Total Antioxidant Capacity andAntioxidant Enzymes in Serum,Saliva, and Gingival CrevicularFluid of Preeclamptic Women Withand Without Periodontal DiseaseVarol Canakci,* Abdulkadir Yildirim,† Cenk Fatih Canakci,* Abubekir Eltas,* Yasin Cicek,*and Humeyra Canakci‡

Background: The aim of this study was to investigate thetotal antioxidant capacity, superoxide dismutase and gluta-thione peroxidase activities, and malondialdehyde levels inserum, saliva, and gingival crevicular fluid (GCF) in pre-eclamptic and normotensive pregnant women with and with-out periodontal disease.

Methods: Forty pregnant women, consisting of 10 pre-eclamptic subjects with periodontal disease, 10 preeclampticperiodontally healthy subjects, 10 normotensive subjectswith periodontal disease, and 10 normotensive periodontallyhealthy subjects, were included in this study. After clinicalmeasurement and samplings, total antioxidant capacity, su-peroxide dismutase, glutathione peroxidase activities, andmalondialdehyde levels in serum, saliva, and GCF of pre-eclamptic and normotensive pregnant women were deter-mined, and the data were tested by non-parametric tests.Total antioxidant capacity of the clinical samples was mea-sured using a novel automated colorimetric measurementmethod. Superoxide dismutase and glutathione peroxidaseactivities and malondialdehyde levels were determined spec-trophotometrically.

Results: Superoxide dismutase and glutathione peroxidaseactivities in GCF and serum and total antioxidant capacity insaliva, GCF, and serum were the lowest in preeclampticwomen with periodontal disease. However, serum and GCFlevels of malondialdehyde were the highest in this group ofpregnant women.

Conclusions: Systemic and local antioxidant and total anti-oxidant capacities are affected by periodontal disease in addi-tion to the impact of preeclamptic status. Similar commentsmay be made for the increases in systemic and local malon-dialdehyde levels. J Periodontol 2007;78:1602-1611.

KEY WORDS

Antioxidant; glutathione peroxidase; malondialdehyde;periodontal disease; preeclampsia; superoxide dismutase.

Recent studies1-4 suggest that peri-odontal disease, as a source ofsubclinical and persistent infec-

tion, may induce systemic inflammatoryresponses that increase the risk for ad-verse pregnancy outcomes. Since 1996,there has been an increase in researchevidence suggesting associations be-tween periodontal disease and increasedrisk for systemic diseases and adversepregnancy outcomes, including pretermbirth, low birth weight, miscarriage orearly pregnancy loss, and preeclamp-sia.1-7 Preeclampsia is a challengingdisease of human pregnancy that affectsthe mother and her fetus.8 It is a com-mon obstetric syndrome of undefinedetiology affecting ;7% to 10% of preg-nant women and remains one of the twomost common causes of maternal mor-tality in the developed world.9,10 Re-cently, preeclampsia was proposed tobe a syndrome caused by an excessivesystemic inflammatory response topregnancy. It has been postulated thatpreeclampsia is associated with greaterperiodontal destruction,5 and maternalperiodontal disease during pregnancyis associated with an increased risk forthe development of preeclampsia.7 Peri-odontal disease may burden pregnant

* Department of Periodontology, School of Dentistry, Ataturk University, Erzurum, Turkey.† Department of Biochemistry, School of Medicine, Ataturk University.‡ Department of Obstetrics and Gynecology, School of Medicine, Ataturk University. doi: 10.1902/jop.2007.060469

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women systemically with endotoxin, inflammatorycytokines, and oxidative stressors at the maternal–fetal interface.6 Thus, it may be a vascular stressorthat plays a role in the development of preeclampsiain pregnant women. Preeclamptic women were found tohave higher levels of cytokines in gingival crevicularfluid (GCF),5 and their periodontal disease was asso-ciated with some microorganisms.6

Reactive oxygen species play an important role incell signaling and metabolic processes and contributeto pathogenic processes in a variety of inflammatorydisorders. All organisms possess a range of enzymaticand non-enzymatic antioxidant systems to protectthem against harmful oxidative reactions.11,12 Undercertain conditions, an increase in oxidants and a de-crease in antioxidants cannot be prevented, and theoxidant/antioxidant balance shifts toward the oxida-tive state. There is increasing evidence that oxidativestress is an important contributing factor in the path-ogenesis of preeclampsia11 and periodontal dis-ease.13-16 Antioxidant enzymes, such as glutathioneperoxidase and superoxide dismutase, protect tissuesagainst oxidative injury from free oxygen radicalsgenerated by various metabolic processes. Antioxi-dant molecules are present in all body fluids and tis-sues. The function of superoxide dismutase is toremove damaging reactive oxygen species from thecellular environment by catalyzing the dismutationof two O2

.- to H2O2. Three superoxide dismutase iso-enzymes are known in humans: Cu,Zn-superoxidedismutase, found in the cytoplasm and nucleus; mito-chondrial Mn-superoxide dismutase; and extracellularsuperoxide dismutase (Cu,Zn-superoxide dismu-tase).17,18 Glutathione peroxidase is a family of anti-oxidant enzymes that reduces hydrogen peroxideand/or lipid hydrogen peroxides by the oxidationof reduced glutathione or s-nitrosoglutathione.19,20

Two forms of glutathione peroxidase are known: clas-sical cellular glutathione peroxidase and extracellularglutathione peroxidase, which serves as an impor-tant antioxidant in many extracellular surfaces andspaces.

Malondialdehyde is an indicator of lipid peroxida-tion and one of the final decomposition products oflipid peroxidation, which has numerous deleteriouseffects on biologic systems.21 Free radicals releasedfrom a poorly perfused fetoplacental unit initiate lipidperoxidation by attacking polyunsaturated fatty acidsin cell membranes. The endothelial cells lining thehigh-pressured arterial vessels are particularly vul-nerable to oxygen free radical–initiated lipid peroxida-tion.22,23

Preeclampsia, characterized by hypertension, pro-teinuria, and edema, is an important cause of fetoma-ternal morbidity and mortality24 and is associatedwith greater periodontal destruction. A relationship

between preeclampsia and periodontitis has beenshown in selected observational (i.e., case-controland cohort) studies.4,7 The number of studies aboutthe effect of reactive oxygen species or antioxidantstatus on periodontal tissues is limited. To our knowl-edge, no study has been performed on the oxidant/antioxidant status in pregnant women with or with-out periodontal disease.

The aim of this study was to investigate the total an-tioxidant capacity; superoxide dismutase and gluta-thione peroxidase activities; and malondialdehydelevels in serum, saliva, and GCF in preeclampticand normotensive pregnant women with and withoutperiodontal disease.

MATERIALS AND METHODS

Patient SelectionThe study was carried out in 40 pregnant women: 20preeclamptic and 20 normotensive pregnant women.Preeclamptic and normotensive (without the compli-cation of preeclampsia) subjects were recruitedamong pregnant women at the Department of Obstet-rics and Gynecology, School of Medicine, AtaturkUniversity, from April 2004 to April 2006. Womenwere diagnosed with preeclampsia if they had bloodpressure ‡140/90 mm Hg on two or more occasions‡4 hours apart after 20 weeks’ gestation and protein-uria, which was considered present when one 24-hoururine collection showed a total protein excretion ‡300mg or when there was a urine dipstick value ‡1+ (30mg/dl) on two occasions ‡6 hours apart if a 24-hoururine specimen was unavailable.25 The preeclampticpatients were healthy before the onset of preeclampsia.

Normotensive pregnant women were selected frompregnant women who delivered 48 hours before or af-ter the preeclamptic pregnant women. Normotensivewomen were selected from the same hospital and thesame population as the preeclamptic women. Pre-eclamptic and normotensive women were matchedindividually for age (within 1 year), periodontal dis-ease status, parity, gravidity, and prenatal care. Thus,40 pregnant women, including 10 preeclamptic sub-jects with periodontal disease, 10 preeclamptic peri-odontally healthy subjects, 10 normotensive subjectswith periodontal disease, and 10 normotensive peri-odontally healthy subjects, were included in this study.

Age, parity, gravidity, smoking, alcohol use, pre-natal care, marital status, and medical histories wereobtained from the hospital records. Education level ofthe mother and household income were obtainedthrough a personal interview conducted by a trainedphysician at the first prenatal visit. None of thesepatients had a history of endocrine disorders (e.g., di-abetes mellitus, pheochromocytoma, and thyrotoxico-sis), polycystic ovary syndrome, uterinemalformation,

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renal disease, collagen vascular disease (lupus, scle-roderma), coarctation of the aorta, chronic hyperten-sion, heart murmur, or valvular heart disease. Theydid not receive antibiotic prophylaxis for dental treat-ment or any medication that may have influenced sexsteroid metabolism. They also had not received peri-odontal therapy for at least 6 months. This study wasapproved by our ethics committee, and a written in-formed consent was obtained from each woman.

Dental ExaminationThe number of teeth, the number of restorations, de-cay on all tooth surfaces, and clinical periodontal pa-rameters were determined within 48 hours beforedelivery. All examinations were performed by twotrained and calibrated examiners (periodontists)who were not aware of the information that the womenhad given in the interview. Each tooth was measuredand examined for probing depth (PD) in millimetersand clinical attachment level (CAL) in millimeters atsix sites per tooth (mesio-buccal, buccal, disto-buccal,mesio-lingual, lingual, and disto-lingual) with a Williamsprobe with Michigan markings.§ The PD was deter-mined by measuring the distance from the free gingivalmargin to the base of the pocket. CAL was measuredas the distance between the cemento-enamel junctionand the base of the pocket. Dental plaque was scoredas being present or absent at four points (mesial, buc-cal, lingual, and distal)oneach tooth.Bleeding onprob-ing (BOP) was assessed at the six sites at which PDwas determined and was deemed positive if it occurredwithin 15 seconds after probing. BOP was expressedas the percentage of sites showing bleeding. Exam-iners were thoroughly trained and calibrated in therecording of the PD and CAL measurements. Intraexa-miner variability in using the dental examination crite-ria was tested by performing duplicate examinationson12 randomlyselectedmothersonconsecutive days.Agreement was 91% for PD and bleeding and 93% forCAL. Periodontal health was defined as the absence ofgingival pockets ‡4 mm and the absence of attachmentloss ‡3 mm with no BOP. Periodontal disease wasdefined as two or more tooth sites with PD ‡4 mm orCAL of 4 mm that bled on probing.26 No dental radio-graphs were taken because of the pregnant conditionof the subjects.

SamplesAll samples were obtained in the morning after anovernight fast. Subjects were asked not to drink (ex-cept water) or chew gum for the same period, andabstention was checked prior to biologic sample col-lection.27 All chemicals used were obtained from onecompanyi and were of ultrapure grade; type I reagent-grade deionized water was used. All spectrophotometricmeasurements were carried out using a spectropho-tometer.¶

Serum preparation. Blood samples from 40 pregnantpatients were collected in vacutainer tubes withoutadditive and were centrifuged at 3,500 · g for 5 min-utes to get serum. Serum aliquots were stored at-80�C until analysis.

Saliva preparation. Whole saliva samples were ob-tained by expectorating into disposable tubes beforeclinical measurements. About 2 ml of whole salivawas collected in disposable tubes and centrifugedimmediately to remove cell debris (1,000 · g for 10minutes at 4�C). The supernatant was removed andstored in small aliquots at -80�C until analysis.

GCF preparation and collection of samples. GCFwas obtained from the pregnant patient within 48hours before delivery. Before sample collection, theindividuals were checked for protocol adherence.GCF samples were collected from a mesio-buccaland disto-palatal site on each tooth (molars, premo-lars, canines/incisors). To avoid irritation, the sampleswere obtained before the clinical measurements andbetween 8:00 am and 10:00 am. The area was iso-lated with cotton rolls, saliva contamination elimina-tion was ensured, and it was air dried slightly. Thesamples were obtained in 30 seconds with stan-dardized paper strips# by using the orifice method,and volume was measured on a precalibrated elec-tronic gingival fluid measuring device.** Twenty-fourpaper strips that absorbed GCF were removed andplaced in microcentrifuge tubes containing phosphate-buffered saline (pH 7) consisting of 0.1% Tween 20for glutathione peroxidase assays,28 20 mM tris-HClbuffer (pH 6.5) for superoxide dismutase assays,29

or phosphate-buffered saline (pH 7.4) for malondial-dehyde and total antioxidant capacity assays. Tubeswere mixed by vortexing and were centrifuged at1,500 · g for 10 minutes. The supernatants weretransferred to Eppendorf tubes and stored at -80�Cuntil analysis. Samples that were visually hemolyticwere discarded. Samples were defrosted and assayedimmediately to ensure minimal deterioration, and eachpatient’s samples were assayed at the same time asthe matched control’s samples.

Assay of Glutathione Peroxidase ActivitiesGlutathione peroxidase activity was measured ac-cording to Paglia and Valentine.30 A total of 2.65 ml50 mM potassium phosphate buffer (pH 7.0), includ-ing 5 mM EDTA, 100 ml glutathione (GSH) (150 mM),20 ml glutathione reductase (30 U/ml), 20 ml NaN3

(0.12 M), and 100 ml nicotinamide adenine dinucleotidephosphate, reduced form (NADPH; 8 mM), and 50 mlsample (serum, saliva, or GCF) were mixed, and the

§ Hu-Friedy, Chicago, IL.i Sigma, St. Louis, MO.¶ Beckman DU 530, Beckman, Fullerton, CA.# Periopaper, Proflow, Amityville, NY.** Periotron 8000, Oraflow, Plainview, NY.

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tubes were incubated for 30 minutes at 37�C. Thereaction was started by the addition of 100 ml H2O2

solution (2 mM), mixed rapidly by inversion, andthe conversion of NADPH to nicotinamide adeninedinucleotide phosphate (NADP) was measured spec-trophotometrically for 5 minutes at 340 nm. Theenzyme activity was expressed as U/l using an extinc-tion coefficient for NADPH at 340 nm of 6.22 · 10-6

M-1�cm-1.

Assay of Superoxide Dismutase ActivitiesCu,Zn-superoxide dismutase activity was measuredusing the method described by Sun et al.31 A total of2.45 ml assay reagent (0.3 mM xanthine, 0.6 mMNa2EDTA, 0.15 mM nitroblue tetrazolium [NBT], 0.4M Na2CO3, and 1 g/l bovine serum albumin) was com-binedwith0.5mlsample (serum,saliva,orGCF).Xan-thine oxidase (50 ml, 167 U/l) was added to initiate thereaction; the reduction of NBT by superoxide anionradicals,whichareproducedbythexanthine–xanthineoxidase system, was determined by measuring theabsorbance at 560 nm. Cu,Zn-superoxide dismutaseactivity was expressed as U/ml, where 1 U is definedas that amount of enzyme causing half-maximal in-hibition of NBT reduction.

Assay of Malondialdehyde LevelsMalondialdehyde levels were measured in the clinicalsamples by the method of Jain et al.32 This method isbased on the reaction of malondialdehyde with thio-barbituric acid to produce a complex that can be de-termined spectrophotometrically; 0.2 ml sample wasmixed thoroughly with 0.8 ml phosphate-bufferedsaline (pH 7.4) and 0.025 ml butylated hydroxyto-luene solution (0.88%). After addition of 0.5 ml 30%trichloroacetic acid, the samples were placed on icefor 2 hours and then centrifuged at 2,000 · g at 25�Cfor 15 minutes. One milliliter of supernatant was mixedwith 0.075 ml 0.1 M EDTA and 0.25 ml 1% thiobarbi-turic acid in 0.05 N NaOH. The samples were placed inboiling water for 15 minutes, cooled to room temper-ature, and the absorbance was determined at 532 nm.Total thiobarbituric acid–reactive substances were ex-pressed as malondialdehyde, using a molar extinctioncoefficient formalondialdehydeof1.56 · 105 cm-1�M-1.The results were expressed as nanomoles per milliliter.

Assay of Total Antioxidant CapacityThe total antioxidant status of the clinical samples wasmeasured using a novel automated colorimetric mea-surement method for total antioxidant capacity devel-oped by Erel.33 We adapted this method (which wasused on blood plasma in that study) to saliva andGCF extracts. The assay results are expressed as mil-limolar Trolox†† equivalent (mMTeq). This method iscapable of measuring endogenous and exogenousantioxidants, such as Trolox, vitamin C, glutathione,

uric acid, and bilirubin. Several oxidative and anti-oxidative parameters were studied to understand themechanism completely. These parameters areclosely related, and thus, the results support eachother. This approach is estimated to decrease anymeasurement error in each parameter.

Statistical AnalysesThe normality of the data’s distribution was examinedusing the Shapiro-Wilk test. The difference in the fourgroups of normally distributed variables was assessedusing two-factor analysis of variance with an interac-tion effect and Tukey multiple comparison tests. TheKruskal-Wallis analysis of variance was used, followedby the Mann-Whitney U test using the Bonferroni cor-rection, to evaluate the differences among the groupsin other variables (non-normal distribution). Correla-tions between variables were determined by Spearmanrank test. A value of P <0.05 was considered to be sig-nificant. All values are expressed as mean – SD. Forthese procedures, a statistical program‡‡ was used.

RESULTS

The demographic and pregnancy-related character-istics of the four groups are shown in Table 1. Therewere no significant differences in mean age, gravidity,or parity among the study groups (P >0.05). However,there were significant differences in the duration ofgestation, birth weight, maternal body weight, andsystolic and diastolic blood pressure in the third tri-mester between the preeclamptic and normotensivepatients. None of the pregnant patients used alcoholor smoked, and all were married. Prenatal care, edu-cation level, and household income were similaramong the groups. Education levels were 8.7 – 2.5years for preeclamptic groups and 8.3 – 2.5 yearsfor normotensive pregnant groups. Household in-come was $430 – $90 per month for preeclampticgroups and $424 – $77 per month for normotensivepregnant groups (data not shown). Maternal bodyweight was evaluated as covaried in the model; how-ever, maternal body weight, education level, andhousehold income were adjusted. No confounding ef-fects of body weight and other factors among preg-nant women were observed in the biochemicalanalyses (P >0.05).

Table 2 displays the clinical dental and periodontalvariables in the study groups. The numbers of teethpresent, decayed teeth, and restorations were similarin the four groups (P >0.05). As expected, mean PDand CAL were higher in the pregnant women withperiodontal disease (P <0.001). They also exhibitedhigher percentages of sites with PD ‡4 mm, sites with

†† Trolox, Sigma-Aldrich Chemical, St. Louis, MO.‡‡ SPSS 11.0 for Windows, SPSS, Chicago, IL.

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CAL ‡ 4 mm, sites with plaque, and sites exhibitingBOP (P <0.001).

Laboratory FindingsTotal antioxidant capacity, malondialdehyde levels,and superoxide dismutase and glutathione peroxi-dase activities in the serum, saliva, and GCF of pre-eclamptic and normotensive women, regardless of

periodontal status, are shownin Table 3. There were signifi-cant differences in the meantotal antioxidant capacities andglutathione peroxidase activi-ties in serum and GCF betweenthe two groups of pregnantwomen; however, mean totalantioxidant capacity and gluta-thione peroxidase activity insaliva did not differ signifi-cantly between the preeclamp-tic and normotensive pregnantwomen (P >0.05). We foundsignificant differences in themean local and systemic su-peroxide dismutase activitiesbetween the preeclamptic andnormotensive pregnant women(P <0.05). Mean malondialde-hyde levels in serum and GCFwere higher in the preeclamp-tic women than in the normo-tensive pregnant women (P<0.05); however, the malon-dialdehyde level in saliva didnot differ significantly betweenthe preeclamptic and normo-tensive women (P >0.05).

Total antioxidant capacity,malondialdehyde levels, and su-peroxide dismutase and gluta-thione peroxidase activities inthe serum, saliva, and GCF ofthe periodontally healthy preg-nant women and the pregnantwomen with periodontal dis-ease, regardless of preeclamp-sia status, are shown in Table 3.Significant differences in themean local and systemic totalantioxidant capacities and su-peroxide dismutase activitieswere found between the womenwith periodontal disease andperiodontally healthy controls(P <0.01 for serum; P <0.05for saliva and GCF). We found

significant differences in the malondialdehyde levelsand glutathione peroxidase activities in serum andGCF, but not in saliva, between the two groups (P<0.05 and P <0.01, respectively).

Comparisons Between GroupsThe comparisons of GCF, saliva, and serum superox-ide dismutase and glutathione peroxidase activities,

Table 1.

Demographic and Pregnancy-Related Characteristics

Variable

Preeclamptic Women

With Periodontal

Disease

(n = 10)

Preeclamptic

Periodontally

Healthy

Women

(n = 10)

Normotensive

Women With

Periodontal

Disease

(n = 10)

Normotensive

Periodontally

Healthy

Women

(n = 10)

Age (years) 24.6 – 5.1 25.5 – 4.8 24.9 – 6.9 25.6 – 5.7

Gravidity (N) 2.2 – 1.0 2.4 – 1.0 2.3 – 0.9 2.4 – 1.0

Parity (N) 0.9 – 0.7 1.0 – 0.8 1.0 – 0.8 1.0 – 0.8

Gestation (weeks) 34.2 – 0.8† 33.9 – 0.8† 38.9 – 0.3 39.3 – 0.3

Body weight (kg) 81.0 – 14.4* 80.0 – 22.4* 66.6 – 16.2 66.9 – 13.2

SBP (mm Hg) 172.2 – 4.7† 169.9 – 4.3† 106.2 – 2.9 105.4 – 2.1

DBP (mm Hg) 117.2 – 3.3† 116.9 – 3.6† 61.6 – 2.2 60.9 – 2.4

Birth weight (g) 2,168 – 104.9† 2,207 – 97.4† 3,401 – 72.6 3,399 – 82.9

Data are mean – SD.SBP = systolic blood pressure; DBP = diastolic blood pressure.* Significant differences between normotensive and preeclamptic women (P <0.01).† Significant differences between normotensive and preeclamptic women (P <0.001).

Table 2.

Clinical Dental and Periodontal Variables

Variable

Preeclamptic

Women With

Periodontal

Disease

(n = 10)

Preeclamptic

Periodontally

Healthy Women

(n = 10)

Normotensive

Women With

Periodontal

Disease

(n = 10)

Normotensive

Periodontally

Healthy

Women

(n = 10)

Dental parametersTeeth (N) 24.1 – 6.2 24.9 – 6.1 24.4 – 5.9 25.4 – 4.9Decayed teeth (N) 4.3 – 3.7 4.9 – 3.2 3.9 – 4.1 3.8 – 3.9Restored teeth (N) 8.9 – 6.6 8.4 – 5.3 7.4 – 6.1 8.4 – 4.9

Periodontal parameters at deliveryPD (mm) 4.24 – 0.42* 1.74 – 0.21 4.12 – 0.38* 1.68 – 0.27CAL (mm) 3.98 – 0.42* 1.31 – 0.25 3.87 – 0.51* 1.27 – 0.21PD ‡4 mm (% sites) 20.4 – 10.1* 1.2 – 0.3 19.2 – 11.2* 1.4 – 0.4CAL ‡4 mm (% sites) 27.3 – 10.9* 0.3 – 0.1 26.6 – 10.9* 0.3 – 0.1Plaque (% sites) 69.7 – 21.7* 17.9 – 20.8 67.7 – 23.6* 19.9 – 18.7BOP (% sites) 47.6 – 14.2* 6.7 – 2.2 45.6 – 13.4* 5.3 – 1.8

Data are mean – SD.* Significant differences between periodontally healthy women and women with periodontal disease (P <0.001).

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malondialdehyde levels, and total antioxidant capac-ity values among the four groups of women are shownin Figure 1.

Local and systemic total antioxidant capacitiesamong the four groups were significantly different.The lowest values were observed in the preeclampticswith periodontal disease, whereas the highest valueswere present in the normotensive periodontallyhealthy subjects. The serum and GCF superoxide dis-mutase activities were significantly different betweenall groups, except between preeclamptic periodon-tally healthy women and normotensive women withperiodontal disease and between preeclamptic peri-odontally healthy women and normotensive peri-odontally healthy women (in GCF only; P >0.05).The serum and GCF glutathione peroxidase activitieswere significantly different between all groups, exceptbetween preeclamptic periodontally healthy womenand normotensive women with periodontal disease(in serum only; P >0.05). The serum and GCF malon-dialdehyde levels were different among all groups,except between preeclamptic periodontally healthywomen and normotensive women with periodontaldisease (in GCF only; P >0.05). Superoxide dismutaseand glutathione peroxidase activities and malondial-dehyde levels in saliva showed no significant differ-ences between groups (P >0.05).

CorrelationsThe correlations between GCF, saliva, and serum ac-tivities of superoxide dismutase and glutathione per-oxidase and values of malondialdehyde and totalantioxidant capacity of all groups were analyzed bySpearman rank test. Correlations between serum ma-londialdehyde and GCF malondialdehyde levels andsaliva total antioxidant capacity and GCF total antiox-idant capacity were positive and statistically signifi-cant for all four groups (r = 0.38 for preeclampticwomen with periodontal disease, r = 0.39 for pre-eclamptic periodontally healthy women, r = 0.41 fornormotensive women with periodontal disease, andr = 0.44 for normotensive periodontally healthywomen for malondialdehyde levels; P <0.05 and r =0.37 for preeclamptic women with periodontal dis-ease, r = 0.43 for preeclamptic periodontally healthywomen, r = 0.35 for normotensive women with peri-odontal disease, and r = 0.46 for normotensive peri-odontally healthy women for total antioxidantcapacity values; P <0.05). In contrast, a negative cor-relation between saliva superoxide dismutase andserum superoxide dismutase activities was noted forall four groups (r = 0.56 for preeclamptic womenwith periodontal disease, r = 0.61 for preeclamptic peri-odontally healthy women, r = 0.49 for normoten-sive women with periodontal disease, and r = 0.39 for

Table 3.

Serum, Saliva, and GCF Superoxide Dismutase and Glutathione Peroxidase Activities;Malondialdehyde Levels; and Total Antioxidant Capacity

Variable

Preeclamptic

Pregnant Women

(n = 20)

Normotensive

Pregnant Women

(n = 20)

Pregnant Women

With Periodontal

Disease

(n = 20)

Periodontally Healthy

Pregnant Women

(n = 20)

SerumTotal antioxidant capacity (mmTeq) 0.75 – 0.18 1.15 – 0.29† 0.83 – 0.28 1.07 – 0.29†

Superoxide dismutase (U/ml) 2.61 – 0.72 3.26 – 0.69* 2.80 – 0.31 3.06 – 0.39†

Glutathione peroxidase (U/l) 132.1 – 18.8 165.7 – 21.9† 127.2 – 26.8 177.1 – 49.9†

Malondialdehyde (nmol/ml) 4.50 – 0.98 3.29 – 0.89* 4.21 – 0.88 3.46 – 0.94*

SalivaTotal antioxidant capacity (mmTeq) 0.33 – 0.14 0.49 – 0.23 0.36 – 0.11 0.46 – 0.10*Superoxide dismutase (U/ml) 2.81 – 0.32 3.11 – 0.31* 2.76 – 0.37 3.21 – 0.21*Glutathione peroxidase (U/l) 54.1 – 23.9 62.2 – 27.4 55.2 – 27.3 59.6 – 35.9Malondialdehyde (nmol/ml) 6.61 – 1.70 6.51 – 1.20 6.71 – 1.69 6.41 – 1.77

GCFTotal antioxidant capacity (mmTeq) 0.11 – 0.03 0.16 – 0.04* 0.12 – 0.03 0.15 – 0.04*Superoxide dismutase (U/ml) 1.11 – 0.31 1.44 – 0.37* 0.97 – 0.39 1.56 – 0.56*Glutathione peroxidase (U/l) 73.2 – 12.7 98.1 – 23.5† 68.3 – 32.3 110.9 – 36.9†

Malondialdehyde (nmol/ml) 3.42 – 0.36 2.99 – 0.32* 3.37 – 0.23 3.06 – 0.32*

Data are mean – SD.* Significant differences both between normotensive and preeclamptic women and between women with periodontal health and disease (P <0.05). Data were

adjusted for maternal body weight, education, and income.† Significant differences both between normotensive and preeclamptic women and between women with periodontal health and disease (P <0.01). Data were

adjusted for maternal body weight, education, and income.

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normotensive periodontally healthy women; P <0.05;data not shown).

DISCUSSION

To the best of our knowledge, this is the first reportedinvestigation of possible differences in local and sys-temicsuperoxidedismutaseandglutathioneperoxidaseactivities, malondialdehyde levels, and total antioxi-dant capacities in periodontally healthy and diseasedpregnant women (preeclamptic and normotensive).

The results of the present study indicated that super-oxide dismutase and glutathione peroxidase activitiesin serum and GCF and total antioxidant capacities inserum, saliva, and GCF were the lowest in preeclamp-tic women with periodontal disease, whereas serumand GCF levels of malondialdehyde were the highestin these women. Systemic and local antioxidant activ-ities (superoxidedismutaseandglutathioneperoxidase)and total antioxidant capacity decreased in relation topreeclampsia and periodontal disease. This suggeststhat systemic and local antioxidant activities, total an-tioxidant capacity, and malondialdehyde levels are af-fected by periodontal disease in addition to the impactof preeclampsia.

Knowledge about the total responses of the serumantioxidant systems in preeclampsia is limited.34

There are conflicting results on this topic. Kharb35,36

found significantly higher total antioxidant capacity in

womenwithpreeclampsia,whereasShaarawy et al.34 found signifi-cantly lower total antioxidant ca-pacities in preeclamptic patients.Harma et al.11 observed a signifi-cant difference between the nor-motensive women and those withpreeclampsia upon assessmentof their total antioxidant capacity.The mean total antioxidant capac-ity of the preeclamptic patientswas 1.06 mM Teq equivalent,whereas that of the normotensivepatients was 1.31 mM Teq equiva-lent. There has not been any studyof GCF and saliva total antioxidantcapacity in preeclampsia.

Moore et al.37 measured the an-tioxidant capacity of saliva in peri-odontally diseased and healthyindividuals using the Trolox equiv-alent assay and failed to find anysignificant difference between thegroups. In a similar study, Chappleet al.38 studied serum and salivasamples in diseased and controlgroups using an enhanced chemi-luminescent assay. Serum antiox-

idant capacity in the two groups was similar; however,saliva antioxidant capacity and saliva and serum anti-oxidants were significantly lower in diseased patientscompared to controls.

Recently, the possible association of gingivitis andperiodontitis with impaired salivary antioxidant statusand increased oxidative injury was investigated bySculley and Langley-Evans.39 These researchers re-ported that periodontal disease was associated withreduced salivary antioxidant status and increasedoxidative damage within the oral cavity. They statedthat lower antioxidant concentrations in the GCF con-tributed to increased damage to the gingival andsurrounding structures by activated neutrophils.

Buduneli et al.40 evaluated the possible effects ofsmoking and gingival inflammation on salivary anti-oxidants in gingivitis patients. They reported that nostatistically significant difference was found in anyof the antioxidant indices between any of the groups.Brock et al.27 determined that GCF antioxidant con-centration was significantly lower in periodontitissubjects compared to healthy controls. Althoughthe mean peripheral and total salivary antioxidantcapacities also were lower in periodontitis, the differ-ence was only significant for plasma. Thus, periodon-tal disease has been suggested to be associated withreduced salivary antioxidant status and increasedoxidative damage within the oral cavity.27,39-41

Figure 1.Comparison of serum, saliva, and GCF superoxide dismutase and glutathione peroxidaseactivities; malondialdehyde levels; and total antioxidant capacity among preeclamptic womenwith periodontal disease (solid bars), preeclamptic periodontally healthy women (open bars),normotensive women with periodontal disease (gray bars), and normotensive periodontallyhealthy women (bars with diagonal lines). *Significantly different from preeclamptic periodontallyhealthy women (P <0.05); †significantly different from normotensive women with periodontaldisease (P <0.05); ‡significantly different from normotensive periodontally healthy women(P <0.05).

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The results from the present study show that localand systemic total antioxidant capacity is decreasedsignificantly (for GCF and serum) in pregnant womenwith periodontal disease compared to periodontallyhealthy controls. Furthermore, this decrease was re-flected systemically by lower mean antioxidant ca-pacity in serum obtained from pregnant women withperiodontitis. That alterations in antioxidant capac-ity might be detectable locally and systemically issupported by recent studies indicating that chronicperiodontal disease is associated with peripheral neu-trophils that are hyperreactive with respect to theproduction of reactive oxygen species in responseto Fc gamma-receptor stimulation.42-44

Although some studies45,46 documented an imbal-ance between increased oxidant status and a decreasedantioxidant system in women with preeclampsia, con-tradictory results were reported in other studies.Increased,47,48 decreased,49 or unaltered50,51 gluta-thione peroxidase activity and decreased50 or unal-tered48 superoxide dismutase activity in the serumof pregnant women with preeclampsia have been re-ported. In the present study, malondialdehydelevels were elevated significantly in serum from pre-eclamptic women, whereas superoxide dismutaseand glutathione peroxidase activities were decreased.Superoxide dismutase specifically detoxifies O2

.- rad-icals.52 This association is crucial because glutathi-one peroxidase is the primary antioxidant thatinactivates lipid peroxides and thereby reduces theiractivities. Higher serum and tissue lipid peroxides inthe present study emphasize the fact that additionaloxidative processes take place in preeclamptic preg-nancies.53 These results have been supported byother reports.5,9,14 The decrease in these antioxidantmarkers suggests that this is associated with an im-balance between lipid peroxidation and the antioxi-dant system.53-55 Our findings are consistent withprevious studies14,46,54 suggesting that lipid peroxi-dation is an important factor in the pathogenesis ofpreeclampsia. An insufficiency in antioxidative de-fense mechanisms is another factor that was sug-gested to be responsible for the increase in lipidperoxides in preeclamptic pregnancies.53 Therefore,the low superoxide dismutase activities may be the re-sult of an intracellular attack. However, other factorsmay work in combination with lipid peroxidation tocontribute to such changes.52 In recent years, moreattention has been focused on the role of reactive oxy-gen species, lipid peroxidation products, and antiox-idant systems in the pathology of periodontitis. Recentmedical and dental research in this area is gearedtoward the prevention of free radical–mediated dis-eases by specific nutrient antioxidants.56,57 Elevatedlipid peroxidation and disturbed antioxidant statushave been reported in experimental periodontitis. Tsai

et al.14 found higher lipid peroxidation concentrationsin patients with chronic periodontitis. It was reportedthat the increased GCF lipid peroxidation levels re-flected the increased reactive oxygen species’ dam-age to the periodontal tissue as well as periodontalinflammation. Our results are in agreement with Tsaiet al.’s study14 and indicate that lipid peroxidationconcentration is increased markedly in periodontal in-flammation.

Panjamurthy et al.57 demonstrated that the activi-ties of superoxide dismutase and glutathione peroxi-dase were increased significantly in the plasma,erythrocytes, and gingival tissues in patients with peri-odontitis compared to healthy subjects. In a recentstudy, Wei et al.28 demonstrated that the total amountof glutathione peroxidase is higher in periodontitissites and gingivitis sites than in healthy sites, suggest-ing further investigation is necessary to resolve thisenigma. In contrast, GCF and serum glutathione per-oxidase activities were higher in pregnant women withperiodontal disease relative to periodontally healthypregnant women in our study. This difference maybe due to a number of factors, such as a differentmethodology, inclusion of non-pregnant patients,and sites with periodontal disease and healthy sitesin the same patient. The results of previous studieson superoxide dismutase activity in relation to peri-odontal tissues and GCF are contradictory. Jacoby andDavis58 reported that human periodontal ligamentpossesses superoxide dismutase, which might affordbiologic protection against reactive oxygen species,especially O2

.-, during the inflammatory response.Recently, Akalin et al.29 showed higher superoxidedismutase activity in inflamed gingiva from chronicperiodontitis patients than in healthy gingiva fromcontrols. In the same study, GCF superoxide dismut-ase activity was higher in periodontal health than inchronic periodontitis, but the difference was not statis-tically significant. The investigators did not explainthe negative correlation of superoxide dismutase ac-tivity between gingival tissue and GCF in chronic peri-odontitis patients. Baltacioglu et al.41 reported thatserum and GCF superoxide dismutase activities werelower in the postmenopausal group than in the pre-menopausal group and were lower in the periodontitisgroup than in their matched control group. Our serumand GCF superoxide dismutase activities are consis-tent with the study carried out by the above-mentionedresearchers.29,41

Periodontal disease clearly is a chronic inflamma-tory condition, often underestimated by health profes-sionals and the general population. Based on theresults of the present study and data available fromthe literature, there is increasing evidence that oxida-tive stress (oxidant/antioxidant balance shifts towardthe oxidative state) may be an important contributing

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factor in the pathogenesis of preeclampsia and perio-dontal disease.11,12 However, it is not clear whetherthis oxidative stress is the cause or the result of thesediseases. Therefore, the relationship between thesediseases and oxidative stress is an important areaof study that may provide some explanations for themechanistic links between periodontal disease andpreeclampsia.

CONCLUSIONS

When we consider these data together with the resultsof our study, we hypothesize that periodontal diseasemay be a potential risk factor for the severity, progres-sion, and even the initiation of preeclampsia becauseof reduced antioxidant capacity and/or increased ox-idative stress. However, studies of larger groups andthe analysis of oxidant/antioxidant status are requiredto address this hypothesis. Further study of the rela-tionship between preeclampsia and periodontal dis-ease is needed. A clearer understanding of thisrelationship may assist health care providers in theirefforts to detect both of these diseases earlier. In ad-dition, preventive strategies may be developed thatimpact the prevalence of both diseases. Increased di-alog among medical and dental professionals will beincreasingly important in achieving and maintainingthe optimal health of patients.

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Correspondence: Dr. Varol Canakci, Department of Peri-odontology, School of Dentistry, Ataturk University, 25240Erzurum, Turkey. Fax: 90-442-2360945; e-mail: varol@atauni.edu.tr.

Submitted November 23, 2006; accepted for publicationMarch 1, 2007.

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