The only highly positive correlations were for amino acids that have different disposal pathways like urinary excretion (tau-methylhistidine and hydroxyproline) (39–41), renal metabolism (citrulline) (1), and the negative correlation between glutamate whole body production and several amino acids.

Comparing ICU patients with moderate and high SOFA score (Table 6)

Table 6 -.

Whole Body Metabolism of moderate and several ill critically ill populations

	SOFA 1–7 (n=31)	SOFA 8–16 (n=22)	p (t-test)	p (ANCOVA)
Age (years)	59.1 [52.1, 66.9]	61.4 [52.8, 71.4]	0.687
Sex (male/female)	20/11	18/4	0.222
Body Weight (kg)	80.5 [74.5, 87.0]	87.4 [80.3, 95.2]	0.155
BMI	28.2 [26.1, 30.6]	30.1 [28.1, 32.2]]	0.252
Fat-free Mass (kg)	59.8 [55.3, 64.6]	66.5 [59.5, 74.4]	0.097
Fat Mass (kg)	14.5 [10.2, 20.7]	13.9 [10.0, 19.1]	0.832
Intracellular H2O (L)	26.1 [23.7, 28.7]	29.1 [26.4, 32.1]	0.100	0.151
Extracellular H2O (L)	17.7 [16.1, 19.5]	21.0 [18.7, 23.5]	0.021	0.021
Phase Angle	4.10 [3.69, 4.55]	3.29 [3.00, 3.62]	0.004	0.0005
CRP (mg/L)	144.7 [109.9, 190.5]	153.0 [114.1, 205.2]	0.770	0.802
Glucose (mM)	7.36 [6.78, 8.00]	7.53 [6.80, 8.35]	0.712	0.725
REE (kcal/day)	1822 [1678, 1979]	1663 [1480, 1869]	0.184	0.025
Blood Pressure Systole (mm Hg)	121 [115, 127]	111 [101, 121]	0.050	0.078
Blood Pressure Diastole (mm Hg)	56.7 [53.6, 60.0]	59.2 [54.4, 64.4] [54.2, 64.5]	0.359	0.162
Heart Rate (beats/min)	82.4 [75.7, 89.6]	93.7 [85.5, 102]	0.042	0.004
Plasma Amino Acids
Aspartate	1.9 [1.6, 2.3]	1.3 [1.0, 1.7]	00.023	0.0562
Glutamate	45.4 [37.6, 54.7]	28.1 [21.7, 36.5]	0.003	0.0067
Hydroxyproline	5.18 [4.43, 6.06]	8.41 [6.06, 11.7]	0.004	0.0028
Asparagine	58.0 [50.1, 67.1]	65.6 [53.6, 80.3]	0.304	0.6710
Glutamine	414 [375, 457]	366 [321, 418]	0.124	0.0679
Citrulline	19.4 [16.6, 22.7]	16.6 [12.5, 22.2]	0.298	0.0869
Serine	62.2 [55.0, 70.4]	54.9 [45.8, 65.7]	0.223	0.0655
Glycine	132 [115, 151]	136 [112, 166]	0.783	0.9020
Arginine	51.1 [43.2, 60.6]	42.9 [34.5, 53.2]	0.188	0.0647
Threonine	102 [87.2, 120]	90.7 [73.4, 112]	0.348	0.1498
Alanine	132.6 [110, 159]	184 [158, 215]	0.012	0.0220
Taurine	34.8 [27.7, 43.8]	26.5 [19.1, 36.7]]	0.149	0.1470
Proline	117.8 [101, 137]	130 [100, 169]	0.460	0.7419
tau-methyl histidine	5.18 [3.98, 6.73]	10.2 [7.37, 14.0]	0.002	0.0215
Valine	177 [157, 198]	128 [106, 156]	0.003	0.0011
Methionine	19.7 [15.8, 24.6]	23.7 [17.5, 32.3]	0.303	0.578
Isoleucine	65.8 [58.2, 74.3]	49.8 [37.6, 66.0]	0.041	0.0152
Leucine	112 [97.2, 129]	88.0 [69.2, 112]]	0.063	0.0201
Tryptophan	24.0 [21.1, 27.3]	19.8 [15.3, 25.6]	0.129	0.084
Phenylalanine	61.0 [53.1, 70.0]	70.0 [56.3, 87.1]	0.247	0.545
Ornithine	52.0 [43.5, 62.2]	49.1 [37.0, 65.1]	0.706	0.544
Histidine	42.3 [38.7, 46.1]	49.5 [43.2, 56.8]	0.040	0.0877
Lysine	126 [110, 145]	131 [109, 157]	0.756	0.955
Tyrosine	48.0 [40.8, 56.3]	49.6 [37.4, 65.8]	0.819	0.667
BCAA	356 [315, 401]	270 [218, 334]	0.015	0.0043
EAA	736 [663, 816]	679 [574, 802]	0.379	0.160
NEAA	1228 [1110, 1358]	1241 [1079, 1428]	0.894	0.752
LNAA	494 [440, 554]	420 [343, 513]	0.126	0.0337
SumAA	1816 [1656, 1991]	1793 [1572, 2045]	0.869	0.441
	ICU (n=51) estimated difference with healthy group	P value	% change
TRP, corrected for LNAA	−1.197 [−4.623 to 2.229]	0.4854	-
TYR, corrected for LNAA	12.69 [2.233 to 23.14]	0.0185	24.1%
Whole body production and conversions
Arginine	11596 [10242, 13128]	11422 [9852, 13243]	0.873	0.4582
Glycine	21893 [19437, 24660]	18422 [16211, 20933]]	0.053	0.0266
Hydroxyproline	314 [266, 370]	450 [335, 606]	0.021	0.0155
Citrulline	880 [780, 993]	712 [531, 955]	0.125	0.0275
Phenylalanine	5965 [5425, 6560]	6228 [5447, 7121]	0.580	0.9582
tau-methyl histidine	98.8 [78.3, 125]	195 [134, 286]	0.002	0.0244
Methionine	2650 [2243, 3131]	2646 [2114, 3312]	0.990	0.7562
Tyrosine	5084 [4582, 5641]	5309 [4546, 6199]	0.621	0.9950
Tryptophan	2058 [1861, 2277]	1800 [1604, 2020]	0.082	0.0800
Glutamate	175317 [146233, 210185]	131683 [101450, 170926]	0.061	0.0501
Isoleucine	5746 [4943, 6678]	4972 [4109, 6017]	0.223	0.2268
Histidine	4425 [3923, 4992]	4435 [3784, 5197]	0.982	0.9050
Glutamine	59549 [54404, 65180]	49636 [42979, 57323]	0.024	0.0320
Taurine	3361 [2809, 4022]	3296 [2748, 3952]	0.871	0.8506
Valine	17361 [15904, 18952]	16510 [14698, 18546]	0.473	0.1692
Leucine	17341 [15824, 19003]	17022 [15267, 18978]	0.790	0.5994
Ornithine	3167 [2791, 3594]	2938 [2334, 3698]	0.526	0.2871
Essential Amino Acids	56420 [51785, 61470]	55571 [49714, 62119]	0.822	0.4464
Branched-Chain Amino Acids	40881 [37598, 44450]	38864 [34843, 43350]	0.447	0.2235
Glutamate ➨ Glutamine	60670 [49503, 74356]	43849 [32001, 60082]	0.067	0.0860
Citrulline ➨ Arginine	785 [678, 909]	423 [298, 602]	0.0004	0.0001
Arginine ➨ Citrulline	92.5 [70.5, 121]	83.3 [60.3, 115]	0.611	0.8116
Arginine ➨ Ornithine	3926 [3321, 4641]	3558 [2833, 4468]	0.486	0.3184
Ornithine ➨ Citrulline	86.9 [59.7, 126]	78.8 [57.7, 108]]	0.675	0.6795
Citrulline ➨ Ornithine	266 [213, 331]	133 [94.8, 188]	0.0006	0.0004
Phenylalanine ➨ Tyrosine	263 [214, 324]	138 [93.6, 204]	0.0020	0.008
Whole Body Clearance
	SOFA 8–16 estimated difference with SOFA 1–7 group		P value	% change
Arginine	−529 [−2732, 1674]		0.6312	-
Glycine	−4183 [−7677, −689]		0.0201	−18.1%
Hydroxyproline	−64.3 [−145, 15.9]		0.1135	-
Citrulline	−22.8 [−161, 115]		0.7408	-
Phenylalanine	81.7 [−582, 745]		0.8054	-
tau-methyl histidine	−12.8 [−20.8, −4.91]		0.0021	−10.4%
Methionine	−191 [−1057, 675]		0.6581
Tyrosine	−41.0 [−806, 724]		0.9145
Tryptophan	−113 [−324, 98.6]		0.2883	-
Glutamate	−36258 [−87057, 14542]		0.1574	-
Isoleucine	−591 [−2075, 894]		0.4269	-
Histidine	−217 [−945, 512]		0.5527	-
Glutamine	−8490 [−15315, −1665]		0.0160	−14.0%
Taurine	−89.2 [−1033, 854]		0.8482	-
Valine	193 [−1506, 1892]		0.8201	-
Leucine	155 [−1722, 2031]		0.8689	-
Ornithine	−251 [−908, 407]		0.4465	-

Values are geometric mean [95% CI] or mean (estimated differences), plasma concentration in μM, whole body production and conversions in μmol/h. Statistics are on logtransformed data by unpaired t-test (p ttest) (except sex: Mann-Whitney test) or ANCOVA (p ANCOVA) when data are corrected for confounders sex, age and BMI. For clearance calculation, ANCOVA with WBP as dependent and plasma concentration as independent. Values (μmol/hour) are estimated differences with SOFA 1–7 group. % change is estimated difference mean divided by WBP mean of the SOFA 1–7 group. Bold is p<0.05.

There was no significant difference in the patients’ general characteristics when the critically ill patients were stratified on the basis of this score (SOFA-moderate 1–7 and SOFA-high 8–16), except for a higher extracellular water content, and heart rate and a lower phase angle and REE.

Plasma Amino Acids

We found that only for a few amino acids did the plasma concentration differ between patients with a moderate and high SOFA score with a lower plasma glutamate, leucine, isoleucine and valine concentration in patients with a high SOFA score. We found higher plasma concentrations of hydroxyproline, alanine, tau-methylhistidine, and LNAA. In addition, an increase in the ratio between tyrosine and LNAA was found.

Whole-body production and conversions

Whole-body production differences were characterized by smaller differences between patients with a moderate and high SOFA score. Higher whole body productions in patients with a high SOFA score were observed for hydroxyproline and tau-methylhistidine and lower whole-body productions of glycine, citrulline, and glutamine.

The conversion of glutamate to glutamine was not different, while in the arginine-ornithine-citrulline pathway, a reduction of citrulline to ornithine conversion and further reduction in citrulline to arginine were observed in patients with a high SOFA score.

Also, a further reduction in the phenylalanine to tyrosine conversion was observed indicating a further reduction of the net whole-body protein breakdown.

Whole-body clearance

We only observed that the whole body clearance was significantly reduced for glycine, tau-methylhistidine, and glutamine.

Discussion

The present study confirms previous observations in critically ill patients regarding changes in plasma concentrations and whole body production and conversions of some specific amino acids. We added new observations in whole body productions and conversions that were only possible due to our innovative stable tracer pulse approach. By simultaneous infusion of 18 amino acid stable tracers, we are now able to reveal the large upregulation of the glutamate-glutamine pathway in ICU patients.

Mechanism and consequences of the upregulated metabolism

The well-known observation that metabolism is upregulated in critical illness is most likely related to the increase in inflammation, induced either by sepsis or trauma (42). We now show that this upregulation occurs in the metabolism of many amino acids with a prominent upregulation of the glutamate-glutamine pathway. This pathway most likely is driven by the increased muscle glutamine production because of increased protein breakdown, subsequent uptake by the liver, deamidation to provide nitrogen for the upregulated urea synthesis and release again of glutamate into the circulation, and uptake by muscle (1).

General characteristics

The careful selection of the healthy matched controls to compare to the ICU patients shows evident and previously reported differences like a low phase angle (43, 44), blood pressure and increased plasma CRP, glucose and heart rate (45). BIA-derived phase angle has long been related to health status (43, 44) and its reduction in ICU patients has been linked to 90-day mortality (43). The ICU group in this study exhibited the expected changes, therefore we believe that the studied patients are a good representation of the general patient population admitted to the ICU.

The ICU patients were all characterized by a lower plasma amino acid concentration that relates to the increased clearance of amino acids from the circulation as was reported many decades ago (46). However, we found that the concentrations of phenylalanine and methionine were higher in the ICU group (Table 3) although their clearances (Table 5) were also higher. The increase in plasma tau-methylhistidine was not related to a change in clearance, probably related to the fact that the disposal is via urinary excretion (39–41). Although many of these changes in plasma concentrations were observed before (4), the interpretation remained difficult, which could be explained by the observation in the present study that changes in plasma concentration by and large do not reflect very closely the production of these amino acids (Figure 5).

Whole-body protein synthesis and breakdown

Increased whole-body protein breakdown can be deducted from the increased whole-body production of essential amino acids (Table 4) like phenylalanine (57%), valine (71%), leucine (76%), or the sum of all essential amino acids (62%). Protein synthesis was also increased, as calculated from the difference between protein breakdown and phenylalanine hydroxylation to tyrosine (phenylalanine >> tyrosine).

Remarkably, net protein breakdown (the difference between protein breakdown and synthesis=phenylalanine hydroxylation) was reduced in the ICU patients (Table 4), confirming our earlier observations (9). Others have found an increase (47) of net protein breakdown in ICU patients. The pulse approach to measuring net protein breakdown (36) is likely to be more sensitive for the detection of changes in phenylalanine hydroxylation as there is no need for priming. In ICU patients, there can be large differences in the plasma concentration, and priming of the tyrosine pool can therefore lead to severe over- and under-priming and thus a non-steady-state condition of the tyrosine product of phenylalanine hydroxylation, thus reducing the precision of this calculation. The finding that net protein breakdown is reduced in critically ill patients is, although not new, in contrast to what is generally expected. One explanation of this intracellular process could be that although protein breakdown is stimulated and that the released amino acids stimulate protein synthesis, fewer amino acids are lost extracellularly. This could indicate an amino acid-saving process in critical illness. Remarkably, following patients at the ICU for many days shows that net protein breakdown is even more reduced when patients are longer at the ICU and maybe in a better condition (48). Our hypothesis is that the reduction in the net protein breakdown in the critically ill relates to the large reduction in muscle mass and available protein that can be lost and possibly to an intracellular amino acid-saving process. There is a certain amount of protein in the human body that can be lost during disease or starvation before it will affect the survival rate.

We also measured the breakdown of myofibrillar proteins from the whole body production of tau-methylhistidine (Table 4). We observed a 158% increase that relates to the 171% increase in the plasma tau-methylhistidine concentration. In line with previous studies showing an increase in the muscle breakdown and urinary (49) tau-methylhistidine in ICU patients, our data indicate that there is a large increase in muscle myofibrillar protein breakdown in the critically ill.

Whole-body glutamate/glutamine related metabolism

In the human body, glutamate is used for glutamine production, which is subsequently reused in the liver for glutamate production (glutamate-glutamine cycle (1)) or citrulline production in the gut (1). The arguments that this pathway is downregulated in ICU patients could be based on the observation that ICU patients have lower plasma glutamate (37%), glutamine (36%), and citrulline (40%) concentrations (Table 4). We and other researchers have previously observed that these reductions in plasma glutamate (5, 50), glutamine (51), and citrulline (50) are related to mortality. Many researchers, therefore, have concluded that this pathway is reduced in ICU patients (17, 20) with also a reduction of muscle glutamine concentration (52), but unchanged whole body (47) or muscle glutamine production (52).

We have reported the results in several studies that the whole body citrulline production is reduced in ICU patients (7, 9). Our present study contributes to this knowledge by confirming reduced whole body citrulline (31%) but also an increased whole-body glutamine production (46%) and the new observation that also whole body glutamate production is substantially increased by 160%. Also, the calculated conversion of glutamate to glutamine is increased. Clearly, this large increase relates to the stimulation of this pathway because of increased protein breakdown that will increase the intracellular availability of amino acids that will be transaminated like leucine, valine, and isoleucine making nitrogen available for the glutamate to glutamine conversion or the increased availability of ammonia from the purine-nucleotide cycle in muscle (53, 54). This large increase of whole body glutamate production will strain metabolism in the ICU patients and it seems that reducing protein breakdown could reduce whole body glutamate and glutamine production. These changes in glutamine breakdown and synthesis are likely to be linked to the endogenous glucose production that persists for at least 10 days in critically ill patients (55), as glutamine is directly involved in the glucose metabolic pathways (56).

Whole-body arginine/citrulline/ornithine related metabolism

As mentioned in relation to the increased whole-body production of glutamine, we (7, 9) and others (14) have reported and now confirmed in the present study using the pulse tracer approach a reduction of the whole body citrulline production and the de novo production of arginine from citrulline. These reductions can be explained by a reduced conversion of glutamine to citrulline in the gut (57), which subsequently results in a decrease in the percentage of de novo arginine synthesis in ICU patients (5.3%) compared to healthy subjects (12%). We previously have concluded that therefore in ICU patients arginine becomes an essential amino acid as its turnover mainly depends on the availability of arginine from protein breakdown (58).

In the past, we also showed that nitric oxide production, which equals the direct arginine to citrulline conversion, was reduced in ICU patients (7, 9). In the present study, we also observed a 55% reduced whole body production that related to the reduction to the arginine de novo production.

In the present study, we were able to measure the conversion of arginine to ornithine that can be used as a proxy for urea synthesis (7). As expected, the arginine to ornithine conversion was increased by 173% and seemed to be related to the increase in the whole body glutamine and glutamate production. As expected also reduced conversions of ornithine to citrulline were found, but unchanged citrulline to ornithine conversion.

Other amino acid metabolic pathways

We calculated the ratio between the plasma concentrations of tryptophan and tyrosine, corrected for the concentration of the large neutral amino acids. The tryptophan ratio has been related to the cerebral serotonin production and a lower ratio was found in depressed patients, while the tyrosine ratio has been related to cerebral dopamine production (59). Although we were not able to measure neuropsychological function, others have observed more delirium in ICU patients that could be related to the observed lower ratios (60).

As we discussed above, many of the essential amino acids showed increased whole body productions and clearances. We also measured this for several non-essential amino acids. The whole body productions of glycine, tyrosine, taurine, and ornithine were also increased as well as the clearances, suggesting an overall upregulated amino acid metabolism that is even higher than that of the protein breakdown.

Comparing low and high SOFA score groups of critically ill patients

As indicated, above, the metabolism of the amino acids is substantially upregulated in ICU patients. Because of the large size of the studied ICU group (51 critically ill patients), we were able to create 2 subgroups on the basis of their organ failures (Table 6). We created a group with a moderate organ failure severity (SOFA score 1–7) that had expected mortality of between 0–20% and a group of elevated SOFA score (8 to 16) with an expected mortality rate between 25 and 80% (29).

General characteristics and plasma amino acid concentrations

The lower phase angle in the severe SOFA 8–16 group was comparable to what was described previously (43, 44) and indicate a further deterioration of the condition of the ICU patients. When comparing the plasma amino acid concentrations, the more depressed plasma concentration of glutamate and leucine, valine, and isoleucine suggest that the pathways that provide the nitrogen for glutamine in muscle and then cycling back to the muscle of glutamate were compromised in the sickest critically ill patients. This seems to be supported by the increase of hydroxyproline and tau-methylhistidine, products of slowly turning over proteins (collagen and myosin). We also observed an increase of tyrosine, corrected for the large neutral amino acids, suggesting a higher dopamine level in the brain, but for now, it is unclear what the meaning is of this observation.

Protein synthesis and breakdown and WBP of other amino acids

The large reduction in the plasma glutamate and smaller reduction in glutamine concentration seems to be related to a reduction of their whole body productions. This observation is important as it could indicate that in severely ill ICU patients this pathway is starting to fail. Also, the product of glutamine breakdown in the gut, citrulline, and the downstream arginine de novo production, were reduced. These observations could be linked to the lower whole body net protein breakdown. This observation seems to be in contrast to the observed further increase of the breakdown of slowly turning over proteins as indicated by the increased whole-body tau-methylhistidine and hydroxyproline (41) production. We have no mechanistic explanation for this observation, but it could indicate that certain fast turning over protein pools in the body become depleted and that in the severely ill ICU patients more the structural proteins are broken down at an increased rate over time as it has been observed in long-term (fatal) starvation.

Could plasma amino acid concentration constitute a proxy of whole body production?

We compared the plasma concentrations in the ICU patients with the whole body production. Until now it was not possible to have both measured simultaneously. Our data show that only for a few amino acids like tau-methylhistidine, hydroxyproline, and citrulline measuring the plasma concentrations can be used as a proxy for the whole body production. We suggest that a comprehensive metabolic flux analysis that can be done with our tracer pulse approach will generate more accurate data on the up and down regulation of metabolic pathways.

How can our findings help with new nutritional support approaches?

Feeding critically ill patients remains a challenge (21). ICU patients have a large increase in protein breakdown that releases high levels of amino acids into the circulation that need to be cleared. The plasma concentration in ICU patients is lower for essential amino acids. On the other hand, reducing protein breakdown with protein or amino acid nutrition will probably increase the essential amino acid concentration and improve protein anabolism, but at the same time increase the appearance of amino acids into the circulation. The simultaneous presence of major endocrine stress changes of inflammation and the balance between increasing the plasma concentration, stimulating the appearance in plasma, reducing protein breakdown, and stimulating synthesis is probably the reason why it so difficult to achieve protein anabolism in ICU patients (21).

Why using the pulse tracer approach?

Most studies in critically ill patients are done with the primed-constant infusion approach (7–12, 15, 16). This approach is valid when the tracer/tracee ratio (TTR) is in steady-state (28, 61). Steady-state can be obtained by waiting until steady-state is reached (which can take many hours) or by giving a prime dose at the start of the experiment. The calculation of the amount in the prime dose depends on the size of the pool in which the tracers will distribute and usually is estimated from previous research protocols. This is however very difficult to assess in individual ICU patients as plasma concentrations (used to estimate the pool amount) can differ greatly between patients (62). This is especially a problem when the body pool is large in relation to the whole body production rate, as in the case for glutamine (47, 63) and tau-methylhistidine (28) and other amino acids like glycine. For that reason, pulse administration of a small volume mixture of stable isotope amino acids tracers and subsequent following the decay of the TTR for at least 2 hours is needed to enable the measurement of the actual whole body production rates without the need of assumption of existing concentrations or whole body production (27, 64).

Study limitations

We did not perform a power calculation, as we did not have a single primary endpoint in the study and had no data available to calculate the power towards the least sensitive measurement. We therefore have to identify the present study as an exploratory study. However, to be more confident about the results, we included as many subjects in the study as possible by recruiting from several sites. We recruited critically ill patients from RMC and CUHV. Although the patients of RMC had a lower SOFA score and some small differences in plasma concentrations, we believe that these small differences do not affect the main conclusions of the paper and could also indicate that the metabolic condition of ICU patients is somewhat comparable.

Because nutrition can affect the plasma concentrations and whole body productions, we measured the healthy subjects in the postabsorptive condition and the critically ill patients 6 hours after stopping enteral or parenteral nutrition. We therefore believe that the influence of nutrition on the data in the critically ill patients will be minimal.

Although critically ill patients are treated with different drugs and insulin that can affect amino acid metabolism, our study gives insight into these changes as possible targets for future drug development.

We are aware of the difficulties to obtain reliable data of body composition in critically ill patients. However, the results of body composition differences are not crucial in the interpretation of our study as we chose to analyse the data on a subject level and use only BMI as a correction approach towards differences in body composition for the whole body production and conversions.

Conclusions

Our study in ICU patients and the comparison with matched healthy subjects show that amino acid metabolism, measured with both plasma concentrations and their whole body productions, is severely disturbed in critically ill patients.

Supplementary Material

1

Supplemental Figure 1A Consort diagram of all subjects recruited.

Supplemental Figure 1B Consort diagram of subjects recruited at Texas A&M University.

Supplemental Figure 1C Consort diagram of subjects recruited at Lausanne University Hospital (CHUV).

Supplemental Figure 1D Consort diagram of subjects recruited at Rabin Medical Center.

List of abbreviations

BCAA

branched-chain amino acids

BIA

bioimpedance analysis

BMI

body mass index

FFM

fat-free mass

hsCRP

high sensitivity C-reactive protein

ICU

intensive care unit

PHE

phenylalanine

REE

resting energy expenditure

SOFA

systemic organ failure assessment

TRP

tryptophan

TTR

tracer/tracee ratio

TYR

tyrosine

WBP

Whole-body production rate