ABSTRACT
Per iod on t al d isease an d in f lam m at or y b lood
cyt okines in pat ient s w it h st able coronary art ery
disease
Cassio KAMPITS1, Marlon M. MONTENEGRO1, Ingrid W. J. RIBEIRO10DULDQD9)857$'22&DULVL$32/$1&=<.2, Cassiano K. RÖSING1, Alex. N HAAS1
1- Universidade Federal do Rio Grande do Sul, Faculdade de Odontologia, Departamento de Periodontia, Porto Alegre, RS, Brasil. 2- Universidade Federal do Rio Grande do Sul, Faculdade de Medicina, Divisão de Cardiologia, Porto Alegre, RS, Brasil.
Corresponding address: Cassio Kampits - Ramiro Barcelos, 2492 - Porto Alegre - RS - Brazil - 90035-003 - Phone/fax: +55 51 97629030 - e-mail: cassiokampits@gmail.com
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eriodont al disease has been associat ed w it h elevat ions of blood cyt okines involved in at herosclerosis in syst em ically healt hy individuals, but lit t le is know n about t his associat ion in st able cardiovascular pat ient s. The aim of t his st udy was t o assess t he associat ion bet w een periodont al disease ( exposure) and blood cyt okine levels ( out com es) in a t arget populat ion of pat ient s w it h st able coronary art ery disease ( CAD) . Mat erial and Met hods: This cross- sect ional st udy included 91 pat ient s w it h st able CAD w ho had beenXQGHURSWLPL]HGFDUGLRYDVFXODUFDUH%ORRGOHYHOVRI,/ǃ,/,/,/,)1DŽDQG
TNF-D w ere m easured by Lum inex t echnology. A full- m out h periodont al exam inat ion was conduct ed t o record probing dept h ( PD) and clinical at t achm ent ( CA) loss. Mult iple linear r egr ession m odels, adj ust ing for gender, body m ass index , oral hy poglycem ic dr ugs, sm oking, and occurrence of acut e m yocardial infarct ion were applied. Result s: CAD pat ient s
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vs. 3.01 pg/ m L; p= 0.01) , I L- 10 ( m edian: 2.33 pg/ m L vs. 1.01 pg/ m L; p= 0.03) , and TNF-D ( m edian: 9.17 pg/ m L vs. 7.47 pg/ m L; p= 0.02) . Higher num bers of t eet h w it h at least 6
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I L- 10 concent rat ions. Elevat ed concent rat ions of TNF-D w ere associat ed w it h higher m ean CA loss ( R2= 0.07) . Conclusion: Periodont al disease is associat ed w it h increased syst em ic
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support ing t he idea t hat periodont al disease can be a prognost ic fact or in cardiovascular pat ient s.
Keyw ords: Cyt okines. Car diovascular diseases. Per iodont al diseases. At her oscler osis. I nt erleukins.
I N TRODUCTI ON
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in it iat ion an d dev elopm en t of at h er oscler osis1 9. At her oscler ot ic lesions ar e com pr ised of var ious
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cyt okines, which part icipat e in t he plaque form at ion cascade in vessel walls12 6SHFL¿FDOO\ LQWHUOHXNLQ ( I L) - 6, t um or necrosis fact or ( TNF) - a, and int erferon
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blood from cardiovascular pat ient s22,29, and t he I
L-10 concent rat ion is increased in unst able com pared wit h st able at herom as18. Moreover, I L- 6 and TNF- a have direct effect s on t he product ion and release of C- react ive prot ein ( CRP)20 and, consequent ly, have been used as pr edict or s of fut ur e car diovascular event s in cardiac pat ient s11.
be expressed using various clinical param et ers and
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fact or s and sy st em ic condit ions26. Pr obing dept h and clinical at t achm ent loss are t he m ost frequent
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healt h/ disease st at us, and t hey m ay be expressed as individual averages or by t he num ber of t eet h affect ed per individual.
One of the possible m echanism linking periodontal diseases t o cardiovascular diseases ( CVD) is t he
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per iodon t al et iopat h ogen ic pr ocess1 4 , 2 2. St u dies have dem onst rat ed t hat periodont it is, which is t he dest ruct ive form of periodont al disease, increases sy st em ic lev els of som e of t h e afor em en t ion ed CVD- relat ed cyt okines3,15. Most evidence support ing t his claim has been provided by st udies conduct ed i n ot h er w i se sy st em i cal l y h eal t h y i n d i v i d u al s, su g g est in g t h at p er iod on t al d isease m ay b e a r isk f act or f or CVD. On t h e ot h er h an d , t h er e is lit t le in f or m at ion abou t t h e sy st em ic ef f ect s of p er iod on t al d isease on p at ien t s w it h st ab le CVD, wit h few st udies evaluat ing t he associat ion bet ween periodont al disease and a sm all num ber of
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pat ient s2,8,10,17,28.
To t he best of t he aut hors’ knowledge, no st udy has evaluat ed t he associat ion bet ween periodont al param et ers and blood levels of a broad range of cy t ok ines, assessed by Mult iplex t echnology, in cardiovascular pat ient s. Such st udies m ay indicat e
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a n d d e st r u ct i o n i n t h e o ccu r r e n ce o f f u t u r e cardiovascular event s on t hese pat ient s. Therefore, t he aim of t his st udy was t o assess t he associat ions bet w een per iodont al disease and blood cy t ok ine levels in pat ient s wit h st able CAD who were under st andard cardiovascular care.
MATERI AL AN D METHODS
This cr oss- sect ional obser vat ional st udy w as conduct ed wit h st able CAD pat ient s who had been receiving cardiovascular care for at least six m ont hs in a t ert iary care cardiovascular clinic at a universit y hospit al in Brazil. To be included in t he st udy, all
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occurrence of at least one of t he following event s six m ont hs before ent ering t he st udy: docum ent ed h i st o r y o f m y o ca r d i a l i n f a r ct i o n ( MI ) , st a b l e angina, or ischem ia in noninvasive t est s; surgical or percut aneous m yocardial revascularizat ion and lesion size great er t han 50% in at least one m aj or cor on ar y ar t er y, as assessed b y an g iog r ap h y ; an d pr esen ce of an gin a an d posit iv e r esu lt s of noninvasive t est ing of ischem ia. Addit ional inclusion crit eria included t he presence of at least four t eet h, no periodont al t reat m ent , and no use of ant ibiot ics
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t he st udy.
Overall, 239 consecut ive pat ient s were evaluat ed fr om Apr il t o Decem ber 2011. Am ong t hem , 78 ( 32.6% ) individuals had less t han four t eet h, 30 ( 1 2 . 6 % ) r ef u sed p ar t icip at ion , an d 1 7 ( 7 . 1 % ) lived out side t he cit y and w ere not available for clin ical ex am in at ion . Mor eov er, 1 6 ( 6 . 7 % ) d id not at t end t he clinical exam inat ion appoint m ent . Clinical exam inat ions were perform ed in 98 ( 41.0% ) i n d i v i d u a l s; h o w ev er, sev en d i d n o t u n d er g o
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present invest igat ion com prised 91 individuals. Th e st u d y p r o t o co l w a s a p p r o v e d b y t h e inst it ut ional review boards of t he Universit y Hospit al and t he Federal Universit y of Rio Grande do Sul. All part icipant s read and signed an inform ed consent form before ent ering t he st udy.
Cardiovascular care
All pat ient s received cardiovascular t ert iary care including m edicat ion and counseling. Regar ding drug prescript ion, t he prot ocol of t he cardiovascular care in t his clinic includes st at ins for t he m aj orit y of patients. When appropriate, oral hypoglycem ic drugs, insulin, acet ylsalicylic acid, and ant ihypert ensives were also prescribed. Counseling m ainly includes d aily ex er cise, sm ok in g cessat ion , an d d iet ar y t herapy. Most pat ient s in t his sam ple were using st at in s ( 9 2 . 3 % ) , acet y lsalicy lic acid ( 9 0 . 1 % ) , and ant ihypert ensive drugs ( 93.4% ) . Only 13.1% and 9.9% were using insulin and ant idepressives, respect ively.
Dat a collect ion
We applied a st ruct ured quest ionnaire t o collect dat a about age, gender, and sm oking exposure. We obt ained m edical hist ory, m edicat ion use, weight , and height from t he hospit al records of t he pat ient s.
Clinical periodont al param et ers were assessed wit h a m anual periodont al probe ( William s probe, Newm ar, São Paulo, SP, Brazil) by t wo calibrat ed exam iners ( weighted kappa values ranging from 0.80 and 0.90) who were unaware of t he cardiovascular
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Visible plaque ( VP) , gingival recession ( GR) , probing dept h ( PD) , and bleeding on probing ( BOP) were recorded at six sit es per t oot h in all present t eet h, excluding t hird m olars. Clinical at t achm ent ( CA) loss was obt ained as t he sum of GR and PD.
Blood sam ples
for analy sis. Plasm a fr om t he r em aining 10 m L
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Sam ples were st ored for t wo years on average.
Cardiovascular st at us
Th e ca r d i o v a scu l a r st a t u s o f p a t i en t s w a s assessed by ident ifying know n blood m ar ker s of risk for cardiovascular event s. High- sensit ivit y CRP, glucose, glycat ed hem oglobin, t riglycerides ( TG) , t ot al cholest erol ( TC) , and high- densit y lipoprot ein cholest erol ( HDL- C) were m easured as previously d escr ib ed8. Low - d en sit y lip op r ot ein ch olest er ol ( LDL- C) was calculat ed by t he Friedwald form ula.
Par t icipan t s w er e f u r t h er div ided in t o t h ose h av in g m ediu m - an d lon g- t er m past h ist or y of m aj or cardiovascular event s using a com binat ion of inform at ion about t he occurrence of cardiovascular event s and t im e elapsed since t he event . Pat ient s t hat have experienced acut e m yocardial infarct ion or m y ocar dial r ev ascu lar izat ion ( an gioplast y or surgery) in t he last nine years were included in t he high st at us group, whereas if t he event s occurred bef or e 1 0 y ear s or m or e, t h e in div idu als w er e
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Syst em ic cyt okines
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m et hodology in com binat ion wit h t he 3.1 Xponent soft ware package ( Lum inex Corp., Aust in, TX, USA ) . The MI LLI PLEX MAP Hum an Cyt okine/ Chem okine Magnet ic Bead Panel- 07 kit ( HCYTOMAG- 60k, EMD Millipore, Saint Charles, USA) was used following t he m anufact urer ’s inst ruct ions. Result s were expressed as st andard curve unit s in pg/ m L. All sam ples were an aly zed at t h e sam e t im e u n der st an dar dized experim ent al condit ions.
I n sum m ary, before st art ing t he im m unoassay, sa m p l e s w e r e co m p l e t e l y t h a w e d , m i x e d b y vort exing, and cent rifuged at 1000g for 10 m inut es t o r em ov e p ar t i cu l at es. Al l k i t r eag en t s w er e
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of wash buffer was added t o each of t he 96 wells in t he plat e, which was t hen sealed, m ixed on a plat e shaker for 10 m inut es at room t em perat ure, invert ed, and t apped several t im es ont o absorbent t ow els t o decant and r em ove t he r esidual wash buffer. The st andard curve and cont rol wells received
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Assay buffer alone was used as t he background ( 0
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Each ant ibody- bead vial was sonicat ed for 30
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each ant ibody- bead vial were added t o t he m ixing
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t o each well. The plat e was sealed and incubat ed overnight ( 18 hours) at 4° C wit h agit at ion on a plat e shaker. Well cont ent s were gent ly rem oved, and t he
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aliquot of det ect ion ant ibodies was added t o each well and incubat ed for 1 hour at room t em perat ure wit h agit at ion on a plat e shaker. Wit hout furt her
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added t o each w ell, f ollow ed by in cu bat ion f or anot her 30 m inut es wit h agit at ion on a plat e shaker. Wash in g w it h w ash bu f f er w as r epeat ed t w ice.
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The beads were re- suspended on a plat e shaker for 5 m inut es, and t he plat e was run on Lum inex. The
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calculat ing cyt okine concent rat ions in t he sam ples.
St at ist ical analysis
Th e o u t co m e s o f t h e p r e se n t st u d y w e r e
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Num bers of t eet h wit h PD and CA loss of 6 m m or
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as having severe periodont it is in t he presence of t wo or m ore int erproxim al sit es wit h CA loss of 6 m m or great er and at least one int erproxim al sit e wit h PD of 5 m m or great er in nonadj acent t eet h7.
Mult iple linear regression m odels were used t o st udy t he associat ion bet ween periodont al st at us and syst em ic levels of cyt okines. I n t hese m odels,
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m ean CA loss, m ean PD, and num ber s of t eet h wit h CA loss of at least 6 m m and PD of at least 6 m m . Cyt okine concent rat ions were log- t ransform ed
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periodont al st at us as t he m ain independent variable and adj ust ing t hem for gender, body m ass index ( BMI ) , use of oral hy pogly cem ic dr ugs, lifet im e sm oking exposure ( in packyears) , and t im e elapsed since t he occurrence of acut e m yocardial infarct ion.
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( SE) , and adj ust ed R2 values were det erm ined. I t has been suggest ed t hat t he levels of t riglycerides m ay m odify t he product ion of cyt okines5. Thus, an
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com par isons accor ding t o t he TG cont r ol st at us, wit h 150 m g/ dL as t he cut- off.
dich ot om ized by t h e m edian v alu e. Sen sit iv it y,
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values ( PPV and NPV, respect ively) were calculat ed for severe periodont it is and each cyt okine.
St at ist ical an aly ses w er e p er f or m ed w it h a st at ist ical package ( STATA SE for Macint osh version 13, St at aCorp, College St at ion, USA) . The individual was t he unit of analysis. The alpha level was set at 5% .
RESULTS
The m ean age of t he sam ple was 62.9 ( SD: 9.9) years and t he average lifet im e sm oking exposure equaled 21.8 ( SD: 31.9) packyears. Table 1 describes t he charact erist ics of t he sam ple. Considering t he obser v ed m ean v alu es, car diov ascu lar pat ien t s from t he present st udy had elevat ed levels of CRP and TG, whereas TC, HDL, and LDL were m ost ly
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obser v ed. Ov er all, 6 8 ( 7 6 . 4 % ) in div idu als h ad severe periodont it is.
A t ot al of 5 2 ( 5 7 . 1 % ) pat ien t s ex per ien ced acut e m yocardial infarct ion, and m ore t han half of t hese pat ient s had t his m aj or event during t he nine years before t he st udy ( m edium - t erm past hist ory of event s) . CAD pat ient s in t he m edium - t erm past hist ory, com pared wit h t he long- t erm group, had
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and TNF-D ( Table 2) .
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loss ( R2= 0.07) and PD of at least 6 m m ( R2= 0.06)
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concent rat ions. Mean CA loss ( R2= 0.05) and PD ( R2 ZHUH VLJQL¿FDQWO\ UHODWHG WR WKH ,/
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associat ed w it h higher m ean CA loss ( R2= 0.07) .
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Table 4 shows m ult iple linear regression m odels of t h e associat ion bet w een clin ical at t ach m en t l o ss a n d se r u m cy t o k i n e s ( l o g t r a n sf o r m e d ) accor din g t o t r igly cer ide con t r ol. I n in div idu als
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num ber of t eet h wit h CA loss of at least 6 m m was
Characteristic n (%)
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Non-smoker 35 (38.4)
Former smoker 46 (50.6)
Smoker 10 (11.0)
BMI (Kg/m2) 27.84±4.56
Periodontal status Mean ± SD 9LVLEOHSODTXH 67.9±20.3
PD (mm) 3.0±0.7
CA loss (mm) 4.8±1.6
%23 74.7±24.2
Tooth loss (number of teeth) 12.9±7.0 Cardiovascular variables Mean ± SD
CRP (mg/L) 4.50±5.25
Triglycerides (mg/dL) 179±132
Total cholesterol (mg/dL) 167±30
LDL-C (mg/dL) 89±28
HDL-C (mg/dL) 42±12
Glucose (mg/dL) 122±46 *O\FDWHGKHPRJORELQ 6.98±2.07
Cytokines Mean ± SD
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IL-10 (pg/mL) 4.79±12.97
,/ȕSJP/ 1.26±2.79
IL-6 (pg/mL) 2.81±2.61
IL-8 (pg/mL) 6.65±5.64
TNF-D (pg/mL) 10.09±5.80 Table 1- Demographic and behavioral characteristics of the study sample (n=91)
Past history of cardiovascular events
Cytokines Long-term Medium-term
,)1ȖSJP/ 3.01 (0.76-34.13) 5.06 (0.90-84.89) IL-10 (pg/mL) 1.01 (0.05-20.06) 2.33 (0.05-88.47) ,/ȕSJP/ 0.66 (0.05-4.7) 0.71 (0.05-26.46) IL-6 (pg/mL) 1.92 (0.59-15.60) 2.13 (0.38-11.70)
IL-8 (pg/mL) 4.45 (1.45-30.00) 4.97 (1.53-27.82)
TNF-D (pg/mL) 7.47 (4.00-34.02) 9.17 (2.81-63.18)
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found for I L- 6, I L- 8, and TNF-D.
Diagnost ic t est s of sev er e per iodont it is as a pr edict or of cy t ok in e con cen t r at ion s abov e t h e m edian are shown in Table 5. A posit ive diagnosis of periodont it is indicat ed sensit ivit y values ranging bet ween 77.8 and 84.4, whereas t he absence of
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( range: 20.0–31.8) . PPVs were close t o 50.0, and
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The present st udy dem onst rat ed t hat periodont al
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periodont al condit ion, as m easured by CA loss and PD, was associat ed wit h increased blood levels of
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not ion t hat periodont al disease m ay be a prognost ic fact or in cardiovascular pat ient s.
Mean CA loss Mean PD 7HHWKZLWK&$ORVVPP 7HHWKZLWK3'PP
Cytokine beta±SE R2 beta±SE R2 beta±SE R2 beta±SE R2
,)1Ȗ 0.02±0.02 0.03 0.05±0.04 0.04 0.02±0.01 0.07 0.02±0.01 0.06 IL-10 0.07±0.03 0.05 0.12±0.06 0.06 0.02±0.01 0.04 0.03±0.02 0.05 ,/ȕ 0.001±0.03 0.03 0.002±0.06 0.03 -0.01±0.01 0.04 -0.01±0.02 0.03 IL-6 0.02±0.02 0.02 0.03±0.04 0.02 0.003±0.01 0.01 0.01±0.01 0.02
IL-8 0.01±0.02 0.05 -0.03±0.03 0.06 0.01±0.01 0.05 -0.01±0.01 0.06
TNF-D 0.02±0.01 0.07 0.02±0.02 0.04 -0.001±0.01 0.03 -0.002±0.01 0.03
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Table 3- Multiple linear regression models of the association between periodontal status and serum cytokines (log transformed), with adjustment for gender, body mass index, use of oral hypoglycemic drugs, smoking exposure, and time elapsed since major cardiovascular event
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7ULJO\FHULGHVPJG/ Triglycerides <150 mg/dL
Cytokine beta±SE R2 beta± SE R2
,)1Ȗ 0.04±0.02 0.19 0.01±0.01 0.08
IL-10 0.03±0.01 0.1 0.01±0.02 0.02
,/ȕ 0.06±0.03 0.1 0.01±0.01 0.17
IL-6 0.001±0.01 0.04 0.003±0.01 0.03
IL-8 0.001±0.01 0.18 0.01±0.01 0.06
TNF-D 0.01±0.01 0.24 0.001±0.01 0.04
Table 4- Multiple linear regression models of the association between clinical attachment loss and serum cytokines (log transformed) according to triglyceride control
Sensitivity 6SHFL¿FLW\ Positive predictive value
Negative predictive value ,)1Ȗ 84.4 (70.5-93.5) 31.8 (18.6-47.6) 55.9 (43.3-67.9) 66.7 (43.0-85.4) IL-10 77.8 (62.9-88.8) 25.0 (13.2-40.3) 51.5 (39.0-63.8) 52.4 (29.8-74.3) ,/ȕ 73.5 (58.9-85.1) 20.0 (9.1-35.6) 52.9 (40.4-65.2) 38.1 (18.1-61.6) IL-6 77.8 (62.9-88.8) 25.0 (13.2-40.3) 51.5 (39.0-63.8) 52.4 (29.8-74.3)
IL-8 82.6 (68.6-92.2) 30.2 (17.2-46.1) 55.9 (43.3-67.9) 61.9 (38.4-81.9)
The role of I L- 10 in t he cascade of at herosclerosis and cardiovascular event s rem ains cont roversial, w i t h so m e st u d i es i n d i ca t i n g t h a t h i g h I L- 1 0 concent rat ions m ay hav e a pr ot ect iv e effect on MI9,23 and ot her longit udinal observat ional st udies4,16 report ing t hat baseline elevat ed blood I L- 10 levels increase t he risk for MI . This cont roversy seem s t o h av e been clar if ied by a r ecen t ly pu blish ed m et a- analysis13, which dem onst rat ed t hat high I
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new car diovascular event s over t im e in pat ient s w it h acut e cor onar y sy ndr om e. I f I L- 10 is t r uly relat ed t o new m aj or cardiovascular event s, t hen periodont al disease m ight have a det rim ent al effect on at herosclerosis by increasing t he levels of I L- 10, as dem onst rat ed in t his st udy. Nevert heless, t he possible effect of ot her confounders not assessed in t he present st udy and which m ay be relat ed t o t he associat ion found bet ween periodont al st at us and I L- 10 should not be discarded.
,)1DŽDQG71)D are proat herogenic cyt okines21.
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collagen product ion and sm oot h cell proliferat ion, t h er eb y a l t er i n g t h e p l a q u e co m p o si t i o n a n d st abilit y3071)ĮLVLQYROYHGLQWKHVWLPXODWLRQRI I L- 6 pr odu ct ion2 7. I n t h e pr esen t st u dy, clin ical p a r a m e t e r s o f p e r i o d o n t a l i n f l a m m a t i o n a n d
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disease m ay act in early st ages of t he cascade of at her ogenic event s in car diovascular pat ient s by
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Th e I L- 6 cy t ok in e is dir ect ly inv olv ed in t h e hepat ic pr oduct ion of CRP20. Per iodont al disease was not associat ed wit h blood levels of I L- 6 in t he present sam ple of cardiovascular pat ient s. However, very low levels of t his cyt okine were det ect ed in our pat ient s, probably due t o t he cardiovascular care received by using st at ins. Such low levels m ay have hindered possible associat ions bet ween periodont al st at us and t his cyt okine.
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associat ion bet ween periodont al t issue breakdown and som e cy t ok ines in t his st udy. The sy st em ic
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have also suggest ed som e m odulat or y effect of elev at ed TG lev els on t h e p r od u ct ion of som e cyt okines when st im ulus cam e from P. gingivalis
lipopoly sacchar ides5. Taken t oget her, t hese dat a m ay indicat e t hat t he effect of periodont al disease
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levels of cardiovascular cont rol. Nevert heless, t his effect should be explored in t he fut ure for bet t er
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of t his pat ient populat ion.
A diagnosis of severe periodont it is was correlat ed
wit h above- m edian concent rat ions of cyt okines by
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in an at t em pt t o provide a bet t er est im at ion of t he clinical relevance of t he associat ions found in t he linear regression m odels t hat used log- t ransform ed dat a ( which cannot be direct ly int erpret ed) . I t was evidenced t hat perform ing a periodont al exam inat ion for est ablishing t he presence of periodont it is m ay be recom m ended because sensit ivit y values were close t o 8 0 % , in d icat in g t h at t h e p r esen ce of periodont it is will m ost oft en be relat ed t o above-m edian cyt okine concent rat ions. On t he ot her hand, w hen t he diagnosis of per iodont it is was alr eady
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abov e- m edian concent rat ions of cy t ok ines w er e low ( PPV range: 51.5–55.9) . When t he diagnosis
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( 66.7) com pared wit h ot her cyt okines.
Th er e is ex t en siv e ev iden ce su ggest in g t h at periodont al disease m ay be a risk fact or for CVD26. I n cont rast , evidence is st ill em erging t o det erm ine if periodont al disease m ay be a prognost ic fact or in pat ient s already diagnosed wit h coronary disease. Risk fact or s and pr ognost ic fact or s differ in t hat
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w her eas t he second r elat es t o t he cour se of an exist ing disease1:HREWDLQHGWKH¿QGLQJVRIWKH present st udy in chronic CAD pat ient s. Thus, t he m ain focus here was t o st udy periodont al disease in t he presence of cardiovascular disease, pot ent ially at t r ibu t in g a per spicu ou s f act or in t h e disease process. I t will be reasonable t o assum e t hat such m arker ( periodont al disease) could play a prognost ic role in t he hist ory of CVD. Not ewort hy, t his st udy is
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b y Lu m in ex in CAD p at ien t s. Pr ev iou s st u d ies support a possible role of periodont al disease in
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periodont al param et ers and I L- 6 and TNF-D10,17,25. Findings of t he present st udy should be analyzed in view of it s possible lim it at ions. The result s of t he
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because of t he loss of st at ist ical pow er. Anot her lim it at ion m ay include t he cross- sect ional nat ure, which precludes causal inferences from being drawn f r om t h e associat ion s. Lu m in ex an aly ses w er e perform ed in uniplicat e because of cost s, and t his m ay have lowered t he precision of t he readings. The st orage t im e of t he sam ples of t wo years m ay be a lim it at ion; however, t here is evidence t hat t he
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during t his period of t im e6,31.
This study applied well-established m ethodologies,
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biom arkers in t he blood. The analyses cont rolled f o r p o ssi b l e co n f o u n d i n g e f f e ct s b y a p p l y i n g m u l t i v a r i a b l e m o d e l s. Mo r e o v e r, p e r i o d o n t a l exam inat ions were conduct ed using a full- m out h prot ocol of six sit es per t oot h t o avoid assessm ent bias24. A broad range of periodont al param et ers was
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and t o associat e param et ers wit h blood levels of cyt okines.
CON CLUSI ON S
Periodont al disease is associat ed wit h increased
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Great er CA loss and PD were associat ed wit h higher
EORRGOHYHOVRI,)1DŽ,/DQG71)D, even aft er cont rolling for im port ant confounders.
ACKN OW LEDGMEN TS
This st udy was funded by CNPq – Nat ional Council
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( 476387/ 2010- 8) .
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