Serum Antibody Levels to Actinobacillusactinomycetemcomitans Predict the Risk for

Coronary Heart DiseasePirkko J. Pussinen, Kristiina Nyyssonen, Georg Alfthan, Riitta Salonen,

Jari A. Laukkanen, Jukka T. Salonen

Objective—The association between serum antibody levels to major periodontal pathogens and coronary heart disease(CHD) was analyzed in a prospective population-based study.

Methods and Results—The population comprised 1023 men (aged 46 to 64 years) in the Kuopio Ischemic Heart DiseaseStudy. The subjects with CHD at baseline (n�113) were more often seropositive for Porphyromonas gingivalis IgA(38.9% versus 28.5%, P�0.021) and IgG (60.2% versus 46.7%, P�0.007) than those without CHD. During the 10-yearfollow-up, 109 men free from CHD at baseline experienced an acute myocardial infarction or CHD death. The men withan end point were more often seropositive for Actinobacillus actinomycetemcomitans IgA (15.5% versus 10.2%,P�0.019) than those who remained healthy. In the highest tertile of A. actinomycetemcomitans IgA-antibodiescompared with the lowest one, the relative risk (RR) for an end point adjusted for CHD risk factors was 2.0 (95%confidence interval [CI], 1.2 to 3.3). In the Porphyromonas gingivalis IgA-antibody tertiles, the highest RR of 2.1 (1.3to 3.4) was observed in the second tertile. All antibody levels correlated positively with the carotid artery intima-mediathickness.

Conclusions—High-serum antibody levels to major periodontal pathogens are associated with subclinical, prevalent, andfuture incidence of CHD. Periodontal pathogens or host response against them may contribute to the pathogenesis ofCHD. (Arterioscler Thromb Vasc Biol. 2005;25:833-838.)

Key Words: atherosclerosis � cardiovascular diseases � infection � inflammation � periodontitis

Periodontitis is a common bacterial infection of the tooth-supporting tissues. The prevalence of severe periodontitis

is 10% to 15% in most populations and it increases with age.1

The host response to the persistent bacterial insult in peri-odontitis leads to chronic inflammation, which causes thesymptoms of the disease: gingival bleeding, formation ofdeepened periodontal pockets, destruction of tooth attach-ment, and eventually loss of tooth. Bacteria, their compo-nents, and local inflammation may act as triggers leading tosystemic inflammation. This can be observed as elevatedconcentrations of acute-phase reactants and inflammatorymediators, especially C-reactive protein, which has beenshown to be directly associated with atherosclerosis.2

A selection of epidemiological studies from the pastdecade suggests that periodontitis is an important risk factorfor coronary heart disease (CHD).3 Most of the literature isbased on clinical periodontal examinations or self-reportedperiodontitis. Therefore, interpretation of the results mayexperience the absence of standard measures for periodontal

disease. However, data on the role of periodontal pathogensin atherogenesis is limited, although some bacterial specieshave been identified as etiologic agents in periodontitis.4 In 3recent serological studies, however, high antibody levels to aperiodontal pathogen, Porphyromonas gingivalis, have beenfound to associate with prevalent CHD,5 high degree ofstenosis in plaque segments,6 and incidence of acute myocar-dial infarction (AMI) in a small study population.7

The aim of the study was to analyze if antibody levels tothe major periodontal pathogens, Actinobacillus actinomyce-temcomitans and P. gingivalis, are associated with the risk ofCHD and carotid intima-media thickness (IMT) in a prospec-tive population-based study.

Subjects and MethodsA total of 2682 men were enrolled in the Kuopio Ischemic HeartDisease Risk Factor (KIHD) study conducted in Finland between1984 and 1989.8 Data on socioeconomic status were collected duringthis examination. The Research Ethics Committee of the Universityof Kuopio approved the study, and all study subjects gave theirwritten informed consent.

Original received November 5, 2004; final version accepted January 11, 2005.From the Institute of Dentistry (P.J.P.), University of Helsinki, and Department of Oral and Maxillofacial Diseases, Helsinki University Central

Hospital, Helsinki; the Research Institute of Public Health (K.N., R.S., J.A.L., J.T.S.), University of Kuopio; the Department of Health and FunctionalAbility (G.A.), National Public Health Institute, Helsinki; Savonlinna Central Hospital (J.A.L.), Savonlinna; and Oy Jurilab Ltd (J.T.S.), Kuopio, Finland.

Correspondence to Pirkko Pussinen, Institute of Dentistry, University of Helsinki, and Department of Oral and Maxillofacial Diseases, HelsinkiUniversity Central Hospital, P.O. Box 63, FI-00014 Helsinki, Finland. E-mail pirkko.pussinen@helsinki.fi

© 2005 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000157982.69663.59

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Study Design, Population, and OutcomeThe present study population comprises 1023 men aged 46 to 64years, who were enrolled in 1987 to 1989 and re-examined after 4years in 1991 to 1993 (baseline).9

The subjects visited the study site of re-examination (1991 to1993) twice with a 7-day interval. Blood pressure and body massindex were measured at the first visit. The IMT of the commoncarotid artery was scanned ultrasonographically and blood sampleswere drawn at the second visit.9 A self-administered questionnaireprovided information on sociodemographic background, diseases,medication, and smoking habits.

Data on deaths were obtained from the National Death CertificateRegister, and underlying cause of death was taken as that assigned atthe Central Statistical Office of Finland. Data on nonfatal AMI werederived from the National Hospital Discharge Data Register. Atbaseline, 113 men reported to have CHD, angina pectoris, coronarybypass, or other coronary disease. Of the 910 men free from CHD atbaseline, a total of 109 subjects experienced a cardiac end point,AMI, or CHD death during a mean follow-up of 10 years until year2002.

Serum and Plasma DeterminationsVenous blood samples were taken after fasting for 12 hours atbaseline (1991 to 1993). Lipoproteins were separated from freshserum samples by combination of ultracentrifugation and precipita-tion.10 Serum total, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride concentra-tions were determined by enzymatic methods, and apolipoprotein B(apoB) and apoA-I by immunoturbidimetry. The concentrations ofserum glutamyltransferase were measured by the kinetic methodfollowing the recommendation of the European Committee forClinical Laboratory Standards. Concentrations of plasma fibrinogenwere determined based on clotting of diluted plasma with excessthrombin by a coagulometer.

Serum IgG and IgA class antibodies to A. actinomycetemcomitansand P. gingivalis were determined by multiserotype enzyme-linkedimmunosorbent assay (ELISA) from serum samples stored at�20°C.11 Two dilutions of each serum in duplicate were used andthe results (ELISA units [EU]) consisting of mean absorbances werecalculated as continuous variables. We included on each plate a highand a low reference serum in duplicates to monitor the interassayvariations. The interassay coefficients for variation as calculatedfrom values of the low reference serum were 6.2% and 4.5% for A.actinomycetemcomitans and 5.0% and 4.7% for P. gingivalis IgAand IgG, respectively. A correction coefficient was calculated fromto the mean of the high reference values and the ELISA results ofeach plate were normalized by this coefficient after the wholematerial was analyzed. The subjects were considered seropositive forA. actinomycetemcomitans and P. gingivalis when the correspondingIgG value was �5.0 EU or the IgA value was �2.0 EU, whichrepresent the mean antibody levels plus 1.5�SD of periodontallyhealthy subjects.11 Combined antibody response in dentate subjectswas computed by summing IgG levels to A. actinomycetemcomitansand P. gingivalis, and the value was considered “high” when itexceeded 14.0 EU, which represents the corresponding mean anti-body level of periodontally healthy subjects plus 3�SD.5,11

Statistical AnalysesThe significance of differences between cases and controls weretested using t test or �2 test. The significance of differences betweenIMT values in the tertiles of antibody levels was tested by t test.Cumulative survival as well as the relative risks and their 95%confidence intervals of cardiac end points in tertiles of A. actinomy-cetemcomitans and P. gingivalis antibody levels were estimatedusing Cox regression models. Confounding factors included in themodel were age, smoking, plasma fibrinogen, diabetes, body massindex, medication for hypertension, socioeconomic status, and serumLDL and HDL cholesterol concentrations. The statistical analyseswere performed using SPSS program version 10.0 for Windows.

ResultsBoth IgA and IgG antibody levels correlated positively withmean IMT, especially among dentate subjects (n�459): thecorrelation coefficients (r) for mean IMT and A. actinomyce-temcomitans IgA and IgG levels, and for P. gingivalis IgAand IgG levels were 0.116 (P�0.013), 0.097 (P�0.038),0.166 (P�0.001), and 0.146 (P�0.002), respectively. In allsubjects free from CHD at baseline (n�907), mean IMTincreased in the tertiles of A. actinomycetemcomitans IgA (Pfor trend 0.021), and P. gingivalis IgA (P�0.001) and IgGantibody levels (P�0.023) (Figure 1). The same trend wasobserved in the tertiles of combined antibody response indentate subjects free from CHD (n�418) (P�0.005) (Figure1). The edentulous subjects (n�481), who were free fromCHD at baseline, had higher mean IMT than the subjects withnatural teeth (Figure 1). The combined antibody responsecorrelated negatively with serum HDL cholesterol(r��0.073, P�0.019) and positively with HDL triglycerideconcentration in the dentate population (r�0.133, P�0.004).

At baseline, a total of 113 men had already signs of CHD.They were older (60.6�5.1 versus 55.7�6.6 years), had higherserum cholesterol (5.61�1.08 versus 5.52�0.92 mmol/L), apoB(1.04�0.28 versus 0.96�0.25 g/L), triglyceride (1.96�1.18versus 1.57�1.01 mmol/L), and plasma fibrinogen (3.41�0.66versus 3.13�0.56 g/L) concentrations, and lower serum HDLcholesterol (1.00�0.29 versus 1.11�0.29 mmol/L) and apoA-I(1.15�0.18 versus 1.21�0.18 g/L) concentrations than the menfree from CHD (n�910), respectively (for all differencesP�0.001). They also more frequently had diabetes (13.3%versus 5.8%, P�0.003) and hypertension (81.4% versus 22.0%,P�0.001), and were reported to more often have claudication(22.1% versus 2.5%, P�0.001) and history of any CVD in thefamily (96.5% versus 84.5%, P�0.001), respectively. The menwith CHD less often had their own teeth (34.8% versus 46.6%,P�0.019), and they were more often seropositive for P. gingi-valis IgA (38.9% versus 28.5%, P�0.021) and IgG (60.2%versus 46.7%, P�0.007) than the men free from CHD. Further-more, the dentate subjects with CHD (n�39) had more fre-

Figure 1. Mean IMT in the tertiles of antibody levels to peri-odontal pathogens. The mean carotid artery intima-media thick-ness was determined in the subjects free from CHD at baseline(n�907). The subjects were divided according to their dentatestatus, and tertiles of IgA and IgG class antibody levels to peri-odontal pathogens A. actinomycetemcomitans (Aa) and P. gingi-valis (Pg). The dentate subjects (n�418) were also dividedaccording to the tertiles of combined antibody response (Aa �Pg), which is the IgG class antibody levels to A. actinomycetem-comitans � P. gingivalis. The error bars indicate SEM. *P�0.05,**P�0.01, and ***P�0.001 compared with the first tertile using ttest.

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quently a high combined antibody response compared with thedentate subjects without CHD (n�420) (35.9% versus 18.8%,P�0.011).

During the mean follow-up of 10 years, 46 (40.7%) menwith a history of CHD at baseline experienced a secondarycoronary event, an AMI, or CHD death. Antibody levels toperiodontal pathogens were not associated with this coronaryevent. However, the men who had a secondary event morefrequently wore dentures than the men who remained freefrom a coronary event (76.1% versus 57.6%, P�0.046; RRfor an end point 2.3, 95% CI, 1.02 to 5.40).

A total of 109 men free from CHD (n�910) at baselineexperienced an AMI or CHD death during the follow-up timeof 10 years. The characteristics of these subjects with andwithout an end point are summarized in Table 1. The subjectswith an end point were more frequently seropositive for A.actinomycetemcomitans IgA than subjects without an endpoint (15.5% versus 10.2%, P�0.019). During the follow-up,46 (5.1%) men with a high IgA class antibody level to A.

actinomycetemcomitans (third tertile) and 23 (2.5%) menwith a low antibody level (first tertile) experienced an endpoint (P�0.004 for difference) (Figure 2). The correspondingrates for the second and first tertiles of P. gingivalis IgA classantibody levels were 48 (5.3%) and 24 (2.6%), respectively(P�0.002 for difference) (Figure 2). The risk for CHD endpoint increased with increasing tertiles of A. actinomycetem-comitans IgA antibody levels (Table 2). The multivariate RRfor the second tertile of P. gingivalis IgA antibody levels wasalso significant (P�0.004) compared with the first tertile, butthe association was not completely linear (P for trend 0.015).

DiscussionWe found that elevated serum IgA class antibody levels toperiodontal pathogens, P. gingivalis and especially to A.actinomycetemcomitans, were associated with CHD inci-dence in a population-based prospective study among menfree from CHD at baseline. The risk ratio for CHD was 2.0 to2.1, which is of the same range as reported earlier for

TABLE 1. Characteristics of Subjects Free From CHD at Baseline, With andWithout an End Point (AMI or CHD death) During the Follow-Up of 10 Years

Parameter With an End Point Without an End Point P *

Mean (SD)

Age, y 57.4 (6.5) 55.4 (6.6) 0.003

BMI, kg/m2 28.1 (3.7) 27.4 (3.5) NS

Waist-to-hip ratio 1.00 (0.04) 0.98 (0.04) 0.005

Alcohol/wk, g 89.2 (116.6) 79.4 (115.1) NS

GGT, U/L 32.7 (26.8) 31.3 (39.2) NS

S-chol, mmol/L 5.77 (0.86) 5.48 (0.92) NS

S-apoB, g/L 1.05 (0.29) 0.95 (0.24) �0.001

S-HDL, mmol/L 1.07 (0.31) 1.11 (0.28) NS

S-apoA-I, g/L 1.21 (0.19) 1.21 (0.18) NS

S-TG, mmol/L 1.83 (1.30) 1.54 (0.96) 0.004

P-fibrinogen, g/L 3.29 (0.53) 3.11 (0.57) 0.001

Mean IMT, mm 0.94 (0.20) 0.86 (0.19) �0.001

Mean maximal IMT, mm 1.30 (0.30) 1.18 (0.27) �0.001

A. actinomycetemcomitans

IgA, EU 2.02 (1.31) 1.77 (1.27) NS

IgG, EU 3.70 (2.30) 3.49 (2.21) NS

P. gingivalis

IgA, EU 1.89 (1.25) 1.82 (1.47) NS

IgG, EU 6.18 (3.29) 5.74 (3.23) NS

N, % P†

N 109 (12.0) 801 (88.0)

Diabetic 11 (10.1) 42 (5.2) 0.043

Hypertensive 39 (35.8) 161 (20.1) �0.001

Smokers 38 (34.9) 208 (26.0) 0.050

Any CVD in family 98 (89.9) 671 (83.8) NS

Claudication 5 (4.6) 18 (2.2) NS

Own teeth 50 (45.9) 370 (46.2) NS

*t test.†�2 test.CVD indicates cardiovascular disease; EU, ELISA units; GGT, glutamyltransferase; TG, triglycerides.

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clinically diagnosed periodontitis.3 In addition, edentulous-ness and seropositivity for P. gingivalis were associated withprevalent CHD at baseline.

Both of these pathogens are connected to aggressive formsof periodontal disease. A. actinomycetemcomitans occursparticularly in periodontal diseases manifesting at young age

(younger than 35 years), but periodontitis caused by P.gingivalis develops usually among middle-aged or elderlypeople. Both A. actinomycetemcomitans and P. gingivalismay also be found in low proportions among periodontallyhealthy subjects and can therefore be considered part of theendogenous oral flora. DNA of these pathogens has beenfound in atherosclerotic plaques,12 and certain clones of A.actinomycetemcomitans may exert potency to cause nonoralinfections.13

Periodontitis is accompanied by a strong immune responseto periodontal pathogens, which can be measured from salivaand serum. Individual IgG class antibody levels are quitestable over time and do not necessarily display activity orseverity of the disease; for example, periodontal treatmentdecreases IgG class antibody levels only temporarily.14 Thecutoff level to seropositivity in our assay is relatively low(mean antibody level plus 1.5�SD of periodontally healthysubjects) and validated using results from polymerase chainreaction detection.11 Therefore, seropositivity indicates thatthe individual is exposed to the pathogens, but that individualmay be in a state of “carrier” rather than with an activedisease. Another cutoff limit, which we have validated for ourassay, specifies individuals with “a high combined antibodyresponse.” Combining the IgG antibody levels of A. actino-mycetemcomitans and P. gingivalis results in an assay withsensitivity of 71% and specificity of 90% for finding peri-odontitis in dentate subjects.11 In the present population, thepercentage of subjects with “serologically diagnosed” peri-odontitis was 36% among men with CHD versus 19% amongmen free from CHD. The proportions are in the same range asin our earlier cross-sectional study conducted in 1997, inwhich percentages were 38% and 27%, respectively.5 Thesepercentages are of the same magnitude as the earlier radio-graphically determined prevalence of severe periodontitis inthe Mini-Finland Oral Health Study (32%),15 suggesting thatthe multiserotype ELISA may be a suitable method toidentify subjects with periodontitis in large-scale studies, inwhich only serum samples are available.

Figure 2. Cumulative survival rates for coronary events. Themen free from CHD at baseline were divided into tertilesaccording to serum A. actinomycetemcomitans and P. gingivalisIgA class antibody levels. The cumulative survival of AMI or car-diac death was analyzed by Cox regression model adjusted forage, smoking, plasma fibrinogen, diabetes, hypertension, socio-economic status, and serum LDL and HDL cholesterolconcentration.

TABLE 2. Relative risk for AMI and CHD Death in Tertiles of Serum AntibodyLevels for Periodontal Pathogens

Tertile

Antibody to 1st 2nd 3rd P for Trend

A. actinomycetemcomitans

IgG Univariate 1.0 1.2 (0.74–1.90) 1.3 (0.79–2.00) 0.616

Multivariate* 1.0 1.2 (0.72–1.86) 1.2 (0.76–1.94) 0.695

IgA Univariate 1.0 1.8 (1.09–3.04) 2.2 (1.32–3.60) 0.009

Multivariate* 1.0 1.5 (0.91–2.58) 2.0 (1.21–3.33) 0.025

P. gingivalis

IgG Univariate 1.0 1.6 (1.01–2.60) 1.5 (0.91–2.39) 0.120

Multivariate* 1.0 1.3 (0.79–2.08) 1.2 (0.70–1.88) 0.606

IgA Univariate 1.0 2.2 (1.36–3.62) 1.8 (1.10–3.06) 0.006

Multivariate* 1.0 2.1 (1.26–3.37) 1.5 (0.87–2.47) 0.015

*Cox regression model adjusted for age, smoking, plasma fibrinogen, diabetes, medication forhypertension, socioeconomic status, and serum LDL�HDL cholesterol.

Relative risk for AMI and CHD death (95% CI).

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High IgA class antibody levels to periodontal pathogens insaliva indicate persistent periodontitis with active tissuedestruction,14 whereas the significance of high IgA antibodylevels in serum is not fully understood. However, local andsystemic antibody levels correlate strongly with each other.Therefore, our results suggest that prevalent CHD and futurerisk of CHD are associated with active forms of periodontitis.Persistently elevated antibodies belonging to IgA class alsoappear to be one of the best serological markers of chronicChlamydia pneumoniae infection.16

The serum antibody levels to periodontal pathogens had amoderate positive correlation with IMT at baseline, indicatingthat exposure to periodontal pathogens may be related alreadyto subclinical atherosclerosis. In 3 earlier studies, carotidartery plaque has been more common in subjects with severeor moderate periodontitis,17 high P. gingivalis IgG titers,6 or�10 missing teeth.18 Edentulousness or missing teeth hasbeen found to be associated with CHD in several studies,5,19,20

and they can be considered as markers of past periodontaldisease.18 Also in the present study, the use of dentures wasassociated with CHD, but it did not predispose to futurecoronary events in CHD-free subjects. However, the meanIMT was higher among the CHD-free men wearing denturesthan those without dentures. These observations imply that inthe final stage of periodontitis, when the teeth are already lost,“the damage is done” regarding atherosclerosis. However,denture-related mucosal lesions are also important determi-nants of CVD risk in edentulous subjects and associated withelevated C-reactive protein concentrations.21 Furthermore,edentulousness is strongly associated with low socioeco-nomic status and thereby also with other chronic infections,which have been recognized as risk factors for CHD sepa-rately and as contributors to the “infectious burden.”22,23

Because chronic infections and CHD usually share commonetiopathogenic factors, eg, smoking and low socioeconomicstatus, the nature of their association is difficult to explore inhumans.

The underlying mechanisms explaining the associationbetween periodontitis and increased risk for CHD are stillunclear. In periodontitis, the local inflammatory state and/orthe long-term systemic exposure to the pathogens or theirvirulence factors act as triggers for systemic inflammation,which can be detected as elevated concentrations of inflam-mation markers.3 This is supported by a recent study showingthat mice administered via oral and anal application with P.gingivalis displayed local periodontal infection and exacer-bated early atherosclerotic lesions.24 Furthermore, recentstudies on human subjects indicate that periodontitis in-creases the proatherogenic properties of LDL25 and decreasesthe antiatherogenic properties of HDL in vitro,26 therebydirecting lipoprotein metabolism in an unfavorable direction.Similarly, as in our earlier study,5 in the present study thecombined antibody response correlated inversely with serumHDL cholesterol concentration. Furthermore, here the anti-body response correlated directly with HDL triglycerideconcentration, which is the first to be affected by inflamma-tion/infection.27,28 These observations suggest that periodon-titis may interfere with HDL metabolism and thereby de-crease proper removal of excess peripheral cholesterol.

Recent studies on the role of periodontal pathogens inatherosclerosis have concentrated on P. gingivalis. In addi-tion to studies on animal models,24,29–31 cell culture studiesdemonstrate that vesicles shed from the surface of P. gingi-valis promote binding of LDL to macrophages, LDL modi-fication, and macrophage-derived foam cell formation, a signof early atherosclerosis.32,33 Periodontitis is, however, amultibacterial disease, and studies on the role of otherpathogens than P. gingivalis in CHD are scarce. To ourknowledge, the present study is the first to suggest that aninfection by A. actinomycetemcomitans may be associatedwith the incidence of CHD. Consistent with our findings,high serum levels of IgA antibodies to A. actinomycetem-comitans have been found to predispose to cerebrovascularstroke in subjects free from CVD at baseline.34

Our study suggests that high IgA antibody levels to both P.gingivalis and A. actinomycetemcomitans are associated withCHD. These results also indicate that serum antibodies toperiodontal pathogens might be worth screening when map-ping individual risk factors for CVD.

AcknowledgmentsThe Academy of Finland (grants 75953, 77613, and 211129), thePaulo Foundation, and the Paavo Nurmi Foundation supported thestudy financially by grants to P.J.P., and the Juho Vainio Foundationby a grant to J.A.L. We thank the staff of the Research Institute ofPublic Health, University of Kuopio, for data collection in theKuopio Ischemic Heart Disease Risk Factor Study (KIHD). TheKIHD study was supported by grants from the Academy of Finland(grants 41471, 1041086, 2041022, and 45155) and the Ministry ofEducation of Finland (grants 167/722/96, 157/722/97, 156/722/98)to J.T.S., and the National Heart, Lung, and Blood Institute of theUSA (grant HL44199) to Professor George A. Kaplan.

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