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Table of Contents   
ORIGINAL RESEARCH  
Year : 2014  |  Volume : 25  |  Issue : 6  |  Page : 794-796
Estimation of salivary neopterin in chronic periodontitis


1 Department of Periodontics, Rajah Muthiah Dental College, Annamalainagar, Tamil Nadu, India
2 Department of Periodontics, Meenakshi Ammal Dental College, Maduravoyal, Chennai, India
3 Department of Biochemistry, Meenakshi University, Chennai, India

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Date of Submission23-Nov-2013
Date of Decision20-Feb-2014
Date of Acceptance12-Jan-2015
Date of Web Publication02-Mar-2015
 

   Abstract 

Background: Periodontal diseases are the most common bacterial infection predominantly associated with Gram-negative microorganisms that exist in the subgingival biofilm. Analysis of saliva provides a noninvasive means of evaluating the role of the host response in periodontal disease. Though salivary enzymes can be used as the biomarkers, neopterin has been recently used as one of the important diagnostic tools in the field of periodontics. Hence, we aimed to identify the neopterin levels in unsimulated saliva from the chronic periodontitis patients and compare them with the periodontally healthy subjects.
Materials and Methods: Twenty subjects participated in the study and were categorized as the experimental group (chronic periodontitis patients) and control groups (healthy subjects). Unstimulated saliva samples were collected from both the groups for neopterin estimation. Neopterin in saliva was estimated using Shimadzu High Performance Liquid Chromatography with LC-20AD pump system, equipped with RF-10 AXL fluorescence detector.
Statistical Analysis Used: Data were expressed as mean ± SD and analyzed using GraphPad Prism version 6.0 (California, USA). Statistical analysis was done by Student's t-test
Results: The neopterin level in unstimulated saliva was found to be higher in the experimental group than the control group with P ≤ 0.05.
Conclusions: The chronic periodontitis patients showed higher neopterin level in unstimulated saliva as compared to control. Hence, neopterin can be used as a potential biomarker for identification of the periodontal disease in its initial stage can help in preventing the disease progression.

Keywords: Biomarkers, chronic periodontitis, neopterin, salivary enzymes

How to cite this article:
Mahendra L, Mahendra J, Borra SK, Nagarajan A. Estimation of salivary neopterin in chronic periodontitis. Indian J Dent Res 2014;25:794-6

How to cite this URL:
Mahendra L, Mahendra J, Borra SK, Nagarajan A. Estimation of salivary neopterin in chronic periodontitis. Indian J Dent Res [serial online] 2014 [cited 2019 Oct 13];25:794-6. Available from: http://www.ijdr.in/text.asp?2014/25/6/794/152207
Periodontal diseases are the most common bacterial infection predominantly associated with Gram-negative microorganisms that exist in the subgingival biofilm. [1] It results from the interaction of the host defense mechanisms with the microbial dental plaque. Analysis of saliva provides a noninvasive means of evaluating the role of the host response in periodontal disease. In periodontal diseases, though salivary enzymes can be used as the biomarkers, neopterin has been recently used as one of the important diagnostic tools in the field of periodontics. According to Vrecko et al. [2] its concentration is increased in the saliva and depends on the number of affected teeth. Ozmeriη et al. [3] evaluated neopterin levels in gingival crevicular fluid (GCF). Neopterin, a pteridine derivative, is produced and released by human monocytes/macrophages and microglia cells in disease states associated with natural cell response involving macrophages and natural killer cells, which intensify specific immunity by producing cytokines. Neopterin can be detected in various body fluids such as serum, cerebrospinal fluid, synovial fluid, pancreatic juice, urine, saliva, and ascites fluid. [4] The levels of neopterin in body fluids are elevated in infections, autoimmune diseases, malignancies, allograft rejection, cardiac and renal failure, coronary artery disease, and myocardial infarction.

Neopterin receptor is known to undergo extravasation, which makes it a good indicator of the disease status. Hence, neopterin is regarded as a biochemical marker of cell-mediated immunity. [5] Neopterin measurements not only provide an insight into the present state of cell-mediated immune response but also allow monitoring and prognosis of periodontal diseases. [6] Hence, the aim of the present study was to identify the neopterin levels in unsimulated saliva from the chronic periodontitis patients and compare them with the periodontally healthy subjects.


   Materials and methods Top


Fifty subjects were recruited with approval from ethical review board, following the declarations of Helsinki [7] for the study. Twenty-five subjects were excluded due to the history of systemic diseases and five subjects refused to participate. The patients were informed about the study, and the informed consent was obtained from the willing participants. Finally, twenty subjects met the eligibility for the study and were categorized as the experimental and control groups based on the following inclusion and exclusion criteria [Figure 1].
Figure 1: Flow chart of the study design

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Inclusion criteria of the experimental group included the subjects between 35 and 55 years of age with generalized chronic periodontitis having ≥ 20 teeth, presence of bleeding on probing (BOP) and presence of 7 teeth with ≥5 mm probing depth. [8] Subjects with the presence of any systemic disease or conditions that could affect periodontal tissues were excluded from the investigation. Control group included subjects with a healthy and intact periodontium.

Sample collection

One milliliter of whole saliva sample was collected by unstimulated passive drool in a sterile disposable plastic container. The patients were asked to collect saliva in their mouth by avoiding swallowing until a small amount of saliva was collected. Then the patients tilted their head forward allowing the saliva to drool passively. Patients were instructed not to brush or eat 8 h before collection of sample. [9] No dental examination was carried out 48 h prior to saliva collection. Samples were stored at 4°C. Freezing may result in loss of activity and is not recommended. [10] The samples were analyzed in central research lab, and neopterin level was estimated.

Biochemical analysis

For neopterin estimation, each sample was centrifuged at 10,600 ×g for 10 min at 4°C. 200 μL ethanol was added to 100 μL of saliva sample to precipitate the proteins and vortexed for 1 min. The supernatant in each sample was collected for estimation after centrifugation. Separations were carried out at room temperature with a 5 mm reverse-phase high performance liquid chromatography (HPLC) Phenomenex C18 column (4.6 mm × 250 mm). The flow rate of 1.5 ml/min with water: Acetonitrile (99:1 v/v) as the mobile phase, and the fluorescence detector was set at 353 nm of excitation and 438 nm of emission for detection. 20 μl of supernatant was injected into HPLC for analysis. Neopterin in saliva was estimated using Shimadzu HPLC with LC-20AD pump system, equipped with RF-10 AXL fluorescence detector.

Statistical analysis

Data were analyzed using SPSS software version 16. Statistical analysis was done using Mann-Whitney U-test for comparison of the neopterin level among the experimental and control group. Pearson's correlation was done to compare the periodontal parameters with the neopterin level.


   Results Top


A total of 20 subjects were selected for the study and were divided into two groups (experimental and control group). Each group consisted of 10 subjects [Table 1]. On comparing the neopterin levels among the two groups, and the correlation was found to be significant at P ≤ 0.01 [Table 2]. On comparing the neopterin levels with the periodontal parameters such as, periodontal pocket depth (PPD), clinical attachment loss (CAL), and BOP, the correlation was also found to be significant at 0.01 level within experimental and control groups [Table 3].
Table 1: Demographic characteristics of the study subjects

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Table 2: Mann-Whitney U‑test for comparison of the neopterin level among the experimental and control group

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Table 3: Pearson's correlation between clinical parameters and neopterin level

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   Discussion Top


Numerous studies have evaluated the use of various host-derived factors in saliva for diagnosis of periodontal disease [11] as it is simple, non-invasive and can be easily collected. Inflammatory biomarkers associated with periodontal disease have been detected in saliva, such as interleukins, tumor necrosis factor-alpha, matrix metalloproteinases. [12] This is one of the few studies that identified the neopterin levels in unsimulated saliva from the chronic periodontitis patients and compared them with the periodontally healthy subjects. In our study, neopterin levels was found at a higher concentration in the chronic periodontitis subjects as compared to the healthy controls [Table 2]. It is said that activated macrophages are shown to produce neopterin, peroxidase, several acid hydrolases, and collagenases, [13] thus playing a direct and important function in cell-mediated immunity. Macrophage collagenase may have a significant role in collagen breakdown in the periodontal disease process. Hence, the neopterin level may be an indicator of these host mechanisms leading to tissue destruction. Our study is in agreement with Pradeep et al. [14] who also reported an increase in neopterin levels in GCF with progression of periodontal disease. However, Bodur et al. [6] reported no statistically significant correlation between the clinical parameters and neopterin concentration in GCF and saliva. Comparison between the neopterin levels and the clinical parameters such as PPD, BOP, CAL also significantly correlated [Table 3]. This shows that with an increase in the severity of periodontal destruction, the neoptrerin level was also elevated among the experimental group.

The present study was done among South Indian Asian population and showed statistically significant relationship between salivary neopterin levels and chronic periodontitis. Thus, an increase in the neopterin levels in saliva may act as a potential biomarker for the chronic periodontitis in its initial stage which can further help in preventing the progression of the disease. However, further clinical investigations are to be conducted on a larger population to determine the role of neopterin levels in periodontal diseases.

 
   References Top

1.
Darveau RP, Tanner A, Page RC. The microbial challenge in periodontitis. Periodontol 2000 1997;14:12-32.  Back to cited text no. 1
    
2.
Vrecko K, Staedtler P, Mischak I, Maresch L, Reibnegger G. Periodontitis and concentrations of the cellular immune activation marker neopterin in saliva and urine. Clin Chim Acta 1997;268:31-40.  Back to cited text no. 2
    
3.
Ozmeriç N, Baydar T, Bodur A, Engin AB, Uraz A, Eren K, et al. Level of neopterin, a marker of immune cell activation in gingival crevicular fluid, saliva, and urine in patients with aggressive periodontitis. J Periodontol 2002;73:720-5.  Back to cited text no. 3
    
4.
Zeimet AG, Widschwendter M, Knabbe C, Fuchs D, Herold M, Müller-Holzner E, et al. Ascitic interleukin-12 is an independent prognostic factor in ovarian cancer. J Clin Oncol 1998;16:1861-8.  Back to cited text no. 4
    
5.
Müller MM, Curtius HC, Herold M, Huber CH. Neopterin in clinical practice. Clin Chim Acta 1991;201:1-16.  Back to cited text no. 5
    
6.
Bodur A, Baydar T, Ozmeric N, Engin AB, Uraz A, Eren K, et al. Neopterin profile to evaluate the effectiveness of treatment in aggressive periodontitis. Pteridines 2003;14:77-81.  Back to cited text no. 6
    
7.
World Medical Association Declaration of Helsinky. Ethical Principles for Medical Research Involving Human Subjects. 18 th WMA General Assembly, Helsinky; 1964.  Back to cited text no. 7
    
8.
Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodontol 1999;4:1-6.  Back to cited text no. 8
    
9.
De La Peña VA, Diz Dios P, Tojo Sierra R. Relationship between lactate dehydrogenase activity in saliva and oral health status. Arch Oral Biol 2007;52:911-5.  Back to cited text no. 9
    
10.
Alonso de la Peña V, Diz Dios P, Lojo Rocamonde S, Tojo Sierra R, Rodríguez-Segade S. A standardised protocol for the quantification of lactate dehydrogenase activity in saliva. Arch Oral Biol 2004;49:23-7.  Back to cited text no. 10
    
11.
Axtelius B, Edwardsson S, Theodorsson E, Svensäter G, Attström R. Presence of cortisol in gingival crevicular fluid. A pilot study. J Clin Periodontol 1998;25 (11 Pt 1):929-32.  Back to cited text no. 11
    
12.
Rathnayake N, Akerman S, Klinge B, Lundegren N, Jansson H, Tryselius Y, et al. Salivary biomarkers of oral health: A cross-sectional study. J Clin Periodontol 2013;40:140-7.  Back to cited text no. 12
    
13.
Roitt I, Brostoff J, Male D. Immunology. London: CV Mosby Company; 1996.  Back to cited text no. 13
    
14.
Pradeep AR, Kumar MS, Ramachandraprasad MV, Shikha C. Gingival crevicular fluid levels of neopterin in healthy subjects and in patients with different periodontal diseases. J Periodontol 2007;78:1962-7.  Back to cited text no. 14
    

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Correspondence Address:
Jaideep Mahendra
Department of Periodontics, Meenakshi Ammal Dental College, Maduravoyal, Chennai
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.152207

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