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ORIGINAL ARTICLE
Year : 2017  |  Volume : 8  |  Issue : 3  |  Page : 99-104

Comparative estimation of salivary and serum C-reactive protein levels in chronic periodontitis with or without Type II diabetes mellitus: A clinico-biochemical study


1 Department of Periodontics, Haldia Institute of Dental Sciences and Research, Purba Medinipur, West Bengal, India
2 Department of Dentistry, College of Medicine, J.N.M. Hospital, Kalyani, West Bengal, India
3 Department of Pharmacology, ICARE Institute of Medical Sciences and Research, Purba Medinipur, West Bengal, India

Date of Web Publication18-Sep-2017

Correspondence Address:
Mrinal Kanti Dholey
Department of Periodontics, Haldia Institute of Dental Sciences and Research, Banbishnupur, P.O. Balughata, Haldia, Purba Medinipur - 721 645, West Bengal
India
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DOI: 10.4103/srmjrds.srmjrds_16_17

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  Abstract 

Background: Periodontal diseases are characterized by chronic inflammatory destruction of periodontal connective tissues and extensive alveolar bone destruction as a result of complex interactions between periodontal pathogens and host defense mechanism. Systemic disease such as diabetes mellitus further aggravated the inflammatory condition. Creactive protein (CRP) elevation is a part of the acute phase response to inflammation. CRP, a proinflammatory mediator level has shown to be elevated in patients with chronic periodontitis. Hence, this study was carried out to compare the level of salivary and serum CRP levels in chronic periodontitis with or without typeII diabetes mellitus. Materials and Methods: A total of 60 patients were selected and divided into three groups: 20 periodontally healthy patients, 20 patients with periodontitis, and 20 periodontitis patients associated with diabetes mellitus. The saliva and serum sample were collected from all patients and estimation of CRP level was done using AVITEX® TURBO CRP kit, and latex particle quantitative turbid metric method was employed. Results: The result showed that salivary and serum CRP level was highest in diabetes mellitus with periodontitis patients as compared to chronic periodontitis and healthy patients. Conclusion: We can conclude that CRP is a potential proinflammatory biomarker in patients with chronic periodontitis and diabetes mellitus.

Keywords: C-reactive protein level, diabetes mellitus, periodontal diseases, saliva, serum


How to cite this article:
Dholey MK, Kole D, Rambabu D, Sen S. Comparative estimation of salivary and serum C-reactive protein levels in chronic periodontitis with or without Type II diabetes mellitus: A clinico-biochemical study. SRM J Res Dent Sci 2017;8:99-104

How to cite this URL:
Dholey MK, Kole D, Rambabu D, Sen S. Comparative estimation of salivary and serum C-reactive protein levels in chronic periodontitis with or without Type II diabetes mellitus: A clinico-biochemical study. SRM J Res Dent Sci [serial online] 2017 [cited 2017 Dec 11];8:99-104. Available from: http://www.srmjrds.in/text.asp?2017/8/3/99/215014


  Introduction Top


Periodontitis is a term used to describe an inflammatory process, initiated by the plaque biofilm, that lead to loss of periodontal attachment to the root surface and adjacent alveolar bone, and which ultimately results in tooth loss.[1] Periodontal diseases are among the most common chronic infectious and inflammatory diseases in the world. Their classification is complex and takes into account the clinical presentation, age at diagnosis, rate of disease progression, and systemic and local factors that may increase risk. Periodontal diseases include gingivitis (in which the inflammation is confined to the gingiva, and is reversible with good oral hygiene) and periodontitis (in which the inflammation extends and results in tissue destruction and alveolar bone resorption).[2] Gingivitis, characterized by inflammation of the gums, affects around 50% of the adult population.[3]

More severe destructive periodontitis associated with gum recession, loss of gingival tissue and underlying alveolar bone, affects 10%–15% of the world population and is the major cause of tooth loss in adults.[4] A study from Southern India showed a high prevalence of periodontitis among diabetics (87.2%) and 52.1% had destructive periodontitis with teeth mobility.[5] Interactions between microbial plaque and host immune system play a critical role in the initiation and progression of periodontal diseases.[6] On the other hand, periodontal inflammation is significantly pronounced in the presence of diabetes.[2],[7]

Infection or inflammation activates leukocytes, which further induce the synthesis and secretion of C-reactive protein (CRP). CRP is an acute-phase reactant protein which is synthesized by the liver in response to the inflammatory cytokines interleukin-6 (IL-6), IL-1, and tumor necrosis factor-alpha. Thus, persistent inflammation in the body and at their acute stage induces the elevation of CRP level in blood serum and saliva.[8],[9] Therefore CRP is useful as a proinflammatory marker in patients of chronic periodontitis with or without diabetes mellitus.

Aims and objectives

The aim of the present study was to find out the levels of CRP in saliva and in serum And also to carry out a comparative study of salivary and serum CRP levels in chronic periodontitis with or without type-II diabetes mellitus.


  Materials and Methods Top


The study was carried out in the Department of Periodontology, Haldia Institute of Dental Sciences and Research, Haldia and Department of Biochemistry, ICARE Institute of Medical Sciences and Research. Approval for the study was taken from the Institutional Ethics Committee. A total of 60 patients were selected for this study, and they were divided into three groups with twenty patients in each group. Written informed consent was taken from individual study participants. Group I (Control Group) having clinically healthy periodontium. Group II (Test Group A) were suffering from chronic periodontitis, and Group III (Test Group B) were suffering from chronic periodontitis with diabetes mellitus.

Inclusion criteria

Subjects were placed into three groups according to the following definitions:

  1. Group I: Non-periodontitis group-clinically healthy periodontal status with probing depth (PD) ≤2 mm along with no evidence of attachment loss
  2. Group II: Chronic generalized periodontitis patients without diabetes mellitus-PD of ≥5 mm and/or clinical attachment loss (CAL) >30% sites with varying degree of disease severity
  3. Group III: Chronic generalized periodontitis patients with type-II diabetes mellitus-patients with the age of more than 45 years having PD of 5 mm with moderate to severe CAL (within 4–7 mm). This group includes patients with type II diabetes mellitus for more than 3 years duration and were on oral anti-diabetic pharmacotherapy.


Exclusion criteria

Current smokers; pregnant and lactating women; individuals with acute or chronic medical disorders; patient under any medication for the past 3 months; Patients who had undergone any dental treatment for the past 6 months were excluded from the study.

Periodontal assessment

Periodontal Disease status was evaluated at 4 sites per tooth (mesiobuccal, buccal, distobuccal, lingual/palatal) by measuring the PD, CAL, gingival index by Loe and Silness), plaque index (PI) (Silness and Loe) using the same periodontal probe (UNC-15 probes Hu-Friedy's, USA) and by the same examiner to avoid bias. The PD was measured as the distance from the gingival margin to the base of the periodontal pocket in millimeters. The CAL were calculated by adding the PD to the gingival margin level.

Loe and Silness gingival index

  • Score 0 = Normal gingiva
  • Score 1 = Mild inflammation-slight change in color, slight edema. No bleeding on probing
  • Score 2 = Moderate inflammation-redness, edema, glazing. Bleeding on probing
  • Score 3 = Severe inflammation-marked redness and edema, ulceration. The tendency toward spontaneous bleeding.


The gingival index (GI) may be scored for all surfaces of all or selected teeth or for selected areas of all or selected teeth. The GI may be used for the assessment of prevalence and severity of gingivitis in populations, groups, and individuals. A score from 0.1 to 1.0 = mild inflammation; 1.1–2.0 = moderate inflammation from, and 2.1–3.0 signifies severe inflammation. The GI has been used frequently in clinical trials of therapeutic agents. The sensitivity and reproducibility are good provided the examiner's knowledge of periodontal biology and pathology is optimal.[10]

Blood investigation was done to estimate blood sugar level for all the patients. AVITEX TURBO CRP (Omega Diagnostic Limited, Scotland, UK) kit was used for this study to measure the CRP level in serum and saliva.[11],[12]

Study procedures

  1. Clinical parameters measure: The purpose of the present investigation was to evaluate the periodontal disease activity by measuring the clinical parameters. In this examination, the GI and PI, PD, gingival recession, and clinical attachment level for all teeth were measured. These measurements were taken at four different locations for each tooth (mesial, buccal, lingual, and distal) and recorded on a proper chart. All the measurements were performed with the aid of a UNC-15 probe [Figure 1]
  2. Radiographical findings: After clinical evaluation, radiographical investigation was done by orthopantomogram X-ray for all suspected subjects to evaluate the periodontal disease activity
  3. Sample collection:
  4. Figure 1: Examination of the gingival index and plaque index, probing depth, gingival recession and clinical attachment level for all teeth

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    1. Blood collection: Two milliliters of blood was collected from the antecubital fossa by venipuncture using a 20-gauge needle with a 2-mL syringe and immediately transferred to the laboratory. The blood sample was allowed to clot at room temperature, and after 1 h serum was separated from blood by centrifuging at 3000 rpm for 5 min
    2. Saliva collection: Unstimulated saliva was collected after oral cavity irrigation with water. After the irrigation, the subjects took 5 ml of water and held it for 5 min in the oral cavity, after which the sample was taken. Unstimulated saliva was obtained by passive drooling during 10 min with the subjects sitting in a relaxed position. About 4–5 ml of saliva was placed in an ice chilled test tube.
  5. All collected samples were then transported to the Department of Clinical Biochemistry, ICARE Institute of Medical Sciences and Research
  6. AVITEX TURBO CRP turbidimetric test kit was used for the measurement of CRP level in blood serum and saliva
  7. Working reagent = 1 ml concentrated Latex reagent + 9 ml diluent, (working reagent is stable for 30 days at 2°C–8°C. Distilled water used as diluent
  8. The working reagent and photometer cuvette holder is brought to 370C. Subsequently sample is prepared and calibrated. Analysis was performed using semi-autoanalyzer machine.


Statistical analysis

All the available data were tabulated; sample's mean and standard deviation for the result were determined. Between each study groups, the results were compared using “paired Student's t-test” for the determination of statistical significance. All statistical analysis was performed using the standard statistical software. P < 0.05 was considered statistically significant.


  Results Top


A total of 60 patients were selected for this study, and they were divided into three groups with twenty patients in each group. Written informed consent was taken from individual study participants. Group I (Control Group) having clinically healthy periodontium. Group II was suffering from chronic periodontitis, and Group III was suffering from chronic periodontitis with diabetes mellitus. The mean age of patients in Group I, II, III were 29.28 ± 7.69 years, 39.56 ± 09.05 years, and 37.22 ± 6.77 years.

C-reactive protein

The mean baseline serum CRP concentrations in the Groups I, II, and III were - 4.490 ± 10.223 mg/L, 18.245 ± 25.680 mg/L, and 32.555 ± 13.773 mg/L, respectively. A significant difference (P< 0.05) was found in the CRP levels between Groups I and II (P = 0.0007) and between Groups I and Group III (P ≤ 0.0001) and between Group II and III (P = 0.0343) [Table 1].
Table 1: Mean serum C-reactive protein concentrations of the patients and control subjects

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A highly significant difference (P ≤ 0.0001) was found in the CRP levels in saliva between Groups I and III [Table 2].
Table 2: Mean C-reactive protein concentrations in saliva of the patients and control subjects

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Plaque index

The plaque control in all the patients was satisfactory. At baseline, the mean PI in the Groups I, II and III was 0.380 ± 0.466, 1.294 ± 0.656, and 1.535 ± 0.608, respectively. A significant difference (P ≤ 0.0001) was found in the PI between Groups I and II (P = 0.0002*) and between Groups I and III (P = 0.0001**) but no significant difference (P = 0.1361***) was found between Group II and Group III [Table 3].
Table 3: Plaque index in patients and control subjects

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Periodontal parameters

Gingival index

At baseline, the mean GI scores in the Groups I, II and III were 0.308 ± 0.355, 1.156 ± 0.503, and 1.450 ± 0.639, respectively. A significant difference (P< 0.0001) was found in the GI between Groups I and II and between Groups I and III but not between Groups II and III (P = 0.1451) [Table 4].
Table 4: Gingival index in patients and control subjects

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Probing pocket depth

The mean PDs for the entire mouth at the beginning of the study for Group I, II and III were 1.450 ± 0.686 mm, 4.570 ± 1.394 mm and 4.900 ± 1.177 mm, respectively. A significant difference (P< 0.0001) was found in the mean PDs between Groups I and II and Groups II and III but not between Groups II and III (P = 0.4235) [Table 5].
Table 5: Probing pocket depth in patients and control subjects

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Probing pocket depth

Comparison of data between Group I and Group II and Group I with Group III showed extremely statistically significant, but the comparison between Group II and Group III is considered to be not quite statistically significant [Figure 2] and [Figure 3].
Figure 2: Bar chart represents the C reactive protein level in saliva in different study group Category 1- Comparison between group 1 (blue colour bar) and group 2 (brown colour bar). Category 2- Comparison between group 1 (blue colour bar) and group 3 (brown colour bar). Category 3- Comparison between group 2 (blue colour bar) and group 3 (brown colour bar)

Click here to view
Figure 3: Bar chart represents the C reactive protein level in blood serum in different study groups Category 1- Comparison between group 1 (blue colour bar) and group 2 (brown colour bar). Category 2- Comparison between group 1 (blue colour bar) and group 3 (brown colour bar). Category 3- Comparison between group 2 (blue colour bar) and group 3 (brown colour bar)

Click here to view



  Discussion Top


We had used AVITEX ® TURBO CRP kit and followed a latex turbidimetric serology test for the measurement of CRP level in serum and saliva of all the subjects. We decided to investigate the level of CRP in patients with chronic periodontitis with or without diabetes mellitus type-II by comparing and correlating its levels as a pro-inflammatory marker. Tillet and Francis (1930) first discovered the presence of CRP in the serum of patients with pneumonia, but it was not isolated until 1941. The name is derived due to the ability of the CRP to react with C polysaccharide isolated from pneumococcal cell walls. The CRP gene is located on the first chromosome. Pepys and Baltz (1983)[13] suggested that CRP is synthesized by the liver in response to diverse inflammatory stimuli, including heat, trauma, infection, and hypoxia. Recent evidence has indicated that patients with severe periodontitis have increased serum levels of CRP compared to unaffected control population said by Gomes-Filho et al.[14]

Ramamoorthy et al.[8] have found that the total volume of inflamed periodontal tissue may also play a role and there is a tendency for higher CRP levels in generalized periodontitis compared to localized periodontitis. The plasma levels of CRP in most healthy subjects is usually 1 mg/L, with the normal being defined as <10 mg/L. CRP levels may fluctuate with various factors such as highblood pressure, alcohol use, smoking, chronic fatigue, diabetes, sleep disturbances, depression, many other systemic diseases, and pregnancy or lactation said by Graziani, et al.[15]

A Swedish study by Fredriksson et al.,[16] showed median CRP of 2 mg/L in periodontitis. In the Netherlands, a study reported by Loos, et al.,[17] the highest CRP levels (1.45 mg/L) in patients with generalized form of periodontitis and CRP levels of 1.30 mg/L in patients with localized form. Another study from India conducted by Chopra, et al.,[18] showed CRP levels of 7.49 mg/L in aggressive periodontitis patients and CRP levels of 4.88 mg/L in chronic periodontitis patients.

There are very few studies which correlate periodontal disease with or without diabetes mellitus and salivary CRP levels and even fewer which study the effect of the intervention on the CRP levels. Studies by Pradeep et al. have shown increased GCF levels of CRP in obese and nonobese patients with chronic periodontitis.[19] Another study by the same group of authors showed a decrease in both serum and GCF CRP levels following short term nonsurgical periodontal therapy in type 2 diabetes mellitus patients.[20] Afrah and Al-Jubouri in 2013[21] examined the salivary CRP levels in diabetic and non-diabetic patients with periodontitis. They showed that salivary CRP levels in the control group were lower than the other CRP Levels in Periodontal Diseases and Normal Subjects two groups and there were no significant differences between the diabetic and non-diabetic patients with periodontitis. The results of this study indicate that diabetes as a metabolic disease, had no effect on salivary CRP levels but the periodontitis due to its inflammatory nature, increases salivary CRP. The results of this study are also in agreement with our findings and others. In 2009, Giannobile et al. examined the serum and salivary levels of CRP and the results showed that CRP levels in serum and saliva of patients with periodontitis and patients with chronic and progressive periodontitis have increased.[22]

Pitiphat et al. in 2008 investigated the association between serum CRP levels and periodontitis. The results showed that CRP levels were higher in groups of generalized periodontitis and localized periodontitis than in healthy individuals and their results are consistent with the findings of the present study.[23]

In this study, it was observed that significantly low levels of CRP were observed in the healthy group (Group I) (in saliva = −4.9550 ± 6.8032 and in serum = −4.490 ± 10.2226) compared to the chronic periodontitis groups (Group II) (in saliva = 0.4735 ± 8.2554 and in serum = 18.2155 ± 25.6550), whereas the highest level seen in patients with chronic periodontitis with diabetes mellitus (Group III) (in saliva = 6.0520 ± 6.8979 and in serum = 32.555 ± 13.773) [Table 2]. This observation was in accordance with the previous study, where the levels of CRP were significantly lower in healthy patients compared to chronic periodontitis patients and in patients having chronic periodontitis with diabetes mellitus.


  Conclusion Top


We can conclude by saying that the study has examined the CRP activity in blood serum as well as in saliva in relation to chronic periodontitis with or without diabetes mellitus type-II which supports the hypothesis that biochemical analysis of CRP level might be an important way of studying disease processes and definitely having diagnostic value and it can be used as a tool to assess the patient's periodontal status. Further studies are warranted to confirm the reliability of CRP level in saliva and serum for screening of periodontitis with or without diabetes mellitus.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Genco R, Kornman K, Williams R, et al. Consensus report. Periodontal diseases: Pathogenesis and microbial factors. Ann Periodontol 1996;1:926-32.  Back to cited text no. 1
    
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Preshaw PM, Alba AL, Herrera D, Jepsen S, Konstantinidis A, Makrilakis K, et al. Periodontitis and diabetes: A two-way relationship. Diabetologia 2012;55:21-31.  Back to cited text no. 2
    
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Pepys MB, Hirschfield GM. C-reactive protein: A critical update. J Clin Invest 2003;111:1805-12.  Back to cited text no. 13
    
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Gomes-Filho IS, Freitas Coelho JM, da Cruz SS, Passos JS, Teixeira de Freitas CO, Aragão Farias NS, et al. Chronic periodontitis and C-reactive protein levels. J Periodontol 2011;82:969-78.  Back to cited text no. 14
    
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Graziani F, Cei S, Tonetti M, Paolantonio M, Serio R, Sammartino G, et al. Systemic inflammation following non-surgical and surgical periodontal therapy. J Clin Periodontol 2010;37:848-54.  Back to cited text no. 15
    
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[PUBMED]  [Full text]  
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Pradeep AR, Priyanka N, Prasad MV, Kalra N, Kumari M. Association of progranulin and high sensitivity CRP concentrations in gingival crevicular fluid and serum in chronic periodontitis subjects with and without obesity. Dis Markers 2012;33:207-13.  Back to cited text no. 19
    
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Jayaprakash D, Aghanashini S, Vijayendra RR, Chatterjee A, Rosh RM, Bharwani A. Effect of periodontal therapy on C-reactive protein levels in gingival crevicular fluid of patients with gingivitis and chronic periodontitis: A clinical and biochemical study. J Indian Soc Periodontol 2014;18:456-60.  Back to cited text no. 20
[PUBMED]  [Full text]  
21.
Afrah AA, Al-Jubouri R. Evaluation of salivary levels of proteinaceous biomarkers matrix metalloproteinase (MMP-8) and C-reactive protein (CRP) in type 2 diabetic patients with periodontitis. J Baghdad Coll Dent 2013;25:63-9.  Back to cited text no. 21
    
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Giannobile WV, Beikler T, Kinney JS, Ramseier CA, Morelli T, Wong DT. Saliva as a diagnostic tool for periodontal disease: Current state and future directions. Periodontol 2000 2009;50:52-64.  Back to cited text no. 22
    
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Pitiphat W, Savetsilp W, Wara-Aswapati N. C-reactive protein associated with periodontitis in a Thai population. J Clin Periodontol 2008;35:120-5.  Back to cited text no. 23
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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