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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 9  |  Issue : 2  |  Page : 103-109

Effect of nonsurgical periodontal therapy on the salivary ghrelin and serum alkaline phosphatase levels in health and chronic periodontal diseases: A clinico-biochemical study


1 Department of Periodontics, Oxford Dental College and Hospital, Bengaluru, India
2 Department of Orthodontics and Dentofacial Orthopaedics at ESIC Dental College, Gulbarga, Karnataka, India

Date of Submission06-Mar-2021
Date of Decision17-May-2021
Date of Acceptance20-May-2021
Date of Web Publication10-Aug-2021

Correspondence Address:
Dr. G Devika
Department of Periodontics, Oxford Dental College and Hospital, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_5_21

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  Abstract 


Context: Ghrelin is a peptide hormone that is secreted predominantly in the stomach having effects on immunomodulation and bone metabolism. Alkaline phosphatase (ALP) is synthesized by the osteoblasts and is presumed to be involved in the calcification of the bone matrix. Objective: The objective of the study is to compare and correlate the salivary ghrelin and serum ALP levels in health and chronic periodontal disease before and after 2 months of nonsurgical periodontal therapy (NSPT). Materials and Methods: A total of 50 systemically healthy subjects were included in the study and divided into healthy, chronic gingivitis, and chronic periodontitis groups. Clinical parameters such as plaque index, gingival index, pocket depth, and clinical attachment level were recorded along with the collection of saliva and serum samples at baseline and also after 2 months of NSPT and subjected to ELISA and colorimetric test. Statistical Analysis: Student independent t-test, Pearson's correlation, and analysis of variance with post-hoc Games Howell test were used for analysis. Results: Results demonstrated that salivary ghrelin and serum ALP were detected in all the groups. There was a statistically significant increase in salivary ghrelin and a reduction in serum ALP levels as wells as the clinical parameters following NSPT in both the treated groups. Conclusion: This study concludes that salivary ghrelin and serum ALP levels may represent as a diagnostic biomarker for periodontal disease and also have the potential for therapeutic assessment of responses to NSPT.

Keywords: Alkaline phosphatase, chronic periodontitis, ghrelin, nonsurgical periodontal therapy


How to cite this article:
Devika G, Savitha A N, Pradeep A R, Shirley AJ, Laishram B, Kondody RT. Effect of nonsurgical periodontal therapy on the salivary ghrelin and serum alkaline phosphatase levels in health and chronic periodontal diseases: A clinico-biochemical study. Niger J Exp Clin Biosci 2021;9:103-9

How to cite this URL:
Devika G, Savitha A N, Pradeep A R, Shirley AJ, Laishram B, Kondody RT. Effect of nonsurgical periodontal therapy on the salivary ghrelin and serum alkaline phosphatase levels in health and chronic periodontal diseases: A clinico-biochemical study. Niger J Exp Clin Biosci [serial online] 2021 [cited 2021 Dec 4];9:103-9. Available from: https://www.njecbonline.org/text.asp?2021/9/2/103/323673




  Introduction Top


Human periodontal diseases result from heterogeneous etiologies, which include complex subgingival biofilm, social and behavioral modulations, and genetic and epigenetic traits of the host, each of which is modulated by the host's immune and inflammatory responses.[1] Although microorganisms are the primary etiologic agents, chemical mediators of inflammation are responsible for the progression and destruction of periodontal tissues.[2]

Increased levels of proinflammatory cytokines are seen in the serum and tissue fluids which contribute to gingival inflammation, periodontal pocketing, clinical attachment loss, and alveolar bone loss.[3] The pathogenesis of periodontitis affects not only the tooth-supporting apparatus but also the systemic health.[4]

Ghrelin is a peptide hormone that is secreted predominantly by the stomach.[5],[6] Small amounts have been detected in other organs, tissues, and cells such as the pituitary gland, salivary glands, teeth, heart, cells of the immune system, and osteoblasts. Ghrelin modulates the immune system by downregulating the lipopolysaccharide (LPS)-induced proinflammatory cytokine production (including interleukin-1β and tumor necrosis factor-α).[7],[8] Ghrelin also exhibits a strong anti-inflammatory activity, and thus, potent activity of ghrelin is dependent on its level of acylation.[9] Ghrelin stimulates the differentiation and proliferation of osteoblastic cells, thereby enhancing bone formation and also expresses an increase in alkaline phosphatase (ALP), osteocalcin, collagen type-1.[10]

Chronic inflammatory diseases such as ankylosing spondylitis, Crohn's disease, and inflammatory bowel disease have shown increased ghrelin levels.[8],[11] Similarly, chronic periodontitis that leads to bone destruction has also shown an increase in the levels of ghrelin as it acts as an anti-inflammatory mediator and is involved in bone metabolism and tissue repair.[12],[13]

ALP is a membrane-bound glycoprotein produced by many cells within the area of the periodontium and gingival crevice.[14] It is released from polymorphonuclear neutrophils during inflammation. ALP is stored in specific granules and secretory vesicles of neutrophils and is mainly released during their migration to the site of infection.[15],[16]

ALP allows bone mineralization by releasing an organic phosphate and by hydrolyzing inorganic pyrophosphate, a potent inhibitor of hydroxyapatite crystal formation and dissolution.[17] This takes place when it is released from osteoblasts during bone formation and from periodontal ligament fibroblasts during periodontal regeneration.[18]

Further, in the periodontium, ALP is a part of the normal turnover of the periodontal ligament, root cementum formation, maintenance, and alveolar bone homeostasis.[19] It is also associated with the calcification process, and so its elevated levels commensurate with active bone remodeling.[20] Hence, it has dual involvement in the process of periodontal inflammation and healing/regeneration.

In addition to anti-inflammatory action, ghrelin has a role in the proliferation and differentiation of osteoblasts.[21] It has also been shown to induce gene expression of bone biomarkers such as osteoprotegrin, ALP. The bone destruction in periodontitis is episodic, and the bone resorption is followed by bone apposition.[22] As ghrelin and ALP both have their role in bone metabolic activity, its correlation has been explored in this study.

However, to date, the effect of nonsurgical periodontal therapy (NSPT) on the salivary ghrelin levels with its correlation to serum ALP has not been explored. Thus, this study aimed to assess and correlate the salivary ghrelin and serum ALP levels in health and chronic periodontal diseases before and after 2 months of NSPT.


  Materials and Methods Top


A total of 50 patients were selected based on inclusion and exclusion criteria. All patients were given a detailed verbal and written description of the study and signed a written informed consent form before commencement of the study. The study protocol was approved by the Institutional Ethics Committee (258/202-18), The Oxford Dental College, Bangalore. The study was conducted following the Helsinki Declaration (1975).

The participants were selected based on inclusion and exclusion criteria. The inclusion criteria included participants aged 20–60 years with a minimum of 20 permanent teeth.[23] Participants with moderate (probing depth of 5–7 mm with clinical attachment loss of 3–4 mm) to severe (probing depth of >7 mm with clinical attachment loss of >5 mm) chronic periodontitis (Update 1999 Classification Taskforce Report 2015)[24] who fulfilled the inclusion criteria were randomly assigned into three groups: Group 1 (healthy periodontium), Group 2 (chronic gingivitis), and Group 3 (chronic periodontitis).

However, the exclusion criteria included systemically compromised patients and those on medications (corticosteroids/bisphosphonate, statin therapy/antiplatelet drugs or blood thinners), Grade II and Grade III tooth mobility, Grade III furcation, tobacco and alcohol users, pregnant and lactating women, and participants who have undergone periodontal treatment within a period of past 1 year.

Clinical parameters to be assessed

Gingival index (GI),[25] plaque index (PI),[26] probing pocket depth (PD),[24] and clinical attachment level (CAL)[27] using Manual University of North Carolina-15 Probe (Hu-Friedy, Chicago, IL, USA) were assessed. A full-mouth scaling and root planing (SRP) procedure was performed after the initial examination. Re-evaluation was done after 1 week, and if the tissue response was satisfactory, the patient was scheduled for surgery after 4 weeks. OPG was taken to check the amount of bone loss.

Evaluation of the serum levels of alkaline phosphatase

A total of 3–5 ml of blood was aspirated by venipuncture in the antecubital fossa (from the median cubital vein) and was collected in the sterile disposable syringe. The syringe was kept undisturbed for 15–30 min at 37°C and then transferred to a sterile plastic vial which was centrifuged at 10,000 rpm for 2–4 min to get a clear serum. The sample was refrigerated immediately after the serum was separated. The serum ALP levels were determined using the histochemical (colorimetric, kinetic, increasing reaction method, IFCC) method.[15]

Phase I periodontal therapy

After collection of the saliva samples and serum ALP at baseline, patients in Group II and Group III underwent NSPT which included patient education and motivation, SRP, and oral hygiene instructions. Patients were put on oral hygiene maintenance which included oral hygiene instructions and recalls every 2 weeks for 2 months.

Following NSPT, at the end of 2 months, saliva and serum samples were collected from Group II and Group III patients, and all the clinical parameters were recorded in the same manner as mentioned above.

Detection of ghrelin molecule

The BIOCODON-Sandwich Elisa kit is developed for quantitative estimation of human ghrelin levels in various human sample types, especially serum, plasma, tissue lysates, and their biological fluids. BIOCODON-Sandwich Elisa kits are precoated with capture primary antibody. For assay, saliva samples were added to antibody-precoated wells followed by incubation for 1 h. Postincubation, an anti-ghrelin antibody labeled with biotin and streptavidin-HRP was added to the plate, resulting in the formation of an immune complex. The unbound enzyme was removed by washing the plate. After incubation, substrates A and B were added to the plate. The solution turned blue and changed to yellow with the effect of acid. The shades of solution and the concentration of human ghrelin were positively correlated.[28]

Statistical analysis

Student independent t-test

A t-test is any statistical hypothesis test in which the test statistic follows a Student's t-distribution if the null hypothesis is supported. It can be used to determine if two sets of data are significantly different from each other and is most commonly applied when the test statistic would follow a normal distribution if the value of a scaling term in the test statistic were known. When the scaling term is unknown and is replaced by an estimate based on the data, the test statistic (under certain conditions) follows a Student's t distribution.

Pearson's correlation

Pearson's correlation coefficient is the covariance of the two variables divided by the product of their standard deviations. Pearson's correlation coefficient when applied to a sample is commonly represented by the letter r and may be referred to as the sample correlation coefficient or the sample Pearson correlation coefficient.

Paired t-test

A paired t-test was used to compare two population means of two samples in which observations in one sample can be paired with observations in the other sample.

Analysis of variance with post-hoc Games Howell test

This test was used with variances that are unequal and also take into account unequal group sizes. Severely unequal variances can lead to increased Type I error, and with smaller sample sizes, more moderate differences in group variance can lead to increases in Type I error. The Games Howell test, which is designed for unequal variances, is based on Welch's correction to df with the t-test and uses the student range statistic.


  Results Top


A total of fifty participants are selected and distributed into Groups I, II, and III. In the age among the study groups, the age range was between 20 and 60 years, with a mean age range of 34.50 ± 2.80 in Group I, 35.60 ± 4.26 in Group II, and 37.20 ± 5.62 in Group III, with P = 0.95 [Graph 1].



Student's paired t-test was used to compare different clinical parameters before and 2 months after treatment in Group II. The results showed that PI was 1.53 ± 0.16 before treatment and reduced to 0.92 ± 0.34 2 months after treatment. The GI was 2.00 ± 0.54 before treatment and reduced to 1.30 ± 0.56 2 months after treatment. PD was 2.13 ± 0.17 and reduced to 2.01 ± 0.14 after treatment. The difference was statistically significant at P < 0.001 [Table 1].
Table 1: Comparison of mean values of different clinical parameters between pre- and post-treatment

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Student's paired t-test was used to compare different clinical parameters before and 2 months after treatment in Group III. The results showed that PI was 2.27 ± 0.42 before treatment and reduced to 1.67 ± 0.27 2 months after treatment. The GI was 2.57 ± 0.31 before treatment and reduced to 1.62 ± 0.23 2 months after treatment. PD was 6.80 ± 0.37 and reduced to 5.43 ± 0.70 after treatment. The CAL was 5.95 ± 0.52 before treatment and reduced to 5.28 ± 0.51 2 months after treatment. The difference was statistically significant at P < 0.001 [Table 2].
Table 2: Comparison of mean values of different clinical parameters between pre- and post-treatment in Group 3

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Group I showed a moderate negative correlation of ‒0.42, Group II showed a very weak positive correlation of 0.04 (pretreatment) and a very weak negative correlation of ‒0.1 (posttreatment), and Group III showed a very weak positive correlation of 0.05 (pretreatment) and a weak positive correlation of 0.30 (posttreatment) [Table 3] and [Table 4].
Table 3: Correlation between salivary ghrelin, serum alkaline phosphatase and other clinical parameters at pretreatment period in all the 3 groups

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Table 4: Correlation between salivary ghrelin, serum alkaline phosphatase and other clinical parameters at 2 months after treatment in all the 3 groups

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Group I showed a weak negative correlation of ‒0.29, Group II showed a very weak negative correlation of ‒0.02 (pretreatment) and a weak negative correlation of ‒0.23 (posttreatment), and Group III showed a weak negative correlation of ‒0.29 (pretreatment) and a very weak negative correlation of ‒0.11 (posttreatment). Group I showed a very weak positive correlation of 0.14, Group II showed a very weak negative correlation of ‒0.01 (pretreatment) and a weak negative correlation of ‒0.38 (posttreatment), and Group III showed a very weak negative correlation of ‒0.06 (pretreatment) and a weak negative correlation of ‒0.26 (posttreatment). Group III showed a weak negative correlation of ‒0.31 (pretreatment) and a very weak negative correlation of ‒0.06 (posttreatment) [Table 3] and [Table 4].

The reduction in levels of ALP was statistically significant with P < 0.001 when the Student's paired t-test was applied. Group I showed a moderate negative correlation of ‒0.58, Group II showed a weak positive correlation of 0.38 (pretreatment) and a weak positive correlation of 0.30 (posttreatment), and Group III showed a weak positive correlation of 0.23 (pretreatment) and a very weak positive correlation of 0.11 (posttreatment). Group I showed a weak negative correlation of ‒0.31, Group II showed a very weak negative correlation of ‒0.03 (pretreatment) and a very weak negative correlation of ‒0.08 (posttreatment), and Group III showed a very weak negative correlation of ‒0.14 (pretreatment) and a very weak negative correlation of ‒0.04 (posttreatment) [Table 3] and [Table 4].

Group I showed a weak positive correlation of 0.34, Group II showed a very weak negative correlation of ‒0.1 (pretreatment) and a very weak negative correlation of ‒0.12 (posttreatment), and Group III showed a correlation very weak negative of ‒0.1 (pretreatment) and a weak negative correlation of ‒0.28 (posttreatment). Group III showed a moderate negative correlation of ‒0.43 (pretreatment) and a weak negative correlation of ‒0.31 (posttreatment) [Table 3] and [Table 4].


  Discussion Top


Chronic periodontitis is well known for the changes it induces in bone activity. Therefore, it was hypothesized that total (serum plus salivary) ALP levels will show a rise in patients suffering from chronic periodontitis.[29] The potential value of ALP as a marker of periodontal disease activity has been studied by various authors, markedly Ishikawa and Cimasoni. They stated that ALP plays a role in bone metabolism and their activity has correlated with PD and bone resorption.[30]

In a study done by Jeyasree et al., the mean baseline and after NSPT values of salivary and serum ALP levels of healthy and chronic periodontitis group were assessed. The levels of salivary and serum ALP were significantly reduced in patients after NSPT.[31]

The study by McNabb et al.,[32] showed that the effective control of supragingival microbial plaque combined with professional subgingival therapy is critical for long-term control of the inflammatory periodontal disease. They stated that there was a decrease in putative periodontal pathogens such as Porphyromonas gingivalis and spirochetes after effective SRP, which is similar according to our study.

In our study, there was a reduction in gingival redness and bleeding after NSPT as the red complex microorganisms - P. gingivalis, Treponema denticola, and Tannerella forsythus in the subgingival environment, which are responsible for gingival bleeding reduced following NSPT. This was similar to the study done by Haffajee et al.,[33] which reported a rapid reduction of redness and bleeding after NSPT.

In Group I, the mean levels of ghrelin measured in saliva were 1.35 ± 0.30 ng/ml. This could be because of the presence of ghrelin-positive oral epithelial cells and lamina propria contributing to its production. This was similar to the study done by Aydin et al.,[34] who showed that in the saliva of periodontally healthy subjects, the mean ghrelin level was 0.19 ng/ml. According to their study, the immunohistochemical analysis indicated that ghrelin positivity was found in ductus epithelium of the salivary gland, and the levels are higher than in plasma.

In addition, in another study done by Pradhan et al.,[35] the ELISA indicated ghrelin positivity as it is found to be a potent releaser of growth hormone and actively participate in controlling energy balance and also has immunoregulatory effects.

In our study, the levels of ALP in Group I were 109.60 ± 11.96 IU/L, in Group II (gingivitis) were 118.90 ± 13.62 IU/L, and Group III were 131.30 ± 20.26 IU/L. Group III showed the maximum levels of serum ALP at baseline. This was supported by Agrawal et al.,[36] where they stated that the increased activity in the serum is probably the consequence of a destructive process in the alveolar bone in periodontitis that resulted in an increase in ALP levels in chronic periodontitis patients.

The levels of ghrelin in Group II were 0.96 ± 0.30 ng/ml and Group III was 0.83 ± 0.21 ng/ml and were significantly lower than those obtained in Group I. This is under the study done by Hataya et al.,[37] who reported that administration of LPS caused a reduction in ghrelin levels during the early phase of inflammation.

According to our study, there was a statistically significant increase in ghrelin levels in both Group II and III 2 months post-NSPT, with a mean increase of 1.58 ± 0.42 ng/ml and 1.20 ± 0.30 ng/ml, respectively. The present study showed a statistically significant reduction in the levels of ALP in both Group II and III 2 months post-NSPT with a mean reduction of 105.95 ± 14.85 IU/L and 118.15 ± 18.30 IU/L, respectively.

Following this study, the changes in levels of ALP were positively correlated with changes in clinical parameters such as PI, GI, PD, and CAL, which was clinically significant. This is following the study done by Binder et al. in which he stated that serum ALP was positively correlated with periodontal disease activity and that it reduced 2 months after NSPT.[38]

The limitations of this study are the clinical parameters and levels of salivary ghrelin and serum ALP being assessed only once after NSPT. Confounding factors such as smokers, diabetes, and postmenopausal women influencing the study should be considered. A longer period of follow-up with larger sample size is to be considered. Furthermore, matching of age and gender comparing plasma ghrelin levels with GCF, saliva, and gingival tissue levels in different types and severity of periodontitis may be useful in assessing the activity of ghrelin.

Within the limitations of the study, salivary ghrelin and serum ALP levels have shown a significant change in their levels after NSPT. Therefore, according to the results of this study, these can be considered as diagnostic markers of periodontal disease.


  Conclusion Top


The following conclusions were drawn from the study:

  1. Salivary ghrelin and serum ALP levels were detected in all three groups
  2. The levels of salivary ghrelin were highest in the healthy group followed by gingivitis. Lower levels of ghrelin were detected in chronic periodontitis patients
  3. The levels of serum ALP were highest in the chronic periodontitis group followed by the gingivitis group. Lower levels of serum ALP were detected in the healthy group
  4. A statistically significant negative correlation was established between levels of salivary ghrelin and clinical parameters
  5. A statistically significant positive correlation was established between levels of serum ALP and clinical parameters
  6. A negative correlation was established between serum ALP and salivary ghrelin.


Thus, within the limitations of the study, salivary ghrelin and serum ALP can be considered as a diagnostic and prognostic marker of periodontal disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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