Oxidative And Nitrosative Modulators In Neuroinflammation

OXIDATIVEANDNITROSATIVE
MODULATORSIN
NEUROINFLAMMATION

OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION

OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION

OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION
Clinicalevaluationofthedisease:ForalKISandRRMSpatients,theclinicalpresentationwasevaluated
clinicalimagesforfurthertestingpurposes.
-Biochemicalanalyzes:
usingtheKurtzke'sExtendedDisabilityStatusScale(EDSS)scale(Kurtzke,1983).ComparedtothefrequencyofEDSSvaluesobtained,alpatientsweresubdividedintosubgroupswithmildtomoderateorsevere
BloodsamplingandCSTBloodandliverweresampledatthesametime.Inalpatients,bloodwassampledbyvenipunctureofaneccubicveinafter12hoursofstarvation,inthemorning,intubescontaining500
mM EDTAasananticoagulantforthepurposesofdeterminingbiological-biochemicalinflammatoryparametersoftheinflammation,orinheparinizedtubes(foralremaininganalyzes)centrifugedat4500gfor10
minutes,afterwhichtheplasmawasseparatedandstoredat-80Cuntiltheplannedbiochemicalanalyzes
werecariedout.TheremainingerythrocyteswerewashedthreetimesincoldFR,andthenhemolizedby
theadditionof9equivalentamountsofcolddemineralizedwaterafterwhichthehemolysateswerestored
at-80°Cuntiltheanalysiswascariedout.Nosamplewasolderthan6monthsatthetimeofconducting
wereobtainedbylumbarpunctureofthesubarachnoidspaceinthelying,lateralposition,atthelevel
L3-L4.Aportionofthesampleswasusedforcytologicalanalyzesandisoelectricfocusingneeds,andthe
partwasimmediatelycentrifugedat10,000gfor3minutesat4°Ctoremovecelularelements,andthen
frozenat-80°Cuntiltheplannedbiochemicalanalyzeswerecariedout.Therewerenosignsofbleeding
inthesamples.Theresultsofisoelectricfocusingwereinterpretedinrelationtothepresenceorabsence
ofoligoclonaltapeinthecerebrospinalfluidwiththecorespondingserum finding.PermeabilityofKMB
wasassessedbytheratioofalbuminconcentrationintheserum ortothecerebrospinalfluid.
sulfanilamidein1M HClplus0.15% N-(1-naphthyl)ethylenediaminedihydrochlorideindistiledwater)in
plasmaandcerebrospinalfluid(NavaroGonzalvezetal,1998).
theanalysis.Alsampleswereonlyexposedtoroom temperatureduringtheanalysis.AlbiochemicalanalyzeswerecariedoutattheInstituteofBiochemistryoftheMedicalFaculty.Cerebrospinalfluidsamples
ConcentrationofnitrateandnitriteConcentrationofNO.wasdeterminedbymeasuringtheconcentrationofNO2andNO3(µmol/l),usingtheGriessdirectspectrophotometricmethod(Griessreagent:1.5%
MalondialdehydeconcentrationMDAconcentrationwasdeterminedinplasma,haemolysatesandcerebrospinalfluidsbyspectrophotometricmethodwiththiobarbutyricacid(TBA).TheconcentrationofTBARS
wasexpressedastheconcentrationofMDA(µmol)perliter(plasmaandliver),orgHb(hemolysate)(Andreevaetal.,1988).

OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION
ConcentrationofadvancedproteinoxidationproductsTheconcentrationofAOPPwasdeterminedin
plasma,haemolysatesandcerebrospinalfluidsusingH2O,KIandaceticacidbyspectrophotometricmethod
at340nm (Witko-Sarsatetal1996).Thevaluesobtainedwereexpressedasµmolperliter(plasmaand
cerebrospinalfluid),orgHb(hemolysate).
ConcentrationofsulfhydrylgroupsThetotalamountof(proteinandnon-protein)SHgroupswas
determinedinplasmaandliver,whileinthehemolysatestheconcentrationofGSHwasdeterminedusing
thespectrophotometricmethodusingDTNB(SedlakandLindsay,1968).Thevaluesobtainedwere
expressedasµmolperliter(plasmaandcerebrospinalfluid),orgHb(hemolysate).
ActivityofsuperoxidedizmutaseSODactivitywasmeasuredbythemethodofMinamiandYoshikawa
(1979),whichisbasedontheinhibitionofautoxidationofpirogalol.Asuperoxideanionformedby
autoxidationofpyrogalolformsacoloredcompoundwithNBT.AstheO2-Purifier,SODinhibitsthis
reaction,whereoneunitofenzymaticactivityrepresents50% inhibition.Thevaluesobtainedare
expressedasUperliter(plasmaandcerebrospinalfluid),orgHb(hemolyzate).
RadiologicalfindingTheCNStissuewasexaminedwithamagneticresonancewith1.5T(Avanto,
Siemens,Erlangen,Germany).TheMRprotocolincludedthefolowingconventionalspinsequences:axial
T1-weighted(VR)=500miliseconds,EVA=78miliseconds,numberofexcitations(BE)=2)and
T2-weighted(VR=4700miliseconds,EV=93miliseconds,BE=2)withacross-sectionthicknessof5mm,
andaspacebetweenthecross-sectionof0.5mm andapixelsizeof0.9x0.9mm.Anintravenously
administeredGd-ContrastAgent(Gadovist,Schering,Berlin,Germany)atadoseof0.1mmol/kgbody
weight.Thenumberand/orvolumeofhyperintensechangesseenonT2sequencesaswelasthevolume
ofGdbindingchangesseenonT1sequencesweretakenforanalysispurposes.AlMRfindingswere
interpretedbyaneuroradiologistwhodidnothaveaninsightintoaclinicalfinding,noradiagnostic
classificationofpatients.Inrelationtothenumberofdescribedchanges,divisionwasmadewithinboth
groupsofpatientstothosewithlowerandthosewithagreaternumberofradiologicalchangesthanthe
meanwithineachgroup.

OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION

OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION
Thepresenceofoligoclonaltape(OKT+)ontheisoelectricfocusingofthecerebrospinalfluidandserum in
relationtothenumberofKISpatients(15patients)withOKT+,p=0.025,wasdemonstratedinalarge
numberofRRMSpatients(54patients).TheKMBdisorder,definedasanincreaseinthealbuminconcentrationinthedrugrelativetoserum
levels>7.0x10-3,wasobservedin7KISand11RRMSpatients.Inother
patients,thediferencesintheassessmentofpermeabilityofKMBbetweentheKIS(5.9±0.95)andRRMS
(6.2±0.7)patientswerenotstatisticalysignificant(p=0.08).Thetotalproteinconcentrationsinthecere-
brospinalfluidwerehigherinRRMS(0.42±0.23g/L)comparedtopatientsoftheKISgroup(0.32±0.13
g/L),p=0.032.Nostatisticalysignificantdiferenceinalbuminconcentrationinthecerebrospinalfluid
wasobservedbetweenKIS(0.25±0.1g/L)andRRMSpatients(0.27±0.1g/L),p=0.07.Also,comparing
totalplasmaproteinconcentrationsbetweenKIS(64.2±12g/L)andRRMS(60.2±10.2g/L)groups,and
plasmaconcentrationsofalbuminKIS(42.3±9.5g/L)andRRMS(43.9±11.1g/L)groups,statisticaly
significantdiferenceswerenoticed,p=0.072ip=0.084.
-NeurologicalfindingsofKISandRRMSpatients:Themajorityofpatientswerefoundtohavesymptoms
andsignsofafectionofmorethanoneCNSfunctionalsystem,inbothKIS(35patients)andRRMSgroup
(47patients),p=0.035(KISvs.RRMS).Inotherpatients,bothgroupsofthedominantneurologicalfindings
werepyramidal(KIS-11,RRMS-9patients),p=0.075,andsensory(KIS-3,RRMS-1patient)damage,as
welasvisualimpairment(KIS-1patient).Statisticalsignificancewasobservedinthediferenceinthedu-
rationofthesymptomsuntilthepatientarivedatahospitaltreatmentinaKISpatientwithalarger(me-
dianvalueof1month(1-12months)comparedtoKISpatientswithasmalerEDSS(Medianvalue4
months(1-12months),p=0.025.Bycomparingthedurationofthedisease(from themomentofdiagnosis
ofthedefinitiveRRMS),itwasfoundthatRRMSpatientswithahigherEDSShavealongerdurationofthe
disease(medianvalue96months(8-396months)comparedtothosewithalowerEDSS(medianvalue46
months(1-250months)(p=0.0013).ComparisonofotherobtainedvaluesinsubgroupsofKISandRRMS
patientsdividedbyEDSSfindingsdidnotshowstatisticalysignificantdiferences(p>0.05).
-RadiologicalfindingsofKISandRRMSpatients:Datarelatedtotheradiologicalfindingindicatethatthe
existenceofT2hyperintensesignals(p=0.035,RRMSvs.KIS)wasobservedin37patientsintheKISgroup
andalRRMSpatients.ThesedatawereobtainedbasedontheresultsoftheMRconductedduringthecurrenthospitalization,aswelastheinsightintotheearliermedicalrecordsofpatients.Forthepurposesof
moredetailedanalysisinthisstudy,onlyMRfindingsthatweremadeduringthecurenthospitalization
wereusedin16patientswithKISand15patientsintheRRMSgroup.Itwasfoundthatthetotalnumber
andnumber

OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION
ofsupranuclearhyperintensesignalsseenontheT2sequencewasstatisticalysignificantlyhigherinthe
RRMSgroupofpatients(medianvalueforthetotalnumberoflesionsandthenumberofsupratentorallo-
numberofsupratentorallesions-8(0-80),p=0.023ip=0.03.ThevolumeofGdbindinglesionsseenon
theT1sequencewasstatisticalysignificantlyhigherintheRRMSgroup,bothinthebrain(277.7±109.1
mm3)andinthebiomass(641.3±210.2.1mm3),incomparisonwiththesamefindinginthebrain(146.5±
46.8mm3)andcysticmoss(446.6±120.1mm3)ofpatientsintheKISgroup,p=0.031(forthebrain),p=
0.04.
-Concentrationsofexaminedparametersinrelationtopatientage:Itwasfoundthatolderpatientshad
calizationlesions-40(5-84supratentoral,3-70infratentionallocalization)comparedtothesamecharac-
teristicsinKISpatients(medianvalueforthetotalnumberoflesions-9(0-56),medianvalueforthetotal
higherconcentrationsofNO2andNO3comparedtoyoungerpatients,andinKIS(plasma-p=0.034,cere-
brospinalfluid-p=0.04),andintheRRMSgroup(plasma-p=0.03;cerebrospinalfluid-p=0.025).Simi-
larly,theconcentrationofMDAinalinvestigatedmediawashigherintheelderlygroupcomparedto
youngerpatients,inbothexaminedgroups,KIS(plasma-p=0.07;cerebrospinalfluid-p=0.06;hemolysate-p=0.09),andintheRRMSgroup(plasma-p=0.055;cerebrospinalfluid-p=0.065;hemolysate-p=
0.06).AOPPconcentrationsincreasedinthegroupofelderlypatientsandinKIS(plasma-p=0.05;cerebro-
observedinelderlypatientscomparedtoyoungerandKIS(plasma-p=0.025,cerebrospinalfluid-p=0.04,
inKIS(plasma-p=0.065;cerebrospinalfluid-p=0.031;haemolysate-p=0.05)andinRRMSpatients
(plasma-p=0.025;-p=0.07).
-Concentrationsofexaminedparametersinrelationtohalfofpatients:Analysisoftheobtainedvaluesof
thetestedparameters,comparedtohalfofpatients,itwasfoundthatmenhavehighervaluesofNO2and
NO3concentrationsinrelationtowomen,andinKIS(plasma-p=0,06,cerebrospinalfluid-p=0,075),and
spinalfluid-p=0.06;hemolysate-p=0.055),andintheRRMSgroup(plasma-p=0.06;=0.04;hemoly-
sate-p=0.095).Atthesametime,thedecreaseinSHvaluesinplasmaandliver,GSHinhemolysateswas
hemolysate-p=0.9)andinRRMSpatients(plasma-p=0.015;cerebrospinalfluid-p=0.035;hemolysatep=0.75).ThetrendofdeclineintheactivityofSODwiththeincreaseintheyearsoflifewasalsoobserved
intheRRMSgroup(plasma-p=0,065;cerebrospinalfluid-p=0,023).TheconcentrationofMDAinalinvestigatedmediawasselectivelyhigherinwomencomparedtomen,inbothexaminedgroups,KIS(plasma
-p=0.078,cerebrospinalfluid-p=0.045,hemolysate-p=0.9),andintheRRMSgroup(plasma-p=0.85;
cerebrospinalfluid-p=0.95;hemolysate-p=0.6).

OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION
ConcentrationsofAOPPshowedanincreaseinconcentrationinmenversuswomenandinKIS(plasma-p=
0.58,cerebrospinalfluid-p=0.6;haemolysate-p=0.55),andinRRMSpatients(plasma-p=0,06;
cerebrospinalfluid-p=0.06;hemolysate-p=0.55).ThedecreaseinthevalueofSHgroupsinplasmaand
liver,ieGSHinhemolysates,inwomenversusmenandinKIS(plasma-p=0.095;cerebrospinalfluid-p=
0.055;hemolysate-p=0.65)andRRMSpatients(plasma-p=0.025;cerebrospinalfluid-p=0.025;
hemolysate-p=0.07).ThedeclineinSODactivitywasmoredominantinwomencomparedtomenandin
KIS(plasma-p=0.075,cerebrospinalfluid-p=0.3;hemolysate-p=0.05)andinRRMSpatients(plasmap=0.065;=0.03;hemolysate-p=0.045).
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