1) The document reports on a study examining oxidative and nitrosative modulators in patients with multiple sclerosis. 2) Clinical evaluations, biochemical analyses of blood and cerebrospinal fluid samples, and radiological findings were collected from patients with different forms and severities of multiple sclerosis. 3) The results indicate higher levels of oxidative stress and inflammation markers in blood and cerebrospinal fluid of patients with the relapsing-remitting form of multiple sclerosis compared to the initial isolated form.

1. OXIDATIVEANDNITROSATIVE
MODULATORSIN
NEUROINFLAMMATION

2. OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION

3. OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION

4. Clinicalevaluationofthedisease:ForallKISandRRMSpatients,theclinicalpresentationwasevaluated
usingtheKurtzke'sExtendedDisabilityStatusScale(EDSS)scale(Kurtzke,1983).Comparedtothefrequen-
cyofEDSSvaluesobtained,allpatientsweresubdividedintosubgroupswithmildtomoderateorsevere
clinicalimagesforfurthertestingpurposes.
-Biochemicalanalyzes:
BloodsamplingandCSTBloodandliverweresampledatthesametime.Inallpatients,bloodwassam-
pledbyvenipunctureofaneccubicveinafter12hoursofstarvation,inthemorning,intubescontaining500
mMEDTAasananticoagulantforthepurposesofdeterminingbiological-biochemicalinflammatoryparam-
etersoftheinflammation,orinheparinizedtubes(forallremaininganalyzes)centrifugedat4500gfor10
minutes,afterwhichtheplasmawasseparatedandstoredat-80Cuntiltheplannedbiochemicalanalyzes
werecarriedout.TheremainingerythrocyteswerewashedthreetimesincoldFR,andthenhemolizedby
theadditiontheadditionof9equivalentamountsofcolddemineralizedwaterafterwhichthehemolysateswerestored
at-80°Cuntiltheanalysiswascarriedout.Nosamplewasolderthan6monthsatthetimeofconducting
theanalysis.Allsampleswereonlyexposedtoroomtemperatureduringtheanalysis.Allbiochemicalana-
lyzeswerecarriedoutattheInstituteofBiochemistryoftheMedicalFaculty.Cerebrospinalfluidsamples
wereobtainedbylumbarpunctureofthesubarachnoidspaceinthelying,lateralposition,atthelevel
L3-L4.Aportionofthesampleswasusedforcytologicalanalyzesandisoelectricfocusingneeds,andthe
partwasimmediatelycentrifugedat10,000gfor3minutesat4°Ctoremovecellularelements,andthen
frozenat-80°Cuntiltheplannedbiochemicalanalyzeswerecarriedout.Therewerenosignsofbleeding
inthesamples.Theresultsofisoelectricfocusingwereinterpretedinrelationtothepresenceorabsence
ofoligoclonaltapeinthecerebrospinalfluidwiththecorrespondingserumfinding.PermeabilityofKMB
wasassessedbytheratioofalbuminconcentrationintheserumortothecerebrospinalfluid.
ConcentrationofnitrateandnitriteConcentrationofNO.wasdeterminedbymeasuringtheconcentra-
tionofNO2andNO3(µmol/l),usingtheGriessdirectspectrophotometricmethod(Griessreagent:1.5%
sulfanilamidein1MHClplus0.15%N-(1-naphthyl)ethylenediaminedihydrochlorideindistilledwater)in
plasmaandcerebrospinalfluid(NavaroGonzalvezetal,1998).
MalondialdehydeconcentrationMDAconcentrationwasdeterminedinplasma,haemolysatesandcere-
brospinalfluidsbyspectrophotometricmethodwiththiobarbutyricacid(TBA).TheconcentrationofTBARS
wasexpressedastheconcentrationofMDA(µmol)perliter(plasmaandliver),orgHb(hemolysate)(An-
dreevaetal.,1988).
OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION

5. 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
autoxidationofpyrogallolformsacoloredcompoundwithNBT.AstheO2-Purifier,SODinhibitsthis
reaction,whereoneunitofenzymaticactivityrepresents50%inhibition.Thevaluesobtainedare
expressedasUperliter(plasmaandcerebrospinalfluid),orgHb(hemolyzate).
RadiologicalfindingTheCNStissuewasexaminedwithamagneticresonancewith1.5T(Avanto,
Siemens,Erlangen,Germany).TheMRprotocolincludedthefollowingconventionalspinsequences:axial
T1-weighted(VR)=500milliseconds,EVA=78milliseconds,numberofexcitations(BE)=2)and
T2-weighted(VR=4700milliseconds,EV=93milliseconds,BE=2)withacross-sectionthicknessof5mm,
andaspacebetweenthecross-sectionof0.5mmandapixelsizeof0.9x0.9mm.Anintravenously
administeredGd-ContrastAgent(Gadovist,Schering,Berlin,Germany)atadoseof0.1mmol/kgbody
weighweight.Thenumberand/orvolumeofhyperintensechangesseenonT2sequencesaswellasthevolume
ofGdbindingchangesseenonT1sequencesweretakenforanalysispurposes.AllMRfindingswere
interpretedbyaneuroradiologistwhodidnothaveaninsightintoaclinicalfinding,noradiagnostic
classificationofpatients.Inrelationtothenumberofdescribedchanges,divisionwasmadewithinboth
groupsofpatientstothosewithlowerandthosewithagreaternumberofradiologicalchangesthanthe
meanwithineachgroup.
OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION

6. OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION

7. Thepresenceofoligoclonaltape(OKT+)ontheisoelectricfocusingofthecerebrospinalfluidandserumin
relationtothenumberofKISpatients(15patients)withOKT+,p=0.025,wasdemonstratedinalarge
numberofRRMSpatients(54patients).TheKMBdisorder,definedasanincreaseinthealbuminconcentra-
tioninthedrugrelativetoserumlevels>7.0x10-3,wasobservedin7KISand11RRMSpatients.Inother
patients,thedifferencesintheassessmentofpermeabilityofKMBbetweentheKIS(5.9±0.95)andRRMS
(6.2±0.7)patientswerenotstatisticallysignificant(p=0.08).Thetotalproteinconcentrationsinthecere-
brospinalfluidbrospinalfluidwerehigherinRRMS(0.42±0.23g/L)comparedtopatientsoftheKISgroup(0.32±0.13
g/L),p=0.032.Nostatisticallysignificantdifferenceinalbuminconcentrationinthecerebrospinalfluid
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,statistically
significantdifferenceswerenoticed,p=0.072ip=0.084.
-NeurologicalfindingsofKISandRRMSpatients:Themajorityofpatientswerefoundtohavesymptoms
andsignsofaffectionofmorethanoneCNSfunctionalsystem,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
wellasvisualimpairment(KIS-1patient).Statisticalsignificancewasobservedinthedifferenceinthedu-
rationofthesymptomsuntilthepatientarrivedatahospitaltreatmentinaKISpatientwithalarger(me-
diandianvalueof1month(1-12months))comparedtoKISpatientswithasmallerEDSS(Medianvalue4
months(1-12months)),p=0.025.Bycomparingthedurationofthedisease(fromthemomentofdiagnosis
ofthedefinitiveRRMS),itwasfoundthatRRMSpatientswithahigherEDSShavealongerdurationofthe
disease(medianvalue96months(8-396months))comparedtothosewithalowerEDSS(medianvalue46
months(1-250months))(p=0.0013).ComparisonofotherobtainedvaluesinsubgroupsofKISandRRMS
patientsdividedbyEDSSfindingsdidnotshowstatisticallysignificantdifferences(p>0.05).
-RadiologicalfindingsofKISandRRMSpatients:Datarelatedtotheradiologicalfindingindicatethatthe
existenceofT2hyperintensesignals(p=0.035,RRMSvs.KIS)wasobservedin37patientsintheKISgroup
andallRRMSpatients.ThesedatawereobtainedbasedontheresultsoftheMRconductedduringthecur-
renthospitalization,aswellastheinsightintotheearliermedicalrecordsofpatients.Forthepurposesof
moredetailedanalysisinthisstudy,onlyMRfindingsthatweremadeduringthecurrenthospitalization
wereusedin16patientswithKISand15patientsintheRRMSgroup.Itwasfoundthatthetotalnumber
andnumber
OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION

8. ofsupranuclearhyperintensesignalsseenontheT2sequencewasstatisticallysignificantlyhigherinthe
RRMSgroupofpatients(medianvalueforthetotalnumberoflesionsandthenumberofsupratentorallo-
calizationlesions-40(5-84supratentoral,3-70infratentionallocalization))comparedtothesamecharac-
teristicsinKISpatients(medianvalueforthetotalnumberoflesions-9(0-56),medianvalueforthetotal
numberofsupratentorallesions-8(0-80)),p=0.023ip=0.03.ThevolumeofGdbindinglesionsseenon
theT1sequencewasstatisticallysignificantlyhigherintheRRMSgroup,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
higherconcentrationsofNO2andNO3comparedtoyoungerpatients,andinKIS(plasma-p=0.034,cere-
brospinalfluid-p=0.04),andintheRRMSgroup(plasma-p=0.03;cerebrospinalfluid-p=0.025).Simi-
larly,theconcentrationofMDAinallinvestigatedmediawashigherintheelderlygroupcomparedto
youngerpatients,inbothexaminedgroups,KIS(plasma-p=0.07;cerebrospinalfluid-p=0.06;hemoly-
sate-p=0.09),andintheRRMSgroup(plasma-p=0.055;cerebrospinalfluid-p=0.065;hemolysate-p=
0.06)0.06).AOPPconcentrationsincreasedinthegroupofelderlypatientsandinKIS(plasma-p=0.05;cerebro-
spinalfluid-p=0.06;hemolysate-p=0.055),andintheRRMSgroup(plasma-p=0.06;=0.04;hemoly-
sate-p=0.095).Atthesametime,thedecreaseinSHvaluesinplasmaandliver,GSHinhemolysateswas
observedinelderlypatientscomparedtoyoungerandKIS(plasma-p=0.025,cerebrospinalfluid-p=0.04,
hemolysate-p=0.9)andinRRMSpatients(plasma-p=0.015;cerebrospinalfluid-p=0.035;hemolysate-
p=0.75).ThetrendofdeclineintheactivityofSODwiththeincreaseintheyearsoflifewasalsoobserved
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
intheRRMSgroup(plasma-p=0,065;cerebrospinalfluid-p=0,023).TheconcentrationofMDAinallin-
vestigatedmediawasselectivelyhigherinwomencomparedtomen,inbothexaminedgroups,KIS(plasma
-p=0.078,cerebrospinalfluid-p=0.045,hemolysate-p=0.9),andintheRRMSgroup(plasma-p=0.85;
cerebrospinalfluidcerebrospinalfluid-p=0.95;hemolysate-p=0.6).
OXIDATIVEANDNITROSATIVEMODULATORSINNEUROINFLAMMATION

9. 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(plasmaKIS(plasma-p=0.075,cerebrospinalfluid-p=0.3;hemolysate-p=0.05)andinRRMSpatients(plasma-
p=0.065;=0.03;hemolysate-p=0.045).
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