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ORIGINAL ARTICLE
Year : 2018  |  Volume : 7  |  Issue : 4  |  Page : 265-271

Estimation of midkine levels in serum and gingival crevicular fluid in smokers and non-smokers of chronic gingivitis patients – A clinico-biochemical study


1 Department of Dentistry, Sri Padmavathi Medical College for Women, SVIMS, Andhra Pradesh, Tirupati, India
2 Department of Periodontics, CKS Teja Institute of Dental Sciences, Andhra Pradesh, Tirupati, India

Date of Web Publication10-Jan-2019

Correspondence Address:
Dr. Dandu Subramanyam Madhu Babu
Department of Dentistry, Sri Padmavathi Medical College for Women, SVIMS, Tirupati
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JDRNTRUHS.JDRNTRUHS_43_18

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  Abstract 


Objectives: To detect the presence of Midkine in serum and gingival crevicular fluid (GCF) of healthy non-smokers, chronic gingivitis smokers, and non-smoker individuals and to evaluate the role of Midkine in the progression of gingival inflammation.
Materials and Methods: Periodontal clinical parameters examination including gingival index, pocket probing depth, clinical attachment level, and collection of GCF and serum was performed on 60 subjects, categorized into three groups with 20 subjects in each group: Group I (healthy non-smokers); Group II (chronic gingivitis non-smokers), and Group III (chronic gingivitis smokers). GCF and serum were collected from 20 patients of each group. Midkine levels were estimated using the enzyme-linked immunosorbent assay.
Results: Midkine was detected in all samples. The results of this study suggest that mean GCF and serum Midkine levels were the highest in Group III and varied significantly from those of Groups I and II. Furthermore, GCF and serum Midkine levels increased proportionally with the progression of inflammation.
Conclusion: As the gingival disease progresses, there is a substantial increase of Midkine concentrations in serum and GCF. Since, Midkine levels in the GCF and serum correlated positively with clinical parameters, Midkine may be considered as a “novel biomarker” in disease progression. However, controlled longitudinal studies are required to confirm this possibility.

Keywords: Gingival crevicular fluid, gingivitis, midkine, serum, smoking


How to cite this article:
Babu DS, Narahari S, Vemuri V, Prasuna E, Reddy DS, Reddy NR. Estimation of midkine levels in serum and gingival crevicular fluid in smokers and non-smokers of chronic gingivitis patients – A clinico-biochemical study. J NTR Univ Health Sci 2018;7:265-71

How to cite this URL:
Babu DS, Narahari S, Vemuri V, Prasuna E, Reddy DS, Reddy NR. Estimation of midkine levels in serum and gingival crevicular fluid in smokers and non-smokers of chronic gingivitis patients – A clinico-biochemical study. J NTR Univ Health Sci [serial online] 2018 [cited 2019 Mar 23];7:265-71. Available from: http://www.jdrntruhs.org/text.asp?2018/7/4/265/249829




  Introduction Top


Smoking is an environmental factor that places individuals at high risk for negative effects on the periodontal health.[1] However, the biological mechanisms behind these detrimental effects are still obscure. Periodontal diseases (PDs) are considered to be initiated by bacteria that activate pathogenic processes leading to tissue destruction. Basically, same subgingival microflora is observed in smoker and non-smoker patients with PD. Hence, the effects of smoking on periodontal health seem unrelated to the composition of subgingival microflora.[2] In order to further elucidate the role of smoking in PD, investigations of its influence on host response are needed. Cytokines such as interleukins-1, -6 (IL-1, IL-6) and tumor necrosis factor alpha (TNF-α) are considered to be involved in the host response of PD as mediators of tissue destruction. Increased levels of these cytokines have been observed in gingival crevicular fluid (GCF) of patients with PD.[3]

However, the underlying mechanisms by which smoking is associated with the pathological conditions of the periodontium are still not fully understood. For example, conflicting results have been reported on the subgingival microbiota in smoker and non-smoker patients with PD.[4] To further elucidate the role of smoking in PD, investigations of its influence on host response are needed. Among many inflammatory and immune mediators identified in GCF, cytokines have attracted particular attention and are involved in both inflammation-related alteration and repair of the periodontal tissues. Certain cytokines have been proposed as potentially useful diagnostic or prognostic markers of periodontal destruction.[5]

Midkine (MK) and pleiotrophin (PTN) comprise a distinct family of heparin-binding growth factors[6] that are multifunctional, acting both as growth factors and activators of neutrophils and also both MK and PTN have antibacterial activity against a wide variety of bacteria.[7] MK is induced by retinoic acid and is a novel multifunctional heparin-binding growth factor. MK is structurally unrelated to fibroblast growth factors or other heparin-binding growth factors and is the initial member of a new cytokine/growth factor family.[8] In adults, significant MK expression is observed only in restricted sites such as the kidney, gut, epidermis, bronchial epithelium, lymphocytes, and macrophages, while MK expression is induced in many tissues after injury. MK exerts pleiotropic effects, including cell proliferation, cell migration, angiogenesis, and fibrinolysis, in a variety of tissues. MK also plays an important role in the induction of oncogenesis, inflammation, and tissue repair. Importantly, MK exhibits another activity related to an inflammatory response, namely, enhancement of fibrinolytic activity. MK will cause leukocyte migration in two distinct steps: one is the attraction of leukocytes through a hepatotactic mechanism, and the other is the infiltration of leukocytes from the bloodstream to a tissue by virtue of degradation of the basement membrane through the enhancement of fibrinolytic activity. The dual activities of MK will explain the physiological and pathological significance of the expression of MK in early stages of tissue damage and inflammation. MK, by promoting tissue infiltration with proinflammatory cells, contributes to chronic inflammation and angiogenesis, supported further by its mitogenic and differentiating properties.[9] Elevation of circulating MK in disease with an inflammatory background, mostly in malignancies, has been frequently observed and the utility of MK as a disease marker has been suggested. The purpose of this study was to estimate MK levels in serum and GCF in smokers and non-smokers of gingivitis patients.


  Materials and Methods Top


The study population consisted of 60 subjects attending the outpatient clinic of our department. written informed consent was obtained from the participants who agreed to participate voluntarily. all eligible candidates were thoroughly informed of the nature, potential risks, and benefits of the study. ethical clearances were obtained from the institution's ethical committee and review boards (no. cks/ethics/0054/2014) on 12/02/2014. inclusion criteria included who were within age range of 20–60 years and presence of ≥15 functional teeth, smoking. people who have diabetes, rheumatoid arthritis, pregnancy or lactation, hiv infection, bleeding disorders, who were taking immunosuppressive chemotherapy and presence of other infections, refusal of informed consent and periapical pathology, orthodontic appliances periodontal or antibiotic therapies previous 6 months, and who were using mouth rinses containing antimicrobials in the preceding 2 months are excluded from the study.

  • The subjects selected randomly and categorized into three groups, each group comprising of 20 patients based on gingival index (GI) and smoking. Smoking history will be evaluated by means of self-reporting, following a standardized questionnaire. Subjects were classified as smokers based on criteria established by the center for disease control and prevention (CDC). Current smokers were defined as those who had smoked 100 or more cigarettes over their lifetime and smoked at the time of interview. Non-smoker subjects were those who had never smoked
  • GROUP-1 (HEALTHY NON-SMOKERS) Consisted of 20 subjects (non-smokers) with clinically healthy periodontium and with no evidence of disease, GI = 0, probing pocket depth (PPD) ≤3 mm, and clinical attachment loss (CAL) =0 mm, with no radiographic evidence of bone loss. Subject should not be a smoker
  • GROUP-2 (CHRONIC GINGIVITIS NON-SMOKERS) Consisted of 20 subjects (non-smokers) with clinically healthy periodontium and with evidence of disease, GI ≥ 1, PPD ≤ 3 mm, and CAL ≤ 1 mm, with no radiographic evidence of bone loss. Subject should not be a smoker
  • GROUP-3 (CHRONIC GINGIVITIS SMOKERS) Consisted of 20 smoker subjects with clinically healthy periodontium and with evidence of disease, GI ≥ 1, PPD ≤ 3 mm, and CAL ≤ 0 mm, with no radiographic evidence of bone loss.


Clinical parameters

The participants were subjected to clinical examinations or the following periodontal clinical parameters: GI, PPD, and CAL. A single examiner (SR) recorded all measurements from patients using a UNC15 periodontal probe to ensure adequate intraexaminer reproducibility. The difference between the examinations was within 1 mm in 82% PPD measurements and 90% CAL measurements. Marginal gingival bleeding was recorded using GI, PPD, and CAL. The GI, PPD, CAL, and MK level assessments in the GCF were conducted.

Collection of GCF

Samples were collected on the subsequent day by second examiner to ensure the masking of the sampling examiner and to avoid the contamination of GCF with blood-associated probing at the inflamed sites. In this study, we selected only one site in each Group II and Group III, whereas in Group I, multiple sites without inflammation were selected to collect adequate GCF. In Groups II and III, the site with the highest clinical signs of inflammation was selected for GCF collection. The selected site was gently air-dried, and clinically detectable supragingival plaque was removed using a curette without touching the marginal gingiva. GCF was collected by placing a micropipette at the entrance of the gingival sulcus. A standardized volume of 1 μL GCF was collected from each site after calibrating white color-coded 1–5 μL-calibrated volumetric micropipettes (Sigma Aldrich). The sites that did not express any GCF and the micropipettes that were contaminated with blood and saliva were excluded from the study. The GCF samples were expelled from the micropipettes with a jet of air by using a blower provided with the pipettes. The micropipettes were further flushed with an affixed amount of the diluents to ensure that no GCF was stuck to the walls of the pipettes. The collected GCF samples were placed immediately into individual microcentrifuge tubes containing 300 μL of phosphate-buffered saline. The samples were stored at −70°C until the enzyme-linked immunosorbent assay (ELISA) was performed. During the 12 weeks, subjects were seen at 1-week intervals and plaque control measures were performed.

Collection of serum

Blood (5 mL) was collected from antecubital fossa through venipuncture by using a 20-gauge needle with a 5-mL syringe and was immediately transferred to the laboratory. The blood sample was allowed to clot at room temperature, and the serum was separated from the blood after 1 h by centrifuging the blood at 3000 × g for 10 min. The serum was immediately transferred to a plastic vial and stored at − 70°C until ELISA was performed.

Determination of Midkine in GCF and serum

MK levels in GCF and serum samples were determined using a solid-phase sandwich ELISA (catalog no. RHF911CKC, Antigenix America Inc., USA). The samples were run in triplicate to ensure accuracy and to provide sufficient data for the statistical validation of the results. An ELISA reader (Biorad, USA) with primary and reference wavelengths of 450 and 655 nm, respectively, was used to measure the absorbance of the substrate. The MK levels in the tested samples were evaluated using a standard curve plot for which the absorbance values of standards were provided along with the kit. The absorbance readings were converted into definite volumes (ng/μL) by using a standard reference curve. The MK level in each sample was calculated by dividing the amount of MK with the volume of sample (ng/μL).


  Statistical Analysis Top


Data were analyzed using the SPSS version 11.5 (SPSS Inc., Chicago, IL, USA). A sample size of 20 was sufficient to achieve >80% power at a 0.1 level of significance. Validity tests for normality assumption were conducted using standardized range statistics to confirm that the assumption is valid. In addition, pairwise comparisons were conducted using the Mann–Whitney U test to determine the pair(s) that differed. The Spearman's correlation analysis was used to identify an association between the GCF MK level and clinical parameters.


  Results Top


Samples in all groups tested positive for the presence of MK. Mean GCF MK levels in Groups I, II, and III were 0.136, 0.316, and 0.342, respectively. As observed, the GCF MK level in Group II appeared between the highest and lowest values. In addition, mean serum MK levels in Groups I, II, and III were 0.143, 0.331, and 0.336 ng/μL, respectively. As observed, the serum MK level in Group II appeared between the highest and lowest values [Table 1].
Table 1: Descriptive Statistics of Baseline Parameters in The Study Population

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The Mann–Whitney U tests were conducted to determine if significant differences exist between GCF and serum MK levels and between the study groups. The results indicated that the GCF MK levels increased progressively from Group I to III. The results indicated that the serum MK levels in Group III was between Groups I and II [Table 2] and [Table 3].
Table 2: Pairwise Comparison Using Mann-Whitney U Test for Gingival Crevicular Fluid (GCF) Midkine

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Table 3: Pairwise Comparison Using Mann-Whitney U Test For Serum Midkine

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The Spearman's correlation analysis was conducted to determine a correlation between GCF and serum MK levels and the clinical parameters for all groups. The results revealed a significant positive correlation between GCF and serum MK levels and the clinical parameters [Table 4], [Table 5], [Table 6].
Table 4: Spearman Correlation Test between Gingival Crevicular Fluid (GCF) and Serum and Clinical Parameters in Group I

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Table 5: Spearman Correlation Test between GCF and Serum and Clinical Parameters in Group II

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Table 6: Spearman Correlation Test between GCF And Serum and Clinical Parameters in Group III

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The confidence interval was calculated for differentiating the limits of GCF and serum MK levels in different groups before considering MK as an inflammatory biomarker [Table 7] and [Table 8].
Table 7: Differentiating Values for Different Groups for Gingival Crevicular Fluid (GCF) Midkine (ng/μl)

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Table 8: Differentiating Values for Different Groups for Serum Midkine (ng/μl)

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  Discussion Top


PD is a biofilm-induced chronic inflammatory disease that leads to the destruction of the periodontium i.e., the tooth-supporting structures.[10] The host response in periodontitis has traditionally been considered to be mediated mainly by B- and T-lymphocytes, neutrophils, and monocytes/macrophages. These are triggered to produce inflammatory mediators including cytokines, chemokines, arachidonic acid metabolites, and proteolytic enzymes, which collectively contribute to tissue degradation and bone resorption by activation of several distinct host degradative pathways.[11]

MK and PTN comprise a distinct family of heparin-binding growth factors that are multifunctional, acting both as growth factors and activators of neutrophils and also both MK and PTN have antibacterial activity against a wide variety of bacteria.[12]

MK is a heparin-binding protein of 13 kDa, and was found as the product of a retinoic acid-responsive gene, which becomes activated at the early differentiation stage of embryonal carcinoma cells.[13]

In previous studies, the expression of MK has been noted in several systemic inflammatory diseases in humans. Narita et al.[14] conducted a study to demonstrate MK is expressed by infiltrating macrophages in in-stent restenosis in hypercholesterolemic rabbits. The results suggested that macrophages are the major source of MK in the atherosclerotic neointima. Ludwig et al.[15] conducted a study to demonstrate that MK was critically involved in the recruitment of PMNs during acute inflammation, playing a key role for adhesion, and subsequent extravasation.

Increasing evidence has led to the hypothesis that the dysregulation of the MK pathway during bacterial infections is a conceivable determinant of PD activity. This stimulated several investigations into the potential role of MK in the pathogenesis of PD. However, no studies have investigated the effects of smoking on GCF and serum MK levels in gingival health and disease. Therefore, we assessed the effects of smoking on MK levels in GCF and serum.

In this study, an extra crevicular (unstimulated) method of GCF collection by using micropipettes was conducted to ensure atraumatism and in turn obtain undiluted samples of native GCF, the volume of which could be accurately assessed to avoid the non-specific attachment of the analyte to the filter paper fibers. In this study, GCF and serum MK levels were analyzed using the ELISA (sensitivity level for MK detection is 0.020 ng/μL).

McLaughlin et al.[16] conducted a study to examine the immediate effects of smoking on gingival fluid flow. The results indicate that smoking produces a marked transient increase in GCF flow rate, which might reflect changes in blood flow known to be produced by nicotine. In the present study, mean GCF and serum MK levels increased proportionally from Group I (0.136–0.143 ng/μL) to Group III (0.342–0.333 ng/μL), whereas mean GCF and serum MK levels in Group II were between those of two groups (0.316–0.331 ng/μL). In the present study, mean GCF and serum MK levels followed the order Group III > Group II > Group I. The MK levels increased proportionally with the severity of the disease in Groups II and III, thus confirming a positive correlation with the clinical parameters.

The variability in MK levels among patients of each group could be attributed to the role of MK in the different stages of the disease during GCF and serum sample collection. The increase in the MK levels in the GCF of two participants (0.213 and 0.186 ng/μL) and in the serum of two participant (0.218 and 0.186 ng/μL) of Group I was possibly because of subclinical inflammation, allergy, and infection not reported by these patients. The large variation in the MK levels in Groups I and II was partially because of differences in the disease activity and crevicular fluid flow during sample collection, variations in the number of defense cells migrating to the crevice, differences in the expression of MK receptors, and the interaction between MK and MK receptor.


  Conclusion Top


According to the results, the GCF and serum MK levels increased proportionally with the severity of inflammation. The data indicated that the high GCF and serum MK levels cause a significantly greater risk of gingivitis progression. This study is useful in assessing the health, disease status of gingival tissues, with low MK levels. Therefore, MK can be considered an inflammatory biomarker in gingivitis condition.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Bergström J, Eliasson S. Noxious effect of cigarette smoking on periodontal health. J Periodontal Res 1987;22:513-7.  Back to cited text no. 1
    
2.
Preber H, Bergström J, Linder LE. Occurrence of periopathogens in smoker and non-smoker patients. J Clin Periodontol 1992;19:667-71.  Back to cited text no. 2
    
3.
Rossomando EF, Kennedy JE, Hadjimichael J. Tumour necrosis factor alpha in gingival crevicular fluid as a possible indicator of periodontal disease in humans. Arch Oral Biol 1990;35:431-4.  Back to cited text no. 3
    
4.
Boström L, Linder LE, Bergström J. Influence of smoking on the outcome of periodontal surgery. A 5-year follow-up. J Clin Periodontol 1998;25:194-201.  Back to cited text no. 4
    
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Genco RJ. Host responses in periodontal diseases: Current concepts. J Periodontol 1992;63:338-55.  Back to cited text no. 5
    
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Boström L, Linder LE, Bergström J. Smoking and GCF levels of IL-1beta and IL-1ra in periodontal disease. J Clin Periodontol 2000;27:250-5.  Back to cited text no. 6
    
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Giannopoulou C, Cappuyns I, Mombelli A. Effect of smoking on gingival crevicular fluid cytokine profile during experimental gingivitis. J Clin Periodontol 2003;30:996-1002.  Back to cited text no. 7
    
8.
Nordin SL, Sonesson A, Malmsten M, Mörgelin M, Egesten A. The epithelium-produced growth factor midkine has fungicidal properties. J Antimicrob Chemother 2012;67:1927-36.  Back to cited text no. 8
    
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Takada T, Toriyama K, Muramatsu H, Song XJ, Torii S, Muramatsu T. Midkine, a retinoic acid-inducible heparin-binding cytokine in inflammatory responses: Chemotactic activity to neutrophils and association with inflammatory synovitis. J Biochem 1997;122:453-8.  Back to cited text no. 9
    
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Taba M Jr, Kinney J, Kim AS, Giannobile WV. Diagnostic biomarkers for oral and periodontal diseases. Dent Clin North Am 2005;49:551-71.  Back to cited text no. 10
    
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Yucel-Lindberg T, Båge T. Inflammatory mediators in the pathogenesis of periodontitis. Expert Rev Mol Med 2013;15:e7.  Back to cited text no. 11
    
12.
Takada T, Toriyama K, Muramatsu H, Song XJ, Torii S, Muramatsu T. Midkine, a retinoic acid-inducible heparin-binding cytokine in inflammatory responses: Chemotactic activity to neutrophils and association with inflammatory synovitis. J Biochem 1997;122:453-8.  Back to cited text no. 12
    
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Muramatsu T. Midkine: A promising molecule for drug development to treat diseases of the central nervous system. Curr Pharm Des 2011;17:410-23.  Back to cited text no. 13
    
14.
Narita H, Chen S, Komori K, Kadomatsu K. Midkine is expressed by infiltrating macrophages in in-stent restenosis in hypercholesterolemic rabbits. J Vasc Surg 2008;47:1322-9.  Back to cited text no. 14
    
15.
Weckbach LT, Muramatsu T, Walzog B. Midkine in inflammation. ScientificWorldJournal 2011;11:2491-505.  Back to cited text no. 15
    
16.
McLaughlin WS, Lovat FM, Macgregor ID, Kelly PJ. The immediate effects of smoking on gingival fluid flow. J Clin Periodontol 1993;20:448-51.  Back to cited text no. 16
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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Introduction
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