Arginine deficiency in sepsis may impair nitric oxide (NO) production for local perfusion and add to the catabolic state. In contrast, excessive NO production has been related to global haemodynamic instability. Therefore, the aim of the present study was to investigate the dose–response effect of intravenous arginine supplementation in post-absorptive patients with septic shock on arginine-NO and protein metabolism and on global and regional haemodynamics. Eight critically ill patients with a diagnosis of septic shock participated in this short-term (8 h) dose–response study. L-Arginine-HCl was continuously infused [intravenously (IV)] in three stepwise-increasing doses (33, 66 and 99 μmol·kg−1·h−1). Whole-body arginine-NO and protein metabolism were measured using stable isotope techniques, and baseline values were compared with healthy controls. Global and regional haemodynamic parameters were continuously recorded during the study. Upon infusion, plasma arginine increased from 48±7 to 189±23 μmol·l−1 (means±S.D.; P<0.0001). This coincided with increased de novo arginine (P<0.0001) and increased NO production (P<0.05). Sepsis patients demonstrated elevated protein breakdown at baseline (P<0.001 compared with healthy controls), whereas protein breakdown and synthesis both decreased during arginine infusion (P<0.0001). Mean arterial and pulmonary pressure and gastric mucosal-arterial partial pressure of carbon dioxide difference (Pr-aCO2) gap did not alter during arginine infusion (P>0.05), whereas stroke volume (SV) increased (P<0.05) and arterial lactate decreased (P<0.05). In conclusion, a 4-fold increase in plasma arginine with intravenous arginine infusion in sepsis stimulates de novo arginine and NO production and reduces whole-body protein breakdown. These potential beneficial metabolic effects occurred without negative alterations in haemodynamic parameters, although improvement in regional perfusion could not be demonstrated in the eight patients with septic shock who were studied.

CLINICAL PERSPECTIVES

  • Although there is evidence for sepsis to be an arginine-deficiency state that contributes to impaired NO production for local perfusion and adds to the catabolic state, the therapeutic role of arginine supplementation in severe sepsis patients is controversial. This is especially so since excessive NO production has been related to global haemodynamic instability.

  • In the eight patients with septic shock who were studied, escalating doses of arginine under post-absorptive conditions increased NO production without a negative impact on haemodynamic instability. Protein breakdown was reduced during arginine infusion.

  • Larger study groups are needed to confirm that arginine can safely be administered in sepsis in general; however these results show the potential significance of arginine infusion in a condition of severe protein wasting such as sepsis.

INTRODUCTION

Severe sepsis is a major cause of mortality in the intensive care unit (ICU) [1]. It is characterized by a systemic inflammatory response to infection and is associated with multiple organ failure, haemodynamic instability [2] and increased protein turnover [3]. Although global volume-related haemodynamic variables can be stabilized with fluids and vasopressors [4], regional disturbances in organ perfusion are the most important predictors of worse outcome [1,5]. Dysfunctional organ perfusion occurs at the microcirculatory level [6] and contributes to the systemic and regional inflammatory response, organ failure, cell damage and impaired tissue oxygenation [710]. Inadequate nitric oxide (NO) production in endothelial cells is suggested to be of key importance to maintain microcirculation. This is supported by the observed decrease in endothelium-dependent NO in the micro-circulation during experimental endotoxaemia in humans [11]. We and others have suggested that observed changes in the arginine pathway during sepsis is the underlying metabolic cause for impaired NO production [12] and dysfunctional microcirculation [13,14]. Plasma arginine concentrations are reduced substantially in sepsis patients in the absence of trauma or surgery, whereas insufficient data are available for sepsis of other origin [15]. The mechanism of the reduced plasma arginine levels is most likely to be a combination of impaired endogenous arginine production [3,12] and increased arginine clearance [3,16]. The reduced arginine availability in sepsis is related to impaired NO production [17], which is further deprived by elevated levels of endogenous NO synthase (NOS) inhibitors [18].

Manipulation of arginine-NO metabolism in animal models of endotoxaemia by parenteral and enteral L-arginine improved mucosal microvascular and hepatosplanchnic perfusion [19,20] and increased NO production in various organs [20,21]. Moreover, reduced whole-body protein turnover with differential alterations at the organ level was observed [22]. Similarly, intravenous nitroglycerin improved microvascular flow in patients with septic shock [23]. In contrast, others have reported harmful effects of parenteral arginine therapy in animals with endotoxaemia [24], and arginine-containing immunonutrition was unfavourable in meta-analyses of studies in sepsis patients [25]. Thus, although there is evidence for sepsis to be an arginine-deficiency state [13], the therapeutic role of arginine supplementation in severe sepsis patients is controversial [2630]. Timing of arginine supplementation during the progression of sepsis, the amount of arginine infused that far exceeds the endogenous arginine production rate, or the complex nutritional matrix of immunonutrition may underlie these controversial findings. However, there is no study that has measured NO production and protein metabolism in patients with sepsis during arginine administration in the post-absorptive state.

Therefore, the present study aimed at investigating the dose–response effect of intravenous arginine supplementation in patients with septic shock in the post-absorptive state on arginine-NO and protein metabolism and on global and regional haemodynamics. As a reference, metabolic data were compared with those in healthy control subjects.

MATERIALS AND METHODS

Patients and treatments

Eight critically ill patients with a diagnosis of septic shock admitted to the general ICU of the University Hospital Maastricht, The Netherlands, were studied within 48 h of diagnosis (Figure 1), according to a single-arm study design. All patients met the criteria for severe sepsis and septic shock {sepsis-induced hypotension not responding to adequate fluid resuscitation and requiring noradrenaline (norepinephrine) at a dose of >0.1 μg·kg−1·min−1 [2]}. A systemic and a pulmonary arterial catheter (Edwards Lifesciences) were in place with continuous pressure monitoring. Exclusion criteria included unresponsiveness to inotropes and vasopressors, life expectancy <24 h, prolonged use or high-dose corticosteroids, known insulin-dependent diabetes mellitus, metastasis or haematological malignancies or the use of chemotherapy, known pre-existing liver or renal failure, and haemodialysis or continuous veno-venous haemofiltration (CVVH) treatment. Sixteen healthy older adults, recruited in the local community, served as a reference group for normal metabolism. The present study has been carried out in accordance with the Declaration of Helsinki (2013) of the World Medical Association. Written informed consent was obtained, and the study was approved by the Medical Ethical Committee of the University Hospital Maastricht. The clinical trial is registered at ClinicalTrials.gov as NCT01775020 (sepsis patients) and NCT01774994 (healthy subjects).

Consort 2010 flow diagram

Figure 1
Consort 2010 flow diagram
Figure 1
Consort 2010 flow diagram

The present study in sepsis patients involved an L-arginine infusion protocol (illustrated in Supplementary Figure S1 at http://www.clinsci.org/cs/128/cs1280057add.htm). Primary outcome measure of the present study was whole-body NO production. Secondary outcome measures were parameters of haemodynamics, blood parameters and gastric perfusion by tonometry. Parenteral nutrition was stopped 4 h before the present study. After 2-h baseline measurements, L-arginine-HCl (Fresenius) was continuously infused [intravenously (IV)] in three stepwise-increasing doses (33, 66 and 99 μmol·kg−1·h−1), each dose for 2 h. Systemic haemodynamics, body temperature and requirement for fluid resuscitation and vasoactive medication were monitored continuously. Blood gases and plasma arginine levels were measured at 30-min intervals. Electrolytes, lactate and ammonia were measured at hourly intervals. Renal function (urine production), lung function [alveolar partial pressure of oxygen (PAO2)/fraction of inspired oxygen (FiO2) ratio] and fluid balance were determined at 2-h intervals. A gastric tonometric catheter (TRIP Tonometry catheter 14F, Datex-Ohmeda) was introduced and connected to the Tonometer device (Tonocap monitor, Datex-Ohmeda) for measurements of regional haemodynamics at 30-min intervals. All patients were treated with oesomeprazol (40 mg of Nexium® IV).

Arginine, citrulline, NO, urea and protein metabolism was studied in the post-absorptive state using a primed-constant and continuous stable isotope infusion protocol with regular blood sampling, as described previously [3,31]. Blood was treated and analysed as reported before [3,31]. All metabolic data were determined under steady-state conditions (Supplementary Figure S2 at http://www.clinsci.org/cs/128/cs1280057add.htm) and subsequently calculated [3,31].

Substantiation and preparation of the L-arginine doses

In a previous pig study in our laboratory [21], L-arginine was infused at 5.3 μmol·kg−1·min−1 to obtain a 3-fold increase in whole-body arginine production. This dose was chosen in relation to the habitual protein intake of the growing pigs (7.2 g of protein·kg−1·day−1). To obtain the same 3-fold increase in humans, L-arginine at 2.3 μmol·kg−1·min−1 should be infused, regarding a whole-body arginine production of 1.17 μmol·kg−1·min−1 in sepsis [3]. However, in relation to the recommended protein intake for critically ill humans (1.5–2.0 g of protein·kg−1·day−1), which is much lower than in growing pigs, an L-arginine infusion at 1.1 μmol·kg−1·min−1 (66 μmol·kg−1·h−1, equal to 21 g per day for a 75-kg adult) seemed more appropriate. Since 30 g of L-arginine is generally accepted as a daily maximal intake [32], L-arginine was infused at a maximum of 99 μmol·kg−1·h−1 (equals 31 g per day) and at a minimum of 33 μmol·kg−1·h−1 (equals 10 g per day). Two ampoules of L-arginine-HCl (21.07% solution in 20 ml ampoules; Fresenius) were diluted in 250 ml of sterile 0.9% NaCl (resulting in a 161.28 mmol·l−1L-arginine-HCl solution) and infused at the desired infusion rates. In total, ~184 ml was infused over 8 h (for a 75-kg adult).

Stable isotope infusion protocol for arginine, citrulline, NO, urea and protein metabolism

Whole-body arginine, citrulline, NO and urea metabolism in sepsis patients was assessed via combined infusion of stable isotopes L-[guanidino-15N2]arginine ([15N2]Arg: prime, 3.65 μmol·kg−1; infusion, 3.51 μmol·kg−1·h−1), L-[ureido-13C-2H2]citrulline ([13C-2H2]Cit: prime, 0.58 μmol·kg−1; infusion, 0.27 μmol·kg−1·h−1) and 13C-urea (prime, 36.5 μmol·kg−1; infusion, 7.42 μmol·kg−1·h−1). Whole-body protein metabolism was assessed via combined infusion of stable isotopes L-[ring-2H5]phenylalanine ([2H5]Phe: prime, 2.19 μmol·kg−1; infusion, 2.26 μmol·kg−1·h−1) and L-[ring-2H2]tyrosine ([2H2]Tyr: prime, 0.95 μmol·kg−1; infusion, 0.77 μmol·kg−1 ·h−1), and a bolus dose of L-[ring-2H4]TYR ([2H4]Tyr: prime, 0.32 μmol·kg−1) was administered to prime the phenylalanine (PHE)-derived plasma tyrosine pool. Same isotopes and infusion rates were applied to the healthy subjects, except for [15N2]Arg (prime, 5.48 μmol·kg−1; infusion, 5.27 μmol·kg−1·h−1). Stable isotopes were purchased from the Cambridge Isotopes. Sterile stable isotope solutions were prepared by Mrs G. ten Have (Metabolic Research Centre, Department of Surgery, Maastricht University, Maastricht, The Netherlands) in collaboration with the Department of Clinical Pharmacology (University Hospital Maastricht, Maastricht, The Netherlands).

Before administration of the priming dose, arterial blood was collected for measurement of the natural (baseline) enrichment of amino acids. Subsequent blood samples were collected at 60, 90 and 120 min at baseline and during each arginine dose to reassure a tracer steady state. For infusion of isotopes, a peripheral line already in place for clinical care was used, and the arm was positioned ~10 cm above the level of the heart to deliver a uniform dose of tracers to the heart throughout the experiment. In total, 500 ml of stable isotope solution was administered during the 8-h dose–response study in sepsis patients.

In healthy subjects, a comparable 2-h stable isotope infusion protocol in the post-absorptive state after an overnight fast was performed. A catheter was placed in the antecubital vein of the arm for isotope infusion. Venous blood sample was collected at baseline before isotope infusion, and arterialized blood samples [33] were taken at 60, 90, 105 and 120 min after the start of infusion. In healthy subjects, 300 ml was administered during the 2-h study.

Blood chemistry and analyses

Promptly after sampling, blood was distributed in pre-chilled tubes on ice, containing either heparin (Hep; 4-ml lithium-heparin vacutainer, Greiner Bio-One) or EDTA (4-ml EDTA-K3 vacutainer, Greiner Bio-One). Additional blood was sampled at baseline only [serum, 5-ml vacutainer, and NaF, 2-ml vacutainer (Greiner Bio-one)] and placed at room temperature for 30-min prior to centrifugation (12 min, 239 000 g, 4°C). The rest of the blood was centrifuged immediately (10 min, 312000 g, 4°C), and plasma was collected on ice. For plasma urea concentration analysis, 900 μl of Hep-plasma was added to 90 μl of 50% trichloroacetic acid solution, ensuring stability of the substances. For analysis of amino acid concentrations and tracer enrichments, Hep-plasma was deproteinized by mixing 500 μl with 20 mg of dry sulfosalicylic acid. Hep- and EDTA-plasma samples were frozen in liquid nitrogen and stored at −80°C until further analysis.

Arterial plasma amino acid levels were determined by HPLC analysis [34]. Analysis for enrichment was done by LC-ESI-MS (QTrap 5500 MS; AB Sciex) with ExpressHT Ultra LC (Eksigent Div.; AB Sciex) after derivatization with 9-fluoren-9-ylmethoxycarbonyl (Fmoc) [35]. Fmoc was fragmented to obtain specific and high-sensitivity fragments. Plasma urea concentrations were determined by using commercially available kits on a Cobas Mira S (Roche Diagnostica). Haematological parameters, blood gasses, lactate, albumin, creatinine, alanine aminotransferase (ALAT) and C-reactive protein (CRP) were analysed using validated methods at the Maastricht University Hospital laboratories.

Calculations for arginine, citrulline, NO, urea and protein metabolism

Tracer tracee ratios (TTRs) were used to calculate whole-body production (Wb Ra) of arginine (ARG), citrulline (CIT), urea, phenylalanine (PHE) and tyrosine (TYR): Wb Ra=infusion rate (tracer)/TTR. Endogenous arginine production is the total arginine production, corrected for intravenous arginine infusion. Conversion of arginine into citrulline (Qarg>cit) was used as a measure of NO production, calculated as: Wb RaCIT×TTR15N-CIT/TTR15N2-ARG. Conversion of citrulline into arginine (Qcit>arg) was used as a measure of de novo arginine production, calculated as: Wb RaARG×TTR13C-2H2-ARG/TTR13C-2H2-CIT. Conversion of arginine into urea (Qarg>urea) was calculated as: Wb RaUREA×TTR15N2-UREA/TTR15N2-ARG. Conversion of phenylalanine into tyrosine (Qphe>tyr) was used as a measure of PHE hydroxylation (PHE hydrox), calculated as Wb RaTYR×TTR2H4-TYR/TTR2H5-PHE. To calculate whole-body protein synthesis (PS), the following definition was used: PS=Wb RaPHE−PHE hydrox, where Wb RaPHE represents protein breakdown.

These metabolic values were averaged for the period from 1 to 2 h after start of isotope infusion in both subject groups and similarly after each alteration in arginine infusion rate in the sepsis patients.

Statistical analyses

Results are means±S.D. Baseline values between groups were compared using an unpaired Student's t test (for normally distributed variables) or Mann–Whitney's U test (for non-parametric parameters). Welch's correction was applied in case of unequal variances. Effects of arginine infusion in sepsis patients were evaluated by using repeated-measures one-way ANOVA with arginine-dose as within-subject factor with post-hoc Bonferroni's multiple comparison test (for normally distributed variables) or Friedman's test with post-hoc Dunn's multiple comparison test (for non-parametric variables). Metabolic data were analysed by mixed model analysis with multiple imputations to account for missing values. Normality was evaluated by Shapiro–Wilk's normality test. Sample size calculation (eight sepsis patients) was based on an estimated 9% change in NO production between baseline and each consecutive arginine dose, using an S.D. of 6.4% (based on [36], measuring urinary nitrate), a significance level α of 0.05, and a power of 80%.

The statistical package within GraphPad Prism 5 for WINDOWS (version 5.01; GraphPad Software) was used for statistical analysis, except for metabolic data analyses using SAS Proc MI. Statistical significance was defined as a two-tailed P<0.05.

RESULTS

Subject characteristics

Characteristics for healthy controls and sepsis patients are listed in Table 1. All patients were mechanically ventilated, sedated and received vasopressor medication and antibiotics. A functioning pulmonary artery catheter was in position in seven of the eight sepsis patients. Gastric tonometry was performed in only five of the eight patients due to equipment problems or gastric resection in the other three patients. Three patients received parenteral nutrition before the present study, but none of the subjects had received any enteral nutrition before the present study.

Table 1
General characteristics of the sepsis patients and the healthy control group

Data are means±S.D. or as numbers. aPerianal abscess (n=1), suspected intestinal ischaemia (n=1), post-operative instability on day 4 after pancreatoduodenectomy (n=1), post-operative leakage after gastric resection (n=1), instability after laparotomy with resection of necrotic gallbladder (n=1). bLegionella pneumonia (n=1). cIntestinal ischaemia with respiratory insufficiency (n=1), respiratory insufficiency with suspected pneumonia and endocarditis (n=1). dData available in only five of eight patients. P1, significance for comparison between healthy controls and sepsis: unpaired Student's t test (for age and BMI) or non-parametric Mann–Whitney's U test (for CRP). BMI, body mass index; APACHE, Acute Physiology and Chronic Health Evaluation [49]; TISS, Therapeutic Intervention Scoring Systems [50] (P>0.05).

ParameterHealthy controls (n=16)Sepsis patients (n=8)P1
Age (years) 60.9±6.2 66±11 NS 
BMI (kg·m−224.4±2.7 26.8±3.6 0.08 
CRP (mg·l−11.1±1.2 249±132 <0.001 
Sex (male/female) (n8/16 4/4  
Suspected source of sepsis (n   
Abdominal  5a  
Pulmonary  1b  
Other  2c  
Albumin (g·l−1)d  8.3±3.4  
Creatinine (mmol·l−1 227±160  
ALAT (unit·l−1 73±112  
APACHE II score  33±5  
TISS score  47±8  
28 days mortality (n 2 (25%)  
ParameterHealthy controls (n=16)Sepsis patients (n=8)P1
Age (years) 60.9±6.2 66±11 NS 
BMI (kg·m−224.4±2.7 26.8±3.6 0.08 
CRP (mg·l−11.1±1.2 249±132 <0.001 
Sex (male/female) (n8/16 4/4  
Suspected source of sepsis (n   
Abdominal  5a  
Pulmonary  1b  
Other  2c  
Albumin (g·l−1)d  8.3±3.4  
Creatinine (mmol·l−1 227±160  
ALAT (unit·l−1 73±112  
APACHE II score  33±5  
TISS score  47±8  
28 days mortality (n 2 (25%)  

Plasma levels

Plasma levels are listed in Table 2. Baseline plasma levels of arginine and citrulline were significantly lower in sepsis patients than in the healthy controls (P<0.0001). Also, plasma glutamine was significantly lower (P<0.05), whereas plasma urea was elevated in sepsis patients at baseline (P<0.001).

Table 2
Plasma levels at baseline and during stepwise-increased L-arginine-HCl infusion (33, 66 and 99 μmol·kg−1·h−1) in the sepsis patients, and baseline concentrations in the healthy control group

Data as means±S.D. NS, P>0.05. P1, significance for comparison between healthy controls and sepsis: unpaired Student's t test or non-parametric Mann–Whitney's U test (for urea and insulin). P2, significance for comparison between L-arginine doses: repeated-measures one-way ANOVA or Friedman's non-parametric test (for urea and insulin). P<0.05 compared with baseline sepsis with post-hoc Bonferroni's multiple comparison test. P<0.05 compared with the lower preceding dose with post-hoc Bonferroni's multiple comparison test.

Healthy control (n=16)Sepsis patients (n=8)
ParameterBaselineBaseline336699P1P2
ARG (μmol·l−192±17 48±7 83±11 130±16†‡ 189±23†‡ <0.0001 <0.0001 
CIT (μmol·l−140.6±6.5 9.7±3.5 9.9±3.6 10.1±4.7 10.3±4.2 <0.0001 NS 
GLN (μmol·l−1634±70 552±114 521±95 518±105 536±93 <0.05 NS 
ORN (μmol·l−167±14 65±12 103±17 181±35†‡ 288±49†‡ NS <0.0001 
LYS (μmol·l−1169±30 129±20 128±20 127±21 131±23 <0.01 NS 
ARG/ORN+LYS 0.39±0.07 0.25±0.03 0.36±0.04 0.42±0.04†‡ 0.45±0.05 <0.0001 <0.0001 
UREA (mmol·l−13.7±0.9 9.8±6.7 9.4±6.8 9.9±6.7 10.0±6.5 <0.001 NS 
Glucose (mmol·l−15.1±0.5 5.2±1.2 5.1±1.2 5.0±1.3 4.9±1.3 NS NS 
Insulin (m-units·l−15.1±2.9 7.3±5.6 8.1±7.4 8.3±8.2 8.2±5.9 NS NS 
Healthy control (n=16)Sepsis patients (n=8)
ParameterBaselineBaseline336699P1P2
ARG (μmol·l−192±17 48±7 83±11 130±16†‡ 189±23†‡ <0.0001 <0.0001 
CIT (μmol·l−140.6±6.5 9.7±3.5 9.9±3.6 10.1±4.7 10.3±4.2 <0.0001 NS 
GLN (μmol·l−1634±70 552±114 521±95 518±105 536±93 <0.05 NS 
ORN (μmol·l−167±14 65±12 103±17 181±35†‡ 288±49†‡ NS <0.0001 
LYS (μmol·l−1169±30 129±20 128±20 127±21 131±23 <0.01 NS 
ARG/ORN+LYS 0.39±0.07 0.25±0.03 0.36±0.04 0.42±0.04†‡ 0.45±0.05 <0.0001 <0.0001 
UREA (mmol·l−13.7±0.9 9.8±6.7 9.4±6.8 9.9±6.7 10.0±6.5 <0.001 NS 
Glucose (mmol·l−15.1±0.5 5.2±1.2 5.1±1.2 5.0±1.3 4.9±1.3 NS NS 
Insulin (m-units·l−15.1±2.9 7.3±5.6 8.1±7.4 8.3±8.2 8.2±5.9 NS NS 

Plasma arginine concentrations increased stepwise (i.e. different between each increasing dose) during arginine infusion, to ~4-fold the baseline level with the highest dose (P<0.0001). This was similar for plasma ornithine (P<0.0001). The arginine/lysine+ornithine (LYS+ORN) ratio increased significantly during arginine infusion (P<0.0001). No changes were observed in plasma citrulline, glutamine (GLN), lysine and urea. Supplementary Table S1 (http://www.clinsci.org/cs/128/cs1280057add.htm) lists all of the amino acids measured, showing alterations in several other amino acids with sepsis and minor alterations in plasma alanine and glycine with arginine infusion.

Plasma glucose and insulin were not significantly different between sepsis patients and healthy controls at baseline and did not change during arginine infusion in the sepsis patients. Three of the eight sepsis patients received insulin therapy with no major changes during the arginine infusion protocol. Excluding these patients from the analysis of plasma insulin during arginine infusion did not change the outcome.

Arginine, citrulline, NO and urea metabolism

Results are shown in Figure 2 for arginine metabolism, Figure 3 for citrulline metabolism and NO production, and Figure 4 for urea metabolism (see Supplementary Table S2 at http://www.clinsci.org/cs/128/cs1280057add.htm for average values of metabolic parameters). Whole-body total and endogenous arginine production (Ra ARG) and citrulline production (Ra CIT) were significantly lower in sepsis patients than in healthy controls at baseline (P<0.0001). Ra CIT and endogenous Ra ARG were not affected by arginine infusion [not significant (NS)]. NO production (Qarg>cit) was not different between groups at baseline but significantly increased with arginine infusion (P<0.05 for overall time effect; P=0.09 for baseline compared with 99 μmol·kg−1·h−1 arginine infusion, after Bonferroni's correction for multiple comparisons). De novo arginine production (Qcit>arg) was significantly lower in sepsis patients than in healthy controls at baseline (P<0.0001) and increased with arginine infusion (P<0.0001). Urea production (Ra UREA) was significantly higher in sepsis patients than in healthy controls at baseline (P<0.001), with no significant change during arginine infusion. Conversion of arginine into urea (Qarg>urea) was not significantly different at baseline between sepsis patients and healthy controls, but increased stepwise during arginine infusion in the sepsis patients (P<0.0001), significantly different between each adjacent arginine dose (P<0.001).

Arginine metabolism in healthy controls (n=16) and during arginine infusion in sepsis patients (n=8)

Figure 2
Arginine metabolism in healthy controls (n=16) and during arginine infusion in sepsis patients (n=8)

Data are means±S.D. **P<0.01 compared with healthy control group (unpaired Student's t test). #P<0.05 compared with baseline sepsis value (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). $P<0.05 compared with the lower preceding dose (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). BW, body weight.

Figure 2
Arginine metabolism in healthy controls (n=16) and during arginine infusion in sepsis patients (n=8)

Data are means±S.D. **P<0.01 compared with healthy control group (unpaired Student's t test). #P<0.05 compared with baseline sepsis value (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). $P<0.05 compared with the lower preceding dose (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). BW, body weight.

Citrulline, de novo arginine and NO production in healthy controls (n=16) and during infusion in sepsis patients (n=8)

Figure 3
Citrulline, de novo arginine and NO production in healthy controls (n=16) and during infusion in sepsis patients (n=8)

Data are means±S.D. **P<0.01 compared with healthy control group (unpaired Student's t test). #P<0.05 compared with baseline sepsis (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). $P<0.05 compared with the lower preceding dose (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). BW, body weight.

Figure 3
Citrulline, de novo arginine and NO production in healthy controls (n=16) and during infusion in sepsis patients (n=8)

Data are means±S.D. **P<0.01 compared with healthy control group (unpaired Student's t test). #P<0.05 compared with baseline sepsis (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). $P<0.05 compared with the lower preceding dose (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). BW, body weight.

Urea metabolism in healthy controls (n=16) and during infusion in sepsis patients (n=8)

Figure 4
Urea metabolism in healthy controls (n=16) and during infusion in sepsis patients (n=8)

Data are means±S.D. Qarg>urea: arginine to urea conversion rate. **P<0.01 compared with healthy control group [unpaired Student's t test (Qarg>urea) or non-parametric Mann–Whitney's U test (Ra UREA)]. #P<0.05 compared with baseline sepsis (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). $P<0.05 compared with the lower preceding dose (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). BW, body weight.

Figure 4
Urea metabolism in healthy controls (n=16) and during infusion in sepsis patients (n=8)

Data are means±S.D. Qarg>urea: arginine to urea conversion rate. **P<0.01 compared with healthy control group [unpaired Student's t test (Qarg>urea) or non-parametric Mann–Whitney's U test (Ra UREA)]. #P<0.05 compared with baseline sepsis (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). $P<0.05 compared with the lower preceding dose (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). BW, body weight.

Protein metabolism

Results are shown in Figure 5 (see Supplementary Table S2 for average values of metabolic parameters). Protein breakdown (Ra PHE) and PS were significantly higher in sepsis patients than in healthy controls at baseline (P<0.001). Net protein breakdown (Qphe>tyr) was significantly lower in sepsis patients at baseline (P<0.0001). During arginine infusion, protein breakdown and synthesis decreased significantly (P<0.0001), and each arginine dose differed significantly from baseline (P<0.05 for all, except P=0.05 for PS level at an arginine infusion of 33 μmol·kg−1·h−1 compared with baseline).

Protein metabolism in healthy controls (n=16) and during infusion in sepsis patients (n=8)

Figure 5
Protein metabolism in healthy controls (n=16) and during infusion in sepsis patients (n=8)

Data are means±S.D. PB, protein breakdown. **P<0.01 compared with healthy control group (unpaired Student's t test). #P<0.05 compared with baseline sepsis (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). $P<0.05 compared with the lower preceding dose (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). BW, body weight.

Figure 5
Protein metabolism in healthy controls (n=16) and during infusion in sepsis patients (n=8)

Data are means±S.D. PB, protein breakdown. **P<0.01 compared with healthy control group (unpaired Student's t test). #P<0.05 compared with baseline sepsis (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). $P<0.05 compared with the lower preceding dose (mixed model analysis with multiple imputations and pooled post-hoc Student's t test with Bonferroni's correction for multiple comparisons). BW, body weight.

Whole-body tyrosine production (Ra TYR) was not different between the groups at baseline but decreased significantly (P<0.01) during arginine infusion in the sepsis patients, with levels at arginine infusion of 66 and 99 μmol·kg−1·h−1 being significantly different from baseline (P<0.05).

Haemodynamics

The global haemodynamics, systolic pressure, diastolic pressure and mean arterial pressure (MAP), as well as pulmonary pressures, pulmonary capillary wedge pressure (PCWP), cardiac output, cardiac index (CI) and noradrenaline infusion rate did not change during L-arginine infusion (Table 3; Figure 6 for individual haemodynamic values). Heart rate (HR) decreased significantly during L-arginine infusion (P<0.05), whereas stroke volume (SV) increased (P<0.05) and stroke index (SI) tended to increase (P=0.05) (Table 3 and Figure 6). Dobutamine infusion rate did not change in any subject during the present study. Two subjects received an additional 500 ml of hydroxyethyl starch (Voluven®, Fresenius) during the L-arginine infusion period, whereas two others received a similar volume during the present study before arginine infusion had started. No significant differences during L-arginine infusion were observed for central venous pressure (CVP), systemic vascular resistance (SVR), PAO2/FiO2 ratio and arterio-venous partial pressure of carbon dioxide (PCO2) difference (Table 3). A reduction in arterio-venous partial pressure of oxygen (PO2) difference was observed during L-arginine infusion (P<0.05; Table 3).

Table 3
Haemodynamics at baseline and during stepwise-increased L-arginine-HCl infusion (33, 66 and 99 μmol·kg−1·h−1) in the sepsis patients

Data are unweighted means±S.D., i.e. means of all patients after first calculating the within-subject average in case of multiple measurements. NS, P>0.05. P2, significance for comparison between L-arginine doses: repeated-measures one-way ANOVA [for MAP, pulmonary arterial pressure (PAP), HR, CI, SV, SI, CVP, PAO2/FiO2, arterio-venous difference in PO2 (A-V PO2), arterio-venous difference in PCO2 (A-V PCO2), arterial pH, lactate, base excess (BE) and urinary output] or Friedman's non-parametric test (for SVR, PCWP, Pr-aCO2 and bicarbonate). P<0.05 compared with baseline (post-hoc Bonferroni's multiple comparison test). No significant differences between arginine doses.

ParameterBaseline336699P2
Global haemodynamics (n=8; n=7 with pulmonary flotation catheter) 
 MAP (mmHg) 75±21 80±16 79±10 80±12 NS 
 PAP (mmHg) 29±5 29±4 28±5 27±4 NS 
 HR (beats·min−1101±17 101±21 96±16 95±17 <0.05 
 CI (l·min−1·m−23.9±0.7 4.0±0.5 4.0±0.8 4.2±1.0 NS 
 SV (ml·beat−177±8 81±8 83±13 88±10 † <0.05 
 SI (ml·beat−1·m−241±6 43±7 44±9 46±7 † 0.05 
 CVP (mmHg) 12.3±6.5 13.3±6.4 13.8±5.1 12.9±5.0 NS 
 SVR (dyne·s·cm−5672±242 691±229 666±231 707±275 NS 
 PCWP (mmHg) 14.8±4.1 15.0±3.6 14.7±3.8 14.4±3.4 NS 
PAO2/FiO2 (mmHg) 232±105 223±96 220±91 207±69 NS 
 A-V PCO2 (torr) [kPa] 4±2 [−0.5±0.2] 4±1 [−0.5±0.1] 4±1 [−0.5±0.1] 3±1 [−0.4±0.2] NS 
 A-V PO2 (torr) [kPa] 52±14 [6.9±1.9] 49±13 [6.5±1.7] 49±10 [6.5±1.3] 39±11 [5.2±1.5] <0.05 
Regional haemodynamics (n=5 with tonometry catheter) 
Pr-aCO2 (torr) [kPa] 11±3 [1.4±0.4] 12±3 [1.6±0.4] 11±5 [1.5±0.6] 9±2 [1.2±0.3] NS 
Other (n=8)      
 Arterial pH 7.35±0.09 7.36±0.09 7.37±0.09 7.36±0.10 NS 
Lactate (mmol·l−12.4±1.3 2.3±1.2 2.2±1.2 2.1±1.0 † <0.05 
Bicarbonate (mmol·l−117.1±3.8 17.2±3.5 17.3±3.2 17.3±3.3 NS 
BE (mmol·l−1−7.5±4.8 −7.4±4.3 −7.0±4.0 −7.2±4.0 NS 
Urinary output (ml·kg−1·h−10.6±0.5 0.9±0.7 1.1±0.7 1.1±0.9 0.05 
ParameterBaseline336699P2
Global haemodynamics (n=8; n=7 with pulmonary flotation catheter) 
 MAP (mmHg) 75±21 80±16 79±10 80±12 NS 
 PAP (mmHg) 29±5 29±4 28±5 27±4 NS 
 HR (beats·min−1101±17 101±21 96±16 95±17 <0.05 
 CI (l·min−1·m−23.9±0.7 4.0±0.5 4.0±0.8 4.2±1.0 NS 
 SV (ml·beat−177±8 81±8 83±13 88±10 † <0.05 
 SI (ml·beat−1·m−241±6 43±7 44±9 46±7 † 0.05 
 CVP (mmHg) 12.3±6.5 13.3±6.4 13.8±5.1 12.9±5.0 NS 
 SVR (dyne·s·cm−5672±242 691±229 666±231 707±275 NS 
 PCWP (mmHg) 14.8±4.1 15.0±3.6 14.7±3.8 14.4±3.4 NS 
PAO2/FiO2 (mmHg) 232±105 223±96 220±91 207±69 NS 
 A-V PCO2 (torr) [kPa] 4±2 [−0.5±0.2] 4±1 [−0.5±0.1] 4±1 [−0.5±0.1] 3±1 [−0.4±0.2] NS 
 A-V PO2 (torr) [kPa] 52±14 [6.9±1.9] 49±13 [6.5±1.7] 49±10 [6.5±1.3] 39±11 [5.2±1.5] <0.05 
Regional haemodynamics (n=5 with tonometry catheter) 
Pr-aCO2 (torr) [kPa] 11±3 [1.4±0.4] 12±3 [1.6±0.4] 11±5 [1.5±0.6] 9±2 [1.2±0.3] NS 
Other (n=8)      
 Arterial pH 7.35±0.09 7.36±0.09 7.37±0.09 7.36±0.10 NS 
Lactate (mmol·l−12.4±1.3 2.3±1.2 2.2±1.2 2.1±1.0 † <0.05 
Bicarbonate (mmol·l−117.1±3.8 17.2±3.5 17.3±3.2 17.3±3.3 NS 
BE (mmol·l−1−7.5±4.8 −7.4±4.3 −7.0±4.0 −7.2±4.0 NS 
Urinary output (ml·kg−1·h−10.6±0.5 0.9±0.7 1.1±0.7 1.1±0.9 0.05 

Individual haemodynamics in sepsis patients (n=8)

Figure 6
Individual haemodynamics in sepsis patients (n=8)

BW, body weight; norepinephrine, noradrenaline.

Figure 6
Individual haemodynamics in sepsis patients (n=8)

BW, body weight; norepinephrine, noradrenaline.

Regional haemodynamics showed no significant change in mucosal-arterial PCO2 difference (Pr-aCO2) gap (Table 3). No changes were observed in arterial pH, base excess and bicarbonate, whereas lactate decreased significantly during L-arginine infusion (P<0.05) and urinary output tended to increase (P=0.05) (Table 3). L-Arginine infusion did not affect plasma levels of ammonia (99±35 μmol·l−1 at baseline), sodium (139±6 mmol·l−1 at baseline) and chloride (116±4 mmol·l−1), but potassium levels declined from 4.4±0.3 mmol·l−1 at baseline to 4.2±0.4 and 4.2±0.5 mmol·l−1 during infusion of the two highest L-arginine doses (P<0.05).

DISCUSSION

L-Arginine infusion in the present study increased the plasma arginine level up to 4-fold baseline, with increased de novo arginine and NO production, increased arginine to urea conversion and reduced whole-body protein turnover. Global haemodynamic parameters and the need for vasoactive medication were not affected by acute L-arginine treatment. Regional gastric perfusion was not changed significantly. The observed increase in SV and reduced arterial lactate level could point towards improved organ function, but this needs further investigation.

Arginine-NO and protein metabolism

Patients with septic shock demonstrated a lower plasma arginine level, reduced endogenous de novo arginine production and a low arginine/ornithine+lysine ratio. This low ratio is an indicator of relative low arginine bioavailability because arginine, lysine and ornithine are taken up in the cell by the same transporter [37]. We have found, in line with previous studies [3,12,16], that sepsis is a condition of arginine deficiency. The precursor of de novo arginine production, i.e. plasma citrulline and the associated citrulline production rate, and the citrulline precursor glutamine were also lowered. This confirms previous observations [3,38]. During the arginine infusion, plasma arginine and ornithine levels increased ~4-fold at the highest arginine dose, and the arginine/ornithine+lysine ratio improved significantly. However, no alterations in plasma citrulline and glutamine were observed. The observed increase in de novo arginine production and elevated arginine/ornithine+lysine ratio suggest an increase in arginine bioavailability for metabolic conversion by NOS and arginase. This is indeed confirmed in the present study by the increased NO production rate, whereas increased conversion of arginine into urea confirms an increase in arginase activity. Similar observations on arginine-NO metabolism are made in a haemodynamic endotoxaemia pig model of sepsis supplemented with arginine [20,21]. Unfortunately, the stable isotope conversion method cannot specify the enzymatic origin of increased NO, i.e. inducible NOS-II or endothelial NOS-III, which is known to be differently regulated in sepsis [3944]. The observed increase in arginase activity could serve as a stimulus for cell proliferation and tissue regeneration [14] and, therefore, be beneficial in sepsis.

Plasma urea was not significantly elevated in the present study, even though the conversion of arginine into urea was increased during arginine infusion, and ornithine increased from 65 to 288 μmol·l−1. It is entirely possible that an equimolar increase in plasma urea that reflects ~2.5% increase from baseline is within the variance of laboratory analysis and is therefore not detected. Moreover, urinary urea excretion may further limit the increase in plasma urea. Although whole-body urea production was not significantly affected by arginine infusion, whole-body protein breakdown and PS, i.e. protein turnover, decreased during arginine supplementation. This also resulted in a tendency for reduced net protein breakdown and suggests reduced catabolism. This is in line with previous data in endotoxemic pigs treated with arginine, demonstrating reduced whole-body protein turnover, especially in the liver [21]. Since the dramatic muscle wasting that is observed in critically ill patients with sepsis is due to increased protein breakdown with normal PS [45], a potential reduction in muscle protein breakdown with arginine could help to spare muscle protein. The fact that endogenous arginine production did not similarly decrease with the decrease in protein breakdown is probably due to the increase in de novo arginine production.

Global and regional haemodynamics

The data show that the global haemodynamic profile (MAP and systemic vascular pressure) remained stable during continuous arginine infusion, with minimal need for a change in the noradrenaline dose or fluid administration. Moreover, we have also measured cardiovascular function invasively and observed a decline in HR, an increase in SV and a tendency towards increased SI, with maintenance of SVR and pulmonary pressure. We cannot rule out that these cardiovascular effects were independent of the arginine treatment and, for example, related to the ~700 ml of additional volume from arginine and isotope infusion over 8 h, since we had no placebo control in this dose–response study. However, it does indicate that the cardiopulmonary function and haemodynamic instability are at least not deteriorated when arginine is continuously infused at doses between 33 and 99 μmol·kg−1·h−1. One previous acute study in sepsis patients showed transient hypotension, an increase in CI, and decreased systemic and pulmonary vascular resistances after bolus infusion of L-arginine (200 mg·kg−1, ~15 g bolus) [46]. In a canine model of Escherichia coli peritonitis, continuous arginine infusion (10 or 100 mg·kg−1·h−1) resulted in a reduction in MAP with no changes in cardiopulmonary function [24]. A decline in MAP was also observed in the hyperdynamic endotoxemia pig model with continuous L-arginine infusion (5.3 μmol·kg−1·min−1) [21]. However, when arginine was infused as a pre-treatment before endotoxaemia in pigs, MAP was preserved [20].

In the present study, we have observed no significant alteration in the gastric Pr-aCO2 gap. Therefore, the present study provides no indication for altered mucosal perfusion and improved oxygenation with arginine infusion, as we have observed previously in the endotoxaemia pig model where hepatosplanchnic perfusion was improved [20]. The relative short duration of arginine infusion and the small number of sepsis patients with a tonometry catheter in the present study probably contributed to this non-significant finding.

No effects of arginine supplementation were observed on blood pH, PAO2/FiO2 and bicarbonate values. However, a reduction in the arterio-venous PO2 difference was present at the highest arginine dose. This could indicate shunting with reduced uptake of oxygen by the organs, but a relationship with the lowered protein turnover may be an interesting alternative hypothesis. Plasma lactate levels tended to decrease, but the unchanged base excess does not support a reduced metabolic acidosis, which we have observed previously with L-arginine treatment in the hyperdynamic pig model of sepsis in our laboratory [20]. No effects of L-arginine were observed on plasma electrolytes, except for a tendency to a decrease in plasma potassium level. This is in contrast with the previously reported increased blood potassium in normal subjects with intravenous arginine [47]. The decrease in potassium might indicate improvement of renal function, which could fit with the increased urine production. No changes in plasma glucose and insulin were observed, even though intravenous arginine is known for its stimulating effects on the release of insulin [48]. It is entirely possible that this stimulation occurs at a higher arginine dose or at bolus or enteral administration of arginine.

Conclusions, limitations and perspective

This is the first study investigating intravenous arginine supplementation under post-absorptive conditions in eight sepsis patients, demonstrating that, even in the presence of septic shock, escalating doses of arginine increased NO production without a negative impact on haemodynamic instability. Unfortunately, the hypothesized improvement in microcirculation and mucosal oxygenation with increased NO could not be demonstrated, probably due to the limited number of patients with tonometry. The observed reduction in protein breakdown with arginine infusion, as well as the observed increase in arginase activity, is of potential interest in this condition of protein wasting. Since these metabolic effects are already apparent at 66 μmol·kg−1·h−1 arginine infusion (~21 g arginine per day), this seems an appropriate dose for future studies. Placebo-controlled and longer- term studies in adequate sized patient groups are needed to confirm the specificity of metabolic effects for arginine infusion. Moreover, regarding the heterogeneity of critically ill patients and the suggestion that some sub-populations or stages of sepsis are not suitable for arginine supplementation [30], a general statement on the haemodynamic safety of arginine supplementation in sepsis patients requires care and a larger study. Alternatively to arginine, administration of a precursor of endogenous arginine synthesis, e.g. citrulline, is of potential interest. Even more so, since NOS-III and the enzymes for de novo arginine production, i.e. argininosuccinate synthase and lyase, are coupled in endothelial cells [49]. Moreover, citrulline supplementation during endotoxaemia in mice reduced intestinal microcirculatory dysfunction [50], which supports a potential role of citrulline.

AUTHOR CONTRIBUTION

All authors contributed significantly to the study; Yvette Luiking was involved in the study design, data acquisition, analysis and interpretation, and drafting the article; Martijn Poeze was involved in the study design, data acquisition, and interpretation and critical revising the article; and Nicolaas Deutz was involved in the conception and design, data analysis and interpretation, and critical revising the article. All authors read and approved the final paper.

We thank Miranda H. Hendrikx, BSc, for assisting in data collection; P. Breedveld, MD, C.H.C. Dejong, MD, PhD, F. Rubulotta, MD, and P.W. de Feiter, MD, from the Department of Surgery and Intensive Care, Maastricht University Hospital, Maastricht, The Netherlands, for their assistance in patient selection and support during the studies; and Mr Victor Wilson and Ms Myung Hee Im for statistical mixed model analyses. None of the authors had any financial or personal interest in the company sponsoring the research at the time of the study, including advisory board affiliations. Yvette Luiking is currently an employee at Nutricia Research.

FUNDING

This study was supported by a grant from the Novartis Medical Nutrition. The sponsor was not involved in data collection, analysis or interpretation.

Abbreviations

     
  • ALAT

    alanine aminotransferase

  •  
  • CI

    cardiac index

  •  
  • CRP

    C-reactive protein

  •  
  • CVP

    central venous pressure

  •  
  • CVVH

    continuous veno-venous haemofiltration

  •  
  • FiO2

    fraction of inspired oxygen

  •  
  • Fmoc

    9-fluoren-9-ylmethoxycarbonyl

  •  
  • Hep

    heparin

  •  
  • HR

    heart rate

  •  
  • ICU

    intensive care unit

  •  
  • IV

    intravenously

  •  
  • MAP

    mean arterial pressure

  •  
  • NOS

    NO synthase

  •  
  • NS

    not significant

  •  
  • PAO2

    alveolar partial pressure of oxygen

  •  
  • PCO2

    partial pressure of carbon dioxide

  •  
  • PCWP

    pulmonary capillary wedge pressure

  •  
  • PHE

    phenylalanine

  •  
  • PHE hydrox

    PHE hydroxylation

  •  
  • PO2

    partial pressure of oxygen

  •  
  • Pr-aCO2

    mucosal-arterial PCO2 difference

  •  
  • PS

    protein synthesis

  •  
  • SI

    stroke index

  •  
  • SV

    stroke volume

  •  
  • SVR

    systemic vascular resistance

  •  
  • TTR

    tracer tracee ratio

  •  
  • Wb Ra

    whole-body production

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Supplementary data