Correlation of salivary neopterin and plasma fibrinogen levels in patients with chronic periodontitis and/or type 2 diabetes mellitus

Introduction

Periodontitis is a common inflammatory disease affecting the periodontium of the teeth [1]. It has been observed that 10%–15% of adults are affected with periodontitis [2]. Major tissue destruction during periodontal disease results from the activation of various immune cells [3], thereby the proinflammatory mediators and acute phase proteins like capsular reactive protein (C reactive protein/CRP) and plasma fibrinogen are elevated.

Conventional periodontal diagnostic techniques give an idea about the disease severity, whereas biomarkers indicate presence or absence of disease activity. Neopterin is a biomarker of cellular immune activation during which T-lymphocytes secrete interferon γ, which induces human macrophages to produce neopterin [4]. The remarkable value of neopterin as an immunodiagnostic tool is that it reflects multiple cooperations between immunocompetent cells [5]. Neopterin concentrations increase in parallel with the clinical course of infections [6], and it can be determined in human body fluids like serum, plasma, urine, cerebrospinal fluid, saliva, gingival crevicular fluid, synovial fluid and ascetic fluid [7]. Increased neopterin levels are related to increased body mass indices at which state the moderate immune stimulation leads to insulin resistance [8]. Neopterin is also a novel predictor for coronary events [9], and diabetic patients have increased possibility of acquiring cardiovascular disease [10], [11].

Plasma fibrinogen level is a useful predictor of cardiovascular disease risk, and >3.5 g/L is found in atherosclerotic cardiovascular disease and periodontitis. Fibrinogen is an acute phase protein whose excessive levels cause increased blood viscosity and an increase in proinflammatory cytokine production [12].

In this study, we compared the changes in salivary neopterin levels and plasma fibrinogen levels in chronic periodontitis patients, with and without type 2 diabetes mellitus. Levels of neopterin were assessed in these patients after nonsurgical periodontal therapy (NSPT) by comparing the changes that may have occurred after the therapy, thereby reducing the cardiovascular disease risk by assessing plasma fibrinogen levels before and after NSPT in those patients. By doing this study, we intended to find whether any correlation of periodontal clinical parameters and diabetic status with salivary neopterin exists so that salivary neopterin could be considered as a biomarker in the progression of periodontal disease in diabetic patients.

Materials and methods

This study was conducted in the Department of Periodontology in Amrita School of Dentistry, Kochi. Ethical approval for the study was obtained from the Amrita Vishwa Vidhyapeetam University, Kochi, Kerala. Forty subjects between 25 and 75 years of age and who matched the inclusion and exclusion criteria were selected from the outpatient section of the Department of Periodontology, and the control group was selected from among the staff of Amrita School of Dentistry. All the study participants signed the informed consent before the study and were divided into four groups:

• Group 1: patients with both of chronic periodontitis and diabetes mellitus (CP+D)

• Group 2: patients with chronic periodontitis only (CP)

• Group 3: patients with diabetes mellitus only (D)

• Group 4: controls, volunteers neither with chronic periodontitis nor with diabetes mellitus (C).

Inclusion criteria included subjects belonging to age-group 25–75 years and presence of at least 20 natural teeth, with chronic generalized periodontitis in >30% sites of teeth involved with a clinical attachment loss of >5 mm at five or more sites and diagnosed with type 2 diabetes mellitus confirmed by HbA_1c level (>6.5%).

The following subjects were excluded from the study: subjects with a history of habits like tobacco usage or alcohol consumption, having chronic inflammatory disorders of skin and oral mucosa, presence of systemic diseases like hypertension or other cardiovascular disorders, bleeding disorders, hepatitis or human immunodeficiency virus (HIV) infection and immunosuppressive chemotherapy, subjects who have had antibiotics or anti-inflammatory drugs within the past 6 months, subjects who underwent professional cleaning/periodontal treatment within the past 6 months or diagnosed as aggressive periodontitis and females in pregnancy or lactation period.

The following periodontal parameters were analyzed:

• Oral hygiene index-simplified (OHI-S)

• Gingival index (GI)

• Pocket probing depth (PPD)

• Clinical attachment level (CAL).

The periodontal parameters were calculated by an investigator not involved in the study, using mouth mirror and William’s graduated periodontal probe. PPD and CAL were measured with a customized acrylic stent as a reference. Plasma fibrinogen, HbA_1c levels and salivary neopterin levels were evaluated at baseline and 3 months after NSPT. Five milliliter and 2 mL of peripheral venous blood were collected from study subjects to estimate plasma fibrinogen and HbA_1c levels, respectively. HbA_1c levels were estimated using ion-exchange high-performance liquid chromatography, plasma fibrinogen levels were estimated using Plasma fibrinogen kit. Patients were asked to chew on paraffin wax for 2 min before NSPT. Two milliliters of whole stimulated saliva was collected in eppendorf tubes after 30 s of chewing, to evaluate salivary neopterin levels using ELISA kit [Human Neopterin (Np) ELISA kit, Catalogue No: E13651281, Sincere Biotech Co., Ltd, Shunyi District, Beijing, China].

After that, all subjects received oral hygiene instructions and NSPT, which included thorough supragingival and subgingival scaling and root planning. These patients were recalled after 3 months, and the same sample collection procedures were repeated. Total of 80 samples were analyzed for salivary neopterin levels.

Results

This study included four groups: Group CP+D with seven males and three females (47.8±7.011, mean age± standard deviation), Group CP with five males and five females (47.8±8.077), Group D with seven males and three females (49.4±7.011) and Group C with six males and four females (34.6±2.451).

Tables 1–4 show the intragroup comparison of all variables before and after treatment in Group CP+D, Group CP, Group D and Group C, respectively.

All the variables, GI, OHI-S, CAL, PPD, plasma fibrinogen, HbA_1c levels and salivary neopterin levels, were found to be significant (p<0.001^a) after treatment in all the groups except for CAL, HbA_1c and neopterin levels in Group C, PPD in Group D and Group C.

Figure 1 shows plasma fibrinogen in each group before and after treatment. The plasma fibrinogen levels before treatment in Group CP+D was 342.70±23.06 mg/dL, in Group D was 336.70±25.69 mg/dL, in Group CP was 295.10± 18.08 mg/dL and in Group C was 145.40±14.01 mg/dL. It also shows that after treatment, the plasma fibrinogen levels in Group CP+D reduced to 259.60±15.28 mg/dL, in Group CP to 252.50±21.25 mg/dL, in Group D to 259.00±15.78 mg/dL and in Group C to 138.70±14.99 mg/dL.

Figure 1:

Plasma fibrinogen levels (mg/dL) in each group before and after treatment (mean±SD values are shown).

*Denotes the statistical significant difference after periodontal treatment.

Figure 2 shows salivary neopterin levels in each group before and after treatment. Before treatment, the salivary neopterin level in Group CP+D was 38±4 nmol/L, in Group CP was 37.2±3.2 nmol/L, in Group D was 39.2±3.6 nmol/L and in Group C was 9.2±38 nmol/L. Figure 2 also shows that the salivary neopterin levels in all the groups showed significant reduction after treatment, i.e., 30±5.2 nmol/L in Group CP+D, 27.2±6 nmol/L in Group CP, 30.8±6 nmol/L in Group D and 7.6±0.4 nmol/L in Group C.

Figure 2:

Salivary neopterin levels (nmol/L) in each group before and after treatment (mean±SD values are shown).

*Denotes the statistical significant difference after periodontal treatment.

Figures 3 and 4 show the correlation of salivary neopterin levels in Group CP+D, Group CP, Group D and Group C before and after treatment with plasma fibrinogen and HbA_1c levels, respectively. Before treatment, there is positive correlation of salivary neopterin levels with plasma fibrinogen and HbA_1c levels in Group CP+D and in Group CP and with fibrinogen levels in Group D. There is statistically significant correlation of salivary neopterin levels with HbA_1c levels in Group D before treatment. After treatment, there is statistically significant correlation with plasma fibrinogen and neopterin levels in Group D after treatment.

Figure 3:

Intergroup correlation of plasma fibrinogen and salivary neopterin level before and after treatment.

Figure 4:

Intergroup correlation of salivary neopterin and HbA_1c levels before and after treatment.

Discussion

Salivary neopterin is a very sensitive indicator of an ongoing inflammatory state and can reflect minor inflammatory changes as a result of periodontitis. Thus, we intended to assess the possibility of using salivary neopterin and plasma fibrinogen as an early indicator of cardiovascular disease risk in diabetic patients with chronic periodontitis.

With 80% of power and 95% of confidence interval, a total of 40 subjects, who matched the inclusion and exclusion criteria and belonging to 25–75 years of age were selected [13]. Clinical parameters, OHI-S, GI, PPD and CAL, at the baseline and after 3 months of NSPT were recorded by a trained clinician who was not involved in the study. GI, OHI-S, PPD and CAL assess the severity and quantity of gingival inflammation [12], [14].

Forty subjects of the age-group 25–75 years included in the study. Studies [15], [16] assessed age association to periodontitis and diabetes and found that periodontitis and diabetes are more prevalent in the third to sixth decades of life. In our study, there were a higher number of males present in Group CP+D and in Group D. Another study [17] observed that there was a higher incidence of periodontitis and diabetes in older individuals and also higher in males when compared to females. Yet another study assessed the association of age and gender with neopterin levels and found that the changes in neopterin with age were associated with gender and race differences [18].

From our study, GI, OHIS and plasma fibrinogen showed statistically significant difference before and after NSPT in all the groups (p<0.001) (Tables 1–4 and Figure 1). CAL, HbA_1c and neopterin also showed statistically significant difference before and after treatment in Group CP+D, Group CP and Group D (p<0.001) (Figure 2). Also, PPD showed statistical significant difference after treatment in Group CP+D and Group CP (Tables 1 and 2).

Various variables assessed here and their correlation with findings before treatment are similar to several studies where they found that diabetes mellitus and attachment loss were positively associated, with an odds ratio of 2.32 [17], [19], [20].

In our study, a reduction in all the variables was seen after treatment in all the four groups (Tables 1–4). NSPT still remains the treatment of the choice for the initial treatment of periodontal diseases. There is a statistically significant reduction (p<0.05) in all the parameters after NSPT in our study (Tables 1–4), which is similar to various other studies conducted wherein improvement in clinical parameters in 3 months after NSPT has been observed [21], [22].

Periodontal disease has been considered as the sixth complication of diabetes. In our study, HbA_1c levels in Group CP+D were the highest, followed by Group D, Group CP and Group C before treatment (Tables 1–4). Studies [19], [22], [23], [24] report that the inflammatory mediators enter systemic circulation and interfere with actions of insulin receptors and cause insulin resistance and poor glycemic control and confirm the possible biologic link between diabetes and periodontal disease.

After 3 months of NSPT, HbA_1c levels in our study subjects showed a statistical significant reduction (p<0.001) in all the study groups (Tables 1–4). This shows that NSPT has the potential to improve the glycemic control in periodontitis patients with and without diabetes, in patients with only diabetes as well as in the healthy group with no diabetes or periodontitis, which is in accordance with various other studies conducted [23], [24], [25]. Contrary to our study, one study has concluded that NSPT did not improve glycemic control in patients with diabetes mellitus and moderate to advanced chronic periodontitis [26]. Group CP showed significant mean difference in HbA_1c levels before and after treatment (Table 2), which is in accordance with similar studies wherein it was concluded that HbA_1c progression and periodontal disease was related in diabetes-free participants, and the HbA_1c levels of periodontitis subjects without diabetes reduced significantly after 3 months of NSPT [27], [28].

Plasma fibrinogen is one of the acute phase proteins whose levels are increased in diabetes and chronic periodontitis. In our study, plasma fibrinogen levels before NSPT in all the three groups showed statistically significant higher value (p<0.001) when compared to the control group (Tables 1–3). Group D showed a statistically significant greater plasma fibrinogen level, which is similar to studies that showed same results [29], [30]. This rise in plasma fibrinogen in diabetics is due to procoagulant state resulting in hyperfibrinogenemia, presence and association of lipoprotein (a) [Lp(a)] and fibrin at the sites of vascular injury [29]. In our study, baseline plasma fibrinogen levels were also significantly elevated in Group CP (Table 2), which is similar to the results of a study conducted in 2003 [31] where the results concluded that it is the presence of H1H2 or H2H2 genotypes in chronic periodontitis patients, which is associated with higher plasma fibrinogen levels than healthy individuals. Another study [32] concluded that platelet aggregation is induced by the platelet aggregation associated protein expressed on some strains of periodontal bacteria that lead to increased plasma fibrinogen levels during chronic periodontitis. Hence, it can be inferred that plasma fibrinogen levels increase in diabetic patients affected with chronic periodontitis.

In our study, plasma fibrinogen levels after NSPT have shown a significant reduction (p<0.001) when compared to their baseline values (Tables 1–4 and Figure 1). These results from our study are similar to the results from the study conducted in 2013 [33], which showed a reduction in plasma fibrinogen levels after 2 months. Contrary to our study, few studies [34], [35] found no significant reduction in plasma fibrinogen levels after NSPT.

The results of plasma fibrinogen levels from our study showed that there was a significant reduction in plasma fibrinogen levels in all the three study groups and the control group after 3 months. Thus, NSPT is a very successful treatment modality in bringing down plasma fibrinogen levels in all groups of patients irrespective of whether they have diabetes or periodontitis. We have observed that although there was a reduction in plasma fibrinogen levels after 3 months, it has not reached normal levels. Thus, bringing down plasma fibrinogen levels in diabetics, we can bring down the cardiovascular risk in them associated with increased plasma fibrinogen levels.

Neopterin is regarded as a biomarker of cell-mediated immunity [36]. Its estimation determines the total effect of the immunological network and monocytes/macrophage interactions [4]. Whole saliva represents variable amounts of blood, serum, serum products, epithelial and immune cells, etc. [37] so the concentrations of neopterin levels in saliva are similar to that present in serum [38]. Another study [39] concluded that neopterin measurements provide information on a current state of cellular immune response and help to predict the disease progression. Salivary neopterin levels assessed in our study showed significant difference (p<0.001) in the values between all the study groups before treatment (Tables 1–4 and Figure 2). There was no significant difference in neopterin levels between study groups before treatment. High salivary neopterin levels seen in Group D in our study is in accordance with various other studies [5], [8], [43]. Contrary to this, another study in 1999 [40] concluded that there is no increase in neopterin levels in type 2 diabetes mellitus.

Salivary neopterin levels were also high in group CP+D and in Group CP before treatment (Tables 1 and 2 and Figure 2), which are similar to many studies [4], [41], [42] where they concluded that neopterin levels are positively correlated with the periodontal disease and may be an indicator of periodontal disease activity.

Salivary neopterin levels have shown statistically significant reduction after NSPT in three study groups: Group CP+D, Group CP and Group D (p<0.001). There was reduction in neopterin levels in the control group (Group C) although not statistically significant. Our study is perhaps the first study to assess salivary neopterin levels after NSPT in diabetic and chronic periodontitis patients. Our results are in accordance with another study [41] where they analyzed the effectiveness of NSPT on the profile of neopterin in aggressive periodontitis patients, and they concluded that the mean neopterin level was reduced after treatment, but it was not statistically significant. The decrease in salivary neopterin levels after the treatment seems to indicate the effectiveness of the treatment.

In our study, after treatment, there is positive correlation of salivary neopterin and plasma fibrinogen in Group CP+D and Group CP and statistically significant correlation (p<0.05) with plasma fibrinogen and HbA_1c levels in Group D after treatment (Figures 3 and 4). Salivary neopterin levels, HbA_1c levels and plasma fibrinogen levels have been found to reduce after NSPT. Thus, we could infer that neopterin could serve as an effective tool to assess the inflammatory process related to periodontitis and diabetes mellitus and also predict future cardiovascular events in diabetic patients as seen by increased plasma fibrinogen levels in this study.

The limitations of the present study are short duration of the study period and smaller sample size. A longer period of assessment is suggested to highlight the long term variations after NSPT, and a larger sample size would have been more reflective of the parameters investigated in our study. Hence, further research may help to overcome these limitations and validate neopterin association in diabetic subjects with periodontitis.

Conclusion

Based on the results obtained, it can be concluded that plasma fibrinogen levels and salivary neopterin levels show significant reduction after NSPT. The decrease in salivary neopterin levels after the treatment seems to indicate the effectiveness of the treatment and also serve as an effective tool to assess the inflammatory process related to periodontitis and diabetes mellitus and predict future cardiovascular events in diabetic patients.