www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Evaluation
of
serum
prolidase
enzyme
activity
and
oxidative
stress
in
patients
with
tinnitus
夽
Adnan
Ekinci
a,∗,
Kaan
Kamasak
baHititUniversity,FacultyofMedicine,OtorhinolaryngologyDepartment,C¸orum,Turkey bHititUniversity,FacultyofMedicine,NeurosurgeryDepartment,C¸orum,Turkey
Received31October2018;accepted6January2019 Availableonline15March2019
KEYWORDS
Tinnitus; Prolidase; Oxidativestress
Abstract
Introduction:Tinnitusisdefinedastheperceptionofsoundintheheadorintheheadinthe absenceofexternalsounds.Thecauseoftinnitusisstillunknown.
Objective: Weaimedtocomparetheserum levelsoftotal oxidantstatus,total antioxidant status,serumprolidaseenzymeactivityandtheoxidativestressindexinpatientswithtinnitus tothoseofnormalsubjects.
Methods:Twentyfivepatientswithtinnitus(meanage34.3)and25healthycontrols(meanage 37.2)wereincludedinthestudy.
Results:Totaloxidantstatuslevelsinthepatientgroupweresignificantlyhigherthaninthe controlgroup(p=0.037).Themeantotaloxidantstatusvaluewas2.54±0.95mmoL/Linthe patientgroup,and2.06±0.98mmoL/Linthecontrolgroup.Themeanoxidativestressindex levelwas0.22±0.10AUinthepatientgroup,whileitwas0.17±0.08AUinthecontrolgroup. Oxidativestressindexwassignificantlyhigherinthepatientgroup(0.026).Therewasno signi-ficantdifferencebetweenthegroupsintermsoftotalantioxidantstatusvalues(p=0.838).The meanserumprolidaseenzymeactivitylevelwas202.74±33.56U/Linthepatientgroupand 175.46±42.68U/Linthecontrolgroup.Serumprolidaseenzymeactivitylevelsinthepatient groupweresignificantlyhigherthaninthecontrolgroup(0.040).
Conclusion: Wedetectedthatthetotaloxidantstatus,oxidativestressindexandserum proli-daseenzymeactivitylevelswerehigherinpatientswithtinnituswhencomparedtothehealthy controls.Thisfindingsuggeststhatoxidativestressindexandserumprolidaseenzymeactivity mayplayaroleintheetiopathogenesisoftinnitus.
© 2020 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).
夽 Pleasecitethisarticleas:EkinciA,KamasakK.Evaluationofserumprolidaseenzymeactivityandoxidativestressinpatientswith
tinnitus.BrazJOtorhinolaryngol.2020;86:405---10.
∗Correspondingauthor.
E-mail:draekinci@hotmail.com(A.Ekinci).
PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial. https://doi.org/10.1016/j.bjorl.2019.01.009
1808-8694/©2020Associac¸˜aoBrasileiradeOtorrinolaringologiaeCirurgiaC´ervico-Facial.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
PALAVRAS-CHAVE
Zumbido; Prolidase; Estresseoxidativo
Avaliac¸ãodaatividadedaenzimaséricaprolidaseedoestresseoxidativoem
pacientescomzumbido
Resumo
Introduc¸ão:Ozumbidoédefinidocomoapercepc¸ãodeumsomnacabec¸anaausênciadesons externos.Acausadozumbidoaindaédesconhecida.
Objetivo:Compararosníveisséricosdoestadooxidantetotal,estadoantioxidantetotal, ati-vidadedaenzimaprolidaseséricaeoíndicedeestresseoxidativoempacientescomzumbido eemindivíduosnormais.
Método: Foramincluídos no estudo 25 pacientes com zumbido (média de 34,3anos) e 25 controlessaudáveis(médiade37,2anos).
Resultados: Osníveisdoestadooxidantetotalnogrupodepacientesforamsignificantemente maioresdoquenogrupocontrole(p=0,037).Ovalor médiodoestadooxidantetotalfoide 2,54±0,95mmoL/Lnogrupodepacientesede2,06±0,98mmoL/Lnogrupocontrole.Onível médiodeíndicedeestresseoxidativofoide0,22±0,10AUnogrupodepacientes,enquantono grupocontrolefoide0,17±0,08AU.Oíndicedeestresseoxidativofoisignificantementemaior nogrupodepacientes(0,026).Nãohouvediferenc¸asignificanteentreosgrupos emrelac¸ão aosvalores do estadoantioxidantetotal (p=0,838).Onível médio deatividade daenzima prolidaseséricafoide202,74±33,56U/Lnogrupodepacientesede175,46±42,68U/Lno grupocontrole.Osníveisdeatividadedaenzimaprolidaseséricanogrupodepacientesforam significantementemaioresdoquenogrupocontrole(0,040).
Conclusão:Detectamosque osníveis deestadooxidante total,índice deestresse oxidativo e atividade da enzima prolidase séricaforam maiores nos pacientes comzumbido quando comparadosaoscontrolessaudáveis.Esse achado sugerequeoíndice deestresse oxidativo eaatividadedaenzimaprolidaseséricapodemdesempenharumpapelnaetiopatogeniado zumbido.
© 2020 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Publicado por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http:// creativecommons.org/licenses/by/4.0/).
Introduction
Tinnitusis definedastheperceptionof soundinthehead orinthehead intheabsenceofexternalsounds. Tinnitus isamong themost commonsymptoms of thehearing sys-temandadversely affectsthequalityof lifeof patients.1
Theincidence oftinnitusis between10% and15%.2
Tinni-tushasbeenreportedtobecausedbyanumberofdiseases affectingtheauditorynervoussystem.However,thecause oftinnitusisstillunknown.3
Prolidaseisametalloenzymidebelongingtothefamilyof hydrolases,whichplaysanimportantroleincollagen degra-dation and biosynthesis. Prolidase enzymatic activity has beendetectedinmanytissuessuchasstomach,heart,brain, pancreas,kidney,liverandamnioticfluid.4Itisthoughtthat
changesintheactivityofprolidaseenzymemayplayarole inthedevelopmentofmanydiseasessuchasnasalpolyps, schizophrenia,anxiety,bipolar disorder,majordepression, Type2diabetes mellitus, chronic hepatitisC, osteoporosis, bronchialasthmaandliverfibrosis.5---11
Reactive oxygen species (ROS) are reactive molecules containingone or more unpaired electrons in their outer atomic orbitals.12 ROS overproduction is involved in the
pathogenesis of many diseases. These include diabetes mellitus,cancer, rheumatoidarthritis,systemiclupus ery-thematosus,Behc¸et’sdisease,andatherosclerosis.13Various
antioxidantdefense mechanisms have been developed by the organism to protect against the adverse effects of
ROS. Undernormal conditionsthereis abalance between theantioxidantdefense mechanismand freeradicals,but oxidative stress is observed if this balance is impaired in the direction of free radicals.14 Oxidant and antioxidant
molecules canbemeasuredindividually intheplasma but recently total oxidant status (TOS) and total antioxidant status(TAS)measurementshavebeenimproved.15
Wedecidedtodothisstudybecauseofthefactthat oxi-dativestress andprolidaseenzyme arebeinginvestigated in manydiseasesand thereis alimitednumberof studies addressingtheirrelationshipswithetiopathogenesisof tin-nitus,whichinturn,isstillnotfullyexplained.Weaimedto comparetheserumlevelsofprolidase,TASandTOSin tin-nitus patients withnormalhealthy subjects.Although the relationshipbetweentinnitusandoxidativestresshasbeen studiedpreviously,itsrelationshipwithprolidasehasnever been investigated. This is the firststudy in the literature toexaminethelevelsofprolidaseenzymesinpatientswith tinnitus.
Materials
and
methods
ThisstudywascarriedoutattheOtorhinolaryngologyClinic ofHitit Universitybetween10January 2018and31March 2018.ThestudyprotocolwasapprovedbyHititUniversity, Ethics Committee for ClinicalResearch (decree no. 2018-23). Atotalof 25patients diagnosedwithtinnitusand 25
healthy subjects were included in the study. There were 14 were males and 11 were females witha mean age of 34.3yearsinthetinnitusgroup.The controlgroup consis-tedof25subjects,12malesand13females,withamean age of 37.2 years.All participants provided their written informedconsents.Allpatientswereexaminedbyan otola-ryngologistwithdetailedhistory.Inaddition,routineblood tests, audiologic evaluation and neuro-otologic diagnostic evaluationwereperformed.Thecriteriaforinclusioninthe tinnitusgroupwerethepresenceofsubjectivetinnitusforat least1yearattheageof18---65yearsandnomedical treat-mentfortinnitusuntilatleast8weeksbeforeenteringthe study.Thecontrolgroupincludednormalhealthyindividuals whohadnocomplainoftinnitus,betweentheagesof18---65, assistedinourclinicforotherreasons.Inbothgroups,the exclusioncriteriawerethepresenceofotosclerosis, tympa-nosclerosis,chronicotitismedia,vestibularschwannomas, Meniere’sdisease, malignancy,autologoussurgicalhistory, venoushum,ototoxicdrugtaking,temporalbonetrauma, middleor externalearproblems. Patientswithmetabolic, hematologic, cardiovascular, psychiatric and neurological diseaseswerealsoexcludedfromthestudy.Otologic, neu-rological andaudiologic examinationswere normal for all participants.Onlyoneserumsamplewastakenbeforeany type of treatment, frompatients and healthy volunteers. Aninformedconsentwasobtainedfromallpatientsinboth groupsasanindicationoftheirvoluntaryparticipationinthe study.A10mLofvenousbloodsamplewasdrawnfromeach patientbetween8:00AMand10:00AM,intotheclot acti-vatorcontainingtubes(Isotherm,HongyuMedical,Weihai, China)after12hoffasting.Aftertheformationoftheblood clot,thesampleswerecentrifugedfor10minat4000rpm, andtheserumwasseparatedandthenstoredat−80◦Cuntil thedayofanalysis.
TAS,TOSandprolidasemeasurements
Weusedtotalantioxidantandtotaloxidantcapacity mea-surements, developed by Erel.15,16 For the TAS and TOS
analyzes,themethoddevelopedbyErelwasused.Forthis purpose a completely automated RelAssay kit (Rel Assay Diagnostics kit, Mega Tip, Gaziantep, Turkey) was used and colorimetric method using the VitalScientific, Selec-tra/Flexor E(The Netherlands) autoanalyzer.TAS andTOS Results were presented as moL H2O2equivalent/L. SPEA
levels were measured by ELISA (Cat. no. CSB-E16196h, CusaboBiotechCo.Ltd).Intra-assayCoefficientofVariation (CV)andinter-assaycoefficientwere<8%and<10%, respec-tively.Thetestintervalwasbetween93.75and6000mU/mL withasensitivityof 93.75mU/mL.Forthemeasurements, RadimcompanyAL˙ISE˙IautomaticELISAwasused.
Statisticalanalysis
TheSPSSsoftwarepackage(version22.0,SPSSInc.,Chicago, IL, USA) was chosen for statistical analysis. The norma-litydistributionwasdeterminedbythe Shapiro---Wilktest. Continuous variables were presented as mean±standard deviation,median(min---max)accordingtodispersion hypo-theses,andcategoricalvariablespresentedasnumberand percentage.Whencontinuousvariableswereassessed,two
independentfactorswereanalyzedbyStudent’st-testifthe variableswerenormallydistributed.The Mann---WhitneyU
testwasusedwhenindependentsampleswerenotnormally distributed.Significancelevelwasacceptedasp<0.05.
Results
The patient and the control groups were similar in terms of age and gender (p>0.05 for both). The mean TOSvalue was 2.54---0.95mmoL/L and median (min---max) 2.58 (0.37---5.04mmoL/L) in the patient group, and it was 2.06±0.98mmoL/L and median (min---max) 1.92 (0.37---4.81mmoL/L) in the control group. Serum TOS levels in the patient group were significantly higher than in the control group (p=0.037) (Table 1, Fig. 1A). The mean OSI levels was 0.22±0.10AU and median (min---max) 0.20 (0.03---0.49AU) in the patient group, while it was 0.17±0.08AU and median (min---max) 0.17 (0.03---0.44AU) in the control group. OSI was signifi-cantly higher in the patient group (0.026) (Table 1,
Fig. 1B). The mean TAS level was 1.16±0.15mmoL/L and median (min---max) 1.17 (0.71---1.43mmoL/L) in the patientgroupand1.18±0.18mmoLandmedian(min---max) 1.15(0.94---1.70mmoL/L)in the controlgroup. There was nosignificant difference between the groups in terms of TAS values (p=0.838) (Table 1, Fig. 1C). The mean SPEA level was 202.74±33.56U/L and median (min---max) 199 (161---290U/L)inthepatientgroupand175.46±42.68U/L and median (min---max) 188 (112---251U/L) in the control group.SPEA levelsin the patientgroup weresignificantly higherthanthecontrolcroup(0.040)(Table1,Fig.1D).
Discussion
Tinnitusisclassifiedasobjectiveandsubjective.Objective tinnitusisalsoheardbyotherpeopleandthemostcommon causearevascularanomalies.2Subjectivetinnitusisdefined
asthesoundheardonlybythepatient.Thepathology lea-dingtotinnituscanbelocatedanywherefromtheearcanal tothehearing nervouscenter. Subjective tinnitus accom-paniedbysensorineuralhearinglossarethemostcommon symptomsoftinnitussecondarytoacoustictrauma.17Among
thediseasesthatcancausesubjectivetinnitusare otosclero-sis,tympanosclerosis,acoustictrauma,suddenhearingloss, stress,ototoxicdrugs,Meniere’sdisease,headtrauma,and acousticneuroma.18Somemetabolic,neurological,
psycho-logicalandpharmacologicaldiseasescanalsobecausesof subjectivetinnitus.19
The etiopathogenesis of tinnitus hasnot yet been cla-rified.The developmentof tinnituscanoften beduetoa cochlear damage.20 In some studies, oxidative stress has
been reported to disrupt the sensorineural epithelium of thelabyrinthine,acoustic,andvestibularnerves.20---22Some
experimental studies reported that ROS can damage the cochlearsensoryepithelium.23 Some othershave reported
anincreaseofoxidativestressintinnituspatients.24
Oxida-tivestress plays an importantrole in thepathogenesis of tinnitusduetoacoustictrauma.Excessivenoiseexposure, may cause ROS accumulation, which can lead to necro-sis and apoptosis of the outer hair cells.25 Normally, the
Table1 ComparisonofpatientsandcontrolgroupsintermsofTOS,OS˙I,TASandSPEAlevels.
Group n Mean±SD Median(min---max) p-value
TOS Patient 25 2.54±0.95 2.58(0.37---5.04) 0.037a Control 25 2.06±0.98 1.92(0.37---4.81) OS˙I Patient 25 0.22±0.10 0.20(0.03---0.49) 0.026a Control 25 0.17±0.08 0.17(0.03---0.44) TAS Patient 25 1.16±0.15 1.17(0.71---1.43) 0.838 Control 25 1.18±0.18 1.15(0.94---1.70) SPEA Patient 25 202.78±33.56 199(161---290) 0.040a Control 25 175.46±42.68 188(112---251)
SD,standarddeviation;min,minimum;max,maximum. aStatisticallysignificant(p<0.05). 6,00 ,50 ,40 ,30 ,20 ,10 ,00 300,00 250,00 200,00 150,00 100,00 1,75 1,50 1,25 1,00 ,05 5,00 4,00 2,00 1,00 ,00 3,00 TO S OSI Prolidase TA S patient patient patient patient Control Control Control Control Groups Groups Groups Groups 50 22 21 12 20 8 * 50 12 13 6 6 8 36
A
B
C
D
Figure1 BoxplotsofmeanchangesinTOS(A),OS˙I(B),TAS(C)andSPEA(D)accordingtogroups.
andvariousenzymesagainstoxidativemolecules.According tosomereports,theseantioxidantsmaynotbedetoxifying enoughtoplayaroleinprotectingagainstacutetraumaand someothereardiseases.26Thus,sensorineuralepitheliumis
atriskforROS-inducedlesionsincochlea.Oxidativestress hasbeen reportedtodamage the labyrinth, acousticand vestibularnervoussystem.27
Savastano et al., prescribed phospholipidsand various vitaminsasoralantioxidanttherapyforatotalof31patients withidiopathicunilateraltinnitusfor18weeks.Theywere submittedtoapure toneaudiometric evaluation,andthe severity of ear tinnitus was determined by a visual
ana-loguescale(VAS)48hbeforeandafterthetreatment.There wasareduction ofthetinnitus intensitywiththe antioxi-danttreatment.28Similarly,Gopaletal.,reportedthatthe
severityoftinnitusdecreasedafterantioxidanttherapyand concluded that antioxidant agents might be an option in tinnitus treatment.29 On the other hand, Polanski et al.,
reported that antioxidant agents had no benefits in the treatmentoftinnitus.30
Nerietal.,investigatedthelevelsofmalonaldehyde, 4-hydroxynonenal and myeloperoxidase enzymes,which are markersofoxidativedamage,intheirstudyof44patients with tinnitusand 25 healthy volunteers.31 In the patients
withtinnitus,these3parameterswerefoundtobehigher thaninthecontrolgroupandtheantioxidantenzyme glu-tathione peroxidase was found to be lower. According to theauthors,this elevatedoxidative stressmaycause vas-cular endothelial dysfunction leading to a defect in the microcirculationoftheinnerear.Basedonthesefindings, oxidativestresswasfoundtobeimportantintinnitus patho-genesis. Koc et al., studied 54 tinnitus patients and 60 healthsubjects.TheyreportedthatTINandoxidativestress levelswerestatisticallysignificantlyhigherinpatientswith tinnitus. TAS and PON levels were significantly lower in thetinnitusgroupthaninthecontrolgroup.They conclu-ded thatpatients withtinnitus wereexposedtooxidative stress.24Inourstudy,wefoundthatTOSandOSIlevelswere
statisticallyhigherintinnituspatientsthanincontrols.We alsofoundhighlevelsofSPEAinpatientswithtinnitus.
Aubert etal.,in an experimental study carried outon frogs,found thattheinjection ofphenazine methasulfate into the perilymphatic region caused an increase in ROS formationanddeteriorationofendolymphsecretion.They reportedthattheadditionoftrimethasinetothe perilym-phatic region was protective against harmful effects of ROS.22 Theseresults indicatethat thebeneficialeffectof
trimetazineobservedduringtinnitustreatmentmaybedue toantioxidantproperties.
Prolidaseplaysanimportantroleintheregulationof col-lagen biosynthesisanddegradation. Theprolidaseenzyme is the only enzyme that catalyzes the intracellular rapid hydrolysisofprolineandhydroxyprolinethatoccursatthe end of collagen catabolism. So, the collagen metabolism canbedeterminedbytheSPEAlevels.32Inastudy
conduc-tedinrats, itwasshown thatexposureofproline torats’ brainsreducestheantioxidantpotentialandinduces oxida-tivestress.33
Somestudiesintheliteraturehavereportedthatthere is a direct relationship between tinnitusand various psy-chological disorders.34 In our literature review, we found
thattherelationshipbetweentinnitusandprolidaseenzyme hasnotbeenpreviouslyinvestigated.However,ithasbeen reported that the activity of prolidase enzyme increases in diseases such as anxiety disorder, depression, bipolar disorder, which are frequently associated with tinnitus. For this reason, we added the enzyme prolidase in our investigation.
Simsek et al., observed that the incidence of anxiety disorder and depression was significantly increased in patients withsubjective tinnitus.35 Ercanet al. reported
that oxidative stress index and prolidase enzyme activity wereincreasedinanxiouspatientscomparedtothecontrol group. They also reported that levels of oxidative stress severitymayberelatedtoSPEAenzyme levels,thus indi-catingthatSPEAlevelsmaybeindicativeofdiseaseactivity inanxietydisorders.12
Onelimitationofthisstudywasthesmallsizeofboth, patientandcontrolgroups.Regardless,westillbelievethat investigating the relationship between tinnitus symptoms scoresandSPEAandOS˙Ilevelsmaybeuseful.
Conclusion
Wefound that totaloxidantstatus, oxidative stressindex andserumprolidaseenzymeactivitylevelswerestatistically higherinpatientswithtinnitusthanincontrols.Therewas nosignificant differencebetweenthe tinnitusandcontrol groups in terms of total antioxidant status levels. These findings suggest that tinnitus patients were found to be exposedtomoreoxidativestress.Elevatedserumprolidase enzymeactivityandoxidativestressindexlevelsmayhave aroleinthepathogenesis oftinnitus.Thereis aneed for moreextensiveandcomprehensiveworkinthisarea.
Funding
ThisstudywasfundedbyHititUniversityScientificResearch Projects.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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