*Iwona Przywitowska1, Urszula Kaczmarek1, Grzegorz Bartnicki2, Alina Wrzyszcz-Kowalczyk1
Salivary flow rate, total protein and pH in caries-free children and adolescents aged between 5 and 18 years
Szybkość wydzielania śliny, białko całkowite i pH u dzieci wolnych od próchnicy w wieku od 5 do 18 lat
1Department of Conservative and Paediatric Dentistry, Medical University of Wrocław
Head of Department: Professor Urszula Kaczmarek, MD, PhD
2Department of Air Conditioning, Heating, Gas Supply, and Air Protection, Wroclaw University of Technology
Head of Department: Professor Renata Krzyżyńska, PhD, DSc
Wstęp. Nieliczne i nie w pełni zgodne sa? dane dotycza?ce szybkości wydzielania śliny i poziomów jej składników w wieku rozwojowym.
Cel pracy. Celem pracy było porównanie szybkości wydzielania śliny, poziomu białka całkowitego i pH s?liny u osób w wieku od 5 do 18 lat, w celu uzyskania informacji o funkcjonalnym dojrzewaniu gruczołów s?linowych w okresie rozwojowym.
Materiał i metody. Zbadano 90 dzieci i młodzieży obojga płci w wieku od 5 do 18 lat wolnych od próchnicy. Badani byli wolni od próchnicy (wartość zero wskaźnika ICDAS II). Pobierano od nich niestymulowaną ślinę mieszaną, w której oznaczano pH i białko całkowite oraz szybkość wydzielania. Badanych podzielono na trzy grupy wiekowe: 5-6, 13-14 i 18 lat.
Na przeprowadzenie badań uzyskano zgodę Komisji Bioetycznej Uczelni Nr KB-335/2013.
Wyniki. W grupie wiekowej 5-6 lat zaobserwowano istotnie niższe wydzielanie śliny niż u osób w wieku 13-14 i 18 lat. Natomiast poziom pH śliny w najmłodszej grupie był istotnie wyższy w porównaniu ze starszymi grupami wiekowymi. Stężenie białka całkowitego było najniższe w wieku 5-6 lat, wyższe w wieku 13-14 lat i najwyższe w wieku 18 lat (różnica istotna między grupami 5-6 i 18 lat). Między grupami zauważono spadkowy trend poziomu pH, a wzrostowy stężenia białka. Rozpatrując wszystkich badanych, wykazano pozytywną korelację wieku z szybkością wydzielania i stężenia białka, a negatywną z poziomem pH. Ponadto wraz ze wzrostem sekrecji śliny obniżały się poziom pH i stężenie białka.
Wnioski. W wieku od 5 do 18 lat wzrasta spoczynkowa szybkość wydzielania śliny i stężenie białka całkowitego, a maleje poziom pH w spoczynkowej ślinie mieszanej.
Introduction. Data regarding salivary flow and the levels of salivary components in developmental age are scarce and not fully consistent.
Aim. The aim of the study was to compare unstimulated mixed saliva flow rate, pH and total protein in children aged between 5 and 18 years to obtain information on the functional maturation of salivary glands during the developmental period.
Material and methods. A total of 90 children and adolescents (both sexes) aged between 5 and 18 years were examined. All subjects were caries-free (ICDA II score zero). Unstimulated mixed saliva was sampled from all patients to assess pH, total protein and flow rate. The subjects were divided into age groups 5-6, 13-14 and 18 years.
The study was approved by the Bioethics Committee of the University (No. Nr KB-335/2013).
Results. Significantly lower salivary flow rates were observed in 5-6 year olds vs. 13-14 and 18-year-olds. In contrast, pH values were significantly higher in the youngest group compared to older age groups. Total protein was the lowest in 5-6 year olds, higher in 13-14 year olds and the highest at the age of 18 years (significant difference between age groups of 5-6 and 18 years). A decreasing trend in pH values and an increasing one in protein levels were observed between the age groups. Considering the entire group of subjects, a positive correlation between age and salivary flow rate and protein levels, and a negative correlation with pH were found. Moreover, pH and protein levels decreased with increasing salivary flow.
Conclusions. Unstimulated mixed saliva flow rate and total protein increase, while pH levels decrease between the ages of 5 to 18 years.
Mixed or total saliva is a mixture of oral secretions, which come into direct contact with oral anatomical structures. It is a natural oral environment for hard and soft tissue exposure to external environmental factors and interactions between tissues, food, microbes and air. A variety of organic and mineral components contained in saliva allow for the normal course of multiple processes maintaining a healthy oral ecosystem (1, 2). Saliva is produced mainly by three paired large salivary glands, i.e. parotid, sublingual and submandibular glands, as well as, to a minor extent, by multiple (400-1000) small glands found in the oral mucosa. Under physiological conditions, the total daily volume of oral secretions ranges between 0.5 to 1 L in adults, including 80% of saliva stimulated by food. Each type of salivary gland produces secretion with a specific composition and properties, which depend on a number of factors, including diseases and pharmacotherapy (3-6). The major salivary glands produce about 90% of the total salivary volume. The secretions produced under stimulated conditions in parotid glands, which are the largest salivary glands (serous glands), constitute a thin aqueous liquid high in α-amylase and low in organic components and glycoproteins, contributing to about 53% of total saliva (7). Under unstimulated conditions, the amount of produced saliva is significantly lower, accounting for about 20-30% (1). The submandibular gland (SMG) is the second largest gland (8), which produces serous/mucous secretions. The gland produces less than a half of total saliva under stimulated conditions and 1/3 of total saliva under unstimulated conditions (8). Dense and viscous serous/mucous secretion produced by the sublingual glands, both stimulated and unstimulated, accounts for only a small proportion of total salivary volume (1, 8). Minor salivary glands produce mucous saliva high in proteins, which accounts for about 10% of total saliva (1, 8). Normal unstimulated and stimulated salivary flow rate is about 0.25-0.35 mL/min and 1-3 mL/min, respectively. Hyposalivation, i.e. reduced salivary flow, is defined as unstimulated salivary flow rate < 0.1 mL/min and stimulated salivary flow rate < 0.5-0.7 mL/min (1, 9-11). Salivary volume depends, among other things, on the quantity and quality of consumed foods, body hydration, emotional stimuli, age and sex (12, 13). Secretion of saliva follows a circadian rhythm. During sleep, salivary glands produce only about 2-10% of total daily volume, with submandibular and sublingual contributions of about 80 and 20%, respectively, and with arrested secretion in the parotid glands. Salivary flow increases by about 25-30% in the morning. Minor salivary glands do not follow a circadian rhythm, but maintain a steady level of secretion (14, 15). In humans, major salivary glands arise from a thickening of the oral ectoderm at around 4 to 6 weeks of foetal life for parotid glands, at the end of week 6 for submandibular glands, and 7-8 weeks for the sublingual gland. Minor salivary glands arise from ectodermal and endodermal thickening at the end of the 12th week. Further development involves complex interactions between epithelial cells and the adjacent mesenchymal cells, which induces and controls morphogenesis and salivary gland cell differentiation (16). At 16 weeks of gestation, the submandibular gland starts the production of serous secretions, the production of which is reduced at 28 weeks. The parotid gland begins to secrete at 18 weeks of gestation (17). It is assumed that salivary glands are functionally capable of secreting saliva already at the time of birth (18). However, studies indicate that age-related quantitative and qualitative changes in saliva are particularly pronounced in older patients compared to young individuals (19-21). Data regarding salivary flow and the levels of salivary components in developmental age are scarce and not fully consistent.
The aim of the study was to compare salivary flow rate, pH and total protein in children and adolescents to obtain information on the functional maturation of salivary glands during the developmental period.
Material and methods
Non-cavitated and cavitated caries-free children and adolescents (classified based on the ICDAS II, code 0) were randomly selected and examined. A total of 90 subjects of both sexes were included in the study. The participants were classified into 3 age groups: 5-6, 13-14 and 18 years. Inclusion criteria were as follows: age between 5 and 6 years, between 13 and 14 years, and 18 years, full dentition with a code 0 in ICDAS II, no chronic systemic diseases or pharmacotherapy, written consent of parent/legal guardian/18-year-old patient, and patient’s cooperation. Failure to meet one of the above inclusion criteria was the exclusion criterion. Clinical assessment of oral health was performed by two independent researchers (following calibration), with 90% conformity of assessment. Unstimulated mixed saliva was sampled in the morning, at least 1.5 hrs after a meal or about 4 mL of beverage. While collecting saliva, the subjects were placed in a sitting position with the head tilted and the mouth open, and were asked to let saliva gather on the bottom of their mouth and spit into calibrated tubes placed in ice. The time needed to collect saliva was recorded and salivary volume was measured to calculate salivary flow rate (mL/min). Salivary samples were then centrifuged at 3,500 rpm for 10 minutes. The obtained supernatants were used to determine salivary pH (pH-metric method using the ESAgP-301W type combined electrode connected to the pH/lon Meter CPI-551 Microcomputer) and total protein using the Lowry’s micromethod (22) based on measuring the content of tryptophan and tyrosine residues in the protein using the Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate), comparing the measured absorbance of the sample with a standard curve for bovine albumin; protein levels were expressed in mg/mL. Statistica 12.0 (StatSoft, Poland) was used for statistical analysis, using the Kolmogorow-Smirnow test to assess normal distribution of variables, followed by Tukey’s test. A p-value ≤ 0.05 was considered statistically significant. The study was approved by the Bioethics Committee of the University (No. Nr KB-335/2013).
Significantly lower salivary flow was found in 5-6 year olds compared to those aged 13-14 and 18 years (fig. 1). Salivary pH was significantly higher in the youngest age group compared to older groups. A linear downward trend in mean pH levels was observed between the study groups (fig. 2). Total protein levels were significantly lower in 5-6 year olds, increased in 13-14 year olds, to reach peak values in 18-year-olds (significant difference between 5-6 year olds and 18-year-olds) (tab. 1). Furthermore, a linear upward trend in mean protein levels was found between the study groups (fig. 3).
Fig. 1. Age-related trend in salivary flow rate
Fig. 2. Age-related trend in salivary pH
Fig. 3. Age-related trend in salivary total protein
Tab. 1. Salivary parameters in different age groups of caries-free patients
|Parameter||5-6 years||13-14 years ||18 years ||Significance of differences – p-value|
|x ± SD||x ± SD||x ± SD|
|Salivary flow rate mL/min||0.20 ± 0.07a, b||0.27 ± 0.11a, c||0.27 ± 0.08b, c||a-ap = 0.0102*|
b-bp = 0.0055*
c-cp > 0.05 ns
|pH||7.78 ± 0.55a-c||7.13 ± 0.38a, c||6.95 ± 0.48b||a-ap = 0.0000*|
b-bp = 0.0000*
|Total protein mg/mL ||0.65 ± 0.17a, b||0.86 ± 0.28b, c||1.09 ± 0.48a, c||a-ap = 0.0002*|
*statistically significant; ns – not significant
The compared pairs are marked with the same letters.
For example: the difference between 5-6 years (a) and 13-14 years (a) statistically significant (*, p = 0.0102); difference between 5-6 years (b) and 18 years (b) statistically significant (*, p = 0.055); the difference between 13-14 years (c) and 18 years (c) not statistically significant (p> 0.05).
Considering the entire study group, it was found that unstimulated salivary flow rate and total protein significantly increased, whereas salivary pH decreased with age. Furthermore, pH and salivary proteins decreased with increasing unstimulated salivary flow (tab. 2).
Tab. 2. Correlation coefficients between the analysed salivary parameters and patient’s age
|Parameters||Age||Salivary flow rate mL/min||pH|
|Salivary flow rate mL/min||r = 0.338|
p = 0.001*
| || |
|pH||r = -0.576|
p = 0.0000*
|r = -0.373|
p = 0.0002*
|Total protein mg/mL||r = 0.475|
p = 0.0000*
|r = -0.440|
p = 0.0000*
|r = 0.137|
p = 0.196
*statistically significantly dependent correlation coefficient
Unstimulated salivary flow and salivary content of appropriate levels of biochemical components are important for oral health. Saliva has many important functions. Salivary mucins and proline-rich glycoproteins (PRGs) contribute to lubricating effects of saliva, ensuring protection (reducing the effects of mechanical, chemical and thermal mucosal damage – mucins), by wetting the mucous membrane and teeth as well as by the flow itself, which helps remove bacteria, products of their metabolism and residual food (23). High salivary content of water and mucins facilitates phonation, bolus formation, swallowing and chewing. The PRG-albumin complex is another lubricant covering the oral mucosa and reducing friction between food bolus and teeth (23).
A number of reactions involved in taste perception occur upon salivary effects on mucosal chemoreceptors in the oral cavity (24).
Saliva contains multiple enzymes, such as α-amylase, phosphatases and esterases. Alpha-amylase, which is synthesised mainly in the parotid glands, initiates digestion of extrinsic α-glycans, which are later converted to maltose (a disaccharide), which breaks down into glucose. The resulting monosaccharides are either metabolised by bacteria into acids or give rise to bacterial polysaccharides (25-27). Saliva helps maintain mucosal and periodontal integrity in the oral cavity (28), as well as contributes to mucosal wound healing (29, 30), remineralisation (31, 32) and maintaining oral pH (33).
Age-related changes in salivary composition may result from the physiological development of salivary glands. It has been postulated that although human salivary glands develop already in prenatal life, their further functional development continues in childhood and ends in adolescence (34). A number of studies indicate lower unstimulated mixed salivary flow in children compared to adults, as well as increasing salivary flow with age (35-38). Wu et al. (37) observed an increased unstimulated salivary flow in school children compared to preschool children. Our findings support this thesis for unstimulated saliva. We showed a significant increase in unstimulated mixed salivary flow rate between 5-6 year olds and 13-14 year olds and no further increase between 13-14 year olds and 18-year-olds. This may, to some extent, support the thesis presented by Crossner (34), who concluded, based on the assessment of stimulated saliva, that salivary glands reach full maturity at the age of 15 years. Furthermore, a positive co-variability of salivary flow rate and age was found for the entire study group. However, Tulunoglu et al. (39) fund no such a relationship among patients aged between 7 and 15 years, and neither did Forcella et al. (40) for 6 to 15 year-olds or Wu et al. (37) for 3 to 14 year-olds. This may be due to the technique for salivary collection, and thus the accuracy of measurements.
Our study showed a significant negative correlation between salivary pH and age as opposed to Piróg et al. (41) and Forcella et al. (40), who used Saliva Check Buffer for pH measurement. However, we found a positive correlation between age and protein levels, which corresponds to the findings presented by Wu et al. (37).
Hyyppä et al. (42) assessed total protein in unstimulated saliva in toothless children aged between 2 and 6 months (mean age 4.3 months) and in the same children at the age of 12 to 19 months (mean age 12.7 months) with a few erupted teeth and in adults aged between 21 and 31 years (mean age 23.3 years). The authors observed similar protein levels in children with no or a few teeth, which were significantly lower compared to adults. In their study in 3-14 year olds, without considering dental caries, Wu et al. (37) demonstrated the highest protein levels in mixed unstimulated saliva in 12-14 year olds, and the lowest levels in 3-5 year olds. Similarly, we observed the lowest protein levels in 5-6 year olds, and the highest in 18-year-olds. Furthermore, a positive correlation between age and protein levels was reported for the entire study group. We also found a positive co-variability for salivary flow rate and pH, which corresponds to the findings presented by Forcella et al. (40).
Unstimulated salivary flow rate and total protein levels increase, while unstimulated mixed salivary pH values decrease between 5 and 18 years of age.
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