Low plasma citrulline levels are associated with acute respiratory distress syndrome in patients with severe sepsis.

0.65 MB·English

by  Ware, Lorraine B

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RESEARCH Open Access
Low plasma citrulline levels are associated with
acute respiratory distress syndrome in patients
with severe sepsis
Lorraine B Ware1,2*, Jordan A Magarik1, Nancy Wickersham1, Gary Cunningham3, Todd W Rice1,
Brian W Christman1, Arthur P Wheeler1, Gordon R Bernard1 and Marshall L Summar3
Abstract
Introduction: The role of nitric oxide synthase (NOS) in the pathophysiology of acute respiratory distress syndrome
(ARDS) is not well understood. Inducible NOS is upregulated during physiologic stress; however, if NOS substrate is
insufficient then NOS can uncouple and switch from NO generation to production of damaging peroxynitrites. We
hypothesized that NOS substrate levels are low in patients with severe sepsis and that low levels of the NOS
substrate citrulline would be associated with end organ damage including ARDS in severe sepsis.
Methods: Plasma citrulline, arginine and ornithine levels and nitrate/nitrite were measured at baseline in 135
patients with severe sepsis. ARDS was diagnosed by consensus definitions.
Results: Plasma citrulline levels were below normal in all patients (median 9.2 uM, IQR 5.2 - 14.4) and were
significantly lower in ARDS compared to the no ARDS group (6.0 (3.3 - 10.4) vs. 10.1 (6.2 - 16.6), P = 0.002). The rate
of ARDS was 50% in the lowest citrulline quartile compared to 15% in the highest citrulline quartile (P = 0.002). In
multivariable analyses, citrulline levels were associated with ARDS even after adjustment for covariates including
severity of illness.
Conclusions: In severe sepsis, levels of the NOS substrate citrulline are low and are associated with ARDS. Low
NOS substrate levels have been shown in other disease states to lead to NOS uncoupling and oxidative injury
suggesting a potential mechanism for the association between low citrulline and ARDS. Further studies are needed
to determine whether citrulline supplementation could prevent the development of ARDS in patients with severe
sepsis and to determine its role in NOS coupling and function.
Introduction The proximal substrate for NO synthesis by NOS is
Theroleofnitricoxidesynthases(NOS)andtheirproduct L-arginine. L-arginine is synthesized primarily from the
nitricoxide(NO)inthepathophysiologyofclinicalacute urea cycle intermediate L-citrulline by argininosuccinate
respiratory distress syndrome (ARDS) is still not well synthase (ASS) and argininosuccinate lyase (ASL)
understood. The signaling molecule NO can regulate a (Figure 1). Experiments in our laboratories have shown
numberofprocessesimportantinthepathophysiologyof that complexing of ASS and ASL with NOS in several
ARDS [1,2] including vasculartone, platelet aggregation, tissuesresultsinsubstratechannelingofcitrullinethrough
leukocyteadhesion,andmitochondrialoxygenconsump- ASSandASLtoNOStodriveNOproduction[4].Inaddi-
tion[3].ProductionofNOiscatalyzedbythethreenitric tion, we have recently observed that in human vascular
oxide synthases (NOS), and all three isoforms of NOS endothelialcells,extracellularcitrullineandnotarginine,
(NOS-1,NOS-2andNOS-3)areexpressedinthelung. is the effective precursor of NO production. These find-
ings suggest that circulating levels of citrulline may be
*Correspondence:[email protected] morepredictiveofNOSfunctionthanargininelevels.
1DepartmentofMedicine,DivisionofAllergy,PulmonaryandCriticalCare Circulating levels of L-citrulline are dependent on the
MedicineVanderbiltUniversitySchoolofMedicine,T1218MCN,116121st
proximal urea cycle function of the gut and secondarily
AvenueS,Nashville,TN37232-2650,USA
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Wareetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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Figure1Schematicoftheintegrationofnitricoxideproductionwiththeureacycle.ARG1,arginase-1;CPSI,carbamylphosphatesynthase
inhibitor;NADPH,nicotinamideadeninedinucleotidephosphate;NAGS,N-acetylglutamatesynthase;NO,nitricoxide;OTC,ornithine
transcarbamylase;TCA,tricarboxylicacid.
theliver[5]andlowlevelsofcitrullineandargininehave includedinthisanalysis.Plasmawasdrawnfrompatients
beenreportedinadults[6,7]andchildren[8,9]withsep- aspartoftheclinicaltrialprotocolatthetimeofenroll-
sisandothercriticalillnessbuthavenotbeenpreviously ment.Aftercollection,plasmawascentrifugedtoremove
reportedinARDS.Wehypothesizedthatthephysiologic cellsandthesupernatantwasstoredinsmallaliquotsat
stressofsepsiswouldresultinoveralldecreasesincitrul- -80Cuntilthawedforaminoacidanalysis.Forinclusionin
line production to levels that could potentially lead to a theclinicaltrials,documentedorpresumedinfection,sys-
fall in NO production in the lung, and potentiate the temicinflammatoryresponsesyndrome(SIRS),andpre-
development of ARDS. We further hypothesized that sence ofshockordysfunctionoftwoother organswere
arginine levels would not be associated with ARDS required.Exclusioncriteriahavebeenpreviouslypublished
because of substrate channeling from citrulline to NO. [10,11]. The institutional review board or independent
Totestthishypothesis,wemeasuredplasmalevelsofthe ethicscommitteeateachenrollmentsiteapprovedtheclini-
NOprecursorscitrullineandarginine,aswellastheNO caltrials[10,11],asdidtheVanderbiltInstitutionalReview
products nitrate and nitrite in 135 patients with severe Board. Each participant or their surrogate provided
sepsis enrolled in two randomized clinical trials of an informedconsenttoparticipateinthestudy.Patientswere
anti-tumornecrosisfactorantibody.Forcomparison,we assessedforthepresenceofARDSbasedonconsensusdefi-
also measured plasma levels ofornithine, an amino acid nitions[12]onthedayofclinicaltrialenrollment,which
thatisnotadirectsubstrateforNOSbutisanintermedi- wasalsothedayofbaselineplasmacollection.Clinicaldata
ateintheureacycle. includingdemographics,severityofillnessscoring(Acute
PhysiologyandChronicHealthEvaluation,APACHEII)
Materials and methods [13],Brusselsorganfailurescores[14],laboratoryvalues
Patients andoutcomeswereobtainedfromthestudydatabases.
Ofthe141patientsenrolledintwophaseIIrandomized
controlledstudiesofaffinity-purified,anti-tumornecrosis Amino acid, nitrate and nitrite measurements
factor-aovinefabfragments(CytoFab)135hadbaseline PlasmaaminoacidlevelsweremeasuredbytheHitachiL-
plasma samples available for measurements and were 8800Ihighperformanceaminoacidanalyzer(Pleasanton,
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CA, USA). Plasma total nitrate and nitrite (NOx) were were more likely to be mechanically ventilated. They
measuredinduplicatebyaSievers280Ichemiluminescent were more likely to have coagulation failure and less
nitricoxideanalyzer(GE,Boulder,CO,USA). likely to have renal failure, but had a similar overall
severity of illness as measured by the APACHE II
Statistical analysis score.
AllstatisticalanalysiswasdoneusingIBMSPSSStatistics
Version 19.0 for Macintosh. Amino acid levels and NOx Amino acid levels and ARDS
levels were not normally distributed and were compared The median plasma level of citrulline was very low at
betweengroupsusingtheMann-WhitneyU-test.Correla- 9.2 uM (interquartile range (IQR) 5.2 to 14.4) compared
tionswereassessedusingSpearmanrankcorrelationcoef- to a normal range of 40 ± 10 uM in healthy adults [16].
ficients. Categorical variables were compared by chi The median plasma level of arginine was also low at
squareanalysisorFisher’sexacttestasappropriate.Multi- 22.7 uM (IQR 12.8 to 37.9) compared to a normal range
variablelogisticregressionmodelswereusedtoassessthe of 27 to 80 uM. The median level of ornithine was 22.7
potential confounding effects of clinical variables on the uM (IQR 14.3 to 39.0), which was within the normal
association of plasma citrulline levels with diagnosis of range of 13 to 64 uM.
ARDS.Potentialconfounderswereincludedinthemodel Amino acidlevelsarecomparedbetween patientswith
one at a time to assess the confounding effects of each andwithoutARDS inTable 2. Plasma levelsofcitrulline
variableontherelationshipofcitrullinetoARDS[15].In were significantly lowerinpatients withARDSat enroll-
logistic regression equations, citrulline was expressed as mentcomparedtopatientswithoutARDS(Figure2a).By
oddsratio (OR)per5uMincrement.AP-valuelessthan contrast, plasma levels of ornithine and arginine did not
orequalto0.05wasconsideredstatisticallysignificant. differ significantly between patients with and without
ARDS(Figures 2band2c). Amongpatientsinthelowest
Results quartileof plasmacitrullinelevel,theincidenceofARDS
Patients was50% comparedtoanincidenceof15%inthehighest
Patient characteristics are summarized in Table 1. quartile of plasma citrulline (P = 0.002 for trend across
Patients with ARDS at enrollment were younger and quartiles)(Figure3).
Table 1Clinical characteristics of135 patients withsevere sepsis
Characteristic Allpatients1 ARDS NoARDS P-value2
N=44 N=91
Age,years 55±16 49±17 57±16 0.011
Male,% 54 50 65 0.10
Caucasian,% 73 82 69 0.12
APACHEIIscore 24±7 24±7 24±7 0.97
WBC,×103 17.6±12.0 17.0±11.7 18.0±12.1 0.66
Creatinine,mg/dL 2.4±2.1 2.0±2.3 2.6±2.0 0.14
PaO/FiO ratio 155±88 102±51 184±91 <0.001
2 2
Mechanicallyventilatedatenrollment,% 84 95 79 0.014
Liverfailure,% 23 22 24 0.84
Renalfailure,% 41 25 41 0.01
Coagulationfailure,% 28 46 20 0.002
Mortalityat28days,% 36 39 34 0.60
1Dataaremean±SDorpercentofpatientsasindicated.2P-valueforcomparisonofpatientswithARDSorwithoutARDS.APACHEII,AcutePhysiologyand
ChronicHealthEvaluation;WBC,whitebloodcells;PaO2/FiO2,arterialtoinspiredoxygenratio.
Table 2Comparison ofbaseline plasma aminoacid andNOxlevels in patients withandwithout ARDS
Analyte ARDS1 NoARDS P-value
n=44 n=91
Citrulline,uM 6.0(3.3-10.4) 10.1(6.2-16.6) 0.002
Arginine,uM 19.3(9.6-34.5) 22.9(14.7-40.0) 0.18
Ornithine,uM 23.0(8.5-37.1) 22.3(16.0-43.2) 0.22
NOx 53.8(24.2-92.3) 49.8(34.5-80.9) 0.89
1Datapresentedasmedian(IQR).ARDS,acuterespiratorydistresssyndrome;NOx,totalnitrateandnitrite.
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Figure2Boxplotsummaryofplasmalevelsofcitrulline(panelA),arginine(panelB)andornithine(panelC)in135patientswith
severesepsis.Citrullinelevelsweresignificantlylowerinpatientswithacuterespiratorydistresssyndrome(ARDS)comparedtopatientswithout
ARDS.Horizontallinerepresentsmedian,boxencompasses25thto75thpercentile,errorbarsencompass10thto90thpercentile.*P=0.002.
Amino acid levels and non-pulmonary organ failure
Aminoacidlevelswerenotdifferentbetweenpatientswith
andwithoutrenalfailure(median9.4,IQR5.0to12.7vs.
8.3 uM, IQR 5.2 to 15.1; P = 0.68). There were also no
differencesinaminoacidlevelsbetweenpatientswithand
withoutcardiovascularfailure(shock)andliverfailureas
definedbytheBrusselsorganfailurescoringsystem.How-
ever,therewasatrendtowardslowercitrullinelevelsin
patientswithcoagulationfailure(thrombocytopenia)(P=
0.060). Plasma NOx levels were significantly higher in
patientswithrenalfailure(median70.6Um,IQR41.4to
101.1vs.40.6uM,IQR22.6to72.1;P<0.001),andplasma
NOxlevelsweresignificantlyassociatedwithserumcreati-
ninemeasurements(r=0.48;P<0.001).
Multivariable analysis
The association of plasma citrulline levels with ARDS
was assessed after adjustment for potential confounding
Figure 3 Incidence of acute respiratory distress syndrome
variables in multivariable analyses. The unadjusted OR
(ARDS)byquartileofplasmacitrullinelevel.Plasmacitrulline
levelsineachquartilewere:1stquartile0to5.12uM,2ndquartile for ARDS associated with a 5 uM decrease in plasma
5.16to8.56uM,3rdquartile9.15to14.18uM,4thquartile14.35to citrulline level was 1.50 (95% CI, 1.14, 1.98; P = 0.004).
50.07uM.Patientswiththelowestplasmacitrullinelevel Adjustment for other covariates including age, gender,
(1stquartile)hadthehighestpercentageofpatientswithARDS. ethnicity, APACHE II level, and presence of shock did
P=0.002fortrendacrossquartiles.
not alter this relationship (Table 3).
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Table 3Multivariable analyses ofassociation between plasmacitrulline levels andARDS in patients withsevere sepsis
Variable Oddsratioper5uMdecrease P-value
inplasmacitrulline(95%CI)
Unadjusted 1.50(1.14,1.98) 0.004
Adjustedfor:
Age 1.43(1.08,1.89) 0.012
Gender 1.52(1.14,2.02) 0.004
Ethnicity 1.50(1.14,1.97) 0.004
APACHEIIscore 1.50(1.14,1.98) 0.004
Presenceofshock 1.51(1.14,1.99) 0.004
ARDS,acuterespiratorydistresssyndrome;APACHEII,AcutePhysiologyandChronicHealthEvaluation.
Table 4Comparison ofplasma aminoacids and biomarkersoflung injuryby 28-day mortality in patients withARDS
(n = 44)
Analyte Lived1 Died P-value
n=27 n=17
Citrulline 5.7(3.6-9.8) 7.5(1.9-12.6) 0.56
Arginine(uM) 19.9(10.3-32.8) 18.8(7.8-37.8) 0.88
Ornithine(uM) 23.2(11.5-34.9) 20.6(5.3-41.0) 0.74
NOx 34.8(23.4-72.7) 60(42.2-124.2) 0.06
1Dataaspresentedasmedian(IQR).ARDS,acuterespiratorydistresssyndrome;NOx,totalnitrateandnitrite.
Clinical outcomes ASL-NOS system and uncoupling of NOS. This could
In patients with ARDS, amino acid levels were not sig- be an important mechanism contributing to the patho-
nificantly associated with mortality (Table 4) or ventila- genesis of ARDS, since NOS uncoupling leads to an
tor-free days (not shown). increase in superoxide production and oxidant stress.
Chronic uncoupling of NOS is important in a variety of
Discussion disease processes including diabetes [3], hypertension
We hypothesized that the physiologic stress of sepsis [3] and diastolic dysfunction [20].
would result in decreased citrulline production and that
lower circulating levels of citrulline would be associated
with the occurrence of ARDS. We observed markedly
low plasma levels of citrulline in all patients with severe
sepsis, and a significant association between lower
citrulline levels and the occurrence of ARDS in sepsis.
Furthermore, lower levels of plasma citrulline were inde-
pendently associated with a diagnosis of ARDS, even
when controlling for measures of severity of illness. The
finding of a strong association between low citrulline
levels and presence of ARDS in sepsis patients suggests
that NOS substrate deficiency might play a role in the
pathogenesis of ARDS.
NOS are modular enzymes with both a reductase and
oxygenase domain (Figure 4). Coupling of electron
Figure4Schematicofanitricoxidesynthase(NOS)dimerwith
transfer between these domains leads to synthesis of
boththeoxygenaseandreductasedomainsshown.Couplingof
NO. When uncoupled, NOS preferentially catalyzes the
electrontransferbetweenthesedomainsleadstosynthesisofnitric
production of superoxide ion from reduction of molecu- oxide(NO).Whenuncoupled,NOSpreferentiallycatalyzesthe
lar oxygen rather than NO [17]. Uncoupling of NOS productionofsuperoxideionfromreductionofmolecularoxygen
can be caused by oxidative stress [18], ischemia reperfu- ratherthanNO.UncouplingofNOScanbecausedbyoxidative
stress,ischemiareperfusionandsubstratedeficiency.Arg,arginine;
sion [19] and substrate deficiency [17]. In the current
FMN,flavinmononucleotidebindingdomain;FAD,flavinadenine
study, substrate deficiency of citrulline would be
dinucleotidebindingdomain;NADPH,nicotinamideadenine
expected to lead to insufficient precursor in the ASS- dinucleotidephosphate.
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Although we hypothesized that substrate deficiency of NOS.Furthermore,citrullineentersthecellwithrelatively
citrullinewouldleadtodecreasedNOproduction,itwas little competitionthroughneutralaminotransport,while
not possible to measure NO production prospectively in arginineusesdibasicaminoacidtransport,andwedetect
thecurrentstudy.WedidmeasureplasmalevelsofNOx, verylittlefreearginineinsidecells[25],findingsthatalso
thestablebyproductsofNOproduction.Plasmalevelsof favoraprimaryroleforcitrullineavailabilityinregulation
citrulline and arginine were not associated with plasma ofNOScoupling.
levels of NOx, nor were NOx levels associated with the Although it was not possible in this observational
presence of ARDS. Although one might predict that study to determine the mechanisms that lead to low
substratedeficiencywouldleadtoafallinNOproduction, citrulline levels in this critically ill cohort of patients
and a resultant fall in plasma NOx levels, others have with severe sepsis, there are several possible mechan-
reported that circulating NOx levels are strongly modu- isms. Decreased nutritional intake is common in the
latedbyrenalexcretionofNOx,whichisimpairedinthe early stages of critical illness and could be a potential
settingofrenalinsufficiency [21].Concordant withthese mechanism leading to low substrate levels for citrulline
findings, plasma NOx levels in our study were strongly production. However, levels of ornithine and other
and inversely associated with serum creatinine measure- amino acids such as glycine and glutamine (data not
ments,andweresignificantlyhigherinpatientswithrenal shown) were in the normal range, suggesting that
failure. In a separate cohort, we previously reported that decreased nutritional intake is not the primary driver of
lowlevelsofurinenitricoxidespecies(NOx)areaninde- low citrulline and arginine levels in this study. Gut
pendentpredictorofmortalityinpatientswithARDS[22]. enterocytes are an important source of citrulline pro-
Unfortunately,nourinewascollectedinthepresentstudy duction that can be injured in critical illness. Indeed,
foranalysisofurineNOxspecies. plasma citrulline levels have been proposed as a func-
Toourknowledge,therehavenotbeenanypriorstudies tional indicator of small bowel enterocyte mass [26].
of urea cycle products in clinical ARDS, although there Thus, decreased enterocyte production is one potential
have been several small studies in patients with sepsis. mechanism of low citrulline levels that may be relevant
Freund et al. [23] measured plasma amino acid levels in in both sepsis and ARDS [27]. Although we did not
25patientswithsepsisandfoundthatlowargininelevels measure arginase activity in this study, increases in argi-
were associated with mortality; citrulline was not mea- nase activity could also contribute to low circulating
sured in that study. Druml and colleagues [24] also citrulline levels since arginase and NOS compete for
reported low levels of plasma arginine in nine patients arginine substrate. Arginase activity can increase in the
with sepsis. In 59 critically ill children, plasma arginine setting of sepsis and oxidative stress [28]. However, in a
andcitrulline levelswerelowerinpatientswith sepsisor shock model in rats, arginase in the lung was downregu-
trauma compared to patientswithviral disease andwere lated, arguing against arginase upregulation in the lung
inverselyassociatedwithseverityofinflammationasindi- as a major determinant of local levels in the lung [29].
catedbytheplasmaC-reactiveproteinconcentration[8]. The association of low levels of plasma citrulline with
Severalstudieshaveexaminedarginineandcitrullineflux development of ARDS in patients with severe sepsis
in critical illness and sepsis. Plasma arginine levels and might have implications for the prevention and/or treat-
arginine production have been found to be low in six ment of ARDS. Citrulline repletion is already being
adults with septic shock compared to healthy controls tested in other clinical settings that are associated with
[21]. In13adults withsepsis, plasmaarginineandcitrul- NOS substrate deficiency. In pulmonary hypertension
linelevels werefoundtobelowcomparedtocontrols,as after congenital heart surgery, low levels of citrulline
was the rate of citrulline flux [9]. Luiking et al. reported have been shown to predict reduced NO production
similarfindingsofverylowcitrullinelevelsandlowrates and poor clinical outcomes [30]. In an early phase clini-
of citrulline production in 10 patients with septic shock cal trial, intravenous citrulline supplementation was safe
and concluded that the fall in citrulline production was and well tolerated in children undergoing congenital
drivenbydiminisheddenovoarginineandNOproduction heart surgery with cardiopulmonary bypass, and led to
[7].Thecurrentfindingsoflowplasmalevelsofcitrulline increases in plasma NOx and arginine levels as well as a
andarginineinpatientswithseveresepsisareconcordant reduction in post-operative pulmonary hypertension,
withthesepriorstudiesandprovideadditionalnewinfor- without any evidence of systemic changes in blood pres-
mation regarding the independent association of low sure [31]. A larger clinical trial in children undergoing
citrullinelevelswithdevelopmentofARDSinseveresep- cardiac surgery with cardiopulmonary bypass is ongoing.
sis. In light of our recent report of ASS-ASL-NOS com- This study has several strengths. A major strength is
plex formation and substrate channeling [4], citrulline the size of the study with 135 critically ill patients,
substrate availability is likely to be the primary external which to our knowledge is the largest study of NOS
factoraffectingtheabilityofcellstomaintaincouplingof substrate levels in critical illness to date. Because of the
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large size of the study we were able to test the associa- study,enrolledpatients,analyzeddataandeditedthemanuscript.MS
tion of NOS substrate levels with other organ failures conceivedanddesignedthestudy,analyzedthedataandeditedthe
manuscript.Allauthorsreadandapprovedthefinalmanuscript.
and clinical outcomes in addition to ARDS. This study
also has limitations. First, although the association Competinginterests
Theauthorsdeclaretheyhavenocompetinginterests.VanderbiltUniversity
between low levels of citrulline and ARDS was robust, it
hasappliedforandreceivedpatentsforthetherapeuticuseofintravenous
provides only indirect evidence that substrate deficiency citrulline.
leading to NOS uncoupling is important in the patho-
physiology of ARDS; we were not able to directly mea- Acknowledgements
TheresearchwassupportedbyNIHHL103836,NIHHL088263,anAmerican
sure either NO or superoxide production in this study, HeartAssociationEstablishedInvestigatorAwardandanunrestricted
and the plasma NOx measures appeared to be modu- researchgiftfromAsklepionPharmaceuticalsandUcyclydPharmaInc.
lated by renal failure. In addition, ARDS could be a
Authordetails
cause rather than a consequence of low substrate levels. 1DepartmentofMedicine,DivisionofAllergy,PulmonaryandCriticalCare
A second limitation is that we did not measure tetrahy- MedicineVanderbiltUniversitySchoolofMedicine,T1218MCN,116121st
AvenueS,Nashville,TN37232-2650,USA.2DepartmentofPathology,
drobiopterin (BH ) levels. BH is a NOS cofactor that is
4 4 MicrobiologyandImmunology,VanderbiltUniversitySchoolofMedicine,
normally bound to the NOS oxygenase domain [19]. 116121stAvenueS,Nashville,TN37232,USA.3DepartmentofPediatrics,
BH levels can be reduced in the setting of oxidative DivisionofGeneticsandMetabolism,Children’sNationalMedicalCenter,111
4
stress [18] and ischemia reperfusion [19]. However, like MichiganAvenue,NWWashington,DC20010,USA.
NOx, circulating BH4 levels are affected by renal failure Received:11July2012 Revised:26October2012
[32], and it is not clear that circulating levels are a good Accepted:9January2013 Published:17January2013
indicator of potential BH effects on NOS. A final lim-
4
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