J Bagh College Dentistry Vol. 29(3), September 2017 Salivary physicochemical among Pedodontics, Orthodontics and Preventive Dentistry 68 Salivary Physicochemical Characteristics in Relation to Oral Health Status Among Institutionalized Autistic Adolescents in Baghdad/Iraq Ali Hadi F. Al-Fatlawi, B.D.S., M.Sc. (1) Nada Jafer MH. Radhi, B.D.S., M.Sc., Ph.D. (2) ABSTRACT Background: Autism spectrum disorder (ASD) is characterized by impairments in social interaction and communication, restricted patterns of behavior, and unusual sensory sensitivities. Saliva may provide an easily accessible sample for analysis. Some salivary constituents levels altered in adolescents with ASD including antioxidants . This study aimed to investigate salivary physicochemical characteristic in relation to oral health status among adolescents with ASD. Materials and methods: Two groups were included in this study: 40 institutionalized autistic adolescents and 40 apparently healthy school adolescents control group with age range (12-15 years old, only males) selected randomly from Baghdad. Each group subdivided into two groups according to the severity of dental caries: caries free group (20 child, DMFT=0) and high caries group (20 child, DMFT≥6). Decayed, missing and filled surfaces (DMFS), plaque (PlI), Gingival (GI) and calculus (CI) indices were used to measure oral health status for both groups. Copper (Cu), zinc (Zn) and thiocyanate (SCN) in saliva measured by atomic absorption spectrophotometer. Salivary alpha amylase (sAA) and glutathione (GSH) assessed by enzyme-linked immunosorbent assay (ELISA). Salivary pH and flow rate were measured directly. The data of current study was analyzed using SPSS version 21. Results: A higher value of salivary pH, flow rate, sAA, SCN, Cu and Zn were found among study group than control group with significant difference, also higher in caries free subgroup than high caries subgroup. While GSH was significantly higher in control group than study group. Moderate negative correlations between sAA, Cu, Zn and PlI, CI, GI with highly significant and salivary pH correlate moderately with PlI and CI with highly significant. Conclusion: There was alteration in salivary constituents levels which related to oral health status in adolescents with ASD and can act as adjunctive diagnostic aid for diagnosing autism. Keywords: Autism spectrum disorder; sAA; SCN; GSH. (J Bagh Coll Dentistry 2017; 29(3):68-74) INTRODUCTION Autism spectrum disorders (ASD) is a neurodevelopmental disorder in which social interaction, language, behavior and cognitive functions are impaired severely (1). It is characterized by a complex, behaviorally defined, static immature brain disorder that is of great concern to various professions (2). Individuals with ASD may have less learning abilities than healthy individuals. This may affect their oral hygiene (3). Various studies in the past have shown a higher prevalence of periodontal disease and a lower caries in autistics compared to unaffected children (2,4,5). Oral fluid or whole saliva is an important complex chemical milieu of teeth and oral soft tissues, which maintains the homeostasis of the oral cavity (6). The most abundant protein in saliva is alpha amylase (sAA), accounting for approximately 20% of salivary proteins. Thiocyanate (SCN) is an important physiological anion involved in innate defense of mucosal surfaces. ______________________________________________ (1) Ph.D. student, Department of Pediatric and Preventive Dentistry, College of Dentistry, Baghdad University. (2) Assist. Professor, Department of Pediatric and Preventive Dentistry, College of Dentistry, Baghdad University. There are indispensable parts of host defense system that act as a substrate for lactoperoxidase that oxidizes airway surface lipid thiocyanate thereby generating antimicrobial agent hypothiocyanate (7). Copper (Cu) ions have been reported to have an antibacterial effect. It was suggested that there is an altered regulation of ceruloplasmin in a subset of children with autism. Such alterations may lead to abnormal copper metabolism that may play a pathological role in autism (8). Zinc (Zn) can reduce dental calculus formation, control plaque and reduce the solubility of enamel. In fact, some preliminary studies suggested altered serum Cu/Zn ratios in autism (8,9). The antioxidant defense system of saliva has several components. One of these antioxidants is glutathione (GSH), salivary GSH levels decrease in periodontal diseases and dental caries (10). Individuals with ASD showed elevated levels protein, SCN, and sAA when compared to normal children. Antioxidant enzymes, cholesterol and GSH levels were found to be decreased in ASD than in normal children (7). As far as, no previous study was conducted among institutionalized autistic adolescents in Baghdad regarding the relation between physicochemical characteristic of saliva, dental J Bagh College Dentistry Vol. 29(3), September 2017 Salivary physicochemical among Pedodontics, Orthodontics and Preventive Dentistry 69 caries and gingival health. For all these explanation, this study was designed. MATERIALS AND METHODS The study included two male groups: 40 institutionalized autistic children and 40 apparently healthy schoolchildren with age range (12-15 years old, only males). The sample selected according to the severity of dental caries. Each group subdivided into two groups: caries free group (20 child, DMFT=0) and high caries group (20 child, DMFT≥6). For all children intra-oral assessment of caries experience was measured according to criteria of WHO (1997) (11), Oral hygiene status was evaluated by application plaque index (PlI) of Silness and Löe (12), and calculus index (CI) of Ramfjord (13). Gingival inflammation assessed by using Gingival Index (GI) of Löe and Silness (14). For salivary samples each child was asked to sit down and relaxes as much as possible and was asked to chew a piece of Arabic gum for one minute before all the saliva was removed by expectoration; chewing was then continued for ten minutes with the same piece of gum and the collection of saliva by spitting was done during this time (15). The collected saliva was centrifuged at 1000 rpm for 10 minutes; this done after 1 hour after collection to eliminate debris and cellular matter. The centrifuged supernatants stored frozen at (-20°C) in polyethylene tube until assayed. Salivary flow rate assessed by dividing the total volume of saliva collected in milliliter over the time of collection in minute. pH of saliva was measured by using pH meter. Copper and zinc in saliva were measured by atomic absorption spectrophotometer using commercial kits (Spectrum, Germany). Salivary thiocyanate assessed according to the method described by Slowinski et al. using spectrophotometer (16). Salivary alpha amylase and glutathione measured by enzyme-linked immunosorbent assay (ELISA) using special commercial kits (Cloud-Clone Corp., USA). The mean values with SD were measured for all tested salivary variables. The statistical significance, direction and strength of linear correlation between two quantitative normally distributed variables, by Pearson’s rank linear correlation coefficient. P value less than the 0.05 level of significance was considered statistically significant. A receiver operating characteristic (ROC), or simply ROC curve, is a graphical plot which illustrates the performance of a binary classifier system as its discrimination threshold is varied. All analyzed tests were bilateral. RESULTS Table 1 shows mean values of pH and flow rate (ml/min) in stimulated saliva among study and control groups by caries severity. A higher value of salivary pH and flow rate were found among study compared to the control group, which was statistically high significant with pH (P<0.001) and flow rate (P=0.003). A higher value of salivary pH and flow rate were found among caries free subgroup than high caries subgroup, which was statistically high significant with pH (P<0.01) and not significant difference with flow rate (P>0.05). The correlation coefficients of salivary pH and flow rate with PlI, GI and CI among study and control groups are illustrated in table 2. In study group, data of present study shows that a moderate negative correlations between salivary pH and PlI, CI. In control group, a strong negative correlation between salivary pH and PlI, GI, CI. A moderate negative correlation between salivary flow rate and PlI, CI among control group. The mean values of all salivary constituents were found higher in caries free than high caries subgroup among study group with high significant differences for sAA, Cu and Zn. As shown in Table 3. Table 4 demonstrates the mean concentration of salivary constituents in (µg/dl) among study and control groups with statistical differences. A higher mean value of salivary sAA, SCN, Cu, Zn, and Cu/Zn were found among study compared to the control group, with high statistical significant difference for sAA, SCN and Cu, while significant difference with Zn. Mean value of GSH was lower in study group than control group with high significant difference. Figure 1 and Table 5 demonstrate the areas under ROC curve for sAA (0.838), Cu (0.813) and Zn (0.763) were significantly from 0.5 value of an equivocal test (P<0.001 for sAA, Cu and P= 0.005 for Zn), whereas the areas under ROC curve for Cu/Zn (0.621), SCN (0.616) and GSH (0.501) were not significantly different from 0.5 value of an equivocal test. Figure 2 and Table 6 show the areas under ROC curve for salivary GSH (0.858), SCN (0.821), sAA (0.786), Cu (0.652) and Zn (0.648) were significantly from 0.5 value of an equivocal test (P<0.001 for GSH, SCN, sAA; P= 0.019 for Cu; P= 0.023 for Zn), whereas the areas under ROC curve for Cu/Zn (0.538) was not significantly different from 0.5 value of an equivocal test. J Bagh College Dentistry Vol. 29(3), September 2017 Salivary physicochemical among Pedodontics, Orthodontics and Preventive Dentistry 70 Table 7 demonstrates the correlation coefficients of salivary constituents with PlI, GI and CI among study and control groups. In study group, a moderate negative correlations between sAA, Cu, Zn and PlI, GI, CI. A very weak negative correlation between GSH, SCN, Cu/Zn and PlI, GI, CI. In control group, a moderate negative correlation between Zn and PlI as well as CI, while a very weak negative correlation was seen regarding sAA, GSH, SCN. A weak negative correlation between Cu and PlI, CI. DISSCUSION Autism spectrum disorder is a neurodevelopment disorder characterized by impaired social interaction and behavior, which can have adverse influence on dental care and oral health in the affected individuals. Studies on oral health in individuals with ASD have conflicting results. In a population-based sample of autistic children and adolescents, parents reported poorer condition of their children’s teeth and gingiva compared to other children (17,18,19). This study recorded an increasing in the flow rate and pH of stimulated saliva in the study group which was statistically significant compared to control group. Individuals with ASD report more stress in everyday life and they also report a higher level of dental anxiety, both factors that may negatively affect salivary secretion. These results disagreement with other studies (5,18,20). Alpha-amylase is a calcium-dependent metalloenzyme which can hydrolyze starch to glucose and maltose; consequently, having an important role in binding to bacteria (21). In the present study, sAA was higher in study group compared to control group with highly significant difference. In addition, sAA was higher in caries free subgroup than high caries subgroup with highly significant difference. sAA activity is an increasingly investigated biomarker for the activation of the autonomic nervous system. Hence, the high activity of α- amylase in autistic children shows that there is hyperstimulation of autonomic nerves, which may be associated with hyperactive nature of autistic children. This result in line with other studies (7,22). Glutathione is a family of multifunctional enzymes that plays an important role in the detoxification of xenobiotics including carcinogens. In addition, GSH plays a critical role in cellular protection against oxidative stress and toxic foreign chemicals. The level of salivary GSH was lower in ASD group compared to control group with highly significant differences. In addition, there is a slight increase of GSH level in high caries subgroup in relative to caries free subgroup among ASD group with non-statistical differences, and this might be due to lack of the substrate GSH and hence may be accounted for the reduced detoxifying capacity of GSH in autistic children. The activity of the enzyme may possibly be affected, if there is any genetic polymorphism in GST (23). This result was supported by other studies (24,25,26). Thiocyanate is an important physiological anion involved in innate defense of mucosal surfaces. It is a metabolite of cyanide and the product of detoxification of compounds containing cyanide through a reaction catalyzed by the enzyme rhodanese (27). In the current study, the concentration of salivary SCN was higher in study group than control group with highly significant differences. In addition, the level of salivary SCN was slightly higher in caries free subgroup than high caries subgroup among ASD group, but with non-significant differences. The elevated SCN level in ASD group may be attributed to low rhodanese activity as supported by Waring and Klovrza (28), who stated that the raised ratio of thiosulfate and SCN might be due to reduced rhodanese activity. Scientists interested the relationship between salivary trace elements (Zn and Cu) and dental caries activity for many years. Conflicting reports from researchers who have investigated Cu and Zn in saliva indicate that the nature of the role that these elements play in the carious process remains undefined (29). Elevated level of Cu and depressed Zn have been associated with ASD, attention deficit disorders and depression (30). In this study, the concentration of Cu and Zn were higher in study group than control group with significant differences, in addition, the level of Cu and Zn were higher in caries free subgroup than high caries subgroup among study group with highly significant differences. This may be attributed to the antimicrobial properties of Cu and Zn, which include acute loss of bacterial intracellular K+ and inhibition of H—ATP synthase, inhibition of various bacterial metabolic enzymes through oxidation of key thiol groups, and formation of insoluble salts on the tooth surface, thereby increasing its acid resistance (31). REFERENCES 1. Friedlander AH, Yagiela JA, Paterno VI, Mahler ME. The neuropathology, medical management and dental implications of autism. J Am Dent Assoc 2006; 137: 1517–27. J Bagh College Dentistry Vol. 29(3), September 2017 Salivary physicochemical among Pedodontics, Orthodontics and Preventive Dentistry 71 2. Luppanapornlarp S, Leelataweewud P, Putongkam P, Ketanont S. Periodontal status and orthodontic treatment need of autistic children. World J Orthod 2010; 11: 256– 61. 3. Pilebro C, Backman B. Teaching oral hygiene to children with autism. Int J Paediatr Dent 2005; 15: 1–9. 4. Rai K, Hegde AM, Jose N. Salivary antioxidants and oral health in children with autism. Arch Oral Biol 2012; 57: 1116–20. 5. Blomqvist M, Bejerot S, Dahllöf G. A cross-sectional study on oral health and dental care in intellectually able adults with autism spectrum disorder. BMC Oral Health 2015; 5: 81. 6. Amado FM, Vitorino RM, Domingues PM, Lobo MJ, Duarte JA. Analysis of the human saliva proteome. Expert Rev Proteomics 2005; 2(4): 521-39. 7. Suganya V, Umashangeethapriy D, Lakshmi M, Geetha A, Sujatha S. Analysis of salivary components to evaluate the pathogenesis of autism in children. Asian J Pharm Clin Res 2014; 7(4): 205–211. 8. Zahir S, Sarkar S. Study of trace elements in mixed saliva of caries free and caries active children. J Indian Soc Pedod Prev Dent 2006; 24: 27–29. 9. Chauhan A, Chauhan V, Brown WT and Cohen I. Oxidative stress in autism: Increased lipid peroxidation and reduced serum levels of ceruloplasmin and transferrin - the antioxidant proteins. Life Sci 2004; 75 (21): 2539–49. 10. Vojdani A, Mumper E, Granpeessheh D. Low natural killer cell cytotoxic activity in autism: the role of glutathione, IL-2 and IL-15. J Neuroimmunol 2008; 205: 148–154. 11. WHO. Oral health surveys basic methods. 4th ed. World health organization. Geneva, Switzerland, 1997. 12. Silness J, Löe H. Periodontal disease in pregnancy: Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964; 22: 121–135. 13. Ramfjord SP. Indices for prevalence and incidence of periodontal disease. J Perio 1959; 30: 51–59. 14. Löe H and Silness J. Periodontal disease in pregnancy. Acta Odontol Scand 1963; 21: 533–551. 15. Ali R. Odontometric measurements and salivary cortisol among low birth weight 5 years old kindergarten children in relation to dental caries. Master thesis, College of Dentistry, University of Baghdad, 2013. 16. Lahti M, Vilpo J, Hovinen J. Spectrophotometric determination of tiocyanate in human saliva. J Chem Educ 1999; 76: 1281–2. 17. Kopycka-Kedzierawski DT, Auinger P. Dental needs and status of autistic children: results from the National Survey of Children's Health. Pediatr Dent 2008; 30: 54– 8. 18. Bassaoukou IH, Nicolau J, Dos Santos MT. Saliva flow rate, buffer capacity, and pH of autistic individuals. Clin Oral Investig 2009; 13: 23–27. 19. Loo CY, Graham RM, Hughes CV. The caries experience and behavior of dental patients with autism spectrum disorder. JADA 2008; 139: 1518–24. 20. Gunawan I, Wibowo T, Pradopo S. Salivary pH conditions in children patients with mild autism and normal children. Indonesian Pediat Dent J 2010; 2: 2. 21. Harris N, Garcia-Godoy F, Nathe CN. Primary preventive dentistry. 7th ed. London: Asimon and Schuster Company, 2009. 22. Mojarad F, Fazlollahifar S, Poorolajal J, Hajilooi M. Effect of alpha amylase on early childhood caries: a matched case-control study. Braz Dent Sci 2013; 16: 1. 23. Williams TA, Mars AE, Buyske SG, Stenroos ES, Wang R, Factura-Santiago MF, et al. Risk of autistic disorder in affected offspring of mothers with a glutathione S-transferase P1 haplotype. Arch Pediatr Adolesc Med 2007; 161(4): 356–61. 24. Al-Gadani Y, El-Ansary A, Attas O, Al-Ayadhi L. Metabolic biomarkers related to oxidative stress and antioxidant status in Saudi autistic children. Clinical Biochemistry 2009; 42; 1032–40. 25. Geier, Kern, Geier. A prospective study of oxidative stress biomarkers in autistic disorders. Electronic J Applied Psychology 2009; 5(1): 2–10. 26. Rasheed M. Assessment of serum and salivary oxidative stress biomarkers with evaluation of oral health status in a sample of autistic male children. M.Sc. Thesis, College of Dentistry, University of Baghdad, 2011. 27. Wang GF, Li MG, Gao YC, Fang B. Amperometric sensor used for determination of thiocyanate with a silver nanoparticles modified electrode. Sensors 2004; 4(9):147–55. 28. Waring RH, Klovrza LV. Sulphur metabolism in autism. J Nutr Environ Med 2000; 10: 25–32. 29. Irving G, Lantana D, Sunnyvale. Copper and manganese in saliva of children. Den Res J 2010; 49(4): 776–782. 30. Osredkar J, Sustar N. Copper and Zinc. Biological role and significance of copper/zinc imbalance. J Clinic Toxicol 2011; S3: 001. 31. Stipanuk MH. Biochemical, Physiological & Molecular Aspects of Human Nutrition. Saunders Company; 2006. J Bagh College Dentistry Vol. 29(3), September 2017 Salivary physicochemical among Pedodontics, Orthodontics and Preventive Dentistry 72 Table 1: Mean values of salivary pH and flow rate among study and control groups by caries severity with statistical difference. Table 2: Correlation coefficients of salivary pH and flow rate with plaque, gingival and calculus indices among study and control groups. * Significant, ** Highly Significant Table 3: Mean values of salivary constituents (µg/dl) among study group by caries experience with statistical differences. ** Highly Significant Table 4: Mean values of salivary constituents (µg/dl) among study and control subgroups with statistical differences. Salivary parameters Subgroups Study Control Statistical difference (Mean ± SD) (Mean ± SD) t-value P-value pH Caries Free 7.71 ± 0.36 7.29 ± 0.36 3.718 < 0.001** High Caries 7.19 ± 0.44 6.51 ± 0.46 4.757 < 0.001** Total 7.45 ± 0.48 6.90 ± 0.57 4.689 < 0.001** Flow Rate (ml/min) Caries Free 1.75 ± 0.21 1.61 ± 0.22 2.062 0.046* High Caries 1.65 ± 0.29 1.41 ± 0.34 2.476 0.018* Total 1.70 ± 0.25 1.51 ± 0.30 3.103 0.003** Groups Salivary parameters PII GI CI r P r P r P Study pH -0.491 < 0.001** -0.281 0.079 -0.421 0.007** Flow Rate -0.182 0.262 -0.277 0.084 -0.219 0.175 Control pH -0.675 < 0.001** -0.527 < 0.001** -0.556 < 0.001** Flow Rate -0.321 0.043* -0.246 0.126 -0.337 0.033* Salivary Variable Caries free High caries t-value P-value Mean SD Mean SD α-Amylase 10.39 0.80 9.24 0.87 4.311 < 0.001** Glutathione 0.91 0.61 0.87 0.56 0.197 0.845 Thiocyanate 105.39 13.25 100.02 10.75 1.408 0.167 Copper 37.92 11.57 25.24 8.03 4.026 <0.001** Zinc 82.18 14.40 65.15 16.75 3.448 <0.001** Cu/Zn 0.48 0.19 0.40 0.14 1.428 0.162 Groups Study Control Statistical difference (Mean ± SD) (Mean ± SD) t-value P-value α-Amylase 9.81 1.01 8.63 1.07 5.113 <0.001** Glutathione 0.89 ± 0.58 2.20 ± 1.10 6.626 <0.001** Thiocyanate 102.70 ± 12.21 87.21 ± 11.14 5.929 <0.001** Copper 31.58 ± 11.74 25.21 ± 9.15 2.708 0.008** Zinc 73.66 ± 17.67 64.05 ± 16.64 2.505 0.014* Cu/Zn 0.44 ± 0.17 0.41 ± 0.16 0.729 0.468 * Significant, ** Highly Significant * Significant, ** Highly Significant J Bagh College Dentistry Vol. 29(3), September 2017 Salivary physicochemical among Pedodontics, Orthodontics and Preventive Dentistry 73 Figure 1: ROC curve for selected quantitative measurements when used to differentiate caries free subgroup from high caries subgroup among study group. Table 5: Area under ROC curve selected quantitative measurements when used to differentiate caries free subgroup from high caries subgroup among study group. ** Highly Significant Figure 2: ROC curve for selected quantitative measurements when used to differentiate study group from control group. Tested variable Area under the curve P-value Alpha amylase 0.838 < 0.001** Copper 0.813 < 0.001** Zinc 0.763 0.005** Cu/Zn ratio 0.621 0.190 Thiocyanate 0.616 0.199 Glutathione 0.501 0.989 J Bagh College Dentistry Vol. 29(3), September 2017 Salivary physicochemical among Pedodontics, Orthodontics and Preventive Dentistry 74 Table 6: Area under ROC curve selected quantitative measurements when used to differentiate study group from control group. * Significant, ** Highly Significant Table 7: Correlation coefficient between salivary constituents and plaque, gingival, calculus indices among study and control groups. ** Highly Significant الخالصة ( بضعف في التفاعل االجتماعي والتواصل، أنماط محدودة من السلوك وتحسس حسي غير عادي. امراض الفم ASDيتميز اضطراب طيف التوحد ) :المقدمة االسنان وذلك بسبب المظاهر السريرية المعقدة والمتنوعة تكون غير محددة للغاية لدى األفراد الذين يعانون من اضطراب التوحد اال انها تشكل تحد كبير ألطباء الكيميائية للعاب فيما يتعلق بصحة الفم للمراهقين الذين يعانون من اضطراب التوحد في بغداد.-الخاصة بهم. تهدف الدراسة لبحث الخواص الفيزيائية التوحد معاهد رعايةمراهق يعانون من اضطراب التوحد تم اختيارهم من 04مجموعة الدراسة وتتكون من تضمنت الدراسة مجموعتين: :والطرق المواد ( سنة ومن الذكور فقط. كل مجموعة قسمت الى 21-21مراهق سليم من المدارس الثانوية القريبة لمعاهد رعاية التوحد وبعمر ) 04ة وتتكون من سيطرومجموعة ال (، DMFSاستخدام مقاييس تسوس االسنان )(. DMFT≥6( ومجوعة عالية التسوس )DMFT=0مجموعتين وفقا لشدة التسوس: مجموعة خالية التسوس ) ( تم SCN( والثايوسيانيت )Zn(، الزنك )Cu( لتقييم صحة الفم واالسنان للمجموعتين. كال من النحاس )CI( والقلح )GI(، التهاب اللثة )PlIاللويحة الجرثومية ) (. بينما الفا اميليز والجلوتاثيون تم قياسهم باستخدام تقنية قياس االنزيم المناعي المرتبط Spectrophotometerالذري )قياسهم باستخدام جهاز معامل االمتصاص (ELISA.) تم تحليل بيانات الدراسة الحالية باستخدام برنامجSPSS 12النسخة . في مجموعة الدراسة اعلى من أظهرت الدراسة ان قيم كال من االس الهيدروجيني للعاب، سرعة تدفق اللعاب، الفا اميليز، ثايوسيانيت، النحاس والزنك النتائج: في توى الجلوتاثيونة وبفارق معنوي، كذلك هذه القيم في المجموعة خالية التسوس اعلى من المجموعة عالية التسوس وبفارق معنوي. بينما مسسيطرمجموعة ال مقياس (، PlI)ة. وجود عالقة ارتباط متوسطة عكسية بين الفا اميليز، النحاس والزنك مع مقياس اللويحة الجرثومية سيطرمجموعة المجموعة الدراسة كان اقل من القلح ( وPlI)متوسطة بمقياسي اللويحة الجرثومية ( وبفارق معنوي عالي، وكذلك االس الهيدروجيني للعاب يرتبط بعالقةGI( ومقياس صحة اللثة )CIالقلح ) (CI.وبفارق معنوي عالي ) وعة مجماعلى من حد قيم كال من االس الهيدروجيني للعاب، سرعة تدفق اللعاب، الفا اميليز، ثايوسيانيت، النحاس والزنك في مجموعة اضطراب التو ان :االستنتاج د مقارنة مجموعة اضطراب التوح خالية التسوس اعلى من المجموعة عالية التسوس. بينما الجلوتاثيون كان اقل فية، وجميع هذه القيم في المجموعة السيطر .ةلسيطرمجموعة اب Tested variable Area under the curve P-value Glutathione 0.858 < 0.001** Thiocyanate 0.821 < 0.001** Alpha amylase 0.786 < 0.001** Copper 0.652 0.019* Zinc 0.648 0.023* Cu/Zn ratio 0.538 0.564 Groups Salivary constituents PlI GI CI r P r P r P Study Amylase -0.537 < 0.001** -0.460 0.003** -0.491 < 0.001** Glutathione -0.016 0.924 -0.028 0.866 -0.023 0.890 Thiocyanate -0.043 0.790 -0.101 0.534 -0.225 0.162 Copper -0.577 < 0.001** -0.493 < 0.001** -0.539 < 0.001** Zinc -0.526 < 0.001** -0.546 < 0.001** -0.560 < 0.001** Cu/Zn -0.278 0.082 -0.209 0.195 -0.181 0.263 Control Amylase -0.089 0.587 -0.078 0.631 -0.245 0.127 Glutathione -0.097 0.553 -0.094 0.564 -0.162 0.317 Thiocyanate -0.120 0.460 -0.194 0.230 -0.097 0.551 Copper -0.221 0.170 -0.089 0.583 -0.263 0.101 Zinc -0.415 0.008** -0.163 0.315 -0.500 < 0.001** Cu/Zn 0.069 0.670 -0.025 0.877 0.118 0.467