REVISTA
BRASILEIRA
DE
REUMATOLOGIA
www . r e u m a t o l o g i a . c o m . b r
Original
article
Higher
nitric
oxide
levels
are
associated
with
disease
activity
in
Egyptian
rheumatoid
arthritis
patients
Adel
Mahmoud
Ali,
Reem
Abdelmonem
Habeeb
∗,
Noran
Osama
El-Azizi,
Dina
Aziz
Khattab,
Rania
Ahmed
Abo-Shady,
Rania
Hamdy
Elkabarity
DepartamentodeMedicinaInterna,DivisãodeReumatologiaeDepartamentodePatologiaClínica,UniversidadeAinShams,Cairo,Egipt
a
r
t
i
c
l
e
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n
f
o
Articlehistory:
Received10April2013 Accepted6July2014
Availableonline23October2014
Keywords:
Rheumatoidarthritis(RA) Oxidativestress
Nitricoxide(NO)
a
b
s
t
r
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c
t
Background:Oxidative stressgeneratedwithininflammatoryjoints canproduce autoim-munephenomenaandjointdestruction.Radicalspecieswithoxidativeactivity,including reactivenitrogenspecies,representmediatorsofinflammationandcartilagedamage. Objectives:ToassessserumnitricoxideasamarkerofoxidativestressinEgyptianpatients withrheumatoidarthritisanditsrelationtodiseaseactivity.
Methods:Eightypatientswithrheumatoidarthritisweredividedinto2groups,accordingto theDAS-28score:GroupI:42patientswithdiseaseactivity,andGroupII:38patientswith nodiseaseactivity.Fortyage-andsex-matchedindividualswereincludedascontrolgroup (GroupIII).Routinelaboratoryinvestigationsweredone,andnitricoxidewasmeasured usingElisa.Handplainradiographiesweredoneforradiologicalstatusscoringusingthe Sharpmethod.
Results:Acomparisonbetweennitricoxideinallthreegroupsshowedahighlysignificant difference(p<0.001),significantlyhigherlevelswereobtainedamongrheumatoid arthri-tispatientsincomparisontocontrols,andhigherlevelswereobtainedinpatientswith activedisease(mean±SD82.38±20.46)incomparisontopatientswithoutactivedisease (35.53±7.15).NitricoxideinGroupIshowedasignificantpositivecorrelationwithmorning stiffness(r=0.45),arthritis(r=0.43),plateletcount(r=0.46),erythrocytesedimentationrate (r=0.83),C-reactiveprotein(r=0.76)andDiseaseActivityScore(r=0.85).Nitricoxideshowed asignificantpositivecorrelation(r=0.43)withhandradiographies(Sharpscore)inGroupI.
Conclusion:Thereareincreasedlevelsofnitricoxideintheserumofpatientswith rheuma-toid arthritis. Nitric oxide correlates significantly with disease activity, inflammatory markersandradiologicaljointstatus.
©2014ElsevierEditoraLtda.Allrightsreserved.
DOIoforiginalarticle:http://dx.doi.org/10.1016/j.rbr.2014.07.003. ∗ Correspondingauthor.
E-mail:[email protected](R.A.Habeeb). http://dx.doi.org/10.1016/j.rbre.2014.07.002
Níveis
de
óxido
nítrico
mais
elevados
estão
associados
à
atividade
da
doenc¸a
em
pacientes
egípcios
com
artrite
reumatoide
Palavras-chave:
Artritereumatoide(AR) Estresseoxidativo Óxidonítrico(NO)
r
e
s
u
m
o
Introduc¸ão: Oestresseoxidativoproduzidonointeriordearticulac¸õesinflamadaspode pro-duzirfenômenosautoimunesedestruic¸ãoarticular.Asespéciesradicaiscomatividade oxidativa,incluindoespéciesreativasdenitrogênio,representammediadoresdeinflamac¸ão edelesãocartilaginosa.
Objetivos: Avaliaroóxidonítricoséricocomomarcadordeestresseoxidativoempacientes egípcioscomartritereumatoideesuarelac¸ãocomaatividadedadoenc¸a.
Métodos: Oitentapacientescomartritereumatoideforamdivididosemdoisgrupos,de acordocoma pontuac¸ãoDAS28: GrupoI:42pacientescomdoenc¸aativa,eGrupoII:38 pacientescomdoenc¸ainativa.Quarentaindivíduosequiparadosporidadeegêneroforam incluídoscomogrupocontrole(GrupoIII).Foramrealizadosexameslaboratoriaisderotina eoóxidonítricofoimedidousandoElisa.Radiografiassimplesdasmãosforamfeitaspara apontuac¸ãodoestadoradiológicoutilizandoométododeSharpe.
Resultados: Acomparac¸ãodonívelséricodeóxidonítricoentreostrêsgruposmostrou umadiferenc¸aaltamentesignificativa(p<0,001).Obtiveram-seníveissignificativamente maiselevadosentreospacientescomartrite reumatoideem comparac¸ãocomos con-troles.Osníveismaiselevadosforamobtidosempacientescomadoenc¸aativa(média±DP 82,38±20,46)em comparac¸ãocomaqueles coma doenc¸ainativa(35,53±7,15). Oóxido nítriconoGrupoIexibiuumacorrelac¸ãopositivasignificativacomarigidezmatinal(r=0,45), artrite (r=0,43), contagem de plaquetas (r=0,46), velocidade de hemossedimentac¸ão (r=0,83),proteínaC-reativa(r=0,76)eÍndicedeAtividadedeDoenc¸a(r=0,85).Oóxidonítrico mostrouumacorrelac¸ãopositivasignificativa(r=0,43)comasradiografiasdasmãos(índice deSharpe)noGrupoI.
Conclusão: Observa-seumaumentonosníveisséricosdeóxidonítricoempacientescom artritereumatoide.Oóxidonítricosecorrelacionasignificativamentecomaatividadeda doenc¸a,marcadoresinflamatórioseestadoradiológicodasarticulac¸ões.
©2014ElsevierEditoraLtda.Todososdireitosreservados.
Introduction
Nitricoxide(NO)isanendogenouslyproducedsmallmolecule thathascriticalrolesincellularsignalingandisinvolvedin avarietyofphysiological processes. NOcan haveopposite biologicaleffects,dependinguponvariousenvironmentaland pathophysiologicalconditions.1
Oxidant stress generated within an inflammatory joint can produce autoimmune phenomenaand connective tis-sue destruction withinthe synovium. Radicalspecies with oxidativeactivity,whichincludereactivenitrogenspeciesand reactiveoxygenspecies,representthemediatorsandeffectors ofcartilagedamage.2
NOmediatesmanydifferentcell functionsatthesiteof synovialinflammation,including signaltransduction, mito-chondrialfunctionandapoptosis.3
NOhasemergedasanimportantmediatorinrheumatoid arthritis(RA)synovium.IncreasedlevelsofNOinserumand synovialfluidhavebeenreportedinpatientswithRA, anky-losingspondylitisandosteoarthritis.4
Theaimofthiswork isto assessNOlevel asa marker of oxidative stress in Egyptian patients with RA and to correlate it with various disease parameters and disease activity.
Methods
In a cross sectional study, 80 patients with RA, diagnosed accordingtotheAmericanCollegeofRheumatology/European League against Rheumatism (ACR/EULAR) criteria,5 were dividedinto2groupsaccordingtodiseaseactivity:GroupI:42 patientswithdiseaseactivity,andGroupII:38patientswithno diseaseactivity.Athirdgroupwasincluded:GroupIII:40age andsexmatchedhealthyindividualsascontrol.Allpatients wererecruitedfromtheRheumatologyOutpatientClinicand Internal Medicine Ward at AinShams University Hospital. Informedconsentswereobtainedfromallparticipants,and the study was approved by the Ain Shams Medical ethics committee.Thenatureofthepresent studywasexplained, andlaboratoryandradiologicalproceduresrepresentstandard careposingnoethicalconflicts.Forallthepatients,the follow-ingweredone:
I.Detailedmedicalhistoryandthoroughclinicaland mus-culoskeletalexaminationwithassessmentofdiseaseactivity byusingDAS28-ESR:6
Disease activity was assessed using DAS28-ESR, and a DAS28-ESR≤2.6 was considered indicative of no disease activity.6
II.Laboratoryinvestigations: a)Routinelaboratoryinvestigations: Completebloodcount;
ESRinthefirsthour,estimatedbyWesternmethod; CRP(mg/dLwithtiter).
b)Immunologicalinvestigations:
Rheumatoid factor: measured by using bioticRA factor latexagglutinationslideforthequalitativedeterminationof RFinserum.
c)Serumnitricoxide
NOwasperformedusingELISA(R&DSystems,Inc., Min-neapolis, USA). This assay determines NO concentrations based on the enzymatic conversion of nitrate to nitrite by nitrate reductase. A preliminary deproteinization step hasbeen done using ultrafiltration method,the filter used was centristart 1, supplied by vivascience sartorius group (www.sartorius.com).Thereactionisfollowedby colorimet-ricdetection ofnitriteasanazo dyeproductoftheGriess Reaction.7
III.Radiologicalinvestigations:
PlainX-rayhandsandwristsforscoringtheradiological damageusingSharpmethod.8
Statistical
analysis
Thecollected data were coded, tabulated, and statistically analyzedusingSPSSprogram(StatisticalPackageforSocial Sciences)softwareversion17.0.Descriptionofqualitative vari-ablesasnumberand percentagewas done.Chi-square test wasusedtocomparequalitativevariables.Fisherexacttest
wasusedinsteadofchi-squaretestwhenoneexpectedcell ormore≤5.Unpairedt-testwasusedtocomparetwo inde-pendentgroupsasregardsquantitativevariables.Spearman & PersonCorrelationcoefficientranktestwasusedtorank differentvariablesagainsteachother’spositivelyorinversely. P-value=levelofsignificance,wherep>0.05=notsignificant (NS),p<0.05=significant(S),andp<0.001=highlysignificant (HS).
Results
In GroupI,23 were females,and 19 weremales.Their age ranged from 27 to 63 years withmean ± SD 48.78±12.41, and disease duration 5.48±6.63 years.In Group I, 21 were females, and 17 were males. Their age rangedfrom 28 to 65yearswithmean±SD44.10±10.54,anddiseaseduration 6.59±5.81years.Therewasnosignificantdifferencebetween bothgroupsasregardsgender,ageordiseaseduration.
Fatigueandmorningstiffnessweresignificantlymore fre-quentinGroupIthaninGroupIIpatients(p<0.001).Patients fromGroupIalsohadsignificantlyhigherESRandCRPvalues
thanGroupIIpatients(p<0.001)(Table1).
There was asignificant differencebetween GroupI and GroupIIasregardsX-raySharpscore(Table2).
On comparingNOlevel amongthe threegroups, signif-icantly higher levels were detected among RA patients in comparisontocontrols,withhighestlevelsobtainedamong RApatientswithdiseaseactivity(GroupI)(Table3).
CorrelationbetweenserumNOlevelandvariousdisease parametersinGroupIshowedasignificantpositivecorrelation (p<0.05)withmorningstiffness,arthritisandplateletcount, andahighlysignificantpositivecorrelation(p<0.001)with dis-easeactivity,ESRandCRP.(Table4);furthermore,NOshowed
Table1–ComparisonbetweenGroupIandGroupIIasregardsdemographic,clinicalandlaboratorydata.
GroupI GroupII p
Demography
Age,mean(SD) 48.78(12.41) 44.10(10.54) >0.05
Gender,n(%)
Male 19(45) 17(45) >0.05
Female 23(55) 21(55) >0.05
Diseaseduration(years),mean(SD) 5.48(6.63) 6.59(5.81) >0.05
Clinicalmanifestations
Weightloss,n(%) 16(38) 15(39) >0.05
Fatigue,n(%) 21(50) 10(26) <0.001
Fever,n(%) 4(10) 0(0) >0.05
Morningstiffness,n(%) 36(85) 13(34) <0.001
Laboratorydata
Hemoglobin,mean(SD) 11.06(1.64) 11.87±1.61 >0.05
Totalleucocyticcount,mean(SD) 7.68(2.61) 7.12±2.38 >0.05
Platelet,mean(SD) 382.81(102.24) 261.14±74.37 <0.05
Erythrocytesedimentationrate(ESR),mean(SD) 60.69(24.32) 19.76±5.29 <0.001
C-reactiveprotein(CRP),mean(SD) 24.48(12.64) 8.52±3.01 <0.001
Rheumatoidfactor(RF),n(%) 36(86) 30(79) >0.05
Table2–ComparisonbetweengroupIandIIasregards X-raySharpscore.
Sharpscore GroupI GroupII p
n % n %
0 2 5 3 8 <0.05
1 4 10 16 42 <0.001
2 6 14 11 29 <0.01
3 18 43 5 13 <0.001
4 10 23 2 5 <0.001
5 2 5 0 0 <0.01
Table3–ComparisonbetweenGroupsI,II,andIIIas regardsserumNO.
SerumNO GroupI GroupII GroupIII p-value
Mean±SD 82.38±20.46 35.53±7.15 14.25±4.09 <0.001
SignificantlyhigherlevelsofNOweredetectedamongRApatients incomparisontocontrolswithhighestlevelsobtainedamongRA patientswithdiseaseactivity(GroupI).
Table4–CorrelationbetweenserumNOleveland variousdiseaseparametersinGroupI.
Variable r p-value
Age 0.231 >0.05
Diseaseduration -0.252 >0.05
Morningstiffness 0.453 <0.05
Arthritisa 0.432 <0.05
Platelet 0.458 <0.05
ESR 0.832 <0.001
CRP 0.763 <0.001
Diseaseactivity 0.846 <0.001
ESR,erythrocytesedimentationrate;CRP,C-reactiveprotein. NOshowedasignificantpositivecorrelation(p<0.05)with morn-ingstiffness,arthritisandplateletcount,andahighlysignificant positivecorrelation(p<0.001)withdiseaseactivity,ESRandCRP. a 28-tenderand/orswollenjointcount.
asignificantpositivecorrelationwithX-rayhandSharpscore (p<0.05)inGroupI(Fig.1).Nosignificantcorrelationbetween NOandX-rayhandSharpscoreinGroupIIcouldbedetected.
Discussion
Inflammationandtissueinjuryrelatedtooxidativestresshas beenimplicatedinthepathogenesisofRA.Oxidativestress anddecreasedantioxidantstatusarethehallmarksinpatients ofRAasobservedinrecentyears.9NO-dependenttissueinjury hasbeenimplicatedinavarietyofrheumaticdiseases, includ-ingRA.10
ThepresentstudyassessesNOlevelasamarkerof oxida-tive stress in patients with RA and its relation to disease activity.Thestudyincluded80patientswithRA,42patients withdiseaseactivity(GroupI)and38withoutactivity(Group II).
Patients with disease activity complained significantly more(p<0.001)ofmorningstiffnessandfatigueascompared tothose withoutdiseaseactivity.Thisismostlikelydueto thefactthatclinicalmanifestations,suchasmorning
stiff-140
120
100
80
60
40
20
0
XRAY H.
r=0.432 P-value=0.005*
No
0 1 2 3 4 5 6
Figure1–CorrelationbetweenserumNOlevelandhand
X-raySharpscoreinGroupI.NOshowedasignificant positivecorrelationwithX-rayhandSharpscore(p<0.05).
ness,havelongbeenassociatedwithdiseaseactivityinRA, andsoitisexpectedthatsignificantlyhighervaluesaretobe obtainedinactiveRApatients.11
Inthepresentstudy,bothESRandCRPweresignificantly higherintheactivegroup(p<0.001).AlthoughCRPappearsto bethebettertestregardingmeasurementoftheacutephase, yetbecauseESRissensitivetoimmunoglobulinsandRF,itmay measuregeneralseveritybetterthanCRP,eventhoughitisa poorermeasureofinflammation.12Inastudytoassessclinical utilityofmeasurementsofCRPandESRinevaluatingpatients withRA,itwasfoundthatbothESRandCRPweresignificant predictorsofswollenjointcount(p<0.001foreach).13
Inthisstudy,comparisonbetweenserumNOinallthree groups showed a highly significant difference (p<0.001), where significantly higher levels were obtained amongRA patientsthancontrolsandevenhigherlevelswereobtained in active RA patients (mean±SD 82.38±20.46), than the inactive group (mean±SD 35.53±7.15). SerumNO level in GroupIshowedasignificantpositivecorrelationwith morn-ing stiffness (r=0.45), arthritis (r=0.43) and platelet count (r=0.46), and a highly significant positive correlation was foundbetweenNOandeachofESR(r=0.83)andCRP(r=0.76), aswellasDAS(r=0.84).
Oncomparisonbetweenbothgroupsasregards radiologi-caldamageusingSharpscore,therewasahighlysignificant differencebetweenbothgroups(p<0.001).NOlevelsshoweda significantpositivecorrelation(r=0.43)withX-rayhandSharp scoreonlyinGroupI,yetnocorrelationbetweenNOand X-rayhandSharpscoreinGroupIIcouldbedetected.Studies havedemonstratedalongitudinalrelationshipbetween dis-easeactivityandradiographicstatus.14Astudyconcludedthat thelevelofdiseaseactivity,aswellasthedurationof remis-sion,affectsubsequentprogressionofradiographicdamage inRA.15Consequently,sincediseaseactivitymaybeoneof thedriversofjointdamageandradiographicprogressionin RA,itwouldbeexpectedthatsinceNOcorrelateswith dis-easeactivity,itwouldalsocorrelatewithradiographicdamage inpatientswithactivedisease,ashasbeendetectedinthe presentstudy.Incontrast,howeverotherstudieshave con-cluded that,despiteclinicalimprovementandremissionin patients with active RA,the radiographicprogression con-tinues over time because of the underlying inflammatory process.16
control groups,17,18 One study suggested that nitrate and nitriteproductionisenhancedinpatientswithRAcompared withhealthysubjects.4
NOhasbeenshowntoregulateT-cellfunctionsunder phys-iologicalconditions,butoverproductionofNOmaycontribute toT-lymphocytedysfunction.TheincreaseinNOproduction inRApatientsmay beduetotheincrease inNOsynthase activity.19
Similarly,asignificantcorrelationbetweenserumnitrate concentrations and number of tender joints, number of swollenjoints,DASscoreandCRPlevelhasbeendetected.20 Theresultssuggestthatthesefindingscanserveasareliable parameterofdiseaseactivityinpatientswithRA.
Although it has been suggested that reactive nitrogen speciesareproducedwithintheinflamedjointsofRApatients andthatthelevelscorrelatedirectlywithdiseaseactivity,21 severalstudiesconcludedthatNOleveldidnotcorrelatewith ESRneitherwithCRP,andthattherewasanon-significant cor-relationbetweentheserumlevelsofeachofnitrogenspecies and theduration ofdisease or ESRasa markerofdisease activity.18,22
Oxidativestressgeneratedinaninflamedjointcan con-tributetoautoimmunephenomenonand connectivetissue destructioninRA,andtheproductionofNOandprostaglandin E2fromarticularchondrocytesprobablycontributetothe car-tilagedestructionseeninarthritis.23Inthepresentstudy,a significantpositivecorrelationwasdetectedbetweenserum NOlevelandradiologicalstatusinbothRAgroups, suggest-ingaroleforNOnotonlyindiseaseactivity,butalsoinjoint damage.
Inthepresentcrosssectionalstudyconductedonacohort ofEgyptianRApatients,NOwasnotonlydetectedwith sig-nificantlyhigherlevelsinactiveRApatients, butcorrelated significantlywithvariousdiseaseparametersandactivity.The studyalsoincludedradiologicalassessmentofX-rayhands usingSharpscore,whichalsoshowedsignificantcorrelation withNOlevels.However,mainlyduetothelimitationimposed byitscrosssectionaldesign,the resultsofthe studycould eitherdemonstrateacausativeeffectofNOorsimplyreflect theinflammatorystatusofdiseaseactivity.
Finally,it canbeconcludedfrom thepresent studythat thereisanincreasedlevelofNOintheserumofRApatients andthatNOlevelcorrelatessignificantlywithdisease activ-ityaswellasradiologicaljointstatus.Thiscouldsuggestthe involvementoffreeradicalsnotonlyintheinflammatory pro-cessofRA,butalsointhejointdestruction.Newtherapeutic protocolsbasedoncorrectingoxidativestresslevelsmayprove effectiveinrestrictingdiseaseprogressionandlimiting defor-mities.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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1. LiH,WanA.ApoptosisofRheumatoidArthritis
Fibroblast-LikeSynoviocytes:PossibleRolesofNitricOxide
andtheThioredoxin1.MediatorsInflamm.2013:953462.Epub 2013Apr3.
2.VasanthiP,NaliniG,RajasekharG.StatusofOxidativeStress inRheumatoidArthritis.IntJRheumDis.2009;12:
29–33.
3.PhillipsDC,DiasHK,KitasGD,GriffithsHR.Aberrantreactive oxygenandnitrogenspeciesgenerationinRA:causesand consequencesforimmunefunction,cellsurvivaland therapeuticintervention.AntioxidRedoxSignal. 2010;12:743–85.
4.ErsoyY,OzerolE,BaysalO,TemelI,MacWalterRS,MeralU, etal.Serumnitrateandnitritelevelsinpatientswith rheumatoidarthritis,ankylosingspondylitis,and osteoarthritis.AnnRheumDis.2002;61:76–8.
5.AletahaD,NeogiT,SilmanA,etal.Rheumatoidarthritis classificationcriteria:anAmericanCollegeof
Rheumatology/EuropeanLeagueagainstRheumatism collaborativeinitiative.AnnRheumDis.2010;69:1580–8. 6.AletahaD,SmolenJ.TheSimplifiedDiseaseActivityIndex
andtheClinicalDiseaseActivityIndex:areviewofusefulness andvalidityinrheumatoidarthritis.ClinExpRheumatol. 2005;235Suppl39:S100–8.
7.GreenLC,WagnerDA,GlogowskiJ,SkipperPL,WishnokJS, TannenbaumSR.Analysisofnitrate,nitrite,and[15N]nitrate inbiologicalfluids.AnalBiochem.1982;126:131–8.
8.SharpJT,YoungDY,BluhmGB,etal.Howmanyjointsin handsandwristsshouldbeincludedinascoreofradiologic abnormalitiesusedtoassessrheumatoidarthritis?Arthritis Rheum.1985;28:1326–35.
9.AjayK,PranshiM,NeelimaS.Studyofnon-enzymatic antioxidantandlipidperoxidelevelsinrheumatoidarthritis. UJMD.2012;1:10–3.
10.NagyG,KonczA,TelaricoT,FernandezD,ErsekB,BuzásE, etal.Centralroleofnitricoxideinpathogenesisof rheumatoidarthritisandsystemiclupuserythematosus. ArthritisResTher.2010;12:210.
11.TamasMM,FeleaI,RednicS.Howmuchdifferencedoesthe ageatonsetmakeinearlyarthritispatients?Comparison betweentheACR1987andtheACR/EULAR2010classification criteriaforrheumatoidarthritisatthetimeofdiagnosis. RheumatolInt.2013;33:2881–4.
12.WellsG,BeckerJC,TengJ,DougadosM,SchiffM,SmolenJ. Validationofthe28-jointDiseaseActivityScore(DAS28)and EuropeanLeagueAgainstRheumatismresponsecriteria basedonC-reactiveproteinagainstdiseaseprogressionin patientswithrheumatoidarthritis,andcomparisonwiththe DAS28basedonerythrocytesedimentationrate.AnnRheum Dis.2009;68:954–60.
13.CynthiaS,MahboobU,EricL.WhichMeasureof InflammationtoUse?AComparisonofErythrocyte SedimentationRateandC-ReactiveProteinMeasurements fromRandomizedClinicalTrialsofGolimumabin RheumatoidArthritis.JRheumatol.2010;36: 1606–10.
14.LandewéR,VanderHeijdeD.Radiographicprogressionin rheumatoidarthritis.ClinExpRheumatol.2005;23Suppl. 39:S63–8.
15.AletahaD,FunovitsJ,BreedveldFC,SharpJ,SeguradoO, SmolenJS.Rheumatoidarthritisjointprogressionin sustainedremissionisdeterminedbydiseaseactivitylevels proceedingtheperiodofradiographicassessment.Arthritis Rheum.2009;60:1242–9.
16.MarkatseliTE,VoulgariPV,AlamanosY,DrososAA.Prognostic FactorsofRadiologicalDamageinRheumatoidArthritis:A 10-yearRetrospectiveStudy.JRheumatol.2010;38:44–52. 17.SavasG,ArzuS,EdaC.Nitricoxideandsuperoxidedismutase
18.AidaA,MohamedA.SerumProteinCarbonylContent,Total ThiolandNitricOxideinPatientswithRheumatoidArthritis. JournalofAmericanScience.2011;7:683–6.
19.Mäki-PetäjäKM,CheriyanJ,BoothAD,HallFC,BrownJ, WallaceSM,etal.Induciblenitricoxidesynthaseactivityis increasedinpatientswithrheumatoidarthritisand contributestoendothelialdysfunction.IntJCardiol. 2008;129:399–405.
20.OnurO,AkinciAS,AkbiyikF,UnsalI.Elevatedlevelsofnitrate inrheumatoidarthritis.RheumatolInt.2001;20:154–8.
21.KhanF,SiddiquiA.Prevalenceofanti-3-nitrotyrosine antibodiesinthejointsynovialfluidofpatientswith rheumatoidarthritis,osteoarthritisandsystemiclupus erythematosus.ClinChimActa.2006;370:100–7. 22.MarwanS,SamirA,KhalidS.Serumnitricoxideand
peroxynitritelevelsinadultsero-positiverheumatoid arthritistreatedwithdiseasemodifyingantirheumaticrugs: apreliminaryreport.TurkJMedSci.2010;40:191–7.
