Maha.docx J Bagh College Dentistry Vol. 28(1), March 2016 Salivary α-amylase Oral and Maxillofacial Surgery and Periodontics 114 Salivary α-Amylase and Albumin Levels in Patients with Chronic Periodontitis and Poorly or Well Controlled Type II Diabetes Mellitus Maha Abdul Aziz Ahmed, B.D.S., M.Sc. (1) ABSTRACT Background: Recent studies suggest that chronic periodontitis (CP) and type2 diabetes mellitus (T2DM) are bidirectionally associated. Analysis of saliva as a mirror of oral and systemic health could allow identification of α amylase (α-Am) and albumin (A1) antioxidant system markers to assist in the diagnosis and monitoring of both diseases. The present study aims at comparing the clinical periodontal parameters in chronic periodontitis patients with poorly or well controlled Type 2Diabetes Mellitus, salivary α-Am, A1, flow rate (FR) and pH then correlate between biochemical, physical and clinical periodontal parameters of each study and control groups. Materials and Methods: 80 males, with an age range of (35-50) years were divided into four groups, (20 subjects each): two groups had well or poorly controlled Type 2Diabetes Mellitus both of them with chronic periodontitis, group of patients with only chronic periodontitis and control group with healthy periodontium and systemically healthy. From all subjects unstimulated whole salivary samples were collected to measure FR, pH, Al and α – Am, then clinical periodontal parameters (plaque index, gingival index, bleeding on probing, probing pocket depth and clinical attachment level)were recorded. Results: Patients had chronic periodontitis with poorly controlled Type 2Diabetes Mellitus demonstrated the highest median values of all clinical periodontal parameters and highest increase in levels of salivary α-Am and Al with lowest median values of FR and pH, in addition to the highly significant differences among the study and control groups regarding biochemical and physical parameters. Positive correlations were revealed between α-Am with Al and both of them with all clinical periodontal parameters but, they were negative with FR and pH. Conclusion: Patients with poor glycemic control had more severe periodontal tissue break down with decrease in FR and pH also obvious increase in levels of A1 and α- Am so, these biochemical markers will provide an objective phenotype to allow practitioners for early diagnosis, which is essential for improved prognosis and effective delay of clinical complications associated with chronic periodontitis and DM and an important strategy to lower the incidence of both diseases world wide. Keywords: Periodontitis, Type 2Diabetes Mellitus, salivary albumin and α-amylase. (J Bagh Coll Dentistry 2016; 28(1):114-120). INTRODUCTION Periodontitis is irreversible inflammatory disorder of the supporting structures of the tooth leading to progressive attachment loss and destruction of alveolar bone. Chronic periodontitis (CP) is the most prevalent form of periodontitis, hence affects about 10%-15% of adult population world wide. Furthermore in the presence of systemic disease (e.g.DM),which modifies the host response to plaque accumulation, the disease progression may become more aggressive(1). The DM, is a metabolic disorder characterized by hyperglycemia and T2DM which is the most common type is linked to insulin resistance and patients with DM are prone to oral complications such as periodontal disease (PD), dry mouth and abscesses (2). Hence, today various researches are being conducted to evaluate possible compound in the oral fluids through which it may possible to assess the presence and severity as well as, toidentify the patients at risk for these diseases thus, analysis of saliva which is a complex secretory fluid that can be easily collected through non-invasive means (1)Assistant Professor. Department of Periodontics. College of Dentistry, University of Baghdad. for the screening of large samples in addition, saliva contains locally produced microbial and host response mediators, as well as, systemic (serum) markers (3). Thus the investigation of salivary proteins such as Al and α- Am in patients with CP and DM may be useful to enhance the knowledge of their roles in these diseases. So, this study designed to determine the effect of glycemic control in T2DM on periodontal health status as well as, on the levels of salivary Al, α-Am, FR and pH. MATERIALS AND METHODS The participants in this study was 80 males with age range (35-50) years, recruited from specialized center for endocrinology and Diabetes in Baghdad and from periodontics Department, at the teaching hospital in the College of Dentistry, University of Baghdad. They were divided into four groups. 1. Study group of 20 males suffer from CP with well controlled T2DM HbA1c <7%(4) (CP+wT2DM). 2. Study group of 20 males suffer from CP with poorly controlled T2DM,HbA1C> 9%(4)(CP+pT2DM). J Bagh College Dentistry Vol. 28(1), March 2016 Salivary α-amylase Oral and Maxillofacial Surgery and Periodontics 115 3. Study group of 20 males suffer from CP but systemically healthy (CP). 4. Control group of 20 males with clinically healthy periodontium and apparently systemically healthy.Healthy periodontium defined by the absence of any signs and symptoms of gingival inflammation, without periodontal pockets or clinical attachment loss. This group represented a base line data for the salivary Al and α-Am levels. Patients with CP demonstrated the presence of at least four sites with PPD (≥4mm)and clinical attachment loss of (1-2) mm or greater (5). The inclusion criteria were only males with at least 20 teeth present, T2DM ≥ 5 years on oral hypoglycemic therapy only and body mass index within the normal range (18.5-24.9 kg/m2) (6). The exclusion criteria were females, presence of systemic diseases other than T2DM, patients administered medications (anti-inflammatory and anti-microbial) or undergone periodontal treatment in the 3 months prior to the study, smoking, alcohol consumption, T1DM and T2DMadministring insulin, presence of nephropathy, retinopathy and diabetic foot. Unstimulated whole salivary samples were collected from all participants (7). During that salivary (FR) was measured through dividing the volume of the collected sample by the collection time. After this by using DP universal test paper, the salivary pH was measured, then samples were centrifuged for 15min. at 4000rpm and frozen, at -20 C. By using Michigan O periodontal probe, the examination of clinical periodontal parameters was performed on four surfaces (mesial, buccal/labial, distal and lingual / palatal) of all teeth except the 3rd molar, which included: 1. Plaque index system (PLI)(8). 2. Gingival Index system (GI) (9). 3. Bleeding on probing (BOP) (1). 4. Probing pocket Depth (PPD). 5. Clinical Attachment level (CAL). For biochemical analysis of salivary A1, Protein U.S / Syrbio kit was used. While for salivary α- Am, (single Reagent GALG2-CNP) /SPECTRUM kit was used, hence the activities were determined by measuring the absorbace at 598 nm and 405 nm respectively both by the spectrophotometer. Descriptive statistics that include mean and median values and inferential statistics which include kruskal – Wallis H test, Mann- Whitney U test and pearson correlation (r) were used. The level of significance (S) was accepted at P ≤0.05, highly significance (HS) at P < 0.01 and non- significant (NS) at P > 0.05. We certify that this study involving human subjects is in accordance with the Helsinky declaration of 1975 as revised in 2000 and that it has been approved by the relevant institutional ethical committee. RESULTS The highest mean of age was found in CP + pT2DM group (45.85), followed by CP + wT2DM(44.95), then CP group (41.7) while, the least mean of age was detected in control group (38). Patients with CP + pT2DM demonstrated the highest median values of the clinical periodontal parameters, then patients suffer from CP + wT2DM, after that CP patients. Inter study groups comparisons regarding all clinical periodontal parameters revealed, HS differences between CP + pT2DM with both CP + wT2DM and CP groups while, they were NS differences between CP + wT2DM with CP groups (Table -1). Table 2 showed the biochemical analysis of both Al and α – Am presented that have highest increase in median values were revealed in CP + pT2DM group after that patients with CP + wT2DM, then CP group as compared to the control group hence, HS differences were demonstrated among the four groups. On the other hand, the physical parameters analysis showed decrease in median values of both FR and PH in study groups when compared to control group and the lowest median values demonstrated in CP + pT2 DM group. Again, HS differences among the study and control groups were found. The comparisons between all pairs of the study and control groups regarding α-Am, A1, FR and pH demonstrated HS and S differences except the NS differences between CP+wT2DM with CP groups concerning α-Am, A1 and pH(Table -3). The results of correlations (tables 4&5) between α-Am and A1 with clinical periodontal parameters were positive but they were negative with FR and pH at all groups, although α-Am revealed moderate positive correlations with PLI and GI at CP + pT2 DM and CP + wT2DM groups respectively. The correlations between α-Am with Al were positive at all groups (table -6). J Bagh College Dentistry Vol. 28(1), March 2016 Salivary α-amylase Oral and Maxillofacial Surgery and Periodontics 116 Table 1: Median Values of the Clinical Periodontal Parameters and the Inter Groups Comparisons between All Pairs of the Study Groups CP & CP+ wT2DM CP+ pT2DM & CP CP+ pT2DM & CP+ wT2DM Median Groups Clinical periodontal parameters P- value Sig. Mann Whitney U test P- value Sig. Mann Whitney U test P- value Sig. Mann Whitney U test 0.51 NS 1.948 0.00 HS 5.411 0.00 HS 4.735 2.682 CP+ pT2DM PLI 1.815 CP+ wT2DM 1.341 CP 0.232 Control 0.499 NS 0.677 0.00 HS 5.42 0.00 HS 5.410 2.553 CP+ pT2DM GI 1.556 CP+ wT2DM 1.5 CP 0.108 Control 0.217 NS 1.233 0.00 HS 3.993 0.00 HS 4.390 60.5 CP+ pT2DM BOP score1 46 CP+ wT2DM 42 CP 0.409 NS 0.825 0.00 HS 4.255 0.00 HS 4.363 6.67 CP+ pT2DM PPD 6.13 CP+ wT2DM 5.945 CP 0.323 NS 0.989 0.00 HS 4.749 0.00 HS 4.372 4.4 CP+ pT2DM CAL 3.08 CP+ wT2DM 2.435 CP *P<0.01 High significant Table 2 Median Values of Salivary α-Amylase , Albumin ,FR and pH and the Significance of Differences Among the Study and Control Groups. Kruskal-Wallis H test Control CP CP+ wT2DM CP+ pT2DM Parameters P-valueSig. Chi square Median Median Median Median 0.00 HS 43.62 65.47 90.86 99.25 162.14 α-Amylase U/L 0.00 HS 30.568 56.51 75.72 79.18 104.8 Albumin mg/dl 0.00 HS 65.6 1.2 0.75 0.725 0.23 FR ml/min 0.00 HS 24.96 7 6 6 5 pH J Bagh College Dentistry Vol. 28(1), March 2016 Salivary α-amylase Oral and Maxillofacial Surgery and Periodontics 117 Table 3: Inter Groups Comparisons of the Median Values of Salivary α-Amylase, Albumin, FR and pH between All Pairs of the Study and Control Groups CP& Control CP+ wT2DM&Control CP+ wT2DM&CP CP+ pT2DM&Control CP+ pT2DM&CP CP+ pT2DM&CP+ wT2DM Parameters P- value Mann Whitney U test P- value Mann Whitney U test P- value Mann Whitney U test P- value Mann Whitney U test P- value Mann Whitney U test P- value Mann Whitney U test 0.00 HS 4.003 0.00 HS 4.436 0.725 NS 0.352 0.00 HS 5.410 0.00 HS 3.517 0.001 S 3.354 α-Amylase U/L 0.00 HS 4.275 0.044 S 2.332 0.322 NS 0.864 0.00 HS 4.816 0.03 S 2.976 0.021 S 2.998 Albumin mg/dl 0.00 HS 4.809 0.00 HS 5.278 0.00 HS 4.870 0.00 HS 5.444 0.00 HS 5.437 0.00 HS 4.998 FR ml/min 0.014 S 2.453 0.01 S 3.213 0.203 NS 1.274 0.00 HS 5.231 00.00 HS 4.925 0.00 HS 4.275 pH Table 4: Correlations between the Levels of α-Amylase with the Clinical Parameters of Each Study and Control Groups. Control CP CP+ wT2DM CP+ pT2DM Statistical analysis Parameters 0.248 0.254 0.188 0.56 r PLI 0.292 NS 0.281 NS 0.427 NS 0.816 NS p 0.290 0.222 0.57 0.164 r GI 0.214 NS 0.348 NS 0.012 S 0.489 NS p - 0.003 0.227 0.207 r BOP score1 - 0.990 NS 0.330 NS 0.381 NS p - 0.199 0.039 0.173 r PPD - 0.400 NS 0.871 NS 0.466 NS p - 0.068 0.201 0.154 r CAL - 0.775 NS 0.395 NS 0.516 NS p -0.156 -0.009 -0.442 -0.268 r FR 0.511 NS 0.969 NS 0.049 S 0.254 NS p -0.096 -0.144 -0.035 -0.131 r pH 0.687 NS 0.543 NS 0.884 NS 0.582 NS p Table 5: Correlations between the Levels of Albumin with the Clinical Parameters of Each Study and Control Groups. Control CP CP+ wT2DM CP+ pT2DM Statistical analysis Parameters 0.131 0.085 0.134 0.148 r PLI 0.581 NS 0.721 NS 0.573 NS 0.533 NS p 0.224 0.070 0.186 0.327 r GI 0.343 NS 0.771 NS 0.434 NS 0.159 NS p - 0.186 0.157 0.378 r BOP score1 - 0.434 NS 0.508 NS 0.100 NS p - 0.255 0.268 0.121 r PPD - 0.277 NS 0.253 NS 0.611 NS p - 0.107 0.189 0.482 r CAL - 0.653 NS 0.424 NS 0.032 S p -0.046 -0.151 -0.214 -0.321 r FR 0.847 NS 0.526 NS 0.365 NS 0.167 NS p -0.235 -0.045 -0.273 -0.197 r pH 0.318 NS 0.849 NS 0.245 NS 0.406 NS p J Bagh College Dentistry Vol. 28(1), March 2016 Salivary α-amylase Oral and Maxillofacial Surgery and Periodontics 118 Table 6: Correlations between Salivary Levels of (α-Amylase and Albumin) of Each Study and Control Groups. Control CP CP+ wT2DM CP+ pT2DM Statistical analysis Parameters 0.511 0.195 0.103 0.291 r α-amylase and albumin 0.831 NS 0.411 NS 0.665 NS 0.214 NS p DISCUSSION The CP + T2DM patients revealed higher mean of age, this can be explained by the greater incidence of both diseasesin adults(10). In diabetic patients, the vascular changes, neutrophil dysfunction, altered collagen synthesis, accumulation of advanced glycation end products leading to impaired tissue repair capacity (1), as well as increased glucose level in gingival crevicular fluid (GCF) and saliva (11), decrease FR that disrupt the cleaning and buffering capacities and clearance of bacterial substrate which then increase accumulation of plaque and calculus (12), in addition increased levels of α – Am andAl, in which the former favored proliferation of both aerobic and anerobic bacteria in plaque, while the latter considered potential energy sources and enable the attachment of pathogenic bacteria thus alter the composition of plaque (13). So, diabetics had three fold increase in risk of having periodontitis compared to non-diabetics, hence adults with an HbA1c level 0f 9%had significantly higher prevalence of severe periodontitis thus, the gingival inflammation and bleeding are intensified, greater prevalence and extent of pockets with twice as likely a non- diabetics to have attachment loss (2,10,12). Saliva contains numerous defense antioxidant proteins e.g. Al and α- Am which able toinhibit the generation of free radicals (14). The highly significant increase in α-Am level in CP patients as compared to control group revealed by this study are in accordance with other studies (15-19), the same result was found when comparing CP + T2 DM groups with control group, hence different researchers had reported that salivary α-Am concentrations from T2DM patients were higher(11,20-22) or lower (23-26) than its levels in non- diabetics.The response of salivary gland to inflammatory diseases, resulting in enhanced synthesis and secretion of defense proteins (15). The increased basement membrane permeability of salivary glands in diabetics leads to increased passage of proteins into the saliva, moreover the sialosis in the parotid gland in T2 diabetics, hence most of α-Am being synthesized in this gland, could result in variations in the salivary composition (22).Studies showed that α – Am is a major lipopolysaccharide binding protein of Agri, gatibacteractinomycetemcomitansandPorphyromo nasgingivalis (P.gingivalis) and interfere with bacterial adherence and biofilm formation also performs adirect inhibitory effect on the growth of Neisseria Gonorrhoeaand P.gingivalis(13). The notable increase in Al level in CP patients in comparison with control subjects in this study was inconsistent with findings of previous studies (27- 29), while others (14,30)demonstrated decrease in Al levels with deterioration of periodontal tissue condition. Although the significant increase of Al in T2 diabetics found by researchers (31,32) were in agreement with this study, but disagree with other results (33,34). Al accounting for more than 50% of all plasma proteins, thus is regarded as markers for plasma protein leakage occurring as a consequence of inflammatory process, so the high salivary Al level in CP patients due to ulceration in sulcular epithelium confirming the sulcular originof Al from GCF, thus 4-5 times rise in Al level was noted during periodontal tissue destruction when compared with that of the control (28),moreover the presence of TreponemaDenticola seemed to increase Al in periodontitis patients (35). On the other hand, disregulation in the factors that regulate Al synthesis during DM occur which include nutrition, hormonal balance and osmotic pressure and the inflammation of salivary gland causing increased leakage of serum proteins into the saliva (33). Finally, studies measured A1 and α – Am levels in T2 diabetics, they ignor their periodontal health status. The more acidic pH in CP patients was in line with some studies (29,36)hence, significant correlation did exist between pH and PPD on the other hand increase in pH was found (28,37) in CP patients. From the present study the decrease in pH of diabetics was coincide with other reports(12,25,38) hence, significant decrease in pH was demonstrated when comparing uncontrolled T2DM with healthy and controlled T2DM as well as, healthy withcontrolled T2DM (39). The decrease in salivary FR and bicarbonate content consequently contributed to the more acidic saliva(38). The higher concentrations of hydrogenions (from salivary glands or oral microbiota), thelowest the pH, since pH level negatively correlated with proportion of periodontal pathogens, that grow in mildly acidic pH, either utilize or create products that are mild to moderately acidic in nature (29). J Bagh College Dentistry Vol. 28(1), March 2016 Salivary α-amylase Oral and Maxillofacial Surgery and Periodontics 119 The decrease in salivary FR in this study coincide with others concerning CP (15,19,29), and DM (12,24,25,40) but diverge with previous studies about CP (37) and DM (41) who reported increased of FR, on the other hand some researchers found that FR levels not affected by periodontal health status (28) or presence of DM (42). There are multiple causes of salivary hypofunction including inflammation e.g. periodontal disease(19), hydrogen concentration, aging (40) or systemic disease e.g. DM (24), so in this case the decrease in pH, medication given for diabetics, poly urea and dehydration, neuropathies, microvascular changes, metabolic disturbances also, hypertrophy of salivary glands can be attributed to decrease in FR (12, 25). Positive correlations of α –Am and A1 with each other and with clinical periodontal parameters, but they were negative with FR and pH, this can be explained by the presence and increased inflammation with periodontal tissue destruction due to CP and DM which lead to increased levels of α-Am and Al but decrease in FR and pH. These results were in concurrent with other results (16,17,19) who found significant positive correlations between α-AM with PPD and CAL, while significant negative correlation with FR(19) in CP patients. In general, there were correlations between α – Am with glycemic control (23,24,26), but non significant with FR at controlled and uncontrolled T2DM(23). Significant positive correlation was detected between Al levels with GI inT2 diabetes (43). Finally, the results may differ from one study to another these maybe due to e.g. the diversity in selection criteria of samples, metabolic control, wide range of age, different types of salivathat can limit direct comparison. REFERENCES 1. Newman mg, Takei HH, Klokkevold PR, Carranza FA. Carranza's clinical Periodontology. 12th ed. St. Louis MO: SouadersElsvier; 2015. 2. Mealey BL, Oates TW. Diabetes Mellitus and Periodontal disease. J Periodontal 2006; 77:1289-303. 3. Anil KN, Neh B. Saliva as Adetective Biofluid. International J Med App Sci 2015; 4. 4. Diabetes Care. Diagnosis and Classification of DiabetesMellitus.American Diabetes Association. 2014; 37(1):14-80. 5. Lang NP, Bartold PM, Cullinam M, et,al., International Classification Work Shop. Consensus Report: Chronic Periodontitis. Annals Periodontol 1999; 4: 53. 6. World Health Organization. WHO Expert Consultation. Appropriate Body Mass Index for Asian Populations and Its Implications for Policy and Intervention Strategies. The lancet 2004; 363: 157- 163. 7. Tenovuod. Saliva. In Textbook of Clinical Cardiology by thy Lstrup A, Fejerskov O. 2nd ed. Copenhagen: Munksgaard; 1994; 17.43. 8. Silness P, Loe H. Periodontal Disease in Pregnancy. ActaOdontol Sand 1964; 22:121. 9. Loe H. The Gingival Index, the Plaque Index and the Retention Index System. J Periodontal 1967;38(6): 610-6. 10. Abdul-Wahab GA, Ahmed MA. Assessment of Some Salivary Enzymes Levels in Type 2 Diabetic Patients with Chronic Periodontitis (Clinical and Biochemical Study). J Baghdad Coll Dentistry 2015; 27(1):138-43. 11. Prabal P, Desai NT, Kannan N, et al. Estimation of Salivary Amylase, Salivary Total Protein and Periodontal Microflora in Diabetes Mellitus. JIDA 2003;74:143-49. 12. Sabir DA, Ahmed MA. Assessment of Salivary Leptin and Resistin Levels in Type 2 Diabetic Patients with Chronic Periodontitis (A Comparative Study). J Baghdad College Dentistry. J Bagh Coll Dentistry 2015; 27(4):107-14 13. Taylor JJ.Protein Biomarkers of Periodontitis in Saliva.ISRN. 2014, P.18. 14. Miricescu D, Greabu M, Totan A, et al. The Antioxidant Potential of Saliva: Clinical Significance in Oral Diseases. The Pharmacol Clin Toxicol 2011; 15(2):139-43. 15. Sanchez GA, V Miozza, A Delgado, et al. Determination of Salivary Levels of Mucin and Amylase in Chronic Periodontitis Patients. J of Periodontal Res. 2011; 46(2):221-7. 16. Hady H, Bertl K, Laky M, et al. Salivary and Serum Chromogranin A and Amylase in Periodontal Health andDisease. JPeriodontal 2012; 83(10): 1314-21. 17. Sanchez GA, VA Miozza, A Delgado, et al. Relationship between Salivary Mucin or Amylase and the Periodontal Status. Oral Dis. 2013; 19: 585-91. 18. Swati K, Rahul B, Biju T, et al. Estimation of Levels of Salivary Mucin, Amylase and Total Protein in Gingivitis and Chronic Periodontitis Patients. J. Clin. Diag. Res. 2014; 8(10): ZC 56-ZC60. 19. Andrea BA, Aljandra KD, et al. Comparison of Salivary Levels of Mucin and Amylase and Their Relation with Clinical Parameters Obtained from Patients with Aggressive and Chronic Periodontal Disease. J.Appl. Oral Sci 2015; 23(3). 20. Aydin S. AComparison of Ghrelin, Glucose, Alph- Amylase and Protein Levels in Saliva from Diabetics. JBiochemistry and Nuclear Biol 2007; 40: 29-35. 21. Sathyapriyas S, Bharani GO, Nagalingam M, et al. Potential of Salivary Proteins as a Biomarkers in Prognosis of Diabetes Mellitus. J Pharmacy Res 2011;4(7): 2228-29. 22. L Malathi, KMK Masthan, NBalachander, et al. Estimation of Salivary Amylase in Diabletic Patients and Saliva as a Diagnostic Toolin Early Diabetic Patients. J ClinDiagn Res 2013; 7(11): 2634-6. 23. Artis SP, Degwekar SS, Bhwte RR. Estimation of Salivary Glucose, Salivary Amylase, Salivary Total Protein and Salivary Flow Rate in Diabetics in India. J Oral Sci 2010; 52:359-68. 24. Al-Zahawi ShM, Mahmood HA, Al-Qassab ZA. Effects of Diabetes Mellitus Type II on Salivary Flow Rate and Some Salivary Parameters (Total Protein, Glucose and Amylase) in Erbil City.J BaghColl Dentistry 2012; 24(2): 123. J Bagh College Dentistry Vol. 28(1), March 2016 Salivary α-amylase Oral and Maxillofacial Surgery and Periodontics 120 25. Prathibha KM, Johnson P, Ganesh M, et al. Evaluation of Salivary Profile among an Adults Type 2 Diabetes Mellitus in South India. J Clin Diagn Res 2013; 7(8): 1592-5. 26. Indria M, Shekar PC, et al. Evaluation of Salivary Glucose, Amylase and Total protein in Type 2 Diabetes Mellitus Patients. Indian Dental Res 2015; 26: 271. 27. Conclaves LR, Soares MR, Noqueira FC, et al. Comparative Proteomic Analysis of Whole Saliva from Chronic Periodontitis Patients. J of Proteomics 2010; 73(7): 1334-41. 28. Shaila M, Pai GP, Shetty P.Salivary Protein Concentration, Flow Rate, Buffer Capacity and pH Estimation: A Comparative Study among Young and Elderly Subjects both Normal and with Gingivitis and Periodontitis. J Indian Soc Periodontol 2013; 17(1): 42-46. 29. Gazy Y, Mohiadeen B, Al-Kasab Z. An Assessment of some Salivary Biochemical Parameters in Cigarette Smokers with Chronic Periodontitis. J Baghdad Coll Dentistry 2014; 26(1): 144-9. 30. Scully DV, Langley-Evans SC. Periodontal Disease is Associated with Lower Antioxidant Capacity in Whole Saliva and Evidence of Increased Protein Oxidation. Clin Sci 2003;105(2):167-72. 31. Doods MWJ, Chih –Koyeh, Dorthea A Johnson. Salivary Alterations in Type 2 (Non-Insulin Dependent) Diabetes Mellitus and Hypertension. Community Dental Oral Epi. 2000; 28:373-81. 32. Vaziri PB, M Vahedi, Adollahzadeh SH, et al. Evaluation of Salivary Albumin in Diabetic Patients. Iranian J Publ Health 2009; 38(3): 54-9. 33. Carda C, Lioreda NM, Salom L,etal.Structural and Functional Salivary Disorders in Type 2 Diabetic Patients. Med Oral PatolOral Cir Buccal 2006; 11: E 309-14. 34. Hassan HR, Abdul Sattar A. Influence of Diabetes Disease on Concentration of Total Protein, Albumin and Globulins in Saliva and Serum: A Comparative Study. Iraqi National of Chemistry 2015; 15(1). 35. Mahald Y, Tervahartiala T, et al. Association of Periodontal Microorganisms with Salivary Proteins and MMP-8 inGingival Crevicular Fluid. J ClinPeriodontol 2012; 39(3): 256-63. 36. Sharmila B, Sangeeta M, Rahul K. Salivary pH: Adiagnostic biomarker. J of Indian Society of Periodontol 2013;17(4): 461-5. 37. Ali BGh, Ali OH. Detection of Salivary Flow Rate and Minerals in Smokers and Non-Smokers with Chronic Periodontitis (Clinical and Biochemical Study). J Baghdad Coll Dentistry 2012; 24(1):68-71. 38. Eslami H, Fakhrzadeh V, Pakdel F, et al. Comparative Evaluation of Salivary pH Level in Type II Diabetic Patients and Healthy Subjects. VISI J Acemdic 2015(4):144-8. 39. Arul Asrikemath J, Sanjay R, Palaniveluperamachi. Evaluation of Correlation between Salivary pH and Prevalence of Dental Caries in Subjects with and without Diabetes Mellitus. Research J Recent Sci 2014; 3: 224-6. 40. Hamad AI, Alkiaisi RO, Alkaisi IJ. Flow Rates of Resting Whole Saliva of Diabetic Patients in Relation to Age and Gender. Tikrit J Dental Sci 2012; 1: 1-5. 41. Jose Roberto C, Regina Marcia SP, Fernando de OC, et al. Salivary and Microbiological Parameters of Chronic Periodontitis Subjects with and without Type 2 Diabetes Mellitus: A Case-Control Study. RevOdontol UNESP 2014; 43(3). 42. Collin HL, Niskanen L, Uusitupa M, et al. Oral Symptoms and Signs in Elderly Patients with Type 2 Diabetes Mellitus. A Focus on Diabetic Neuropathy. Oral SurgOral Med Oral PatholOral RadiolEndod 2000;90 (3): 299-305. 43. Ben – Aryeh H, Serouya R, Kanter Y, et al. Oral Health and Salivary Composition in Diabetic Patients. J Diabetes Complications 1993; 7(1): 57- 62.