CLINICAL AND LABORATORY EVALUATION OF ADRENOMEDULLIN AS A BIOMARKER IN MODERATE CHRONIC PERIODONTITIS

Shahawi M. Salim 1 , Jilan M. Youssef 2 , Shereen Salah 3 and Mohamed M. Anees 4 . 1. B.D.S. Faculty of Dentistry Sirte University, Libya. 2. Professor of Oral Medicine and Periodontology, Faculty of Dentistry, Mansoura University. 3. Professor of Clinical Pathology, Faculty of Medicine, Mansoura University. 4. Ass. Professor of Oral Medicine and Periodontology, Faculty of Dentistry, Mansoura University. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5 (12), 1622-1628 1624 Collection of gingival crevicular fluid (GCF) sample:-In both groups, GCF samples were collected at the initial visit and in group I after four weeks of treatment. Clinically detected supra gingival plaque was removed carefully without touching the marginal gingiva to minimize contamination of the paper strips by the plaque. The teeth were then gently washed with water, and the sites under study were isolated with cotton rolls to prevent contamination with saliva and gently dried with an air syringe. One sterile paper point was used for each collection site. GCF samples were collected from only 2 sites in each of the diseased participants and the healthy controls. Paper point were carefully inserted into the gingival crevice until mild resistance was felt and left there for 30 seconds. Following collection of GCF the paper point placed in eppendroffs tube contain phosphate buffer saline and stored at -80°C for later processing. Paper points for each participant were pooled, and the GCF was extracted and assayed for the levels of interleukin-1β (IL-1β) and Adrenomedullin (ADM). (8) GCF IL1β and ADM analysis:-The levels of IL1β and ADM were determined with the use of commercially available enzyme-linked immune sorbent assay (ELISA) kit following the manufacturer instructions.

Result:-
Group I (6 males and 9 females). Group II (2 males and 13 females) Table (1) illustrates the difference between study and control groups as regard clinical and laboratory parameters before treatment. Plaque index showed a high statistically significant difference ( p < 0.001 ) between study group and control group at base line with higher mean±SD plaque index in study group (2.37±0.26) than control group (0.531±0.09). Gingival index showed a high statistically significant difference between studied groups (p < 0.001) with higher mean gingival index in study group than control group (2.14±0.1& 0.27±0.08 respectively). Bleeding on probing index illustrated a high statistically significant difference between studied groups (p < 0.001) with higher mean in study group (0.92±0.08) than control group (0.19±0.07). Probing pocket depth illustrated a high statistically significant difference between studied groups (p < 0.001) with higher mean in probing pocket depth in study group (3.07±0.4) than control group (0.92±0.06). Interleukin-1β level (pg/ml) showed statistically significant higher mean value in study group (33.32±3.96) than control group (21.32±2.23) at p < 0.001. Adrenomedullin level (pg/ml) showed statistically significant higher mean value in study group (17.73±1.9) than control group (11.0±1.6) at p < 0.001.  Table (2) illustrate the change in clinical and laboratory indices after treatment in study groups. The mean and standard deviation of plaque index at baseline was 2.37±0.26 and after four weeks of treatment became 0.66±0.14.
There was a highly statistically significant improvement between plaque index values at baseline and four weeks after treatment (P < 0.001). The mean and standard deviation of gingival index at baseline was 2.14 ± 0.1 and after four weeks of treatment became 0.59 ± 0.12. There was a statistically highly significant difference between gingival index values at baseline and four weeks after treatment (P < 0.001). The mean and standard deviation value of bleeding on probing index at baseline was 0.92± 0.08 and after four weeks of treatment became 0.29±0.07. There was a highly significant difference between bleeding on probing index values at baseline and four weeks after treatment (P< 0.001). The mean and standard deviation of probing pocket depth at baseline was 3.07±0.4 and after four weeks of treatment was 1.03±0. 16. There was a statistically highly significant difference between probing pocket depth values at baseline and 4 weeks after treatment (P< 0.001). Regarding the clinical attachment level at baseline, the mean and standard deviation was 3.11±0.38 versus its values after four weeks of treatment 1.054±0. 16. There was a highly significant difference between clinical attachment level values at baseline and four weeks after treatment (P < 0.001). The mean and standard deviation of IL-1β at baseline was 33.32±3.96 and after four weeks of treatment became 23.09±3.8. There was a high statistically significant decrease in the level of IL-1β in GCF 1625 between baseline and four weeks after treatment (p < 0.001. The mean and standard deviation of ADM at baseline was 17.73±1.9 and after 4 weeks of treatment became 11.93±1.99. There was a high statistically significant difference in the level of ADM in GCF between baseline and 4 weeks after treatment (p < 0.001).

Discussion:-
The fundamental event in the transition from gingivitis to periodontitis is the loss of soft tissue attachment of the tooth and subsequent loss of alveolar bone. Periodontitis is an inflammatory disease that leads to local elevation in levels of pro-inflammatory cytokine which plays a vital role in the process of inflammation associated with the destruction of the periodontium. (9) The interaction of micro-organisms with the host immune system determines the course and severity of the resulting disease. It is generally accepted that much of the periodontal tissue destruction observed in periodontitis is host-mediated through release of proinflammatory cytokines by local tissues and immune cells in response to the bacterial flora and its products/metabolites, especially lipopolysaccharides. Antimicrobial proteins and peptides constitute a diverse class of host-defense molecules that act early to combat invasion and infection with bacteria and other microorganisms. (10) Antimicrobial peptides (e.g defensins, cathelicidin and adrenomedullin) are an important part of the host innate immune response in gingival tissues. (11) They are important contributors for maintaining the balance between the healthy and diseased conditions of the oral cavity. (2) Gingival crevicular fluid as well as saliva represent a powerful vehicle, which contains various cellular and biochemical arrays for observation of tissue and cell products and permits a degree of non-invasive accessibility to the periodontium. (12) Thus, the majority of new diagnostic tests for periodontal disease utilize these vehicles, (13) and evaluation of the different tissue and cell markers in gingival crevicular fluid as well as saliva is regarded as a useful manner to determine a person's risk for periodontal disease. (14) As the adrenomedullin concentrations in gingival crevicular fluid is about 30-fold compared to saliva , (15) so in the present study gingival crevicular fluid was selected to assess adrenomedullin level in chronic periodontitis patients (moderate type). Gingival crevicular fluid samples were obtained from each subject by using absorbent filter paper 1626 point. The paper points were inserted into crevices or pockets until minimum resistance was felt for only 30 seconds. (16) Careful isolation and plaque removal was performed prior to gingival crevicular fluid collection to prevent saliva or plaque contamination, and any filter paper contaminated with saliva, plaque or blood was discarded to avoid false results in the gingival crevicular fluid analysis procedure. (17) Furthering, the present study was designed to evaluate if gingival crevicular fluid adrenomedullin could be considered as potential marker of periodontal inflammation and / or destruction in cases of moderate chronic periodontitis.
The evaluation of clinical parameters (GI, PI, BOP, PPD and CAL) revealed significant differences, between moderate chronic periodontitis patients at baseline and healthy subjects. These results were in accordance with Ghodpage (18) ; Lamster and Ahlo (19) . They revealed statistically significant differences between chronic periodontitis patients at baseline and healthy subjects regarding the same clinical parameters.
The clinical parameters (GI, PI & BOP) in moderate chronic periodontitis patients decreased significantly after phase I periodontal therapy versus their level at the baseline. These results were in agreement with Abdul Aziz et al where they found significant reduction in PI, GI, and POB, after phase I periodontal therapy . (20) The significant intragroup differences in the form of the reported improvement of the measured clinical parameters after phase I therapy reinforce the importance of SRP as a basic therapeutic modality for chronic periodontitis. These results clarify the role played by scaling and root planning that eliminate the favorable environment for colonization of periodontopathogen resulting in reduction of the inflammatory response induced by the bacterial load and cessation of periodontal disease progression, manifested as reduction of probing pocket depth and a relative gain of clinical attachment. (21) Regarding interleukin-1β concentration in gingival crevicular fluid, the results of the present study have shown that, there was a highly significant difference in the concentration of gingival crevicular IL-1β between the chronic periodontitis patients and control group at baseline. These findings were consistent with the results of many studies. They demonstrated that the concentration of interleukin-1β in gingival crevicular fluid was higher in patients with periodontitis than in individuals with clinically healthy sites of periodontium. Furthermore, in the present study there was a significant reduction in the level of intrleukin-1 β in gingival crevicular fluid between the baseline and after four weeks of periodontal treatment. In agreement with the current data, other studies demonstrated a decrease in gingival crevicular fluid levels of interleukin-1β after phase I therapy. (22)(23)(24) The noticed difference in IL-1β level among our studied groups (study and control) could be explained as IL-1 β represent a kind of proinflammatory cytokine, which has a central role in regulating the immune system and is responsible for a variety of immune responses. As the IL-1β can influence a large number of cells, such as fibroblasts, chondroblasts, bone cells, neutrophils and lymphocytes, the periodontal destruction and periodontal regeneration can be partially related to this cytokine. (25) Regarding the present study, adrenomedullin level in gingival crevicular fluid of moderate chronic periodontitis was compared with control group, and it was found that the total adrenomedullin levels in gingival crevicular fluid was significantly higher in moderate chronic periodontitis group at baseline. Lundy et al (6) supported our data. They showed that, the total amount of gingival crevicular fluid adrenomedullin in periodontitis sites was higher than that of healthy sites. Similarly, our data was supported by Oya et al. (26) They demonstrated that patients with periodontitis had a higher total amount of GCF adrenomedullin compared to healthy controls. Also, they added the total amount of gingival crevicular fluid adrenomedullin was significantly higher in periodontitis groups than that of the gingivitis group, and although not significant, there was a higher total amount of gingival crevicular fluid adrenomedullin in the gingivitis group compared to that of the healthy group. Other studies have reported higher adrenomedullin level in gingival crevicular fluid of chronic periodontitis patients as compared to periodontal health. (27)(28)(29)(30) The higher gingival crevicular fluid adrenomedullin levels in patient group could be due to the fact that it is constitutively expressed and secreted by oral epithelial cells and its expression increased in response to live oral bacteria, as lipopolysaccharide (LPS) is a potent stimulator of adrenomedullin secretion. (31) Furthermore the increase of gingival crevicular fluid adrenomedullin levels was more prominent in patients with periodontitis 1627 because of the presence of more severe inflammation and periodontal destruction and an increase of bacteriastimulated adrenomedullin production by epithelial cells to protect the host from the bacterial invasion. (6,(32)(33)(34) Moreover, adrenomedullin production is controlled by various humoral factors and physical factors and inflammatory cytokines such as tumor necrosis factor TNF-α, TNF-β, interleukin IL-1α and IL-1β. (35) In the study group, regarding the level of adrenomedullin in gingival crevicular fluid, a significant reduction was found after phase I therapy in comparison to its level at baseline. To the best of our knowledge, no other studies in the literature evaluated the effect of phase I periodontal therapy on the level of adrenomedullinin in crevicular fluid. Thus, the present study was designed to evaluate the effect of phase I therapy on adrenomedullin level and searching for an answer of this question that may serve partly in more understanding of the exact role played by adrenomedullin in the pathogenesis of periodontal diseases. Since, this was the first study concerning this assessment of effect of scaling and root planing on crevicular level of adrenomedullin. So, we cannot know how many studies will be in agreement or disagreement with our results.

Conclusions:-
Scaling and root planing (SRP) is the mainstay of treatment of periodontal diseases as SRP was effective in improving clinical parameters in patients with moderate chronic periodontitis. ADM in gingival crevicular fluid could be used as a potential diagnostic marker for chronic periodontitis.