Erosive effects of different acids on bovine enamel:

Introduction

Dental erosion is defined as irreversible loss of

Archives of Oral Biology (2005) 50, 541552

eight enamel specimens. Amount of titratable acid was determined for all acidic

(HCl, pH 3). LAwas one of the most erosive acids. AAwas very erosive at pH 3. HCl andMAwere shown to have the lowest erosive effects. There was only a weak correlation

pH level, erosive effects mainly depend on pH and type of acid but not on amount ofdental hard tissue due to chemical processes with-out involvement of microorganisms.1 Acids areassumed to be the main etiological factor.2 Erosive

* Corresponding author. Tel.: +49 551 39 2898;fax: +49 551 39 2037.

E-mail address: Christian.hannig@med.uni-goettingen.de(C. Hannig).

00039969/$ see front matter # 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.archoralbio.2004.11.002titratable acid.# 2004 Elsevier Ltd. All rights reserved.(r = 0.28) between P and Ca release and the amount of titratable acid.Themethod of the present study allows investigation of minimal erosive effects via

direct determination of P and Ca dissolution. During short time exposition at constantsolutions.MA, LA, TA, AA and HCl caused linear release of Ca and P, PA of Ca, CA of P. For CA,

MA, LA, TA, AA, PA and HCl mineral loss was shown to be pH-dependent. Ca dissolutionvaried between 28.6 4.4 (LA, pH 2) and 2.4 0.7 nmol mm2 min1 (HCl, pH 3), Pdissolution ranged between 17.2 2.6 (LA, pH 2) and 1.4 0.4 nmol mm2 min1release of calcium and phosphate in vitro

Christian Hannig*, Arne Hamkens, Klaus Becker,Rengin Attin, Thomas Attin

Department of Operative Dentistry, Preventive Dentistry and Periodontology,University of Gottingen, Robert-Koch-Str. 40, D-37075 Gottingen, Germany

Accepted 2 November 2004

KEYWORDSErosion;Calcium;Phosphate;Demineralisation

Summary The present study intended to investigate minimal erosive effects ofdifferent acids on enamel during short time incubation via determination of calciumand phosphate dissolution. Bovine enamel specimens were eroded for 15 min witheight different acids of pH 2, 2.3 and 3 (citric (CA), maleic (MA), lactic (LA), tartaric(TA), phosphoric (PA), oxalic (OA), acetic (AA) and hydrochloric acid (HCl)). Calcium(Ca) and phosphate (P) release were determined photometrically using arsenazo III(calcium) and malachite green (phosphate) as substrates. Each subgroup containedwww.intl.elsevierhealth.com/journals/arob

mens were stored in artificial saliva for 7 days for24

542 C. Hannig et al.tooth destruction is classified to be intrinsic orextrinsic according to the source of the acids.3

Main intrinsic factor inducing dental erosions isgastric juice which comes in contact with the denti-tion especially in patients suffering from anorexianervosa, bulimia or chronic regurgitations.4,5 Ero-sive potential of gastric juice is mainly determinedby hydrochloric acid: gastric juice has been shown tohave a significantly higher erosive potential com-pared to carbonated acidic drinks.6

An external factor creating dental erosions isrepresented by acidic fumes in chemical industry,for example, in production of batteries.7,8 Also,some orally applied medicaments, such as acetylicsalicylic acid, may cause erosions.8 However, inmodern western societies diet can be regarded asmost important factor for erosions.3,9,10 Increasingconsumption of acidic beverages, such as sportdrinks or lemonades, is one main reason.11 Alsofruits, vegetables and especially juices which areregarded as healthy food, may cause significanterosive effects.2,3 Thereby, citric acid representsthe main acid in fruits and vegetables.10 Vinegar andvinegar conserves of vegetables contain acetic acid.Oxalic acid occurs mainly in rhubarb, tartaric acid inwine and grapes, maleic acid in apples and appledrinks.12,13 Phosphoric acid is used in numerous coladrinks.13 Lactic acid which is usually associated withcaries is present in sauerkraut or yoghurt as meta-bolic product of lactobacilli. Organic acids, in par-ticular citric, maleic and tartatic acid, are deemedto be extremely erosive to dental hard tissues due tothe ability of some of them to form chelate com-plexes with calcium released from the tooth.14

Many in vitro investigations were performed toelucidate the erosive capabilities of different foodsor beverages which represent usually a mixture ofdifferent acids. Typical methodical approaches areelectron microscopy, profilometric evaluation ofsurface loss or determination of microhardness.1520 Other studies already investigated erosive poten-tial of acidic solutions measuring calcium and phos-phate dissolution.21,22 This was done with variousdrinks but not with pure solutions of certain acids. Inthese studies, there was no clear relationshipbetween acidityeither described by pH or thetitratable amount of acidand demineralization.This may be explained by other minerals or mole-cules present in the drinks altering the erosivepotential. As yet, the erosive potential of puresolutions of different acids at constant pH valuestypically occurring in acidic drinks has not beencharacterized systematically. This is especially truefor short time incubation. Typical incubation timesin the existing studies range between 10 and

120 min, but usually incubation for 30 or 120 minstandardized remineralization. Five hundredmilli-litres of artificial saliva contained 0.001 g ascorbate,0.015 g glucose, 0.290 g NaCl, 0.085 g CaCl2, 0.080 gNH4Cl, 0.635 g KCl, 0.080 g NaSCn, 0.165 g KH2PO4,0.100 g carbamide and 0.170 g Na2PO4.

Eight enamel specimens were included in eachsubgroup.

Acids

Enamel specimens were incubated in the acidic solu-tions of acetic, citric, maleic, tartaric, phosphoric,oxalic, hydrochloric and lactic acid (Merck, Darm-stadt, Germany) at pH 2, 2.3 and 3, respectively.Concentrations of the certain acids at the three pHlevels are given in Table 1. pH was checked with a pHis quite common.13,1520 These time periods aremuch longer than the contact of the teeth withan erosive substrate during consumption of erosivefood or beverages.14,23 Furthermore, erosive effectswere often evaluated without any standardizationof pH of the test solutions.13,1520

Therefore, the present study aimed to quantifydemineralization of enamel samples via highly sen-sitive determination of calcium and phosphate dis-solution caused by certain acids after short timeerosion for 15 min. This was done systematicallyfor different solutions of pure acids typically occur-ring in foods and drinks at constant pH levels toprovide standardized basic parameters for directcomparison of the erosive effects.

Materials and methods

Specimens

Cylindrical enamel specimens (diameter 5 mm)were gained from bovine incisors of 2-year-old cat-tle. Specimens were etched laterally and at thebottom for 30 s with 37% phosphoric acid gel (Etch-ing gel, DMG, Hamburg, Germany) and treated for30 s with primer (OptiBond FL, bottle 1, Kerr, Karls-ruhe, Germany). Bonding of OptiBond (bottle 2) wasapplied afterwards and light cured for 30 s. Applica-tion of bonding was performed three times. In thefollowing, unsealed enamel surfaces were groundflat and polished (grit 4000). In a standardizedgrinding procedure, ca. 200 mm of enamel wasremoved. This was controlled with a micrometermeasurement device (HHW, Hommel, Schwennin-gen, Germany). Specimens with structural altera-tions of enamel were eliminated.

Before incubation with the different acids, speci-meter (RadiometerA/S, Copenhagen, Denmark).

ratio which was found for all other acids. Oxalic acid

Erosive effects of different acids on bovine enamel 543Incubation of specimens with the differentacids

Enamel samples were incubated for 5 min in 1000 mlacid (pH 2 and 2.3) and in 5000 ml (pH 3), respec-tively. Due to the lower acid concentration at pH 3,the higher incubation volume was chosen at this pHlevel to avoid consumption of acid. Stability of pHunder the chosen conditions was verified in preli-minary trials. During incubation and before deter-mination of calcium and phosphate, samples wereshaken gently.

Photometric determination of calcium andphosphate release

Mineral dissolution caused by the different acids wasdetermined by assessing calcium and phosphaterelease into the solutions photometrically in doubleassays.

Calcium (Ca) assayRelease of calcium was measured for specimensincubated in acetic, maleic, tartaric, phosphoric,hydrochloric and lactic acid using the arsenazo III

Table 1 Concentrations of the acids at the differnt pHlevels

Concentrations of acids[mmol/l] at

pH 2.0 pH 2.3 pH 3,0

Hydrochloric acid 10.5 4.50 0.75Acetic acid 2214 721 34.9Lactic acid 292 71.5 3.60Maleic acid 15.7 5.67 0.86Tartaric acid 50.0 18.4 1.32Phosphoric acid 16.2 5.90 0.88Citric acid 95.2 24.2 1.87Oxalic acid 8.84 4.46 0.75method (Fluitest 1, Ca-A-II, analyticon, Lichten-fels, Germany).

Arsenazo III reacts with calcium in an acid solu-tion to form a blue purple complex. Intensity devel-oped is proportional to the calcium concentration.Absorption can be determined at l = 650 nm.25,26

Reagent for determination of calciumwas composedof 100 mmol/l Imidazol buffer (pH 6.5) and0.12 mmol/l arsenazo III. Determination of calcium,dissolved by oxalic acid and citric acid, was notpossible, since the method did not allow reliablecalcium determination in these acids.

Phosphate (P) assayErosive dissolution of phosphate was tested with allacids except for phosphoric acid as this acid releasesphosphate by itself and thereby interferes with thedetermination of phosphate release from enamel.Malachite green reacts with phosphate to a colouredcomplex which can be determined photometricallyat l = 650 nm. 0.045 mg of malachite green solutedin 100 ml aqua bidest. were admixed to 12.69 gammonium molybdate dissolved in 300 ml HCl(4 mol/l). The reagent was stirred for 30 min after-wards and filtered (pore size 0.22 mm). The reagentswere stored at 4 8C.

For determination of Ca and P loss, individualstandard curves were obtained for each pH of allacids with standardized Ca and P solutions. Dilutedsamples of standardized calcium and phosphatesolutions were admixed to the certain acidic solu-tions. Standard solutions contained 1.46 g P or Caper 100 ml aqua bidest. and the corresponding testvolume of acid, respectively. Precision of the mea-surements was validated with the standard solu-tions.

Phosphate release induced by phosphoric acidand calcium dissolution caused by citric acid werecalculated according to the calcium to phosphate

Table 2 Amount of titratable acid as determined forthe tested acids [mval NaOH/l acid]

Amount of titratable acid[mval NaOH/l acid]

pH 2.0 pH 2.3 pH 3.0

Hydrochloric acid 10.0 4.7 1.0Acetic acid 2210.0 712.5 36.0Lactic acid 242.5 57.5 2.8Maleic acid 30.0 11.0 1.7Tartaric acid 99.3 36.3 2.8Phosphoric acid 27.0 9.5 1.4Citric acid 275.0 67.5 5.3Oxalic acid 18.8 9.3 1.6

Titration performed until pH 7.was not considered for detailed determination ofphosphate release per minute due to the specialkinetics of demineralization. Mineral loss of theenamel samples was calculated by accumulatingmean calcium and phosphate loss for the certainacids.

Preparation and gaining of solutions for thephotometric assaysAt pH 2 and 2.3, after 1, 2, 3, 4, and 5 min, 100 mlwere taken from the acidic solutions and replacedby 100 ml of fresh acid tomaintain the volume and tokeep pH constant. This was controlled with a pHelectrode (RadiometerA/S, Copenhagen, Denmark).The addition of 100 ml in the respective time led to adilution of the samples. This dilution was taken into

544 C. Hannig et al.

Figure 1 Mean calcium (A) and phosphate (B) dissolution from enamel specimens during immersion in hydrochloric acid(HCl) adjusted to pH 2, 2.3 and 3 [nmol/mm2]. S.D.: standard deviation; (&): pH 2; (~): pH 2.3; (^): pH 3.0.

Figure 2 Mean phosphate release from enamel specimens during 5 min incubation in oxalic acid at pH 2, 2.3 and 3. Thephosphate release is given as [nmol/mm2] of enamel surface. S.D.: standard deviation; (&): pH 2; (~): pH 2.3; (^): pH3.0.

Erosive effects of different acids on bovine enamel 545

Figure 3 Mean phosphate release of enamel immersed in oxalic acid for 5 min, followed by admixture of fresh oxalicacid (after 5 min) and of hydrochloric acid (after 10 min) to the oxalic acid solution.

Figure 4 Mean calcium release [nmol mm2 min1] from bovine enamel specimens stored for 5 min in different acids ofpH 2.0 (A), 2.3 (B), 3.0 (C), (HCl: hydrochloric acid; AA: acetic acid; LA: lactic acid; MA: maleic acid; TA: tartaric acid; PA:phosphoric acid). Significantly different data are signed with different letters (p < 0.05). n = 8 enamel specimens persubgroup (MV S.D.).

546 C. Hannig et al.

Figure 5 Mean phosphate release [nmol mm2 min1] from bovine enamel specimens stored for 5 min in different acidsof pH 2.0 (A), 2.3 (B), 3.0 (C), (HCl: hydrochloric acid; AA: acetic acid; LA: lactic acid; MA: maleic acid; TA: tartaric acid;CA: citric acid). Significantly different data are signed with different letters (p < 0.05). n = 8 enamel specimens persubgroup (MV S.D.).

Figure 6 Correlation of mineral loss and proton concentration for all acids and pH levels. Mineral loss as calculated byaccumulation of calcium and phosphate release. Correlation coefficient: r = 0.96.

consideration in later performed calculation of cal-cium and phosphate concentration in the solutions.

From the volume taken from the solution, 5, 10 or20 ml were admixed to 100 ml of the calcium test orto 200 ml of the phosphate assay, respectively. Theamount admixed to the assays depended on thecalcium or phosphate concentration which was tobe expected according to preliminary trials for therespective acid.

At pH 3, after 1, 2, 3, 4 and 5 min, 1000 ml were

volume and pH level. Due to the low ion concentra-tions, gained samples were evaporated at 80 8C for5 h and resolved in 100 ml HCl (pH 2). According tothe concentration of Ca and P which was to beexpected, 5, 10 or 20 ml of the solution were addedto calcium or phosphate assays.

Titration

Amount of titratable acid in the different acidic

Erosive effects of different acids on bovine enamel 547

Figure 7 Correlation of mineral loss and the amount of titratable acid for all acids and pH levels. Mineral loss ascalculated by accumulation of calcium and phosphate release. Correlation coefficient: r = 0.26.taken from the incubated volume and replacedimmediately by 1000 ml of fresh acid to maintainFigure 8 Erosive alterations caused by 1 min immersion in (acid (all pH 2).solutions at pH 2, 2.3 and 3 was determined bytitration with NaOH (1 mol/l) until pH 7 (Table 2).a) oxalic acid, (b) lactic acid, (c) acetic acid and (d) citric

548 C. Hannig et al.

n (aFigure 9 Erosive alterations caused by 1 min immersion ihydrochloric acid (all pH 2).Scanning electron microscopic (SEM)evaluation of erosive effects

For SEM investigation, samples were treated withthe different acids for 5 min (pH 2). Morphology ofetched surfaces was evaluated with SEM (960, Zeiss,Oberkochen, Germany, 2.4 kV, 20 mA, 270 s). Airdried samples were sputtered with gold (FissonsInstruments, Typ SC 510, Uckfield, UK) resulting ina gold coating of 7.5 1011 m thickness.

Statistics

Data were evaluated with MannWhitney U-test(Statistica, StatSoft, Hamburg, Germany). Levelof significance was set at p < 0.05. Where appro-priate, tests for correlations were carried out withExcel 5.0 (Microsoft Office 2000, USA).

Results

Kinetics of calcium and phosphate releasefrom the enamel during exposition to acidsfor 15 min at constant pH

Maleic, lactic, acetic, hydrochloric, phosphoric andtartaric acid led to a linear dissolution of calcium in) maleic acid, (b) tartaric acid, (c) phosphoric acid and (d)a time dependent manner during incubation for 15 min at constant pH. One example is given forhydrochloric acid (Fig. 1A).

For all acids, except for phosphoric acid, a photo-metric determination of phosphate release wasperformed. With exception of oxalic acid, all acidsled to a linear phosphate dissolution in a time and pHdependent manner during short time incubation.For hydrochloric acid, linear kinetics of phosphaterelease are depicted exemplarily (Fig. 1B).

Oxalic acid featured a special characteristicwhen incubated with enamel specimens. Determi-nation of erosive phosphate dissolution yielded non-linear kinetics for pH 2 and 2.3 during incubation for15 min (Fig. 2). In order to investigate the reasonfor this observation, specimens were incubated inoxalic acid for another 5 min. The additional oxalicacid did not lead to further phosphate dissolution;phosphate release reached a plateau. After admix-ture of hydrochloric acid (pH 2) a new increase ofphosphate dissolution was observed (Fig. 3).

Calcium and phosphate release per minute

Linearity of mineral loss during incubation of theenamel samples for 15 min with the different acidsallowed experimental determination of calcium andphosphate dissolution per minute (Figs. 4 and 5). For

erosive at higher pH levels, only.The ratio of calcium to phosphate release [Ca:P]

Erosive effects of different acids on bovine enamel 549ranged between 1.45 0.07 for tartaric acid, pH 3and 1.94 0.35 for acetic acid, pH 3. Over all acidsand pH values, the ratio was 1.68 0.13 as average.

Correlation of mineral loss with titratableacid and proton concentration

There was a strong correlation of the mineral lossand the proton concentration which is determinedby pH (r = 0.96, Fig. 6). Also, correlation of theamount of titratable acid (Table 2) and the mineralloss was calculated (Fig. 7). Only a weak correlationwas recorded (r = 0.28) under the given conditions.Therefore, it can be concluded that erosive impactof acids on bovine enamel is predominantly deter-mined by the pH of the acid during short timeexposition at constant pH level.

Scanning electron microscopic evaluation oferosive effects

All acids caused distinct erosive alterations of theenamel surfaces. Electron microscopic pictures aregiven in Figs. 8 and 9. Oxalic acid yielded precipita-tions on the enamel surface (Fig. 8a).

Discussion

In the present study, a photometric method fordirect sensitive and specific determination of cal-cium and phosphate loss during erosive attacks wasadopted.25,26 This allowed to investigate erosivecharacteristics of certain acids during short timeincubation in detail. With this method, even mini-mal erosive effects occurring within 1 min can beanalysed quantitatively. Therefore, it seems to beacceptable to talk about nano erosive effectsbecause nmol or ng of Ca and P dissoluted fromenamel can be detected precisely.26 Remineralisa-each acid, the erosive calcium and phosphaterelease at the different pH levels differed signifi-cantly (p < 0.05) (Figs. 4 and 5). At pH 2, lactic acidcaused significantly strongest release of calcium andphosphate (p < 0.05). At this pH, acetic acid fea-tured weakest erosive effects. At pH 2.3, lactic acidled to strongest calcium and phosphate loss fol-lowed by acetic acid and tartaric acid. Hydrochloricacid was least erosive. At pH 3, acetic acid was mosterosive compared to all the other acids. Lowesterosive effects were observed with hydrochloricacid. In summary, at all pH levels lactic acid wasone of the most erosive acids. Acetic acid was veryble but softened areas in the eroded lesions are notaffected. This is an advantage of the method com-pared to other methods for evaluation of erosiveeffects such as profilometry or determination ofmicrohardness.13,18,27

In previous studies, amount of mineral dissolvedby erosive drinks, acids, juices, and foodstuffs wasshown to be associated with their titratable amountof acid, exposure time, temperature, concentra-tion, character of the certain acid and pH underdifferent test conditions.19,2831 However, to ourknowledge, the present study is the first systematicattempt to elucidate erosive character of differentpure acids at three certain pH levels in detail viaphotometric determination of Ca and P loss duringshort time incubation.

Bovine teeth were used as a substitute for humanenamel as done in other investigations.32,33 Enamelsamples were incubated with an excess of acidproviding constant pH levels. This resembles thesituation during consumption of acidic drinks. Afterconsumption of a low pH drink, the pH on the tongueand tooth surfaces stays low for about 2 min.14,23 pHlevels between 2 and 3 were chosen as many acidicdrinks range in this level.2,11 In other studies com-paring erosive effects of different acids incubationwas performed with acidic solutions of same con-centrations.13,27 Due to the different levels of dis-sociation this will of course lead to different pHvalues. In the present study, for direct comparison ofdifferent acids, three standardized pH levels werechosen, since various beverages of same pH oftencontain different acids.

Under the given conditions, there was a clearinfluence of pH and type of acid on erosive effects.The different erosive character of the acids foundfor Ca and P dissolution was illustrated by thescanning electron microscopic evaluation of acidtreated enamel samples. A strong correlationbetween mineral loss and proton concentrationwas found for all acids. Correlation of pH and erosiveeffects has already been proven in other studies byprofilometry. However, in these studies sampleswere eroded for 10 or 30 min.13,27

In a profilometric study with 1030 min incuba-tion of the enamel samples, strongest erosionswere observed with PA, less erosive effects werecaused by CA, LA and MA.13 It is noteworthy that inthis experimental setup acidic solutions of sameacid concentrations but of different pH levels wereused for characterization of erosive capacity of thecertain acids. Due to the differing test conditions,it is not unexpected that grading of the erosivecapacity of the certain acids is different from thepresent results where lactic acid and acetic acidwere found to have strongest erosive impact, HCL

was least erosive. In contrast to the present data

calcium.3941 If enamel specimens are treated with

550 C. Hannig et al.using pure acids, Bartlett et al. found that gastricjuice, which contains mainly HCl was more erosivethan acidic carbonated beverages usually madewith phosphoric or citric acid.4 Under differentconditions also, pure HCL was more erosive thanphosphoric acid.27

One possible reason for the different erosiveeffects caused by the acids are specific interactionsof organic acids with hydroxyapatite.34,35 Mono-, di-, and tri-carboxylic acids are chemisorbed andbonded to hydroxyapatite via ionic interactions.34

Thereby, decalcification of and chemisorption tohydroxyapatite depend on the solubility of the car-boxylic salt in its own acidic solution. These chemi-sorption processes are not influenced by pH orconcentration of the acid. Carboxylic groups areassumed to substitute hydroxy or phosphate groupson the surface of enamel or hydroxyapatite, respec-tively.34,35 Due to the different structures of theorganic acids, different behavior was observed foroxalic, lactic, citric, and maleic acid. Oxalic acidwas chemically bonded to hydroxyapatite, whereasmaleic, lactic, and citric acid decalcify hydroxya-patite after adsorbing chemically.34,35

Apart from pH or type of acids present in acidicdrinks, also titratable amount of acid is assumed tohave a strong influence on the erosive capacity.2,28

The total acid level as determined via the amount oftitratable acid is considered even more importantthan pH of acidic solutions, since it determines theactual amount of protons available to interact withthe tooth surface.3 However, under the chosen con-ditions with abundance of acid, the amount oftitratabe acid was not well correlated with dissolu-tion of calcium and phosphate during short timeexposition at constant pH.

Other investigations were based on longtimeexposition of enamel specimens to acidicreagents.36,37 It should be noted that if a limitedamount of acid is present, erosive effects of thestrong acids are distinctly restricted due to theirhigh grade of dissociation. Weak acids with highamount of titratable acid and low level of dissocia-tion have the capability for further dissociationreleasing additional protons if acid is consumed.

In the present study, which was based on a photo-metric assay, determination of calcium dissolutioninduced by oxalic acid and citric acid was not pos-sible. A potential reason for this may be their capa-city to form chelating complexes with calcium.38

These chelating processes are influenced by pH andtend to increase as pH rises since the metal ionscompete directly with hydrogen ions. The chelationof calcium by oxalate and citrate may detract ionsfrom the chromogenous molecules applied in the

photometric test.oxalic acid, initial dissolution of enamel is followedby deposition of calcium oxalate crystals (mono- anddi-hydrate forms) as shown in a previous electronmicroscopic study.41 This calcium oxalate layer wasfound to prevent further dissolution of phosphate byoxalic acid, but offers no protection against erosiveattacks by other acids: the addition of HCl increasedphosphate loss from the samples pretreated withoxalic acid.

Dental enamel consists of about 95 wt.% inor-ganic mineral which is predominantly characterizedas calcium-phosphate crystals. Hydroxyapatite isdissolved according to the chemical reactionCa10(PO4)6(OH)2 + 8H

+$ 10Ca2+ + 6HPO42 + 2H2-2H2O.

42 The ratio of calcium to phosphate in aver-age amounted to about 1.7 which was confirmed bythe present experimental results underlining preci-sion of the photometric assays applied.42

In interpretation of the present data it must beregarded that they are basic in vitro results. In the invivo situation, the effects of acids on dental hardtissue are modulated by several factors, such asother components of acidic beverages like bufferingagents, fluoride, calcium and phosphate.2,29 Also,salivary flow rate,43 mode of drinking2,3 and espe-cially the aquired salivary pellicle have great impacton erosive effects.19,44,45 These aspects wereneglected in order to establish a method for numer-ical description of nano erosion via loss of Ca and Pper mm2 under standardized and reproducible con-ditions. Further investigations are necessary to elu-cidate impact of other factors, such as pellicle onerosive Ca and P dissolution induced by differentacids. Also, mineral content of the surroundingsolution and fluoride content of the enamel speci-mens may influence the erosive effects. Therefore,superficial enamel layers containing 20-fold morefluoride than deeper layers46 were removed in thestandardized grinding procedure to minimize possi-ble fluoride effects.

However, knowledge of the distinct erosivepotential of different acids given as calcium andphosphate release and their concentrations in bev-erages allows to use them as variables in a multipleregression calculating erosive potential of bev-erages and foodstuffs.28

Conclusion

The highly sensitive approach used in the presentthe surface of the samples treated with oxalic acid.It is known that oxalic acid forms precipitates withSEM analysis showed formation of a precipitate onstudy allows investigation of minimal erosive

19

Erosive effects of different acids on bovine enamel 551potential of some beverages. Caries Res 1995;29:34954.. Amaechi BT, Higham SM, Edgar WM. Factors influencingthe development of dental erosion in vitro: enamel type,temperature and exposure time. J Oral Rehabil 1999;26:18.

2000;108:1104.Lussi A, Jaeggi T, Jaeggi-Scharer S. Prediction of the erosiveeffects during short time exposition of enamel toerosive substrates via photometric determinationof Ca and P loss.

Different acids expose different erosive characteron enamel as shown via Ca and P release.

During short time exposition of enamel to variousacids at constant pH, erosive capacity is mainlydetermined by pH and type of acid, but not byamount of titratable acid or concentration of thecertain acid.

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552 C. Hannig et al.

Erosive effects of different acids on bovine enamel: release of calcium and phosphate in vitroIntroductionMaterials and methodsSpecimensAcidsIncubation of specimens with the different acidsPhotometric determination of calcium and phosphate releaseCalcium (Ca) assayPhosphate (P) assayPreparation and gaining of solutions for the photometric assays

TitrationScanning electron microscopic (SEM) evaluation of erosive effectsStatistics

ResultsKinetics of calcium and phosphate release from the enamel during exposition to acids for 1-5min at constant pHCalcium and phosphate release per minuteCorrelation of mineral loss with titratable acid and proton concentrationScanning electron microscopic evaluation of erosive effects

DiscussionConclusionReferences