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Egyptian Journal of Oral & Maxillofacial Surgery:
doi: 10.1097/01.OMX.0000405286.13210.e3
Clinical Papers

The mutual effect of hyperlipidemia and proinflammatory cytokine related to periodontal infection

Al Bahrawy, Mohammed M.a; Abdel Ghaffar, Khaled K.a; El-Mofty, Mohamed S.a; Abdel Rahman, Ahmedb

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Author Information

aDepartments of Oral Medicine and Periodontology

bCardiology, Ain Shams University, Cairo, Egypt

Correspondence to M. Al Bahrawy, Demonstrator, Department of Oral Medicine and Periodontology, Ain Shams University, Cairo, Egypt; Tel: 0020111988517 e-mail: doc_bahrawy@hotmail.com

Received May 12, 2011

Accepted June 30, 2011

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Abstract

Hyperlipidemia is a highly prevalent worldwide disease. Moreover, it is a known risk factor for cardiovascular diseases. In contrast, periodontitis is an inflammatory disease in which Gram-negative microorganisms and their products are the principal etiologic agents. They produce endotoxins in the form of lipopolysaccharides, which are instrumental in generating a host-mediated tissue destructive immune response and have been connected to several systemic health changes, including an altered lipid metabolism. The aim of this study was to evaluate the bidirectional effect of hyperlipidemia and pharmacological treatment of hyperlipidemia on proinflammatory cytokines related to the severity of periodontal disease. Twenty-four individuals aged above 20 years seen at the clinics of Ain Shams University (Cairo, Egypt), were recruited into this study and were divided into three groups. All of them suffered from chronic periodontitis, and two individuals suffered from hyperlipidemia. Only one group received lipid-lowering treatment and was paired according to sex and age. Their levels of total cholesterol, low-density cholesterol and high-density cholesterol, and triglycerides, as well as serum level of interleukin-1β and tumor necrosis factor α, were determined at baseline, after 3 weeks, and after 6 months of mechanical and chemical periodontal treatments. Variables related to high-cholesterol levels, including age and sex, were evaluated. The values recommended by the World Health Organization were considered to classify hyperlipidemia. The results showed that the change between baseline and week 3 showed no significant change except in mean levels of total cholesterol, with a significance of 0.022 or less, and low-density lipoprotein, with a significance of 0.022 or less, in individuals who received lipid-lowering treatment with periodontitis treatment. However, the group with hyperlipidemia that did not receive lipid-lowering drugs showed a decrease in triglyceride levels, but it was a nonsignificant change, which may show a trend. None of the other lipid and cytokine profiles showed a significant change. Therefore, in this studied population, although a relationship was found between periodontal disease treatment and blood lipid levels it was not significant.

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Introduction

Periodontitis is defined as an inflammatory disease of the supporting tissues of the teeth caused by specific microorganisms or groups of specific microorganisms, resulting in progressive destruction of the periodontal ligament and alveolar bone with pocket formation, recession, or both 1. Studies have indicated that periodontal disease may have profound effects on systemic health. According to the Garlet review [2], patients with periodontal disease may have higher risk for cardiovascular disease when compared with individuals with a healthy periodontium. In a reappraisal of Host Defense and Tissue Destruction Viewpoints, it was postulated that, with the discovery of several T-cell subsets bearing distinct immunoregulatory properties, this proinflammatory versus anti-inflammatory scenario has become more complex, and a series of studies has hypothesized protective or destructive roles for Th1, Th2, Th17, and Treg subpopulations of polarized lymphocytes. Interestingly, the ‘protective versus destructive’ archetype is usually considered in a framework related to tissue destruction and disease progression. However, it is important to remember that periodontal diseases are infectious inflammatory conditions, and recent studies have demonstrated that cytokines [tumor necrosis factor α (TNF-α) and interferon-γ] considered harmful in the context of tissue destruction play important roles in the control of periodontal infection.

Studies on humans and animals have shown that a number of cytokines such as TNF-α and interleukin-1β (IL-1β) are produced in response to systemic Gram-negative lipopolysaccharide (LPS) exposure. It is believed that these cytokines exert effects on lipid metabolism by influencing the production of other cytokines, altering hemodynamics/amino acid utilization of various tissues involved in lipid metabolism, or by modifying the hypothalamic–pituitary–adrenal axis, by increasing plasma concentrations of adrenocorticotropic hormone, cortisol, adrenaline, noradrenaline, and glucagon 3–5. In contrast, lipids may interact with the macrophage cell membrane, interfer with membrane-bound receptors and enzyme systems, alter macrophage gene expression for proinflammatory cytokines such as TNF-α and IL-1β and essential polypeptide growth factors such as platelet-derived growth factor, transforming growth factor β1, and basic fibroblast growth factor 6,7. In addition, serum lipids, whether induced by diabetes or diet, increase polymorphonuclear leukocyte (PMNL) production of proinflammatory cytokines and inhibit macrophage production of essential polypeptide factors, impairing the wound-healing process 8–10.

Thus, it is hypothesized that a relationship exists between adult periodontitis and hyperlipidemia, which involves the inflammatory response to LPS from periodontal pathogens such as Porphyromonas gingivalis.

Data of this study suggest that periodontitis may trigger lipid level alterations, perhaps by eliciting an increased systemic inflammatory burden. Periodontitis is a chronic, inflammatory, destructive disease that affects the supporting tissues of the teeth. It is often associated with enhanced concentrations of proatherogenic plasma lipids, that is, total and low-density lipoprotein cholesterol (LDL-C) and triglycerides 11–15. Although the role of periodontitis-associated dietary behavior cannot be excluded, periodontitis itself was shown to be accompanied by a proatherogenic lipid profile 12,13,16. Bacterial toxins such as LPS, β2-glycoprotein, and modified phospholipids can induce changes in cholesterol concentrations 16–18. The results of these studies show that local periodontal treatment results in a significant decrease in total and LDL-C levels.

Statins, which are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors used in lipid-lowering treatments, are potent inhibitors of cholesterol biosynthesis. Cholesterol-lowering therapy using statins significantly reduces the risk of coronary heart disease. However, extensive use of statins leads to an increase in other undesirable, and beneficial, effects, so-called pleiotropic effects. With respect to these effects, statins augment the expression of bone morphogenetic protein-2, a potent simulator of osteoblast differentiation and its activity, and promote mineralization by cultured osteoblasts, indicating that statins have an anabolic effect on bone. Chronic administration of statins in ovariectomized rats modestly increases bone mineral density of cancellous bone but not of compact bone. In clinical studies, there are conflicting results with regard to the clinical benefits of this therapy for the treatment of osteoporosis. Observational studies suggest an association between statin use and reduction in fracture risk. Clinical trials reported no effect of statin treatment on bone mineral density in hip and spine and on bone turnover. Statins may also influence oral osseous tissues. Administration of statins in combination with osteoporosis therapy appears to improve alveolar bone architecture in the mandibles of ovariectomized rats with maxillary molar extraction. Statins continue to be considered as potential therapeutic agents for patients with osteoporosis and possibly with periodontal disease. Development of new statins that are more specific and potent for bone metabolism will greatly increase the usefulness of these drugs for the treatment of bone diseases 19.

In contrast, many studies showed no significant difference between lipid profile and periodontitis: for example, Machado et al’s. study 20 that aimed to verify the relationship between severe and moderate periodontal diseases and blood lipid levels. This study demonstrated that there is no significant relationship between periodontal disease, regardless of its intensity, and blood lipid levels in the studied population. Banihashemrad et al. 21 studied the relationship between cholesterol and triglyceride blood values and periodontal parameters in patients at the Mashhad Health Center. Although there was a direct correlation between periodontal disease and the measured biochemical parameters, none of these correlations were significant.

In Moeintaghavi et al.’s study 22 on hyperlipidemia in patients with periodontitis, the results showed that hyperlipidemia may be associated with periodontitis in healthy people. However, it is controversial whether periodontitis causes an increase in levels of serum lipids, and whether hyperlipidemia is a risk factor for both periodontitis and cardiovascular diseases.

It is obvious from previous literature that there is a significant relationship between hyperlipidemia and periodontal diseases based on the host-immune response. The aim of this study was to elucidate this relationship, which would lead to an understanding of an important mutual etiologic factor of hyperlipidemia and periodontal disease.

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Patients and methods

Patients’ selection

This study was conducted on 24 patients (13 men and 11 women) aged 28–67 years. All patients were selected from the Cardiology and Oral Medicine Department Clinics of the Faculty of Medicine and Faculty of Dentistry, Ain Shams University (Cairo, Egypt).

Eligible patients fulfilled the following criteria:

They had 10 or more natural teeth;

They were 28 years of age or older to avoid puberty hormonal bias;

They were suffering from moderate-to-severe chronic periodontitis in at least one tooth, with attachment loss (AL) of more than or equal to 4 mm 23–25;

They were free from systemic diseases known to influence the periodontal condition;

The enrolled individuals had not received any periodontal therapy for a minimum of 6 months before the study.

Exclusion criteria were as follows:

Smoking history;

History of systemic disease that affects lipid metabolism, such as impaired glucose tolerance, diabetes mellitus. or other endocrine diseases, nephrotic syndrome, chronic renal disease, and cardiovascular disease 25;

History of drug treatment of hyperlipidemia;

Pregnancy or lactation;

History of systemic antibiotic administration within the last 3 months;

Known hypersensitivity or allergy to one of the used medications;

Participation in other clinical trials related to the selection criteria of our study;

History of alcohol abuse;

Were receiving any medication known to affect the periodontal status.

A specially designed sheet (a copy is included after references) for the study was created and printed that included patients’ personal history and a questionnaire pertaining to hypertension history, smoking status, cardiac problems, diabetes mellitus, hypersensitivity, and daily brushing habit. A table was also used to fill in the results of the different laboratory and clinical tests conducted on the patient.

Patients were divided into three groups:

Group I: Patients with no history of dyslipidemia or lipid-lowering treatment who had undergone only periodontal treatment;

Group II: Patients with a history of dyslipidemia who did not undergo any lipid-lowering treatment during the study and underwent only periodontal treatment;

Group III: Patients with a history of dyslipidemia who received lipid-lowering treatment during the study in addition to periodontal treatment.

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Study design

The study used a randomized, single, blind, case–controlled, parallel technique.

During the first visit, patients who met the inclusion/exclusion criteria were assigned numbers in ascending order. After being informed of the nature and purpose of the study, the patients signed an informed consent form, which included a questionnaire about their personal history and medical history and finally tables containing all laboratory and clinical examinations carried out on the patient. The top of the form contained the group number and patient number, where a numbering system had been introduced for the patients and the groups. Thereafter, a 4-cc blood sample was taken from the antecubital vein, which was held undisturbed for 0.5 h, allowing the blood to clot. A comprehensive periodontal examination was then conducted by a single periodontist who motivated the patients to exercise oral hygiene measures. The patients were given detailed instructions on how to self-perform plaque control. Full-mouth supragingival and subgingival scalings were performed using an ultrasonic scaler that had been specially modified to permit scaling in the Cardiology Department. This was followed by root planning for all the enrolled patients. Finally, a mouthwash was prescribed for all the patients enrolled in the study.

Patients in group III received suitable lipid-lowering treatment during the initial visit. A pharmacologically controlled drug was prescribed to group III individuals (Lipantyl; Solavy Alexandria Chem., Alexandria, Egypt, Lipinorm; Tabuk Pharmaceuticals, Saudi Arabia) and they were followed periodically to ensure that the drug prescribed was being taken by the individuals as instructed by the cardiologist.

After the first visit, patients were recalled weekly for three successive weeks during which time their oral hygiene was checked and reinforcement of oral hygiene was carried out. After 3 weeks, a comprehensive periodontal examination was conducted. It was ensured that the patients were taking their prescribed medication if any. A 4-cc blood sample was taken from the antecubital vein and oral hygiene was checked and reinforced.

The final visit was 6 months after the initial visit. A comprehensive periodontal examination was carried out and it was ensured that the patient was taking his prescribed medication if any. A 4-cc blood sample was taken from the antecubital vein and oral hygiene was checked and reinforced.

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Clinical parameters

Dental examinations were assessed at six points (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual) around each tooth of 10 teeth (four molars and six anteriors in the upper and lower arches equally) at baseline and 3 weeks and 6 months after the start of the study.

Clinical measurements of periodontal parameters were recorded and consisted of the following: plaque index (PI), pocket depth (PD), AL, and bleeding index (BI). All assessments were carried out with a Williams graduated periodontal probe 26.

PI 27;

BI 28;

PD 29;

Clinical attachment levels (CAL) 29.

A fixed-point determination was made by using the anatomical Cemento Enamel Junction or the apical extent of the existing restoration margin on each tooth. PD and CAL were measured at six following sites on each tooth: mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual.

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Treatment protocol

All three study groups were subjected to a treatment protocol that had the following criteria:

Oral hygiene measures, in which a modified Stillman tooth-brushing technique was adopted. Complete instructions and a demonstration were given to all individuals in the study;

Supragingival and subgingival scaling using ultrasonic scaler and root planning;

Chlorhexidine mouth washes (0.12%) four times/day for 2 weeks were prescribed for 2 weeks for all study individuals.

All individuals of the study were subjected to a therapeutic periodontal protocol comprising chemotherapy (chemical control) and mechanotherapy (mechanical control). The chemotherapy consisted of chlorohexidine mouth wash (0.12%) four times per day for 2 weeks to avoid teeth discoloration and superinfection. In addition, oral hygiene methods were stressed upon by training the patients on proper oral hygiene, providing them with a hygienic tooth brush and tooth paste. The mechanotherapy consisted of supragingival and subgingival scaling comprising two types of mechanical techniques: a peizostrictive ultrasonic scaler, besides manual scaling and root planning. This ensured full control of the periodontal infection. Apart from adoption of these techniques, the oral hygiene status was followed using the same indices during the study every week for 3 weeks and then after 6 months.

Results of all laboratory and periodontal examinations were collected and the means of the results of the periodontal examinations were obtained and compared with those of laboratory investigations conducted throughout the follow-up period. All the results were studied statistically to determine whether there was statistical significance between the different results throughout the study.

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Laboratory parameters

Criteria of dyslipidemia were as follows:

Plasma triglyceride value of more than 200 mg/dl;

Plasma LDL-C value of more than 130 mg/dl;

Plasma high-density lipoprotein cholesterol (HDL-C) value of less than 35 mg/dl.

The patients with hyperlipidemia who participated in the study were suffering from type IIa hyperlipidemia.

Venous blood samples were obtained from the antecubital vein for the measurement of

Plasma triglycerides;

Total cholesterol;

LDL-C;

HDL-C;

Proinflammatory cytokines [analyzed by enzyme-linked immunosorbent assay (ELISA)].

In the clinical biochemistry laboratory, blood was centrifuged using a Universal centrifugation machine (Universal Inc., USA) with a speed of 1300 rpm for 15 min. Approximately 2 cm3 of serum was obtained from it, which was separated into two separate tubes: one for lipid profile examination and the other for the ELISA test. The tubes were marked with the patients’ numbers and the date of the test and stored at −70° centigrade until assays were performed. Repeated freeze–thaw cycles were avoided. Plasma lipids were determined in the Clinical Biochemistry Laboratory using the Lipid Research Clinics Program 30,31.

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Results

In our study, mean baseline PD of group I was 2.36, of group II was 2.16, and of group III was 2.61. The mean baseline BI of group I was 2.36, of group II was 2.14, and of group III was 2.20. The mean baseline PI of group I was 2.37, of group II was 2.19, and of group III was 2.24. The mean baseline CAL of group I was 3.67, of group II was 2.97, and of group III was 4.46.

The mean baseline serum level of IL-1β was 5.44 pg/ml for group I, 19.51 pg/ml for group II, and 8.21 pg/ml for group III. These data show that the highest baseline serum level of IL-1β was in group II, followed by group III and then by group I.

The mean baseline serum level of TNF-α was 43 pg/ml for group I, nil for group II, and 19.13 pg/ml for group III, which shows that the baseline serum level of TNF-α was the highest in group I compared with groups II and III. The levels of TNF-α in the whole examination were very low, showing nil result in most of the wells.

In group I, there was an increase in the mean value of triglycerides through all follow-up periods, which was statistically nonsignificant. There was a decrease in the mean value of cholesterol through all follow-up periods, which was also statistically nonsignificant. There was a decrease in the mean value of LDL from week 0 to week 3, followed by a slight increase at 6 months. There was a decrease in the mean value of HDL from week 0 to week 3, followed by a slight increase at 6 months. There was a decrease in the mean value of IL-1β from week 0 to week 3, followed by a slight increase at 6 months at P<0.14, which was very close to P<0.05. There was a decrease in the mean value of TNF-α from week 0 to week 3, followed by a slight increase at 6 months at P<0.06, which was very close to significance. There was a decrease in the mean values of PD through all follow-up periods. This decrease was statistically significant between week 0 and week 3 and between week 0 and month 6. There was a decrease in the mean value of BI from week 0 to week 3, followed by a slight increase at 6 months. This result was statistically significant between week 0 and week 3 and between week 0 and month 6. There was a decrease in the mean value of PI from week 0 to week 3, followed by a slight increase at 6 months. This result was statistically significant between week 0 and week 3. There was an increase in the mean value of CAL through all follow-up periods. This result was statistically nonsignificant between all follow-up periods.

In group II there was decrease in the mean value of triglycerides through all follow-up periods. This decrease was statistically nonsignificant. There was an increase in the mean value of cholesterol from week 0 to week 3, followed by a slight decrease at 6 months. This result was statistically nonsignificant. There was an increase in the mean value of LDL through all follow-up periods. This increase was statistically nonsignificant. There was an increase in the mean value of HDL through all follow-up periods, which was again statistically non-significant. There was a decrease in the mean value of IL-1β from week 0 to week 3, followed by no change at 6 months. This result was statistically nonsignificant. The result for TNF-α was the same for all follow-up periods. There was a decrease in the mean values of PD through all follow-up periods. This decrease was statistically significant between week 0 and week 3 and between week 0 and month 6. There was a decrease in the mean value of BI from week 0 to week 3, followed by no change at 6 months. This result was statistically significant between week 0 and week 3 and between week 0 and month 6. There was a decrease in the mean value of PI in all follow-up periods. This result was statistically significant between week 0 and week 3 and between week 0 and month 6. There was a slight increase in the mean value of CAL from week 0 to week 3, followed by no change at 6 months. This result was statistically nonsignificant between all follow-up periods.

In group III there was an increase in the mean value of triglycerides from week 0 to week 3, followed by a decrease at 6 months. This increase was statistically nonsignificant. There was a decrease in the mean value of HDL from week 0 to week 3, followed by a slight increase at 6 months, which was also statistically nonsignificant. This can be explained by the high value of standard deviation and/or small sample size. There was a decrease in the mean value of cholesterol from week 0 to week 3, followed by a slight increase at 6 months. This result was statistically significant between week 0 and week 3. There was a decrease in the mean value of LDL from week 0 to week 3, followed by a slight increase at 6 months. This result was also statistically significant between week 0 and week 3. There was a decrease in the mean value of IL-1β from week 0 to week 3, followed by a slight increase at 6 months at P<0.07, which was very close to significance. This result was statistically nonsignificant. There was a decrease in the mean value of TNF-α from week 0 to week 3, followed by a slight increase at 6 months. This result was statistically nonsignificant. There was a decrease in the mean values of PD from week 0 to week 3, followed by a slight increase at 6 months. This decrease was statistically nonsignificant. The change was greater during the first period (week 0–week 3), and the difference between the first (week 0–week 3) and second periods (week 3–month 6) was statistically significant. There was decrease in the mean value of BI from week 0 to week 3, followed by a slight increase at 6 months. This result was statistically significant between week 0 and week 3. There was a decrease in the mean value of PI from week 0 to week 3, followed by a slight increase at 6 months. There was a slight increase in the mean value of CAL from week 0 to week 3, followed by a slight decrease at 6 months. This result was statistically nonsignificant between all follow-up periods.

At baseline, group III showed higher mean values of triglycerides, cholesterol, HDL, LDL, and TNF-α compared with group II and group I, and the differences were statistically nonsignificant except for triglycerides. This is because group III was a hyperlipidemic group. Triglyceride values were statistically significant between group I and group III, which could be because group I was normolipidemic. Group II showed higher mean values of IL-1β compared with group I and group C, and the differences were statistically nonsignificant.

At week 3, triglycerides showed higher values for group III, followed by group II and then by group I. The differences were statistically nonsignificant. Cholesterol showed higher values for group II, followed by group III and then by group I, and the differences were statistically nonsignificant. LDL showed higher values for group II, followed by group III and then by group I. The differences were statistically significant between group I and group II and between group III and group II. This was because they were hyperlipidemic groups. HDL showed higher values for group I, followed by group II and then by group III, and the differences were statistically nonsignificant. IL-1β showed higher values for group II, followed by group I and then by group III, and the differences were statistically nonsignificant. TNF-α showed similar values (0) for all groups.

At 6 months, triglycerides showed higher values for group III, followed by group I and then by group II, and the differences were statistically nonsignificant. Cholesterol showed higher values for group II, followed by group III and then by group I, and the differences were statistically nonsignificant. LDL showed higher values for group II, followed by group III and then by group I, and the differences were statistically nonsignificant. HDL showed higher values for group II, followed by group I and then by group III, and the differences were statistically nonsignificant. IL-1β showed higher values for group I, followed by group II and then by group III, and the differences were statistically nonsignificant. TNF-α showed higher values for group I, followed by group III, and the differences were statistically nonsignificant (Tables 1–3).

Table 1
Table 1
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Table 2
Table 2
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Table 3
Table 3
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Correlation between interleukin-1β and the lipid profile

The correlation between IL-1β and triglycerides, total cholesterol, and HDL was weak and negative and nonsignificant, whereas it showed a weak positive correlation with LDL, which was also nonsignificant.

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Correlation between tumor necrosis factor α and lipid profile

The correlations between TNF-α and triglycerides, total cholesterol, and LDL were weak and positive and all of them were nonsignificant, whereas it showed a weak negative correlation with HDL, which was also nonsignificant.

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Discussion

The association between periodontal health and hyperlipidemia has been discussed widely in the dental literature 12,13,17,22,24,25,32–42. Although very limited data have been reported on the periodontal status of patients with hyperlipidemia who underwent medical evaluation, it has been reported that hyperlipidemic patients manifested poor periodontal status compared with healthy controls 25,38. The findings in these studies seem to conclude that successful management of periodontal infection may lead to a reduction in local periodontal symptoms and may have a beneficial effect on the metabolic control of hyperlipidemia.

IL-1β and TNF-α can be considered as suitable serum parameters in studying the effect of hyperlipidemia on the clinical results of conventional periodontal treatment while monitoring the changes in their serum levels induced by the treatment.

We wished to determine the effect of chemical and mechanical conventional periodontal treatment on the clinical and laboratory response of lipid profile and proinflammatory cytokines in normolipidemic and hyperlipidemic individuals with and without lipid-lowering treatment.

The study population was relatively small because of the stringent eligibility requirements. We have eliminated a number of potential confounders such as impaired glucose tolerance, diabetes mellitus or other endocrinal diseases, nephritic syndrome, chronic renal disease, and cardiovascular disease, which are believed to be involved in the development of periodontal disease, in addition to smoking status, use of antibiotics, and use of dyslipidemia-lowering drugs, which to our knowledge have not been suggested except in one study 43.

The study was designed to determine the exact change in the serum lipid level before, during, and after 6 months of periodontal treatment. We also sought to determine whether it is due to the direct effect of periodontal treatment, due to the body metabolic hemostasis, or due to the direct effect of lipid-lowering treatment. We also wanted to determine whether all these factors act together and, if so, which factor is the most effective among them. Furthermore, we wanted to determine whether periodontal treatment has any significant effect on lipid metabolism.

Ethically, it's accepted that group II comprising individuals diagnosed with hyperlipidemia who did not receive lipid-lowering treatment. Because periodontal treatment has an effect on lowering unfavorable lipids in many papers, as shown in the review of literature. In addition, triglyceride levels were shown to decrease in this group in our study, although this decrease was nonsignificant. This may be because of methodological problems or the small sample size or the short study duration. Beside all patients were informed of the nature of the study before its start.

Smokers were excluded from the study as previous studies have shown that smokers exhibit altered inflammatory response to LPSs 44.

A minimum of 10 remaining teeth was one of the inclusion criteria to minimize the influence of tooth number on the total number of periodontal pockets, with at least one tooth with AL more than or equal to 4 mm. This was not considered a weak number of affected teeth, as has been shown in the literature 23–25.

Any patient who did not follow the oral hygiene instructions was excluded from the study to avoid confounding factors related to the patient.

Besides serum lipid analysis, ELISA investigation of serum for proinflammatory cytokines of IL-1β and TNF-α (RayBio Human ELISA kit [RayBiotech, Inc., Norcross GA, USA]) according to the manufacturer’s protocol was performed. The sensitivity of ELISA was reported to be more than the immunofluorescence and gives identical results to those of radioimmunoassay without the hazards of radioactive agents 45–47.

Samples were collected in sterilized sealed tubes to avoid bacterial contamination, which may lead to further release of proinflammatory cytokines by immunological cells in the sample.

Serum samples were taken at baseline, after 3 weeks, and after 6 months. This was to determine whether periodontal treatment and/or the serum lipid level had a direct effect on proinflammatory cytokines in serum. We also wanted to determine whether the change in proinflammatory cytokines in serum was due to the periodontal treatment or because of serum lipid control, by comparing the change in proinflammatory cytokines within the normolipidemic group with that in the hyperlipidemic group with and without lipid-lowering treatment.

The highest baseline serum level of IL-1β was seen in group II, followed by group III and then group I, showing that levels of IL-1β was least in the normolipidemic group. This is in accordance with previous studies that stated that LPS-stimulated monocytes from dyslipidemic patients released significantly more TNF-α (types IIa and IIb dyslipidemias) and IL-1β (type IIa dyslipidemia) in comparison with monocytes in 59 age-matched, sex-matched, and weight-matched control individuals 48.

The highest baseline serum level of TNF-α was in group I compared with groups II and III. The results of TNF-α in the whole examination was very weak, showing nil result in most of the wells. This may be attributed to methodological discrepancy and/or small sample size and/or high value of standard deviation, or because of the low sensitivity of the kit for TNF-α (30 pg/ml) in comparison with IL-1β (0.3 pg/ml).

In group I, the nonsignificant change in lipid profile throughout the study could be attributed to the fact that it was a normolipidemic group before and throughout the study. Therefore, these changes were within the normal ranges of metabolic control. The IL-1β and TNF-α results were statistically nonsignificant, which could be explained by the high value of standard deviation and/or small sample size.

In group II, all the lipid profile parameters showed a nonsignificant difference in changes throughout the study. These results are in accordance with those of previous studies that indicated that hyperlipidemia may be associated with periodontitis in healthy people. However, it is unclear whether periodontitis causes an increase in levels of serum lipids or that hyperlipidemia is a risk factor for both periodontitis and cardiovascular disease (Pussinen et al. 16, Moeintaghavi et al. 22, D’Aiuto et al. 49, Banihashemrad et al. 21). IL-1β showed a nonsignificant change throughout the study. The result for TNF-α was the same for all follow-up periods. This can be explained by the high value of standard deviation and/or small sample size.

In group III, the change in triglyceride and HDL levels showed a nonsignificant change, which could be explained by the high value of standard deviation and/or small sample size. In the meantime, LDL and total cholesterol levels showed a significant decrease in values, which could be explained by the effect of the lipid-lowering treatment. Whether adjunctive periodontal treatment had an effect could not be confirmed, which is in accordance with Fentoglu et al.’s study 50. There was a decrease in the mean value of IL-1β at P<0.07, which was very close to significance but was statistically nonsignificant. There was a decrease in the mean value of TNF-α, which was also statistically nonsignificant. These results, although nonsignificant, show a decrease in the levels of proinflammatory cytokines when lipid-lowering treatment was used in conjunction with periodontal treatment, in accordance with previous studies 50.

A statistically significant reduction in all periodontal parameters except attachment level at a P value of less than 0.05 was observed in our results in all three groups after periodontal treatment. The professional mechanical instrumentation reduces the number of pathogens populating the subgingival biofilms of the infected periodontium and removes the environment conducive to microbial colonization. Added to this was the antimicrobial effect of 0.12% cholorohexidine gluconate 51.

The difference between the lipid profile changes within each group and between different groups did not show significant difference, except in group III in which the difference was observed only in LDL-C and total cholesterol levels at P<0.05. These results show that only lipid-lowering drugs could statistically significantly decrease the lipid clinical parameters. We could not prove whether periodontal parameters had an adjunctive role and more research work is needed to prove or disprove this effect.

Although group II, which is the group with hyperlipidemia that did not receive lipid-lowering drugs, showed a decrease in triglyceride levels, it was a nonsignificant change. However, this result may show a trend, which means that it may show more significant results with a larger study population or with a longer study period.

Although there are several studies with regard to the association between periodontal disease and systemic lipid levels, the results are extremely controversial. Some studies 12,13,26 have suggested that there is a relationship between cholesterol levels and periodontitis, whereas other studies 12,24,26 have shown a relationship between triglyceride levels and periodontitis. Some other studies have reported that there is a relationship between periodontal disease, cholesterol, and triglycerides 26,37; however, it was also reported that lipid marker changes were insignificant between standard periodontal treatment and control groups, and some reductions of total and LDL cholesterols were present only in the intensive periodontal treatment group 49. A study has also shown that periodontitis is associated with a reduction in HDL cholesterol level and that periodontal therapy results in an increase in this antiatherogenic lipid fraction 16. This discrepancy may arise from the methodological difficulties associated with the complexity of lipid metabolism and with the variety in the lipid-measuring parameters. Consequently, a homogenous hyperlipidemic population that is categorically divided (e.g. as mild or moderate) by the physician is required for the evaluation of the association between periodontal disease and hyperlipidemia. Another possible cause is the genetic cause of hyperlipidemia, as approximately one in every 500 people have familial hypercholesterolemia.

Finally, although longitudinal evaluations with larger populations may be an important step in revealing the causal relationship between periodontal disease and impaired lipid metabolism, our study may provide a different viewpoint for clinicians and it may shed light on the clarification of this association in future studies.

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Conclusion

This study reached the following conclusions: the clinical outcome of periodontal treatment did not show a significant relationship with the laboratory outcome of the lipid profile. The laboratory outcome of proinflammatory cytokines in serum did not show a significant relationship with the lipid profile laboratory outcome. The only significant improvement in the lipid profile was in total cholesterol and LDL-C in the group receiving lipid-lowering treatment and the study could not prove or deny whether periodontal treatment outcome could have an effect on this significant change. The laboratory outcome of proinflammatory cytokines in serum did not show a significant relationship with periodontal treatment outcome.

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Clinical relevance
Rationale of the study

A high odds ratio has been reported for hyperlipidemia and periodontal diseases in humans, and the severity of periodontitis seems to correlate with the hyperlipidemic status of the patients. Early studies have indicated that the lipoprotein-containing fraction of the serum enhances the leukotoxic activity of the periodontopathogen Actinobacillus actinomycetemcomitans against human PMNL. The protease inhibitors of normal serum account for this enhancement, whereas delipidated serum has no effect on leukotoxin-dependent PMNL cytolysis. No information exists for the effect of serum lipoproteins or hyperlipidemic serum.

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Principal findings and practical implications

Previously, it was thought that serum lipid alterations were related to the underlying pathological conditions rather than to the infectious process. However, recent studies have demonstrated that lipid metabolism may be altered by chronic local and acute systemic infections, which are involved in the plasma concentrations of unregulated cytokines and hormones. The main features of this catabolic state are lipid oxidation and elevated free fatty acids, triglycerides, and LDL-C.

It is obvious from previous literature that there is a significant relationship between hyperlipidemia and periodontal diseases based on host immune response and proinflammatory cytokines.

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Acknowledgements

The study was self-funded by the authors and their institution.

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Conflicts of interest

There are no conflict of interest.

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