Sepsis: An Arginine Deficiency State?

Yvette C. Luiking, PhD; Martijn Poeze, MD, PhD; Cornelis H. Dejong, MD, PhD; Graham
Ramsay, MD, PhD; Nicolaas E. Deutz, MD, PhD

Crit Care Med 32(10):2135-2145, 2004. © 2004 Lippincott Williams & Wilkins

Posted 10/27/2004

Abstract and Introduction

Abstract

Objective: Sepsis is a major health problem considering its significant morbidity and mortality rate.
The amino acid l-arginine has recently received substantial attention in relation to human sepsis.
However, knowledge of arginine metabolism during sepsis is limited. Therefore, we reviewed the
current knowledge about arginine metabolism in sepsis.

Data Source: This review summarizes the literature on arginine metabolism both in general and in
relation to sepsis. Moreover, arginine-related therapies are reviewed and discussed, which includes
therapies of both nitric oxide (NO) and arginine administration and therapies directed toward inhibition
of NO.

Data: In sepsis, protein breakdown is increased, which is a key process to maintain arginine delivery,
because both endogenous de novo production from citrulline and food intake are reduced. Arginine
catabolism, on the other hand, is markedly increased by enhanced use of arginine in the arginase and
NO pathways. As a result, lowered plasma arginine levels are usually found. Clinical symptoms of
sepsis that are related to changes in arginine metabolism are mainly related to hemodynamic
alterations and diminished microcirculation. NO administration and arginine supplementation as a
monotherapy demonstrated beneficial effects, whereas nonselective NO synthase inhibition seemed
not to be beneficial, and selective NO synthase 2 inhibition was not beneficial overall.

Conclusions: Because sepsis has all the characteristics of an arginine-deficiency state, we

hypothesise that arginine supplementation is a logical option in the treatment of sepsis. This is
supported by substantial experimental and clinical data on NO donors and NO inhibitors. However,
further evidence is required to prove our hypothesis.

Introduction

Sepsis is defined as a systemic response to an infection.[1,2] It is a major health problem because of its
significant morbidity and overall mortality rate of about 30% and generally requires intensive care
treatment.[3] Considerable efforts have been undertaken to understand the pathogenesis of sepsis and
to improve its therapeutic modalities.[4] The amino acid arginine has recently received substantial
attention in relation to human sepsis. However, from this discussion it became clear that knowledge of
arginine metabolism during sepsis is limited. Moreover, therapeutic interventions based on both
stimulation and inhibition of arginine metabolism have produced seemingly contradictory results.
Therefore, we will review the current knowledge about arginine metabolism in sepsis, which indicates
that sepsis is an arginine-deficiency state. This hypothesis regarding sepsis as an arginine deficient
disease makes arginine supplementation a logical option in the treatment of sepsis.

Arginine Metabolism in Sepsis

In this section, we will shortly review the metabolism of arginine in general and describe in more detail
arginine metabolism in sepsis. Subsequently, we will analyze how these changes in arginine
metabolism in sepsis are related to clinical symptoms and functional effects.
Short Resume of Arginine Metabolism Under Normal Conditions

Arginine is a semi-essential amino acid, which is mainly synthesized endogenously in the proximal
renal tubule by conversion of citrulline to arginine (Fig. 1).[5-9] Citrulline is mainly derived from intestinal
conversion of arterial (mainly muscle derived) and luminal glutamine through the glutamate-to-
ornithine pathway,[6,10-13] and about 83% of the intestinal release of citrulline is taken up by the
kidneys.[6] Under normal conditions, this pathway contributes about 10-15% to whole-body arginine
production.[13,14] Besides endogenous production from citrulline, arginine is also available from protein
breakdown and food intake, with the jejunum as the major site of intestinal absorption.

Figure 1. Metabolic pathways of arginine. Pathways are compartmentalized to different organs and to cytosolic and
mitochondrial locations. ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; ODC, ornithine decarboxylase; OTC,
[12]
ornithine carbamoyltransferase; NO, nitric oxide; NOS, nitric oxide synthase. Partly derived from van de Poll et al., Wu and
[18] [20] [70]
Morris, Flynn et al., and Luiking and Deutz.

Four major metabolic pathways for arginine exist (refer to Morris,[15] Kelm,[16] Boger and Bode-Boger,[17]
Wu and Morris,[18] Cynober et al.,[19] and Flynn et al.[20] for recent reviews). First, arginine is degraded
to urea and ornithine by isoforms of the enzyme arginase. Type I cytosolic arginase is expressed in
the liver and is involved in detoxification of ammonia and urea synthesis, and type II mitochondrial
arginase is expressed at low levels in extrahepatic tissues and is involved in synthesis of ornithine,
proline, and glutamate.[21] Via ornithine and the polyamines, arginine is important for cell growth and
differentiation[22] and for synthesis of connective tissue. Due to the arginase activity in the intestinal
mucosa (type II arginase), approximately 40% of arginine absorbed from the intestinal lumen is
degraded in the first pass.[23] A second metabolic pathway of arginine is the synthesis of nitric oxide
(NO) by isoforms of the enzyme NO synthase (NOS) with concomitant formation of citrulline.[24,25]
Basically, three isoforms of NOS exist. NO synthesized by NOS-1 (neuronal NOS) and NOS-3
(endothelial NOS) enzymes acts as a neurotransmitter and as vasodilator, respectively.[25] NO
synthesized by NOS-2 (inducible NOS) at high levels has immunoregulatory functions, such as
control or killing of infectious pathogens, modulation of cytokine production, and T-helper cell
development, and has cytoprotective action as a free radical scavenger[26] when induced by elevated
circulating cytokine concentrations (mainly tumor necrosis factor-α, and interleukin-1, interleukin-6,
and interleukin-8) or microbial products (e.g., lipopolysaccharide) during inflammatory
processes.[24,25,27-29] This has led to the suggestion that arginine could have a great potential as an
immunomodulator[30,31] and may prove useful in catabolic conditions such as severe sepsis.[32] Apart
from the arginase and NO breakdown route, a large part of arginine is used for protein synthesis and
hence disappears from the circulation, and arginine is also involved in the biosynthesis of creatine
(the precursor of creatinine) and the synthesis of agmatine.[18] Under normal conditions, about 1.2% of
plasma arginine production is used for NO synthesis, whereas this percentage is about 15% for urea
synthesis.[14] Arginine metabolism is highly compartmentalized, which is due to the fact that the
enzymes involved in arginine metabolism are expressed to a different extent in the various organs
involved.[18] Finally, arginine stimulates secretion of several hormones.[33]

Arginine Availability in Sepsis

In septic patients, plasma and intracellular muscle arginine levels were found to be markedly
decreased compared with control values of healthy subjects or control hospital patients,[34-36] although
other amino acids besides arginine may also decrease,[35] and a decrease may also exist in nonseptic,
stressed patients[37] (Table 1). An additional important factor is that plasma arginine concentrations
were found to be significantly lower in those patients who died of sepsis compared with patients
surviving sepsis.[34] In well-controlled animal models of sepsis, induction of sepsis decreased the total
blood amino acid concentration, including arginine.[38,39]

Reduced arginine levels in sepsis suggest that arginine metabolism has changed or transport across
the cell membrane is increased. Plasma arginine production, which is the total production of arginine
from protein breakdown and from de novo synthesis, was not different between pediatric septic
patients and healthy adults[40] or between adult septic patients and nonseptic intensive care unit
controls.[36] However, arginine production during endotoxemia in pigs was increased, mainly from
muscle protein breakdown.[39] In particular, the intestinal-renal pathway resulting in de novo arginine
synthesis from citrulline has been considered as the primary pathway responsible for maintenance of
the plasma arginine level.[12,13,32,41] We recently observed a diminished de novo arginine synthesis in
septic patients when compared with healthy adults and with nonseptic intensive care unit patients with
moderate inflammation, in line with lowered plasma arginine level in the septic patients.[36] This may
point to lack of adaptation to the enhanced arginine need in sepsis, which is normally through up-
regulation of endogenous arginine production, and may be due to lack of citrulline[9,36] or renal
failure.[42]

Exogenous daily arginine supply by nutritional intake is normally about 5-6 grams,[43,44] which is still a
substantial amount compared with the endogenous daily arginine production of about 15-20 gram.[14,45]
Because septic patients are often not fed during their initial stay in the intensive care unit, arginine
supply relies completely on endogenous arginine synthesis. The importance of endogenous arginine
synthesis is also demonstrated by the normal physiologic adaptation to a low-protein diet. It has been
suggested that large amounts of arginine are then metabolized into citrulline in the small bowel to
bypass the liver, and citrulline is subsequently converted back to arginine in the kidney.[12,22] This
saves arginine from being converted to urea and therefore being wasted. However, even when septic
patients are fed, arginine availability may still be compromised due to impaired intestinal absorption[46]
or impaired intestinal function through citrulline production[36] when nutrition is given enterally.

In conclusion, diminished endogenous de novo production of arginine is probably an important factor

that reduces the availability of arginine in sepsis. This may be worsened by reduced arginine intake
and increased arginine catabolism, as is discussed in detail in the next paragraph.

Arginine Catabolism in Sepsis

Arginine catabolism involves multiple organs and compartmentalization of different catabolic

pathways[14,18,47] as described above. Substrate availability for arginine-requiring catabolic enzymes
also depends on arginine transport systems. Several arginine transporters exist, of which system y+ is
the most important and high-affinity transport mechanism, ascribed on the molecular level to cationic
amino acid transporters (CATs). Of these CATs, CAT-1, CAT-2(B), and CAT-3 have been identified
and differ in their tissue distribution.[18] These transport systems are often co-localized with the
catabolic enzymes and can thereby modulate cellular arginine metabolism.[18] For example, CAT-1
arginine transporter and endothelial NOS enzyme are co-localized in plasma membrane caveolae.[48]
By this way, arginine is specifically channelled to NO production and does not mix with the total
intracellular pool.[47] The arginine transport systems can be modulated by bacterial endotoxins and
inflammatory cytokines, which can up-regulate CAT-2 arginine transporters[49,50] and down-regulate
CAT-1 arginine transporters.[50] As a result, transport of arginine to NOS-2 (inducible NOS) is
increased, whereas transport to NOS-3 (endothelial NOS) is decreased. Lysine, ornithine, and certain
endogenous NOS inhibitors use the same transporter as arginine and may thereby compete for
transporter capacity in conditions of low arginine.[47] These mechanisms may control arginine catabolic
pathways in sepsis and may also explain the arginine paradox[51,52]: the fact that endothelial NO
synthesis can be regulated by varying extracellular arginine concentrations despite the fact that
intracellular arginine concentration far exceeds the Km of NOS-3 for arginine.[18] This, in fact, may also
explain some of the contradictory results seen in the experimental intervention studies. Therefore, the
question arises of how the different arginine catabolic pathways are affected in sepsis?

Arginase Pathways

The enzyme arginase is a large arginine consumer. Because arginase activity is increased in sepsis,
arginase activity may be an important regulatory factor for arginine availability and function.[53] In
septic children, the fraction of urea produced from plasma arginine was increased three-fold, which
indicates enhanced arginase activity in sepsis.[40]

By depleting the body of arginine, arginase activity is an important determining factor regarding the
availability of arginine for NO synthesis and for other metabolic pathways of arginine.[18,53] The
arginase-dependent depletion of arginine in interferon-γ/lipopolysaccharide-stimulated macrophages
causes an anti-inflammatory cytokine interleukin-13-mediated down-regulation of NOS-2 protein,
which can be restored by l-arginine administration.[54] Because not only macrophages but also bacteria
express arginase, this may be a mechanism whereby infectious pathogens might shut down an
important effector arm of the immune response locally and prolong their own survival.[54]

Arginine-NO Pathway

Elevated NO synthesis has been suggested in sepsis, based on elevated plasma levels of the
elimination products nitrate/nitrite,[55-63] and has been ascribed to NOS-2 stimulation by endotoxins and
cytokines.[64-66] However, discrepancies between the degree of plasma nitrate/nitrite elevation and
actual NO production have been described,[39,61,67,68] which may partly be due to an effect of renal
failure on plasma nitrate levels.[69] Using stable isotope techniques to measure arginine to citrulline
conversion as a measure of NO production,[70] a 1.6-fold increase in NO synthesis rate in critically ill
septic pediatric patients was described,[40] which we recently confirmed in adult septic patients when
compared with healthy control values.[36] Interestingly, such an increase could not be detected when
comparing septic patients with nonseptic intensive care unit controls with moderate inflammation.[36] In
hyperdynamic septic pigs, increased NO synthesis was quantitatively matched by increased arginine
disposal,[39] which confirms the link between arginine availability and NO synthesis.

Reduced arginine availability may limit NO synthesis because provision of the arginine pool for NO
synthesis depends for >50% on extracellular sources of arginine.[14,66,67,71-73] Although NOS-2 activity
increases during sepsis, activity of the other NOS isoforms seems down-regulated.[69,74-77] This may
reduce NO production enzyme specifically.

Endogenous NOS Inhibitors

Asymmetric dimethylarginine (ADMA) is the most powerful endogenous and competitive NOS inhibitor
because it competes with l-arginine for the active site of NOS (not NOS isoform specific) and for y+-
mediated uptake into cells (refer to Leiper and Vallance[78] for review). ADMA is derived from the
catabolism of posttranslationally modified proteins that contain methylated arginine residues. ADMA is
metabolized by dimethylaminohydrolase to citrulline and methylamines, and it is excreted in urine.[79]
Increased protein catabolism and impaired renal function could therefore contribute to elevated ADMA
levels. High expression of dimethylaminohydrolase in the liver makes this organ important in the
metabolism of ADMA and hepatic dysfunction a prominent determinant of ADMA concentration.[79-82]

In critically ill patients, elevated ADMA levels have been described and were considered a strong and
independent risk factor for intensive care unit mortality.[80] Accumulation of ADMA therefore was
hypothesized to be a causative factor in the development of multiple organ failure by interfering with
physiologic functions of NO production.[82] Through this pathway, ADMA may contribute to impaired
blood flow in sepsis.[79] NO production could benefit from increasing the l-arginine/ADMA ratio by
additional l-arginine supplementation.

Arginine for Protein Synthesis and as an Energy Source

Arginine is also an important constituent of proteins and therefore is required for protein synthesis.
Increased synthesis of acute-phase proteins and up-regulated enzymes occurs in sepsis. Elevated
muscle protein synthesis was also observed in an experimental model of sepsis in pigs.[83] Moreover,
increased arginine oxidation is observed during sepsis in pediatric patients[40] and indicates increased
use of arginine as an energy source, at least in children with sepsis.

In conclusion, arginine consumption is enhanced in sepsis, mainly due to increased catabolism by

arginase and NOS enzymes and increased protein synthesis. Due to the limited arginine availability
and competition for arginine transporter or enzyme activity (e.g., by the endogenous NOS inhibitor
ADMA), arginine is to an important extent the limiting factor for NO production.

Arginine-Related Hormonal Release in Sepsis

Another aspect regarding a role of arginine in sepsis involves arginine-stimulated hormonal release,
including insulin release.[84] Failure of insulin production has been described in sepsis.[85] Oral
ingestion of arginine was recently attributed a role in glucose metabolism, although not by way of
stimulated insulin secretion but by attenuating the increase in glucose and glucagon release in
healthy subjects.[86] Plasma glucose levels are in general elevated in septic patients, and recent
studies pointed at maintaining normal glucose levels by intensive insulin therapy as a therapeutic
strategy to reduce morbidity and mortality in critically ill patients.[87] It could therefore be suggested
that reduced arginine availability may affect glucose homeostasis in sepsis.

Relevance of Changes in Arginine Metabolism to Characteristics of Sepsis

As described above, changes in arginine metabolism are present in sepsis, and arginine availability is
probably limited. This raises the question: what are the clinical symptoms that may be related to these
changes in arginine metabolism?

Sepsis, particularly septic shock, is characterized by elevated cardiac output and hypotension caused
by vasodilatation, associated with maldistribution of blood flow and low peripheral vascular resistance.
Other rheologic abnormalities in sepsis are aggregation of neutrophils and platelets, which may
reduce blood flow and release active oxygen species that can directly damage cells. The balance
between oxygen delivery and oxygen use may be disturbed and may contribute to a rise in blood
lactate, disturbed acid base balance, and increased gastric CO2.[88,89] These characteristics of sepsis
have been attributed to increased NO production by NOS-2,[28,90] as has been described in many
reviews.[26,64-66,69,90-93] Moreover, elevated NO production in critically ill patients was suggested to impair
substrate/oxygen utilization by enhanced protein nitrosylation and inhibition of mitochondrial
respiration[94,95] and should therefore correlate inversely with global oxygen extraction ratios, and relate
directly with blood lactate levels.[27]

An alternative aspect and hypothesis for the NO-related pathogenesis is the reduction of NOS-3
activity. NOS-3 expression is, under normal conditions, related to maintenance of organ perfusion.
During sepsis, NOS-3 expression is decreased.[75,96] Increased NOS-2 expression could then be
considered as an adaptive response to limit tissue injury in the acute setting by redistribution of blood
flow.[65] Microcirculatory shutdown and shunting in sepsis contributes to reduced blood flow and tissue
oxygen delivery, with impaired oxygen extraction and increased venous Po2 as a result.[88,97-100] An
indication of diminished microcirculation in clinical sepsis is the reduced microvascular sublingual
blood flow, which was related to outcome.[101] Correspondingly, in porcine endotoxemia, liver blood
flow and oxygen delivery are both significantly reduced, with a marked increase in the hepatic oxygen
extraction ratio and development of acidosis, indicative of inadequate organ perfusion.[98]
Hyperperfusion and subsequent ischemia can therefore be considered present in sepsis. Because
NOS-3-derived NO suppresses vascular smooth-muscle proliferation, inhibits platelet adhesion and
aggregation, and interferes with leukocytes-endothelial cell interaction,[102,103] reduction of NOS-3
activity could contribute to aggregation of leukocytes and platelets in capillaries, characteristic of
sepsis. NO also seems to be important for regulation of plasma volume and albumin escape in septic
shock.[104] Moreover, disruption of gastrointestinal myoelectrical activity in sepsis[105,106] could be linked
to diminished NO.[107] Thus, several features of sepsis refer back to reduced local NO synthesis,
probably through reduced NOS-3 activity.

The general therapy in sepsis is directed toward correction of global hemodynamic variables as an
important clinical target, but global hemodynamics is not an indication of adequate tissue flow.[108]
There is ample evidence from experimental animal studies for the beneficial effects of vasodilators in
models of sepsis in terms of vascular dilation with microcirculatory recruitment and improved tissue
oxygenation.[89] Only a few clinical studies on the use of vasodilators with microcirculatory monitoring
are available, but the studies indicate that promotion of blood flow may also be due to improved
hemorrheology.[89]

In conclusion, clinical symptoms of sepsis that are related to changes in arginine metabolism in this
condition are mainly hemodynamic alterations and diminished microcirculation. Increased NO
production by NOS-2 and diminished NO production by NOS-3 seem to be important factors.

Arginine-related Therapies in Sepsis

As a result of the different hypotheses regarding the role of NO in the pathogenesis of sepsis,
different arginine-related therapies have been used (Table 2). These therapies are directed on one
side at inhibition of excessive NO production by nonselective and selective NOS-2 inhibitors and, on
the other side, at arginine supplementation or administration of NO. But what are the consequences
of these therapies, and which is the most beneficial in sepsis?

NO Administration

NO can be administered by NO inhalation or by administration of NO donors, but few data are

available from clinical studies. NO inhalation in an animal model of sepsis inhibited the decrease in
cardiac output[109] and selectively reduced pulmonary hypertension and improved arterial oxygenation
and pH, with a marked attenuation of sympathetic activation.[110,111] The NO donor nitroglycerin
markedly increased sublingual microvascular flow in septic patients, even though arterial and central
venous pressure dropped temporarily, whereas pressure-guided resuscitation resulted in corrected
blood pressure but depressed microcirculatory flow.[112] In experimental animal models of sepsis, NO
donors increase portal, mesenteric, and liver blood flow,[113-117] prevent lactic acidosis,[113] and increase
cardiac index and left ventricular stroke work index.[114,116] Arterial and pulmonary pressures were,
however, not affected by the NO donor 3-morpholinosydnonimine (SIN-1), whereas renal blood flow
was decreased.[114]

Thus, beneficial effects of NO administration have been described on microcirculatory flow, but
whether this results in better clinical outcome is still unknown. Therefore, we wondered whether the
same beneficial effects of NO could also be applied to supplementation of the NO precursor arginine
in sepsis?

Arginine Supplementation

Data on enteral l-arginine supplementation in septic patients are limited to immunonutrition (IMPACT
[Novartis Nutrition, Minneapolis, MN], Immun-Aid [B Braun, Irvine, CA], and Perative [Abbott
Laboratories, Abbott Park, IL]) containing l-arginine as a component.[118-121] Several reviews on
immunonutrition in critical illness have been published.[122-128] However, conclusions regarding the
benefits and potential use in sepsis are not uniform, and recently published Canadian guidelines for
nutritional support in critically ill patients recommended against the use of arginine-supplemented
diets in critically ill patients due to lack of treatment effect with respect to mortality and infections.[126,129]
However, the observation that specifically low-arginine nutrition coincides with higher mortality
whereas this effect was not observed for high-arginine nutrition[126,129] questions the validity of the
recommendation against use of arginine-supplemented diets. Second, the study of Bower et al.[118] is
probably confounded by the fact that not all patients tolerated enteral nutrition, and that among these
patients, the Acute Physiology and Chronic Health Evaluation score was higher in the
immunonutrition group. Third, the major difficulty of drawing conclusions about arginine from arginine-
containing immunonutrition is that, in addition to arginine, the formulae also contain nucleotides and
ω-3 polyunsaturated fatty acids. Moreover, immunonutrition is, in general, supplied enterally and
therefore depends on adequate intestinal function. Well-controlled studies with l-arginine as a
monotherapy or as supplied in comparison with an isonitrogenous placebo are therefore needed. The
only study using l-arginine infusion in septic patients as a monotherapy involved a bolus l-arginine
injection, which induced transient systemic and pulmonary vasodilatory actions only at 1 min after
administration, without further adverse effects.[130] An increase in oxygen delivery and consumption
was also observed.[130] Further data on l-arginine treatment involve only animal studies. In these
studies, l-arginine induced both systemic and pulmonary vasodilatation,[110,131] and, at least when given
before the onset of sepsis, reduced production of inflammatory variables, enhanced cellular
immunity,[132-134] and improved survival by modulating macrophage bacterial clearance mechanisms.[135]
Moreover, l-arginine increased albumin and liver protein synthesis,[136] restored endothelial histologic
injury,[137] and slightly limited the increase in pulmonary arterial pressure and concomitant edema
formation.[138] In a hyperdynamic pig model, l-arginine increased muscle protein turnover, and protein
turnover was reduced in the liver.[139] We[139] therefore suggested that l-arginine reduces the severity of
the hepatic response to tissue injury and inflammation. When l-arginine infusion was begun even
before initiation of endotoxin infusion in the same pig model,[139a] liver blood flow and oxygen delivery
and consumption increased. l-arginine also normalized the intestinal motility pattern in endotoxemic
pigs (Bruins et al., unpublished). However, effects on survival are not uniformly positive.[134,140] In Table
3, the potential metabolic pathways that may benefit from arginine supplementation in sepsis are
summarized. Beneficial effects of arginine supplementation that were observed in other clinical
diseased states[141-144] may also apply to sepsis.

In conclusion, the previous discussion may point to a beneficial effect of arginine and NO, although
the effect on survival is still questionable. Does the information about the effects of NO inhibition
therefore help us in further understanding of the beneficial effects of arginine in sepsis?

NO Inhibition

In human sepsis, only nonselective NOS inhibitors have been used. Prolonged inhibition of NO
synthesis in sepsis with NG-nitro-l-arginine methyl ester or NG-monomethyl-l-arginine increased
systemic and pulmonary blood pressure and vascular resistance, decreased cardiac output,[145-151] did
not change plasma proinflammatory cytokines,[149] and reduced the requirement for
norepinephrine.[150,152] A decrease in plasma nitrate and increase in plasma arginine was found during
NG-monomethyl-l-arginine treatment by Watson et al.,[150] but the decrease in plasma nitrate was not
confirmed by Avontuur et al.[149] Moreover, oxygen delivery decreased, arterial oxygenation and
oxygen extraction improved,[146,150,152] and pulmonary gas exchange improved.[148] However, no effects
of these NOS inhibitors were seen on renal, pulmonary, and liver function.[151-156] It was suggested that
tissue oxygenation was not compromised because oxygen consumption and splanchnic oxygenation
(as measured by tonometry)[146] were unaffected and because arterial lactate and pH did not
change.[146,153] Although short-term resolution of shock was higher in septic patients treated with NG-
monomethyl-l-arginine,[156] this did not improve long-term survival.[146,153-157] A subsequent international,
multiple-center, phase III study investigating nonselective NOS inhibition (NG-monomethyl-l-arginine)
in septic patients was terminated because of increased mortality in the treatment group.[151] The use of
nonselective NOS inhibitors in septic patients is therefore not recommended. Animal studies indicated
improved systemic vascular response[109,131,138,158] but impaired organ perfusion[114,159-162] or reduced
oxygen supply[98] with tissue damage.[163] Increased mortality was reported in several studies (see
Symeonides and Balk[64] and Kirkeboen and Strand[69] for reviews). Using nonselective NOS-inhibitors,
NO was suggested to have beneficial effects on microcirculation by anti-adhesive and improved
erythrocyte deformability.[102,164] Due to the absence of an overall benefit of nonselective NOS
inhibitors, selective NOS-2 inhibitors came into use, but they have only been applied in animals.
These inhibitors in general prevent hypotension, but the increase in pulmonary pressure and vascular
resistances were not influenced by NOS-2 inhibition.[165] Moreover, cardiac contractility and tissue
oxygen delivery and uptake improved, and the increase in monocyte reactive oxygen species
production and metabolic derangements was prevented.[165-168] In addition, tyrosine nitration is
prevented,[166] the endotoxin-induced impairment in liver microvascular and in portal and hepatic
arterial blood flow is reduced, and liver injury is prohibited.[169] However, tissue hypoperfusion,
ischemia and decreased oxygen delivery,[170] and acidosis[165] have also been reported. In addition,
absence of changes in liver morphology and in hepatocellular injury, intestinal mucosal injury, renal
dysfunction, and pancreatic injury were observed by others.[167,171-173] Short-term survival (6 to 7
hrs)[165,171,174] and long-term survival improved,[175] but increased late mortality (2 days) has also been
observed.[176]

Differences between animal (mainly small vs. large mammals) and sepsis models (time and dose of
administration) may contribute to the divergent results in experimental studies and contribute to
differences between animal and human studies. Moreover, effectiveness of NO blockade may depend
on the precise stage at which treatment is given.[91] Thus, a uniform strategy for the use of NOS
inhibitors is not present at the moment.

In conclusion, nonselective NOS inhibition seems not to be beneficial, and selective NOS-2 inhibition
also did not induce overall beneficial effects. In summary, l-arginine administration as a monotherapy
has beneficial effects in animal models, and more data in septic patients with regard to its metabolic
effects and outcome are required. In general, the majority of experimental and clinical data regarding
NO-donors do not support the suggested drawback of increasing NO production with l-arginine, and
also, NOS inhibition is not beneficial overall.

Conclusion and Hypothesis

Sepsis is an Arginine Deficiency State

In sepsis, arginine availability is reduced by increased arginine catabolism, mainly through arginase
pathways, and reduced availability through diminished endogenous arginine synthesis[36] and food
intake (Fig. 2). This makes arginine a near essential amino acid in sepsis, as was suggested
before,[177] and limits arginine availability for NO production[178] and other arginine-consuming
pathways. This is illustrated by the observation that exogenous arginine administration can increase
NO production.[68]

Figure 2. Hypothesis: sepsis is an arginine deficiency state. During sepsis, arginine (Arg) catabolism by the major arginine
catabolic pathways (arginase, nitric oxide synthase [NOS] and protein breakdown) is elevated. As a result of mainly increased
arginase activity, plasma arginine level drops. The endogenous arginine synthesis pathway from citrulline in the kidney does
not respond adequately because de novo arginine production is lowered. This results in inadequate substrate delivery for nitric
oxide (NO) synthesis and may impair microcirculation (NOS-3 mediated). Arginine supplementation could restore this
deficiency.
Although increased NO production by NOS-2 in sepsis is linked to systemic hypotension, many
characteristics of sepsis could be linked to locally diminished NO availability. For example, impaired
NO synthesis by NOS-3 may be related to loss of ability to autoregulate microcirculation.[179] Increased
NO from NOS-2 could then be regarded as a compensatory mechanism to improve blood flow. This
could also explain why nonselective NOS inhibitors, which inhibit both NOS-3 and NOS-2, are not
beneficial. Arginine might improve microcirculation by increasing NO production, mainly by delivering
adequate amounts of substrate for NOS-3. The fact that in our hypothesis the reduction of NOS-3
expression is important in the pathophysiology of sepsis makes the supplementation of arginine a
more logical step than selective inhibition of NOS-2. Although this latter therapy may reduce the
exaggerated induction of NOS-2 and therefore treat the sepsis-induced hypotension, it will not
compensate for the prolonged reduction in arginine availability, which causes a considerable number
of sepsis-related features.

The importance of adequate arginine levels and NO production is further supported by the
observations that marked reduction in serum arginine is a predictor of mortality in patients with
sepsis[34] and that patients surviving septic shock had higher plasma nitrate levels than
nonsurvivors.[180] However, debate exists concerning this last argument.[27,55] Adequate arginine levels
may also reduce the production of superoxide and peroxynitrite by NOS, as this occurs in conditions
of reduced levels of arginine or cofactor tetrahydrobiopterin.[181] Moreover, arginine has anabolic
effects and contributes to protein synthesis (e.g., acute phase proteins).[139] Although arginine-
containing immunonutrition did not improve survival, further studies are needed with arginine
supplementation as a monotherapy. It would be interesting to focus on the effect of arginine
supplementation (both intravenously and enterally) on microcirculation and subsequent organ
function, on protein metabolism, and ultimately, on survival. Well-designed and controlled studies are
therefore needed.

Tables

Table 1. Summary of Factors That Affect Arginine Availability in Sepsis

Table 2. Arginine-related Therapies in Septic Patients
Table 3. Potential Pathways That Benefit From Arginine Supplementation in
Sepsis

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