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Table of Contents   
ORIGINAL RESEARCH  
Year : 2013  |  Volume : 24  |  Issue : 5  |  Page : 582-586
Comparative analysis of gingival crevicular fluid neopterin levels in health and periodontal disease: A biochemical study


1 Department of Periodontics, Saveetha Dental College, Chennai, India
2 Department of Periodontics, Thai Moogambigai Dental College, Chennai, India
3 Department of Periodontics, New Horizon Dental College and Research Institute, Bilaspur, Chattisgarh, India

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Date of Submission05-Oct-2012
Date of Decision01-Jul-2013
Date of Acceptance31-Oct-2013
Date of Web Publication21-Dec-2013
 

   Abstract 

Background: Periodontitis involves intricate interactions of the biofilm with the host immunoinflammatory response and subsequent alterations in bone and connective tissue homeostasis. Neopterin is a marker, belonging to the class of pteridines, which is associated with cell-mediated immunity. It is produced by interferon-γ stimulated macrophages. The levels of neopterin in body fluids are elevated in infections, autoimmune diseases, malignancies and allograft rejections. This investigation was designed to estimate the levels of neopterin in gingival crevicular fluid (GCF) in health, chronic gingivitis and chronic periodontitis.
Materials and Methods: Thirty subjects were divided into three groups of ten subjects each as with healthy periodontium (Group 1), chronic gingivitis (Group 2) and chronic periodontitis (Group 3). The GCF samples were obtained from the subjects by placing color-coded calibrated, volumetric, micro-capillary pipettes extracrevicularly. The samples were placed in plastic vials and stored at −70°C until the time of neopterin estimation using enzyme immunoassay.
Results: The mean neopterin level in Group 3 (126.28 ± 37.70 nmol/L) is significantly higher than the mean neopterin level in Group 1 (48.66 ± 18.82 nmol/L) and Group 2 (70.68 ± 18.26 nmol/L) (P < 0.05). However, there is no significant relationship between neopterin levels and various clinical parameters in each study group (P > 0.05).
Conclusions: The results of our study indicate that the neopterin levels in GCF are positively associated with periodontal disease, which may provide a useful tool in monitoring its progression. Nevertheless, further longitudinal studies are required with larger sample sizes in which neopterin levels are progressively estimated and compared to baseline values.

Keywords: Gingival crevicular fluid, neopterin, periodontal disease

How to cite this article:
Arjunkumar R, Sudhakar U, Jayakumar P, Arunachalam L, Suresh S, Virupapuram P. Comparative analysis of gingival crevicular fluid neopterin levels in health and periodontal disease: A biochemical study. Indian J Dent Res 2013;24:582-6

How to cite this URL:
Arjunkumar R, Sudhakar U, Jayakumar P, Arunachalam L, Suresh S, Virupapuram P. Comparative analysis of gingival crevicular fluid neopterin levels in health and periodontal disease: A biochemical study. Indian J Dent Res [serial online] 2013 [cited 2019 Nov 12];24:582-6. Available from: http://www.ijdr.in/text.asp?2013/24/5/582/123376
The host response in periodontal disease can be considered in many ways, that is, as the processes that render a subject susceptible to disease or as the pathology of the lesions or as the processes involved in the acute and chronic inflammatory lesion of periodontitis. As reported by Page et al., [1] bacteria are essential in, but not sufficient to cause periodontitis and host factors, such as heredity, and environmental factors, such as smoking, are important determinants of periodontal disease occurrence and severity. An intermediate mechanism that lies between bacterial stimulation and tissue destruction is the production of cytokines, which stimulates inflammatory events that activate effector mechanisms. Upon stimulation by interferon-γ (IFN-γ), a typical Th1-cytokine, preferentially, human monocytes/macrophages produce and release large amounts of neopterin, [2] 6-D-erythro-19,29,39-trihydroxypropylpterin, which is synthesized from guanosine triphosphate (GTP) by GTP-cyclohydrolase I. Determinations of neopterin levels reflect the stage of activation of the cellular immune system, which is of importance in the pathogenesis and progression of various diseases, e.g., in viral infections, in autoimmune or inflammatory diseases, rejection episodes following allograft transplantation and in several malignant diseases. Neopterin can be determined in serum, plasma and other body fluids such as urine, cerebrospinal fluid, saliva, synovial fluid, ascitic fluid. [3]

Gingival crevicular fluid (GCF) is an inflammatory exudate that seeps into the gingival crevices or periodontal pockets around teeth with inflamed gingiva. [4] The detection of some biochemical markers can provide current information about tissue destruction in periodontitis. Because most forms of periodontal disease are site specific, one approach is to measure the substances in the GCF that correlate with the level of disease activity and tissue destruction. [5] It is believed that the measurement of neopterin in GCF might be useful for the diagnosis and prediction of the prognosis of periodontitis because it has been well documented that T cells, in addition to other inflammatory infiltrates, mediate the immunopathological events in periodontal disease. [6] This study was undertaken to estimate the levels of neopterin in gingival crevicular fluid in health, gingivitis and chronic periodontitis and assess the relationship of neopterin levels with the increase in severity of periodontal disease.


   Materials and Methods Top


The study population consisted of 30 subjects belonging to both sexes and all the subjects selected were from the outpatient clinics of the Department of Periodontics, in our institution. Exclusion criteria were aggressive forms of periodontal disease, history of periodontal treatment received in the past six months, medications that would affect the periodontal status, history of underlying systemic disease, anomalies of the immune system, smoking and tobacco chewing habits.

Approval from the ethical committee of the institution was obtained, the nature and purpose of the study was explained to the subjects and written consent was taken. All data was recorded in a standard proforma. The following parameters were evaluated for the subjects: Simplified oral hygiene index (OHI-S) by Greene and Vermillion, [7] the gingival index (GI) by Loe and Silness, [8] probing depth and clinical attachment level, full mouth intraoral periapical radiographs (IOPA). Oral examination was carried out with proper illumination using mouth mirror and manual graduated William's periodontal probe.

To assess the oral hygiene status, simplified oral hygiene Index (OHI-S) was performed on each of the 30 subjects. The OHI-S has two components, the simplified debris index (DI-S) and the simplified calculus index (CI-S). The two scores can be used independently or may be combined for the OHI-S. GI was measured by assessing, each of the four gingival areas of the tooth (facial, mesial, distal and lingual) for inflammation and given a score from 0 to 3. Bleeding is assessed by running a probe along the soft tissue wall of the gingival crevice. The probing depth was recorded by probing each tooth at six sites from gingival margin to base of the pocket (buccal, mesiobuccal, distobuccal, lingual, mesiolingual and distolingual). Readings were taken to nearest millimeter. The clinical attachment was recorded as the distance from the cementoenamel junction (CEJ) to the base of the pocket. [9] Total score of all teeth divided by the number of sites examined gave the average clinical attachment loss for the patient per tooth. Full mouth intraoral periapical radiographs were taken for each patient. Radiographic bone loss was recorded dichotomously (presence or absence) to differentiate chronic periodontitis patients from other groups. No further delineation was attempted within the chronic periodontitis group based on the extent of alveolar bone loss.

Ten subjects with good oral hygiene, no bleeding on probing, no visual signs of gingival inflammation, gingival index = 0, no clinical attachment loss and no radiographic evidence of bone loss were included in Group 1. The chronic gingivitis group (Group 2) consisted of 10 subjects with presence of visual signs of inflammation, gingival index ≥1, no clinical attachment loss, no radiographic evidence of bone loss. The chronic periodontitis group (Group 3) consisted of 10 subjects› presence of inflammatory changes in the periodontal tissues gingival index ≥1 probing depth and clinical attachment loss ≥4mm and radiographic evidence of bone loss.

Selection of test site, detailed case history, clinical examination, radiographic examination and supragingival scaling were done one day before the collection of gingival crevicular fluid. On the subsequent day after drying the area with a blast of air, supragingival plaque was removed without touching the marginal gingiva, and the GCF was collected. A standardized volume of 1 μL was collected from each test site with an extracrevicular approach (unstimulated), using volumetric capillary pipettes (Sigma Aldrich, St. Louis, MO) that were calibrated from 1 to 5 μL. Each sample collected was allowed a maximum of 20 min. The collected GCF was transferred immediately to plastic vials and stored at −70°C until the time of assay. Neopterin was determined using a commercially available ELISA kit according to the manufacturer's instructions.


   Statistical Analysis Top


The data was collected for various parameters such as age, gender, oral hygiene index, gingival index, number of teeth present and clinical attachment loss and recorded on a previously prepared proforma for this study. The data was analyzed statistically, to find the mean, standard deviation and test of significance of mean values for the various parameters between the groups. The Kruskal-Wallis one-way ANOVA was used to calculate. The P < 0.05 was considered statistically significant. The Mann-Whitney U test followed by Bonferroni Correction method was employed to identify the significant groups at 5% level. The Spearman Rank Correlation Coefficient was calculated to assess the relationship between the various clinical parameters and neopterin levels in each study group.


   Results Top


The age and sex distribution of the subjects involved in the study is depicted in [Table 1]. Mean age of the subjects in group 1 was 25.0 ± 4.7 years, 26.3 ± 5.3 years in Group 2 and 35.7 ± 7.2 years in Group 3. A comparison of the mean values of the clinical parameters in the various study groups is depicted in [Table 2]. The neopterin level in the study groups is described in [Table 3]. The neopterin levels in Group 1 ranged between 21.13 nmol/L and 79.65 nmol/L, 48.45nmol/L and 95.12 nmol/L in Group 2 and between 70.59 nmol/L and 184.62 nmol/L in Group 3. Comparison of the levels of neopterin in the three different groups showed that the mean neopterin level in Group 3 (126.28 ± 37.70) is significantly higher than the mean neopterin level in Group 1 (48.66 ± 18.82) and Group 2 (70.68 ± 18.26) (P < 0.05). However, there is no significant difference in mean neopterin level between Group 1 and Group 2 (P > 0.05). The relationship between neopterin level and various clinical parameters within each study group is shown in [Table 4]. There is no significant relationship between neopterin levels and various clinical parameters in each study group (P > 0.05).
Table 1: Mean age, number of teeth present and gender distribution in the three groups


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Table 2: Comparison of mean values of clinical parameters in various study groups


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Table 3: Comparison of mean neopterin levels in various study groups


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Table 4: Relationship between neopterin level and various clinical parameters within each study group


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   Discussion Top


Periodontitis is an inflammatory disease characterized by the destruction of the periodontal ligament and the alveolar bone. [10] It results from the interaction of the host defense mechanisms with the plaque microorganisms. In health, host immune responses are sufficient to hold in check, the pathogenic potential of both the normal resident microbial flora and exogenous microbial pathogens. Complex inflammatory and immune reactions are involved in the progression of periodontitis. [11]

Elevated levels of lysosomal acid hydrolase, β-glucuronidase and prostaglandin E was found in patients with periodontitis. [11],[12],[13] There are articles showing promise on diagnostic use of pro-inflammatory markers in certain body fluids. The methods comprised of saliva, gingival sulcus fluid and traditionally serum. [14] It was thought that a simple and robust marker for the activation state of the immune system in patients with periodontally affected teeth would be helpful for management of such patients, particularly for control of the efficacy of therapeutic measures.

Neopterin is regarded as a biochemical marker of cell-mediated immunity. Due to its chemical structure, it belongs to the group of pteridines, pyrazino-(2, 3-d-) -pyrimidine compounds commonly found in living cells. Neopterin can be detected in various body fluids such as serum, cerebrospinal fluid, synovial fluid, pancreatic juice, urine, saliva, ascites fluid and gingival crevicular fluid. [15],[16],[17],[18] Neopterin is stable for a few weeks when kept below -20°C. [19] It is measured by enzyme-linked immunosorbent assay, radioimmunoassay [20] and high-performance liquid chromatography. Increased serum and urinary neopterin levels can be of clinical value in the diagnosis and prognosis of conditions associated with cell-mediated immunity.

Many studies have looked at the association between total amount or concentration levels of different constituents of GCF and periodontal health status. The collection of GCF is a minimally invasive procedure and the analysis of specific constituents in the GCF provides a quantitative biochemical indicator for the evaluation of the local cellular metabolism that reflects a person's periodontal health status. Since host response is a critical determinant in periodontal disease pathogenesis, the measure of inflammatory mediator levels in the GCF had been used to evaluate 'risk': Risk for a tooth, or more precisely a site, to lose clinical attachment and alveolar bone, or risk for an individual to develop periodontal disease. Results from a pilot longitudinal study indicate that GCF inflammatory mediator levels increase with time in periodontitis patients, both in sites with increased bone loss and stable sites with no bone loss. [21] It was thought that neopterin concentrations might be elevated, locally, due to ongoing immunological processes in the course of periodontal disease. In particular, possible correlations between neopterin concentrations and progression of periodontal disease were studied.

The present study was aimed at investigating neopterin concentration in GCF samples from patients with healthy and diseased periodontium and assessed the relationship between neopterin and periodontal disease. Most of the previous investigations have estimated neopterin levels in either serum or saliva. Saliva consists of a mixture of oral fluids, [22] and includes secretions of the major and minor salivary glands, in addition to constituents of non-salivary origin. [23] Serum, as a clinical tool, has its issues with regards to collection, storing and shipping. It causes anxiety and discomfort to the subjects. Nevertheless, both saliva and serum cannot be used as true representatives of the diseased condition in the gingival crevice. There have been only two studies in the past that have estimated neopterin levels in the gingival crevicular fluid and assessed its relationship with periodontal disease. [5],[18] To our knowledge, this is the third investigation, which has attempted to estimate neopterin levels in GCF and compared it with periodontal disease activity.

The results of our study are in accordance with the results of Ozmeric et al., [18] who reported that the neopterin levels were higher in a diseased periodontium (18+/- 12.75 nmol/L) compared to healthy sites (2.51+/- 1.72 nmol/L). The results of our study are also in concordance with the results of Pradeep et al., [5] who reported that the mean neopterin concentrations in the gingival crevicular fluid increased with the progression of periodontal disease. However, the results were contradictory to our study when the relationship between neopterin level and various clinical parameters within each study group was studied. There is no significant relationship between neopterin levels and various clinical parameters (OHI, GI, CAL and mean number of teeth present) in each study group in our study. This disparity in the results could be explained by the fact that although the disease progresses differently at each local site because of site-specific characteristics, [24] the host response is more global, and follows the same pattern at all sites within an individual's mouth. This was confirmed by Figueredo et al., [25] who found that levels of GCF interleukin-1 were increased in samples from periodontitis patients, regardless of the severity of disease at the sample site. These authors suggest that GCF interleukin-1 levels are characteristic of the patient. Some studies report positive correlations between GCF inflammatory mediators levels and site clinical status, [21],[26] while other studies report poor correlations between GCF inflammatory mediator levels and site clinical status. [21],[25],[27]

The results of our study indicate that neopterin levels in GCF are positively associated with periodontal disease and may be an indicator of periodontal disease activity. However, the neopterin levels did not correlate with the clinical parameters of the sampled site. These findings fit into the more general view and further underscores the relevance of measuring neopterin concentrations not only systemically but also locally. Possible long-term monitoring of GCF neopterin levels in patients with periodontitis is also advisable as levels are said to decline after periodontal treatment. [5]


   Conclusions Top


Neopterin levels in GCF are associated with increase in severity of periodontal disease and can be a useful tool in monitoring disease status in a patient. Because of the lack of correlation between GCF neopterin and site clinical parameters, further longitudinal, well-controlled clinical studies are required with larger sample sizes in which neopterin concentrations are progressively estimated and compared with baseline values are advocated. Such studies will help to confirm the significance of specific GCF markers for diagnosis and monitoring of periodontitis.

 
   References Top

1.Page RC, Offenbacher S, Schroeder HE, Seymour GJ, Kornman KS. Advances in the pathogenesis of periodontitis: Summary of developments, clinical implications and future directions. Periodontol 2000 1997;4:216-48.  Back to cited text no. 1
    
2.Wachter H, Fuchs D, Hausen A, Reibnegger G, Werner ER. Concentrations of neopterin in serum of recipients of renal allografts. Adv Clin Chem 1989;27:81-141.  Back to cited text no. 2
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3.Plata-Nazar K, Kozielska E, £uczak G. Clinical value of neopterin. Part I. Med Wieku Rozwoj 2004;8:433-7.  Back to cited text no. 3
    
4.Armitage GC. Analysis of gingival crevice fluid and risk of progression of periodontitis. Periodontol 2000 2004;34:109-19.  Back to cited text no. 4
    
5.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. 5
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6.Celenligil H, Kansu E, Ruacan S, Eratalay K, Qaglayan G. Immunohistological analysis of gingival lymphocytes in adult periodontitis. J Clin Periodontol 1990;17:542-8.  Back to cited text no. 6
    
7.Greene JC, Vermillion JR. The simplified oral hygiene index. J Am Dent Assoc 1964;68:7-13.  Back to cited text no. 7
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8.Loe H, Silness J. Periodontal disease in pregnancy. Acta Odontol Scand 1963;21:533-51.  Back to cited text no. 8
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9.Newman MG, Takei H, Klokkevold PR, Carranza FA. Carranza's Clinical Periodontology; 10 th Ed, Saunders: Elsevier; 2006  Back to cited text no. 9
    
10.Van Dyke TE, Lester M, Shapira L. The role of host response in periodontal disease progression: Implications for future treatment strategies. J Periodontol 1993;64:792-806.  Back to cited text no. 10
    
11.Lamster IB, Oshrain RL, Celenti RS, Fine JB, Grbic JT. Indicators of the acute inflammatory and humoral immune response in gingival crevicular fluid: Relationship to active periodontal disease. J Periodontal Res 1991;26:261-3.  Back to cited text no. 11
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12.Pippin DJ. Increased intracellular levels of β-Glucuronidase in polymorphonuclear leukocytes from humans with rapidly progressive periodontitis. Arch Oral Biol 1990;35:325-88.  Back to cited text no. 12
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13.Garrison SW, Nichols FC. LPS-elicited secretory responses in monocytes: Altered release of PGE2 but not IL-1 beta in patients with adult periodontitis. J Periodontal Res 1989;24:88-95.  Back to cited text no. 13
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14.Chomyszyn-Gajewska M. Evaluation of chosen salivary periodontal disease markers. Przegl Lek 2010;67:213-6. Review (Polish)  Back to cited text no. 14
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15.Berdowska A, Zwirska-Korczala K. Neopterin measurement in clinical diagnosis. J Clin Pharm Ther 2001;26:319-29.  Back to cited text no. 15
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16.Millner MM, Franthal W, Thalhammer GH, Berghold A, Aigner RM, Füger GF, et al. Neopterin concentrations in cerebrospinal fluid and serum as an aid in differentiating central nervous system and peripheral infections in children. Clin Chem 1998;44:161-7.  Back to cited text no. 16
    
17.Katoh S, Sueoka T, Matsuura S, Sugimoto T. Biopterin and neopterin in human saliva. Life Sci 1989;45:2561-8.  Back to cited text no. 17
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18.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. 18
    
19.Muller MM, Curtius HC, Harold M, Huber CH. Neopterin in clinical practice. Clin Chim Acta 1991;14:1-16.  Back to cited text no. 19
    
20.William J, Saad N, Salib M, Riad H, Mahran KS, Iskander I, et al. The acute effect of intravenously administered recombinant human erythropoietin on the immune response of uremic patients maintained on regular hemodialysis. Artif Organs 1998;22:192-6.  Back to cited text no. 20
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21.Champagne CM, Buchanan W, Reddy MS, Preisser JS, Beck JD, Offenbacher S. Potential for gingival crevice fluid measures as predictors of risk for periodontal diseases. Periodontol 2000 2003;31:167-80.  Back to cited text no. 21
    
22.Kaufman E, Lamster IB. Analysis of saliva for periodontal diagnosis. A review. J Clin Periodontol 2000;27:453-65.  Back to cited text no. 22
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23.Mandel ID, Wotman S. The salivary secretions in health and disease. Oral Sci Rev 1976;8:25-47.  Back to cited text no. 23
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24.Tanner A, Kent R, Maiden MF, Taubman MA. Clinical, microbiological and immunological profile of health, gingivitis and putative active periodontal subjects. J Periodontal Res 1996;31:195-204.  Back to cited text no. 24
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25.Figueredo CM, Ribiero MS, Fischer RG, Gustafsson A. Increased interleukin-1β concentration in Gingival crevicular fluid as a characteristic of Periodontitis. J Periodontol 1999;70:1457-63.  Back to cited text no. 25
    
26.Nakashima K, Roehrich N, Cimasoni G. Osteocalcin, prostaglandin E2, and alkaline phosphatase in gingival crevicular fluid: Their relations to periodontal status. J Clin Periodontol 1994;21:327-33.  Back to cited text no. 26
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27.Gonzales JR, Herrmann JM, Boedeker RH, Francz PI, Biesalski H, Meyle J. Concentration of interleukin-1β and neutrophil elastase activity in gingival crevicular fluid during experimental gingivitis. J Clin Periodontol 2001;28:544-9.  Back to cited text no. 27
    

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Correspondence Address:
Radhika Arjunkumar
Department of Periodontics, Saveetha Dental College, Chennai
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.123376

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