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KEYWORDS
Erosion;
Calcium;
Phosphate;
Demineralisation
Introduction
* 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.2004.11.002
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C. Hannig et al.
is quite common.13,1520 These time periods are
much longer than the contact of the teeth with
an erosive substrate during consumption of erosive
food or beverages.14,23 Furthermore, erosive effects
were often evaluated without any standardization
of pH of the test solutions.13,1520
Therefore, the present study aimed to quantify
demineralization of enamel samples via highly sensitive determination of calcium and phosphate dissolution caused by certain acids after short time
erosion for 15 min. This was done systematically
for different solutions of pure acids typically occurring in foods and drinks at constant pH levels to
provide standardized basic parameters for direct
comparison of the erosive effects.
Acids
Enamel specimens were incubated in the acidic solutions of acetic, citric, maleic, tartaric, phosphoric,
oxalic, hydrochloric and lactic acid (Merck, Darmstadt, Germany) at pH 2, 2.3 and 3, respectively.
Concentrations of the certain acids at the three pH
levels are given in Table 1. pH was checked with a pH
meter (RadiometerA/S, Copenhagen, Denmark).
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Concentrations of acids
[mmol/l] at
Hydrochloric acid
Acetic acid
Lactic acid
Maleic acid
Tartaric acid
Phosphoric acid
Citric acid
Oxalic acid
pH 2.0
pH 2.3
pH 3,0
10.5
2214
292
15.7
50.0
16.2
95.2
8.84
4.50
721
71.5
5.67
18.4
5.90
24.2
4.46
0.75
34.9
3.60
0.86
1.32
0.88
1.87
0.75
Hydrochloric acid
Acetic acid
Lactic acid
Maleic acid
Tartaric acid
Phosphoric acid
Citric acid
Oxalic acid
pH 2.0
pH 2.3
pH 3.0
10.0
2210.0
242.5
30.0
99.3
27.0
275.0
18.8
4.7
712.5
57.5
11.0
36.3
9.5
67.5
9.3
1.0
36.0
2.8
1.7
2.8
1.4
5.3
1.6
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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. The
phosphate release is given as [nmol/mm2] of enamel surface. S.D.: standard deviation; (&): pH 2; (~): pH 2.3; (^): pH
3.0.
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Figure 3 Mean phosphate release of enamel immersed in oxalic acid for 5 min, followed by admixture of fresh oxalic
acid (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 of
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; PA:
phosphoric acid). Significantly different data are signed with different letters (p < 0.05). n = 8 enamel specimens per
subgroup (MV S.D.).
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C. Hannig et al.
Figure 5 Mean phosphate release [nmol mm2 min1] from bovine enamel specimens stored for 5 min in different acids
of 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 per
subgroup (MV S.D.).
Figure 6 Correlation of mineral loss and proton concentration for all acids and pH levels. Mineral loss as calculated by
accumulation of calcium and phosphate release. Correlation coefficient: r = 0.96.
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Figure 7 Correlation of mineral loss and the amount of titratable acid for all acids and pH levels. Mineral loss as
calculated by accumulation of calcium and phosphate release. Correlation coefficient: r = 0.26.
consideration in later performed calculation of calcium and phosphate concentration in the solutions.
From the volume taken from the solution, 5, 10 or
20 ml were admixed to 100 ml of the calcium test or
to 200 ml of the phosphate assay, respectively. The
amount admixed to the assays depended on the
calcium or phosphate concentration which was to
be expected according to preliminary trials for the
respective acid.
At pH 3, after 1, 2, 3, 4 and 5 min, 1000 ml were
taken from the incubated volume and replaced
immediately by 1000 ml of fresh acid to maintain
volume and pH level. Due to the low ion concentrations, gained samples were evaporated at 80 8C for
5 h and resolved in 100 ml HCl (pH 2). According to
the concentration of Ca and P which was to be
expected, 5, 10 or 20 ml of the solution were added
to calcium or phosphate assays.
Titration
Amount of titratable acid in the different acidic
solutions at pH 2, 2.3 and 3 was determined by
titration with NaOH (1 mol/l) until pH 7 (Table 2).
Figure 8 Erosive alterations caused by 1 min immersion in (a) oxalic acid, (b) lactic acid, (c) acetic acid and (d) citric
acid (all pH 2).
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C. Hannig et al.
Figure 9 Erosive alterations caused by 1 min immersion in (a) maleic acid, (b) tartaric acid, (c) phosphoric acid and (d)
hydrochloric acid (all pH 2).
Statistics
Data were evaluated with MannWhitney U-test
(Statistica, StatSoft, Hamburg, Germany). Level
of significance was set at p < 0.05. Where appropriate, tests for correlations were carried out with
Excel 5.0 (Microsoft Office 2000, USA).
Results
Kinetics of calcium and phosphate release
from the enamel during exposition to acids
for 15 min at constant pH
Maleic, lactic, acetic, hydrochloric, phosphoric and
tartaric acid led to a linear dissolution of calcium in
549
affected. This is an advantage of the method compared to other methods for evaluation of erosive
effects such as profilometry or determination of
microhardness.13,18,27
In previous studies, amount of mineral dissolved
by erosive drinks, acids, juices, and foodstuffs was
shown to be associated with their titratable amount
of acid, exposure time, temperature, concentration, character of the certain acid and pH under
different test conditions.19,2831 However, to our
knowledge, the present study is the first systematic
attempt to elucidate erosive character of different
pure acids at three certain pH levels in detail via
photometric determination of Ca and P loss during
short time incubation.
Bovine teeth were used as a substitute for human
enamel as done in other investigations.32,33 Enamel
samples were incubated with an excess of acid
providing constant pH levels. This resembles the
situation during consumption of acidic drinks. After
consumption of a low pH drink, the pH on the tongue
and tooth surfaces stays low for about 2 min.14,23 pH
levels between 2 and 3 were chosen as many acidic
drinks range in this level.2,11 In other studies comparing erosive effects of different acids incubation
was performed with acidic solutions of same concentrations.13,27 Due to the different levels of dissociation this will of course lead to different pH
values. In the present study, for direct comparison of
different acids, three standardized pH levels were
chosen, since various beverages of same pH often
contain different acids.
Under the given conditions, there was a clear
influence of pH and type of acid on erosive effects.
The different erosive character of the acids found
for Ca and P dissolution was illustrated by the
scanning electron microscopic evaluation of acid
treated enamel samples. A strong correlation
between mineral loss and proton concentration
was found for all acids. Correlation of pH and erosive
effects has already been proven in other studies by
profilometry. However, in these studies samples
were eroded for 10 or 30 min.13,27
In a profilometric study with 1030 min incubation of the enamel samples, strongest erosions
were observed with PA, less erosive effects were
caused by CA, LA and MA.13 It is noteworthy that in
this experimental setup acidic solutions of same
acid concentrations but of different pH levels were
used for characterization of erosive capacity of the
certain acids. Due to the differing test conditions,
it is not unexpected that grading of the erosive
capacity of the certain acids is different from the
present results where lactic acid and acetic acid
were found to have strongest erosive impact, HCL
was least erosive. In contrast to the present data
Discussion
In the present study, a photometric method for
direct sensitive and specific determination of calcium and phosphate loss during erosive attacks was
adopted.25,26 This allowed to investigate erosive
characteristics of certain acids during short time
incubation in detail. With this method, even minimal erosive effects occurring within 1 min can be
analysed quantitatively. Therefore, it seems to be
acceptable to talk about nano erosive effects
because nmol or ng of Ca and P dissoluted from
enamel can be detected precisely.26 Remineralisable but softened areas in the eroded lesions are not
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C. Hannig et al.
Conclusion
The highly sensitive approach used in the present
study allows investigation of minimal erosive
551
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