Comparative analysis of gingival crevicular fluid β-glucuronidase levels in health, chronic gingivitis and chronic periodontitis.

BACKGROUND AND OBJECTIVES: Current methods available for periodontal disease diagnosis are seriously deficient in terms of accuracy, in the ability to predict ongoing or future disease activity and indeed in determining whether previously diseased sites are in an arrested phase or still active. One area that is receiving a great deal of attention is the biochemical investigation of gingival crevicular fluid (GCF). β-glucuronidase (βG) is one of the enzymes found in GCF that is involved in degradation of the ground substance and fibrillar components of host connective tissue. GCF βG activity might be a good indicator or predictor of periodontal disease activity. This study was conducted to estimate and compare the GCF βG levels in patients with healthy periodontium, chronic gingivitis, and chronic periodontitis.
METHODOLOGY: Subjects were classified into three groups of 20 patients each; healthy individuals, chronic gingivitis, and chronic periodontitis. After recording the plaque index, gingival index and probing pocket depth, 1 μL GCF was collected by placing a calibrated microcapillary pipette extracrevicularly and transferred to sterile plastic vials containing 350 μL of normal saline with 1% bovine serum albumin. Analysis of βG was done by spectrophotometry.
RESULTS: βG levels in GCF were significantly higher in chronic periodontitis group (mean value - 2.04743), followed by chronic gingivitis group (mean - 1.11510) and healthy group (0.53643).
CONCLUSION: Increased βG levels were observed in patients with increased periodontal destruction, hence GCF βG levels can be used as biochemical marker for periodontal disease activity.

Periodontitis is an inflammatory disease of the supporting tissues of the teeth caused by specific microorganism, or group of specific microorganisms, resulting in progressive destruction of the periodontal ligament and alveolar bone with pocket formation, gingival recession or both.[1] The interaction of host defense mechanisms and the etiologic agent is an important determinant of the onset and progression of the disease.[2]

Gingival crevicular fluid (GCF) plays a special role in maintaining the structure of junctional epithelium and in the antimicrobial defense of periodontium. GCF is a complex mixture of substances derived from serum, leukocytes, structural cells of the periodontium and oral bacteria. These substances possess a great potential for serving as indicators of periodontal disease and healing after therapy. The host-derived substances in GCF include antibodies, cytokines, enzymes and tissue degradation products. The antibodies in GCF are comprised of both locally and systemically synthesized molecules, and they reflect periodontal colonization by particular microbial species.[3]

β-glucuronidase (βG) is an important component of the primary granules of polymorphoneutrophils (also found in other cells and bacteria). βG, together with hyaluronidase, is involved in the catabolism of proteoglycans.[4] Studies have examined the relationship of βG in GCF to periodontal attachment loss (PAL). The data suggested that persistently elevated levels of this acid glycohydrolase could identify patients experiencing periodontal attachment loss, as well as identify the risk for PAL for a period of 3–6 months in the future.[5]

Aims and objectives

To estimate the levels of GCF βG in patients with healthy periodontium, chronic gingivitis and chronic periodontitis.

To compare the levels of GCF βG in health, and in patients with chronic gingivitis and chronic periodontitis.

β-glucuronidase

β-glucuronidase is one of the hydrolases found in the azurophilic or primary granules of polymorphonuclear leukocytes (PMNs). It's specific substrates are represented by the aliphatic and aromatic p-D-glucuronides. In the catabolism of mucopolysaccharides βG is probably responsible for the final degradation of the oligosaccharides produced initially by the action of hyaluronidase (Goggins and Billups, 1971).

β-glucuronidase has often been used as lysosomal marker to show the release or lysosomal hydrolases from phagocytizing cells in vitro. For instance, release of βG from neutrophils occurs in the presence of phagocytosable or nonphagocytosable immune complexes and antineutrophil antibodies (Hawkins 1972). The enzyme was demonstrated by histochemical techniques in macrophages, fibroblasts and endothelial cells of healthy or chronically inflamed human gingivae.[6]

Lamster et al.[7] conducted a study on βG activity in GCF, a multicenter trial examining the relationship of the enzyme in GCF to the traditional clinical parameter of periodontal disease and probing attachment loss (PAL). In this report, the baseline data were used to evaluate the relationship of βG activity in GCF to traditional parameters of periodontal disease. They found sites and teeth with increased severity of periodontal disease demonstrate greater βG activity quantitative assessment of this enzyme in GCF provides a different measure of periodontal pathology than traditional clinical parameters of disease.

Methodology

Source of data

The patients for this study were selected from the Department of Periodontics, Yenepoya Dental College Hospital, Mangalore, who gave informed consent to participate in the study.

Methods of collection of data

Patient population

60 patients were selected for the study.

Study consisted of three groups:

Group 1: 20 patients with healthy periodontium

Group 2: 20 patients with chronic gingivitis

Group 3: 20 patients with chronic periodontitis.

Inclusion criteria

Age group: 20–50 years.

Clinically healthy gingiva

Gingival index score of less than or equal to 1

Probing pocket depth of less than or equal to 3 mm.

Chronic gingivitis

Gingival index score of more than or equal to 2

Probing pocket depth of less than or equal to 3 mm.

Chronic periodontitis

Gingival index score of more than or equal to 2

Probing pocket depth of more than or equal to 4mm.

Exclusion criteria

Patients suffering from any systemic diseases

Patients who had received previous periodontal therapy within 6 months

Smokers

Patients taking medications known to influence the periodontal disease

Pregnant and lactating women.

Clinical parameters

Plaque index (Silness and Loe)

Gingival index (Loe and Silness)

Probing pocket depth.

Procedure

Gingival crevicular fluid collection [Figure 1].

Figure 1

Gingival crevicular fluid collection using calliberated volumetric micropipettes

The patient was seated comfortably on a dental chair. The patient was asked to rinse the mouth vigorously with the chlorhexidine mouthwash. Prior to collection the selected site was thoroughly isolated with cotton rolls, gently dried with warm air and an obvious supragingival plaque was removed carefully with gauze so as not to stimulate bleeding from the sulcular epithelium. 1 μL GCF was collected by placing a calibrated microcapillary pipette extracrevicularly. The volumetric microcapillary pipettes were calibrated with a calibration mark after each microliter. If GCF is contaminated with blood, the collection was repeated. The collected GCF was transferred to sterile plastic vials containing 350 μL of normal saline with 1% bovine serum albumin. Samples were taken for biochemical analysis (spectrophotometric analysis) of βG.

Biochemical analysis

Principle

Phenolphthalein glucuronic acid + βG phenolphthalein + glucuronic acid (one unit = 1 μg of phenolphthalein released at 56° C).

The βG activity in the diluted GCF sample was determined in a spectrophotometer. 1 μL of collected GCF was transferred to the small sterile plastic vial that contained 350 μL of normal saline with 1% bovine serum albumin. 100 μL of 0.075 M acetate buffer at pH 4.9, 50 μL of 0.03 M phenolphthalein glucuronic acid at pH 4.5, 5.0 μL of saline and 50 μL of sample fluid were incubated at 56° C for 2 h. The reaction was terminated by adding 350 μL of 0.1 M 2-amino-2-methyl-1-propanol buffer at pH 11 [Figure 2]. The absorbance was measured at a wave length of 550 nm in a spectrophotometer and compared to phenolphthalein standard curve to obtain concentration result. Phenolphthalein standard curve was constructed into eight concentration of phenolphthalein ranging from 4.0 to 0.03 μg/mL in 1: 2 serial dilution (4.0, 2.0, 1.0, 0.5, 0.25, 0.12, 0.06, 0.03 μg/mL) [Figure 3] and were plotted against absorbance at 550 nm in spectrophotometer [Figure 4].

Figure 2

Incubator

Figure 3

Standardized phenolphthalein concentrations

Figure 4

Spectrophotometer

Results

The aim of the present study was to estimate and compare the GCF βG levels in patients with healthy periodontium, chronic gingivitis, and chronic periodontitis. This study was carried out in the Department of Periodontics, Yenepoya Dental College Hospital, Mangalore.

The patients for this study were selected from the Department of Periodontics, Yenepoya Dental College Hospital, Mangalore, who gave informed consent to participate in the study.

Test used

Data were statistically analyzed using SPSS ver 17 and MS Excel (SPSS Statistics for windows version 17 Chicago SPSS incorporation). One-way ANOVA and Tukey multiple comparison test were used to analyze the data. P < 0.05 was considered to be statistically significant.

Table 1 depicts the levels of phenolphthalein concentration in different groups.

Table 1

Phenolphthalein concentration (μg/ml) in different groups

The mean value of Group I was 1.53265 and standard deviation of 0.099739 with the minimum value of 1.280 and maximum value of 1.710.

The mean value of Group II was 3.18600 and standard deviation of 0.402115 with the minimum value of 2.500 and maximum value of 3.850.

The mean value of Group III was 5.84980 and standard deviation of 0.175272 with the minimum value of 5.524 and maximum value of 6.124.

The mean values when subjected to statistical analysis by the ANOVA test, the F-ratio of phenolphthalein concentration was 1406.727 with P < 0.0005, which was statistically significant.

Table 2 depicts the intergroup comparison of phenolphthalein concentration by the Tukey multiple comparison test. The P value was statistically significant when Group I compared with Group II, when Group I compared with Group III, and when Group II compared with Group III.

Table 2

Multiple comparisons of phenolphthalein concentration between three groups

The intergroup comparison of the amount of phenolphthalein concentration and βG concentration between healthy, chronic gingivitis and chronic periodontitis showed statistically significant differences. The values in chronic periodontitis are highest followed by chronic gingivitis and healthy respectively. Graph 1 shows the phenolphthalein concentration in different groups.

Graph 1

Comparison of mean phenolphthalein concentration between different groups

Table 3 depicts the levels of βG concentration in different groups. Mean value of Group I was 0.53643 and standard deviation of 0.034909 with the minimum value of 0.448 and maximum value of 0.599.

Table 3

Concentration of β.glucuronidase (μg/μL) in different groups

The mean value of Group II was 1.111510 and standard deviation of 0.140740 with the minimum value of 0.875 and maximum value of 1.348.

The mean value of Group III was 2.04743 and standard deviation of 0.061345 with the minimum value of 1.933 and maximum value of 2.143.

The mean values when subjected to statistical analysis by the ANOVA test, the F-ratio of βG concentration was 1406.727 with P < 0.0005, which was statistically significant.

Table 4 depicts the intergroup comparison of βG concentration by the Tukey multiple comparison test. The P value was statistically significant when Group I compared with group II, when Group I compared with Group III, and when Group II compared with Group III. The Graph 2 shows the βG concentration levels in different groups.

Table 4

Multiple comparisons of β-glucuronidase concentration between three groups

Graph 2

Comparison of mean β-glucuronidase concentration in different groups

Data were statistically analyzed using SPSS ver 17 and MS Excel. One-way ANOVA and Tukey Multiple comparison test were used to analyze the data. P < 0.05 was considered to be statistically significant.

Discussion

Tissue destruction as a consequence of host bacteria interaction is a well-described process in the pathogenesis of periodontal diseases. During the periodontal destruction, host cells mainly PMNs release their granular enzymes that are capable of attacking all extracellular matrix components. Thus extracellular presence of enzymes seems to play an important role in connective tissue damage.

β-glucuronidase is an important component of the primary granules of PMN (also found in other cells and bacteria). βG, together with hyaluronidase, is involved in the catabolism of proteoglycans. The endoglycosidase hyaluronidase cleaves hexosaminidic linkages, producing tetrasaccharides. This tetrasaccharide is further degraded by βG and β-N-acetyl hexosaminidase. βG is an exoglycosidase, and its substrates include dermatan sulfate, heparan sulfate, chondroitin sulfate, and hyaluronic acid. Therefore, βG most likely contributes to non-collagenous matrix degradation in periodontal diseases. In fact, the relationship between βG activity and periodontal disease has been clearly shown. Furthermore, GCF βG activity might be a good indicator or predictor of periodontal disease activity and its potential for indicating primary granular release from PMNs has been observed.[4]

Lamster et al.[8] found that GCF βG level was increased sharply with an increase in probing pocket depth. Lamster et al.[9] showed a highly significant correlation between βG activity and, mean gingival index and sites with probing depth ≥5mm.

The findings of the present study confirm the relationship between βG level and periodontal disease since increased enzyme levels in GCF was observed when clinical periodontal destruction was present. However, the disease groups presented higher enzyme concentration than the periodontally healthy group. Therefore, these findings support the role for increasing βG levels in the pathogenesis of periodontal disease. These results are in agreement with the findings of Lamster et al.,[7] Layik et al.,[4] who concluded that βG levels were significantly higher in patients with chronic generalized periodontitis.

Lysosomal release from host cells in response to plaque in the absence of phagocytosis and the relatively increased production of oxygen metabolites acting as inactivators of inhibitors and activator of enzymes and PMN priming in the gingival crevice can also contribute to the enzymatic profile of GCF.[4] Lamster et al.[5] have suggested that elevated βG activity could be valuable in the identification of patients at risk for active periodontal disease.

The relationship of bleeding on probing to enzyme activity was also evaluated in the earlier studies while the percent of sites that bled increased with increasing βG activity. While βG activity in GCF appears to increase with increasing severity of traditional signs of periodontal pathology, both the probing depth and bleeding on probing data suggest that the measurement of this enzyme in the fluid is a different measure of the pathologic process than traditional clinical parameters of periodontal disease.[7]

Lamster et al.[10] examined the presence of enzymes (lactate dehydrogenase, βG, and arylsulfatase) in GCF. The data were analyzed in terms of enzyme activity/30-s sample and as concentration of enzyme in a standard volume of GCF. Enzyme activity/30-s sample was a different and possibly more sensitive indicator of periodontal pathology than standard clinical parameters. There was a disassociation between clinical parameters and the data for enzyme analysis when it was reported as concentration.

The standard assay for βG based on phenolphthalein being generated from phenolphthalein glucuronic acid was not adequate for use in GCF analysis. The modification used by Lamster et al.[11] increased assay sensitivity five-fold and allowed smaller samples to be used. In assay the absorbance at 550 nm was measured against a reagent blank, and compared to a phenolphthalein standard curve to obtain concentration results which could then be used to calculate the βG activity. (one unit = 1μg of phenolphthalein released at 56° C).

The βG activity in GCF and gingival tissue homogenates was measured by Layik et al.[4] using the synthetic substrate phenolphthalein glucuronic acid. The intensity of red color of phenolphthalein was measured at 550 nm. Commercial phenolphthalein was used as the standard.

Kaul et al.[12] examined the relationship between βG enzyme which is an indicator of acute inflammatory response in GCF and specific bacterial species or group of species in subgingival plaque to provide an indication as to which bacterial species or group of species may be associated with destructive host-derived processes with the principle that the βG acts on phenolphthalein mono-β-glucuronic acid (substrate) liberating free phenolphthalein. The intensity of the resulting pink color in alkali was proportional to the enzyme activity.

Gingival crevicular fluid βG was assayed using a time-dependent fluorimetric procedure by Lamster et al.[7] The assay results were comparable to what were obtained for βG analysis in GCF using a spectrophotometric procedure (Lamster et al. 1988).

The present study also showed increased phenolphthalein concentration in diseased groups.

The highest βG concentration in GCF was found in periodontitis group. This increase in βG can be attributed to the hyperactive state and pronounced response of PMN as a consequence of severity of microbial virulence factors and also to the lytic effect of more pathogenic subgingival bacteria and host cells leading to an intense host enzyme release. So, Lamster et al.[7] have suggested that elevated βG level could be valuable in the identification of patients at risk for active periodontal disease.

Findings in this study showed that βG concentration is a volume based measurement, it was precise enough to demonstrate the statistical differences among all the groups. Therefore, results of this study further strengthened the fact that as periodontal destruction increases, the concentration of βG in GCF also increases. Hence from this study, it may be deduced that βG is a potential biochemical marker of disease activity.

Summary and Conclusion

The present study was conducted in the department of Periodontics, Yenepoya Dental college Mangalore. The objective of the present study was to evaluate the levels of βG enzyme in periodontally healthy individuals, patients with chronic generalized gingivitis and chronic generalized periodontitis.

In this study 60 subjects were selected according to the inclusion and exclusion criteria. Plaque index (Sillness and Loe), gingival index (Loe and Sillness), and probing pocket depth were assessed. In all the three groups GCF was collected and βG levels were assessed using spectrophotometric analysis.

From the observations in this study following conclusions were drawn.

The βG levels in GCF were significantly higher in chronic gingivitis and chronic periodontitis groups when compared with healthy individuals. In the chronic periodontitis group βG levels were significantly higher than chronic gingivitis group

The phenolphthalein concentration was significantly higher in chronic gingivitis and chronic periodontitis groups when compared with healthy individuals. In the chronic periodontitis group the phenolphthalein concentration was significantly higher than chronic gingivitis group

Increased βG levels were observed in patients with increased gingival index scores and increased probing pocket depth

Gingival crevicular fluid βG levels can be used as biochemical marker for periodontal disease activity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.