Rihab F.doc J Bagh College Dentistry Vol. 25(4), December 2013 Salivary cortisol Pedodontics, Orthodontics and Preventive Dentistry126 Salivary cortisol among low birth weight 5 years old kindergarten children in relation to dental caries (comparative study) Rihab A. Ali, B.D.S. (1) Ban S. Diab B.D.S., M.Sc., Ph.D. (2) ABSTRACT Background: Birth weight is a powerful predictor of infant growth and survival. Evidence now shows that children born with low birth weight face an increased risk of chronic diseases and have many health problems including oral health. The aims of this study were to assess the salivary flow rate, viscosity, and salivary cortisol among low birth weight kindergarten children aged 5 years old in Hilla centre, in relation to dental caries and compares them with the normal birth weight children of the same age and gender. Materials and methods: The total sample involved 80 children (40 low birth weights and 40 normal birth weights) aged 5 years old. The diagnosis and recording of severity of dental caries was recorded through the application of d1-4mfs index according to the criteria described by Mühlemann (1976). The stimulated saliva was collected from the total sample under standardized conditions and then analyzed for measuring salivary flow rate and viscosity, in addition to estimation of salivary cortisol by special cortizol kit using VIDAS® Cortisol S. Results: The mean rank of dmfs, ds, ms and fs were found to be higher among low birth weight than normal birth weight groups, with a statistically significant difference for dmfs, ds (P<0.05), highly significant difference for ms (P<0.01) and non significant difference for fs (P>0.05). Concerning the ds grade, data analysis showed a significant difference only for d1 grade (P<0.05). Salivary analysis demonstrated that the mean rank of salivary flow rate was found to be lower among the low birth weight than the normal birth weight groups with non significant difference (P>0.05). The viscosity of saliva was found to be highly significantly higher among low birth weight than normal birth weight groups (P<0.01). Concerning salivary cortisol, data analysis showed that the mean rank was higher among low birth weight than normal birth weight groups. However, the difference was not significant (P>0.05). Conclusion: The results of the current research revealed that low birth weight status affect oral health conditions. Key words: Low birth weight, dental caries, salivary flow rate, salivary viscosity, salivary cortisol. (J Bagh Coll Dentistry 2013; 25(4):126-133). الخالصة الدالئل تشیر اآلن أن األطفال الذین یولدون مع انخفاض الوزن عند الوالدة یواجھون خطرا متزایدا من . وزن الوالدة یشكل مؤشرا قویا لنمو الرضع و البقاء على قید الحیاة : خلفیة ذه الدراسة تقییم معدل تدفق اللعاب ، واللزوجة ، والكورتیزول اللعابي بین انخفاض الوزن وكانت أھداف ھ. األمراض المزمنة ولھا العدید من المشاكل الصحیة بما في ذلك صحة الفم سنوات من العمر في مركز الحلة ، وعالقتھ بتسوس األسنان ومقارنتھا مع الوزن الطبیعي عند الوالدة لألطفال من نفس العمر 5عند الوالدة لریاض األطفال الذین تتراوح أعمارھم بین .والجنس وتم تسجیل شدة تسوس . سنوات 5الذین تتراوح أعمارھم بین ) عند الوالدة الطبیعي الوزن 40انخفاض الوزن عند الوالدة و 40(طفال 80تضمنت العینة الكلیة : المواد و الطرق ب المحفز من العینة الكلیة في ظل ظروف موحدة ومن ثم تحلیلھا لقیاس خالل تطبیق مؤشر وقد تم جمع اللعاd1- 4mfs وفقا للمعاییر التي وصفھا Mühlemann (1976) .األسنان من معدل تدفق اللعاب واللزوجة ، باإلضافة إلى تحلیل الكورتیزول اللعابي باستخدام كت خاص بالكورتیزول .VIDAS® Cortisol S طفال الوزن الوزن عند الوالدة عنھ أل اطفال انخفاضلتكون أعلى عند ، وجدتعند الوالدة الطبیعيdmfs ،ds ،ms وfs وفرق معنوي كبیر للغایة msوفرق غیر معنوي فیما یخص fs وفیما یتعلق ds ، أظھر تحلیل البیانات فرقا معنویا فقط للرتبة تسوس السطوح لألسنان اللبنیةرتبة متوسط : النتائج معدل تدفق اللعاب وجدت لتكون أقل بین مجموعة انخفاض الوزن عند الوالدة من تحلیل اللعاب أن رتبة متوسط أظھر d1 . , مع وجود فرق دال إحصائیا dmfs, ds, فیما یخص ن لزوجة اللعاب وجدت لتكون أعلى بكثیر مع فارق معنوي كبیر للغایة بین مجموعة انخفاض الوزن عند الوالدة م. مجموعة الوزن الطبیعي عند الوالدة مع وجود فارق غیر معنوي موعة وفیما یتعلق بالكورتیزول اللعابي ، أظھر تحلیل البیانات أن رتبة متوسط الكورتیزول أعلى بین مجموعة انخفاض الوزن عند الوالدة من مج. مجموعة الوزن الطبیعي عند الوالدة .ومع ذلك ، كان الفارق غیر معنوي . الوزن الطبیعي عند الوالدة .ؤثر على األوضاع الصحیة للفمتلحالي أن حالة انخفاض الوزن عند الوالدة كشفت نتائج البحث ا: االستنتاج .زوجة اللعاب ، الكورتیزول اللعابيلانخفاض الوزن عند الوالدة، تسوس األسنان ، معدل تدفق اللعاب ، : الكلمات الرئیسیة INTRODUCTION Low birth weight (LBW) is a major determinant of mortality, morbidity and disability in infancy and childhood and also has a long-term impact on health outcomes in adult life. The consequences of poor nutritional status and inadequate nutritional intake for women during pregnancy not only directly affect women’s health status, but may also have a negative impact on birth weight and early development. Low birth weight also results in substantial costs to the health sector and imposes a significant burden on society as a whole (1). (1) M.Sc student Department of Pedodontics and Preventive Dentistry, College of Dentistry, University of Baghdad. (2) Assistant Professor, Department of Pedodontics and Preventive Dentistry, College of Dentistry, University of Baghdad. Low birth weight defines a heterogeneous group of infants: some are born early, some are born growth restricted, and others are born both early and growth restricted. It is generally recognized that being born with low birth weight is a disadvantage for the baby (2). Some authors defined low birth weight as baby weighting less than or equal to (2500 g) (3,4) while others defined LBW as a baby that weighs less than (2500 g) (up to and including 2499 g) regardless of gestational age (5,6). Children with low birth weight are at risk for medical problems in childhood and adulthood and often have poor oral health. Dental caries remain one of the major global public health problems. It is considered as multifactorial since it is influenced by dietary as well as host factors (7). However, the literatures contain contradictory J Bagh College Dentistry Vol. 25(4), December 2013 Salivary cortisol Pedodontics, Orthodontics and Preventive Dentistry127 results concerning the relation between LBW and dental caries. Saraiva et al. (8), Javadinejad et al. (9), and Rajshekar and Laxminarayan (10), reported that children born with LBW have higher caries experience in the primary dentition, whereas Gravina et al. (11), and Cruvinel et al. (12), found a greater prevalence of carious lesions in children born at full term than in children born prematurely. They suggested that low birth weight, including preterm births, predisposes to high levels of streptococcal colonization due to reduced immunofunction, in addition that the LBW favoring the development of enamel hypoplasia and salivary disorders (13,14). In contrast, Burt and Pai (15), Peres et al. (16), and Shulman et al. (17), concluded that no significant association is observed between low birth weight and dental caries. Saliva is an ideal translational research tool and diagnostic medium as saliva is mirror of the serum and is being used in novel ways to provide molecular biomarkers for a variety of oral and systemic diseases and conditions (18-20). Saliva contains various microbes and host biological components that could be used for caries risk assessment (21). Saliva represents the first line of defense against dental caries after tooth eruption (22,23). In addition, saliva displays physical properties related to the maintenance of a healthy oral environment, including secretion rate and viscosity (24). This finding tends to support the hypothesis that the mechanism behind the increased risk of dental pathology in preterm, low birth weight and retarded children is centered at structural and functional immaturity of salivary gland which can lead to hyposalivation and low salivary flow in addition to reducing water content in the saliva and thus increasing salivary viscosity (25,26,27). Pre- and peri-natal risk factors such as low birth weight and prematurity are presumed to shape the developing brain and predict the development of stress-related problems. In premature infants, a number of complications are considered to be associated with adrenal insufficiency and cortisol values have been examined by a number of investigators in an attempt to identify infants with relative adrenal insufficiency (28-30). Cortisol is produced in response to stress and is easily measured in saliva (31). There have been relatively few studies that have examined the impact of pre- and peri-natal risk factors on salivary cortisol level. Bettendorf et al. (32) found that healthy full-term neonates exhibited higher salivary cortisol concentrations than healthy preterm neonates. While Wüst et al. (33) reported that birth weight was associated with salivary cortisol responses to psychosocial stress in adult life. Low birth weight predicted higher cortisol reactivity in young adulthood. However, the development of cortisol circadian rhythm in premature infants is not necessarily retarded and that low gestational age is not critical for the timing of emergence of the hypothalamus- pituitary- adrenal (HPA) axis circadian activity (34). Grunau et al. (35) reported that extremely low gestational age (ELGA) infants show significantly higher salivary cortisol levels many months past their expected date of delivery, suggesting possible (re-setting) of basal cortisol levels, and long term (programming) of the HPA axis. On the other hand, Schmidt et al. (36) concluded that ELBW adults exhibited not significantly lower salivary cortisol at baseline compared with adults born at normal birth weight. Cortisol is capable of affecting local, mucosal immunity and oral microbial flora, and that mucosal immune competence affects bacterial colonization and growth (37). It affects the performance of some immune system cells (38). Basal salivary cortisol and cariogenic bacteria were the strongest predictors of dental caries, and from a theoretical perspective, salivary cortisol could plausibly suppress mucosal immunity against cariogenic bacteria (39). The present study was conducted among a group of kindergarten children with low birth weight aged 5 years old in comparison to control group to evaluate the following variables: the occurrence and severity of dental caries, the changes in the physicochemical characteristics of stimulated saliva and these including (salivary flow rate, salivary viscosity, salivary cortisol), in addition to determine the relation between these variables. MATERIALS AND METHODS In the present investigation, the study group included 40 children with low birth weight aged 5 years old of both gender. They were examined in their kindergarten during the period from the third of December 2012 till the end of January 2013. The control group included 40 children with normal birth weight who possess as much similarity as possible to the study group with regard to age, gender, social structure and geographic position except in birth weight condition. Both study and control groups should not have any systemic disease that could affect on the salivary analysis. The assessment and recording of caries experiences were done through J Bagh College Dentistry Vol. 25(4), December 2013 Salivary cortisol Pedodontics, Orthodontics and Preventive Dentistry128 the application of decayed (d), missing (m) and filled (f) surface index (dmfs) index for primary teeth. In this study, the decayed fraction of the index was recorded according to the lesion severity using the criteria described by Mühlemann (40). The collection of the stimulated salivary samples from the children was performed under standardized conditions according to the instructions cited by Tenovuo and Lagerlöf (41) and Farsi (42). Immediately after saliva collection, salivary flow rate was measured freshly and without centrifuged (after foam had all disappeared) by dividing the volume of the collected stimulated saliva in milliliter (ml) on the collection time in minute (min) (25). Salivary viscosity was determined by measuring the volume rate of flow through a tube of known dimensions. This was done by using Ostwald's viscometer which is simple device for measuring the viscosity of liquid (43). In this study, salivary viscosity was measured at room temperature and before centrifuging of salivary samples (44). The viscosity of the saliva is measured according to a liquid which have a known coefficient of viscosity, and usually the distilled water is used for this purpose. The flow times of two liquids (saliva and distilled water) which have equal volumes passing through a capillary of the same viscometer are measured and the coefficient of the viscosity of the saliva is determined. The salivary samples were then taken to the laboratory for biochemical analysis and centrifuged at 3000 r.p.m. for 10 minutes. The clear supernatant was separated by micropipette and was stored at (- 20°C ) in a deep freeze and further assessment for cortizol level in saliva was done by special cortizol kit (VIDAS® Cortisol S) using MiniVIDAS technique which is multiparametric immunoassay system (45). At the end of the assay, results are automatically calculated by the instrument in relation to the calibration curve. These results were obtained in traditional unit (ng/ml). To be converted from traditional to SI units, they had been converted to (nmol/L) by multiplying them by Endocrinology Conversion Factor (2.759) (46). Intra and inter calibration were performed to overcome any problem that could be faced during the research, and to ensure proper application of diagnostic criteria used in recording dental status through inter calibration. Statistical Analysis and processing of the data were carried out using SPSS version 19. After exploring the data, it had been found that they were not normally distributed. The non-parametric Mann-Whitney U test was utilized for the parameters of the data which were not normally distributed and in this test the median and mean rank were used to analyze and determine the differences between the study and control groups. The correlation coefficient tests between the variables were done by using Pearson correlation. The confidence level was accepted at the level of less than or equal to 5%. The highly confidence level was accepted at the level of less than or equal to 1%. RESULTS The distribution of the low birth weight and their matching normal birth weight children by gender are shown in Table (1). Dental caries experience in primary dentition represented by dmfs and its components (ds,ms,fs) among low and normal birth weight groups are illustrated in Table (2). Results revealed that the mean rank of dmfs for the total sample was found to be higher among low birth weight than normal birth weight and the difference was significant (Mann Whitny=566.000, Z=-2.280,P=0.023). The mean rank of decay fraction (ds), filling (fs) and missing (ms) surface were found to be higher among low birth weight than normal birth weight with significant difference for ds (Mann Whitny=586.000, Z=-2.087, P=0.037) and highly significant difference for ms (Mann Whitny=625.000, Z=-2.622, P=0.009) while for fs, the difference was not significant (P> 0.05). The grades of decay fraction among low and normal birth weight groups are represented in Table (3). Concerning low birth weight, the d1 fraction of severity was the highest, followed by d3, d2. While the d4 showed the least fraction of decay severity. For normal birth weight group, the d4 fraction of severity was the highest, followed by d2 and d3. While the d1 showed the least fraction of decay severity. For the total sample, data analysis showed that the mean rank of d1 was higher among low birth weight than normal birth weight with statistically significant difference (Mann Whitny=616.000, Z=-2.193, P=0.028).However, the same picture was observed for d2, d3, and d4 with statistically non significant difference (P> 0.05). The physicochemical characteristics of the stimulated whole salivary flow rate among low and normal birth weight groups are illustrated in Table (4). The mean rank of salivary flow rate was found to be lower among low birth weight than normal birth weight with no significant difference (P> 0.05). Apposite finding was found concerning salivary viscosity as the mean rank was highly significantly higher among low birth weight group than normal birth weight group (Mann Whitny=387.500, Z=-3.970, P=0.000). J Bagh College Dentistry Vol. 25(4), December 2013 Salivary cortisol Pedodontics, Orthodontics and Preventive Dentistry129 The same result was found for salivary cortisol with no significant difference (P>0.05). Table (5) shows the correlation coefficient between caries experience in primary dentition represented by dmfs and ds components and salivary variables among low and normal birth weight groups. For low birth weight group, the relation between salivary flow rate and caries experience was weak and not significant in positive direction concerning dmfs while in negative direction concerning ds. The same relations were found between salivary viscosity and both dmfs and ds in positive direction while the relations between salivary cortisol and both dmfs and ds were found to be weak, and non significant in negative direction. Concerning normal birth weight, the relations between salivary flow rate and both dmfs and ds were weak and non significant in positive direction. The same relations were found between salivary cortisol and both dmfs and ds while the relations between salivary viscosity and both dmfs and ds were found to be weak, and non significant in negative direction. Table (6) represents the correlation coefficient between salivary cortisol and both salivary flow rate and salivary viscosity among low and normal birth weight groups. Concerning low and normal birth weight groups, data analysis for the total sample showed that the relation between salivary flow rate and cortisol was found to be weak and non significant in negative direction, while the relation between salivary viscosity and cortisol was found to be weak and non significant in positive direction. DISCUSSION Infants born preterm and with low birth weight are at greater risk for mortality, disability and a variety of health and developmental problems compared with infants born at term (47,48). A fetal programming hypothesis proposed that stressful events during fetal development may program the developing brain for how it handles subsequent stress (49). In terms of caries experience among the low birth weight group, the present study showed that the mean rank of dmfs and decay fraction ds were significantly higher than the normal birth weight group. This result was in agreement with the results reported by many previous studies (8- 10,50,51), and was in disagreement with the results that concluded by others (11,12,52-55). These results might be due to neonatal malnutrition which increases the risk of poor pregnancy outcomes including low birth weight infants. The greater systemic infant malnutrition associated with low birth weight children is said to result in systemic insults to the developing primary teeth which can lead to disturbances in the mineralization resulting in hypoplasia (13,56) and thus predisposing the teeth to caries (14,57). Other causes can be attributed to maternal lactation failure, so that lack of breast feeding or exclusive breast feeding for shorter duration in these children leads to undernourishment during maturation phase of teeth and this can lead to hypoplasia and enamel defects which are more susceptible areas to dental caries (58). The higher caries experience could be attributed to the lower social class of those children with low birth weight as reported by some previous studies (59,60). This can be attributed to the low family income and the degree of education in the lower social classes which can affect food selection, nutrient intake, health values, life style, oral hygiene practices, access to health care information and susceptibility to childhood infections (17,52). The lower flow rate that was found in the present study (although not significant) among low birth weight children could give another explanation for high caries experience. This can be explained by the fact that many evidences suggest that intrauterine growth restriction (IUGR) leads to impaired growth and maturation of the salivary gland (structural immaturity and lack of differentiated parenchymal elements of the organ may form the basis of its secretory function’s lesion) which causes hyposalivation and low salivary flow. This is in agreement with previous study (27). Although, dental decay is a multifactorial disease, the salivation’s insufficiency is believed to have harmful effect on oral health, this is also shown in the present study as data analysis showed that the correlation between the salivary flow rate and decay fraction (ds) and grades of dental caries (d1-4) were found to be inverse relations among the low birth weight children. This could be attributed to the fact that the higher the flow rate, the faster the clearance, the higher the buffer capacity and the lower dental caries (61,62). Increased dental caries might be due to increased saliva viscosity as data of the current study showed high viscosity among low birth weight children. This can be explained by the fact that preterm and low birth weight children are affected by structural and functional immaturity of the salivary gland. This gives reasons for increased viscosity that may be due to reduced water content in the saliva. This finding is in correlation with other study (63) and this is also found by data of the present study that showed an J Bagh College Dentistry Vol. 25(4), December 2013 Salivary cortisol Pedodontics, Orthodontics and Preventive Dentistry130 inverse relation exists between salivary viscosity and salivary flow rate which was in agreement with other study (25). Increased salivary viscosity plays a role in increasing caries incidence. Among low birth weight children, data analysis of the present study revealed that the correlation of the salivary viscosity with dental caries was found to be a positive relation, which could give another explanation for high caries experience in present study as well as in previous studies (64-66) among other study groups. This may be attributed to the fact that saliva that is more viscid is less effective in clearing the mouth from food derbies, reduced bacterial co-aggregation, and played a role in increasing caries incidence (65). Other causes for high caries experience could be attributed to high salivary cortisol level among low birth weight group (although not significant) and this was in agreement with previous study (33) and can be explained as the higher cortisol level may suppress the oral immunity and induce the proliferation of the cariogenic bacteria leading to increased incidence of dental caries. Basal salivary cortisol secretion was positively associated with dental caries. From a theoretical perspective, salivary cortisol could suppress mucosal immunity against cariogenic bacteria. Basal salivary cortisol secretion and cariogenic bacteria bore strong and independent associations with counts of dental caries (39). Concerning the correlation of the salivary cortisol with flow rate, data analysis of the present study reported an inverse correlation among both low and normal birth weight groups. This is in agreement with some previous studies (67,68) while disagrees with others (69,70) among other study groups. This may be explained by the fact that as the flow rate increased, the pH increased but the protein level decreased (68) and as cortisol is a salivary protein, therefore; its level may be decreased. The correlation of the salivary cortisol with viscosity was found to be a positive relation in both low and normal birth weight children. 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Table 1: Distribution of the low birth weight and their matching normal birth weight children according to gender Low birth weight Normal birth weight Gender No. % No. % Males 21 52.5 21 52.5 Females 19 47.5 19 47.5 Total 40 100 40 100 Table 2: Dental caries experience (dmfs) and components (ds, ms and fs) among low birth weight and normal birth weight children Statistical differences Normal Birth Weight Low Birth Weight P-value Z- value U-Test df Mean Rank Median No. Mean Rank Median No. 0.023 -2.280* 566 78 34.65 2.5 40 46.35 6 40 dmfs 0.037 -2.087* 586 78 35.15 2.5 40 45.85 4 40 ds 0.009 -2.622** 625 78 36.13 0 40 44.88 0 40 ms 0.096 -1.665 721 78 38.53 0 40 42.48 0 40 fs (* P<0.05 Significant, ** P<0.01 Highly Significant) J Bagh College Dentistry Vol. 25(4), December 2013 Salivary cortisol Pedodontics, Orthodontics and Preventive Dentistry133 Table 3: Grades of dental caries among low birth weight and normal birth weight children Statistical differences Normal Birth Weight Low Birth Weight P-value Z- value U-Test df Mean Rank Median No. Mean Rank Median No. 0.028 -2.193* 616 78 35.9 0 40 45.1 0 40 d1 0.414 -0.834 715.5 78 38.39 1 40 42.61 2 40 d2 0.12 -1.555 661.5 78 37.04 0 40 43.96 0 40 d3 0.572 -0.565 763.5 78 39.59 0 40 41.41 0 40 d4 (* P<0.05 Significant) Table 4: Salivary physicochemical characteristic among low birth weight and normal birth weight children Statistical differences Normal Birth Weight Low Birth Weight ` P-value Z- value U-Test df Mean Rank Median No. Mean Rank Median No. 0.532 -0.626 735 78 42.13 0.347 40 38.88 0.307 40 Flow rate (ml/min) 0.000 -3.970** 387.5 78 30.19 0.012 40 50.81 0.012 40 Viscosity (dyne.sec/cm2) 0.835 -0.208 780 78 40.01 5.52 40 40.99 5.52 40 Cortisol (nmol/L) (** P<0.01 Highly Significant) Table 5: Correlation coefficient (r) between dmfs, ds components of dental caries and salivary flow rate (ml/min), salivary viscosity (dyne.sec/cm2), and salivary cortisol (nmol/L) among low birth weight and normal birth weight children Normal Birth Weight Low Birth Weight ds dmfs ds dmfs Salivary variables P r P r P r P r 0.328 0.159 0.28 0.175 0.461 -0.12 0.934 0.014 Flow rate 0.545 -0.099 0.501 -0.11 0.741 0.054 0.427 0.129 Viscosity 0.863 0.028 0.955 0.009 0.098 -0.265 0.081 -0.279 Cortisol Table 6: Correlation coefficient (r) between salivary cortisol (nmol/L) and flow rate (ml/min) and viscosity (dyne.sec/cm2) among low birth weight and normal birth weight children Salivary cortisol Normal Birth Weight Low Birth Weight P r P r 0.273 -0.178 0.725 -0.057 Flow rate 0.152 0.231 0.365 0.147 Viscosity