Evaluation of Gingival Crevicular Fluid and Serum Tartrate-resistant Acid Phosphatase Levels in Subjects with Clinically Healthy Periodontium and Chronic Periodontitis - A Clinico-biochemical Study.

BACKGROUND: Periodontitis is a chronic inflammatory disease with conglomerate etiology making it difficult to diagnose at the early stages. Potential biomarkers in gingival crevicular fluid (GCF) would determine the presence of the current disease activity, predict sites vulnerable for future breakdown, and assess the response to periodontal interventions. Merely elevated levels of inflammatory soft-tissue markers do not indicate bone destruction. Since there is no single ideal biomarker established, bone-related biomarkers such as telopeptide of type I collagen, osteocalcin, calprotectin, osteopontin, and tartrate-resistant acid phosphatase (TRAP) seem to hold great promise as predictive markers to determine bone destruction and active phases in the disease progression. The present study is intended to explore the biologic plausibility of the levels of TRAP in health and chronic periodontitis.
MATERIALS AND METHODS: The present cross-sectional clinico-biochemical study comprised 30 systemically healthy subjects with 15 periodontally healthy and 15 chronic periodontitis subjects who were age and gender matched. GCF and blood samples were collected from all the patients. TRAP estimation was done in both the samples using an enzyme-linked immunosorbent assay kit. The data were analyzed using independent t-test and Pearson correlation test.
RESULTS: Serum and GCF TRAP levels in chronic periodontitis subjects were significantly higher when compared to the periodontally healthy group. There were no significant correlations found among serum and GCF TRAP levels with increasing age and gender in both the groups. An increase in disease severity, i.e., increase in probing pocket depth and clinical attachment level, did not show correlation with the GCF and serum TRAP levels in the chronic periodontitis group.
CONCLUSION: Based on the findings of the present study, increased GCF TRAP levels in chronic periodontitis seem to be a potential marker for identifying ongoing periodontal destruction. Copyright:

INTRODUCTION

Periodontitis being a chronic inflammatory disease has composite etiology which includes biofilm, environmental, behavioral factors, and genetic attributes of the host. In periodontitis, though bacteria are the primary agents, the chemical mediators of inflammation resulting from microbial thrust play a crucial role in initiation and progression.[1] If this is left untreated, there would be continuous bone destruction causing tooth mobility and loss.[2] Chronic inflammation causes bone resorption with cycles of remissions and progressions.[3] Understanding the molecular basis of pathogenesis of periodontitis helps in development of more efficient diagnostic procedures by furnishing information about the location and severity. These findings furnish indispensable data for treatment planning, maintenance, and prognosis.[4] Recent advances in research are incorporating methods to determine and gauge periodontal risk by objective measures such as biomarkers.[4] Biomarkers play a prime role in diagnosis, evaluating treatment outcome and drug discovery. Molecules present in gingival crevicular fluid (GCF), saliva, and in blood products like plasma or serum have been explored in an endeavor to accord a marker both specific and sensitive for the periodontal destruction. Saliva and GCF are remarkably propitious as they carry both locally and systemically derived products and can be easily obtained from the patient.[5] The inflammatory stimulus/response which is triggered by the periodontal pathogens can be evaluated in serum or plasma.[6] The estimation of the levels of inflammatory mediators in the GCF is used to evaluate the “risk” for periodontal disease.[7] Tartrate-resistant acid phosphatase (TRAP) is an metallo-phosphodiesterase which plays a part in osteoclast-mediated bone resorption. The TRAP enzyme is expressed abundantly by bone-resorbing cells such as osteoclasts and few subpopulations of macrophages/monocytes and dendritic cells.[8] GCF TRAP levels are probable indicators for the disease activity and the progression of periodontitis. The present study evaluates TRAP levels in serum and GCF of healthy and chronic periodontitis subjects and explores the biologic plausibility of considering TRAP as a biomarker in periodontitis.

MATERIALS AND METHODS

Fifty-one patients were recruited from the Outpatient Department of Periodontics, out of which 10 patients did not satisfy the criteria and 11 patients dropped in between and 30 patients finally completed the study. This is a cross-sectional study and patients were enrolled based on the criteria using the convenience sampling method. Ethical clearance was issued from the Institutional Review Committee Board (SSCDS/2018/59).

Inclusion criteria

Group A (healthy group) consisted of 15 systemically healthy subjects aged between 30 and 60 years with healthy periodontium and absence of clinical inflammation. Group B (chronic periodontitis group) consisted of 15 systemically healthy subjects aged between 30 and 60 years having a minimum of 14 teeth, with severe chronic generalized periodontitis with probing pocket depth (PPD) ≥6 mm in each quadrant.

Exclusion criteria

Exclusion criteria included subjects with known systemic disease (diabetes, hypertension, etc.) and osteoporosis; history of any recent infections; subjects consuming alcohol, and smokers; pregnancy/lactation; subjects on anti-inflammatory medications and antibiotics in the past 3 months; and history of any periodontal therapy in the past 6 months prior to the study and aggressive periodontitis. After recruitment, informed written consent was procured from all the subjects. After a thorough clinical examination, parameters were recorded in a proforma which is specifically designed for the study.

Clinical parameters

Clinical parameters included plaque index (PI), bleeding index, modified gingival index (MGI), PPD, and clinical attachment level (CAL). Case history, clinical parameters recording, and selection of sampling sites were performed on the 1st day. After 2 days, subsequent appointments were preferred to collect the GCF and blood samples to avoid contamination of GCF with blood. In chronic periodontitis subjects, GCF samples were procured from the site with the deepest PPD. In the periodontally healthy group, pooled GCF samples were procured due to their meager quantity in health.

Sample collection

Standardized volume of 3 μl GCF was collected with calibrated microcapillary pipette€ by placing at the gingival sulcus (un-stimulated) for 5–20 min. In subjects with healthy periodontium, pooled GCF samples were gathered from multiple sites to attain minimum required amount (3 μl). In chronic periodontitis, sample collection involved less time as it could be easily obtained from the sites with the deepest PPD. GCF samples were adulterated with saliva or blood and air bubbles were disposed and the other samples collected were wrapped in aluminum foil. Serum is separated by centrifuging the blood samples at 3000 rpm for 10 min, transferred to a vial, and both the samples were stored at −80°C until they were processed. GCF and serum TRAP levels were measured employing a commercially available enzyme-linked immunosorbent assay (ELISA) Kit¥. Phosphate buffer saline was used to dilute GCF samples to a volume of 1 ml.

Statistical analysis

The data analysis was done using statistical analysis software¶. Intergroup analysis for age, gender, MGI, PI, PPD, CAL, serum, and GCF TRAP was done using the Student's independent t-test. Correlation of serum and GCF TRAP levels with age and gender was evaluated in both Groups A and B and with clinical parameters, namely MGI, PI, PPD, and CAL in Group B using Pearson's correlation coefficient test. P < 0.01 are noted as statistically significant.

RESULTS

The mean age of the subjects in Group A and in Group B was 43.40 ± 6.54 and 43.13 ± 7.84 years, respectively [Table 1]. There were 8 males (53.3%) and 7 females (46.7%) in both the groups [Table 1].

Table 1

Intergroup comparison of clinical parameters and tartrate-resistant acid phosphatase concentration in gingival crevicular fluid and serum

	Group (mean±SD)		P
	A	B
Age (years)	43.40±6.54	43.13±7.84	0.92 (NS)
Sex (%)
Female	46.7	46.7	-
Male	53.3	53.3	-
Overall PPD (mm)	1.76±0.59	6.72±0.39	<0.001 (S)
Overall CAL (mm)	0.00±0.00	6.93±0.35	-
MGI	0.00±0.00	1.68±0.41	-
PI	0.22±0.08	2.07±0.26	<0.001 (S)
Serum TRAP (ng/ml)	107.71±44.65	430.48±135.49
Minimum	35.92	148.1	<0.001 (S)
Maximum	188.51	566.29
GCF TRAP (ng/ml)	2531.94±344.84	3483.46±884.52
Minimum	2040.6	2251.5	0.001 (S)
Maximum	3096.05	5481.5

NS: Nonsignificant, S: Significant P≤0.05, SD: Standard deviation, PPD: Probing pocket depth, CAL: Clinical attachment level, MGI: Modified gingival index, PI: Plaque index, TRAP: Tartrate-resistant acid phosphatase, GCF: Gingival crevicular fluid

Gingival inflammation

Gingival status as assessed by MGI was 0.0 in the Group A and 1.68 ± 0.41 in the Group B [Table 1].

Probing pocket depth

The mean PPD was 1.76 ± 0.59 mm in Group A and 6.72 ± 0.39 mm in Group B, respectively.

Clinical attachment level

Mean CAL was 0.00 mm in the Group A and 6.93 ± 0.35 mm in Group B respectively.

Tartrate-resistant acid phosphatase concentration

In serum

The maximum TRAP levels in Group A subjects were 188.51 ng/ml and 566.29 ng/ml in Group B subjects. The mean serum concentration was 107.714 ± 44.65 ng/ml in Group A and 430.48 ± 135.49 ng/ml in Group B [Figure 1].

Figure 1

Intergroup comparison of subject-wise tartrate-resistant acid phosphatase concentration in serum

In gingival crevicular fluid

The mean concentration in Group A subjects was 2531.94 ± 344.84 ng/ml and 3483.46 ± 884.52 ng/ml in Group B, while the maximum TRAP levels in healthy subjects were 3096.05 ng/ml and 5481.5 ng/ml in the diseased group [Figure 2]. Evaluation of the correlation between GCF and serum TRAP concentration with age and gender along with clinical parameters, i.e., mean PPD, mean CAL, MGI, and PI, was done by Pearson correlation coefficient test. No significant correlation was found among serum and GCF TRAP levels with gender and increasing age in both the groups [Table 2] and with the overall mean PPD and CAL in the chronic periodontitis group [Table 3].

Figure 2

Intergroup comparison of subject-wise tartrate-resistant acid phosphatase concentration in gingival crevicular fluid

Table 2

Correlations between serum and gingival crevicular fluid tartrate-resistant acid phosphatase concentration with age and gender in the Groups A and B using Pearson correlation

With age
Group (age)	Serum TRAP		GCF TRAP
A
Pearson correlation	−0.220		−0.046
P	0.431 (NS)		0.870 (NS)
B
Pearson correlation	−0.184		0.065
P	0.512 (NS)		0.819 (NS)
With gender
Group	Sex (mean±SD)		P
	Female	Male
A
Serum TRAP	102.75±53.17	112.06±38.96	0.702 (NS)
GCF TRAP	2517.91±329.46	2544.22±380.02	0.889 (NS)
B
Serum TRAP	410.78±142.17	447.72±136.60	0.617 (NS)
GCF TRAP	3563.99±871.43	3412.99±949.57	0.755 (NS)

NS: Nonsignificant, SD: Standard deviation, TRAP: Tartrate-resistant acid phosphatase, GCF: Gingival crevicular fluid

Table 3

Correlation between serum and gingival crevicular fluid tartrate-resistant acid phosphatase concentration with clinical parameters of Group B using Pearson correlation

	Mean PPD	Mean CAL	MGI	PI
Serum TRAP
Pearson correlation	−0.065	−0.110	0.084	−0.289
P	0.819 (NS)	0.696 (NS)	0.765 (NS)	0.296 (NS)
GCF TRAP
Pearson correlation	−0.052	0.455	−0.240	−0.136
P	0.854 (NS)	0.089 (NS)	0.390 (NS)	0.629 (NS)

NS: Nonsignificant, TRAP: Tartrate-resistant acid phosphatase, GCF: Gingival crevicular fluid, PPD: Probing pocket depth, CAL: Clinical attachment level, MGI: Modified gingival index, PI: Plaque index

DISCUSSION

In periodontitis, there is documented evidence stating that inflammation also affects the immune system besides causing resorption of alveolar bone.[9] Since GCF is closely approximated with periodontal tissues where the disease process initiates, it seems to provide a hot tip than other biological fluids. Several components of GCF such as inflammatory mediators, enzymes, inhibitors, modifiers, and disintegrating products reflect the course and predictability of the disease progression and their qualitative changes could have diagnostic and therapeutic significance. Since there is no single nonpareil biomarker established, bone biomarkers such as telopeptide of type I collagen (ICTP), calprotectin, osteocalcin, osteopontin, and TRAP seem to hold great promise as predictive markers to determine bone destruction and active phases in the disease progression. TRAP is induced by osteoclasts in ample amounts.[8] Studies reported a positive association between TRAP in rheumatoid arthritis and osteodystrophy in primary hyperparathyroidism patients.[10] In dentistry, numerous immunohistochemical and assay studies have been done to evaluate the correlation of TRAP with bone resorption and were concluded that TRAP enzyme can be a useful biomarker of bone resorption in endodontic lesions[11] and bone remodeling in orthodontic tooth movement.[12] Increased activity of TRAP was evident in ligature-induced periodontitis through histochemical studies.[13] Gursoy et al. inferred the increased TRAP levels in saliva of periodontitis patients.[14] This study is presumed to be the first to evaluate the values of TRAP both in GCF and serum in healthy subjects and periodontitis. A sensitive sandwich ELISA commercially available kit is used to quantify and detect TRAP in the samples. The mean GCF concentration of TRAP levels was significantly elevated in chronic periodontitis subjects in comparison to the periodontally healthy group and did not correlate with the disease severity, measured by PPD and CAL, both in serum and GCF and this is in accordance with other studies that estimated different bone markers such as TRAP, ICTP, and osteopontin.[1415] The increase in TRAP levels can be attributed to the increase in osteoclast activity which indicates the active periodontal destruction. Contrary to this, Sharma and Pradeep reported that the levels of osteopontin, a noncollagenous protein, were proportionately higher in periodontitis and correlated with an increase in PPD and CAL.[16] The mean serum concentration of TRAP was remarkably increased in chronic periodontitis subjects in comparison to the periodontally healthy group and no significant correlation with age and gender was found in serum and GCF in both the groups and this is in harmony with the findings of Cheung et al. in an immunoassay study.[17] Several studies published the elevated levels of inflammatory mediators, connective tissue breakdown products, and host-derived enzymes such as MMP 8 ,9 , and 13, etc., in serum of chronic periodontitis subjects.[1819] Existing paradigms have supported the elevated levels of these markers and suggested their possible role in a periosystemic link and thereby substantiate the element of evaluating these markers in serum.[2021]

CONCLUSION

Serum and GCF TRAP levels were markedly elevated in the periodontitis group in comparison to the healthy group. No significant correlations were found among GCF and serum TRAP levels with gender and increase in age in both the groups. The GCF and serum TRAP levels did not correlate with the disease severity, measured by an increase in PPD and CAL in the chronic periodontitis group.

As it is difficult to diagnose a case of early periodontitis in advanced gingivitis patients, potential biomarkers would ascertain the status of disease activity, anticipate sites pregnable for breakdown, and evaluate the prognosis.[22] On the basis of the present study, estimation of GCF TRAP levels in chronic periodontitis seems to be a plausible indicator of ongoing periodontal disease progression. Further longitudinal studies are required to assess the TRAP levels pre- and posttreatment and correlate the levels with bleeding on probing to ascertain both active and inactive sites and also estimate the disease progression rate and also to gauge it as a probable marker for periodontitis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.