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Table of Contents   
ORIGINAL RESEARCH  
Year : 2022  |  Volume : 33  |  Issue : 3  |  Page : 301-306
Evaluation of serum and gingival crevicular fluid levels of Vitamin D binding protein in subjects with clinically healthy periodontium and chronic periodontitis: A clinico bio-chemical study


1 Department of Periodontics, Sri Balaji Dental College, Moinabad, Telangana, India
2 Department of Periodontics, RVS Dental College, Coimbatore, Tamil Nadu, India
3 Department of Periodontics, Sri Sai College of Dental Surgery, Vikarabad, Telangana, India
4 Department of Laboratory Medicine, Care Hospital, Hyderabad, Telangana, India

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Date of Submission29-Jun-2021
Date of Decision03-Sep-2022
Date of Acceptance05-Sep-2022
Date of Web Publication17-Jan-2023
 

   Abstract 


Background: Advances in oral and periodontal disease diagnostic research are moving towards methods wherein periodontal risk can be identified and quantified by objective measures such as bio-markers. Given the roles of vitamin D binding protein (DBP) in modulating the immune response and in the transport of vitamin D, it is hypothesised that quantitative changes of vitamin DBP are associated with periodontal disease. Aim: The aim of the current study is to measure DBP levels in serum and gingival crevicular fluid (GCF) of patients with generalised chronic periodontitis, in comparison to healthy controls. Materials and Methods: The present cross-sectional clinico-bio-chemical study includes 30 systemically healthy subjects with 15 periodontally healthy and 15 chronic periodontitis subjects who were recruited from the out-patient Department of Periodontics. GCF and blood samples were collected from all the patients. DBP estimation was performed in both the samples using a commercially available ELISA kit. Results: Serum and GCF DBP 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 DBP levels with gender and increasing age in both the groups. An increase in disease severity measured by the increase in probing pocket depth and clinical attachment loss did not show correlation with the GCF and serum DBP levels in the chronic periodontitis group. Conclusion: Based on the findings of the present study, increased serum and GCF DBP levels in chronic periodontitis seem to be a probable marker for identifying ongoing periodontal destruction.

Keywords: Bio-marker, chronic periodontitis, gingival crevicular fluid, serum, vitamin D binding protein

How to cite this article:
Chakravarthy Y, Mishra A, Krishnan P, Pathakota KR, Vijaya V, Kamatham SN. Evaluation of serum and gingival crevicular fluid levels of Vitamin D binding protein in subjects with clinically healthy periodontium and chronic periodontitis: A clinico bio-chemical study. Indian J Dent Res 2022;33:301-6

How to cite this URL:
Chakravarthy Y, Mishra A, Krishnan P, Pathakota KR, Vijaya V, Kamatham SN. Evaluation of serum and gingival crevicular fluid levels of Vitamin D binding protein in subjects with clinically healthy periodontium and chronic periodontitis: A clinico bio-chemical study. Indian J Dent Res [serial online] 2022 [cited 2023 Feb 5];33:301-6. Available from: https://www.ijdr.in/text.asp?2022/33/3/301/367888



   Introduction Top


The explicit role of the microbial challenge in the aetiology of periodontal disease is undisputed. However, it is the impact of the susceptible host's inflammatory response to this microbial challenge that ultimately leads to the destruction of the periodontal structures.[1]

Early diagnosis of periodontal disease and the identification of susceptible individuals or sites at risk are crucial owing to the irreversible nature of this disease. Conventional clinical diagnostic measures have limitations in their diagnostic and prognostic utility. Advances in periodontal disease diagnostic research are inclining towards methods wherein periodontal risk can be assessed by objective measures, namely, bio-markers.[2]

Despite the wealth of information in the literature regarding bio-markers for periodontal disease, we are far from finding unambiguous markers of soft and hard tissue destruction which can complement the clinical gold standards.[3] This clearly emphasises the need to explore newer bio-markers associated with periodontal disease to enable precise diagnosis and effective management.

Vitamin D binding protein (DBP), previously known as the group-specific component (Gc-globulin), is a 51–58 kDa multi-functional serum glycoprotein that is produced by hepatic parenchymal cells and released into circulation as the main transport protein of vitamin D.[4] Additionally, this protein also possesses anti-inflammatory and immuno-modulatory functions such as actin scavenging, binding of fatty acids, chemotaxis, influence on T-cell response, and influence on bone metabolism.[5]

These manifold functions of DBP, including its association with vitamin D levels, indicate its potential importance in a range of diseases such as diabetes mellitus, rheumatoid arthritis, and so on.[6],[7] These findings provide a rationale for further research to determine the clinical relevance of this bio-marker in periodontal disease. Serum and GCF levels of DBP are likely to be useful indicators of disease activity, and their estimation might give useful information for the diagnosis of active periodontal disease.

There are some prior studies, analysing DBP levels associated with periodontal disease.[5],[8],[9],[10] However, there is a dearth of studies assessing both the local and systemic levels of DBP in periodontal health and disease. Therefore, the present study aims to comparatively evaluate DBP levels in gingival crevicular fluid (GCF) and serum of healthy and chronic periodontitis subjects.


   Materials and Methods Top


Study design and groups

The present study was cross-sectional in design, conducted on patients attending the out-patient Department of Periodontics. The study was conducted from January 2016 to July 2016. The study protocol was approved by the Institutional Ethical Committee and Review Board (Ref.No. 403/SSCDS/IRB-E/2014).

The subjects were recruited and allocated into two groups. Group A was composed of 15 systemically healthy subjects aged between 30 and 60 years, having at least a total of 14 teeth, diagnosed with severe chronic generalised periodontitis with at least two sites with a probing pocket depth (PPD) ≥6 mm and clinical attachment loss (CAL) ≥5 mm in each quadrant and positive bleeding on probing,[11] and group B was composed of 15 systemically healthy subjects with clinically healthy periodontium aged between 30 and 60 years with modified gingival index (MGI) scores of ≤2.

Subjects with any known systemic disease, which can alter the course of periodontal disease, pregnancy, lactation, habit of smoking or alcohol consumption, aggressive periodontitis, history of any recent infections, or antibiotic or anti-inflammatory medication within the past 3 months; patients taking vitamin D supplements; and those who underwent periodontal therapy in the past 6 months were excluded from the study. A detailed STROBE flowchart describing the study design is mentioned in [Figure 1] (STROBE statement is included as the supplementary material).
Figure 1: STROBE flowchart

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Periodontal status evaluation

Written informed consent was obtained from all recruited patients. They were then subjected to a comprehensive clinical examination, and all the relevant parameters were recorded. Clinical parameters included PPD, CAL, plaque index (PI), (Sillness and Loe),[12] bleeding index (BI) (Saxton),[13] and modified gingival index (MGI) (Lobene).[14] PPD and CAL were assessed using UNC-15 probe (Hu-Friedy®, Mgf. Co., Inc., Chicago, USA) at six sites around the teeth. Additionally, radiographic examination was performed to confirm the clinical diagnosis of chronic periodontitis.

Sample procurement

Case history, clinical parameter recording, and sampling site selection were performed on the first day. After 2 days, on recall evaluation, GCF and blood samples were collected. Subsequent appointments were preferred to avoid the contamination of GCF with blood associated with the probing of inflamed sites. In the chronic periodontitis group, GCF samples were collected from the site with the deepest PPD. In the periodontally healthy group, pooled GCF samples were collected because of the meagre quantity.

GCF collection

The selected test site was air-dried and isolated with cotton rolls. Without touching the marginal gingiva, supragingival plaque was removed with a scaler to avoid contamination and blocking of the microcapillary pipette. GCF was collected using a black colour-coded 1–5 μl calibrated volumetric micro-capillary pipette (Sigma Aldrich Chemical Company, USA Catalog No. p0549) by placing the tip at the entrance of gingival crevice (unstimulated) for 5–20 minutes. A standardised volume of 3 μl GCF was collected using the calibration on the micro-pipette. Samples contaminated with blood or saliva and those with air bubbles were discarded. The collected samples were stored at -80°C till the time of assay.

Serum preparation

3 ml of blood was collected by venepuncture using a 20-gauge needle. The collected blood sample was allowed to clot at room temperature for about 30 minutes. The sample was then centrifuged at 3000 rpm for 10 minutes to separate the serum component, transferred to a plastic vial, and stored at -80°C till the time of assay.

Laboratory analysis

GCF samples were diluted with phosphate buffer saline to a volume of 100 μl. GCF and serum DBP levels were measured using a commercially available Human Vitamin D Binding Protein ELISA kit (Qayee Biotechnology Co., Ltd, China; Catalogue No- QY-E01060). This assay employed the quantitative sandwich enzyme immunoassay technique. The minimum detectable dose (MDD) of DBP ranged from 3 to 120 ng/ml.

Statistical analysis

Data were expressed in terms of mean ± SD. Statistical tests were employed to analyse the parameters using SPSS version 20.0 software (IBM Corp., Armonk, N.Y., USA). The sample size was estimated using the data from previous studies. A total of 13 subjects per group were required for an 80% power, with a confidence interval of 95%. Variables were tested for normality using the Shapiro–Wilk test. As per the normality test, inter-group analysis for age, gender, MGI, PI, PPD, and CAL was performed using Student's independent t-test, and inter-group comparison of BI, serum, and GCF DBP levels was performed using the non-parametric test, that is, Mann–Whitney U test. Correlation of serum and GCF DBP levels with age and gender was evaluated in both the groups and with clinical parameters, that is, MGI, PI, PPD, and CAL, in group A using Pearson's correlation coefficient test.


   Results Top


The mean age of the subjects in groups A and B was 40.9 ± 5.82 and 39 ± 5.42 years, respectively. There was an equal distribution of males and females in both groups. Serum DBP concentration was expressed in nanograms/millilitre. DBP was detectable in all the subjects of groups A and B. The maximum DBP level in serum of group A subjects was 57.5 ng/ml and 49.5 ng/ml in group B. In group A, the mean serum concentration was 53.3 ± 4.03 ng/ml, and in group B, it was 40.2 ± 7.47 ng/ml. This difference of serum DBP levels was found to be statistically significant between the two groups [Figure 2].
Figure 2: Inter-group comparison of mean DBP concentration in serum and GCF

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GCF concentrations of DBP were expressed in nanograms/millilitre. Again, DBP was detected in all the subjects of groups A and B. The mean concentration in group A subjects was 66.05 ± 52.21 ng/ml and 9.63 ± 9.07 ng/ml in group B. Although the maximum DBP levels of the chronic periodontitis group in GCF was 181.5 ng/ml, it was 19.8 ng/ml in the periodontally healthy group. This difference in GCF levels of DBP between the groups was statistically significant [Figure 2]. Inter-group comparisons of DBP levels in serum and GCF are presented in [Table 1].
Table 1: Inter-group comparison of DBP levels in serum and GCF

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Pearson correlation coefficient test was performed to evaluate correlation between GCF and serum DBP concentration with age, gender, and clinical parameters, that is, mean PPD, mean CAL, MGI, and PI. There was no significant correlation found among serum and GCF DBP levels with gender and increasing age in both the groups [Table 2].
Table 2: Correlations between serum and GCF DBP concentration with age and gender in groups A and B using Pearson correlation

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The overall mean PPD and CAL did not exhibit a significant correlation with the serum and GCF DBP levels in the chronic periodontitis group [Table 3].
Table 3: Correlation between serum and GCF DBP concentration with clinical parameters of group A using Pearson correlation

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   Discussion Top


The present study attempted to evaluate the possible association of chronic periodontitis and DBP by obtaining the subject-based and site-specific information, with the help of serum and GCF assays. The available literature on DBP levels with respect to chronic periodontitis is scanty and mostly comprise studies evaluating DBP levels in serum and saliva. The present study is, to the best of our knowledge, first of its kind to investigate both the serum and GCF levels of DBP in chronic periodontitis, which could reflect the true representation of disease burden.

The mean serum concentration of DBP levels in the present study was found to be significantly higher in chronic periodontitis subjects when compared to periodontally healthy subjects. These findings are in accordance to the observations of Rafique et al.,[15] who noticed increased levels of DBP and decreased levels of vitamin D in the serum of chronic periodontitis patients. Additionally, Zhang et al.[5] observed elevated levels of plasma DBP levels in aggressive periodontitis.[13]

Considering the diverse functions of DBP, an alteration in circulating levels can influence pathophysiology of periodontitis in different ways. DBP is considered to be an acute phase reactant whose levels could be increased with an increase in proinflammatory cytokines such as IL-6. As the levels of proinflammatory cytokines are elevated in the case of chronic periodontitis, a concomitant rise in DBP levels can be expected.[16]

A dual role of DBP in the immunological process has been proposed: functioning as a direct positive regulator of C 5a-mediated neutrophil chemotaxis and functioning to neutralise endogenous inhibitors of chemotaxis. Excessive C 5a-mediated leukocyte recruitment is known to be a critical step in the pathogenesis of many inflammatory disorders including chronic periodontitis.[10]

Moreover, DBP can be secreted by neutrophils, and this level of secretion appears to be enhanced after C 5a stimulation. Evidence also reveals that neutrophils activated by LPS have an increased number of DBP-binding sites. This could explain the heightened DBP levels in periodontitis patients.[10]

Additionally, studies are suggesting that DBP-MAF (DBP macrophage activating factor) might have a more generalised regulating role in bone remodelling, which indicates yet another way of participation of DBP in the modulation of periodontal disease.[17]

The effect of DBP in alteration of circulating levels of vitamin D is also put forward as a mechanism to explain its role in the pathogenesis of periodontal disease. In a recent study, Rafique et al.[15] observed that periodontitis subjects had elevated levels of DBP while having low serum 1,25(OH)2D. They postulated that DBP provides an increased amount of 25(OH)D to the kidney to be converted into 1,25(OH)2D. The activated vitamin D, which is protective to the periodontium, is then consumed by the immune cells while combating periodontal pathogens.

The mean GCF concentration of DBP levels in the present study was found to be significantly higher in chronic periodontitis subjects when compared to periodontally healthy subjects. This is in accordance with the observations of Silva-Boghossian et al.[18] and Tsuchida et al.,[19] who evaluated the proteome composition of the GCF in periodontal health and chronic periodontitis patients. The authors reported that DBP is one of the numerous proteins which showed significantly higher relative abundance in periodontitis sites when compared to the healthy group.

Evidence suggests that DBP levels in local fluids in certain systemic diseases which share common immunoinflammatory pathogenesis were also found to be elevated. Further corroborative evidence in this regard can be obtained from Li et al.,[17] who systematically determined the expression and distribution of DBP in periodontal tissues. They concluded that tissue cells, including gingival epithelial cells and periodontal ligament cells, were able to synthesise DBP, which may point towards their role in local host defence and hard tissue metabolism.

On the other hand, the findings of the present study are contrary to Zhang et al.,[10] who demonstrated that decreased GCF levels of DBP are associated with generalised aggressive periodontitis. However, comparisons with this study should be interpreted carefully, given that aggressive periodontitis represents a more severe condition and is modulated by different factors when compared to chronic periodontitis.

It is also fascinating to note that there are certain counter-regulated proteins such as neutrophil defensin 1, annexin 1, plastin 2, and protein S100-A9, whose levels are increased in chronic periodontitis but decreased in aggressive periodontitis.[20] Despite the differential expression, there is no doubt that the general defence mechanisms are definitely compromised in both the diseases. It can be hypothesised that DBP could be one such protein. This, however, has not yet been substantiated and requires further investigation.

It is also important to understand that the difference observed in the serum DBP levels in the present study between the two groups, although statistically significant, was relatively lower when compared to the difference in GCF DBP values. This signifies the greater importance of local levels of DBP as a valuable predictor of periodontal disease than serum values.

In the present study, an increase in disease severity as measured by PPD and CAL did not correlate with the DBP levels in both GCF and serum in the chronic periodontitis group. This could be because the correlations between GCF DBP levels and clinical parameters in the present study focused on mean values rather than site-specific data. Other probable reasons for not having any correlation in the present study might be the smaller sample size and varying statuses of disease activity at different sites.

In chronic periodontitis, host susceptibility is seen to be a result of mutual interactions among bacteria, the immune system, and the environment among which the host factor plays an important role. Therefore, the estimation of host response markers might help evaluate the disease advancement. Based on the findings of the present study, estimation of serum and GCF levels of DBP in chronic periodontitis seems to be a possible indicator of active periodontal disease.

Howbeit, it is unlikely that a standalone biomarker could explain the nexus of factors involved in periodontal disease pathogenesis. Therefore, future research needs to be directed at the development of “marker collections”.[2]

It should be noted that the present study has some limitations, which include a small sample size and consideration of mean values of attachment loss instead of site-specific analysis. Also, DBP has variant genotypes which could have affected the DBP detection. Another possible limitation could be that the one site selected for GCF sample collection and analysis might not represent the true nature of severity of periodontal disease.


   Conclusion Top


Within the limitations of the study, a significant difference is seen in serum and GCF DBP levels between chronic periodontitis patients and periodontally healthy subjects. Further cross-sectional and interventional studies with a larger sample size are recommended to evaluate DBP levels in different forms of periodontal diseases and their correlation with clinical parameters of disease and thereby gauge its relevance as a potential marker for periodontal disease activity.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Acknowledgements

We would like to sincerely thank Mr. Surya Deep Pratap, Biochemist, Department of Laboratory Medicine, Care Hospitals, Hyderabad, for extending support and guidance in processing the samples and conducting the procedure using ELISA kit.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Zhang X, Meng H, Sun X, Xu L, Zhang L, Shi D, et al. Elevation of vitamin D-binding protein levels in the plasma of patients with generalized aggressive periodontitis. J Periodont Res 2013;48:74–9.  Back to cited text no. 5
    
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Correspondence Address:
Dr. Yashaswini Chakravarthy
H-No- 2-2-18/18/5/2, DD Colony, Baghamberpet, Hyderabad, Telangana - 500 013
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.ijdr_646_21

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