Untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8651896 Volume 17 2018 e18015 Original Article 1 PhD. In Oral Biochemistry Lecturer in Basic science Department / College of Nursing- Hawler Medical University /Erbil/ Iraq. Corresponding author: Jwan Ibrahim. Jawzal Emails: jwanjaw@yahoo.com, jwanjawzali@gmail.com College email: jwanjaw@ nur.hmu.edu.krd Received: August 26, 2017 Accepted: December 01, 2017 Salivary Interleukin 6 and Sialic acid in Periodontitis Jwan Ibrahim. Jawzali Aim: Periodontitis is the major multi –factorial chronic infectious oral diseases in dentate people. Sialic acid regulates innate immunity response that release cytokines. The study aimed to evaluate interleukin-6 levels in periodontittsis and its relation to clinical features, total sialic acid and its fraction and total proteins to clarify its role. Material and Methods: The study was observational case-control study, carried out in periodontology clinic, College of Dentistry / Erbil /Iraq. A total of 60 participants were recruited in this study, They were divided into three groups: control group represent systemically and periodontally healthy subjects, clinically, diagnosed dentate periodontitis  group and partial edentulous group.The data was collected through interview questionnaire, clinical periodontal examination, and biochemical tests for salivary; IL-6.total sialic acid and its fraction, and salivary total proteins Statistical analysis was done by statistical Package for Social Sciences. Results: Statistical analysis showed a significant (P≤ 0.01) highest value of total sialic acid in periodontitis. While the highest value of IL-6 was in partial edentulous group. Old ages increased salivary IL-6 significantly. In periodontitis there was significantly association of IL-6 with probe pocket depth, mobility scores of teeth, protein bonund sialic acid and significant negative association with lipid bound sialic acid in the precipitate. While in partial edentulous IL-6 associated significantly with gingival index and free sialic acid in precipitate and negatively with probe pocket depth. Conclusions: Salivary sialic acid and IL-6 are periodontitis biomarkers in dentate. Pleiotropic role of IL-6 can be diagnosed by sialic acid levels. It depends on age, (which affects number of teeth and salivary flow rate), and treatment conditions. Keywords: Sialic acid. Periodontal inflammation and IL-6. mailto:jwanjaw@yahoo.com mailto:jwanjawzali@gmail.com 2 Jwan Ibrahim Introduction Periodontitis is the major chronic inflammatory oral disease in dentate people. It is a multi -factorial influenced by genetics as well as by the environment1. It is initiated by gram negative bacteria that secrete various cytokines from lipo-polysaccharides2. Improper or exuberant immune response lead directly toward over production of inflammatory cytokines that lead to deleterious inflammatory processes and destruction of the peri- odontal tissue and alveolar bone, consequently periodontal attachment loss3. Interleukin-6 (IL-6) is an important cytokine involved in the regulation of host response to tissue injury and infection4. IL-6, produced locally in bone following stimulation by IL-1 and tumor necrosis factor (TNF)5, by a variety of cells, such as monocytes, fibroblasts, osteoblasts, and vascular endothelial cells in response to inflammatory challenges. IL-6 activity in inflammation is considered double-edged (a pleiotropic cytokine), that may enhance or suppress inflammatory bone destruction. Traditionally IL-6 has been considered to be a pro-inflammatory mediator, because it is induced by IL-1 and TNF-α early in the inflammatory cascade and stimulates expression of acute-phase proteins that have anti-inflammatory properties6. Previous studies reported anti-inflammatory properties of IL-6; it can increase the production of tissue inhibitors of matrix metalloproteinases (TIMP), suppresses IL-1 expression, can induce the synthesis of IL-1 receptor antagonist (IL-1Ra) and the release of soluble TNF receptors4. Recent studies demonstrate that IL-6 deficient in many typical pro-inflammatory properties and exert a number of anti-inflammatory activities such as; indirect stimulation and production of; collagenase, and matrix metalloproteinase, or stromelysin, inhibition superoxide production, and suppression spontaneous IL-1-mediated degradation of cartilage matrix5. Therefore individual variability in the ability to synthesize and release of IL-6 may mod- ulate the susceptibility, development, and progression of a number of autoimmune and inflammatory diseases (periodontal diseases) which are recently reported to be associated with IL-6 deregulation6. Sialic acids are a family of nine carbon acidic monosaccharide. An important function of host sialic acid is to regulate innate immunity7. Bacteria with capsular polysac- charide (such as gram negative bacteria) containing sialic acid interacts with com- ponent of the hosts nonspecific immune response, may modulate the ability of the host to mediate an immune response by affecting the release of cytokine molecules thereby disrupting the coordination of the hosts cell-mediated immune response8. Pro-inflammatory cytokines such as interleukin-1, interleukin-6, and tumor necrosis factor-α stimulated to produce acute-phase glycol-proteins with sialic acid as a com- ponent of the oligosaccharide side chain9. Sialic acid is a stable and representative marker of the overall acute-phase response10. The current criteria for assessment of periodontal tissues surrounding teeth were based on clinical and radiographic changes. Saliva is a non-invasive suitable environ- ment for biochemical and immunological analysis and can be an early indicator for detection of active periodontal disease11. 3 Jwan Ibrahim Imbalance between pro- and anti-inflammatory cytokines could be involved in the initiation and progression of chronic periodontitis12.There are conflicting reports on differences in salivary IL-6 levels between chronic periodontitis and healthy subjects. Studies are required for clarifications the roles of IL-6 in periodontitis progression and identifying factors that may affect the role of IL-6. Therefore this study conducted to evaluate interleukin-6 levels in periodontitis and its relation to clinical features and inflammable salivary biomarkers; total sialic acid and its fraction and total proteins to identify its role in disease progression. Methods and patients: The study was observational case-control study. It was arried out in the dental teaching clinic - College of Dentistry / Hawler Medical University. During the period of 1st August 2009 up to 30th September 2009. Participants (Study populations): A total  of 60 convenient samples recruited in this study, with age ranged between 18 and 70 years of both genders. They were screened for periodontal health status and divided into three groups; first were 20 participants free from periodontal disease and regarded as control group, second 32 dentate out patients group, diagnosed with chronic periodontitis disease,(had >3-≥7mm pock- ets depth), according to the classification of the periodontal diseases issued by the American Academy of Periodontology13, in 1999, third group were 8 partial edentulous periodontitis patients with history of treatment. Inclusion criteria: Healthy participants systemically and with no history of periodontal and antibiotic treatment prior to the study for three month. Exclusion criteria: Pregnant and lactating women and patients with systemic diseases. Sampling technique: A structured questionnaire (interview form) was used to collect data by asking the studied population about social and behavior factors included; age, sex, occupation, educational levels, smoking status, oral hygiene habits (frequency of tooth brushing, use of dental floss), and use of medications. All participants were informed on the procedure and their consents had been taken. Clinical measurement: The clinical periodontal examinations were carried out by the trained dentist and calibrated by the supervisor of the teaching clinic. The measurements included; bleeding on probing by Saxer and Mühlemann14, probe pocket depth (PD) was measured with (Williams probe), for all teeth from gingival margin to the base of pocket at four sides (labial/buccal, lingual/palatal, mesial and distal surfaces), per teeth, mobility of teeth classified by Carranza and Takei15, and miss of teeth. Classifications of The (AAP)13 were used for identifying the severity of disease; probing depths >3 & <5 mm (mild pock- ets), ≥ 5 & <7 mm (moderate pockets) and ≥ 7 mm (sever pockets). The gingival index (GI) and plaque index (PI) scores were recorded, using criteria of (Löe, 1987)16. Saliva collection Saliva samples (prior to the clinical measurements) were collected from all subjects between 9 and 11 hours am. Spitting method17 was used for collection un-stimulated whole saliva. Patients were prevented from eating, drinking, and oral hygiene for 2 h before collection. The samples were stored at -200 C for one hour, then centrifuged immediately at (10000) g and at 40C for 20 minutes to obtain supernatant and precip- itate. Both fractions were stored at -20C0 for analysis. 4 Jwan Ibrahim Biochemical tests: This included salivary IL-6 Enzyme-linked immunosorbent assay (ELISA) kits (Abeckman coulter manufacture). Total sialic acid (TSA) and free sialic acids (FSA) were measured with modified thiobarbituric acid method of Skoza and Mohos18. Extraction and determination of lipid -bound sialic acid (LSA) by Masami19. Determination of sialic acid bound to total proteins (PSA) by Shetty and Pattabira- man20, salivary total proteins by Lowry method Davidson College21. Statistical analysis: The statistical Package for Social Sciences (SPSS, Version 18) was used for data analysis by parametric and non parametric tests for normal and abnormal distribution of variables checked by shapiro-wilk test. Analysis of vari- ance (ANOVA-test) to compare between three or more means and t-test to compare between two means. Correlation coefficient for measuring correlation between quan- titative data and Eta test for measuring association between nominal and interval. Binary logistic regression for odds ratios measurement. A p-value of ≤ 0.05, and ≤ 0.01 were considered statistically significant and high significant respectively. Results The studied population composed of 32 (53.3%) periodontitis, 20 (33.3%) participant as control and 8 (13.3%) partial edentulous patients with teeth miss more than (10). Salivary mean levels of IL-6, total sialic acid and total protein Statistical analysis showed a significant (P≤ 0.01) difference in the means of total sialic acid and total protein between periodontitis, control, and partial edentulous group. Post Hoc Least Significant Difference (LSD) test showed significant (P≤ 0.01) difference of periodontitis with control and partial edentulous groups in sialic acid, while partial edentulous showed similarity with control group. Total salivary proteins, in periodontitis differ significantly (P≤ 0.05) with control but not with partial edentu- lous. There was also a significant (P≤ 0.01) difference in the means of IL-6 between the groups. Post Hoc (LSD) test showed significant highest value (P≤ 0.01) value of IL-6 in partial edentulous compare to periodontitis and control groups. Table 1. Mean levels of salivary; IL-6, protein, and total sialic acid Groups No. IL-6 (pg/ml) Mean ± S.E F-value P-value Periodontitis 32 26.13± 7.4 18.7 0.00** Control 20 1.7 ± 8.3 Partial edentulous 8 66.35± 8.3 Groups No Salivary protein Mean ± S.E. F-value P-value Periodontitis 32 6.6± 0.34 11.5 0.00** Control 20 4.12 ±0..27 Partial edentulous 8 5.7± 0.47 Groups No. Total sialic acid (mg/l) Mean ± S.E. F value P-value Periodontitis 32 156.84± 3.9 16.96 0.00** Control 20 96.80± 0.32 Partial edentulous 8 100.36± 19.5 **(P≤ 0.01), *(P≤ 0.05), No = Number 5 Jwan Ibrahim Relation of ages with salivary IL-6 and cases: Statistical analysis showed a sig- nificant correlation of IL-6 with ages and significant differences of IL-6 among age groups. There was also significant relation and differences of ages among cases. Binary Logistic regression showed a significant little increase risk of IL-6 levels in old ages and in partial edentulous, Table (2). Relation between IL-6 and periodontal health status in periodontitis: Table (3) shows significant positive correlations of IL-6 and protein bound to sialic acid (PSA) with probe pocket depth (PD). There was also high relations of (IL-6) and total sialic acid with mobility of teeth. Statistical analysis showed significant differences in the means of IL-6 and PSA among groups of probe depth (PD), and TSA among teeth mobility scores. Table 2. Relation and differences of ages with salivary (IL-6) and cases Age groups (Years) IL-6 (pg/ml) Mean± S.E. No. Value of R P-value F-value P-value B 95% CI 18-44 14.9 ± 3.7 36 0.37 0.003** - 2.3 0.028* 1.024* 1.0 - 1.1 45-75 35.9 ± 8.3 24 Groups of the study (cases) Mean of ages ± SE No Eta test Degree F-value P-value B 95% CI Periodontitis 39.5 ± 2.23 0.79 Strong 3.5 0.037* 1.03* 1.0 - 1.06 Control 34.9 ± 3.5 Partial ed. 49.5 ± 2.8 Total 39.3 ± 1.8 (P≤ 0.01)**,(P≤ 0.05)*, R=Pearson’s correlation coefficient, B = odds ratio, CI = Confident interval, ed. = edentulous Table 3. Association of IL-6 and sialic acid with probe pocket depth and teeth mobility in periodontitis Prop depth of teeth (PD) in mm IL-6 (pg/ml) Means± S.E. No. Value of R P-value F-Test Value P-value of Difference Mild > 3- < 5 20.7 ± 4.9 17 0.37 0.039* 6.5 0.005* Moderate ≥5-6 26.1 ± 4.9 13 Sever ≥ 7 53.0 ± 37.75 2 Total 26.14 ± 3.9 32 Prop depth (PD) in mm PSA Means± S.E.(mg/l) No. Value of R P-value F-Test Value P-value of Differences Mild > 3- < 5 26.5 ± 2.6 17 0.52 0.002** 15.1 0.001** Moderate ≥5-6 30.2. ± 2.9 13 Sever ≥ 7 79.5 ± 29.4 2 Total 31.3 ± 17.9 32 Mobility scores IL-6 (pg/ml) Means± S.E. No. Value of R P-value F-value P-value of difference. 0 16.44±5.3 9 0.37 0.038* 1.76 0.176 1 23.9±21.6 14 2 40.3± 10.9 7 3 36.0± 15.0 2 Total 26.13±3.9 32 Mobility scores Total sialic acid (mg/l) Means± S.E. No. Value of R P-value F-value P-value of difference. 0 100.6 ± 28.9 9 0.35 0.047* 6.0 0.003** 1 87.7 ± 4.4 14 2 130.3 ± 15.5 7 3 145.1 ± 18.7 2 Total 156.8 ± 41.6 32 * = (P≤ 0.05), ** = (P≤ 0.01), R- Pearson’s correlation 6 Jwan Ibrahim Assosiation of IL-6 in partially edentulous with periodontal health status Table (4) shows a negative correlation of IL-6 with probe pocket depth, and signifi- cant positive correlation of IL-6 with gingival index GI and significant difference of IL-6 among GI groups. There was also negative correlation of probe pocket depth with TSA (-0.85, P value = 0.1) and its fractions, while GI showed positive significant correlation (0.78, P value = 0.05*) with FSA in sediment of saliva. Correlation of IL-6 with total sialic acid and its fractions among groups of studied population: Statistical pearson’s correlation showed a significant correlation of (IL-6) with protein bound sialic in supernatant (r=0.468**), (Fig 1) and total salivary protein (r=0.451**) in periodontitis,. While lipid bound sialic acid in sediment showed significant (P≤ 0.05) negative correlation (r= -0.382*) with (IL-6) in periodontitis (Fig 2) as well as free sialic acid in sediment of saliva showed a significant positive correlation (Spearman cor- relation value= 0.820*) with salivary IL-6 among partial edentulous group. Table 4. Association of IL-6 with probe pocket depth, and gingival scores Prop depth of teeth (PD) in mm IL-6 (pg/ml) Means± S.E. No. Value of Spearman P-value of R Kruskal-Test Value P-value Sig. of Difference Mild > 3- < 5 79.5± 35.7 4 - 0.22 No sig. 0.33 No sigModerate ≥5-6 53.3±19.9 4 Total 66.4± 19.5 8 Groups of Gingival Index Means± S.E Spearman P-value of spearman Mann-Whitney Moderate (1.1-2) 42.8 ± 14.2 6 0.93 0.01** 0.047*Sever (2.1-3) 136.9 ± 31.9 2 Total 66.4 ± 19.5 8 * = (P≤ 0.05); ** = (P≤ 0.01) Figure 1. Correlation between salivary IL-6 and protein bound sialic acid in supernatant of saliva of periodontitis 120.00 Le ve ls o f p ro te in b ou nd s ia lic a ci d in su pe rn at an t o f s al iv e (m g/ l) R2 Linear = 0.219 100.00 80.00 60.00 40.00 20.00 0.00 0.00 20.00 40.00 60.00 Levels of salivary IL-6 in periodotitis (pg/ml) 80.00 100.00 7 Jwan Ibrahim Discussion Salivary levels of IL-6, total sialic acid and total protein in periodontitise and controls Total salivary; sialic acid, protein and IL-6 showed significant high concentration in periodontitis patients compare to controls. This view pathogenesis role of increased salivary TSA levels in periodontal disease suggested by Shinohara et al.22 and can differentiate between periodontal disease and normal condition. High level of IL-6 is in line with previous studies (Elbersol and Cappelli23, Miller et al.24) who found that periodontitis patients have higher (IL-6) levels when compared to the periodontally healthy population. This result is in contrast with studies of Nibali et al.6 and Shaker and Hashem12 who found no significant difference in the levels of IL-6 between chronic periodontitis and periodontally healthy subjects. Significant difference in the level of total; protein, sialic acid and (IL-6) between peri- odontitis and control may be returned to virulence type of bacteria and its products in periodontitis, and change in biosynthesis and post translational glycosylation pro- cesses of the acute-phase glycoprotein in the liver as indicated a by significant pos- itive and negative correlation of (IL-6) with, protein bound to sialic acid (PSA), and lipid bound sialic acid (LSA) in sediment respectively. Bacteria with virulence factor included either serotype lipopolysaccharide or specific antigen stimulates secretion of cytokines from monocytes that are modulated with sialic acid. This was in agreement with Soell et al.25 who stated that structurally related cell surface proteins from strep- tococcus mutans (major bacteria of oral cavity) binds to monocyte surface receptors via sialic acid residues and exerts immune-modulator effects on human monocytes like induction of (TNF-α), IL-1β, and IL-6). Mean level of salivary (IL-6) in control in this study was in agreement with other inves- tigators26,27 who found low levels (1.4±1.0 pg/ml) and (1.8± 4.25 pg/ml) for salivary (IL-6) respectively. Figure 2. Inverse correlation between salivary IL-6 and lipid bound sialic acid in sediment of saliva of periodontitis patients 60.00 Li pi d bo un d si al ic a ci d in s ed im en t of s al iv a (m g/ l) R2 Linear = 0.146 50.00 40.00 30.00 20.00 10.00 0.00 0.00 20.00 40.00 60.00 Levels of salivary II-6 in periodotitis (pg/ml) 80.00 100.00 8 Jwan Ibrahim Salivary levels of IL-6, total sialic acid and total protein in partial edentulous Majority (75%) of partial edentulous group were female former smokers, in age group (45-70) years. Low number of teeth caused lower intensity of inflammation compared to dentate periodontitis group as indicated by lower levels of total salivary sialic acid. This result conforms the hypothesis that intensity of periodontal inflammation has been associated with the number of teeth affected28 and ensured by no significant difference in the level of sialic acid between partial edentulous group and controls. Similarity of partial edentulous group in total protein with periodontitis. may relate to history of accu- mulation effects of periodontitis and treatments process and increase in salivary anti- microbial agents. This result consistence with Jawzaly1 and Shetty and Pattabiraman20 who found high concentration of total protein in periodontitis and gingivitis. High value of IL-6 may due to; sex and old ages, that affect salivary secretion and flow rate29. This result consistence with Alwan et al.30 who found significant difference in the volume of gingival crevicular fluid between chronic periodontitis and healthy control and with Slade et al.31 who suggested that C-reactive protein levels among edentulous not similar to periodontal healthy individuals and could be raised by other risk factors; ages, and smoking, Additionally history of periodontitis and accumulative effects of treatments may affect the volume of saliva and change the equilibrium between the activities of pro-inflamma- tory and anti-inflammatory cytokines and determine the stage of severity and dissolution of inflammation as stated by Shaker and Hashem12 and reported that the total amount of cytokine might be more representative of the disease condition as compared to its concentration. This result also accompanied with Nibali et al.6 who reported that IL-6 increase associated to the short-term inflammatory response to therapy and long-term reductions when a clinical improvement in the periodontal status is obtained. This idea was more abundant among former smokers who had history of compromised outcome of periodontal therapy and conform Goutoudi et al.4 who found higher concentration of IL-6 in gingival cervicular fluid in diseased sites following treatment, and better clinical result in nonsmokers following treatment of periodontitis. Relation between ages and Interleukin 6 (IL-6) Poor periodontal status in old ages reveals a cumulative effects of periodontal micro- bial challenges and periodontal treatment, which cause severe diseases in old ages as indicated by high PD and mobility scores in periodontitis and more teeth miss in partial edentulous. This result agrees with Goutoudi et al.32 who reported deterioted periodontal status (according to periodontal indices) with age. Also Jawzaly et al.1 reported that old age individuals had received more therapy with selective extractions of teeth affected by periodontitis Relation between (IL-6) and periodontal health status among periodontitis and partial edentulous Association both IL-6 and sialic acid with periodontitis indices reveals predictor roles of both for severity of periodontitis. Results of IL-6 consistence with Shaker and Hashem12 who found a significant positive correlation between periodontal param- 9 Jwan Ibrahim eters and serum IL-6, also Alwan et al.30 who found a positive significant correla- tion between quantities of IL-6 in crevicular fluid and tissues inflammation (GI) and destruction (Pd), and with Ng et al.33 who identified significant correlation between alveolar bone loss score and (IL-6). Accompanying association of (IL-6) and sialic acid with its fraction with indices of peri- odontitis can be explained by interaction of sialic acid of salivary glycoprotein in pellicle and products of bacteria and immune system. This view the finding of Murray et al.34 that tissues destruction and pocket formation and mobility are the result of long accu- mulation of plaque, and bacteria toxic products and immune system mediators; such as collagenase, metalloproteinase, that are stimulated and produced by IL-6. It is online also with Gani et al.35 who proposed releases of inflammatory cytokines, including inter- leukins IL 1α and IL-6 and tumor necrosis factor- α, as a result of the recruitment and activation of the monocyte/T-lymphocyte axis by bacterial proliferation and / or bacte- rial products in periodontal pockets. This in turn leads to periodontal tissue destruction. Negative correlation of IL-6 and TSA with PD and significant positive correlation of IL-6 with GI in partial edentulous conforms moderate to severe gingivitis and history of irre- versible tissues destruction and pocket formation among partial edentulous group. This result supports Geivelis et al.36 who found significant positive correlations between GCF IL-6 levels in sites with gingivitis than in healthy ones and Goutoudi et al.4 who found negative correlation between total IL-6 in GCF and PD among patients with chronic peri- odontitis, and Murata et al.37 who found no association between severity of periodontitis and the number of teeth and circulating IL-6 in the elderly. However it is in contrast with previous results suggested a positive correlation of total IL-6 with disease activity and bleeding as well as PD (Lin et al.38). Different results in different studies support the idea that the production of inflammatory mediators differs by type of sample and from sub- ject to subject and other several factors; genetic and bacterial composition4. Correlation of IL-6 with total sialic acid and its fractions in periodontitis and partial edentulous High correlation of (IL-6) with PSA and total salivary protein may due to the role of (IL-6) in inducing synthesis of other mediator and enzymes and agrees with Gani et al.35 who reported that (IL-6) increased hepatic protein synthesis of acute phase pro- teins (such as richily sialylated α-acid glycoprotein, carbon reactive protein, and oth- ers) and decreased synthesis of negative reactant proteins. LSA and FSA in sediment of saliva represent fraction of cells, and high molecular weight mucin (MG1)39. Negative correlations LSA and positive FSA with (IL-6) in peri- odontitis and partial edentulous respectively may explore significant role of terminal sialic acid mucin in both adherence and aggregation by cleaving sialc acid and using it by bacteria depending on the severity of inflammation. Salivary sediment LSA in peri- odontitis may represent incorporation terminal sialic acid of MG1 with lipo-polysac- charide of gram negative bacteria that can hinder the function of the host defenses as reported by1. Additionally degradation of MG1 causes precipitation of its glycoprotein which has sequestering effect for soluble (IL-6)33, and may cause decrease in the level of soluble (IL-6). These results combine also with Gibbons et al.40 that terminal sialic acid mucine glycoprotein cleavage create a variety of carbohydrate linkages. 10 Jwan Ibrahim Correlation of salivary IL-6 with FSA in sediment of partial edentulous may reveal role of IL-6 in regulation of the innate immune response to inflammation by change in glycosylation of glycoprotein of free mucin that induce IL-6 secretion. This result explores the suggestion of41 that terminal sialic acid of free glycoprotein mucin is important component of interaction with bacteria protein and prevents colonization by aggregation and swallowing and McBride and Gisslow42 who showed a correlation between the amounts of sialic acid released from normal saliva and its aggregating activity. Association may due to IL-6 in modulation protein secretion and glycosylation as suggested by previous studies Groux-Degroote et al.43, and Chaudhury et al.44 that changes in glycosylation of mucin may induce interleukin 6 secretion. These observations may reveal anti-inflammatory role of IL-6 and consistence with Tilg et al.45 that (IL-6) regulated acute phase proteins that have anti-inflammatory and immuno-suppressive properties, and may regulate the acute phase response nega- tively. Also46 concluded that IL-6 suppresses IL-Iβ and TNF production induced by LPS and may provide negative feedback effect. Limitation of the study: Small sample size and lack of longitudinal monitoring the changes in salivary biomarkers from onset of periodontitis, progression and treatment The findings concluded that salivary sialic acid and IL-6 are oral inflammatory biomarkers in dentate periodontitis. Direction balance of IL-6 toward pro and anti-inflammation can be diagnosed by sialic acid fraction, and affected by age (which determine the number of teeth, and salivary; flow rate and volume), and treatment history. Acknowledgment Thanks first to all study participants for their contributions and periodontic clinic staff of for their support during data collection. Thanks to college of Dentistry / Hawler medical University for their permission to collect data. Funding: none. Conflicts of interest: none. References 1. Jawzaly JI, Hasan HG, Ahmed BM. Levels of salivary biochemical’s in periodontitis and related diseases. Duhok Med J. 2012;6(4):86. 2. Murata T, Mizaki H, Senpuku H, Hanada N. Periodontitis and Serum Interleukin-6 in The Elderly. Jpn J Infect Dis. 2001 Apr;54(2):69-71. 3. Gemmell E, Seymour GJ. Immunoregulatory control of Th1/Th2 cytokine profiles in periodontal disease. Periodontol 2000. 2004;35:21-41. 4. Goutoudi P, Diza E, Arvanitidou M. Effect of periodontal therapy on crevicular fluid interleukin-6 and interleukin-8 levels in chronic periodontitis. Int J Dent. 2012;2012:362905. doi: 10.1155/2012/362905. 5. Balto K, Sasaki H, Stashenko P. Interleukin-6 deficiency increases inflammatory bone destruction. Infect Immun. 2001 Feb;69(2):744-50. 11 Jwan Ibrahim 6. Nibali L, Fedele S, D’Aiuto F, Donos N. Interleukin-6 in oral diseases: a review. Oral Dis. 2012 Apr;18(3):236-43. doi: 10.1111/j.1601-0825.2011.01867.x. 7. Jawzali JI. Association between salivary sialic acid and periodontal health status among smokers. Saudi Dent J. 2016 Jul;28(3):124-35. doi: 10.1016/j.sdentj.2016.05.002. 8. Robertst IS. Bacterial polysaccharides in sickness and in health. Microbiology. 1995 Sep;141(Pt 9):2023-31. 9. Shahid SM, Mahboob T. Correlation Between Frequent risk Factors of Diabetic Nephropathy and Serum Sialic Acid. Asian J Biochem. 2006;1:244-50. doi: 10.3923/ajb.2006.244.250. 10. Browning LM, Jebb SA, Mishra GD, Cooke JH, O’Connell MA, Crook MA, et al. Elevated sialic acid, but not CRP, predicts features of the metabolic syndrome independently of BMI in women. Int J Obes Relat Metab Disord. 2004 Aug;28(8):1004-10. 11. Yaghobee S, Khorsand A, Rasouli Ghohroudi AA, Sanjari K, Kadkhodazadeh M. Assessment of interleukin-1beta and interleukin-6 in the crevicular fluid around healthy implants, implants with peri- implantitis, and healthy teeth: a cross-sectional study. J Korean Assoc Oral Maxillofac Surg. 2014 Oct;40(5):220-4. doi: 10.5125/jkaoms.2014.40.5.220. 12. Shaker ZF, Hashem BH. Study the role of proinflammatory and anti- inflammatory cytokines in Iraqi chronic periodontitis patients. J Bagh Col Dent. 2012;24(1):164-9. 13. American Academy of Periodontology Task Force Report on the Update to the 1999 Classification of Periodontal Diseases and Conditions. J Periodontol. 2015 Jul;86(7):835-8. doi: 10.1902/jop.2015.157001. 14. Saxer UP, Mühlemann HR. Epidemiology of periodontal diseases. In periodontology: Muelle HP. (2004) editor: Thiem, Stuttgart: New York:; 2004. p.38-46. 15. Carranza FA, Takei HH. Clinical diagnosis. In: Carranza’s Clinical Periodontology. Michael G. Newman, Henry H. Takei, Fermin A. Carranza, Perry R. Klokkevold (editor). Saunders Elsevier, 2006. Middle East and African Edition. P. 540-560 16. Löe H. The gingival index, the plaque index and the retention index systems. J Periodontol. 1967 Nov- Dec;38(6):Suppl:610-6. 17. Dowen B, Heintze U. Salivary secretion rate, Buffer capacity, and pH. In: Jorma T, editor. Human Saliva: Clinical Chemistry and Microbiology. Boca Raton: CRC Press; 1989. Vol. 1; p. 25-73. 18. Skoza L, Mohos S. Stable Thiobarbituric Acid Chromophor with Dimethyl Sulphoxide. Biochem J. 1976 Dec 1;159 (3):457-62. 19. Masami S. Method of measuring lipid bound sialic acid. United states Patent 4837144; 6/6/1989 20. Shetty PK, Pattabiraman TN. Salivary Glycoproteins As Indicators of Oral Diseases. Indian J Clin Biochem. 2004 Jan;19(1):97-101. doi: 10.1007/BF02872400. 21. Davidson College. Protein determination – Lowry Procedure. Biology 371. Davidson NC 28036; 2000. 22. Shinohara M, Ohura k, Ogata K, Inoue H, Miyata T, Yoshioka M. Relationship between the Sialic Acid Concentration in the Serum and Whole saliva in Rats with Naturally Occuring Gingivitis. J Jpn J Pharmacol. 1994 Jan;64(1):61-3. 23. Elbersol JL, Cappelli D. Acute phase reactants in infection and inflammatory diseases. Periodontol 2000. 2000 Jun;23:19-49. 24. Miller CS, King CP Jr, Langub MC, Kryscio RJ, Thomas MV. Salivary biomarkers of existing periodontal disease. J Am Dent Assoc. 2006 Mar;137(3):322-9. 25. Soell M, Holveck F, Schöller M, Wachsmann RD, Klein JP. Binding of Streptococcus mutans SR protein to human monocytes: production of tumor necrosis factor, interleukin 1, and interleukin 6. Infect Immun. 1994 May;62(5):1805-12. 26. Stuart AD, Brown TD. Alpha2,6-linked sialic acid acts as a receptor for Feline calicivirus. J Gen Virol. 2007 Jan;88(Pt 1):177-86. 12 Jwan Ibrahim 27. Rhodus NL, Ho V, Miller CS, Myers S, Ondrey F. NF-kappaB dependent cytokine levels in saliva of patients with oral preneoplastic lesions and oral squamous cell carcinoma. Cancer Detect Prev. 2005;29(1):42-5. 28. Persson RE, Persson GR. The elderly at risk for periodontitis and systemic diseases. Dent Clin North Am. 2005 Apr;49(2):279-92. 29. Al-Azzawi SI, Alwan AM, Salal RH. Influence of age and gender on salivary flow rate in completely edentulous patients. MDJ.2013;10(1):64-8. 30. Alwan AH, Taher MG, Getta HA, Hussain AA. Estimation of the level of Salivary Interleukin 6 (IL-6) and its’ correlation with the clinical parameters in patients with periodontal diseases. IOSR J Dent Med Sci.2015 Sep;14(9):82-8. 31. Slade GD, Offenbacher S, Beck JD, Heiss G, Pankow JS. Acute- phase inflammatory Response to Periodontal Disease in the US Population. J Dent Res. 2000 Jan;79(1):49-57. 32. Goutoudi P, Diza E, Arvanitidou M: Effect of periodontal therapy on crevicular fluid interleukin-1beta and interleukin-10 levels in chronic periodontitis. J Dent. 2004;32(7):511-20. 33. Ng PY1, Donley M, Hausmann E, Hutson AD, Rossomando EF, Scannapieco FA. Candidate Salivary biomarkers associated with alveolar boine loss. FEMS Immunol Med Microbiol. 2007 Mar;49(2):252-60. 34. Murray PA, Levine MJ, Reddy MS, Tabak LA, Bergey EJ. Preparation of a sialic Acid-Binding Protein from Streptococcus mitis KS32AR. Infect Immun. 1986 Aug;53(2):359-65. 35. Gani DK, Lashmi D, Kirshman R, Eramad R. Evolution of C-reactive protein and interleukin-6 in the peripheral blood of patients with chronic periodontitis. J Indian Soc Periodontol. 2009 May-Aug;13(2):69-74. 36. Geivelis M, Turner DW, Pederson ED, Lamberts BL. Measurements of interleukin-6 in gingival crevicular fluid from adults with destructive periodontal disease. J Periodontol. 1993 Oct;64(10):980-3. 37. Murata T, Miyazaki H, Senpuku H, Hanada N. Periodontitis and serum interleukin-6 levels in the elderly. Jpn J Infect Dis. 2001 Apr;54(2):69-71. 38. Lin SJ1, Chen YL, Kuo MY, Li CL, Lu HK. Measurement of gp130 cytokines oncostatin M and IL-6 in gingival crevicular fluid of patients with chronic periodontitis. Cytokine. 2005 May 21;30(4):160-7. 39. Zhang YF, Zheng J, Zheng L, Zhou ZR. Influence of centrifugation treatment on the lubricating properties of human whole saliva. Biosurface Biotribol. 2016;2(3):95-101. doi: 10.1016/j.bsbt.2016.09.001. 40. Gibbons RJ, Etherden I, Moreno EC. Association of neuraminidase-sensitive receptors and putative hydrophobic interactions with high-affinity binding sites for Streptococcus sanguis C5 in salivary pellicles. Infect Immun. 1983 Dec;42(3):1006-12. 41. Marcotte H, Lavoie MC. Oral microbial ecology and the role of salivary immunoglobulin A. Microbiol Mol Biol Rev. 1998 Mar;62(1):71-109. 42. McBride BC, Gisslow MT. Role of sialic acid in saliva-induced aggregation of Streptococcus sanguis. Infect Immun. 1977 Oct;18(1):35-40. 43. Groux-Degroote S, Krzewinski-Recchi MA, Cazet A, Vincent A, Lehoux S, Lafitte JJ, et al. IL-6 and IL-8 increase the expression of glycosyltransferases and sulfotransferases involved in the biosynthesis of sialylated and/or sulfated Lewisx epitopes in the human bronchial mucosa. Biochem J. 2008 Feb 15;410(1):213-23. 44. Chaudhury NMA, Proctor GB, Karlsson NG, Carpenter GH, Flowers SA. Reduced Mucin-7 (Muc7) Sialylation and Altered Saliva Rheology in Sjögren’s Syndrome Associated Oral Dryness. Mol Cel Proteomics. 2016;15(3):1048-59. 45. Tilg H, Dinarello CA, Mier JW. IL-6 and APPs: anti-inflammatory and immunosuppressive mediators. Immunol Today. 1997 Sep;18(9):428-32. 46. Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC, Dinarello CA. Correlations and Interactions in the Production of Interleukin-6 (IL-6), IL-1, and Tumor Necrosis Factor (TNF) in Human Blood Mononuclear Cells: IL-6 Suppresses IL-1 and TNF. Blood. 1990 Jan 1;75(1):40-7. file:///S:/__REVISTAS/bjos/v017/Editora%c3%a7%c3%a3o/Produ%c3%a7%c3%a3o/0015/consolidado/javascript:void(0); file:///S:/__REVISTAS/bjos/v017/Editora%c3%a7%c3%a3o/Produ%c3%a7%c3%a3o/0015/consolidado/javascript:void(0); file:///S:/__REVISTAS/bjos/v017/Editora%c3%a7%c3%a3o/Produ%c3%a7%c3%a3o/0015/consolidado/javascript:void(0);