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Year : 2019  |  Volume : 22  |  Issue : 5  |  Page : 707-712

Changes in essential salivary parameters in patients undergoing fixed orthodontic treatment: A longitudinal study

1 Department of Pediatric Dentistry and Orthodontic Sciences, College of Dentistry, King Khalid University, Abha, Kingdom of Saudi Arabia
2 Department of Diagnostic Sciences and Oral Biology, College of Dentistry, King Khalid University, Abha, Kingdom of Saudi Arabia
3 Department of Clinical Biochemistry, College of Medicine, King Khalid University, Abha, Kingdom of Saudi Arabia

Date of Acceptance12-Feb-2019
Date of Web Publication15-May-2019

Correspondence Address:
Dr. M S Hameed
Department of Diagnostic Sciences and Oral Biology, College of Dentistry, King Khalid University, Post Box 3263, Abha - 61471
Kingdom of Saudi Arabia
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/njcp.njcp_606_18

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Objective: Orthodontic treatment using fixed appliances is known to alter the oral environment and encourage plaque retention around orthodontic brackets and bands, resulting in enamel demineralization and gingival inflammation. This study aimed to evaluate the changes in essential salivary parameters in patients undergoing fixed orthodontic treatment. Materials and Methods: Saliva samples were collected from 60 patients before and 2 months after commencing fixed orthodontic treatment. The salivary flow rate, pH, buffering capacity, and levels of amylase, total protein, and glucose were determined. Parametric and nonparametric tests for paired samples were used for comparing the mean differences before and after commencing treatment. Results: Significant reductions in the salivary flow rate, pH, and buffering capacity were noted 2 months after commencing treatment (P < 0.05). Total protein concentrations and calcium levels decreased significantly and amylase and glucose levels increased after commencing treatment (P < 0.05). Significant correlations were observed between salivary total protein concentrations and buffering capacity as well as calcium levels (P < 0.05). Conclusion: These findings indicate that the biochemical properties of saliva are altered after introducing fixed orthodontic appliances into the oral cavity, thereby promoting plaque retention and increasing the susceptibility to tooth demineralization and gingival inflammation.

Keywords: Buffering capacity, demineralization, fixed appliance, plaque, saliva

How to cite this article:
Alshahrani I, Hameed M S, Syed S, Amanullah M, Togoo R A, Kaleem S. Changes in essential salivary parameters in patients undergoing fixed orthodontic treatment: A longitudinal study. Niger J Clin Pract 2019;22:707-12

How to cite this URL:
Alshahrani I, Hameed M S, Syed S, Amanullah M, Togoo R A, Kaleem S. Changes in essential salivary parameters in patients undergoing fixed orthodontic treatment: A longitudinal study. Niger J Clin Pract [serial online] 2019 [cited 2022 Jul 2];22:707-12. Available from:

   Introduction Top

Orthodontic treatment has gained increasing popularity owing to increased self-awareness of oral health-related quality of life and facial esthetics. However, treatments using fixed appliances may induce the formation of bacterial biofilms in healthy oral cavities, and these can compromise oral hygiene and lead to enamel demineralization and gingival inflammation.

Areas around metal brackets are difficult to clean and are prone to the adhesion of bacteria and debris, whereas in the case of orthodontic bands, biofilm formation occurs mostly at the gingival margin, leading to periodontal inflammation.[1],[2] Several studies have demonstrated the development of white spot lesions on the tooth surface following orthodontic treatment.[3] A recent study by Ren et al. (2014) showed that approximately 60% of all patients developed one or more biofilm-related complications due to orthodontic treatment.[4] Tooth brushing and the natural cleansing action of saliva in the oral cavity are generally insufficient for removing the biofilm. Furthermore, introducing foreign objects, such as orthodontic appliances, can alter the normal functioning of the oral cavity.

Saliva is produced and secreted by the salivary glands. It consists mainly of water (99%) and other organic and inorganic components that contribute to the major functions of the salivary glands.[5] In addition, saliva contains antibacterial, antiviral, and antifungal components that help maintain the normal oral flora. Saliva plays a vital role in maintaining oral health by performing several functions such as lubrication, antimicrobial activity, maintenance of homeostasis, and control of demineralization/remineralization of the teeth.[5] Qualitative and quantitative changes in salivary parameters may lead to serious complications and override the overall benefits of orthodontic treatments. Saliva quality is generally defined by its protein content, viscosity, pH, and buffering capacity, whereas the quantitative properties of saliva are related to its flow rate.[6],[7] Decreased salivary flow rates, acidic pH, and the presence of electrolytes such as fluorides and calcium in the oral cavity have been associated with cariogenicity.[1],[8],[9]

Salivary proteins are adsorbed onto the surface of the enamel and form a protective layer called the pellicle, which regulates the demineralization and remineralization of the enamel in conjunction with calcium and phosphate ions in saliva.[5]

Previous studies on changes in the quality and quantity of saliva following orthodontic treatment have yielded conflicting results.[7],[10] Therefore, this prospective study aimed to evaluate the flow rate, pH, and buffering capacity of saliva and to determine salivary amylase activity, total protein concentration, and calcium and glucose levels in patients undergoing fixed orthodontic treatment.

   Materials and Methods Top

The Institutional Review Board Committee at College of Dentistry, King Khalid University, Abha, Saudi Arabia approved this longitudinal prospective study (IRB approval number: SRC/ETH/2017-18/067), and the procedures followed were in accordance with the Declaration of Helsinki of 1975, as revised in 2000. Informed consent was obtained from all participants prior to enrollment.


Sixty patients (age range, 18–30 years; mean age, 21.7 years) scheduled to undergo fixed orthodontic treatment at King Khalid University, College of Dentistry Clinics, Abha, Saudi Arabia were included in this study. Patients with skeletal malocclusions, poor prognosis, known systemic diseases and undergoing treatment via medication, smokers, and those using dental prostheses were excluded. Angle's Class I malocclusion was found in 41 (68%) patients and Class II malocclusion in 19 (32%) patients.

Collection of saliva

Before collection of the first salivary sample and prior to start of orthodontic treatment, all the patients received full dental prophylaxis and restoration of active caries lesions. The saliva samples were collected in the morning (between 8 am and 11 am) to avoid the effects of the circadian rhythm. The patient was asked to sit on the dental chair in an upright position, and 2 mL of whole unstimulated saliva was collected into a graduated tube using the spitting method. Two samples of saliva were collected from each patient; the first sample was collected 1 week before commencing fixed orthodontic treatment and the second one 2 months after commencing treatment.

Salivary flow rate

The salivary flow rate was recorded in each patient until 2 mL of whole unstimulated saliva was collected.

Salivary pH and buffering capacity

Salivary pH was measured directly using a small handheld pH meter (dimensions, 165 × 29 × 19 mm; weight, 53 g; Horiba Ltd., Tokyo, Japan). Salivary buffering capacity was also quantitatively determined using the compact pH meter, immediately after the collection of the samples. In this method, 0.5 mL of saliva was added to 1.5 mL of hydrochloric acid (HCl; 5 mmol/L), and the mixture was vigorously shaken. Carbon dioxide was removed from the sample by passing a stream of nitrogen through the mixture for approximately 20 min. The final pH was measured after allowing the mixture to stand for 10 min. Salivary buffering capacities were divided into the following three categories as described in a previous study: high (above pH 5.5), medium (from pH 5.5–4.5), and low (below pH 4.5).[11]

After collecting the saliva sample, the container was sealed and labeled for identification, placed in a Styrofoam box containing ice, and sent to the Biochemistry Laboratory at the College of Medicine, King Khalid University, for storage (at −80°C) and further analysis.

Estimation of the biochemical constituents in the saliva samples

Sodium fluoride was added to an aliquot of the collected saliva samples in a separate tube and preserved at −80°C for estimating glucose levels. The samples were thawed to room temperature prior to estimation and centrifuged at 3500 rotations per minute (RPM) for 10 min. The clear supernatant was collected for glucose estimation. After estimating total proteins and salivary amylase, the collected saliva samples were centrifuged at 3500 RPM for 10 min to remove the proteins, and the clear supernatant was used for calcium estimation. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was performed for assessing glucose, total proteins, amylase, and calcium levels in the saliva samples. Commercially available ELISA kits for total proteins (product catalog No. 201-12-1150), glucose (product catalog No. 201-12-0751), calcium (product catalog No. 201-12-2123), and amylase (product catalog No. 201-12-9154) were procured from Shanghai Sunred Biological Technology Co., Ltd. (Shanghai, China). The levels of the constituents were estimated according to the manufacturer's instructions with some modifications, because the kits were primarily designed for serum samples wherein the concentrations of these constituents are generally high. Saliva contains relatively minute quantities of proteins, glucose, calcium, and amylase; hence, the standards were diluted to very low concentrations, whereas double the quantities of saliva and reagents were used to obtain favorable results.

Briefly, the standard protein in the stock solution provided with the kit (16 mg/L) was serially diluted using the “standard diluent solution” to prepare standard solutions of various concentrations (8, 4, 2, 1, 0.5, and 0.05 mg/mL). The concentrations of glucose (100 mg/dL) and calcium (10 mg/dL) in the stock solution provided in the kit were diluted to 80, 60, 40, 20, and 10 mg/dL and 8, 6, 4, 2, and 1 mg/dL, respectively.

The other reagents from each kit were prepared separately according to the manufacturer's instructions. Eighty microliters of the saliva samples was added to precoated wells in the ELISA plate followed by the addition of 20 μL of the respective biotin-labeled antibodies into each well. Finally, 100 μL of streptavidin–horseradish peroxidase was added to each of the wells to form the immune complex. The plate was sealed with a sealing membrane and incubated at 37°C for 60 min on a benchtop shaker. Uncombined enzyme was removed by washing the wells thrice with an automatic ELISA washer. Subsequently, 100 μL each of chromogen solutions “A” and “B” was added to each well, gently mixed, and incubated for another 10 min at 37°C, away from light. The reactions were stopped by adding 100 μL of the stop solution to each well. The color developed by each constituent was read using an automatic ELISA reader (Roche-Hitachi 917D, USA) at 450 nm. Optical density values were plotted against the standard concentration, and a linear curve was drawn from which the concentration of each constituent in saliva was derived.

Statistical analysis

All variables in the study were analyzed for normality using Kolmogorov–Smirnov test. Paired-samples t- test for dependent samples was used for variables with normal distribution, whereas nonparametric Wilcoxon signed-rank matched pair t-test was used for those that were not normally distributed. All statistical analyses were conducted using IBM SPSS Statistics for Windows/Macintosh, Version 20.0 (IBM Corp., Armonk, NY, USA). A P value of <0.05 was considered significant.

   Results Top

The results of the paired t-test for dependent samples using the variables pH, flow rate, amylase, and glucose are shown in [Table 1]. Significant differences in all four parameters were noted before and after commencing orthodontic treatment (P < 0.001). Salivary pH and flow rate decreased, whereas glucose and amylase levels significantly increased 2 months after commencing treatment. Salivary buffering capacity, total protein concentration, and calcium levels were compared before and after commencing treatment using Wilcoxon matched pairs t-test [Table 2]. Significant reductions in all three parameters were observed after commencing treatment (P < 0.001).
Table 1: Comparison of salivary pH, flow rate, and both glucose and amylase levels before and after treatment using the paired t-test

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Table 2: Comparison of salivary buffering capacity and total protein as well as calcium levels before and after treatment using Wilcoxon matched pair t-test

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Total salivary protein concentration significantly correlated with the buffering capacity after commencing orthodontic treatment (r = 0.34; P < 0.05) [Table 3]. Moreover, significant correlations (P < 0.05) were observed between salivary calcium and total protein levels, as well as salivary amylase and glucose levels.
Table 3: Correlations among the salivary parameters after commencement of orthodontic treatment using Spearman's correlation coefficient method

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The percentage distribution of patients in the three buffering capacity groups before and after commencing treatment is shown in [Figure 1]. The results of Wilcoxon matched pair t test indicated an increase in the number of patients with low and medium buffering capacity after commencing treatment; however, a significant decrease was noted in the percentage of patients in the high buffering category (P = 0.002).
Figure 1: Histogram showing differences in salivary buffering capacity before and after commencement of orthodontic treatment. The percentages of patients with low and medium buffering capacity were increased after treatment commencement; However, a significant decrease in the percentage of patients with high buffering capacity was noted after the start of treatment (P = 0.002)

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   Discussion Top

Saliva is involved in myriad functions, such as mechanical cleansing, demineralization and remineralization of the enamel, protection against oral microbial flora, and buffering of acids in the oral cavity. Maintenance of oral hygiene is difficult in individuals with fixed orthodontic appliances, and this leads to plaque accumulation, gingival inflammation, dental caries, and other periodontal conditions. White spot lesions usually develop within a month of starting fixed orthodontic treatment.[12] Previous studies have reported changes in oral microbial counts and in the properties of saliva following fixed orthodontic treatment.[10],[13],[14] However, the results of these studies are conflicting and inconsistent. In this study, we evaluated salivary flow rate, pH, buffering capacity, amylase activity, total protein concentration, and calcium and glucose levels in patients before and 2 months after commencing orthodontic treatment.

Salivary flow rate plays a vital role in oral health; an increased flow rate increases the cleansing action and antimicrobial activities of saliva, whereas a decreased flow rate promotes plaque retention, demineralization, and caries formation.[13],[14] Several studies have reported increased flow rates in patients with fixed orthodontic appliances,[7],[13] which may be attributed to the increase in mechanosensation. Alternatively, the absence of any changes in flow rates after commencing orthodontic treatment has also been documented.[15] In this study, significant reductions in salivary flow rates were noted 2 months after commencing treatment, which may be due to the acclimatization of the patients to the appliance as reported previously.[16] This finding is in accordance with that of an in vitro study, wherein a computational fluid dynamic model was used to characterize and quantify the salivary flow pattern around fixed appliances.[17] A decrease in the velocity of salivary flow around brackets was observed, indicating that orthodontic appliances may hinder the flow of saliva. A recent study by Goje et al. (2107) also reported significant reductions in salivary flow rates 45 days after beginning orthodontic treatment.[18] Similarly, a significant reduction in unstimulated salivary flow was observed in 25 subjects after the placement of an orthodontic appliance.[8]

Minerals such as calcium and phosphate are essential for the remineralization of teeth with initial caries. Decrease in calcium levels renders the oral environment conducive for the development of dental caries.[19] In line with the findings of previous studies, this study showed that calcium levels were significantly decreased in patients after orthodontic treatment.[15],[20] The concentrations of total proteins, sodium, calcium, and bicarbonate are reported to increase with the increase in salivary flow.[5] Correlations between salivary calcium levels and flow rates have yielded conflicting results.[10],[15],[21],[22] Low calcium levels in saliva reflect a low pH, predisposing the enamel to demineralization.[23] Decreased salivary pH in patients with fixed orthodontic appliances has been reported previously.[10],[15],[16] In this study, both salivary pH and calcium levels were significantly decreased 2 months after commencing treatment. However, no significant correlations were noted between salivary calcium levels and flow rate or pH.

Saliva contains a variety of proteins that perform various specific biological functions. Components such as bicarbonates, phosphates, and several proteins contribute toward the buffering capacity of saliva,[24] thus aiding in the neutralization of acidic products and restoration of normal pH balance in the oral cavity.[25] The bicarbonate, phosphate, and protein systems have been associated with salivary flow rate; moreover, a decrease in unstimulated salivary flow has been attributed to a reduction in the buffering capacity of saliva.[26] In this study, salivary flow rate, pH, buffering capacity, and total protein concentrations were significantly lower in the patients 2 months after commencing treatment. These findings are consistent with those of previous studies, wherein high flow rates of unstimulated and stimulated saliva indicated the presence of a high bicarbonate concentration, more alkaline pH, and high buffering capacity.[27] The higher the salivary flow rate, the higher the buffering capacity and clearance within the oral cavity, resulting in better antimicrobial protection.[5] Total protein concentration in saliva was significantly correlated with the buffering capacity (r = 0.3427; P < 0.05; [Table 3]) in this study. Interestingly, similar findings have been reported previously in saliva at an acidic pH.[27] Similarly, salivary calcium levels were also found to be significantly correlated with the total protein levels in this study. The proteins involved in enamel pellicle formation, which include the proline-rich proteins and statherin, are known to attract calcium ions and facilitate enamel remineralization. Thus, decreased levels of total protein and calcium may promote the demineralization of the enamel.

Glucose and amylase levels were significantly increased 2 months after commencing treatment in this study (P < 0.001), thus corroborating the results of previous studies.[10],[18] Amylase binds to certain oral microorganisms and plays an important role in the formation of dental plaque, resulting in the development of caries.[28] Moreover, it is thought to contribute to the hydrolysis of dietary starch, leading to the production of additional glucose in the oral cavity. Higher levels of glucose have been reported in subjects using orthodontic appliances.[10],[29]

The effects of variables such as age and gender were diminished owing to the prospective nature of this study wherein the same subjects before commencement of treatment were used as controls. Based on previous reports, gender does not appear to influence the salivary parameters after orthodontic treatment; in one study, significant differences in stimulated salivary flow and salivary buffering capacity after placement of orthodontic appliances were noted only during the initial stages of treatment.[16] Similarly, no significant differences in salivary parameters have been observed between males and females after placement of fixed orthodontic appliances.[7],[30] In a recent study evaluating age-related changes in salivary parameters, significant decreases in salivary flow rate and calcium levels were noted in the elderly group (age, 60–80 years) when compared with the young group (age, 20–30 years); alternatively, no significant differences in salivary pH, buffering capacity, or amylase levels were reported.[31]

The results of this study are limited by the small sample size and short observation period. Nonetheless, it is clear that the physical properties of saliva are altered after commencing fixed orthodontic treatment, favoring plaque retention and increasing the susceptibility to tooth demineralization and gingival inflammation. Further long-term studies using larger sample sizes are warranted to clearly evaluate and understand the changes that occur within the oral cavity after the placement of fixed orthodontic appliances.

   Conclusion Top

In this study, significant changes in the salivary flow rate, pH, buffering capacity, and total protein concentration as well as amylase, calcium, and glucose levels were observed before and after commencing treatment, indicating that the introduction of orthodontic appliances altered the properties of saliva in the oral cavity. Hence, patients undergoing fixed orthodontic treatment must adopt additional measures to maintain oral hygiene and reduce their susceptibility to developing caries and other periodontal conditions.

Financial support and sponsorship

The authors extend their appreciation to the Maxillofacial Center at College of Dentistry, King Khalid University for funding this work under grant number MRMC 01-017-004.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1]

  [Table 1], [Table 2], [Table 3]


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