STUDY ON THE RISK MARKERS FOR ATHEROSCLEROSIS IN PATIENTS WITH SEVERE PERIODONTITIS

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1 Periodontology STUDY ON THE RISK MARKERS FOR ATHEROSCLEROSIS IN PATIENTS WITH SEVERE PERIODONTITIS Ovidiu NICOLAICIUC 1, Irina-Georgeta URSĂRESCU 2, Alexandra MÂRŢU-ŞTEFANACHE 1, Liliana PĂSĂRIN 3, Diana TATARCIUC 1, Cornelia OANŢĂ 1, Silvia MÂRŢU 4 1 PhD Student, Dept. Periodontology, Gr.T.Popa UMPh Iaşi 2 Univ. Assist., Dept. Periodontology, Gr.T.Popa UMPh Iaşi 3 Lecturer, Dept. Periodontology, Gr.T.Popa UMPh Iaşi 4 Univ. Prof., Dept. Periodontology, Gr.T.Popa UMPh Iaşi Corresponding author: irina_ursarescu@yahoo.com Abstract Aim: To provide insight on the association between periodontitis and atherosclerosis by determining the plasma levels of a series of risk markers for cardiovascular disease in cases with periodontitis. Materials and method: The study was conducted on 29 subjects without diagnosed systemic diseases (17 with severe periodontitis and 12 with healthy periodontium). Fasting blood plasma was analysed for glucose, lipids and markers of systemic inflammation (C-Reactive protein and fibrinogen). The associations between periodontitis and the various substances analysed in plasma were calculated using a multivariate logistic regression model, which compensated for age, gender, smoking and body mass index. Results and discussion: Regression analyses revealed a significant association between periodontitis and the high levels of C-reactive protein (CRP) [odds ratio (OR) 4.0, confidence interval (CI) ] and fibrinogen (OR 8.7, CI ). Conclusions: Severe periodontitis was associated with increased levels of CRP, glucose and fibrinogen. Keywords: periodontitis, atherosclerosis, inflammation, risk factors. 1. INTRODUCTION Cardiovascular disease and periodontal disease are both chronic inflammatory diseases. Numerous epidemiological studies have provided evidence on the association between periodontitis and the elevated risk for cardiovascular diseases [1,2]. Some of these studies demonstrated that periodontitis is an independent risk factor for cardiovascular disease even after adjusting for traditional cardiovascular factors such as age, gender, smoking, obesity and blood lipids. Moreover, experimental evidence has shown that the periodontal pathogens, mainly Porphyromonas gingivalis, play a role in atherogenesis [3,4]. A number of systematic reviews and meta-analyses described the relationship between periodontal infection and cardiovascular disease, suggesting that periodontitis may contribute to cardiovascular disease and stroke in susceptible subjects [5-7]. Periodontitis and cardiovascular disease share a number of common risk factors, such as age, male gender, socio-educational status, and, most importantly, smoking. Therefore, the question arises as to what the nature of the association between periodontitis and cardiovascular disease is. It is well accepted that hyperlipidemia represents a risk factor for coronary heart disease. Serum total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides are conventional lipid biomarkers used to evaluate the lipid profiles of an individual. LDL-C, which contains a single apolipoprotein (apob-100), is the major atherogenic cholesterol. An elevated serum LDL-C level is recognized as a major cause of coronary heart disease [8]. HDL-C is considered to be antiatherogenic. The major apolipoproteins in HDL are apoa-i and apoa-ii. The levels of HDL-C are inversely correlated with the risk of coronary heart disease [8]. Several meta-analyses have shown that a raised triglyceride is also an independent risk factor for coronary heart disease [9,10], while elevated triglyceride levels are commonly associated with other lipid and non-lipid factors [8]. C-reactive protein is an acute-phase reactant that is primarily produced by the liver in response to infection or trauma. It is an important marker for systemic inflammation, and has been consistently found to be elevated in patients with coronary syndromes [11,12]. Serum International Journal of Medical Dentistry 301

2 Ovidiu NICOLAICIUC, Irina-Georgeta URSĂRESCU, Alexandra MÂRŢU-ŞTEFANACHE, Liliana PĂSĂRIN, Diana TATARCIUC, Cornelia OANŢĂ, Silvia MÂRŢU C-reactive protein concentration is significantly increased in patients with coronary heart disease and myocardial infarction [13]. In a large sample population study, it was noted that increasing levels of serum high-sensitivity C-reactive protein were associated with the risk of cardiovascular events, and that high-sensitivity C-reactive protein was the strongest predictor for the risk of such events [11]. Recently, evidence has been accumulated, demonstrating the association between periodontitis and C-reactive protein. The serum C-reactive protein concentration is increased in systemically healthy subjects with periodontitis [14]. Several studies have assessed the serum C-reactive protein levels in patients with cardiovascular disease or cardiovascular risk factors. The results show that periodontitis patients with cardiovascular disease or hypertension have significantly higher serum high sensitivity C-reactive protein concentrations than those without periodontitis [15,16]. These studies indicate that periodontal disease is a chronic inflammatory disease resulting in elevated serum C-reactive protein levels. Fibrinogen, the main coagulation protein in plasma, is a co-factor for platelet aggregation and an acute phase reactant. An association between elevated plasma fibrinogen levels and coronary heart disease has been reported [17]. 2. MATERIALS AND METHOD The periodontitis group consisted of 17 subjects (9 men, 8 women; group mean age 54.7 years, range years) with a minimum of seven sites exhibiting at least 6 mm loss of clinical attachment. All individuals presented severe periodontitis, in which horizontal loss of the supporting tissue by 1/3 or more of the root length with bleeding on probing, furcation involvements of the multirooted teeth and/or angular bony defects with suppuration are present. The healthy, non-periodontal (control) group included 12 subjects (7 men, 5 women; group mean age 55.6 years, range years), none of whom exhibiting clinical signs of periodontitis or extensive gingivitis. All controls were performed within the same age range as those of the experimental cases. Participants were excluded from the study if 302 they had a known history of CVD or any other chronic disease. Cardiovascular health was assessed on the basis of medical history. None of the individuals included in this study were taking statins or any other medication for CVD. 10 of the cases and 8 of the controls were former or current smokers (15.2±2.5 and 17.1±1.8 cigarettes/day, respectively). All former and current smokers had a history of smoking for 10 years or more (mean 22.1±8.2 years). The cases underwent a comprehensive periodontal examination, including radiographs. The oral health status of the control group was subjected to a thorough clinical examination. Prior to the study, examiners were given detailed instructions and information with regard to the categorization of results. Measurements of the periodontal parameters, gingival probing, BOP, oral plaque, extent of furcations and mobility were standardized among the examiners. Teeth and gingival status were evaluated, and pocket depth was measured using a periodontal probe. Probing depth is the distance between the gingival margin and the bottom of the gingival pocket, measured from six angles for each tooth. The presence or absence of dental plaque at the gingival margin along the mesial, buccal, distal and lingual aspects was determined and the hygiene index (HI index, percentage) was calculated. The dental plaque was made visible by gently moving the tip of the probe along the gingival margin of four sides of each tooth. Gingival pockets that were 4 mm or deeper were considered to be pathogenic. Gingival inflammation was noted as bleeding on probing, and data were expressed as the ratio of bleeding sites in dentition. After overnight fasting, blood was collected into EDTA Vacutainer tubes and the haematological indices were determined using a Coulter STKS analyzer (Coulter Electronics Inc., Hialeah, FL, USA). Plasma was obtained after centrifugation at 1500 g for 10 min. and stored at C until analysis. Cholesterol and high-density lipoprotein (HDL) levels were determined using standard clinical chemistry procedures, with a Hitachi 917 analyzer (Roche AG Diagnostics, Mannheim, Germany). The lipid profile was calculated as the ratio between total cholesterol and HDL. CRP levels were determined using a high-sensitivity volume 19 Issue 4 October / December 2015 pp

3 STUDY ON THE RISK MARKERS FOR ATHEROSCLEROSIS IN PATIENTS WITH SEVERE PERIODONTITIS commercial assay kit (DADE Behring, Deerfield, IL, USA), and fibrinogen levels were measured on a BNA nephelometer (Behringwerke AG Diagnostica, Marburg, Germany). Glucose levels were measured in a similar manner. Statistical analyses were performed using PASW software. The p-values reported are based on a univariate comparison between cases and controls, as calculated by Student t-test or Mann Whitney U-test. Confidence intervals were calculated using Wald s method. The w2-test was used to calculate the significance of the dichotomized variables (HDL and glucose). Associations between periodontitis and surrogate markers for the atherosclerotic process (CRP, HDL cholesterol and glucose) were calculated using a multivariate logistic regression model adjusted for age, gender, body mass index (BMI) and smoking. These variables were selected as they are known risk factors for CVD. The smoking variable was also tested for interaction effects with gender, age and the primary variables (CRP, HDL cholesterol and glucose). The study was conducted in accordance with the Helsinki Declaration. All participants gave their informed consent. All blood sample analyses were performed blindly. 3. RESULTS AND DISCUSSION Data on patient demographics, smoking habits and clinical periodontal status are provided in Table 1. Within the (non-medicated) cases, smokers exhibited a more serious periodontal status and had fewer teeth (21.6 versus 25.2) than their nonsmoking counterparts. Cases with periodontitis tended to have a higher BMI compared with the control subjects, the difference being most prominent when non-smokers in the two groups were compared (p<0.01). In contrast to the BMI, the waist/hip ratio was similar among all participants. A univariate comparison between the groups showed that HDL cholesterol levels were lower in the experimental cases (p<0.05), this difference being particularly evident in the non-smoking group (p<0.01). No difference was observed among smokers. HDL cholesterol levels were below 0.9 mmol/l in nine (13%) cases with periodontitis and in only one individual (2%) in the control group. This difference was also apparent in the two subgroups (smokers: 12% versus 0%, non-smokers: 14% versus 4%). Fasting plasma glucose levels were higher () in cases with periodontitis, a difference observed in both smokers and nonsmokers (Table 2). Glucose levels were above 5.5 mmol in 35% of the experimental cases and in 4% of controls (). The levels of the acute-phase reactants CRP and fibrinogen were also elevated (p<0.015,, respectively; Table 2), even if the CRP levels in the smoking sub-groups were not significantly different. Table 1. Characteristics of groups Experimental group (n=17) Control group (n=12) Gender (M/F) 9/8 7/5 Mean age (yrs) (range) 54.7 (41 82) 55.6 (43 69) Mean number of teeth 24.2± ±2.9 Mean number of sites over 4 mm 47±17 8±6 Mean depth of periodontal pockets ±1.0 Mean BOP (%) 46±16 18±11 Mean HI (%) 51±11 27±14 BOP: Bleeding on Probing Index; HI: Hygiene Index International Journal of Medical Dentistry 303

4 Ovidiu NICOLAICIUC, Irina-Georgeta URSĂRESCU, Alexandra MÂRŢU-ŞTEFANACHE, Liliana PĂSĂRIN, Diana TATARCIUC, Cornelia OANŢĂ, Silvia MÂRŢU Table 2. Mean values (SD) of plasma lipoproteins, body mass index, glucose and acute phase markers in both groups Experimental group (n=17) Control group (n=12) Odd ratio (95% CI) (n=29) Glucose (mmol/l) 5.28±0.67 p1< ± ( ) p2<0.001 Triglycerides (mmol/l) 1.46± ± ( ) Total cholesterol (mmol/l) 5.44± ± ( ) HDL-C (mmol/l) 1.37±0.39 p= ± ( ) LDL-C (mmol/l) 3.41± ± ( ) Lipid profile 4.29± ± ( ) Waist/hip ratio 0.90± ±0.11 BMI 25.9± ±4.10 CRP (mg/l) 135±2.10 p < ± ( ) p2=0.009 Fibrinogen (g/l) 2.54± ± ( ) p2<0.001 The values are expressed for both groups as mean±sd; for the odd ratio they are described as mean and range. p1:significance between cases with periodontitis and periodontally healthy subjects p2: significance of odd ratio Regression analyses revealed that the patients with the highest glucose levels were significantly more likely to belong to the periodontitis group, odds ratio (OR) 11.6 [95% confidence interval (CI) ]. A similar result was seen for CRP, OR 4.0 (CI ). Although the reported association between periodontitis and CVD remains controversial, a mechanistic or a causal relationship cannot be excluded. This study has shown an association among the levels of three prominent risk factors for CVD (HDL cholesterol, hyperglycaemia and CRP) and periodontitis. The observed association between periodontitis and the low HDL cholesterol level (OR 3.22) is important, as mortality in individuals with HDL cholesterol levels below 0.9 mmol/l is higher than in individuals with levels over 0.9 mmol/l, regardless of the total cholesterol levels (18). In this study, the HDL cholesterol concentration was 40.9 mmol/l in nine (13%) of the cases, and in only one (2%) of the individuals in the control group, which agrees with earlier studies [19,20]. However, the absence of any difference in HDL cholesterol in the smoking groups indicates that periodontal inflammation might have an impact only on non-smokers. A possible explanation for the relationship between periodontitis and low-hdl cholesterol levels might be that chronic inflammation in the periodontium leads to the release of lipopolysaccharide and pro-inflammatory cytokines, such as IL-1b and TNF-a, which have the capacity to influence lipid metabolism [21]. Fasting glucose levels were 0.5mmol/l higher in the experimental group, regression analysis showing that those with the highest glucose levels belonged to the periodontitis group (OR 304 volume 19 Issue 4 October / December 2015 pp

5 STUDY ON THE RISK MARKERS FOR ATHEROSCLEROSIS IN PATIENTS WITH SEVERE PERIODONTITIS 15.5). The American Diabetes Association defines pre-diabetes as fasting plasma glucose levels above 5.5 mmol/l (100 mg/dl). According to this definition, significantly more cases were prediabetic (35% versus 4%). Although diabetes is an established risk factor for periodontitis, it is not currently known whether pre-diabetes or impaired glucose tolerance also increases the risk. Several studies have also indicated that periodontitis per se is a risk factor for diabetes; however, conclusive evidence has yet to appear. Diagnosed diabetes was an exclusion criterion, however 10 cases with periodontitis and 1 control had a fasting glucose of 6.1 mmol/l or more. This is in line with a recent study of Borrell et al. [22], which showed that the risk of undiagnosed diabetes increases in the presence of periodontitis. The cases also had a tendency to weigh more in the current study, although the difference was significant only for nonsmokers. Increased levels of CRP and fibrinogen indicated a more pronounced systemic inflammation in the patient group. The mean CRP was 0.9 mg/l higher in the experimental cases, and the differences in magnitude have been shown to influence the risk for CVD [23]. Higher CRP levels in cases with periodontitis have been evidenced in several earlier studies [19, 24-26]. Even if the elevated levels of CRP are relatively small, this could be of clinical importance, as slightly elevated levels of CRP can be considered as an independent risk factor for developing CVD. This is especially true, given the chronicity of generalized periodontitis and the subsequent exposure to its systemic inflammatory consequences for a prolonged period of time. Using surrogate markers for atherosclerosis (e.g., HDL cholesterol and CRP) also has limitations; however, endpoints for CVD, such as stroke or death, are not practical and feasible in a case control study such as this, due to time constraints. In studies involving associations, it is important to assess the confounding and effect modification factors, which is the reason for which the subjects with systemic diseases were excluded from this study. Smoking habit is another confounding factor and also an effect modification factor, so that subjects were therefore divided into different smoking status groups. A case control study such as this has its weak point - the study was not blinded and the cases originate from four different areas, whereas the controls originate from only one. These shortcomings resulted from the practicalities of undertaking the study during ordinary dental care visits, which could mean some selection bias, as discussed by Lopez et al. [27]. However, there is always a possibility that other factors or patterns could influence the findings, and associations between common diseases should be therefore interpreted with caution. 4. CONCLUSIONS The present study shows that cases with periodontitis have aberrant levels of some serological risk factors for CVD. Glucose and CRP were significantly higher, and HDL cholesterol was significantly lower in the experimental cases. These differences might contribute to the increased risk for CVD in cases with periodontitis, which has been reported in earlier epidemiological studies. References 1. Skalsky K, Yahav D, Bishara J, Pitlik S, Leibovici L, Paul M. Treatment of human brucellosis: systematic review and meta-analysis of randomised controlled trials. BMJ Mar 29;336(7646): Andriankaja OM, Genco RJ, Dorn J, Dmochowski J, Hovey K, Falkner KL, Trevisan M. Periodontal disease and risk of myocardial infarction: the role of gender and smoking. Eur J Epidemiol. 2007;22(10): Beck JD, Eke P, Lin D, Madianos P, Couper D, Moss K, Elter J, Heiss G, Offenbacher S. Associations between IgG antibody to oral organisms and carotid intima-medial thickness in community-dwelling adults. Atheroscler. 2005;183(2): Brodala NME, Bellinger DA, Damrongsri D, Offenbacher S, Beck J, Madianos P, Sotres D, Chang YL, Koch G, Nichols TC. Porphyromonas gingivalis bacteremia induces coronary and aortic atherosclerosis in normocholesterolemic and hypercholesterolemic pigs. Arterioscler Thromb Vasc Biol. 2005;25(7): Khader YS, Albashaireh ZS, Alomari MA. Periodontal diseases and the risk of coronary heart and cerebrovascular diseases: a meta-analysis. J Periodontol. 2004;75(8): International Journal of Medical Dentistry 305

6 Ovidiu NICOLAICIUC, Irina-Georgeta URSĂRESCU, Alexandra MÂRŢU-ŞTEFANACHE, Liliana PĂSĂRIN, Diana TATARCIUC, Cornelia OANŢĂ, Silvia MÂRŢU 6. Meurman JH, Sanz M, Janket SJ. Oral health, atherosclerosis and cardiovascular disease. Crit Rev Oral Biol Med. 2004;15(6): Mustapha IZ, Debrey S, Oladubu M, Ugarte R. Cardiovascular disease markers of systemic bacterial exposure in periodontal disease and risk: a systematic review and metaanalysis. J Periodontol. 2007;78(12): Persson GR, Persson RE. Cardiovascular disease and periodontitis: an update on the associations and risk. J Clin Periodontol. 2008;35(8): National Cholesterol Education Program (NCEP) Expert Panel. Third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III). Final report. Circulation. 2002;106(25): Assmann G, Schulte H, Funke H, von Eckardstein A. The emergence of triglycerides as a significant independent risk factor in coronary artery disease. Eur Heart J. 1998;19:M8 M Austin MA, Hokanson JE, Edwards KL. Hypertriglyceridemia as a cardiovascular risk factor. Am J Cardiol. 1998;81(4A):7B 12B. 12. Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med. 2000; 342(12): Ridker PM, Buring JE, Shih J, Matias M, Hennekens CH. Prospective study of C-reactive protein and the risk of future cardiovascular events among apparently healthy women. Circulation. 1998;98(8): Anderson JL, Carlquist JF, Muhlestein JB, Horne BD, Elmer SP. Evaluation of C-reactive protein, an inflammatory marker, and infectious serology as risk factors for coronary artery disease and myocardial infarction. J Am Coll Cardiol. 1998;32(1): Blum A, Front E, Peleg A. Periodontal care may improve systemic inflammation. Clin Invest Med. 2007;30(3):E114 E Higashi Y, Goto C, Hidaka T, Soga J, Nakamura S, Fujii Y, Hata T, Idei N, Fujimura N, Chayama K, Kihara Y, Taguchi A. Oral infection inflammatory pathway, periodontitis, is a risk factor for endothelial dysfunction in patients with coronary artery disease. Atheroscler. 2009;206(2): Higashi Y, Goto C, Jitsuiki D, Umemura T, Nishioka K, Hidaka T, Takemoto H, Nakamura S, Soga J, Chayama K, Yoshizumi M, Taguchi A. Periodontal infection is associated with endothelial dysfunction in healthy subjects and hypertensive patients. Hypertens. 2008;51(2): Green D, Foiles N, Chan C, Schreiner PJ, Liu K. Elevated fibrinogen levels and subsequent subclinical atherosclerosis: the CARDIA study. Atheroscler. 2009;202(2): Goldbourt U, Yaari S., Medalie JH. Isolated low HDL cholesterol as a risk factor for coronary heart disease mortality. A 21-year follow-up of 8000 men. Arterioscler Thromb Vasc Biol. 1997;17(1): Buhlin K, Gustafsson A, Pockley AG, Frostegard J, Klinge B. Cardiovascular disease, periodontitis and the monocyte relationship. Eur Heart J. 2003;25: Nibali L, Ready DR, Parkar M, Brett PM, Wilson M, Tonetti MS, Griffiths GS. Gene polymorphisms and the prevalence of key periodontal pathogens. J Dent Res. 2007;86(5): Iacopino AM, Cutler CW. Pathophysiological relationships between periodontitis and systemic disease. Recent concepts involving serum lipids. J Periodontol. 2000;71(8): Borrell LN, Kunzel C, Lamster I, Lalla E. Diabetes in the dental office: using NHANES III to estimate the probability of undiagnosed disease. J Periodontal Res. 2007;42(6): Ridker PM, Rifai N, Rose L, Buring JE, Cook NR. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002;347(20): Noack B, Genco RJ, Trevisan M, Grossi S, Zambon JJ, De Nardin E. Periodontal infections contribute to elevated systemic C-reactive protein level. J Periodontol. 2001; 72(9): Surdu-Macovei A, Pasarin L, Potarniche O, Martu S. Identification of C-Reactive Protein from Gingival Crevicular Fluid in Systemic Disease. Romanian J. of Oral Rehabilitation. 2012;4(2): Surdu A, Martu I, Ursarescu I, Scutariu M, Martu S, Balan A. The influence of dyslipidemia on the periodontal status in type 2 diabetes mellitus patients. Romanian J. of Oral Rehabilitation. 2014;6(4): Lopez R, Scheutz F, Errboe M, Baelum V. Selection bias in case control studies on periodontitis: a systematic review. Eur J Oral Sci. 2007;115(5): volume 19 Issue 4 October / December 2015 pp