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Sepsis: An Arginine Deficiency State?

Yvette C. Luiking, PhD; Martijn Poeze, MD, PhD; Cornelis H. Dejong, MD, PhD; GrahamRamsay, 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 thecurrent knowledge about arginine metabolism in sepsis.

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

Data: In sepsis, protein breakdown is increased, which is a key process to maintain arginine delivery,because both endogenous de novoproduction from citrulline and food intake are reduced. Argininecatabolism, on the other hand, is markedly increased by enhanced use of arginine in the arginase andNO pathways. As a result, lowered plasma arginine levels are usually found. Clinical symptoms ofsepsis that are related to changes in arginine metabolism are mainly related to hemodynamicalterations and diminished microcirculation. NO administration and arginine supplementation as amonotherapy demonstrated beneficial effects, whereas nonselective NO synthase inhibition seemednot 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, wehypothesise that arginine supplementation is a logical option in the treatment of sepsis. This issupported 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 itssignificant morbidity and overall mortality rate of about 30% and generally requires intensive caretreatment. [3] Considerable efforts have been undertaken to understand the pathogenesis of sepsis andto improve its therapeutic modalities.[4] The amino acid arginine has recently received substantialattention in relation to human sepsis. However, from this discussion it became clear that knowledge ofarginine metabolism during sepsis is limited. Moreover, therapeutic interventions based on bothstimulation and inhibition of arginine metabolism have produced seemingly contradictory results.

Therefore, we will review the current knowledge about arginine metabolism in sepsis, which indicatesthat sepsis is an arginine-deficiency state. This hypothesis regarding sepsis as an arginine deficientdisease 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 detailarginine metabolism in sepsis. Subsequently, we will analyze how these changes in argininemetabolism in sepsis are related to clinical symptoms and functional effects.


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Short Resume of Arginine Metabolism Under Normal Conditions

Arginine is a semi-essential amino acid, which is mainly synthesized endogenously in the proximalrenal tubule by conversion of citrulline to arginine (Fig. 1).[5-9] Citrulline is mainly derived from intestinalconversion 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 thekidneys.[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 proteinbreakdown 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 andmitochondrial locations. ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; ODC, ornithine decarboxylase; OTC,ornithine carbamoyltransferase; NO, nitric oxide; NOS, nitric oxide synthase. Partly derived from van de Poll et al.,

[12]Wu and

Morris,[18]

Flynn et al.,[20]

and Luiking and Deutz.[70]

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 degradedto urea and ornithine by isoforms of the enzyme arginase. Type I cytosolic arginase is expressed inthe liver and is involved in detoxification of ammonia and urea synthesis, and type II mitochondrialarginase 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 anddifferentiation[22] and for synthesis of connective tissue. Due to the arginase activity in the intestinalmucosa (type II arginase), approximately 40% of arginine absorbed from the intestinal lumen isdegraded 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] NOsynthesized by NOS-2 (inducible NOS) at high levels has immunoregulatory functions, such ascontrol or killing of infectious pathogens, modulation of cytokine production, and T-helper celldevelopment, and has cytoprotective action as a free radical scavenger[26] when induced by elevatedcirculating cytokine concentrations (mainly tumor necrosis factor-, and interleukin-1, interleukin-6,and interleukin-8) or microbial products (e.g., lipopolysaccharide) during inflammatoryprocesses.[24,25,27-29] This has led to the suggestion that arginine could have a great potential as animmunomodulator[30,31] and may prove useful in catabolic conditions such as severe sepsis. [32] Apartfrom the arginase and NO breakdown route, a large part of arginine is used for protein synthesis and


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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% ofplasma arginine production is used for NO synthesis, whereas this percentage is about 15% for ureasynthesis.[14] Arginine metabolism is highly compartmentalized, which is due to the fact that theenzymes involved in arginine metabolism are expressed to a different extent in the various organsinvolved.[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 markedlydecreased compared with control values of healthy subjects or control hospital patients,[34-36] althoughother 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 concentrationswere found to be significantly lower in those patients who died of sepsis compared with patientssurviving sepsis.[34] In well-controlled animal models of sepsis, induction of sepsis decreased the totalblood amino acid concentration, including arginine. [38,39]

Reduced arginine levels in sepsis suggest that arginine metabolism has changed or transport acrossthe cell membrane is increased. Plasma arginine production, which is the total production of argininefrom protein breakdown and from de novo synthesis, was not different between pediatric septicpatients 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 frommuscle protein breakdown.[39] In particular, the intestinal-renal pathway resulting in de novoargininesynthesis from citrulline has been considered as the primary pathway responsible for maintenance ofthe plasma arginine level.[12,13,32,41] We recently observed a diminished de novoarginine synthesis inseptic patients when compared with healthy adults and with nonseptic intensive care unit patients withmoderate inflammation, in line with lowered plasma arginine level in the septic patients. [36] This maypoint 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 renalfailure. [42]

Exogenous daily arginine supply by nutritional intake is normally about 5-6 grams, [43,44] which is still asubstantial 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, argininesupply 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 beensuggested that large amounts of arginine are then metabolized into citrulline in the small bowel tobypass the liver, and citrulline is subsequently converted back to arginine in the kidney. [12,22] Thissaves arginine from being converted to urea and therefore being wasted. However, even when septicpatients 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 novoproduction of arginine is probably an important factorthat reduces the availability of arginine in sepsis. This may be worsened by reduced arginine intakeand 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 catabolicpathways[14,18,47] as described above. Substrate availability for arginine-requiring catabolic enzymesalso depends on arginine transport systems. Several arginine transporters exist, of which system y+ isthe most important and high-affinity transport mechanism, ascribed on the molecular level to cationicamino acid transporters (CATs). Of these CATs, CAT-1, CAT-2(B), and CAT-3 have been identifiedand differ in their tissue distribution.[18] These transport systems are often co-localized with thecatabolic enzymes and can thereby modulate cellular arginine metabolism.[18] For example, CAT-1arginine 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 totalintracellular pool.[47] The arginine transport systems can be modulated by bacterial endotoxins andinflammatory cytokines, which can up-regulate CAT-2 arginine transporters [49,50] and down-regulate


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CAT-1 arginine transporters.[50] As a result, transport of arginine to NOS-2 (inducible NOS) isincreased, whereas transport to NOS-3 (endothelial NOS) is decreased. Lysine, ornithine, and certainendogenous NOS inhibitors use the same transporter as arginine and may thereby compete fortransporter capacity in conditions of low arginine.[47] These mechanisms may control arginine catabolicpathways in sepsis and may also explain the arginine paradox [51,52]: the fact that endothelial NOsynthesis can be regulated by varying extracellular arginine concentrations despite the fact thatintracellular arginine concentration far exceeds the Kmof NOS-3 for arginine.[18] This, in fact, may alsoexplain some of the contradictory results seen in the experimental intervention studies. Therefore, thequestion 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] Inseptic children, the fraction of urea produced from plasma arginine was increased three-fold, whichindicates enhanced arginase activity in sepsis. [40]

By depleting the body of arginine, arginase activity is an important determining factor regarding theavailability of arginine for NO synthesis and for other metabolic pathways of arginine. [18,53] Thearginase-dependent depletion of arginine in interferon-/lipopolysaccharide-stimulated macrophagescauses 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 bacteriaexpress arginase, this may be a mechanism whereby infectious pathogens might shut down animportant 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 theelimination products nitrate/nitrite,[55-63] and has been ascribed to NOS-2 stimulation by endotoxins andcytokines.[64-66] However, discrepancies between the degree of plasma nitrate/nitrite elevation andactual NO production have been described, [39,61,67,68] which may partly be due to an effect of renalfailure on plasma nitrate levels.[69] Using stable isotope techniques to measure arginine to citrullineconversion as a measure of NO production,[70] a 1.6-fold increase in NO synthesis rate in critically illseptic pediatric patients was described,[40] which we recently confirmed in adult septic patients whencompared with healthy control values.[36] Interestingly, such an increase could not be detected whencomparing septic patients with nonseptic intensive care unit controls with moderate inflammation. [36] Inhyperdynamic septic pigs, increased NO synthesis was quantitatively matched by increased argininedisposal,[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 NOsynthesis depends for >50% on extracellular sources of arginine.[14,66,67,71-73] Although NOS-2 activityincreases during sepsis, activity of the other NOS isoforms seems down-regulated.[69,74-77] This mayreduce NO production enzyme specifically.

Endogenous NOS Inhibitors

Asymmetric dimethylarginine (ADMA) is the most powerful endogenous and competitive NOS inhibitorbecause 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 thecatabolism of posttranslationally modified proteins that contain methylated arginine residues. ADMA ismetabolized 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 ADMAlevels. High expression of dimethylaminohydrolase in the liver makes this organ important in themetabolism 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 andindependent risk factor for intensive care unit mortality. [80] Accumulation of ADMA therefore washypothesized to be a causative factor in the development of multiple organ failure by interfering with


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physiologic functions of NO production. [82] Through this pathway, ADMA may contribute to impairedblood flow in sepsis.[79] NO production could benefit from increasing the l-arginine/ADMA ratio byadditional 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. Elevatedmuscle 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 increaseduse 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 byarginase and NOS enzymes and increased protein synthesis. Due to the limited arginine availabilityand competition for arginine transporter or enzyme activity (e.g., by the endogenous NOS inhibitorADMA), 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]

Oralingestion of arginine was recently attributed a role in glucose metabolism, although not by way ofstimulated insulin secretion but by attenuating the increase in glucose and glucagon release inhealthy subjects.[86] Plasma glucose levels are in general elevated in septic patients, and recentstudies pointed at maintaining normal glucose levels by intensive insulin therapy as a therapeuticstrategy to reduce morbidity and mortality in critically ill patients. [87] It could therefore be suggestedthat 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 isprobably limited. This raises the question: what are the clinical symptoms that may be related to thesechanges in arginine metabolism?

Sepsis, particularly septic shock, is characterized by elevated cardiac output and hypotension causedby vasodilatation, associated with maldistribution of blood flow and low peripheral vascular resistance.Other rheologic abnormalities in sepsis are aggregation of neutrophils and platelets, which mayreduce blood flow and release active oxygen species that can directly damage cells. The balancebetween oxygen delivery and oxygen use may be disturbed and may contribute to a rise in bloodlactate, disturbed acid base balance, and increased gastric CO2.

[88,89] These characteristics of sepsishave been attributed to increased NO production by NOS-2,[28,90] as has been described in manyreviews.[26,64-66,69,90-93] Moreover, elevated NO production in critically ill patients was suggested to impairsubstrate/oxygen utilization by enhanced protein nitrosylation and inhibition of mitochondrialrespiration[94,95] and should therefore correlate inversely with global oxygen extraction ratios, and relatedirectly with blood lactate levels.[27]

An alternative aspect and hypothesis for the NO-related pathogenesis is the reduction of NOS-3activity. 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 beconsidered as an adaptive response to limit tissue injury in the acute setting by redistribution of bloodflow.[65] Microcirculatory shutdown and shunting in sepsis contributes to reduced blood flow and tissueoxygen delivery, with impaired oxygen extraction and increased venous Po2 as a result.

[88,97-100] Anindication of diminished microcirculation in clinical sepsis is the reduced microvascular sublingualblood flow, which was related to outcome.[101] Correspondingly, in porcine endotoxemia, liver bloodflow and oxygen delivery are both significantly reduced, with a marked increase in the hepatic oxygenextraction ratio and development of acidosis, indicative of inadequate organ perfusion.[98]Hyperperfusion and subsequent ischemia can therefore be considered present in sepsis. BecauseNOS-3-derived NO suppresses vascular smooth-muscle proliferation, inhibits platelet adhesion and


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aggregation, and interferes with leukocytes-endothelial cell interaction,[102,103] reduction of NOS-3activity could contribute to aggregation of leukocytes and platelets in capillaries, characteristic ofsepsis. NO also seems to be important for regulation of plasma volume and albumin escape in septicshock.[104] Moreover, disruption of gastrointestinal myoelectrical activity in sepsis[105,106] could be linkedto 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 animportant 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 inmodels of sepsis in terms of vascular dilation with microcirculatory recruitment and improved tissueoxygenation.[89] Only a few clinical studies on the use of vasodilators with microcirculatory monitoringare available, but the studies indicate that promotion of blood flow may also be due to improvedhemorrheology.[89]

In conclusion, clinical symptoms of sepsis that are related to changes in arginine metabolism in thiscondition are mainly hemodynamic alterations and diminished microcirculation. Increased NOproduction 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 oneside at inhibition of excessive NO production by nonselective and selective NOS-2 inhibitors and, onthe other side, at arginine supplementation or administration of NO. But what are the consequencesof 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 areavailable from clinical studies. NO inhalation in an animal model of sepsis inhibited the decrease incardiac output[109] and selectively reduced pulmonary hypertension and improved arterial oxygenationand pH, with a marked attenuation of sympathetic activation.[110,111] The NO donor nitroglycerinmarkedly increased sublingual microvascular flow in septic patients, even though arterial and central

venous pressure dropped temporarily, whereas pressure-guided resuscitation resulted in correctedblood pressure but depressed microcirculatory flow.[112] In experimental animal models of sepsis, NOdonors increase portal, mesenteric, and liver blood flow,[113-117] prevent lactic acidosis,[113] and increasecardiac 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 flowwas decreased.[114]

Thus, beneficial effects of NO administration have been described on microcirculatory flow, butwhether this results in better clinical outcome is still unknown. Therefore, we wondered whether thesame beneficial effects of NO could also be applied to supplementation of the NO precursor argininein 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 [AbbottLaboratories, Abbott Park, IL]) containing l-arginine as a component.[118-121] Several reviews onimmunonutrition in critical illness have been published.[122-128] However, conclusions regarding thebenefits and potential use in sepsis are not uniform, and recently published Canadian guidelines fornutritional support in critically ill patients recommended against the use of arginine-supplementeddiets 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 mortalitywhereas this effect was not observed for high-arginine nutrition [126,129] questions the validity of therecommendation against use of arginine-supplemented diets. Second, the study of Bower et al. [118] is


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probably confounded by the fact that not all patients tolerated enteral nutrition, and that among thesepatients, the Acute Physiology and Chronic Health Evaluation score was higher in theimmunonutrition 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-3 polyunsaturated fatty acids. Moreover, immunonutrition is, in general, supplied enterally andtherefore depends on adequate intestinal function. Well-controlled studies with l-arginine as amonotherapy or as supplied in comparison with an isonitrogenous placebo are therefore needed. Theonly study using l-arginine infusion in septic patients as a monotherapy involved a bolus l-arginineinjection, which induced transient systemic and pulmonary vasodilatory actions only at 1 min afteradministration, without further adverse effects.[130] An increase in oxygen delivery and consumptionwas also observed.[130] Further data on l-arginine treatment involve only animal studies. In thesestudies, l-arginine induced both systemic and pulmonary vasodilatation,[110,131] and, at least when givenbefore the onset of sepsis, reduced production of inflammatory variables, enhanced cellularimmunity,[132-134] and improved survival by modulating macrophage bacterial clearance mechanisms.[135]Moreover, l-arginine increased albumin and liver protein synthesis, [136] restored endothelial histologicinjury,[137] and slightly limited the increase in pulmonary arterial pressure and concomitant edemaformation. [138] In a hyperdynamic pig model, l-arginine increased muscle protein turnover, and proteinturnover was reduced in the liver.[139] We[139] therefore suggested that l-arginine reduces the severity ofthe hepatic response to tissue injury and inflammation. When l-arginine infusion was begun evenbefore initiation of endotoxin infusion in the same pig model, [139a] liver blood flow and oxygen deliveryand consumption increased. l-arginine also normalized the intestinal motility pattern in endotoxemicpigs (Bruins et al., unpublished). However, effects on survival are not uniformly positive. [134,140] In Table3, the potential metabolic pathways that may benefit from arginine supplementation in sepsis aresummarized. Beneficial effects of arginine supplementation that were observed in other clinicaldiseased states[141-144] may also apply to sepsis.

In conclusion, the previous discussion may point to a beneficial effect of arginine and NO, althoughthe effect on survival is still questionable. Does the information about the effects of NO inhibitiontherefore 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 NOsynthesis in sepsis with NG-nitro-l-arginine methyl ester or NG-monomethyl-l-arginine increasedsystemic and pulmonary blood pressure and vascular resistance, decreased cardiac output, [145-151] did

not change plasma proinflammatory cytokines,[149]

and reduced the requirement fornorepinephrine.[150,152] A decrease in plasma nitrate and increase in plasma arginine was found duringNG-monomethyl-l-arginine treatment by Watson et al.,[150] but the decrease in plasma nitrate was notconfirmed by Avontuur et al. [149] Moreover, oxygen delivery decreased, arterial oxygenation andoxygen extraction improved,[146,150,152] and pulmonary gas exchange improved.[148] However, no effectsof these NOS inhibitors were seen on renal, pulmonary, and liver function. [151-156] It was suggested thattissue oxygenation was not compromised because oxygen consumption and splanchnic oxygenation(as measured by tonometry)[146] were unaffected and because arterial lactate and pH did notchange.[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 ofnonselective NOS inhibitors in septic patients is therefore not recommended. Animal studies indicatedimproved 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 (seeSymeonides 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 improvederythrocyte deformability.[102,164] Due to the absence of an overall benefit of nonselective NOSinhibitors, 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 vascularresistances were not influenced by NOS-2 inhibition. [165] Moreover, cardiac contractility and tissueoxygen delivery and uptake improved, and the increase in monocyte reactive oxygen speciesproduction and metabolic derangements was prevented.[165-168] In addition, tyrosine nitration isprevented,[166] the endotoxin-induced impairment in liver microvascular and in portal and hepaticarterial blood flow is reduced, and liver injury is prohibited. [169] However, tissue hypoperfusion,


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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, renaldysfunction, and pancreatic injury were observed by others.[167,171-173] Short-term survival (6 to 7hrs)[165,171,174] and long-term survival improved,[175] but increased late mortality (2 days) has also beenobserved.[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 todifferences between animal and human studies. Moreover, effectiveness of NO blockade may dependon the precise stage at which treatment is given. [91] Thus, a uniform strategy for the use of NOSinhibitors is not present at the moment.

In conclusion, nonselective NOS inhibition seems not to be beneficial, and selective NOS-2 inhibitionalso did not induce overall beneficial effects. In summary, l-arginine administration as a monotherapyhas beneficial effects in animal models, and more data in septic patients with regard to its metaboliceffects and outcome are required. In general, the majority of experimental and clinical data regardingNO-donors do not support the suggested drawback of increasing NO production with l-arginine, andalso, 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 arginasepathways, and reduced availability through diminished endogenous arginine synthesis [36] and foodintake (Fig. 2). This makes arginine a near essential amino acid in sepsis, as was suggestedbefore,[177] and limits arginine availability for NO production [178] and other arginine-consumingpathways. This is illustrated by the observation that exogenous arginine administration can increaseNO production.[68]

Figure 2. Hypothesis: sepsis is an arginine deficiency state. During sepsis, arginine (Arg) catabolism by the major argininecatabolic pathways (arginase, nitric oxide synthase [NOS] and protein breakdown) is elevated. As a result of mainly increasedarginase activity, plasma arginine level drops. The endogenous arginine synthesis pathway from citrulline in the kidney doesnot respond adequately because de novoarginine production is lowered. This results in inadequate substrate delivery for nitricoxide (NO) synthesis and may impair microcirculation (NOS-3 mediated). Arginine supplementation could restore thisdeficiency.


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Although increased NO production by NOS-2 in sepsis is linked to systemic hypotension, manycharacteristics of sepsis could be linked to locally diminished NO availability. For example, impairedNO synthesis by NOS-3 may be related to loss of ability to autoregulate microcirculation.[179] IncreasedNO from NOS-2 could then be regarded as a compensatory mechanism to improve blood flow. Thiscould also explain why nonselective NOS inhibitors, which inhibit both NOS-3 and NOS-2, are notbeneficial. Arginine might improve microcirculation by increasing NO production, mainly by deliveringadequate amounts of substrate for NOS-3. The fact that in our hypothesis the reduction of NOS-3expression is important in the pathophysiology of sepsis makes the supplementation of arginine amore logical step than selective inhibition of NOS-2. Although this latter therapy may reduce theexaggerated induction of NOS-2 and therefore treat the sepsis-induced hypotension, it will notcompensate for the prolonged reduction in arginine availability, which causes a considerable numberof sepsis-related features.

The importance of adequate arginine levels and NO production is further supported by theobservations that marked reduction in serum arginine is a predictor of mortality in patients withsepsis[34] and that patients surviving septic shock had higher plasma nitrate levels thannonsurvivors.[180] However, debate exists concerning this last argument.[27,55] Adequate arginine levelsmay also reduce the production of superoxide and peroxynitrite by NOS, as this occurs in conditionsof reduced levels of arginine or cofactor tetrahydrobiopterin. [181] Moreover, arginine has anaboliceffects 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 argininesupplementation (both intravenously and enterally) on microcirculation and subsequent organfunction, on protein metabolism, and ultimately, on survival. Well-designed and controlled studies aretherefore needed.

Tables

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


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Table 2. Arginine-related Therapies in Septic Patients


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Table 3. Potential Pathways That Benefit From Arginine Supplementation inSepsis

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