Research ArticleBlood Contamination in Saliva Impact on the Measurement ofSalivary Oxidative Stress Markers
Nataacutelia Kamodyovaacute1 Lenka Ba^asovaacute2 Katariacutena Janšaacutekovaacute1 Ivana Koborovaacute1
7ubomiacutera Toacutethovaacute13 Peter Stanko2 and Peter Celec1345
1 Institute of Molecular Biomedicine Faculty of Medicine Comenius University Sasinkova 4 811 08 Bratislava Slovakia2Department of Stomatology and Maxillofacial Surgery Comenius University Heydukova 10 812 50 Bratislava Slovakia3Center for Molecular Medicine Slovak Academy of Sciences Vlarska 7 831 01 Bratislava Slovakia4Institute of Pathophysiology Faculty of Medicine Comenius University Sasinkova 4 811 08 Bratislava Slovakia5Department of Molecular Biology Faculty of Natural Sciences Comenius University Mlynska Dolina 842 15 Bratislava Slovakia
Correspondence should be addressed to Lrsquoubomıra Tothova tothovalubomiragmailcom
Received 5 March 2015 Accepted 8 July 2015
Academic Editor Kishore Chaudhry
Copyright copy 2015 Natalia Kamodyova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Salivary oxidative stress markers represent a promising tool for monitoring of oral diseases Saliva can often be contaminated byblood especially in patients with periodontitis The aim of our study was to examine the impact of blood contamination on themeasurement of salivary oxidative stress markers Saliva samples were collected from 10 healthy volunteers and were artificiallycontaminatedwith blood (final concentration 0001ndash10) Next saliva was collected from 12 gingivitis and 10 control patients beforeand after dental hygiene treatment Markers of oxidative stress were measured in all collected saliva samples Advanced oxidationprotein products (AOPP) advanced glycation endproducts (AGEs) and antioxidant statuswere changed in 1blood-contaminatedsaliva Salivary AOPP were increased in control and patients after dental treatment (by 457 and 341 119901 lt 001) Salivary AGEswere decreased in patients after microinjury (by 693 119901 lt 0001) Salivary antioxidant status markers were decreased in bothcontrol and patients after dental treatment (119901 lt 005 and 119901 lt 001) One blood contamination biased concentrations of salivaryoxidative stress markers Saliva samples with 1 blood contamination are visibly discolored and can be excluded from analyseswithout any specific biochemic detection of blood constituents Salivary markers of oxidative stress were significantly altered inblood-contaminated saliva in control and patients with gingivitis after dental hygiene treatment
1 Introduction
Markers of oxidative stress in saliva have become an attractivetool for analyzing the pathogenesis and monitoring of oraland dental diseases Cross-reacting substances in the mouthand saliva collection methods could influence assay validityof oxidative stress markers [1ndash3] Blood leakage into saliva asa result of microinjury represents an important factor that isexpected to influence the concentrations of markers of oxida-tive stress in salivaThe blood contamination in saliva ismorecommon in individuals suffering frompoor oral health and inpatients with gingivitis or periodontitis The concentrationsof oxidative stress markers are typically several times higherin plasma than in saliva [4] In patients with gingivitis bloodleakage into saliva could artificially increase concentrations
of salivary markers of oxidative stress Recently the effect ofblood contamination on salivary concentrations of selectedhormones was shown [5ndash7] Despite the rising popularity ofsalivary oxidative stress analyses in patients with periodontaldiseases no reports have been published regarding the effectof blood contamination in saliva on concentrations of oxida-tive stress markers The standardization of methodologicalprocesses is a key step before the implementation of salivarybiomarkers for disease prediction and progression
The aim of our study was to analyze the effect of artificialwhole blood contamination and the effect of contaminationwith individual blood components (plasma red blood cellsand hemoglobin) on salivary concentrations of markers ofoxidative stress in healthy probands In addition the impactof blood contamination should be studied in a case-control
Hindawi Publishing CorporationDisease MarkersVolume 2015 Article ID 479251 10 pageshttpdxdoiorg1011552015479251
2 Disease Markers
study comparing the salivary markers of oxidative stressin patients with gingivitis and healthy controls after dentalhygiene treatment
2 Subjects and Methods
21 Participants In study I 10 young periodontally healthyvolunteers (5 females and 5 males) with an average age of235 plusmn 19 years were enrolled In study II a total of 22subjects were enrolled in the dental ambulance in BratislavaSlovakia Twelve subjects were male patients with gingivitiswith an average age of 353 plusmn 80 years and 10 male subjectswere age-matched healthy controls with an average ageof 382 plusmn 49 years In study II subjects underwent anexamination of their periodontal status using plaque index(PI) [8] sulcus bleeding index (SBI) [9] and bleeding onprobing (BOP) All clinical measurements were performedby a single investigator (LB) Exclusion criteria in both studieswere systematic diseases acute illnesses pregnancy smokingand former smoking The studies were approved by theEthics Committee of the Institute of Molecular BiomedicineComenius University Bratislava Slovakia The clinical partof this study was performed according to the principlesexpressed in the Declaration of Helsinki Written informedconsent was obtained from each participating subject
22 Design and Sampling Whole unstimulated saliva sam-ples were collected in the morning before eating Collectedsaliva samples were stored atminus20∘Cuntil analyses On the dayof testing samples were brought to room temperature andcentrifuged at 1000 g for 10min and the supernatant was usedfor testing
In study I saliva samples were artificially contaminatedwith blood Samples of saliva were divided into aliquots Onealiquot from each individual was used as a control (no bloodadded) The remaining salivary aliquots were contaminatedby venous blood and serially diluted to obtain saliva sampleswith the following concentrations of blood 10 5 251 01 001 and 0001 Similar to contamination ofsaliva with whole blood other aliquots were contaminatedwith plasma red blood cells or hemoglobin (Sigma AldrichSteinheim Germany) ranging from 10 to 0001
In study II a baseline saliva sample was collected fromthe participants Dental hygiene treatment was performedby a dentist (LB) Saliva samples were collected again aftertreatment Dental hygiene treatment was used as a model ofblood leakage due to microinjury
23 Biochemical Analysis of Oxidative and Carbonyl StressMarkers in Saliva All reagents or chemicals used in ourexperiments were purchased from Sigma-Aldrich (Stein-heim Germany) Salivary advanced oxidation protein prod-ucts (AOPP) as markers of protein oxidation were deter-mined using a spectrophotometric method Two hundred 120583Lof saliva was incubated with glacial acetic acid and theabsorbance was read at 340 nm Chloramine T with potas-sium iodide was used as calibrator [10] The intra-assay andinterassay variability are 66 and 124 respectively
Salivary advanced glycation end products (AGEs) asmarkers of carbonyl stress were measured using spectroflu-orometric method Saliva samples were diluted 10-fold withphosphate buffered saline (PBS pH = 72) and measured at120582ex = 370 nm 120582em = 440 nm [11] The specific fluorescenceof AGEs was expressed in arbitrary unitsThe intra-assay andinterassay variability are 89 and 105 respectively
Ferric reducing antioxidant power (FRAP) marker ofantioxidant status was determined according to Benzie andStrain [12] The intra-assay and interassay variability are 17and 9 respectively Briefly prewarmed 37∘C FRAP reagent(1 volume of 3molL acetate buffer pH 36 + 1 volume of10mmolL 246-tripyridyl-S-triazine in 40mmolL HCl +1 vol of 20mmolL FeCl
3) was mixed with 20120583L of saliva
Absorbance was read at 593 nm Ferrous sulphate was usedas standard in calibration curve
Total antioxidant capacity (TAC) marker of antioxidantstatus was measured using spectrophotometric methodSaliva was mixed with acetate buffer (pH = 58) incubatedwith 221015840-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)and oxidized with hydrogen peroxide in acetate buffer (pH =36) Absorbancewasmeasured at 660 nm Troloxwas used asstandard in calibration curve [13] The intra-assay and inter-assay variability are 66 and 124 respectively
Total proteins were quantified using BCA protein assaykit (Sigma Aldrich Steinheim Germany) Briefly 10120583L ofsaliva was mixed with BCA working reagent incubated for30min at 37∘C and measured at 562 nm Concentrations ofsalivary oxidative stress markers were normalized to totalproteins All measurements were done on a Sapphire IIinstrument (Tecan Grodig Austria)
24 Statistical Analysis Analysis was performed with XLSta-tistics 100530 (Carr R XLentWorks Australia) and Graph-Pad Prism 503 (GraphPad Software San Diego California)In study I two-way repeated measures (RM) ANOVA wasused to analyze oxidative stress markers in saliva artificiallycontaminated with blood Based on the results from two-wayRM ANOVA data from both genders were combined andanalyzed using one-way RM ANOVA and Tukeyrsquos multiplecomparison test In study II the effect of microinjury onsalivary markers of oxidative stress before and after den-tal hygiene was determined using Wilcoxon matched-pairssigned rank test for control and gingivitis group separatelyData are presented as mean + SD Level 120572 = 005 was chosenas a limit level of significance
3 Results
31 Study I Saliva samples contaminated by whole bloodwith a final concentration of 01 blood and higher are visiblycolored (Figure 1) The effect of two independent variablesgender and blood contamination on dependent variableoxidative stress markers was analyzed using two-way RMANOVA Separate ANOVAs were used for whole bloodplasma red blood cells and hemoglobin contaminationBecause no major effect of gender was observed data fromboth genders were combined for further analyses (Table 1)
Disease Markers 3
Table 1 The effect of gender and blood contamination on oxidativestress markers was analyzed using two-way RM ANOVA SeparateANOVAs were computed for whole blood plasma red blood cells(RBC) and hemoglobin contamination
Two-way RMANOVA119865
119901 value 119901 valuesummary
Salivary AOPPBlood contamination
Effect of blood 4986 lt00001 lowastlowastlowastlowast
Effect of gender 051 049 ns
Plasma contaminationEffect of plasma 3359 lt00001 lowastlowastlowastlowast
Effect of gender 368 009 ns
RBC contaminationEffect of RBC 2963 lt00001 lowastlowastlowastlowast
Effect of gender 020 066 ns
Hemoglobin contaminationEffect of hemoglobin 2831 lt00001 lowastlowastlowastlowast
Effect of gender 136 028 nsSalivary AGEsBlood contamination
Effect of blood 2139 lt00001 lowastlowastlowastlowast
Effect of gender 542 00483 lowast
Plasma contaminationEffect of plasma 2075 lt00001 lowastlowastlowastlowast
Effect of gender 448 007 ns
RBC contaminationEffect of RBC 6802 lt00001 lowastlowastlowastlowast
Effect of gender 525 005 ns
Hemoglobin contaminationEffect of hemoglobin 1917 lt00001 lowastlowastlowastlowast
Effect of gender 652 00340 lowast
Salivary FRASBlood contamination
Effect of blood 1200 lt00001 lowastlowastlowastlowast
Effect of gender 3878119890minus008 099 ns
Plasma contaminationEffect of plasma 2359 lt00001 lowastlowastlowastlowast
Effect of gender 027 062 ns
RBC contaminationEffect of RBC 9772 lt00001 lowastlowastlowastlowast
Effect of gender 018 068 ns
Hemoglobin contaminationEffect of hemoglobin 1832 lt00001 lowastlowastlowastlowast
Effect of gender 08376 039 ns
Table 1 Continued
Two-way RMANOVA119865
119901 value 119901 valuesummary
Salivary TACBlood contaminationEffect of blood 2431 lt00001 lowastlowastlowastlowast
Effect of gender 020 067 nsPlasma contaminationEffect of plasma 3302 lt00001 lowastlowastlowastlowast
Effect of gender 051 049 nsRBC contaminationEffect of RBC 2437 lt00001 lowastlowastlowastlowast
Effect of gender 025 063 nsHemoglobin contaminationEffect of hemoglobin 4380 lt00001 lowastlowastlowastlowast
Effect of gender 015 071 nsAOPP advanced oxidation protein products AGEs advanced glycation endproducts FRAP ferric reducing antioxidant power TAC total antioxidantcapacity ns nonsignificantlowast
Figure 1 Saliva samples of two probands (columns P1 and P2) con-taminated by venous blood with the following final concentrationsof blood 10 5 25 1 01 001 0001 and 0 Note thevisible discoloration of saliva with blood contamination from 01
One-way RM ANOVA and Tukeyrsquos multiple comparison testwere used for further analyses A significant bias in themeasured concentrations of salivary oxidative stress markerswas caused by 1 blood contamination in saliva (Figure 2)AOPP as a marker of protein oxidation was significantlyhigher in saliva contaminatedwith 1 and 25 venous bloodby 1187 and 1685 respectively (119902 = 1313 and 119902 = 1863119901 lt 00001 Tukeyrsquos test Figure 2(a)) In saliva samplescontaminated with 5 and 10 blood AOPP concentrationswere lower in comparison to samples contaminatedwith 25blood (Figure 2(a)) Salivary carbonyl stress measured asAGEs concentrations decreased proportionally when bloodwas added to saliva at concentrations 1ndash10 (Figure 2(b))
Figure 2 Oxidative stress and antioxidant status markers in saliva contaminated with blood (a) Salivary AOPP concentrations biomarker ofoxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrations biomarkerof antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SD lowastlowastlowastlowast119901 lt 00001lowastlowast119901 lt 001 and lowast119901 lt 005
Concentrations of antioxidant statusmarkers FRAP and TACalso decreased in blood-contaminated saliva (Figures 2(c)and 2(d))
To determine which blood component is responsible forchanges in measured salivary markers the impact of plasmared blood cells and hemoglobin was studied Salivary AOPPconcentrations were decreased proportionally in the pres-ence of 01ndash10 plasma contamination in saliva by 273ndash850 (Figure 3(a)) Salivary AOPP concentrations wereincreased in the presence of 01ndash10 red blood cells by 804ndash4933 (Figure 4(a)) A similar trend was observed afteraddition of 25ndash10 hemoglobin to saliva with increasedAOPP concentrations by 721ndash1094 (Figure 5(a)) SalivaryAGEs were increased in the presence of 25ndash10 plasma insaliva by 506ndash699 (Figure 3(b)) Addition of 1ndash10 redblood cells or hemoglobin resulted in comparable changes(Figures 4(b) and 5(b)) Concentrations of antioxidant statusmarkers FRAP and TAC were decreased in saliva whenplasma (Figures 3(c) and 3(d)) red blood cells (Figures 4(c)and 4(d)) or hemoglobin (Figures 5(c) and 5(d)) were added
32 Study II To study the effect of blood contamination in areal clinical situation the impact of microinjury in gingivitisand age-matched healthy control patients was modeledClinical parameters of both study groups are summarized inTable 2 Clinical parameters were significantly worse in thegingivitis group compared with the control group (Table 2)Dental hygiene asmodel ofmicroinjury caused blood leakagein both patients groups The effect of microinjury on salivarymarkers of oxidative stress before and after dental hygienewas determined using Wilcoxon matched-pairs signed ranktest for control and gingivitis group separately SalivaryAOPPconcentrations were increased in 9 out of 10 control probandsand in 10 out of 12 gingivitis patients after microinjury(Figures 6(a) and 6(e)) Salivary AGEs concentrations weredecreased in 9 out of 10 control probands and in 12 out of12 gingivitis patients (Figures 6(b) and 6(f)) Salivary FRAPconcentrationswere decreased in 7 out of 10 control probandsand in 11 out of 12 gingivitis patients after treatment (Figures6(c) and 6(g)) Salivary TAC concentrations were decreasedin 9 out of 10 in control probands and in 11 out of 12 gingivitispatients after microinjury (Figures 6(d) and 6(h))
Figure 3 Oxidative stress and antioxidant status markers in saliva contaminated with plasma (a) Salivary AOPP concentrations biomarkerof oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrations biomarkerof antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SD lowastlowastlowastlowast119901 lt 00001lowastlowastlowast119901 lt 0001 lowastlowast119901 lt 001 and lowast119901 lt 005
Table 2 Comparison of clinical parameters between control and gingivitis patients
Clinical parameterGroup
Unpaired 119905-test119905
119901 value 119901 value summaryControl Gingivitis(119899 = 10) (119899 = 12)
Schwartz and Granger reported that blood components insaliva invisible to the eye have the potential to bias salivaryanalytes and the control of blood contamination in saliva wassuggested Transferrin enzymatic immunoassaywas designedfor quantitative monitoring of blood contamination [14] Itwas shown that concentrations of testosterone dehydroepi-androsterone (DHEA) and cortisol are increased in salivasamples artificially contaminated with blood [14] On the
other hand microinjury of the oral cavity did not changeDHEA or cortisol and increased salivary testosterone [6]Despite the rising number of studies analyzing oxidativestress in saliva no reports have been published regarding theeffect of occult blood contamination on concentrations of sal-ivary markers of oxidative stress
Four markers of oxidative stress and antioxidant statuswere analyzed in our study Advanced oxidation protein pro-ducts (AOPP) a novel oxidative stress biomarker was discov-ered in the plasma of uremic patients in 1996 [10] Recently
Figure 4 Oxidative stress and antioxidant status markers in saliva contaminated with red blood cells (RBC) (a) Salivary AOPPconcentrations biomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) SalivaryFRAP concentrations biomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presentedas mean + SD lowastlowastlowastlowast119901 lt 00001 and lowast119901 lt 005
AOPPwas suggested as part of the nonenzymatic antioxidantsystem of plasma proteome and oxidized fibrinogenwas indi-cated as key molecule responsible for human plasma AOPPreactivity [15] Advanced glycation end products (AGEs)marker of carbonyl stress are developed during the reactionof protein amino groups with reactive carbonyl compounds[16] Ferric reducing antioxidant power (FRAP) assay indi-rectly reflects the total antioxidant capacity of the sample[12 17] Total antioxidant capacity (TAC) assay developed byErel is directmeasurementmethod for total antioxidants [13]Our results have shown that most of the measured salivarymarkers of oxidative stress and antioxidant status are biasedin the presence of 1blood contamination in salivaHoweveras shown in results section saliva contaminatedwith 1 bloodis visually discolored Blood-contaminated saliva samples canbe easily excluded from the analyses Thus at least for themarkers analyzed in this study there is no need to use salivarytransferrin assay for the monitoring of blood contamination
To determine which blood component is responsiblefor changes in measured salivary markers the impact ofplasma red blood cells and hemoglobin was studied Based
on our results increased AOPP concentrations in blood-contaminated saliva can be explained by the presence ofred blood cells and hemoglobin in saliva Hemoglobin insaliva probably interferes with the colorimetric AOPP assayand artificially increases the AOPP concentrations AOPPconcentrations in plasma of healthy probands were reportedas 3 times as high as AOPP concentrations in saliva [15]we have therefore expected increased AOPP concentrationsin saliva contaminated with plasma But an opposite trendwas observed when plasma was spiked into saliva anddecreased salivary AOPP was detected in the presence of01ndash10 plasma When plasma is spiked into saliva theconcentration of total proteins is increased In our studydecreased concentrations of measured salivary markers afterplasma addition into saliva could be caused by normaliza-tion to the increased total proteins Based on our resultshemoglobin in saliva can mask the detection of AGEs andlead to underestimation of AGEs in saliva Plasma additioninto saliva led to decreased salivary AGEs concentrationsSalivary FRAP and TAC concentrations were decreased inthe presence of RBC hemoglobin and plasma contamination
Disease Markers 7
0
50
100
150
200
250
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
AOPP
(120583m
olg
)
(a)
0
500
1000
1500
2000
2500
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
AGEs
(au
g)
(b)
0
200
400
600
800
lowastlowastlowast
lowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
FRA
P (120583
mol
g)
(c)
0
500
1000
1500
lowastlowastlowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
TAC
(120583m
olg
)
(d)
Figure 5 Oxidative stress and antioxidant status markers in saliva contaminated with hemoglobin (a) Salivary AOPP concentrationsbiomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrationsbiomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SDlowastlowastlowastlowast119901 lt 00001 lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001
in saliva Decreased concentrations of measured markers insaliva contaminated with plasma could be as in the case ofAOPP assay caused by normalization to total proteins
The effect of blood contamination in saliva on concentra-tions of salivary markers of oxidative stress was not studiedin real clinical situation in the past Concerning that bloodcontamination in saliva is common in patients with gingivitisthe impact of microinjury was modeled in this study groupand age-matched healthy controls The results were similar tosaliva artificially contaminated with blood AOPP concentra-tions were increased after microinjury in the visual presenceof blood in saliva AOPP concentrations were increased inboth control and gingivitis group after dental hygiene treat-ment AOPP concentrations were not different between con-trol and gingivitis patients before dental hygiene treatmentAlso other measured salivary markers followed the trendobserved in saliva artificially contaminated with blood Sali-vary AGEs concentrations and also antioxidant markers TACand FRAP were decreased after dental hygiene treatment dueto presence of blood in saliva Our results have confirmedthe concern that dental hygiene treatment could bias the
concentrations of oxidative stress markers in saliva Based onour results we recommend the saliva collection before dentalhygiene treatment or clinical examination of oral cavity
5 Conclusions
Salivary oxidative stress concentrations are significantlyinfluenced by 1 blood contamination in saliva Salivasamples with 1 blood contamination are visibly colored andit is possible to easily exclude such contaminated samplesfrom further salivary oxidative stress analyses Microinjuryto the periodontium caused blood leakage into saliva in bothgingivitis and control group and biased concentrations ofoxidative stress markers in saliva For the purpose of salivaryoxidative stress analyses saliva samples should be collectedbefore dental hygiene treatment or clinical examination of theoral cavity
Conflict of Interests
The paper is original work and is not under consideration byanother journal There is no conflict of interests to declare
8 Disease Markers
Control patients
Control patients
Control patients
(a)
(b)
(c)
Before AfterDental hygiene treatment
0
500
1000
1500
2000
AGEs
(au
g)
Gingivitis patients
Before AfterDental hygiene treatment
0
500
1000
1500
2000
lowastlowastlowast
AGEs
(au
g)
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Before AfterDental hygiene treatment
0
200
400
600
800
lowast
FRA
P (120583
mol
g)
(f)
(g)
Gingivitis patients
Before AfterDental hygiene treatment
0
200
400
600
800
lowastlowast
FRA
P (120583
mol
g)
(e)
Gingivitis patients
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Figure 6 Continued
Disease Markers 9
(d) (h)
Control patients Gingivitis patients
Before Afterminus500
0
500
1000
1500
Dental hygiene treatment
lowast
TAC
(120583m
olg
)
Before AfterDental hygiene treatment
lowastlowastlowast
minus500
0
500
1000
1500
TAC
(120583m
olg
)
Figure 6 The effect of blood leakage after dental hygiene treatment on markers of oxidative stress and antioxidant status in saliva of control(andashd) and gingivitis (endashh) patients (a e) Salivary AOPP concentrations biomarker of oxidative damage to proteins (b f) Salivary AGEsconcentrations biomarker of carbonyl stress (c g) Salivary FRAP concentrations biomarker of antioxidant status (d h) Salivary TACconcentrations biomarker of antioxidant status lowastlowastlowast119901 lt 00001 lowastlowast119901 lt 001 and lowast119901 lt 005
This publication is the result of the implementation of theproject of University Science Park of Comenius University inBratislava (ITMS 26240220086) supported by the Researchand Development Operational Programme funded by theEuropean Regional Development Fund
References
[1] J Hodosy and P Celec ldquoDaytime of sampling tooth-brushingand ascorbic acid influence salivary thiobarbituric acid reactingsubstancesmdasha potential clinical marker of gingival statusrdquoDisease Markers vol 21 no 4 pp 203ndash207 2005
[2] N Kamodyova and P Celec ldquoSalivary markers of oxidativestress and Salivette collection systemsrdquo Clinical Chemistry andLaboratory Medicine vol 49 no 11 pp 1887ndash1890 2011
[3] N Kamodyova L Tothova and P Celec ldquoSalivary markersof oxidative stress and antioxidant status influence of externalfactorsrdquo Disease Markers vol 34 no 5 pp 313ndash321 2013
[4] F A Akalin E Baltacioglu A Alver and E Karabulut ldquoLipidperoxidation levels and total oxidant status in serum saliva andgingival crevicular fluid in patients with chronic periodontitisrdquoJournal of Clinical Periodontology vol 34 no 7 pp 558ndash5652007
[5] D A Granger E A Shirtcliff A Booth K T Kivlighanand E B Schwartz ldquoThe lsquotroublersquo with salivary testosteronerdquoPsychoneuroendocrinology vol 29 no 10 pp 1229ndash1240 2004
[6] K T Kivlighan D A Granger E B Schwartz V Nelson MCurran and E A Shirtcliff ldquoQuantifying blood leakage intothe oral mucosa and its effects on the measurement of cortisoldehydroepiandrosterone and testosterone in salivardquoHormonesand Behavior vol 46 no 1 pp 39ndash46 2004
[7] D A Granger D Cicchetti F A Rogosch L C HibelM Teisl and E Flores ldquoBlood contamination in childrenrsquossaliva prevalence stability and impact on the measurement
of salivary cortisol testosterone and dehydroepiandrosteronerdquoPsychoneuroendocrinology vol 32 no 6 pp 724ndash733 2007
[8] H Loe and J Silness ldquoPeriodontal disease in pregnancy IPrevalence and severityrdquo Acta Odontologica Scandinavica vol21 no 6 pp 533ndash551 1963
[9] H R Muhlemann and S Son ldquoGingival sulcus bleedingmdashaleading symptom in initial gingivitisrdquo Helvetica OdontologicaActa vol 15 no 2 pp 107ndash113 1971
[10] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996
[11] G Munch R Keis A Wessels et al ldquoDetermination ofadvanced glycation end products in serum by fluorescencespectroscopy and competitive ELISArdquo European Journal ofClinical Chemistry and Clinical Biochemistry vol 35 no 9 pp669ndash677 1997
[12] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[13] O Erel ldquoA novel automated direct measurement method fortotal antioxidant capacity using a new generation more stableABTS radical cationrdquo Clinical Biochemistry vol 37 no 4 pp277ndash285 2004
[14] E B Schwartz and D A Granger ldquoTransferrin enzyme immu-noassay for quantitative monitoring of blood contamination insalivardquo Clinical Chemistry vol 50 no 3 pp 654ndash656 2004
[15] Z Qing E Ling-Ling W Dong-Sheng and L Hong-ChenldquoRelationship of advanced oxidative protein products in humansaliva and plasma age-and gender-related changes and stabilityduring storagerdquo Free Radical Research vol 46 no 10 pp 1201ndash1206 2012
[16] T Miyata K Kurokawa and C Van Ypersele De StrihouldquoAdvanced glycation and lipoxidation end products role ofreactive carbonyl compounds generated during carbohydrate
10 Disease Markers
and lipid metabolismrdquo Journal of the American Society of Neph-rology vol 11 no 9 pp 1744ndash1752 2000
[17] R L Prior and G Cao ldquoIn vivo total antioxidant capacity com-parison of different analytical methodsrdquo Free Radical Biologyand Medicine vol 27 no 11-12 pp 1173ndash1181 1999
study comparing the salivary markers of oxidative stressin patients with gingivitis and healthy controls after dentalhygiene treatment
2 Subjects and Methods
21 Participants In study I 10 young periodontally healthyvolunteers (5 females and 5 males) with an average age of235 plusmn 19 years were enrolled In study II a total of 22subjects were enrolled in the dental ambulance in BratislavaSlovakia Twelve subjects were male patients with gingivitiswith an average age of 353 plusmn 80 years and 10 male subjectswere age-matched healthy controls with an average ageof 382 plusmn 49 years In study II subjects underwent anexamination of their periodontal status using plaque index(PI) [8] sulcus bleeding index (SBI) [9] and bleeding onprobing (BOP) All clinical measurements were performedby a single investigator (LB) Exclusion criteria in both studieswere systematic diseases acute illnesses pregnancy smokingand former smoking The studies were approved by theEthics Committee of the Institute of Molecular BiomedicineComenius University Bratislava Slovakia The clinical partof this study was performed according to the principlesexpressed in the Declaration of Helsinki Written informedconsent was obtained from each participating subject
22 Design and Sampling Whole unstimulated saliva sam-ples were collected in the morning before eating Collectedsaliva samples were stored atminus20∘Cuntil analyses On the dayof testing samples were brought to room temperature andcentrifuged at 1000 g for 10min and the supernatant was usedfor testing
In study I saliva samples were artificially contaminatedwith blood Samples of saliva were divided into aliquots Onealiquot from each individual was used as a control (no bloodadded) The remaining salivary aliquots were contaminatedby venous blood and serially diluted to obtain saliva sampleswith the following concentrations of blood 10 5 251 01 001 and 0001 Similar to contamination ofsaliva with whole blood other aliquots were contaminatedwith plasma red blood cells or hemoglobin (Sigma AldrichSteinheim Germany) ranging from 10 to 0001
In study II a baseline saliva sample was collected fromthe participants Dental hygiene treatment was performedby a dentist (LB) Saliva samples were collected again aftertreatment Dental hygiene treatment was used as a model ofblood leakage due to microinjury
23 Biochemical Analysis of Oxidative and Carbonyl StressMarkers in Saliva All reagents or chemicals used in ourexperiments were purchased from Sigma-Aldrich (Stein-heim Germany) Salivary advanced oxidation protein prod-ucts (AOPP) as markers of protein oxidation were deter-mined using a spectrophotometric method Two hundred 120583Lof saliva was incubated with glacial acetic acid and theabsorbance was read at 340 nm Chloramine T with potas-sium iodide was used as calibrator [10] The intra-assay andinterassay variability are 66 and 124 respectively
Salivary advanced glycation end products (AGEs) asmarkers of carbonyl stress were measured using spectroflu-orometric method Saliva samples were diluted 10-fold withphosphate buffered saline (PBS pH = 72) and measured at120582ex = 370 nm 120582em = 440 nm [11] The specific fluorescenceof AGEs was expressed in arbitrary unitsThe intra-assay andinterassay variability are 89 and 105 respectively
Ferric reducing antioxidant power (FRAP) marker ofantioxidant status was determined according to Benzie andStrain [12] The intra-assay and interassay variability are 17and 9 respectively Briefly prewarmed 37∘C FRAP reagent(1 volume of 3molL acetate buffer pH 36 + 1 volume of10mmolL 246-tripyridyl-S-triazine in 40mmolL HCl +1 vol of 20mmolL FeCl
3) was mixed with 20120583L of saliva
Absorbance was read at 593 nm Ferrous sulphate was usedas standard in calibration curve
Total antioxidant capacity (TAC) marker of antioxidantstatus was measured using spectrophotometric methodSaliva was mixed with acetate buffer (pH = 58) incubatedwith 221015840-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)and oxidized with hydrogen peroxide in acetate buffer (pH =36) Absorbancewasmeasured at 660 nm Troloxwas used asstandard in calibration curve [13] The intra-assay and inter-assay variability are 66 and 124 respectively
Total proteins were quantified using BCA protein assaykit (Sigma Aldrich Steinheim Germany) Briefly 10120583L ofsaliva was mixed with BCA working reagent incubated for30min at 37∘C and measured at 562 nm Concentrations ofsalivary oxidative stress markers were normalized to totalproteins All measurements were done on a Sapphire IIinstrument (Tecan Grodig Austria)
24 Statistical Analysis Analysis was performed with XLSta-tistics 100530 (Carr R XLentWorks Australia) and Graph-Pad Prism 503 (GraphPad Software San Diego California)In study I two-way repeated measures (RM) ANOVA wasused to analyze oxidative stress markers in saliva artificiallycontaminated with blood Based on the results from two-wayRM ANOVA data from both genders were combined andanalyzed using one-way RM ANOVA and Tukeyrsquos multiplecomparison test In study II the effect of microinjury onsalivary markers of oxidative stress before and after den-tal hygiene was determined using Wilcoxon matched-pairssigned rank test for control and gingivitis group separatelyData are presented as mean + SD Level 120572 = 005 was chosenas a limit level of significance
3 Results
31 Study I Saliva samples contaminated by whole bloodwith a final concentration of 01 blood and higher are visiblycolored (Figure 1) The effect of two independent variablesgender and blood contamination on dependent variableoxidative stress markers was analyzed using two-way RMANOVA Separate ANOVAs were used for whole bloodplasma red blood cells and hemoglobin contaminationBecause no major effect of gender was observed data fromboth genders were combined for further analyses (Table 1)
Disease Markers 3
Table 1 The effect of gender and blood contamination on oxidativestress markers was analyzed using two-way RM ANOVA SeparateANOVAs were computed for whole blood plasma red blood cells(RBC) and hemoglobin contamination
Two-way RMANOVA119865
119901 value 119901 valuesummary
Salivary AOPPBlood contamination
Effect of blood 4986 lt00001 lowastlowastlowastlowast
Effect of gender 051 049 ns
Plasma contaminationEffect of plasma 3359 lt00001 lowastlowastlowastlowast
Effect of gender 368 009 ns
RBC contaminationEffect of RBC 2963 lt00001 lowastlowastlowastlowast
Effect of gender 020 066 ns
Hemoglobin contaminationEffect of hemoglobin 2831 lt00001 lowastlowastlowastlowast
Effect of gender 136 028 nsSalivary AGEsBlood contamination
Effect of blood 2139 lt00001 lowastlowastlowastlowast
Effect of gender 542 00483 lowast
Plasma contaminationEffect of plasma 2075 lt00001 lowastlowastlowastlowast
Effect of gender 448 007 ns
RBC contaminationEffect of RBC 6802 lt00001 lowastlowastlowastlowast
Effect of gender 525 005 ns
Hemoglobin contaminationEffect of hemoglobin 1917 lt00001 lowastlowastlowastlowast
Effect of gender 652 00340 lowast
Salivary FRASBlood contamination
Effect of blood 1200 lt00001 lowastlowastlowastlowast
Effect of gender 3878119890minus008 099 ns
Plasma contaminationEffect of plasma 2359 lt00001 lowastlowastlowastlowast
Effect of gender 027 062 ns
RBC contaminationEffect of RBC 9772 lt00001 lowastlowastlowastlowast
Effect of gender 018 068 ns
Hemoglobin contaminationEffect of hemoglobin 1832 lt00001 lowastlowastlowastlowast
Effect of gender 08376 039 ns
Table 1 Continued
Two-way RMANOVA119865
119901 value 119901 valuesummary
Salivary TACBlood contaminationEffect of blood 2431 lt00001 lowastlowastlowastlowast
Effect of gender 020 067 nsPlasma contaminationEffect of plasma 3302 lt00001 lowastlowastlowastlowast
Effect of gender 051 049 nsRBC contaminationEffect of RBC 2437 lt00001 lowastlowastlowastlowast
Effect of gender 025 063 nsHemoglobin contaminationEffect of hemoglobin 4380 lt00001 lowastlowastlowastlowast
Effect of gender 015 071 nsAOPP advanced oxidation protein products AGEs advanced glycation endproducts FRAP ferric reducing antioxidant power TAC total antioxidantcapacity ns nonsignificantlowast
Figure 1 Saliva samples of two probands (columns P1 and P2) con-taminated by venous blood with the following final concentrationsof blood 10 5 25 1 01 001 0001 and 0 Note thevisible discoloration of saliva with blood contamination from 01
One-way RM ANOVA and Tukeyrsquos multiple comparison testwere used for further analyses A significant bias in themeasured concentrations of salivary oxidative stress markerswas caused by 1 blood contamination in saliva (Figure 2)AOPP as a marker of protein oxidation was significantlyhigher in saliva contaminatedwith 1 and 25 venous bloodby 1187 and 1685 respectively (119902 = 1313 and 119902 = 1863119901 lt 00001 Tukeyrsquos test Figure 2(a)) In saliva samplescontaminated with 5 and 10 blood AOPP concentrationswere lower in comparison to samples contaminatedwith 25blood (Figure 2(a)) Salivary carbonyl stress measured asAGEs concentrations decreased proportionally when bloodwas added to saliva at concentrations 1ndash10 (Figure 2(b))
Figure 2 Oxidative stress and antioxidant status markers in saliva contaminated with blood (a) Salivary AOPP concentrations biomarker ofoxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrations biomarkerof antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SD lowastlowastlowastlowast119901 lt 00001lowastlowast119901 lt 001 and lowast119901 lt 005
Concentrations of antioxidant statusmarkers FRAP and TACalso decreased in blood-contaminated saliva (Figures 2(c)and 2(d))
To determine which blood component is responsible forchanges in measured salivary markers the impact of plasmared blood cells and hemoglobin was studied Salivary AOPPconcentrations were decreased proportionally in the pres-ence of 01ndash10 plasma contamination in saliva by 273ndash850 (Figure 3(a)) Salivary AOPP concentrations wereincreased in the presence of 01ndash10 red blood cells by 804ndash4933 (Figure 4(a)) A similar trend was observed afteraddition of 25ndash10 hemoglobin to saliva with increasedAOPP concentrations by 721ndash1094 (Figure 5(a)) SalivaryAGEs were increased in the presence of 25ndash10 plasma insaliva by 506ndash699 (Figure 3(b)) Addition of 1ndash10 redblood cells or hemoglobin resulted in comparable changes(Figures 4(b) and 5(b)) Concentrations of antioxidant statusmarkers FRAP and TAC were decreased in saliva whenplasma (Figures 3(c) and 3(d)) red blood cells (Figures 4(c)and 4(d)) or hemoglobin (Figures 5(c) and 5(d)) were added
32 Study II To study the effect of blood contamination in areal clinical situation the impact of microinjury in gingivitisand age-matched healthy control patients was modeledClinical parameters of both study groups are summarized inTable 2 Clinical parameters were significantly worse in thegingivitis group compared with the control group (Table 2)Dental hygiene asmodel ofmicroinjury caused blood leakagein both patients groups The effect of microinjury on salivarymarkers of oxidative stress before and after dental hygienewas determined using Wilcoxon matched-pairs signed ranktest for control and gingivitis group separately SalivaryAOPPconcentrations were increased in 9 out of 10 control probandsand in 10 out of 12 gingivitis patients after microinjury(Figures 6(a) and 6(e)) Salivary AGEs concentrations weredecreased in 9 out of 10 control probands and in 12 out of12 gingivitis patients (Figures 6(b) and 6(f)) Salivary FRAPconcentrationswere decreased in 7 out of 10 control probandsand in 11 out of 12 gingivitis patients after treatment (Figures6(c) and 6(g)) Salivary TAC concentrations were decreasedin 9 out of 10 in control probands and in 11 out of 12 gingivitispatients after microinjury (Figures 6(d) and 6(h))
Figure 3 Oxidative stress and antioxidant status markers in saliva contaminated with plasma (a) Salivary AOPP concentrations biomarkerof oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrations biomarkerof antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SD lowastlowastlowastlowast119901 lt 00001lowastlowastlowast119901 lt 0001 lowastlowast119901 lt 001 and lowast119901 lt 005
Table 2 Comparison of clinical parameters between control and gingivitis patients
Clinical parameterGroup
Unpaired 119905-test119905
119901 value 119901 value summaryControl Gingivitis(119899 = 10) (119899 = 12)
Schwartz and Granger reported that blood components insaliva invisible to the eye have the potential to bias salivaryanalytes and the control of blood contamination in saliva wassuggested Transferrin enzymatic immunoassaywas designedfor quantitative monitoring of blood contamination [14] Itwas shown that concentrations of testosterone dehydroepi-androsterone (DHEA) and cortisol are increased in salivasamples artificially contaminated with blood [14] On the
other hand microinjury of the oral cavity did not changeDHEA or cortisol and increased salivary testosterone [6]Despite the rising number of studies analyzing oxidativestress in saliva no reports have been published regarding theeffect of occult blood contamination on concentrations of sal-ivary markers of oxidative stress
Four markers of oxidative stress and antioxidant statuswere analyzed in our study Advanced oxidation protein pro-ducts (AOPP) a novel oxidative stress biomarker was discov-ered in the plasma of uremic patients in 1996 [10] Recently
Figure 4 Oxidative stress and antioxidant status markers in saliva contaminated with red blood cells (RBC) (a) Salivary AOPPconcentrations biomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) SalivaryFRAP concentrations biomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presentedas mean + SD lowastlowastlowastlowast119901 lt 00001 and lowast119901 lt 005
AOPPwas suggested as part of the nonenzymatic antioxidantsystem of plasma proteome and oxidized fibrinogenwas indi-cated as key molecule responsible for human plasma AOPPreactivity [15] Advanced glycation end products (AGEs)marker of carbonyl stress are developed during the reactionof protein amino groups with reactive carbonyl compounds[16] Ferric reducing antioxidant power (FRAP) assay indi-rectly reflects the total antioxidant capacity of the sample[12 17] Total antioxidant capacity (TAC) assay developed byErel is directmeasurementmethod for total antioxidants [13]Our results have shown that most of the measured salivarymarkers of oxidative stress and antioxidant status are biasedin the presence of 1blood contamination in salivaHoweveras shown in results section saliva contaminatedwith 1 bloodis visually discolored Blood-contaminated saliva samples canbe easily excluded from the analyses Thus at least for themarkers analyzed in this study there is no need to use salivarytransferrin assay for the monitoring of blood contamination
To determine which blood component is responsiblefor changes in measured salivary markers the impact ofplasma red blood cells and hemoglobin was studied Based
on our results increased AOPP concentrations in blood-contaminated saliva can be explained by the presence ofred blood cells and hemoglobin in saliva Hemoglobin insaliva probably interferes with the colorimetric AOPP assayand artificially increases the AOPP concentrations AOPPconcentrations in plasma of healthy probands were reportedas 3 times as high as AOPP concentrations in saliva [15]we have therefore expected increased AOPP concentrationsin saliva contaminated with plasma But an opposite trendwas observed when plasma was spiked into saliva anddecreased salivary AOPP was detected in the presence of01ndash10 plasma When plasma is spiked into saliva theconcentration of total proteins is increased In our studydecreased concentrations of measured salivary markers afterplasma addition into saliva could be caused by normaliza-tion to the increased total proteins Based on our resultshemoglobin in saliva can mask the detection of AGEs andlead to underestimation of AGEs in saliva Plasma additioninto saliva led to decreased salivary AGEs concentrationsSalivary FRAP and TAC concentrations were decreased inthe presence of RBC hemoglobin and plasma contamination
Disease Markers 7
0
50
100
150
200
250
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
AOPP
(120583m
olg
)
(a)
0
500
1000
1500
2000
2500
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
AGEs
(au
g)
(b)
0
200
400
600
800
lowastlowastlowast
lowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
FRA
P (120583
mol
g)
(c)
0
500
1000
1500
lowastlowastlowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
TAC
(120583m
olg
)
(d)
Figure 5 Oxidative stress and antioxidant status markers in saliva contaminated with hemoglobin (a) Salivary AOPP concentrationsbiomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrationsbiomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SDlowastlowastlowastlowast119901 lt 00001 lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001
in saliva Decreased concentrations of measured markers insaliva contaminated with plasma could be as in the case ofAOPP assay caused by normalization to total proteins
The effect of blood contamination in saliva on concentra-tions of salivary markers of oxidative stress was not studiedin real clinical situation in the past Concerning that bloodcontamination in saliva is common in patients with gingivitisthe impact of microinjury was modeled in this study groupand age-matched healthy controls The results were similar tosaliva artificially contaminated with blood AOPP concentra-tions were increased after microinjury in the visual presenceof blood in saliva AOPP concentrations were increased inboth control and gingivitis group after dental hygiene treat-ment AOPP concentrations were not different between con-trol and gingivitis patients before dental hygiene treatmentAlso other measured salivary markers followed the trendobserved in saliva artificially contaminated with blood Sali-vary AGEs concentrations and also antioxidant markers TACand FRAP were decreased after dental hygiene treatment dueto presence of blood in saliva Our results have confirmedthe concern that dental hygiene treatment could bias the
concentrations of oxidative stress markers in saliva Based onour results we recommend the saliva collection before dentalhygiene treatment or clinical examination of oral cavity
5 Conclusions
Salivary oxidative stress concentrations are significantlyinfluenced by 1 blood contamination in saliva Salivasamples with 1 blood contamination are visibly colored andit is possible to easily exclude such contaminated samplesfrom further salivary oxidative stress analyses Microinjuryto the periodontium caused blood leakage into saliva in bothgingivitis and control group and biased concentrations ofoxidative stress markers in saliva For the purpose of salivaryoxidative stress analyses saliva samples should be collectedbefore dental hygiene treatment or clinical examination of theoral cavity
Conflict of Interests
The paper is original work and is not under consideration byanother journal There is no conflict of interests to declare
8 Disease Markers
Control patients
Control patients
Control patients
(a)
(b)
(c)
Before AfterDental hygiene treatment
0
500
1000
1500
2000
AGEs
(au
g)
Gingivitis patients
Before AfterDental hygiene treatment
0
500
1000
1500
2000
lowastlowastlowast
AGEs
(au
g)
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Before AfterDental hygiene treatment
0
200
400
600
800
lowast
FRA
P (120583
mol
g)
(f)
(g)
Gingivitis patients
Before AfterDental hygiene treatment
0
200
400
600
800
lowastlowast
FRA
P (120583
mol
g)
(e)
Gingivitis patients
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Figure 6 Continued
Disease Markers 9
(d) (h)
Control patients Gingivitis patients
Before Afterminus500
0
500
1000
1500
Dental hygiene treatment
lowast
TAC
(120583m
olg
)
Before AfterDental hygiene treatment
lowastlowastlowast
minus500
0
500
1000
1500
TAC
(120583m
olg
)
Figure 6 The effect of blood leakage after dental hygiene treatment on markers of oxidative stress and antioxidant status in saliva of control(andashd) and gingivitis (endashh) patients (a e) Salivary AOPP concentrations biomarker of oxidative damage to proteins (b f) Salivary AGEsconcentrations biomarker of carbonyl stress (c g) Salivary FRAP concentrations biomarker of antioxidant status (d h) Salivary TACconcentrations biomarker of antioxidant status lowastlowastlowast119901 lt 00001 lowastlowast119901 lt 001 and lowast119901 lt 005
This publication is the result of the implementation of theproject of University Science Park of Comenius University inBratislava (ITMS 26240220086) supported by the Researchand Development Operational Programme funded by theEuropean Regional Development Fund
References
[1] J Hodosy and P Celec ldquoDaytime of sampling tooth-brushingand ascorbic acid influence salivary thiobarbituric acid reactingsubstancesmdasha potential clinical marker of gingival statusrdquoDisease Markers vol 21 no 4 pp 203ndash207 2005
[2] N Kamodyova and P Celec ldquoSalivary markers of oxidativestress and Salivette collection systemsrdquo Clinical Chemistry andLaboratory Medicine vol 49 no 11 pp 1887ndash1890 2011
[3] N Kamodyova L Tothova and P Celec ldquoSalivary markersof oxidative stress and antioxidant status influence of externalfactorsrdquo Disease Markers vol 34 no 5 pp 313ndash321 2013
[4] F A Akalin E Baltacioglu A Alver and E Karabulut ldquoLipidperoxidation levels and total oxidant status in serum saliva andgingival crevicular fluid in patients with chronic periodontitisrdquoJournal of Clinical Periodontology vol 34 no 7 pp 558ndash5652007
[5] D A Granger E A Shirtcliff A Booth K T Kivlighanand E B Schwartz ldquoThe lsquotroublersquo with salivary testosteronerdquoPsychoneuroendocrinology vol 29 no 10 pp 1229ndash1240 2004
[6] K T Kivlighan D A Granger E B Schwartz V Nelson MCurran and E A Shirtcliff ldquoQuantifying blood leakage intothe oral mucosa and its effects on the measurement of cortisoldehydroepiandrosterone and testosterone in salivardquoHormonesand Behavior vol 46 no 1 pp 39ndash46 2004
[7] D A Granger D Cicchetti F A Rogosch L C HibelM Teisl and E Flores ldquoBlood contamination in childrenrsquossaliva prevalence stability and impact on the measurement
of salivary cortisol testosterone and dehydroepiandrosteronerdquoPsychoneuroendocrinology vol 32 no 6 pp 724ndash733 2007
[8] H Loe and J Silness ldquoPeriodontal disease in pregnancy IPrevalence and severityrdquo Acta Odontologica Scandinavica vol21 no 6 pp 533ndash551 1963
[9] H R Muhlemann and S Son ldquoGingival sulcus bleedingmdashaleading symptom in initial gingivitisrdquo Helvetica OdontologicaActa vol 15 no 2 pp 107ndash113 1971
[10] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996
[11] G Munch R Keis A Wessels et al ldquoDetermination ofadvanced glycation end products in serum by fluorescencespectroscopy and competitive ELISArdquo European Journal ofClinical Chemistry and Clinical Biochemistry vol 35 no 9 pp669ndash677 1997
[12] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[13] O Erel ldquoA novel automated direct measurement method fortotal antioxidant capacity using a new generation more stableABTS radical cationrdquo Clinical Biochemistry vol 37 no 4 pp277ndash285 2004
[14] E B Schwartz and D A Granger ldquoTransferrin enzyme immu-noassay for quantitative monitoring of blood contamination insalivardquo Clinical Chemistry vol 50 no 3 pp 654ndash656 2004
[15] Z Qing E Ling-Ling W Dong-Sheng and L Hong-ChenldquoRelationship of advanced oxidative protein products in humansaliva and plasma age-and gender-related changes and stabilityduring storagerdquo Free Radical Research vol 46 no 10 pp 1201ndash1206 2012
[16] T Miyata K Kurokawa and C Van Ypersele De StrihouldquoAdvanced glycation and lipoxidation end products role ofreactive carbonyl compounds generated during carbohydrate
10 Disease Markers
and lipid metabolismrdquo Journal of the American Society of Neph-rology vol 11 no 9 pp 1744ndash1752 2000
[17] R L Prior and G Cao ldquoIn vivo total antioxidant capacity com-parison of different analytical methodsrdquo Free Radical Biologyand Medicine vol 27 no 11-12 pp 1173ndash1181 1999
Table 1 The effect of gender and blood contamination on oxidativestress markers was analyzed using two-way RM ANOVA SeparateANOVAs were computed for whole blood plasma red blood cells(RBC) and hemoglobin contamination
Two-way RMANOVA119865
119901 value 119901 valuesummary
Salivary AOPPBlood contamination
Effect of blood 4986 lt00001 lowastlowastlowastlowast
Effect of gender 051 049 ns
Plasma contaminationEffect of plasma 3359 lt00001 lowastlowastlowastlowast
Effect of gender 368 009 ns
RBC contaminationEffect of RBC 2963 lt00001 lowastlowastlowastlowast
Effect of gender 020 066 ns
Hemoglobin contaminationEffect of hemoglobin 2831 lt00001 lowastlowastlowastlowast
Effect of gender 136 028 nsSalivary AGEsBlood contamination
Effect of blood 2139 lt00001 lowastlowastlowastlowast
Effect of gender 542 00483 lowast
Plasma contaminationEffect of plasma 2075 lt00001 lowastlowastlowastlowast
Effect of gender 448 007 ns
RBC contaminationEffect of RBC 6802 lt00001 lowastlowastlowastlowast
Effect of gender 525 005 ns
Hemoglobin contaminationEffect of hemoglobin 1917 lt00001 lowastlowastlowastlowast
Effect of gender 652 00340 lowast
Salivary FRASBlood contamination
Effect of blood 1200 lt00001 lowastlowastlowastlowast
Effect of gender 3878119890minus008 099 ns
Plasma contaminationEffect of plasma 2359 lt00001 lowastlowastlowastlowast
Effect of gender 027 062 ns
RBC contaminationEffect of RBC 9772 lt00001 lowastlowastlowastlowast
Effect of gender 018 068 ns
Hemoglobin contaminationEffect of hemoglobin 1832 lt00001 lowastlowastlowastlowast
Effect of gender 08376 039 ns
Table 1 Continued
Two-way RMANOVA119865
119901 value 119901 valuesummary
Salivary TACBlood contaminationEffect of blood 2431 lt00001 lowastlowastlowastlowast
Effect of gender 020 067 nsPlasma contaminationEffect of plasma 3302 lt00001 lowastlowastlowastlowast
Effect of gender 051 049 nsRBC contaminationEffect of RBC 2437 lt00001 lowastlowastlowastlowast
Effect of gender 025 063 nsHemoglobin contaminationEffect of hemoglobin 4380 lt00001 lowastlowastlowastlowast
Effect of gender 015 071 nsAOPP advanced oxidation protein products AGEs advanced glycation endproducts FRAP ferric reducing antioxidant power TAC total antioxidantcapacity ns nonsignificantlowast
Figure 1 Saliva samples of two probands (columns P1 and P2) con-taminated by venous blood with the following final concentrationsof blood 10 5 25 1 01 001 0001 and 0 Note thevisible discoloration of saliva with blood contamination from 01
One-way RM ANOVA and Tukeyrsquos multiple comparison testwere used for further analyses A significant bias in themeasured concentrations of salivary oxidative stress markerswas caused by 1 blood contamination in saliva (Figure 2)AOPP as a marker of protein oxidation was significantlyhigher in saliva contaminatedwith 1 and 25 venous bloodby 1187 and 1685 respectively (119902 = 1313 and 119902 = 1863119901 lt 00001 Tukeyrsquos test Figure 2(a)) In saliva samplescontaminated with 5 and 10 blood AOPP concentrationswere lower in comparison to samples contaminatedwith 25blood (Figure 2(a)) Salivary carbonyl stress measured asAGEs concentrations decreased proportionally when bloodwas added to saliva at concentrations 1ndash10 (Figure 2(b))
Figure 2 Oxidative stress and antioxidant status markers in saliva contaminated with blood (a) Salivary AOPP concentrations biomarker ofoxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrations biomarkerof antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SD lowastlowastlowastlowast119901 lt 00001lowastlowast119901 lt 001 and lowast119901 lt 005
Concentrations of antioxidant statusmarkers FRAP and TACalso decreased in blood-contaminated saliva (Figures 2(c)and 2(d))
To determine which blood component is responsible forchanges in measured salivary markers the impact of plasmared blood cells and hemoglobin was studied Salivary AOPPconcentrations were decreased proportionally in the pres-ence of 01ndash10 plasma contamination in saliva by 273ndash850 (Figure 3(a)) Salivary AOPP concentrations wereincreased in the presence of 01ndash10 red blood cells by 804ndash4933 (Figure 4(a)) A similar trend was observed afteraddition of 25ndash10 hemoglobin to saliva with increasedAOPP concentrations by 721ndash1094 (Figure 5(a)) SalivaryAGEs were increased in the presence of 25ndash10 plasma insaliva by 506ndash699 (Figure 3(b)) Addition of 1ndash10 redblood cells or hemoglobin resulted in comparable changes(Figures 4(b) and 5(b)) Concentrations of antioxidant statusmarkers FRAP and TAC were decreased in saliva whenplasma (Figures 3(c) and 3(d)) red blood cells (Figures 4(c)and 4(d)) or hemoglobin (Figures 5(c) and 5(d)) were added
32 Study II To study the effect of blood contamination in areal clinical situation the impact of microinjury in gingivitisand age-matched healthy control patients was modeledClinical parameters of both study groups are summarized inTable 2 Clinical parameters were significantly worse in thegingivitis group compared with the control group (Table 2)Dental hygiene asmodel ofmicroinjury caused blood leakagein both patients groups The effect of microinjury on salivarymarkers of oxidative stress before and after dental hygienewas determined using Wilcoxon matched-pairs signed ranktest for control and gingivitis group separately SalivaryAOPPconcentrations were increased in 9 out of 10 control probandsand in 10 out of 12 gingivitis patients after microinjury(Figures 6(a) and 6(e)) Salivary AGEs concentrations weredecreased in 9 out of 10 control probands and in 12 out of12 gingivitis patients (Figures 6(b) and 6(f)) Salivary FRAPconcentrationswere decreased in 7 out of 10 control probandsand in 11 out of 12 gingivitis patients after treatment (Figures6(c) and 6(g)) Salivary TAC concentrations were decreasedin 9 out of 10 in control probands and in 11 out of 12 gingivitispatients after microinjury (Figures 6(d) and 6(h))
Figure 3 Oxidative stress and antioxidant status markers in saliva contaminated with plasma (a) Salivary AOPP concentrations biomarkerof oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrations biomarkerof antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SD lowastlowastlowastlowast119901 lt 00001lowastlowastlowast119901 lt 0001 lowastlowast119901 lt 001 and lowast119901 lt 005
Table 2 Comparison of clinical parameters between control and gingivitis patients
Clinical parameterGroup
Unpaired 119905-test119905
119901 value 119901 value summaryControl Gingivitis(119899 = 10) (119899 = 12)
Schwartz and Granger reported that blood components insaliva invisible to the eye have the potential to bias salivaryanalytes and the control of blood contamination in saliva wassuggested Transferrin enzymatic immunoassaywas designedfor quantitative monitoring of blood contamination [14] Itwas shown that concentrations of testosterone dehydroepi-androsterone (DHEA) and cortisol are increased in salivasamples artificially contaminated with blood [14] On the
other hand microinjury of the oral cavity did not changeDHEA or cortisol and increased salivary testosterone [6]Despite the rising number of studies analyzing oxidativestress in saliva no reports have been published regarding theeffect of occult blood contamination on concentrations of sal-ivary markers of oxidative stress
Four markers of oxidative stress and antioxidant statuswere analyzed in our study Advanced oxidation protein pro-ducts (AOPP) a novel oxidative stress biomarker was discov-ered in the plasma of uremic patients in 1996 [10] Recently
Figure 4 Oxidative stress and antioxidant status markers in saliva contaminated with red blood cells (RBC) (a) Salivary AOPPconcentrations biomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) SalivaryFRAP concentrations biomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presentedas mean + SD lowastlowastlowastlowast119901 lt 00001 and lowast119901 lt 005
AOPPwas suggested as part of the nonenzymatic antioxidantsystem of plasma proteome and oxidized fibrinogenwas indi-cated as key molecule responsible for human plasma AOPPreactivity [15] Advanced glycation end products (AGEs)marker of carbonyl stress are developed during the reactionof protein amino groups with reactive carbonyl compounds[16] Ferric reducing antioxidant power (FRAP) assay indi-rectly reflects the total antioxidant capacity of the sample[12 17] Total antioxidant capacity (TAC) assay developed byErel is directmeasurementmethod for total antioxidants [13]Our results have shown that most of the measured salivarymarkers of oxidative stress and antioxidant status are biasedin the presence of 1blood contamination in salivaHoweveras shown in results section saliva contaminatedwith 1 bloodis visually discolored Blood-contaminated saliva samples canbe easily excluded from the analyses Thus at least for themarkers analyzed in this study there is no need to use salivarytransferrin assay for the monitoring of blood contamination
To determine which blood component is responsiblefor changes in measured salivary markers the impact ofplasma red blood cells and hemoglobin was studied Based
on our results increased AOPP concentrations in blood-contaminated saliva can be explained by the presence ofred blood cells and hemoglobin in saliva Hemoglobin insaliva probably interferes with the colorimetric AOPP assayand artificially increases the AOPP concentrations AOPPconcentrations in plasma of healthy probands were reportedas 3 times as high as AOPP concentrations in saliva [15]we have therefore expected increased AOPP concentrationsin saliva contaminated with plasma But an opposite trendwas observed when plasma was spiked into saliva anddecreased salivary AOPP was detected in the presence of01ndash10 plasma When plasma is spiked into saliva theconcentration of total proteins is increased In our studydecreased concentrations of measured salivary markers afterplasma addition into saliva could be caused by normaliza-tion to the increased total proteins Based on our resultshemoglobin in saliva can mask the detection of AGEs andlead to underestimation of AGEs in saliva Plasma additioninto saliva led to decreased salivary AGEs concentrationsSalivary FRAP and TAC concentrations were decreased inthe presence of RBC hemoglobin and plasma contamination
Disease Markers 7
0
50
100
150
200
250
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
AOPP
(120583m
olg
)
(a)
0
500
1000
1500
2000
2500
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
AGEs
(au
g)
(b)
0
200
400
600
800
lowastlowastlowast
lowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
FRA
P (120583
mol
g)
(c)
0
500
1000
1500
lowastlowastlowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
TAC
(120583m
olg
)
(d)
Figure 5 Oxidative stress and antioxidant status markers in saliva contaminated with hemoglobin (a) Salivary AOPP concentrationsbiomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrationsbiomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SDlowastlowastlowastlowast119901 lt 00001 lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001
in saliva Decreased concentrations of measured markers insaliva contaminated with plasma could be as in the case ofAOPP assay caused by normalization to total proteins
The effect of blood contamination in saliva on concentra-tions of salivary markers of oxidative stress was not studiedin real clinical situation in the past Concerning that bloodcontamination in saliva is common in patients with gingivitisthe impact of microinjury was modeled in this study groupand age-matched healthy controls The results were similar tosaliva artificially contaminated with blood AOPP concentra-tions were increased after microinjury in the visual presenceof blood in saliva AOPP concentrations were increased inboth control and gingivitis group after dental hygiene treat-ment AOPP concentrations were not different between con-trol and gingivitis patients before dental hygiene treatmentAlso other measured salivary markers followed the trendobserved in saliva artificially contaminated with blood Sali-vary AGEs concentrations and also antioxidant markers TACand FRAP were decreased after dental hygiene treatment dueto presence of blood in saliva Our results have confirmedthe concern that dental hygiene treatment could bias the
concentrations of oxidative stress markers in saliva Based onour results we recommend the saliva collection before dentalhygiene treatment or clinical examination of oral cavity
5 Conclusions
Salivary oxidative stress concentrations are significantlyinfluenced by 1 blood contamination in saliva Salivasamples with 1 blood contamination are visibly colored andit is possible to easily exclude such contaminated samplesfrom further salivary oxidative stress analyses Microinjuryto the periodontium caused blood leakage into saliva in bothgingivitis and control group and biased concentrations ofoxidative stress markers in saliva For the purpose of salivaryoxidative stress analyses saliva samples should be collectedbefore dental hygiene treatment or clinical examination of theoral cavity
Conflict of Interests
The paper is original work and is not under consideration byanother journal There is no conflict of interests to declare
8 Disease Markers
Control patients
Control patients
Control patients
(a)
(b)
(c)
Before AfterDental hygiene treatment
0
500
1000
1500
2000
AGEs
(au
g)
Gingivitis patients
Before AfterDental hygiene treatment
0
500
1000
1500
2000
lowastlowastlowast
AGEs
(au
g)
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Before AfterDental hygiene treatment
0
200
400
600
800
lowast
FRA
P (120583
mol
g)
(f)
(g)
Gingivitis patients
Before AfterDental hygiene treatment
0
200
400
600
800
lowastlowast
FRA
P (120583
mol
g)
(e)
Gingivitis patients
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Figure 6 Continued
Disease Markers 9
(d) (h)
Control patients Gingivitis patients
Before Afterminus500
0
500
1000
1500
Dental hygiene treatment
lowast
TAC
(120583m
olg
)
Before AfterDental hygiene treatment
lowastlowastlowast
minus500
0
500
1000
1500
TAC
(120583m
olg
)
Figure 6 The effect of blood leakage after dental hygiene treatment on markers of oxidative stress and antioxidant status in saliva of control(andashd) and gingivitis (endashh) patients (a e) Salivary AOPP concentrations biomarker of oxidative damage to proteins (b f) Salivary AGEsconcentrations biomarker of carbonyl stress (c g) Salivary FRAP concentrations biomarker of antioxidant status (d h) Salivary TACconcentrations biomarker of antioxidant status lowastlowastlowast119901 lt 00001 lowastlowast119901 lt 001 and lowast119901 lt 005
This publication is the result of the implementation of theproject of University Science Park of Comenius University inBratislava (ITMS 26240220086) supported by the Researchand Development Operational Programme funded by theEuropean Regional Development Fund
References
[1] J Hodosy and P Celec ldquoDaytime of sampling tooth-brushingand ascorbic acid influence salivary thiobarbituric acid reactingsubstancesmdasha potential clinical marker of gingival statusrdquoDisease Markers vol 21 no 4 pp 203ndash207 2005
[2] N Kamodyova and P Celec ldquoSalivary markers of oxidativestress and Salivette collection systemsrdquo Clinical Chemistry andLaboratory Medicine vol 49 no 11 pp 1887ndash1890 2011
[3] N Kamodyova L Tothova and P Celec ldquoSalivary markersof oxidative stress and antioxidant status influence of externalfactorsrdquo Disease Markers vol 34 no 5 pp 313ndash321 2013
[4] F A Akalin E Baltacioglu A Alver and E Karabulut ldquoLipidperoxidation levels and total oxidant status in serum saliva andgingival crevicular fluid in patients with chronic periodontitisrdquoJournal of Clinical Periodontology vol 34 no 7 pp 558ndash5652007
[5] D A Granger E A Shirtcliff A Booth K T Kivlighanand E B Schwartz ldquoThe lsquotroublersquo with salivary testosteronerdquoPsychoneuroendocrinology vol 29 no 10 pp 1229ndash1240 2004
[6] K T Kivlighan D A Granger E B Schwartz V Nelson MCurran and E A Shirtcliff ldquoQuantifying blood leakage intothe oral mucosa and its effects on the measurement of cortisoldehydroepiandrosterone and testosterone in salivardquoHormonesand Behavior vol 46 no 1 pp 39ndash46 2004
[7] D A Granger D Cicchetti F A Rogosch L C HibelM Teisl and E Flores ldquoBlood contamination in childrenrsquossaliva prevalence stability and impact on the measurement
of salivary cortisol testosterone and dehydroepiandrosteronerdquoPsychoneuroendocrinology vol 32 no 6 pp 724ndash733 2007
[8] H Loe and J Silness ldquoPeriodontal disease in pregnancy IPrevalence and severityrdquo Acta Odontologica Scandinavica vol21 no 6 pp 533ndash551 1963
[9] H R Muhlemann and S Son ldquoGingival sulcus bleedingmdashaleading symptom in initial gingivitisrdquo Helvetica OdontologicaActa vol 15 no 2 pp 107ndash113 1971
[10] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996
[11] G Munch R Keis A Wessels et al ldquoDetermination ofadvanced glycation end products in serum by fluorescencespectroscopy and competitive ELISArdquo European Journal ofClinical Chemistry and Clinical Biochemistry vol 35 no 9 pp669ndash677 1997
[12] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[13] O Erel ldquoA novel automated direct measurement method fortotal antioxidant capacity using a new generation more stableABTS radical cationrdquo Clinical Biochemistry vol 37 no 4 pp277ndash285 2004
[14] E B Schwartz and D A Granger ldquoTransferrin enzyme immu-noassay for quantitative monitoring of blood contamination insalivardquo Clinical Chemistry vol 50 no 3 pp 654ndash656 2004
[15] Z Qing E Ling-Ling W Dong-Sheng and L Hong-ChenldquoRelationship of advanced oxidative protein products in humansaliva and plasma age-and gender-related changes and stabilityduring storagerdquo Free Radical Research vol 46 no 10 pp 1201ndash1206 2012
[16] T Miyata K Kurokawa and C Van Ypersele De StrihouldquoAdvanced glycation and lipoxidation end products role ofreactive carbonyl compounds generated during carbohydrate
10 Disease Markers
and lipid metabolismrdquo Journal of the American Society of Neph-rology vol 11 no 9 pp 1744ndash1752 2000
[17] R L Prior and G Cao ldquoIn vivo total antioxidant capacity com-parison of different analytical methodsrdquo Free Radical Biologyand Medicine vol 27 no 11-12 pp 1173ndash1181 1999
Figure 2 Oxidative stress and antioxidant status markers in saliva contaminated with blood (a) Salivary AOPP concentrations biomarker ofoxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrations biomarkerof antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SD lowastlowastlowastlowast119901 lt 00001lowastlowast119901 lt 001 and lowast119901 lt 005
Concentrations of antioxidant statusmarkers FRAP and TACalso decreased in blood-contaminated saliva (Figures 2(c)and 2(d))
To determine which blood component is responsible forchanges in measured salivary markers the impact of plasmared blood cells and hemoglobin was studied Salivary AOPPconcentrations were decreased proportionally in the pres-ence of 01ndash10 plasma contamination in saliva by 273ndash850 (Figure 3(a)) Salivary AOPP concentrations wereincreased in the presence of 01ndash10 red blood cells by 804ndash4933 (Figure 4(a)) A similar trend was observed afteraddition of 25ndash10 hemoglobin to saliva with increasedAOPP concentrations by 721ndash1094 (Figure 5(a)) SalivaryAGEs were increased in the presence of 25ndash10 plasma insaliva by 506ndash699 (Figure 3(b)) Addition of 1ndash10 redblood cells or hemoglobin resulted in comparable changes(Figures 4(b) and 5(b)) Concentrations of antioxidant statusmarkers FRAP and TAC were decreased in saliva whenplasma (Figures 3(c) and 3(d)) red blood cells (Figures 4(c)and 4(d)) or hemoglobin (Figures 5(c) and 5(d)) were added
32 Study II To study the effect of blood contamination in areal clinical situation the impact of microinjury in gingivitisand age-matched healthy control patients was modeledClinical parameters of both study groups are summarized inTable 2 Clinical parameters were significantly worse in thegingivitis group compared with the control group (Table 2)Dental hygiene asmodel ofmicroinjury caused blood leakagein both patients groups The effect of microinjury on salivarymarkers of oxidative stress before and after dental hygienewas determined using Wilcoxon matched-pairs signed ranktest for control and gingivitis group separately SalivaryAOPPconcentrations were increased in 9 out of 10 control probandsand in 10 out of 12 gingivitis patients after microinjury(Figures 6(a) and 6(e)) Salivary AGEs concentrations weredecreased in 9 out of 10 control probands and in 12 out of12 gingivitis patients (Figures 6(b) and 6(f)) Salivary FRAPconcentrationswere decreased in 7 out of 10 control probandsand in 11 out of 12 gingivitis patients after treatment (Figures6(c) and 6(g)) Salivary TAC concentrations were decreasedin 9 out of 10 in control probands and in 11 out of 12 gingivitispatients after microinjury (Figures 6(d) and 6(h))
Figure 3 Oxidative stress and antioxidant status markers in saliva contaminated with plasma (a) Salivary AOPP concentrations biomarkerof oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrations biomarkerof antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SD lowastlowastlowastlowast119901 lt 00001lowastlowastlowast119901 lt 0001 lowastlowast119901 lt 001 and lowast119901 lt 005
Table 2 Comparison of clinical parameters between control and gingivitis patients
Clinical parameterGroup
Unpaired 119905-test119905
119901 value 119901 value summaryControl Gingivitis(119899 = 10) (119899 = 12)
Schwartz and Granger reported that blood components insaliva invisible to the eye have the potential to bias salivaryanalytes and the control of blood contamination in saliva wassuggested Transferrin enzymatic immunoassaywas designedfor quantitative monitoring of blood contamination [14] Itwas shown that concentrations of testosterone dehydroepi-androsterone (DHEA) and cortisol are increased in salivasamples artificially contaminated with blood [14] On the
other hand microinjury of the oral cavity did not changeDHEA or cortisol and increased salivary testosterone [6]Despite the rising number of studies analyzing oxidativestress in saliva no reports have been published regarding theeffect of occult blood contamination on concentrations of sal-ivary markers of oxidative stress
Four markers of oxidative stress and antioxidant statuswere analyzed in our study Advanced oxidation protein pro-ducts (AOPP) a novel oxidative stress biomarker was discov-ered in the plasma of uremic patients in 1996 [10] Recently
Figure 4 Oxidative stress and antioxidant status markers in saliva contaminated with red blood cells (RBC) (a) Salivary AOPPconcentrations biomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) SalivaryFRAP concentrations biomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presentedas mean + SD lowastlowastlowastlowast119901 lt 00001 and lowast119901 lt 005
AOPPwas suggested as part of the nonenzymatic antioxidantsystem of plasma proteome and oxidized fibrinogenwas indi-cated as key molecule responsible for human plasma AOPPreactivity [15] Advanced glycation end products (AGEs)marker of carbonyl stress are developed during the reactionof protein amino groups with reactive carbonyl compounds[16] Ferric reducing antioxidant power (FRAP) assay indi-rectly reflects the total antioxidant capacity of the sample[12 17] Total antioxidant capacity (TAC) assay developed byErel is directmeasurementmethod for total antioxidants [13]Our results have shown that most of the measured salivarymarkers of oxidative stress and antioxidant status are biasedin the presence of 1blood contamination in salivaHoweveras shown in results section saliva contaminatedwith 1 bloodis visually discolored Blood-contaminated saliva samples canbe easily excluded from the analyses Thus at least for themarkers analyzed in this study there is no need to use salivarytransferrin assay for the monitoring of blood contamination
To determine which blood component is responsiblefor changes in measured salivary markers the impact ofplasma red blood cells and hemoglobin was studied Based
on our results increased AOPP concentrations in blood-contaminated saliva can be explained by the presence ofred blood cells and hemoglobin in saliva Hemoglobin insaliva probably interferes with the colorimetric AOPP assayand artificially increases the AOPP concentrations AOPPconcentrations in plasma of healthy probands were reportedas 3 times as high as AOPP concentrations in saliva [15]we have therefore expected increased AOPP concentrationsin saliva contaminated with plasma But an opposite trendwas observed when plasma was spiked into saliva anddecreased salivary AOPP was detected in the presence of01ndash10 plasma When plasma is spiked into saliva theconcentration of total proteins is increased In our studydecreased concentrations of measured salivary markers afterplasma addition into saliva could be caused by normaliza-tion to the increased total proteins Based on our resultshemoglobin in saliva can mask the detection of AGEs andlead to underestimation of AGEs in saliva Plasma additioninto saliva led to decreased salivary AGEs concentrationsSalivary FRAP and TAC concentrations were decreased inthe presence of RBC hemoglobin and plasma contamination
Disease Markers 7
0
50
100
150
200
250
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
AOPP
(120583m
olg
)
(a)
0
500
1000
1500
2000
2500
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
AGEs
(au
g)
(b)
0
200
400
600
800
lowastlowastlowast
lowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
FRA
P (120583
mol
g)
(c)
0
500
1000
1500
lowastlowastlowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
TAC
(120583m
olg
)
(d)
Figure 5 Oxidative stress and antioxidant status markers in saliva contaminated with hemoglobin (a) Salivary AOPP concentrationsbiomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrationsbiomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SDlowastlowastlowastlowast119901 lt 00001 lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001
in saliva Decreased concentrations of measured markers insaliva contaminated with plasma could be as in the case ofAOPP assay caused by normalization to total proteins
The effect of blood contamination in saliva on concentra-tions of salivary markers of oxidative stress was not studiedin real clinical situation in the past Concerning that bloodcontamination in saliva is common in patients with gingivitisthe impact of microinjury was modeled in this study groupand age-matched healthy controls The results were similar tosaliva artificially contaminated with blood AOPP concentra-tions were increased after microinjury in the visual presenceof blood in saliva AOPP concentrations were increased inboth control and gingivitis group after dental hygiene treat-ment AOPP concentrations were not different between con-trol and gingivitis patients before dental hygiene treatmentAlso other measured salivary markers followed the trendobserved in saliva artificially contaminated with blood Sali-vary AGEs concentrations and also antioxidant markers TACand FRAP were decreased after dental hygiene treatment dueto presence of blood in saliva Our results have confirmedthe concern that dental hygiene treatment could bias the
concentrations of oxidative stress markers in saliva Based onour results we recommend the saliva collection before dentalhygiene treatment or clinical examination of oral cavity
5 Conclusions
Salivary oxidative stress concentrations are significantlyinfluenced by 1 blood contamination in saliva Salivasamples with 1 blood contamination are visibly colored andit is possible to easily exclude such contaminated samplesfrom further salivary oxidative stress analyses Microinjuryto the periodontium caused blood leakage into saliva in bothgingivitis and control group and biased concentrations ofoxidative stress markers in saliva For the purpose of salivaryoxidative stress analyses saliva samples should be collectedbefore dental hygiene treatment or clinical examination of theoral cavity
Conflict of Interests
The paper is original work and is not under consideration byanother journal There is no conflict of interests to declare
8 Disease Markers
Control patients
Control patients
Control patients
(a)
(b)
(c)
Before AfterDental hygiene treatment
0
500
1000
1500
2000
AGEs
(au
g)
Gingivitis patients
Before AfterDental hygiene treatment
0
500
1000
1500
2000
lowastlowastlowast
AGEs
(au
g)
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Before AfterDental hygiene treatment
0
200
400
600
800
lowast
FRA
P (120583
mol
g)
(f)
(g)
Gingivitis patients
Before AfterDental hygiene treatment
0
200
400
600
800
lowastlowast
FRA
P (120583
mol
g)
(e)
Gingivitis patients
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Figure 6 Continued
Disease Markers 9
(d) (h)
Control patients Gingivitis patients
Before Afterminus500
0
500
1000
1500
Dental hygiene treatment
lowast
TAC
(120583m
olg
)
Before AfterDental hygiene treatment
lowastlowastlowast
minus500
0
500
1000
1500
TAC
(120583m
olg
)
Figure 6 The effect of blood leakage after dental hygiene treatment on markers of oxidative stress and antioxidant status in saliva of control(andashd) and gingivitis (endashh) patients (a e) Salivary AOPP concentrations biomarker of oxidative damage to proteins (b f) Salivary AGEsconcentrations biomarker of carbonyl stress (c g) Salivary FRAP concentrations biomarker of antioxidant status (d h) Salivary TACconcentrations biomarker of antioxidant status lowastlowastlowast119901 lt 00001 lowastlowast119901 lt 001 and lowast119901 lt 005
This publication is the result of the implementation of theproject of University Science Park of Comenius University inBratislava (ITMS 26240220086) supported by the Researchand Development Operational Programme funded by theEuropean Regional Development Fund
References
[1] J Hodosy and P Celec ldquoDaytime of sampling tooth-brushingand ascorbic acid influence salivary thiobarbituric acid reactingsubstancesmdasha potential clinical marker of gingival statusrdquoDisease Markers vol 21 no 4 pp 203ndash207 2005
[2] N Kamodyova and P Celec ldquoSalivary markers of oxidativestress and Salivette collection systemsrdquo Clinical Chemistry andLaboratory Medicine vol 49 no 11 pp 1887ndash1890 2011
[3] N Kamodyova L Tothova and P Celec ldquoSalivary markersof oxidative stress and antioxidant status influence of externalfactorsrdquo Disease Markers vol 34 no 5 pp 313ndash321 2013
[4] F A Akalin E Baltacioglu A Alver and E Karabulut ldquoLipidperoxidation levels and total oxidant status in serum saliva andgingival crevicular fluid in patients with chronic periodontitisrdquoJournal of Clinical Periodontology vol 34 no 7 pp 558ndash5652007
[5] D A Granger E A Shirtcliff A Booth K T Kivlighanand E B Schwartz ldquoThe lsquotroublersquo with salivary testosteronerdquoPsychoneuroendocrinology vol 29 no 10 pp 1229ndash1240 2004
[6] K T Kivlighan D A Granger E B Schwartz V Nelson MCurran and E A Shirtcliff ldquoQuantifying blood leakage intothe oral mucosa and its effects on the measurement of cortisoldehydroepiandrosterone and testosterone in salivardquoHormonesand Behavior vol 46 no 1 pp 39ndash46 2004
[7] D A Granger D Cicchetti F A Rogosch L C HibelM Teisl and E Flores ldquoBlood contamination in childrenrsquossaliva prevalence stability and impact on the measurement
of salivary cortisol testosterone and dehydroepiandrosteronerdquoPsychoneuroendocrinology vol 32 no 6 pp 724ndash733 2007
[8] H Loe and J Silness ldquoPeriodontal disease in pregnancy IPrevalence and severityrdquo Acta Odontologica Scandinavica vol21 no 6 pp 533ndash551 1963
[9] H R Muhlemann and S Son ldquoGingival sulcus bleedingmdashaleading symptom in initial gingivitisrdquo Helvetica OdontologicaActa vol 15 no 2 pp 107ndash113 1971
[10] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996
[11] G Munch R Keis A Wessels et al ldquoDetermination ofadvanced glycation end products in serum by fluorescencespectroscopy and competitive ELISArdquo European Journal ofClinical Chemistry and Clinical Biochemistry vol 35 no 9 pp669ndash677 1997
[12] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[13] O Erel ldquoA novel automated direct measurement method fortotal antioxidant capacity using a new generation more stableABTS radical cationrdquo Clinical Biochemistry vol 37 no 4 pp277ndash285 2004
[14] E B Schwartz and D A Granger ldquoTransferrin enzyme immu-noassay for quantitative monitoring of blood contamination insalivardquo Clinical Chemistry vol 50 no 3 pp 654ndash656 2004
[15] Z Qing E Ling-Ling W Dong-Sheng and L Hong-ChenldquoRelationship of advanced oxidative protein products in humansaliva and plasma age-and gender-related changes and stabilityduring storagerdquo Free Radical Research vol 46 no 10 pp 1201ndash1206 2012
[16] T Miyata K Kurokawa and C Van Ypersele De StrihouldquoAdvanced glycation and lipoxidation end products role ofreactive carbonyl compounds generated during carbohydrate
10 Disease Markers
and lipid metabolismrdquo Journal of the American Society of Neph-rology vol 11 no 9 pp 1744ndash1752 2000
[17] R L Prior and G Cao ldquoIn vivo total antioxidant capacity com-parison of different analytical methodsrdquo Free Radical Biologyand Medicine vol 27 no 11-12 pp 1173ndash1181 1999
Figure 3 Oxidative stress and antioxidant status markers in saliva contaminated with plasma (a) Salivary AOPP concentrations biomarkerof oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrations biomarkerof antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SD lowastlowastlowastlowast119901 lt 00001lowastlowastlowast119901 lt 0001 lowastlowast119901 lt 001 and lowast119901 lt 005
Table 2 Comparison of clinical parameters between control and gingivitis patients
Clinical parameterGroup
Unpaired 119905-test119905
119901 value 119901 value summaryControl Gingivitis(119899 = 10) (119899 = 12)
Schwartz and Granger reported that blood components insaliva invisible to the eye have the potential to bias salivaryanalytes and the control of blood contamination in saliva wassuggested Transferrin enzymatic immunoassaywas designedfor quantitative monitoring of blood contamination [14] Itwas shown that concentrations of testosterone dehydroepi-androsterone (DHEA) and cortisol are increased in salivasamples artificially contaminated with blood [14] On the
other hand microinjury of the oral cavity did not changeDHEA or cortisol and increased salivary testosterone [6]Despite the rising number of studies analyzing oxidativestress in saliva no reports have been published regarding theeffect of occult blood contamination on concentrations of sal-ivary markers of oxidative stress
Four markers of oxidative stress and antioxidant statuswere analyzed in our study Advanced oxidation protein pro-ducts (AOPP) a novel oxidative stress biomarker was discov-ered in the plasma of uremic patients in 1996 [10] Recently
Figure 4 Oxidative stress and antioxidant status markers in saliva contaminated with red blood cells (RBC) (a) Salivary AOPPconcentrations biomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) SalivaryFRAP concentrations biomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presentedas mean + SD lowastlowastlowastlowast119901 lt 00001 and lowast119901 lt 005
AOPPwas suggested as part of the nonenzymatic antioxidantsystem of plasma proteome and oxidized fibrinogenwas indi-cated as key molecule responsible for human plasma AOPPreactivity [15] Advanced glycation end products (AGEs)marker of carbonyl stress are developed during the reactionof protein amino groups with reactive carbonyl compounds[16] Ferric reducing antioxidant power (FRAP) assay indi-rectly reflects the total antioxidant capacity of the sample[12 17] Total antioxidant capacity (TAC) assay developed byErel is directmeasurementmethod for total antioxidants [13]Our results have shown that most of the measured salivarymarkers of oxidative stress and antioxidant status are biasedin the presence of 1blood contamination in salivaHoweveras shown in results section saliva contaminatedwith 1 bloodis visually discolored Blood-contaminated saliva samples canbe easily excluded from the analyses Thus at least for themarkers analyzed in this study there is no need to use salivarytransferrin assay for the monitoring of blood contamination
To determine which blood component is responsiblefor changes in measured salivary markers the impact ofplasma red blood cells and hemoglobin was studied Based
on our results increased AOPP concentrations in blood-contaminated saliva can be explained by the presence ofred blood cells and hemoglobin in saliva Hemoglobin insaliva probably interferes with the colorimetric AOPP assayand artificially increases the AOPP concentrations AOPPconcentrations in plasma of healthy probands were reportedas 3 times as high as AOPP concentrations in saliva [15]we have therefore expected increased AOPP concentrationsin saliva contaminated with plasma But an opposite trendwas observed when plasma was spiked into saliva anddecreased salivary AOPP was detected in the presence of01ndash10 plasma When plasma is spiked into saliva theconcentration of total proteins is increased In our studydecreased concentrations of measured salivary markers afterplasma addition into saliva could be caused by normaliza-tion to the increased total proteins Based on our resultshemoglobin in saliva can mask the detection of AGEs andlead to underestimation of AGEs in saliva Plasma additioninto saliva led to decreased salivary AGEs concentrationsSalivary FRAP and TAC concentrations were decreased inthe presence of RBC hemoglobin and plasma contamination
Disease Markers 7
0
50
100
150
200
250
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
AOPP
(120583m
olg
)
(a)
0
500
1000
1500
2000
2500
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
AGEs
(au
g)
(b)
0
200
400
600
800
lowastlowastlowast
lowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
FRA
P (120583
mol
g)
(c)
0
500
1000
1500
lowastlowastlowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
TAC
(120583m
olg
)
(d)
Figure 5 Oxidative stress and antioxidant status markers in saliva contaminated with hemoglobin (a) Salivary AOPP concentrationsbiomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrationsbiomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SDlowastlowastlowastlowast119901 lt 00001 lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001
in saliva Decreased concentrations of measured markers insaliva contaminated with plasma could be as in the case ofAOPP assay caused by normalization to total proteins
The effect of blood contamination in saliva on concentra-tions of salivary markers of oxidative stress was not studiedin real clinical situation in the past Concerning that bloodcontamination in saliva is common in patients with gingivitisthe impact of microinjury was modeled in this study groupand age-matched healthy controls The results were similar tosaliva artificially contaminated with blood AOPP concentra-tions were increased after microinjury in the visual presenceof blood in saliva AOPP concentrations were increased inboth control and gingivitis group after dental hygiene treat-ment AOPP concentrations were not different between con-trol and gingivitis patients before dental hygiene treatmentAlso other measured salivary markers followed the trendobserved in saliva artificially contaminated with blood Sali-vary AGEs concentrations and also antioxidant markers TACand FRAP were decreased after dental hygiene treatment dueto presence of blood in saliva Our results have confirmedthe concern that dental hygiene treatment could bias the
concentrations of oxidative stress markers in saliva Based onour results we recommend the saliva collection before dentalhygiene treatment or clinical examination of oral cavity
5 Conclusions
Salivary oxidative stress concentrations are significantlyinfluenced by 1 blood contamination in saliva Salivasamples with 1 blood contamination are visibly colored andit is possible to easily exclude such contaminated samplesfrom further salivary oxidative stress analyses Microinjuryto the periodontium caused blood leakage into saliva in bothgingivitis and control group and biased concentrations ofoxidative stress markers in saliva For the purpose of salivaryoxidative stress analyses saliva samples should be collectedbefore dental hygiene treatment or clinical examination of theoral cavity
Conflict of Interests
The paper is original work and is not under consideration byanother journal There is no conflict of interests to declare
8 Disease Markers
Control patients
Control patients
Control patients
(a)
(b)
(c)
Before AfterDental hygiene treatment
0
500
1000
1500
2000
AGEs
(au
g)
Gingivitis patients
Before AfterDental hygiene treatment
0
500
1000
1500
2000
lowastlowastlowast
AGEs
(au
g)
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Before AfterDental hygiene treatment
0
200
400
600
800
lowast
FRA
P (120583
mol
g)
(f)
(g)
Gingivitis patients
Before AfterDental hygiene treatment
0
200
400
600
800
lowastlowast
FRA
P (120583
mol
g)
(e)
Gingivitis patients
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Figure 6 Continued
Disease Markers 9
(d) (h)
Control patients Gingivitis patients
Before Afterminus500
0
500
1000
1500
Dental hygiene treatment
lowast
TAC
(120583m
olg
)
Before AfterDental hygiene treatment
lowastlowastlowast
minus500
0
500
1000
1500
TAC
(120583m
olg
)
Figure 6 The effect of blood leakage after dental hygiene treatment on markers of oxidative stress and antioxidant status in saliva of control(andashd) and gingivitis (endashh) patients (a e) Salivary AOPP concentrations biomarker of oxidative damage to proteins (b f) Salivary AGEsconcentrations biomarker of carbonyl stress (c g) Salivary FRAP concentrations biomarker of antioxidant status (d h) Salivary TACconcentrations biomarker of antioxidant status lowastlowastlowast119901 lt 00001 lowastlowast119901 lt 001 and lowast119901 lt 005
This publication is the result of the implementation of theproject of University Science Park of Comenius University inBratislava (ITMS 26240220086) supported by the Researchand Development Operational Programme funded by theEuropean Regional Development Fund
References
[1] J Hodosy and P Celec ldquoDaytime of sampling tooth-brushingand ascorbic acid influence salivary thiobarbituric acid reactingsubstancesmdasha potential clinical marker of gingival statusrdquoDisease Markers vol 21 no 4 pp 203ndash207 2005
[2] N Kamodyova and P Celec ldquoSalivary markers of oxidativestress and Salivette collection systemsrdquo Clinical Chemistry andLaboratory Medicine vol 49 no 11 pp 1887ndash1890 2011
[3] N Kamodyova L Tothova and P Celec ldquoSalivary markersof oxidative stress and antioxidant status influence of externalfactorsrdquo Disease Markers vol 34 no 5 pp 313ndash321 2013
[4] F A Akalin E Baltacioglu A Alver and E Karabulut ldquoLipidperoxidation levels and total oxidant status in serum saliva andgingival crevicular fluid in patients with chronic periodontitisrdquoJournal of Clinical Periodontology vol 34 no 7 pp 558ndash5652007
[5] D A Granger E A Shirtcliff A Booth K T Kivlighanand E B Schwartz ldquoThe lsquotroublersquo with salivary testosteronerdquoPsychoneuroendocrinology vol 29 no 10 pp 1229ndash1240 2004
[6] K T Kivlighan D A Granger E B Schwartz V Nelson MCurran and E A Shirtcliff ldquoQuantifying blood leakage intothe oral mucosa and its effects on the measurement of cortisoldehydroepiandrosterone and testosterone in salivardquoHormonesand Behavior vol 46 no 1 pp 39ndash46 2004
[7] D A Granger D Cicchetti F A Rogosch L C HibelM Teisl and E Flores ldquoBlood contamination in childrenrsquossaliva prevalence stability and impact on the measurement
of salivary cortisol testosterone and dehydroepiandrosteronerdquoPsychoneuroendocrinology vol 32 no 6 pp 724ndash733 2007
[8] H Loe and J Silness ldquoPeriodontal disease in pregnancy IPrevalence and severityrdquo Acta Odontologica Scandinavica vol21 no 6 pp 533ndash551 1963
[9] H R Muhlemann and S Son ldquoGingival sulcus bleedingmdashaleading symptom in initial gingivitisrdquo Helvetica OdontologicaActa vol 15 no 2 pp 107ndash113 1971
[10] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996
[11] G Munch R Keis A Wessels et al ldquoDetermination ofadvanced glycation end products in serum by fluorescencespectroscopy and competitive ELISArdquo European Journal ofClinical Chemistry and Clinical Biochemistry vol 35 no 9 pp669ndash677 1997
[12] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[13] O Erel ldquoA novel automated direct measurement method fortotal antioxidant capacity using a new generation more stableABTS radical cationrdquo Clinical Biochemistry vol 37 no 4 pp277ndash285 2004
[14] E B Schwartz and D A Granger ldquoTransferrin enzyme immu-noassay for quantitative monitoring of blood contamination insalivardquo Clinical Chemistry vol 50 no 3 pp 654ndash656 2004
[15] Z Qing E Ling-Ling W Dong-Sheng and L Hong-ChenldquoRelationship of advanced oxidative protein products in humansaliva and plasma age-and gender-related changes and stabilityduring storagerdquo Free Radical Research vol 46 no 10 pp 1201ndash1206 2012
[16] T Miyata K Kurokawa and C Van Ypersele De StrihouldquoAdvanced glycation and lipoxidation end products role ofreactive carbonyl compounds generated during carbohydrate
10 Disease Markers
and lipid metabolismrdquo Journal of the American Society of Neph-rology vol 11 no 9 pp 1744ndash1752 2000
[17] R L Prior and G Cao ldquoIn vivo total antioxidant capacity com-parison of different analytical methodsrdquo Free Radical Biologyand Medicine vol 27 no 11-12 pp 1173ndash1181 1999
Figure 4 Oxidative stress and antioxidant status markers in saliva contaminated with red blood cells (RBC) (a) Salivary AOPPconcentrations biomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) SalivaryFRAP concentrations biomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presentedas mean + SD lowastlowastlowastlowast119901 lt 00001 and lowast119901 lt 005
AOPPwas suggested as part of the nonenzymatic antioxidantsystem of plasma proteome and oxidized fibrinogenwas indi-cated as key molecule responsible for human plasma AOPPreactivity [15] Advanced glycation end products (AGEs)marker of carbonyl stress are developed during the reactionof protein amino groups with reactive carbonyl compounds[16] Ferric reducing antioxidant power (FRAP) assay indi-rectly reflects the total antioxidant capacity of the sample[12 17] Total antioxidant capacity (TAC) assay developed byErel is directmeasurementmethod for total antioxidants [13]Our results have shown that most of the measured salivarymarkers of oxidative stress and antioxidant status are biasedin the presence of 1blood contamination in salivaHoweveras shown in results section saliva contaminatedwith 1 bloodis visually discolored Blood-contaminated saliva samples canbe easily excluded from the analyses Thus at least for themarkers analyzed in this study there is no need to use salivarytransferrin assay for the monitoring of blood contamination
To determine which blood component is responsiblefor changes in measured salivary markers the impact ofplasma red blood cells and hemoglobin was studied Based
on our results increased AOPP concentrations in blood-contaminated saliva can be explained by the presence ofred blood cells and hemoglobin in saliva Hemoglobin insaliva probably interferes with the colorimetric AOPP assayand artificially increases the AOPP concentrations AOPPconcentrations in plasma of healthy probands were reportedas 3 times as high as AOPP concentrations in saliva [15]we have therefore expected increased AOPP concentrationsin saliva contaminated with plasma But an opposite trendwas observed when plasma was spiked into saliva anddecreased salivary AOPP was detected in the presence of01ndash10 plasma When plasma is spiked into saliva theconcentration of total proteins is increased In our studydecreased concentrations of measured salivary markers afterplasma addition into saliva could be caused by normaliza-tion to the increased total proteins Based on our resultshemoglobin in saliva can mask the detection of AGEs andlead to underestimation of AGEs in saliva Plasma additioninto saliva led to decreased salivary AGEs concentrationsSalivary FRAP and TAC concentrations were decreased inthe presence of RBC hemoglobin and plasma contamination
Disease Markers 7
0
50
100
150
200
250
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
AOPP
(120583m
olg
)
(a)
0
500
1000
1500
2000
2500
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
AGEs
(au
g)
(b)
0
200
400
600
800
lowastlowastlowast
lowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
FRA
P (120583
mol
g)
(c)
0
500
1000
1500
lowastlowastlowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
0 0001 001 01 1 25 5 10Blood in saliva ()
Saliv
ary
TAC
(120583m
olg
)
(d)
Figure 5 Oxidative stress and antioxidant status markers in saliva contaminated with hemoglobin (a) Salivary AOPP concentrationsbiomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrationsbiomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SDlowastlowastlowastlowast119901 lt 00001 lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001
in saliva Decreased concentrations of measured markers insaliva contaminated with plasma could be as in the case ofAOPP assay caused by normalization to total proteins
The effect of blood contamination in saliva on concentra-tions of salivary markers of oxidative stress was not studiedin real clinical situation in the past Concerning that bloodcontamination in saliva is common in patients with gingivitisthe impact of microinjury was modeled in this study groupand age-matched healthy controls The results were similar tosaliva artificially contaminated with blood AOPP concentra-tions were increased after microinjury in the visual presenceof blood in saliva AOPP concentrations were increased inboth control and gingivitis group after dental hygiene treat-ment AOPP concentrations were not different between con-trol and gingivitis patients before dental hygiene treatmentAlso other measured salivary markers followed the trendobserved in saliva artificially contaminated with blood Sali-vary AGEs concentrations and also antioxidant markers TACand FRAP were decreased after dental hygiene treatment dueto presence of blood in saliva Our results have confirmedthe concern that dental hygiene treatment could bias the
concentrations of oxidative stress markers in saliva Based onour results we recommend the saliva collection before dentalhygiene treatment or clinical examination of oral cavity
5 Conclusions
Salivary oxidative stress concentrations are significantlyinfluenced by 1 blood contamination in saliva Salivasamples with 1 blood contamination are visibly colored andit is possible to easily exclude such contaminated samplesfrom further salivary oxidative stress analyses Microinjuryto the periodontium caused blood leakage into saliva in bothgingivitis and control group and biased concentrations ofoxidative stress markers in saliva For the purpose of salivaryoxidative stress analyses saliva samples should be collectedbefore dental hygiene treatment or clinical examination of theoral cavity
Conflict of Interests
The paper is original work and is not under consideration byanother journal There is no conflict of interests to declare
8 Disease Markers
Control patients
Control patients
Control patients
(a)
(b)
(c)
Before AfterDental hygiene treatment
0
500
1000
1500
2000
AGEs
(au
g)
Gingivitis patients
Before AfterDental hygiene treatment
0
500
1000
1500
2000
lowastlowastlowast
AGEs
(au
g)
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Before AfterDental hygiene treatment
0
200
400
600
800
lowast
FRA
P (120583
mol
g)
(f)
(g)
Gingivitis patients
Before AfterDental hygiene treatment
0
200
400
600
800
lowastlowast
FRA
P (120583
mol
g)
(e)
Gingivitis patients
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Figure 6 Continued
Disease Markers 9
(d) (h)
Control patients Gingivitis patients
Before Afterminus500
0
500
1000
1500
Dental hygiene treatment
lowast
TAC
(120583m
olg
)
Before AfterDental hygiene treatment
lowastlowastlowast
minus500
0
500
1000
1500
TAC
(120583m
olg
)
Figure 6 The effect of blood leakage after dental hygiene treatment on markers of oxidative stress and antioxidant status in saliva of control(andashd) and gingivitis (endashh) patients (a e) Salivary AOPP concentrations biomarker of oxidative damage to proteins (b f) Salivary AGEsconcentrations biomarker of carbonyl stress (c g) Salivary FRAP concentrations biomarker of antioxidant status (d h) Salivary TACconcentrations biomarker of antioxidant status lowastlowastlowast119901 lt 00001 lowastlowast119901 lt 001 and lowast119901 lt 005
This publication is the result of the implementation of theproject of University Science Park of Comenius University inBratislava (ITMS 26240220086) supported by the Researchand Development Operational Programme funded by theEuropean Regional Development Fund
References
[1] J Hodosy and P Celec ldquoDaytime of sampling tooth-brushingand ascorbic acid influence salivary thiobarbituric acid reactingsubstancesmdasha potential clinical marker of gingival statusrdquoDisease Markers vol 21 no 4 pp 203ndash207 2005
[2] N Kamodyova and P Celec ldquoSalivary markers of oxidativestress and Salivette collection systemsrdquo Clinical Chemistry andLaboratory Medicine vol 49 no 11 pp 1887ndash1890 2011
[3] N Kamodyova L Tothova and P Celec ldquoSalivary markersof oxidative stress and antioxidant status influence of externalfactorsrdquo Disease Markers vol 34 no 5 pp 313ndash321 2013
[4] F A Akalin E Baltacioglu A Alver and E Karabulut ldquoLipidperoxidation levels and total oxidant status in serum saliva andgingival crevicular fluid in patients with chronic periodontitisrdquoJournal of Clinical Periodontology vol 34 no 7 pp 558ndash5652007
[5] D A Granger E A Shirtcliff A Booth K T Kivlighanand E B Schwartz ldquoThe lsquotroublersquo with salivary testosteronerdquoPsychoneuroendocrinology vol 29 no 10 pp 1229ndash1240 2004
[6] K T Kivlighan D A Granger E B Schwartz V Nelson MCurran and E A Shirtcliff ldquoQuantifying blood leakage intothe oral mucosa and its effects on the measurement of cortisoldehydroepiandrosterone and testosterone in salivardquoHormonesand Behavior vol 46 no 1 pp 39ndash46 2004
[7] D A Granger D Cicchetti F A Rogosch L C HibelM Teisl and E Flores ldquoBlood contamination in childrenrsquossaliva prevalence stability and impact on the measurement
of salivary cortisol testosterone and dehydroepiandrosteronerdquoPsychoneuroendocrinology vol 32 no 6 pp 724ndash733 2007
[8] H Loe and J Silness ldquoPeriodontal disease in pregnancy IPrevalence and severityrdquo Acta Odontologica Scandinavica vol21 no 6 pp 533ndash551 1963
[9] H R Muhlemann and S Son ldquoGingival sulcus bleedingmdashaleading symptom in initial gingivitisrdquo Helvetica OdontologicaActa vol 15 no 2 pp 107ndash113 1971
[10] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996
[11] G Munch R Keis A Wessels et al ldquoDetermination ofadvanced glycation end products in serum by fluorescencespectroscopy and competitive ELISArdquo European Journal ofClinical Chemistry and Clinical Biochemistry vol 35 no 9 pp669ndash677 1997
[12] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[13] O Erel ldquoA novel automated direct measurement method fortotal antioxidant capacity using a new generation more stableABTS radical cationrdquo Clinical Biochemistry vol 37 no 4 pp277ndash285 2004
[14] E B Schwartz and D A Granger ldquoTransferrin enzyme immu-noassay for quantitative monitoring of blood contamination insalivardquo Clinical Chemistry vol 50 no 3 pp 654ndash656 2004
[15] Z Qing E Ling-Ling W Dong-Sheng and L Hong-ChenldquoRelationship of advanced oxidative protein products in humansaliva and plasma age-and gender-related changes and stabilityduring storagerdquo Free Radical Research vol 46 no 10 pp 1201ndash1206 2012
[16] T Miyata K Kurokawa and C Van Ypersele De StrihouldquoAdvanced glycation and lipoxidation end products role ofreactive carbonyl compounds generated during carbohydrate
10 Disease Markers
and lipid metabolismrdquo Journal of the American Society of Neph-rology vol 11 no 9 pp 1744ndash1752 2000
[17] R L Prior and G Cao ldquoIn vivo total antioxidant capacity com-parison of different analytical methodsrdquo Free Radical Biologyand Medicine vol 27 no 11-12 pp 1173ndash1181 1999
Figure 5 Oxidative stress and antioxidant status markers in saliva contaminated with hemoglobin (a) Salivary AOPP concentrationsbiomarker of oxidative damage to proteins (b) Salivary AGEs concentrations biomarker of carbonyl stress (c) Salivary FRAP concentrationsbiomarker of antioxidant status (d) Salivary TAC concentrations biomarker of antioxidant status Data are presented as mean + SDlowastlowastlowastlowast119901 lt 00001 lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001
in saliva Decreased concentrations of measured markers insaliva contaminated with plasma could be as in the case ofAOPP assay caused by normalization to total proteins
The effect of blood contamination in saliva on concentra-tions of salivary markers of oxidative stress was not studiedin real clinical situation in the past Concerning that bloodcontamination in saliva is common in patients with gingivitisthe impact of microinjury was modeled in this study groupand age-matched healthy controls The results were similar tosaliva artificially contaminated with blood AOPP concentra-tions were increased after microinjury in the visual presenceof blood in saliva AOPP concentrations were increased inboth control and gingivitis group after dental hygiene treat-ment AOPP concentrations were not different between con-trol and gingivitis patients before dental hygiene treatmentAlso other measured salivary markers followed the trendobserved in saliva artificially contaminated with blood Sali-vary AGEs concentrations and also antioxidant markers TACand FRAP were decreased after dental hygiene treatment dueto presence of blood in saliva Our results have confirmedthe concern that dental hygiene treatment could bias the
concentrations of oxidative stress markers in saliva Based onour results we recommend the saliva collection before dentalhygiene treatment or clinical examination of oral cavity
5 Conclusions
Salivary oxidative stress concentrations are significantlyinfluenced by 1 blood contamination in saliva Salivasamples with 1 blood contamination are visibly colored andit is possible to easily exclude such contaminated samplesfrom further salivary oxidative stress analyses Microinjuryto the periodontium caused blood leakage into saliva in bothgingivitis and control group and biased concentrations ofoxidative stress markers in saliva For the purpose of salivaryoxidative stress analyses saliva samples should be collectedbefore dental hygiene treatment or clinical examination of theoral cavity
Conflict of Interests
The paper is original work and is not under consideration byanother journal There is no conflict of interests to declare
8 Disease Markers
Control patients
Control patients
Control patients
(a)
(b)
(c)
Before AfterDental hygiene treatment
0
500
1000
1500
2000
AGEs
(au
g)
Gingivitis patients
Before AfterDental hygiene treatment
0
500
1000
1500
2000
lowastlowastlowast
AGEs
(au
g)
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Before AfterDental hygiene treatment
0
200
400
600
800
lowast
FRA
P (120583
mol
g)
(f)
(g)
Gingivitis patients
Before AfterDental hygiene treatment
0
200
400
600
800
lowastlowast
FRA
P (120583
mol
g)
(e)
Gingivitis patients
Before After0
10
20
30
40
50
Dental hygiene treatment
lowast
AOPP
(120583m
olg
)
Figure 6 Continued
Disease Markers 9
(d) (h)
Control patients Gingivitis patients
Before Afterminus500
0
500
1000
1500
Dental hygiene treatment
lowast
TAC
(120583m
olg
)
Before AfterDental hygiene treatment
lowastlowastlowast
minus500
0
500
1000
1500
TAC
(120583m
olg
)
Figure 6 The effect of blood leakage after dental hygiene treatment on markers of oxidative stress and antioxidant status in saliva of control(andashd) and gingivitis (endashh) patients (a e) Salivary AOPP concentrations biomarker of oxidative damage to proteins (b f) Salivary AGEsconcentrations biomarker of carbonyl stress (c g) Salivary FRAP concentrations biomarker of antioxidant status (d h) Salivary TACconcentrations biomarker of antioxidant status lowastlowastlowast119901 lt 00001 lowastlowast119901 lt 001 and lowast119901 lt 005
This publication is the result of the implementation of theproject of University Science Park of Comenius University inBratislava (ITMS 26240220086) supported by the Researchand Development Operational Programme funded by theEuropean Regional Development Fund
References
[1] J Hodosy and P Celec ldquoDaytime of sampling tooth-brushingand ascorbic acid influence salivary thiobarbituric acid reactingsubstancesmdasha potential clinical marker of gingival statusrdquoDisease Markers vol 21 no 4 pp 203ndash207 2005
[2] N Kamodyova and P Celec ldquoSalivary markers of oxidativestress and Salivette collection systemsrdquo Clinical Chemistry andLaboratory Medicine vol 49 no 11 pp 1887ndash1890 2011
[3] N Kamodyova L Tothova and P Celec ldquoSalivary markersof oxidative stress and antioxidant status influence of externalfactorsrdquo Disease Markers vol 34 no 5 pp 313ndash321 2013
[4] F A Akalin E Baltacioglu A Alver and E Karabulut ldquoLipidperoxidation levels and total oxidant status in serum saliva andgingival crevicular fluid in patients with chronic periodontitisrdquoJournal of Clinical Periodontology vol 34 no 7 pp 558ndash5652007
[5] D A Granger E A Shirtcliff A Booth K T Kivlighanand E B Schwartz ldquoThe lsquotroublersquo with salivary testosteronerdquoPsychoneuroendocrinology vol 29 no 10 pp 1229ndash1240 2004
[6] K T Kivlighan D A Granger E B Schwartz V Nelson MCurran and E A Shirtcliff ldquoQuantifying blood leakage intothe oral mucosa and its effects on the measurement of cortisoldehydroepiandrosterone and testosterone in salivardquoHormonesand Behavior vol 46 no 1 pp 39ndash46 2004
[7] D A Granger D Cicchetti F A Rogosch L C HibelM Teisl and E Flores ldquoBlood contamination in childrenrsquossaliva prevalence stability and impact on the measurement
of salivary cortisol testosterone and dehydroepiandrosteronerdquoPsychoneuroendocrinology vol 32 no 6 pp 724ndash733 2007
[8] H Loe and J Silness ldquoPeriodontal disease in pregnancy IPrevalence and severityrdquo Acta Odontologica Scandinavica vol21 no 6 pp 533ndash551 1963
[9] H R Muhlemann and S Son ldquoGingival sulcus bleedingmdashaleading symptom in initial gingivitisrdquo Helvetica OdontologicaActa vol 15 no 2 pp 107ndash113 1971
[10] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996
[11] G Munch R Keis A Wessels et al ldquoDetermination ofadvanced glycation end products in serum by fluorescencespectroscopy and competitive ELISArdquo European Journal ofClinical Chemistry and Clinical Biochemistry vol 35 no 9 pp669ndash677 1997
[12] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[13] O Erel ldquoA novel automated direct measurement method fortotal antioxidant capacity using a new generation more stableABTS radical cationrdquo Clinical Biochemistry vol 37 no 4 pp277ndash285 2004
[14] E B Schwartz and D A Granger ldquoTransferrin enzyme immu-noassay for quantitative monitoring of blood contamination insalivardquo Clinical Chemistry vol 50 no 3 pp 654ndash656 2004
[15] Z Qing E Ling-Ling W Dong-Sheng and L Hong-ChenldquoRelationship of advanced oxidative protein products in humansaliva and plasma age-and gender-related changes and stabilityduring storagerdquo Free Radical Research vol 46 no 10 pp 1201ndash1206 2012
[16] T Miyata K Kurokawa and C Van Ypersele De StrihouldquoAdvanced glycation and lipoxidation end products role ofreactive carbonyl compounds generated during carbohydrate
10 Disease Markers
and lipid metabolismrdquo Journal of the American Society of Neph-rology vol 11 no 9 pp 1744ndash1752 2000
[17] R L Prior and G Cao ldquoIn vivo total antioxidant capacity com-parison of different analytical methodsrdquo Free Radical Biologyand Medicine vol 27 no 11-12 pp 1173ndash1181 1999
Figure 6 The effect of blood leakage after dental hygiene treatment on markers of oxidative stress and antioxidant status in saliva of control(andashd) and gingivitis (endashh) patients (a e) Salivary AOPP concentrations biomarker of oxidative damage to proteins (b f) Salivary AGEsconcentrations biomarker of carbonyl stress (c g) Salivary FRAP concentrations biomarker of antioxidant status (d h) Salivary TACconcentrations biomarker of antioxidant status lowastlowastlowast119901 lt 00001 lowastlowast119901 lt 001 and lowast119901 lt 005
This publication is the result of the implementation of theproject of University Science Park of Comenius University inBratislava (ITMS 26240220086) supported by the Researchand Development Operational Programme funded by theEuropean Regional Development Fund
References
[1] J Hodosy and P Celec ldquoDaytime of sampling tooth-brushingand ascorbic acid influence salivary thiobarbituric acid reactingsubstancesmdasha potential clinical marker of gingival statusrdquoDisease Markers vol 21 no 4 pp 203ndash207 2005
[2] N Kamodyova and P Celec ldquoSalivary markers of oxidativestress and Salivette collection systemsrdquo Clinical Chemistry andLaboratory Medicine vol 49 no 11 pp 1887ndash1890 2011
[3] N Kamodyova L Tothova and P Celec ldquoSalivary markersof oxidative stress and antioxidant status influence of externalfactorsrdquo Disease Markers vol 34 no 5 pp 313ndash321 2013
[4] F A Akalin E Baltacioglu A Alver and E Karabulut ldquoLipidperoxidation levels and total oxidant status in serum saliva andgingival crevicular fluid in patients with chronic periodontitisrdquoJournal of Clinical Periodontology vol 34 no 7 pp 558ndash5652007
[5] D A Granger E A Shirtcliff A Booth K T Kivlighanand E B Schwartz ldquoThe lsquotroublersquo with salivary testosteronerdquoPsychoneuroendocrinology vol 29 no 10 pp 1229ndash1240 2004
[6] K T Kivlighan D A Granger E B Schwartz V Nelson MCurran and E A Shirtcliff ldquoQuantifying blood leakage intothe oral mucosa and its effects on the measurement of cortisoldehydroepiandrosterone and testosterone in salivardquoHormonesand Behavior vol 46 no 1 pp 39ndash46 2004
[7] D A Granger D Cicchetti F A Rogosch L C HibelM Teisl and E Flores ldquoBlood contamination in childrenrsquossaliva prevalence stability and impact on the measurement
of salivary cortisol testosterone and dehydroepiandrosteronerdquoPsychoneuroendocrinology vol 32 no 6 pp 724ndash733 2007
[8] H Loe and J Silness ldquoPeriodontal disease in pregnancy IPrevalence and severityrdquo Acta Odontologica Scandinavica vol21 no 6 pp 533ndash551 1963
[9] H R Muhlemann and S Son ldquoGingival sulcus bleedingmdashaleading symptom in initial gingivitisrdquo Helvetica OdontologicaActa vol 15 no 2 pp 107ndash113 1971
[10] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996
[11] G Munch R Keis A Wessels et al ldquoDetermination ofadvanced glycation end products in serum by fluorescencespectroscopy and competitive ELISArdquo European Journal ofClinical Chemistry and Clinical Biochemistry vol 35 no 9 pp669ndash677 1997
[12] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[13] O Erel ldquoA novel automated direct measurement method fortotal antioxidant capacity using a new generation more stableABTS radical cationrdquo Clinical Biochemistry vol 37 no 4 pp277ndash285 2004
[14] E B Schwartz and D A Granger ldquoTransferrin enzyme immu-noassay for quantitative monitoring of blood contamination insalivardquo Clinical Chemistry vol 50 no 3 pp 654ndash656 2004
[15] Z Qing E Ling-Ling W Dong-Sheng and L Hong-ChenldquoRelationship of advanced oxidative protein products in humansaliva and plasma age-and gender-related changes and stabilityduring storagerdquo Free Radical Research vol 46 no 10 pp 1201ndash1206 2012
[16] T Miyata K Kurokawa and C Van Ypersele De StrihouldquoAdvanced glycation and lipoxidation end products role ofreactive carbonyl compounds generated during carbohydrate
10 Disease Markers
and lipid metabolismrdquo Journal of the American Society of Neph-rology vol 11 no 9 pp 1744ndash1752 2000
[17] R L Prior and G Cao ldquoIn vivo total antioxidant capacity com-parison of different analytical methodsrdquo Free Radical Biologyand Medicine vol 27 no 11-12 pp 1173ndash1181 1999
Figure 6 The effect of blood leakage after dental hygiene treatment on markers of oxidative stress and antioxidant status in saliva of control(andashd) and gingivitis (endashh) patients (a e) Salivary AOPP concentrations biomarker of oxidative damage to proteins (b f) Salivary AGEsconcentrations biomarker of carbonyl stress (c g) Salivary FRAP concentrations biomarker of antioxidant status (d h) Salivary TACconcentrations biomarker of antioxidant status lowastlowastlowast119901 lt 00001 lowastlowast119901 lt 001 and lowast119901 lt 005
This publication is the result of the implementation of theproject of University Science Park of Comenius University inBratislava (ITMS 26240220086) supported by the Researchand Development Operational Programme funded by theEuropean Regional Development Fund
References
[1] J Hodosy and P Celec ldquoDaytime of sampling tooth-brushingand ascorbic acid influence salivary thiobarbituric acid reactingsubstancesmdasha potential clinical marker of gingival statusrdquoDisease Markers vol 21 no 4 pp 203ndash207 2005
[2] N Kamodyova and P Celec ldquoSalivary markers of oxidativestress and Salivette collection systemsrdquo Clinical Chemistry andLaboratory Medicine vol 49 no 11 pp 1887ndash1890 2011
[3] N Kamodyova L Tothova and P Celec ldquoSalivary markersof oxidative stress and antioxidant status influence of externalfactorsrdquo Disease Markers vol 34 no 5 pp 313ndash321 2013
[4] F A Akalin E Baltacioglu A Alver and E Karabulut ldquoLipidperoxidation levels and total oxidant status in serum saliva andgingival crevicular fluid in patients with chronic periodontitisrdquoJournal of Clinical Periodontology vol 34 no 7 pp 558ndash5652007
[5] D A Granger E A Shirtcliff A Booth K T Kivlighanand E B Schwartz ldquoThe lsquotroublersquo with salivary testosteronerdquoPsychoneuroendocrinology vol 29 no 10 pp 1229ndash1240 2004
[6] K T Kivlighan D A Granger E B Schwartz V Nelson MCurran and E A Shirtcliff ldquoQuantifying blood leakage intothe oral mucosa and its effects on the measurement of cortisoldehydroepiandrosterone and testosterone in salivardquoHormonesand Behavior vol 46 no 1 pp 39ndash46 2004
[7] D A Granger D Cicchetti F A Rogosch L C HibelM Teisl and E Flores ldquoBlood contamination in childrenrsquossaliva prevalence stability and impact on the measurement
of salivary cortisol testosterone and dehydroepiandrosteronerdquoPsychoneuroendocrinology vol 32 no 6 pp 724ndash733 2007
[8] H Loe and J Silness ldquoPeriodontal disease in pregnancy IPrevalence and severityrdquo Acta Odontologica Scandinavica vol21 no 6 pp 533ndash551 1963
[9] H R Muhlemann and S Son ldquoGingival sulcus bleedingmdashaleading symptom in initial gingivitisrdquo Helvetica OdontologicaActa vol 15 no 2 pp 107ndash113 1971
[10] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996
[11] G Munch R Keis A Wessels et al ldquoDetermination ofadvanced glycation end products in serum by fluorescencespectroscopy and competitive ELISArdquo European Journal ofClinical Chemistry and Clinical Biochemistry vol 35 no 9 pp669ndash677 1997
[12] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[13] O Erel ldquoA novel automated direct measurement method fortotal antioxidant capacity using a new generation more stableABTS radical cationrdquo Clinical Biochemistry vol 37 no 4 pp277ndash285 2004
[14] E B Schwartz and D A Granger ldquoTransferrin enzyme immu-noassay for quantitative monitoring of blood contamination insalivardquo Clinical Chemistry vol 50 no 3 pp 654ndash656 2004
[15] Z Qing E Ling-Ling W Dong-Sheng and L Hong-ChenldquoRelationship of advanced oxidative protein products in humansaliva and plasma age-and gender-related changes and stabilityduring storagerdquo Free Radical Research vol 46 no 10 pp 1201ndash1206 2012
[16] T Miyata K Kurokawa and C Van Ypersele De StrihouldquoAdvanced glycation and lipoxidation end products role ofreactive carbonyl compounds generated during carbohydrate
10 Disease Markers
and lipid metabolismrdquo Journal of the American Society of Neph-rology vol 11 no 9 pp 1744ndash1752 2000
[17] R L Prior and G Cao ldquoIn vivo total antioxidant capacity com-parison of different analytical methodsrdquo Free Radical Biologyand Medicine vol 27 no 11-12 pp 1173ndash1181 1999
and lipid metabolismrdquo Journal of the American Society of Neph-rology vol 11 no 9 pp 1744ndash1752 2000
[17] R L Prior and G Cao ldquoIn vivo total antioxidant capacity com-parison of different analytical methodsrdquo Free Radical Biologyand Medicine vol 27 no 11-12 pp 1173ndash1181 1999
