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 Table of Contents  
Year : 2019  |  Volume : 11  |  Issue : 6  |  Page : 363-368

Effect of exposure time of an acidic beverage on the microhardness, mineral weight, and rate of calcium and phosphate ion release of human enamel

1 College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
2 Department of Biomedical Dental Sciences, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
3 Department of Quality Assurance, College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
4 Department of Chemistry, King Fahd University of Petroleum and Minerals, Dhahran, Saudi Arabia

Date of Web Publication26-Nov-2019

Correspondence Address:
Dr. Imran Farooq
Department of Biomedical Dental Sciences, College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam 31441.
Saudi Arabia
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jioh.jioh_147_19

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Aims and Objectives: The aim of this study was to investigate the effect of exposure times of Coca Cola on enamel’s microhardness, mineral weight, and rate of calcium and phosphate ions discharging from it. Materials and Methods: Thirty-two enamel blocks were randomly divided into four groups and exposed to 10mL of their respective solution daily for 7 days, with each group containing eight specimens: Group 1 (enamel blocks exposed to artificial saliva, control group), group 2 (enamel blocks exposed to Coca Cola for 5 minutes), group 3 (enamel blocks exposed to Coca Cola for 10 minutes), and group 4 (enamel blocks exposed to Coca Cola for 30 minutes). Microhardness data (Vickers hardness number [VHN]) and mineral weight were calculated at baseline (sound enamel) and postexposure to Coca Cola. Inductively coupled plasma–optical emission spectrometry (ICP-OES) was utilized to study calcium and phosphate ion percolation from enamel surfaces. Results: Decrease in VHN was directly proportional to exposure time for all experimental groups. Comparison of baseline and postexposure values between control and experimental groups was statistically significant (P < 0.05). Mineral weight of enamel blocks decreased as the exposure time increased for all experimental groups. ICP-OES analysis revealed linear relationship between release of calcium and phosphate ions at 5 and 10 minutes, but at 30 minutes, a decrease in concentration of both ions was observed. Conclusion: The microhardness and mineral weight of enamel decreased linearly with exposure time. The release of calcium and phosphate ions from enamel increased initially but gradually decreased as the exposure time increased.

Keywords: Dental enamel, erosion, ICP-OES, microhardness, mineral weight

How to cite this article:
AlAbdullah AA, AlAbdullah MA, Alkuhl MH, Alnashmi Fh, Farooq I, Siddiqui IA, Alhooshani K. Effect of exposure time of an acidic beverage on the microhardness, mineral weight, and rate of calcium and phosphate ion release of human enamel. J Int Oral Health 2019;11:363-8

How to cite this URL:
AlAbdullah AA, AlAbdullah MA, Alkuhl MH, Alnashmi Fh, Farooq I, Siddiqui IA, Alhooshani K. Effect of exposure time of an acidic beverage on the microhardness, mineral weight, and rate of calcium and phosphate ion release of human enamel. J Int Oral Health [serial online] 2019 [cited 2023 Apr 2];11:363-8. Available from:

  Introduction Top

The human enamel is the stiffest and most mineralized tissue in human body[1] containing calcium and phosphate minerals. The oral cavity suffers from cycles of low and high pH, causing demineralization and remineralization of teeth correspondingly.[2] A lower pH causing loss of minerals from enamel is called demineralization; whereas a higher pH reinstating these minerals back to enamel is known as remineralization.[3] Dental erosion is a common occurrence and is defined as the damage of tooth structure with a chemical, without the association of bacteria.[4] This damage to the tooth structure could range from loss of surface luster to irreversible exposure of dentin,[5] depending on severity and frequency of the chemical challenge. Erosion is still believed to be a significant dental health problem in many industrialized countries because of increased intake of fizzy drinks.[6] Several studies have conveyed that acids present in the food and soft drinks denote a chief etiological risk factor that is accountable for the erosion of enamel.[7]

Various factors in oral environment affect and contribute to the progress of dental erosion.[8] The pH value of liquid is a significant factor, and the dissolution and precipitation of tooth mineral depend directly on pH of exposed liquid.[8] The pH value is defined as being essentially related to the equilibrium amount of concentration of hydrogen ions.[4] Certain habits of individuals during the intake of erosive food are suggested as possible factors of erosion, for example, keeping an acidic liquid in mouth before swallowing, swirling it in the oral cavity, or sucking liquid directly from the teeth.[7] A result of all of these behaviors is an increased contact time of the beverage with teeth, resulting in erosive lesions.[7]

The time period for which a tooth surface is exposed to an acidic drink is a vital determinant of erosion.[8] Prolonged exposure time increases the probability of erosion to occur.[9] Soft drinks possess a low pH due to the availability of citric acid, maleic acid, and phosphoric acid in their composition.[10] The most common worldwide-consumed acidic drinks are cola based having a pH <4 that could dissolve mineral content of the human enamel quite rapidly.[10]

As the use of acidic drinks is on a rise in many countries, gaining information about their erosive potential as a function of time is important for dental and dietary counseling of patients.[11] Therefore, the purpose of this study was to investigate the effect of various exposure times of Coca Cola on the surface microhardness, mineral weight, and discharge of ions (calcium and phosphate) of enamel.

  Materials and Methods Top

The ethical approval (Ref: 2019007) was acquired from Scientific research unit, College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia and ethical protocols were precisely shadowed.

Preparation of enamel blocks

Thirty-two human extracted teeth were obtained from the oral surgery department of the College of Dentistry, Imam Abdulrahman Bin Faisal University. Teeth that were free from caries, any obvious white spot lesions, and restorations or other defects were involved in our study. The teeth were sectioned at cementoenamel junction utilizing diamond saw (Isomet 5000 Linear Precision Saw; Buehler Ltd, IL), and their roots were discarded to obtain enamel blocks. The anatomical crowns of all the teeth were then entrenched in self-cure acrylic resin in such a manner that buccal/labial surface of tooth enamel were exposed. The enamel surfaces to be treated were then polished with 600-grit wet silicon carbide paper. Enamel window of approximately 4 × 4mm was created on the exposed enamel surface by a nail varnish to ensure that all the analyses were performed only in that marked area.

Grouping of specimens and surface treatment with soft drink

A commercially available soft drink (Coca Cola, Saudi Arabia) was selected and used in this study. A new can of Coca Cola was used every day and the specimens were exposed to it daily for 7 days. The enamel blocks (n = 32) were randomly distributed into four groups (based on daily exposure time), with each group receiving eight specimens (n = 8). The grouping was as follows: group 1 (enamel blocks exposed to 10mL of artificial saliva [AS], control group), group 2 (enamel blocks treated with 10mL of Coca Cola for 5 minutes), group 3 (enamel blocks treated with 10mL of Coca Cola for 10 minutes), and group 4 (enamel blocks treated with 10mL of Coca Cola for 30 minutes).

AS preparation

After every exposure, the samples were cleaned with distilled water for 1 minute and then kept in AS. The AS was prepared by mixing NaCl (0.400g), KCl (0.400g), NaH2PO4·H2O (0.69g), CaCl2·H2O (0.795g), and Na2S·9H2O (0.005g) in 1000mL of deionized water, as suggested by Fusayama et al.[12] The pH of freshly prepared AS was 5.5, which was adjusted to a pH of 7 by adding aliquots of NaOH, as proposed by Farooq et al.[13]

Surface microhardness analysis

Before being exposed to Coca Cola, Vickers surface hardness was analyzed using a digital microhardness tester (FM-ARS 9000; Future-Tech Corp, Kawasaki, Japan) to obtain baseline hardness values. Three indents were made on the polished surface of each specimen using a Vickers diamond indenter under a 100-g load applied for 10 seconds. An average of three indents was used for analysis. After 1 week of exposure cycles, the specimens from each group were air-dried for a day and then were again tested to evaluate the changes in the surface microhardness values.

Mineral weight calculation

The weight of samples before and after being exposed to Coca Cola was also calculated. All the samples in one group were placed in a motorized analytical balance scale (SGM Lab Solutions, New Delhi, India) together, as a single enamel block consists of a very small weight that is almost undetectable on the weighing balance. The weight was calculated to the third decimal figure and three times for each group.

Inductively coupled plasma–optical emission spectrometry

The inductively coupled plasma–optical emission spectrometry (ICP-OES) was used to detect the concentration of calcium and phosphate ions leached in AS and acidic drink during exposure times. An inductively coupled plasma spectrometer (PlasmaQuant PQ 9000; Analytik Jena, Germany) was used in this study. The operating parameters of ICP-OES utilized in this study are summarized in [Table 1]. Although other ions such as Na, Mg, Zn, Sr, and Al were detected in analysis, their concentration was not noted as calcium and phosphate ions are main components of dental enamel. The AS in which specimens were stored after exposure cycles from each group was collected safely and analyzed at the end of 1 week. To attain accurate findings, initially pre-cleaned high-clarity polypropylene conical centrifuge tubes (Falcon, NY) with a total capacity of 15 mL were clearly labeled, and 10-mL liquid samples from each group were dispensed to the tubes with a pipette. To remove any potential matrix, 2 mL of nitric acid (TraceMetal Grade; Fisher Chemical, USA) was placed in each sample, and the samples were digested to 150°C for 45 minutes using a 12-position microwave digestion system (MARS 6 iWave; CEM, Matthews, NC). The specimens were then cooled to room temperature before further analysis.
Table 1: Operating procedures used for ICP-OES analysis

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A calibration curve was prepared from 0.05 to 1.0 mg/L with certified ICP-OES standards diluted in a solution of 2% nitric acid (TraceMetal Grade; Fisher Chemical). A total of five points including a calibration blank consisting of 2% nitric acid were prepared. Furthermore, two quality control standards were prepared (at a concentration of 0.5 and 0.7 mg/L) from a different lot of certified ICP-OES standards.

Statistical analysis

Data were analyzed statistically using Kruskal–Wallis test, which was used for the comparison of microhardness among four groups. Pairwise comparison using Wilcoxon Mann–Whitney U test was applied for comparison of baseline and postexposure values between control and experimental groups. Wilcoxon signed rank test was applied to evaluate significance of mean within control and experimental groups comparing baseline versus postexposure results. P value ≤0.05 was considered statistically significant difference of means.

  Results Top

The Vickers micro-indentation was performed to assess surface microhardness of enamel samples [Figure 1]. The results of our study demonstrate a decrease in surface microhardness values postexposure to an acidic beverage for all the groups except control group in which samples were kept in AS and were not exposed to acidic drink. The comparison of baseline and postexposure values between control and experimental groups demonstrated statistically significant differences (P < 0.05 [Table 2]). Descriptive statistics revealed more decrease in hardness values as a function of time (calculated by subtracting the mean of postexposure hardness values from the mean of pre-exposure hardness values [Table 2]).
Figure 1: Vickers micro-indentation performed to analyze the microhardness of the enamel surface

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Table 2: Microhardness values (VHN) for the four groups pre- and postexposure (each value calculated three times)

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Mineral weight calculation in our study revealed a decrease in weight of enamel blocks (except group 1), and intergroup comparisons were statistically significant (P < 0.05 [Table 3]). An increase in weight of enamel blocks of control group was observed, possibly because of remineralization of enamel surfaces by the contents of saliva [Table 3].
Table 3: Mean mineral weight of enamel blocks (in grams) for the four groups pre- and postexposure

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The ICP-OES results revealed a linear relationship between release of calcium and phosphate ions for groups 2 and 3 (at 5 and 10 minutes, respectively), but at 30 minutes in group 4, a decrease in the concentration of both ions was observed [Table 4]. All the intergroup and intragroup comparisons were statistically significant (P < 0.05).
Table 4: Concentration of calcium and phosphate ions (in mg/L) detected on ICP-OES analysis for groups as a function of time

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  Discussion Top

Our results show a linear relationship between exposure time of Coca Cola and decrease in surface microhardness and mineral weight loss of dental enamel. As pH of the acidic drink used in this study was 2 (checked using a pH meter [Precisa; model pH 900, Switzerland]), which is below the critical pH of 5.5, demineralization of enamel was anticipated. According to the manufacturers, Coca Cola contains phosphoric acid in its composition, which can cause superficial etching in enamel surface, creating a surface zone with a damage that could be irrepairable.[14]

The Vickers surface microhardness test was used in our study, and according to previous literature, it is a good indicator to detect early enamel erosive lesions.[3] Alhussain et al.[3] previously used Vickers microhardness test and demonstrated that when enamel comes in contact with an acid, there is decrease in its hardness values. Barac et al.[7] performed a similar study to ours and studied the erosive effect of various soft drinks on enamel by different soft drinks utilizing stylus profilometry. It was reported that erosions created by soft drinks were directly proportional to their contact time with enamel.[7] Suryana et al.[15] and Devlin et al.[16] treated tooth samples with Coca Cola and reported a decrease in their indentation hardness. Our study results have shown similar findings where loss of surface microhardness of enamel was directly proportional to their contact time with Coca Cola.

In this study, mineral weight calculation was also used. Zimmer et al.[17] in an earlier study also used precision balance to evaluate the weight of enamel specimens before and after erosive exposure and reported a weight loss of enamel samples postexposure to different acidic drinks including Coca Cola. This study yielded similar results as well. Von-Fraunhofer and Rogers[18] reported a weight loss of enamel sections that were soaked in sports drinks and other beverages for 14 days. Our study revealed similar results although in this study, enamel samples were treated with acidic beverage for 7 days. The reason for this weight loss in enamel samples is that when the tooth is subjected to an acidic attack, demineralization occurs by chemical dissolution of both organic and inorganic components, resulting in acid transmission in and minerals moving out of the tooth.[19] Acid attack can penetrate into deeper layers of enamel (~120-μm deep from outer surface of enamel),[20] causing mineral loss from the tissue.

Dental enamel is composed of hydroxyapatite crystals that contain calcium and phosphate minerals.[21] When the tooth enamel comes in contact with an acidic medium, the acid mixes with saliva and diffuses in teeth and causes calcium and phosphate ions to leach out.[22] Being the most inorganic tissue of human body, the enamel is more susceptible to acidic attacks.[19] The outer surface layer once decalcified erodes, leaving behind a softer layer that is more susceptible to abrasion, and if detached, leads to a duplicate erosion cycle.[19] In this study, we investigated the relationship between exposure time and percolation amount of calcium and phosphate ions. In our study, it was observed that lowest concentration of calcium and phosphate ions was present in AS group and for all the groups, concentration of these ions increased at 5 and 10 minutes but decreased at 30 minutes after being exposed to the beverage. The credible reason for this could be that enamel loses more minerals initially, but with passage of time, the quantity of remaining minerals in it decreases, leading to a decrease in ions leaching out in saliva. The average concentration of phosphate ion in resting saliva is accepted at 16.8mg/100mL.[23] In our study, it was observed at 21.43mg/100mL (214.30mg/L) at 5 minutes, 29.23mg/100mL (292.30mg/L) at 10 minutes, and 14.57mg/100mL (145.78mg/L) at 30 minutes. The higher concentration of phosphate ions beyond normal limits at 5 and 10 minutes after exposure further confirms the phenomenon of ion release from dental enamel. Hannig et al.[24] carried out a study on bovine enamel and studied the effect of exposure time of various acids on release of calcium and phosphate ions. It was reported in their study that after exposure of 1–5 minutes, a linear release of calcium and phosphate ions was observed.[24] This study results are in agreement with the study results of Hannig et al., and our samples also showed an increase of calcium and phosphate ion release during the first 10 minutes.

In the literature, various studies have used different exposure times to study erosive behavior of dental enamel. Barac et al.[7] exposed enamel specimens daily for 10 days to various acidic drinks (including Coca Cola) for a time period of 15, 30, and 60 minutes.). Torres et al.[8] exposed enamel samples to a beverage for 5 minutes three times a day (total 15min/day) in a study that lasted 60 days. It has been reported earlier that an exposure time of 60 minutes every day for 7 days equals the exposure time of acidic beverage in the mouth per year for most of the people.[25] According to this criteria, we chose a maximum exposure of 30 minutes for 7 days, which equals 6 months exposure time of an acidic drink in vivo.

Although the effect of exposure time of an acidic beverage on enamel has been studied before, to the best of our knowledge this is the first study to use three different investigations (surface microhardness, mineral weight calculation, and ICP-OES) to study this subject. There were certain limitations in our study. The first limitation was the in vitro nature of this study. Certain factors including tongue movement in oral cavity can alter hardness of dental enamel while an acidic drink is being consumed. Another limitation of our study was using only one commercial brand of acidic drink. Conversely, it was important to utilize one brand of commercial drink to standardize the experiments. Future in vivo studies with other brands of acidic drinks should be carried out to obtain more accurate findings. Studies with longer exposure times are also recommended to understand chemical dissolution behavior of dental enamel.

  Conclusion Top

It can be concluded that surface microhardness and mineral weight of enamel decreases linearly with exposure time. The release of calcium and phosphate ions from enamel increases initially but gradually decreases as the exposure time increases.


We are grateful to Deanship of Library Affairs, Imam Abdulrahman Bin Faisal University, for providing us access to the full text of articles included in this study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4]


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