Comparison of the Erosive Potential of Commercially Available Beverages on Human Enamel: An in vitro Study

Objective: To assess and compare the erosive potential of commercially available beverages and to evaluate the relationship between mineral loss and physiochemical properties (pH and titratable acidity) of different beverages. Material and Methods: Seven commercially available beverages [4 soft drinks, 1 chlorhexidine mouthwash and 2 controls (Amul milk and human saliva)] were tested. The pH and tritratable acidity of the beverages was determined. Fifty-six human premolars were sectioned into enamel and root surface (total 112 specimens) by using diamond-cutting machine. The specimens were immersed in each beverage for 24-hour intervals at 370C. The weight loss of the specimens and mineral loss (calcium, phosphorus and fluoride) was determined at pre and post immersion and was subjected to statistical analysis at p<0.05 level of significance. Results: All beverages included in this were acidic. Nimbooz exhibited significant (p<0.05) weight loss compared to all other beverages. Fanta shows significantly (p<0.05) high loss of phosphorus and calcium for enamel and root surface respectively. There was a week correlation with pH and tritratable acidity and loss of minerals. Conclusion: The erosive potential of Fanta and Nimbooz was significant compared to other beverages. However, it must be considered that there are numerous factors, which may influence the erosion rate in the intra oral environment; hence the result of this in vitro study should be applied with caution.


Introduction
Dental erosion is an irreversible loss of tooth structure due to dissolution of the calcified dental tissues by chemicals without the involvement of microorganisms [1,2].Apart from dental caries and periodontal diseases, dental erosion is now considered as the growing oral health problem affecting both children and adults [3,4].This is due to the change in the eating habits, mostly the gaining popularity and increase consumption of acidic beverages over recent years.The severity of erosion is related to several factors, including the chemical properties of the erosive medium, and the frequency and method of contact between acid and tooth.However, the protective mechanisms in the oral cavity like salivary composition, flow rate and buffering capacity, pellicle formation, clearance rates and tooth morphology also influence the rate of erosion [5].Depending upon the severity and length of exposure, the dental erosion varies from reduction in teeth size to total destruction of the dentition.
The etiological factors for tooth erosion are broadly classified into intrinsic and extrinsic categories.Intrinsic erosion occurs from involuntary gastrointestinal disturbances such as gastroesophageal reflux disease while the extrinsic tooth erosion causes include environmental factors, medicaments, lifestyle and diet [2,6].In today's era the extrinsic factor is a subject of interest, due to the increased consumption of acidic drinks as soft drinks, sport drinks and fruit juices [7].The extrinsic etiology of diet has been extensively investigated, but the actual evidence linking a particular acidic food or beverage as the primary agent is limited.The erosive potential of foods and beverages are significantly associated with pH, titratable acidity (TA, i.e. quantity of base required to bring a solution to neutral pH), type of acid, buffer capacity, chelating properties, and concentration of calcium, phosphates and fluoride [8].However, it is now accepted that both pH and TA are indicators of the erosive potential of a foods or beverages [5,9].The pH value corresponds to the equilibrium measure of the hydrogen ion concentration, but it gives no indication of the overall acidic content of the beverage or food [10]; whereas TA gives a measure of all free hydrogen ions available to cause erosion [11].Both the pH and TA can be utilized to analyze the acidic property of foods and beverages by a simple method.Therefore, several previous studies utilized pH and TA measurements to characterize [9,[12][13][14][15].Knowledge of the erosion potential of beverages is important for clinical guidelines regarding beverage consumption practices and development of potentially "safer" beverages.
Through extensive review of literature, it was revealed that the data for the erosive potential of some popular beverages sold in India are scanty.Hence, the aim of this in vitro study was to assess and compare the erosive potential of commercially available beverages.The objectives of this study were to estimate the pH and TA of beverages, to estimate the rates of enamel and root surface dissolution among different beverages and also to derive a regression equation showing the relationship between mineral loss from tooth surface and physiochemical properties (pH and TA) of different beverages.
The study results may provide some insights into a clinically practical and relevant approach to predict the erosive potential of beverages.

Material and Methods
For this experiment 7 commercially available beverages [4 soft drinks (Thums Up, Nimbooz, Red Bull, Fanta), 1 chlorhexidine mouthwash and 2 controls (Amul milk and human saliva)] were tested.The detail of each beverage is shown in Table 1.The beverages were randomly taken from local retail shops.Human saliva was collected from healthy volunteers in the morning, 2 hrs after fasting.The experiment was carried out at department of Chemistry, Hemchandracharya North Gujarat University, Patan, Gujarat, India.

pH and Titratable acidity (TA) Measurement
The pH value of the beverages was determined using pH meter EQ-621 (Equiptronics Instruments, Mumbai, India) connected to an electrode calibrated with standard buffering solutions of pH 4.0 and 7.0.A total of 3 readings were taken of each sample to give a mean measurement of the pH of that beverage.The TA value of the beverages was measured by adding 1M NaOH to 50 ml of each beverage until a pH of 7 was obtained.Titrations were repeated 3 times for each drink and an average value was calculated.

Preparation of Tooth Specimens
A total of 56 human premolars that have been extracted for orthodontic reasons from patients (aged 14-18 years) at Siddhpur Dental College and Hospital were collected.Those premolars having dental caries, fluorosis and any other developmental defects were excluded from the study.Immediately after the extraction, the soft tissue was removed; the teeth were washed by using normal saline and afterwards disinfected by storage in a 5% sodium hypochlorite solution for 24 hrs.Each tooth was cut into crown and root portion by using diamond cutting machine (Isomet 1000; Buehler Ltd., Lake Bluff, IL, USA) under water irrigation.Hence, 112 samples were obtained from 56 teeth.The specimens were ground flat and polished with pumice paste, rinsed with water and dried compressed air.The centers of the polished surfaces were isolated to produce 4 mm x 4 mm flattened window on the tooth specimen with water-resistant band.The residue polished surfaces were covered with nail varnish.After nail varnish dried, the band was removed from the polished tooth surfaces.
Two specimens (each for enamel and root surface) were randomly assigned to a representative beverage from each category including soft drinks, Chlorhexidine, milk and human saliva.Beakers were filled with 200 ml of beverages and the specimens were suspended in the beverages for a total of 24 hrs at room temperature 37 0 C.

Erosive Assessment
The erosive potential of beverages was assessed by measuring the pre and post-immersion weight of the specimen, and by estimating the pre and post-immersion mineral loss (calcium, phosphorus and fluoride) in the beverages.
Initial weights of all tooth specimens were performed prior to beverage immersion.Calcium concentration was measured using Eriochrome Black T as indicator (complexometric titration method) [16] and phosphate concentration was measured using a modification of the Chen method [17] using UV-1800 UV-VIS Spectrophotometer (Shimadzu Scientific Instruments, Kyoto, Japan).
Fluoride content of the beverages was assessed using a selective ion exchange electrode (Orion Model 96-09, Orion Research Inc., Cambridge, MA, USA) combined with ionic strength adjustment and pH, using a 0.5 mol/L citrate buffer, with pH 5.50, ratio 1:1 sample/buffer.The measurements were performed in duplicate.

Statistical Analysis
Descriptive statistics were calculated and physiochemical properties are reported as means and standard deviations.One-way analysis of variance (ANOVA) with post-hoc Tukey's HSD test was performed to compare the physiochemical properties of different beverages.Paired t tests was performed to check the statistically significant differences for erosive potential (pre and post immersion-after 24 hrs mineral loss in the beverages) for each beverages.Relationships between erosive potential and physiochemical properties were evaluate using multiple regression analysis.
Statistical Package for the Social Science (SPSS), version 17 (SPSS Inc., Chicago, IL, USA) was used to conduct analysis.The level of significance was p<0.05.

Results
Table 2 shows the physiochemical properties (pH and TA) of different beverages.All beverages were acidic.The pH value ranges from 2.56 ± 0.07 for Thums Up to 6.77 ± 0.03 for Amul Gold Milk.The quantity of base (1M NaOH) required to neutralize the beverages ranges from 0 mmol/lit for saliva to 126.00 ± 2.00 mmol/lit for Fanta.There was statistically highly significant difference (p<0.001) when the mean value of pH and TA for all the beverages was compared.Tables 3 and 4 shows the erosive potential of different beverages for enamel and root surface respectively.The erosive potential was estimated in terms of weight loss, loss of minerals (calcium, phosphorus and fluoride) from enamel and root surface.The mean values of pre-immersion and postimmersion (after 24 hrs) were compared.Specimens (both enamel and root) immersed in Nimbooz exhibited significant (p<0.05)weight loss compared to all other beverages.The specimens immersed in Fanta show significantly (p<0.05)high loss of phosphorus and calcium for enamel and root surface respectively.Table 5 shows a multiple regression analysis between pH and TA of beverages as independent variables and the loss of weight and minerals from enamel and root surface as dependent variable.There was a weak correlation with pH and TA in enamel (R 2 ranges from 0.17-0.38)or root (R 2 ranges from 0.15-0.31)surfaces in terms of weight, calcium and phosphorus loss.
However, while considering the loss of fluoride there was moderate correlation with pH and TA in enamel (R 2 =0.56) or root (R 2 =0.66) surfaces.

Discussion
In recent years there has been an enormous increase in the beverages consumption, and this is no longer confined to children but is being carried forward into adult life [10,11] hence, in vitro investigation was done to assess the erosive potential of 4 commercially available beverages, chlorhexidine mouthwash, milk and human saliva (negative control) on enamel and root surface.The beverages included in the present study contain mainly either citric acid or phosphoric acid.
Literature shows a mixed result regarding the erosive potential of beverages containing citric acid or phosphoric acid [18] Chlorhexidine mouth wash was included as it is commonly used mouth wash and it is acid based.Milk was included because it has calcium, phosphorus and protein in its composition.Human saliva was included as negative control as it has no erosive substances in its composition.
This study was performed at 37 0 C to represent the temperature in the mouth; however the temperature of beverages may be lower than 37 0 C when it is consumed.Therefore the erosion may be less than that demonstrated in this study, because a decrease in the temperature of drinks would result in less erosion because the solubility of solutes depends on temperature and heat is required to break the bonds holding the molecules in the solid together [19].
The erosive potential of beverages was different this might be due to the fact that different beverages differs in its composition; such as type of acid, pH and amount of titratable acidity.The result of this study revealed that there was no significant loss of weight, calcium and phosphorus (for both enamel and root surface) for Thums Up as compared to Nimbooz and Fanta.This is due to the fact that Thums Up contains phosphoric acid, while the other 2 beverages contained citric acid.This result was in accordance with the previous studies [18,20].The result of previous studies stated that citric acid demonstrate severe demineralizing potential [21,22].The erosive potential of citric acid is pronounced because of the fact that citric acid acts as a chelator able to bind minerals of the apatite, such as calcium [20].
All beverages included in the present study were acidic having wide range of pH and TA.
This result was in accordance with previous studies [9,23,24].Beside pH and TA the rate of erosion is a function of certain mineral contents in the beverages such as calcium, phosphate and fluorides  [25,26].There are conflicting results regarding predictor of erosion in terms of calcium and phosphorus.A previou study showed that phosphorous was a better predictor of enamel demineralization than calcium [22], while opposite results were verified by other authors [27].
Because of the differences in the types of beverages analyzed in previous studies [22,26,27] it is difficult to conclude which minerals in beverages are good variables for predicting the prevention of enamel erosion.Nevertheless, many previous studies [9,[22][23][24] support pH and TA being the most relevant parameters to indicate the erosive potential of beverages.The result of regression analysis erosive potential in this study demonstrated the coefficient of pH was high as compared to coefficient of TA.Hence, it can be concluded that pH was a more important indicator than TA.
The limitations of the present study are as follows: Firstly, the study was design in vitro so it does not completely reflect the actual intra-oral situation.Erosion in vivo is considerably less rapid than in vitro owing to the buffering and remineralizing effect of the saliva [23] and acquired pellicle [28].Besides the anatomic shape and composition of the dental tissues, the frequency and duration of the beverage intake as well as the other dietary habits plays crucial role; therefore, the considerable individual properties have to be taken into account [10].In addition, polished enamel such as used in this study is eroded more rapidly than natural enamel surfaces [23].Hence, the result shows exacerbated erosion potential of beverages.Secondly, the exposure time was limited and third limitation was only 6 beverages were studied and it is possible that other specific beverages could have markedly different erosive effects than those tested.
In order to simulate the oral cavity conditions, future in vivo studies are recommended.In addition, the exposure time was limited; this clearly needs to carry out further studies with long term exposure times in order to obtain more accurate results as it is an important consideration for nutritionists, dentists and physicians counseling patients.Following recommendations can be made for diet modification to reduce the risk of developing dental erosion: Limit acid foods and drinks to mealtimes; reduce frequency of consumption of these drinks; avoid acidic food/beverages last thing at night; finish meals with something alkaline such as a small piece of cheese or milk will neutralize intra-oral acid and avoid tooth brushing after acidic beverages.

Conclusion
The erosive potential of Fanta and Nimbooz was significant compared to other beverages.
Week correlation between pH and TA suggests that erosive potential is determined by the composition rather than by beverage pH and TA.The citric acid plays a significant role in the erosion of tooth surface as compared to phosphoric acid.Paying closer attention to determinants of erosive attack is expected to minimize the harmful effects of beverages through proper counseling and interceptive procedures to the patients.

Table 1 . Composition of different beverages used in the study.
Amul Gold Milk 6% Fat, carbohydrate, protein, calcium, phosphorus, sodium, vitamins A and B complex, folic acid.Amul Dairy Saliva Water, Electrolyte, Enzymes, mucous.

Table 2 . Physiochemical properties (pH and Titratable acidity) of different beverages.
Data were presented as mean ± standard deviation and the significance difference was assessed by using one way ANOVA; **p<0.001highly significant difference.Superscript numbers are significantly different p<0.05;TA= Titratable acidity.

Table 4 . Weight and concentration of Calcium, Phosphorus and Fluoride for root surface immersed in different beverages.
Data were presented as mean ± standard deviation and the significance difference was assessed by using paired t test; *p<0.05significant difference.Post immersion is after 24 hrs.