|Year : 2015 | Volume
| Issue : 1 | Page : 15-19
Total protein in gingival crevicular fluid as indicators of periodontal disease activity: A clinico biochemical analysis
Arati C Koregol1, Shobha P More2, Savita C Koregol3, Nagaraj Kalburgi1
1 Department of Periodontics, PMNM Dental College and Hospital, Bagalkot, Karnataka, India
2 Department of Periodontics, Sinhagad Dental College and Hospital, Pune, Maharastra, India
3 Department of Periodontics, Al-ameen Medical College, Bijapur, Karnataka, India
|Date of Web Publication||8-Jan-2015|
Arati C Koregol
Mahesh Nilay, Near Muchakhandi Cross, Belgaum Road, Bagalkot - 587 101, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Gingival crevicular fluid (GCF) is regarded as a promising medium for detection of markers of periodontal disease activity. Very few investigators have examined concentration of total protein in GCF, but most results are not in agreement to one another. Aim: This study was undertaken to quantitatively estimate total protein concentration of GCF in gingivitis and periodontitis and to find reliability of these as diagnostic markers of disease activity. This will indicate the stage of disease activity, help in early diagnosis, prevention and treatment of periodontal diseases. Materials and Methods: Study included patients between 18 and 55 years, divided into two groups: Gingivitis (Group-I) and periodontitis (Group-II). Using volumetric micro capillary pipette 1 μl GCF was collected for quantitative analysis of total protein using spectrophotometry. Results: The concentration of total protein in GCF and their significant correlation with gingival index, pocket depth measurements, reflects the clinical status of gingival and periodontal tissues. Conclusions: Estimation of proteins may be used as potential diagnostic markers of active disease status in periodontal tissues and to predict effective methods of prevention and treatment.
Keywords: Biochemical markers, disease activity, gingival crevicular fluid, periodontitis, proteins
|How to cite this article:|
Koregol AC, More SP, Koregol SC, Kalburgi N. Total protein in gingival crevicular fluid as indicators of periodontal disease activity: A clinico biochemical analysis. Drug Dev Ther 2015;6:15-9
|How to cite this URL:|
Koregol AC, More SP, Koregol SC, Kalburgi N. Total protein in gingival crevicular fluid as indicators of periodontal disease activity: A clinico biochemical analysis. Drug Dev Ther [serial online] 2015 [cited 2017 Apr 25];6:15-9. Available from: http://www.ddtjournal.org/text.asp?2015/6/1/15/148885
| Introduction|| |
Periodontal diseases are considered to be multifactorial diseases. The origin, composition and clinical significance of gingival crevicular fluid (GCF) have significantly helped us in understanding the pathogenesis of periodontal disease. GCF passes through the tissues before it enter the sulcus. Analysis of very small amount of fluid may reveal important clinical changes taking place within the gingiva. These changes may be valuable in diagnosis, prevention and treatment of periodontal disease. ,
Pioneer work was performed in periodontology by Waerhaug (1952), Brill and Krasse (1958), Egelberg (1966) and others allowing a better understanding of the significance of crevicular fluid. ,
Crevicular fluid in the sulcus apparently performs two conflicting functions. On one hand the fluid delivers host defense factors against colonizing bacteria, yet at the same time it provides the nutrients necessary for bacterial growth. It is one of the first detectable signs of gingival inflammation. Increased crevicular fluid flow begins before the onset of the cardinal signs of gingival inflammation.
Researches on GCF extend over a period of about 50 years. The GCF studies boomed in the 1970s. The studies of Loe et al. started to explore the use of GCF as an indicator of periodontal diseases. Egelberg continued to analyze GCF and focused his studies on the dentogingival blood vessels and their permeability as they relate to GCF flow. The rationale for understanding dentogingival structure and physiology was created by the outstanding electron microscopic studies of Schroeder and Listgartan. Presence and functions of proteins, especially enzymes in GCF were first explored by Sueda et al. It was soon understood that enzymes released from damaged periodontal tissue possessed an enormous potential for periodontal diagnosis. ,
The qualitative and quantitative measurements of its components may act as a gradient for the evaluation of the extent of gingival and periodontal inflammation.  GCF is regarded as a promising medium for the detection of markers of periodontal disease activity. Few attempts have been made to measure the concentration of total protein in GCF. In the last few years, it is evident that inflammation of the marginal gingiva, elicited by any kind of stimuli, was the primary and probably the only reason for the presence of fluid around a tooth.  The collection protocols are straightforward, noninvasive and can be performed at specificities of interest in the periodontium. ,
The potential of using the crevicular fluid as a diagnostic and prognostic marker was realized, and there was an intense interest in the qualitative assessment of GCF. As periodontal diseases are characterized by destruction of tooth supporting tissues, quantitation of tissue breakdown products in GCF has been pursued as a means of identifying sites undergoing active disease. These studies have focused on the most abundant components of the periodontium, that is collagen, proteoglycans, glycoprotein and glycosaminoglycans. It has been suggested that the most specific sign of connective tissue breakdown may be the protein concentration of GCF. ,
A diagnostic test seeks to establish the presence or absence of a disease. Crevicular fluid based diagnostic tests for various periodontal diseases are currently attracting much interest in clinical, academic and industrial circles. This is because the existing clinical diagnostic tests have many shortcomings. By the use of these techniques, it is hoped that treatment will become more effective, and that over treatment will be avoided thus resulting in a more cost effective outcome. In the search for a useful biochemical marker to assess disease activity, various investigators have examined the total protein composition of GCF in health and disease, but most of the results are not in agreement to one another and need confirmation.  Hence, this clinical study of quantitative estimation of the biochemical parameter of GCF total protein was conducted to find the reliability of total protein as a diagnostic marker in gingivitis and periodontitis and also to correlate the total protein concentration in gingivitis and periodontitis patients, which will help in the determination of disease activity, prevention and treatment of periodontal diseases.
| Materials and Methods|| |
The present study included 30 patients of both sexes of age ranging from 18 to 55 years selected from the Department of Periodontics. The selected patients were divided into two groups: Consisting of 15 patients in each group. Group-I (gingivitis group)-consists of 15 patients with gingivitis. Group-II (periodontitis group)-consists of 15 patients with pocket depth of ≥5 to ≤7 mm. Gingival index (Loe and Silness 1963) and probing pocket depth were recorded. The selection of patients was done on the same day before the collection of sample. No prior oral hygiene instructions were given which might change home care habits. Patients who have not received any periodontal treatment during past 6 months, patients who have not taken any antibiotic therapy during past 6 months, and patients who are not suffering from any known systemic diseases were included in this study.
Procedure for gingival crevicualar fluid collection
Selected patients were made to sit in an upright position on the dental chair with proper lighting condition. The test site was dried and isolated with cotton rolls. GCF Samples were obtained by placing calibrated, volumetric micro capillary pipette of internal diameter of 1.1 mm with the capacity of 5 μl extracrevicularly over test sites and a standardized volume of 1 μl was collected. Test sites which did not express appropriate volume of fluid and micropipettes, which were contaminated with blood and saliva were not included in the study ,,
The collected GCF samples were transferred to a volumetric flask containing 2 ml of double distilled water and then centrifuged. The samples were analyzed for GCF total protein concentrations at Department of Pharmachemistry, H.S.K. College of Pharmacy, Bagalkot. Estimation of total protein concentration using ultraviolet (UV) - spectrophotometer. 
The principle involved in this method is that protein reacts with the Folin-Ciocalteau reagent to give a colored complex. The color so formed is due to the reaction of the alkaline copper with the protein and the reduction of phosphomolybdate by tyrosine and tryptophan present in the protein. The intensity of color depends on the amount of these aromatic amino acids present forming a violet purple colored solution.
The intensity of color, which is directly proportional to the protein concentration is measured at 650 nm wave length in UV-spectrophotometer.
Reagents used for biochemical analysis of protein
1. Sodium carbonate and sodium hydroxide
2. Copper sulfate and sodium potassium tartarate
3. Folin-Ciocalteau reagent.
Descriptive data are presented as mean and standard deviations. Students's t-test was used for comparing means between two groups. Pearson's correlation co-efficient was found to assess the relationship between clinical parameters and biochemical measurements. For all the tests, P < 0.05 was considered for statistical significance.
| Results|| |
Comparison of protein level in GCF and serum with gingivitis group [Table 1] showed that the protein in GCF was highly significant than serum protein with t value of 5.02, and the comparison of protein level in GCF and serum with periodontitis group [Table 2] depicted protein in GCF was statistically highly significant than serum values with t value of 21.5. Comparison of protein level in GCF between gingivitis group and periodontitis group [Table 3] showed that the protein levels in Group-II were highly significant than Group-I with t value of 17.6.
|Table 1: Comparison of protein levels and in GCF and serum with gingivitis (Group-I) |
Click here to view
|Table 2: Comparison of protein levels in GCF and serum with periodontitis (Group-II) |
Click here to view
|Table 3: Comparison of protein levels in GCF between gingivitis (Group-I) and periodontitis (Group-II) |
Click here to view
The correlation between gingival index scores protein levels in GCF with gingivitis group [Figure 1] was determined with r value of 0.76, which was statistically significant. The correlation between pocket depth and protein levels in GCF with periodontitis group [Figure 2] indicated a significant increase in protein levels as the pocket depth increases. A correlation co-efficient r value was 0.52.
|Figure 1: Correlation between gingival index scores and protein levels in gingival crevicular fluid with gingivitis group|
Click here to view
|Figure 2: Correlation between pocket depth and protein levels and in gingival crevicular fluid with periodontitis Group-II|
Click here to view
| Discussion|| |
The requirement for a reliable biochemical marker in GCF for disease activity and susceptibility in periodontology was the relative frustration experienced by research workers in this quest for last three decades. Until now, for instance, one of the multiple components analyzed in the fluid has improved clinical judgment of the rate of progress of gingivitis and periodontitis or the rate of repair of these conditions. ,
The flow and composition of GCF serve as a gauge or barometer of the intensity of the inflammation. This fluid contains all the plasma proteins as well as cellular elements such as polymorphonuclears in mild inflammation, and the composition of GCF is characterized by the appearance of bacterial products, degradation products of the host immune system, mediator of inflammation and by-products of the host immune system and the by-products of connective tissue breakdown in severe inflammation. In addition according to "Alfano's theory", this increase in concentration may be attributed to the modulation by the extent of plasma protein exudation. ,
Many research efforts have attempted to use GCF components to detect or diagnose active disease or to predict patients at risk of periodontal disease.  To a certain extent, the method chosen reflects the type of analysis to be performed on the sample subsequently e.g., when cell types and numbers are to be examined the gingival washing procedure  is most suitable. When large volumes of GCF are required the capillary tubing procedure may be the most useful. However, the insertion of a filter paper strip into the sulcus has been shown to cause trauma to the tissues accompanied by increased permeability of the vessels beneath the epithelium. 
Quantitative research on GCF was greatly improved after appropriate standardized techniques of collecting a known volume of fluids were devised. Krasse and Egelberg (1962)  proposed the use of micropipettes. However, the detailed procedure of GCF collection by using glass capillary tubes of known internal diameter and length was described by Kaslick (1968),  Mann (1963).  In the present study, the micropipettes with the internal diameter of 1.1 mm were used for the collection of GCF samples, where the fluid collection takes place through capillary action. These micropipettes were placed at the entrance of the gingival crevice to avoid air entrapment during collection of fluid. As done in the present study, the area should be completely isolated and dried before the collection of GCF to avoid salivary contamination.
Brill and Bromestam (1960) were the pioneers in identifying some of the proteins by immunoelectrophoresis. They identified several different plasma proteins that correspond to those of normal human serum. Brill (1960) and Shultz-Haudt S, Dewar M, Bibby Bg. (1953) discussed the role of these proteins, like they may provide a mechanism of sealing the epithelium of the gingival sulcus to enamel or cementum. 
Previous reports in the literature are in general agreement that the protein concentration of GCF sampled from sites with varying degrees of gingivitis and periodontitis is equivalent to serum as reported by Bang and Cimasoni,  Binder et al. (1987),  Biswas et al. (1977),  Ficara et al. (1975),  Shapiro et al. (1980).
In the present study in periodontitis (Group-II) the GCF total protein concentration level was 14.26 ± 1.24, which was significantly higher than the gingivitis (Group-I) which was 7.99 ± 0.59. These findings support the observations made by Biswas (1977), Dombrowski et al. (1978), Hattingh and Ho (1980) and Mankela et al. (1991). ,,
The correlation between the gingival index, periodontal pocket depth and total protein concentration in GCF was highly significant. These findings support the facts that the total protein level in GCF from inflamed gingiva varies with a degree of gingivitis and periodontitis, which were supported by the studies of Bang and Cimasoni, Bickel et al. (1985), Binder et al. (1987), Biswas et al. (1977), Ficara et al. (1975), and Shapiro et al. (1980). However, Gustafsson et al. (1995) ,,,, presented a contradictory view.
Hence, it can be suggested that the concentration of protein levels in GCF reflects the clinical status of the periodontal tissues and hence that the estimation of this may be used as important diagnostic markers for the periodontal tissues in health and disease.
Even though the following conclusions are drawn in the present study, further investigations need to be done. Diagnostic techniques have advanced since the days, and the goal to instill appropriate oral health is a key concern.
| Conclusion|| |
The present study has given following conclusions:
- The protein levels in GCF with gingivitis and periodontitis group were higher than the serum values of the same patients.
- The protein concentration in GCF was significantly higher in the periodontitis group than gingivitis group.
- The protein levels in GCF showed a significant correlation with pocket depth measurements.
- The protein levels had a significant correlation with gingival index scores.
- This study suggests the possibility that the protein concentration in GCF to be a potential diagnostic marker of active disease status in periodontal tissues.
| References|| |
Kaslick RS, Chasens AI, Mandel ID, Weinstein D, Waldman R, Pluhar T, et al.
Sodium, potassium and calcium in gingival fluid: A study of the relationship of the ions to one another, to circadian rhythms, gingival bleeding, purulence, and to conservative periodontal therapy. J Periodontol 1970;41:442-8.
Manson JD, Eley BM. Diagnostic tests of disease activity. Outline of Periodontics. 3 rd
ed., Ch. 13. Oxford: Reed Educational and Publishing Ltd.; 1995. p. 144-57.
Koregol AC, More SP, Nainegali S, Kalburgi N, Verma S. Analysis of inorganic ions in gingival crevicular fluid as indicators of periodontal disease activity: A clinico-biochemical study. Contemp Clin Dent 2011;2:278-82. Bang JS, Cimasoni G. Total protein in human crevicular fluid. J Dent Res 1971;50:1683.
Page RC. Host response tests for diagnosing periodontal diseases. J Periodontol 1992;63:356-66.
Loe H, Holm-Pedersen P. Absence and presence of fluid from normal and inflamed gingivae. Periodontics 1965;149:171-7.
Cimasoni G. The Crevicular Fluid Update. monographs in Oral Science. 2 nd
ed., Vol. 12. Basel, Munchen, London: Karger S; 1983.
Mann WV Jr. The correlation of gingivitis and pocket depth and exudate from gingival crevice. J Periodontol 1963;34:379.
Curtis MA, Griffiths GS, Price SJ, Coulthurst SK, Johnson NW. The total protein concentration of gingival crevicular fluid. Variation with sampling time and gingival inflammation. J Clin Periodontol 1988;15:628-32.
Egelberg J. Permeability of the dento-gingival blood vessels. II. Clinically healthy gingivae. J Periodontal Res 1966;1:276-86.
Skapski H, Lehner T. A crevicular washing method for investigating immune components of crevicular fluid in man. J Periodontal Res 1976;11:19-24.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with folin phenol reagent. J Biol Chem 1951;193:265-75.
Kaslick RS, Chasens AI, Weinstein D, Waldman R. Ultramicromethod for the collection of gingival fluid and quantitative analysis of its sodium content. J Dent Res 1968;47:1192.
Uitto VJ. Gingival crevice fluid - An introduction. Periodontol 2000 2003;31:9-11.
Curtis MA, Gillett IR, Griffiths GS, Maiden MF, Sterne JA, Wilson DT, et al.
Detection of high-risk groups and individuals for periodontal diseases: Laboratory markers from analysis of gingival crevicular fluid. J Clin Periodontol 1989;16:1-11.
Schacterele GR. Pollack LR. A simplified method for the quantitative assay of small amount of protein in biologic material. Anal Biochem1973;51:654-5.
Theilade J, Egelberg J, Attström R. Vascular permeability to colloidal carbon in chronically inflamed gingiva. J Periodontal Res 1971;6:100-9.
Sueda T, Bang J, Cimasoni G. Collection of gingival fluid for quantitative analysis. J Dent Res 1969;48:159.
Harvey AS, Genco RJ. Gingival fluid and serum in periodontal disease; I. quantitative study of immunoglobulins, compliment components and other plasma proteins. J Periodontol 1977;48:772-7.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]