Dietary Supplementation of Omega-3Fatty Acid and Circulating Levels ofInterleukin-1b, Osteocalcin, andC-Reactive Protein in RatsSaynur Vardar-Sxengul,* Nurcan Buduneli,* Eralp Buduneli,* Levent Kardesxler,*Haluk Baylas,* Gul Atilla,* David Lappin,† and Denis F. Kinane‡

Background: In this study, we evaluated the effects of two different re-gimes of dietary supplementation of omega-3 fatty acid on serum levels ofinterleukin-1 beta (IL-1b), osteocalcin (OC), and C-reactive protein (CRP) inexperimental periodontitis.

Methods: Experimental periodontitis was induced by repeated injections ofEscherichia coli lipopolysaccharide (LPS). Thirty-nine adult male Sprague-Dawley rats were divided into four study groups as follows: an LPS positive controlgroup; a saline (negative) control group; and two different groups with omega-3fatty acid dietary supplementation, one in which we gave the supplement subse-quent to disease induction (TO3) and the other in which the agent was startedprior to and continued subsequent to LPS injections (P + TO3). In the TO3 group,omega-3 fatty acid administration was performed for 14 days following inductionof experimental periodontitis. In the P + TO3 group, omega-3 fatty acid was givenfor 14 days prior to the start of LPS injections and was continued for another 14days subsequent to the induction of experimental periodontitis. On day 15 ofthe first LPS injection, serum samples were obtained and rats were sacrificed. Se-rum samples were analyzed for IL-1b, OC, and CRP concentrations by enzyme-linked immunosorbent assay. Defleshed jaws were analyzed morphometricallyfor alveolar bone loss. Data were evaluated statistically by non-parametric tests.

Results: LPS injection resulted in statistically significantly more bone losscompared to the saline control group (P <0.05). None of the omega-3 fattyacid administration groups showed evidence that this fatty acid was effective inpreventing LPS-induced alveolar bone loss. TO3 and P + TO3 groups revealedsignificantly higher IL-1b and OC levels than the LPS group (P <0.05). The studygroups exhibited no significant differences in the serum CRP levels.

Conclusions: Omega-3 fatty acid administration does not seem to influencecirculating levels of CRP. The significantly increased serum OC level observedin both omega-3 fatty acid regimes is curious and could have an effect on boneturnover, as could the further significant increase in serum IL-1b, which couldcounteract any osteoblastic induction by OC through promotion of osteoclast ac-tivity. The lack of a therapeutic benefit of omega-3 fatty acid in this study, despitethe effects on OC and IL-1b, is difficult to explain, and further studies are requiredto more fully assess the potential role of this fatty acid in periodontal treatment.J Periodontol 2006;77:814-820.

KEY WORDS

C-reactive protein; interleukin-1 beta; omega-3 fatty acid;osteocalcin; rats; serum.

Positive effects foromega-3 fatty acidson bone formation

have been reported inanimal models of variousbone diseases1,2 and alsohuman studies of senileosteoporosis.3 Fish oilis the main source ofomega-3 fatty acids,and the major poly-unsaturated fatty acidcomponents are eicosa-pentaenoic acid (EPA)and docosahexaenoicacid (DHA). EPA andDHA compete with ara-chidonic acid (AA) forthe lipoxygenase (LOX)and cyclooxygenase(COX) pathways, whichresults in the reducedsynthesis of highly activeproinflammatory metab-olites like prostaglandinE2 (PGE2) and leukotri-ene B4 (LTB4).4,5 Lipo-polysaccharide (LPS), amajor virulence factorof Gram-negative peri-odontopathogens, inducesthe release of AA metab-olites via COX and LOXpathways.The requirement

* Department of Periodontology, School of Dentistry, Ege University, _IIzmir, Turkey.† Infection and Immunity Group, Glasgow Dental School, University of Glasgow, Glasgow, U.K.‡ Department of Periodontology, University of Louisville, School of Dentistry, Louisville, KY. doi: 10.1902/jop.2006.050214

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of LOX inhibition has introduced the administrationof polyunsaturated fatty acids in periodontal treat-ment, and omega-3 fatty acid supplementation hasbeen suggested to be beneficial in the treatment ofperiodontal inflammation,6 but the exact molecularmechanisms of these beneficial effects have notbeen elucidated.

Osteocalcin (OC) is a calcium-binding protein ofbone and the most abundant non-collagenous pro-tein of the mineralized tissue.7 The serum level ofOC is considered a marker of bone formation.8-10

Increased serum concentrations of OC have beenproposed to indicate an increase in bone turnover.3

Periodontitis patients have been reported to havelower serum levels of OC than healthy subjects, sug-gesting lower osteoblastic activity and bone forma-tion ability.11

The acute-phase response to injuries or infectionsis initiated by the activation of local macrophagesand other cells leading to the release of mediators liketumor necrosis factor-alpha (TNF-a), interleukin-1beta (IL-1b), and IL-6. In turn, these cause systemicchanges including hepatic release of a range ofplasma proteins like C-reactive protein (CRP).12

CRP, IL-1, IL-6, and TNF-a have been reported tobe associated with various bacterial infections includ-ing periodontitis,13 as patients with periodontitis mayhave elevated circulating levels of these inflammatorymarkers.14-16

Eberhard et al.17 have reported in a human ex-perimental gingivitis study that local application ofomega-3 fatty acid neither improved the patients’clinical condition nor decreased the GCF concentra-tion of LTB4. Our recent study evaluating the effectsof therapeutic versus prophylactic plus therapeuticadministration of omega-3 fatty acid on gingival tis-sue levels of major AA components in rats revealedthat therapeutic omega-3 fatty acid significantlyreduced the local levels of PGE2, PGF2a, LTB4, andplatelet activating factor (PAF).18 Furthermore, pro-phylactic usage provided additional beneficial effectsto the therapeutic administration by decreasing thegingival tissue levels of these mediators to the levelsof healthy tissue. However, to our knowledge, theeffects of omega-3 fatty acid on circulating inflam-matory mediators in periodontal therapy have notbeen investigated before. Our hypothesis was thatomega-3 fatty acid administration may affect cir-culating levels of inflammatory mediators, therebypositively contributing to the control of inflammationand ultimately to the periodontal treatment. The pur-pose of the present study was two-fold: 1) to evaluatethe possible effects of systemic administration ofomega-3 fatty acid on serum levels of IL-1b, OC,and CRP in endotoxin-induced periodontitis in rats;and 2) to investigate whether starting omega-3 fatty

acid administration prior to induction of periodontitisprovides any additional benefits.

MATERIALS AND METHODS

Study DesignThe experimental protocol of the present study wasapproved by the Ethics Committee on animal exper-imentation at Ege University. Thirty-nine adult maleSprague-Dawley rats (203 – 26.9 g) were purchasedfrom the university’s own facilities, housed in tem-perature-controlled rooms, and given water and foodad libitum.

Induction of experimental periodontitis was pur-sued by injection of endotoxin according to the modeldescribed by Ramamurthy et al.19 The rats were in-jected with either 10 ml saline or Escherichia coliendotoxin§ (serotype 055:B5, L2637) (1 mg/ml) intothe palatal gingiva between the first and secondmaxillary molars and into the labial and oral gingivabetween the central incisors in the mandible andmaxilla every other day on 3 separate days. The firstinjection with endotoxin/saline by an insulin syringecarrying a 0.30 · 13-mm needle was performed 24hours before initiation of drug treatment, namely atbaseline.

The rats were distributed into four experimentalgroups as follows: 1) saline controls (N = 12): gingivawere injected with saline, and each rat was orally gav-aged daily with saline solution for 14 days; 2) endo-toxin (LPS) (N = 9): gingiva were injected with LPS,and each rat was orally gavaged with saline solutiondaily for 14 days; 3) treatment with omega-3 acid(TO3) (N = 10): gingiva were injected with LPS, andeach rat was orally gavaged with omega-3 fatty acid(40 mg/kg; 60% EPA and 40% DHA) daily for 14 days;and 4) pretreatment plus treatment with omega-3fatty acid (P + TO3) (N = 8): daily oral gavage withomega-3 fatty acid (40 mg/kg) was started 14 daysbefore baseline, and then LPS injection into the gin-giva was started, and omega-3 fatty acid gavagewas continued for another 14 days after baseline.

On day 15 after baseline, all rats were anesthetized,blood samples were obtained, and the animals weresacrificed by decapitation under ether-induced anes-thesia. The blood samples were centrifuged for 10minutes at ·500 g, separating the serum from thecells. The serum samples were immediately frozenat -40�C. Then, the samples were lyophilized andstored at -20�C until subsequent analysis of theinflammatory mediators (IL-1b, OC, and CRP) byenzyme-linked immunosorbent assay (ELISA). Thewhole heads were removed and frozen for subsequentmorphometric evaluations.

§ Sigma, St. Louis, MO.

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Measurement of Alveolar Bone LossThe heads were boiled for 10 minutes, soft tissueswere removed manually, and the upper and lowerjaws were defleshed and cleaned. The skulls weresoaked in 0.2N NaOH at room temperature for 5 min-utes to remove the remaining soft tissue debris. Thejaws were washed, air dried, and stained with aqueousmethylene blue (1%) to identify the cemento-enameljunction (CEJ). The alveolar bone height was mea-sured at 22 different sites on the palatal aspect inthe left and right maxillary molar regions under a ste-reomicroscope (·40 magnification) by recording thedistance from the CEJ to the alveolar bone crest(Fig. 1). All measurements were made along the longaxis of the roots and perpendicular to the occlusalplane of the maxilla. The mean of the sums of the22 measurements from the left and right sides of themaxilla was used as a measure of alveolar bone lossin each animal. The morphometric measurementsof alveolar bone loss were performed by a single ex-aminer, who was blinded to the study groups, usinga scale adapted to the stereomicroscope.

Laboratory AssaysELISA. Levels of IL-1b, OC, and CRP in serum sam-ples were analyzed by ELISA. Specific ELISA kits wereused for quantitative analysis of IL-1b,i OC,¶ andCRP.# The minimum detection limits were 3 pg/ml,0.5 ng/ml, and 2.5 ng/ml for IL-1b, OC, and CRP, re-spectively. The lyophilized serum samples were ali-quoted and stored at -70�C until further laboratoryprocedures were performed. The serum samples werediluted 1/2, 1/10, and 1/4,000, respectively, for theIL-1b, OC, and CRP assays. The ELISA assays werecarried out according to the manufacturers’ recom-mendations, and 96-well plates precoated with appro-priate antibodies were used. Samples and thestandards were added to duplicate wells and incu-

bated for 3 hours (at room temperature), 18 hours(at 2�C to 8�C), and 30 minutes (at room tempera-ture), respectively, for IL-1b, OC, and CRP assays.The plates were washed, detection antibody wasadded, and the plates were incubated for 60 minutesat room temperature. After washing, conjugated anti-body was added and incubated for 30 minutes atroom temperature. The plates were washed again,and chromogen solution was added and incubatedfor 30 minutes at room temperature. Later, the sub-strate was added and incubated to develop colorchange. Finally, the optical densities were read, andthe samples were compared to the standards. The re-sults were expressed as picograms per milliliter forIL-1b and OC and milligrams per liter for CRP concen-trations.

Statistical AnalysisStatistical analysis was performed using non-parametric tests. Comparisons between the four studygroups were performed using the Kruskal-Wallis test.In case of significant differences, post-hoc two-groupcomparisons were made with the x2 test. P values<0.05 were considered statistically significant. Spear-man rank correlations were used to examine therelationships between the serum levels of various in-flammatory mediators and the amount of alveolarbone loss.

RESULTS

Alveolar Bone LossMeasurement of alveolar bone loss in the maxillarymolar teeth revealed significantly higher bone lossvalues in the LPS, TO3, and P + TO3 groups comparedto the saline control group (P <0.05) (Fig. 2). The al-veolar bone loss in the TO3 and P + TO3 groups wereless than the LPS group, but the differences were notstatistically significant (P >0.05) (Table 1).

IL-1b Levels in SerumThe serum IL-1b levels were very close to or below theminimum detection limit of the assay kit in the salinecontrol and LPS groups. The median levels were 47.0pg/ml and 60.3 pg/ml, respectively, for the TO3 andP + TO3 groups, and these values were significantlyhigher than that of the LPS group (P <0.05) (Fig. 3).

OC Levels in SerumThe lowest serum OC levels were obtained in the LPSgroup, whereas the highest levels were found inthe TO3 group (Fig. 4). Both omega-3 fatty acidtreatment groups exhibited significantly higher serumOC levels compared to the saline control and LPSgroups (P <0.01). No significant differences were

Figure 1.Staining of the CEJ with aqueous methylene blue (1%). Note thepoints of measurement of alveolar bone loss.

i Biosource International, Camarillo, CA.¶ Biomedical Technologies, Stoughton, MA.# Helica Biosystems, Fullerton, CA.

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detected between the two omega-3 fatty acidtreatment groups (P >0.05).

CRP Levels in SerumThe LPS group showed the lowest levels of CRP, but nosignificant differences were found between any of thestudy groups (Fig. 5).

Spearman correlation analysis revealed no signifi-cant correlations between serum levels of the ana-lyzed mediators or between alveolar bone loss andserum levels of the mediators (P >0.05).

DISCUSSION

The present study provides further evidence that E.coli LPS injection into the gingiva of rats caused a sig-nificant amount of alveolar bone loss similar to thevalues reported by Ramamurthy et al.,19 indicatingthe predictability of the experimental periodontitismodel.

Fish oil is a main source of omega-3 fatty acids, andEPA and DHA are the major polyunsaturated fattyacid components of omega-3 fatty acid. These unsat-urated fatty acids compete with AA for the COX andLOX pathways resulting in the production of series-3prostaglandins (PG3) and series-5 leukotrienes(LT5). Through this action, omega-3 fatty acid exertsanti-inflammatory effects and reaches its maximum

Table 1.

Morphometric Data in the Study Groups

Group Alveolar Bone Loss* (mm)

Saline control (N = 12) 0.28 – 0.007†

LPS (N = 9) 0.45 – 0.060

Treatment with omega-3 fattyacid (N = 10)

0.39 – 0.028

Pretreatment plus treatmentwith omega-3 fatty acid (N = 8)

0.38 – 0.013

* All values are mean – SD.† Significantly lower than the other study groups (P <0.05).

Figure 3.Serum levels of IL-1b in the study groups. Dot plots show medians asthick bars and the first and third quartiles as thin bars. Outliers arealso shown but not marked. * Significantly higher than the LPS group(P <0.05).

Figure 2.Severe alveolar bone loss observed in the LPS group.

Figure 4.Serum levels of OC in the study groups. Dot plots show medians asthick bars and the first and third quartiles as thin bars. Outliers arealso shown but not marked. * Significantly higher than the salinecontrol and the LPS groups (P <0.01).

Figure 5.Serum levels of CRP in the study groups. Dot plots show medians asthick bars and the first and third quartiles as thin bars. Outliers arealso shown but not marked. There was no statistically significantdifference between the study groups.

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membrane concentration on day 14.20 Therefore, inthe TO3 group of the present study, we started admin-istration of omega-3 fatty acid on the day following thefirst LPS injection and continued for 14 days, and inthe P + TO3 group, it was started 14 days beforeand continued for another 14 days after the first LPSinjection.

Alam et al.21 have stated that rats fed with a dietrich in omega-3 fatty acid exhibited reductions ingingival tissue levels of AA, PGE2, and LTC4. Fur-thermore, Campan et al.22 have demonstrated in ahuman experimental gingivitis study that the gingivaltissue levels of AA, PGE2, and LTB4 were reduced inthe systemically administered omega-3 fatty acidgroup, whereas they were increased in the placebogroup. No adverse effects have been reported sofar by omega-3 fatty acid application. The anti-inflammatory properties, together with the good toler-ance of omega-3 fatty acids, have justified their use intreatment of periodontal diseases. However, the ef-fects of fatty acids on bone metabolism are unclear.Recently, we demonstrated significant decreasesin gingival tissue levels of PGE2, PGF2a, LTB4, andPAF.18 Our present data suggest that omega-3 fattyacid could influence bone metabolism by significantlyincreasing the serum OC levels compared to thesaline control and LPS groups.

In a pilot study on senile osteoporosis, Krugeret al.3 found significantly lower levels of serum OCin women taking EPA capsules than in controls,which indicated a decrease in bone turnover and sug-gested that EPA has beneficial effects on bone and itis safe to administer for prolonged periods of time. Onthe other hand, Kelly et al.23 demonstrated that nei-ther omega-3 nor omega-6 fatty acids affected circu-lating levels of OC in rats fed with special diets for 8weeks. They concluded that an omega-3 fatty acid–rich diet appeared to enhance calcium absorptionbut had no measurable effect on bone metabolismor bone mass over this time frame. Accordingly, ourpresent findings indicated an increase in serum OClevels signifying an increase in bone turnover, butmorphometric measurements of alveolar bone lossrevealed no significant difference between studygroups. This finding may be explained by the signifi-cant increases in the serum IL-1b levels antagonizingthe induction of OC synthesis.24 It is also likely thatlonger periods of omega-3 fatty acid administrationmay provide clinically detectable effects related withsignificant increases in serum OC level.

Effects of local applicationofomega-3 and omega-6fatty acids were also investigated in a human ex-perimental gingivitis study,17 in which mouthrinsescontaining either omega-3 or omega-6 fatty acid wereused in a double-blind, two-group parallel design.The researchers found that both mouthrinses failed

to improve the patients’ clinical condition during a21-day non-brushing period.

The lack of any increase in the serum IL-1b concen-tration in the LPS group might be explained if the effectof E. coli LPS were localized to the gingiva rather thanbeing reflected in circulation, or if the serum levels ofthis proinflammatory cytokine had subsided after theinitial insult, and the animals (with regards to IL-1b)were no longer responsive to further LPS treatment.Considering the significantly elevated serum IL-1b

levels in omega-3 fatty acid–treated groups, itmay be speculated that the mechanisms by whichomega-3 fatty acid leads to increases in serum levelsof IL-1b, an important cytokine acting in periodontaltissue destruction and OC, a marker of bone forma-tion, are diverse.

From a clinical study in chronic periodontitis pa-tients, Ide et al.25 suggested that an improvement inperiodontal health need not influence the levels ofcirculating inflammatory markers. Similarly, findingsof the present experimental study revealed no signif-icant differences between the study groups in serumCRP levels. Indeed, systemic administration of omega-3fatty acid may modulate local factors rather than thecirculating mediators of bone metabolism. Thus, werecently demonstrated significant inhibitory effectsof omega-3 fatty acid on gingival tissue AA metabo-lites,18 whereas the present study failed to show sig-nificant effects on the circulating levels of CRP andIL-1b, suggesting that local effects are more likelyto occur and should be looked for in periodontaltreatment.

The time period of omega-3 fatty acid or LPS ad-ministration may provide an additional explanationfor the lack of significant differences between thestudy groups in terms of circulating levels of CRP. Inour study, omega-3 fatty acids were used in theTO3 group for a 14-day period only and for 28 daysin the P + TO3 group, and it may be speculated thatadministration of omega-3 fatty acid for longer pe-riods and with different doses may create significanteffects in terms of bone support. However, Moriet al.26 reported that supplementation with purifiedEPA or DHA for 6 weeks in non-smoking diabetic sub-jects resulted in no significant change in serum CRPlevels compared to placebo controls. Madsen et al.27

administered omega-3 fatty acid at two differentdoses (6.6 or 2.0 g daily) in healthy volunteers for12 weeks. According to the findings of their placebo-controlled study, serum CRP concentrations were un-affected by the polyunsaturated fatty acid–containingsupplements. Recently, Geelen et al.28 evaluated theeffects of omega-3 fatty acid in a placebo-controlled,double-blind study in 84 middle-aged subjects. Thesubjects were given capsules of omega-3 fatty acidor placebo for 12 weeks, and the results indicated

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no significant difference in serum CRP concentrations.Further dose-response studies may clarify the optimaldose and administration period of these agents tohave positive effects on bone metabolism and/or sys-temic inflammation markers.

CONCLUSIONS

Thesignificantly increasedserumOClevelobserved inboth of the omega-3 fatty acid administration groupsmay indicate that systemic administration of omega-3 fatty acid increases bone remodeling and thereby in-hibits the progression of alveolar bone resorption.However, the failure to observe any significant changein bone loss levels in omega-3 fatty acid–treated ratsmay be related to the significant increase in serumIL-1b promoting osteoclast activity. These findingsshould be verified by longitudinal clinical human stud-ies investigating clinical and biochemical effects ofomega-3 fatty acids in non-surgical and surgical peri-odontal treatment. In addition, measurements of IL-1b

andothermediators involved inboneremodeling in theGCF and plasma/serum of subjects should also beconsidered to monitor their possible influences on theoutcome.

ACKNOWLEDGMENT

This study was partially supported by grant 2002 Disx015 from the Research Fund, Ege University, Turkey.

REFERENCES1. Li Y, Seifert MF, Ney DM, et al. Dietary conjugated

linoleic acids alter serum IGF-1 and IGF binding pro-tein concentrations and reduce bone formation in ratsfed (n-6) or (n-3) fatty acids. J Bone Miner Res 1999;14:1153-1162.

2. Watkins BA, Li Y, Allen KG, Hoffmann WE, Seifert MF.Dietary ratio of (n-6)/(n-3) polyunsaturated fatty acidsalters the fatty acid composition of bone compart-ments and biomarkers of bone formation in rats. J Nutr2000;130:2274-2284.

3. Kruger MC, Coetzer H, de Winter R, Gericke G, vanPapendorp DH. Calcium, gamma-linolenic acid andeicosapentaenoic acid supplementation in senile os-teoporosis. Aging 1998;10:385-394.

4. Kragballe K, Voorhees JJ, Goetzl E. Inhibition byleukotriene B5 of leukotriene B4-induced activationof human keratinocytes and neutrophils. J InvestDermatol 1987;88:555-558.

5. Kragballe K, Fogh K. A low-fat diet supplemented withdietary fish oil results in the improvement of psoriasisand in formation of leukotriene B5. Acta DermatolVenereol 1989;69:23-28.

6. Rosenstein ED, Kushner LJ, Kramer N, Kazandjian G.Pilot study of dietary fatty acid supplementation inthe treatment of adult periodontitis. ProstaglandinsLeukot Essent Fatty Acids 2003;68:213-218.

7. Lian JB, Gundberg CM. Osteocalcin: Biochemicalconsiderations and clinical applications. Clin OrthopRelat Res 1988;226:267-291.

8. Christenson RH. Biochemical markers of bone metab-olism: An overview. Clin Biochem 1997;30:573-593.

9. Giannobile WV. Crevicular fluid biomarkers of oralbone loss. Curr Opin Periodontol 1997;4:11-20.

10. Shibutani T, Inuduka A, Horiki I, Luan Q, Iwayama Y.Bisphosphonate inhibits alveolar bone resorption inexperimentally-induced peri-implantitis in dogs. ClinOral Implants Res 2001;12:109-114.

11. Shi F, Yu S, Xu L. Analysis of serum osteocalcin ofpatients with periodontitis. Zhonghua Kou Qiang YiXue Za Zhi 1996;31:300-302.

12. Ebersole JL, Capelli D. Acute-phase reactants in in-fections and inflammatory diseases. Periodontol 20002000;23:19-49.

13. Ebersole JL, Machen RL, Steffen MJ, Willmann DE.Systemic acute-phase reactants, C-reactive proteinand haptoglobin, in adult periodontitis. Clin ExpImmunol 1997;107:347-352.

14. Loos BG, Craandijk J, Hoek FJ, Wertheim-van DillenPM, van der Velden U. Elevation of systemic markersrelated to cardiovascular diseases in the peripheralblood of periodontitis patients. J Periodontol 2000;71:1528-1534.

15. Wu T, Trevisan M, Genco RJ, Dorn JP, Falkner KL,Sempos CT. Periodontal disease and risk of cerebro-vascular disease. Arch Intern Med 2000;160:2749-2755.

16. Noack B, Genco RJ, Trevisan M, Grossi S, Zambon JJ,De Nardin E. Periodontal infections contribute toelevated systemic C-reactive protein level. J Peri-odontol 2001;72:1221-1227.

17. Eberhard J, Heilmann F, Acxil Y, Albers HK, Jepsen S.Local application of n-3 or n-6 polyunsaturated fattyacids in the treatment of human experimental gingivi-tis. J Clin Periodontol 2002;29:364-369.

18. Vardar S, Buduneli E, Turkoglu O, et al. Therapeuticversus prophylactic plus therapeutic administration ofomega-3 fatty acid on endotoxin-induced periodontitisin rats. J Periodontol 2004;75:1640-1646.

19. Ramamurthy NS, Golub LM, Gwinnett AJ, Salo T, DingY, Sorsa T. In vivo and in vitro inhibition of metal-loproteinases including MMP-13 by several chemicallymodified tetracyclines (CMTs). In: Davidovitch D, MahJ, eds. Biologic Mechanisms of Tooth Eruption, Re-sorption and Replacement by Implants. Boston: Har-vard Society for the Advancement of Orthodontics;1998:271-277.

20. Croft KD, Sturm MJ, Codde JP, Vandongen R, BeilinLJ. Dietary fish oils reduce plasma levels of plateletactivating factor precursor (lyso-PAF) in rats. Life Sci1986;38:1875-1882.

21. Alam SQ, Bergens BM, Alam BS. Arachidonicacid, prostaglandin E2 and leukotriene C4 levels ingingiva and submandibular salivary glands of ratsfed diets containing n-3 fatty acids. Lipids 1991;26:895-900.

22. Campan P, Planchand P-O, Duran D. Pilot study onn-3 polyunsaturated fatty acids in the treatment ofhuman experimental gingivitis. J Clin Periodontol1997;24:907-913.

23. Kelly O, Cusack S, Jewell C, Cashman KD. The effectof polyunsaturated fatty acids, including conjugatedlinoleic acid, on calcium absorption and bone metab-olism and composition in young growing rats. Br JNutr 2003;90:743-750.

24. Kim CH, Kang BS, Lee TK, et al. IL-1beta regulatescellular proliferation, prostaglandin E2 synthesis, plas-minogen activator activity, osteocalcin production,and bone resorptive activity of the mouse calvarial

J Periodontol • May 2006 Vardar-Sxengul, Buduneli, Buduneli, et al.

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bone cells. Immunopharmacol Immunotoxicol 2002;24:395-407.

25. Ide M, McPartlin D, Coward PY, Crook M, Lumb P,Wilson RF. Effect of treatment of chronic periodontitison levels of serum markers of acute-phase inflamma-tory and vascular responses. J Clin Periodontol 2003;30:334-340.

26. Mori TA, Woodman RJ, Burke V, Puddey IB, Croft KD,Beilin LJ. Effect of eicosapentaenoic acid and doco-sahexaenoic acid on oxidative stress and inflamma-tory markers in treated-hypertensive type 2 diabeticsubjects. Free Radic Biol Med 2003;35:772-781.

27. Madsen T, Christensen JH, Blom M, Schmidt EB. Theeffect of dietary n-3 fatty acids on serum concentra-

tions of C-reactive protein: A dose-response study.Br J Nutr 2003;89:517-522.

28. Geelen A, Brouwer IA, Schouten EG, Kluft C, KatanMB, Zock PL. Intake of n-3 fatty acids from fish doesnot lower serum concentrations of C-reactive proteinin healthy subjects. Eur J Clin Nutr 2004;58:1440-1442.

Correspondence: Dr. Nurcan Buduneli, Department ofPeriodontology, School of Dentistry, Ege University, 35100Bornova, _IIzmir, Turkey. Fax: 90-232-388-03-25; e-mail:nurcan.buduneli@ege.edu.tr.

Accepted for publication November 14, 2005.

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