Dentin is dissolved by high concentrations of L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl) -2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt with or without hydrogen peroxide

Article abstract of DOI:10.1248/bpb.27.831

L-Ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12- trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K₁) is a conjugate of vitamin C and vitamin E that is water-soluble and stable at room temperature. EPC-K₁ has been developed as a hydroxyl radical (·OH) scavenger and antioxidant. In a previous tooth whitening experiment, it was accidentally found that tooth (dentin) blocks were dissolved by EPC-K₁ with H₂O ₂. In the current study, high concentrations of EPC-K₁ (2.5, 25mM) with 3% H₂O₂ dissolved and caused the collapse of dentin blocks. Similar concentrations of EPC-K₁ without 3% H ₂O₂, however, dissolved the dentin blocks without collapse over a 3-week period. In these cases, a ·OH-like signal was detected using an ESR spin-trapping method. The volume of calcium in solution (including the dentin block) increased on the addition of EPC-K₁ in a concentration-dependent manner. In addition, the calcium : phosphorus ratio changed from 2 : 1 in sound dentin to 1 : 2 in the collapsed dentin block. High concentrations of EPC-K₁ are therefore considered to have calcium chelating and dentin dissolving activity. The dentin dissolving activity was enhanced when EPC-K₁ was used with H₂O₂. EPC-K₁ had no protective effect when used in tooth whitening with H₂O₂.

Full text of DOI:10.1248/bpb.27.831

June 2004
Biol. Pharm. Bull. 27(6) 831—834 (2004)
831
Dentin Is Dissolved by High Concentrations of L-Ascorbic Acid 2-[3,4-
Dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-
benzopyran-6-yl-hydrogen Phosphate] Potassium Salt with or
without Hydrogen Peroxide
Masahiro KOZUKA and Yasuhisa TSUJIMOTO*
Research Institute of Oral Science, Department of Endodontics, Nihon University School of Dentistry at Matsudo; 870–1
Sakaecho, Nishi-2, Matsudo, Chiba 271–8587, Japan. Received October 27, 2003; accepted February 17, 2004
L-Ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hy-
drogen phosphate] potassium salt (EPC-K1) is a conjugate of vitamin C and vitamin E that is water-soluble and
stable at room temperature. EPC-K1 has been developed as a hydroxyl radical (·OH) scavenger and antioxidant.
In a previous tooth whitening experiment, it was accidentally found that tooth (dentin) blocks were dissolved by
EPC-K1 with H2O2. In the current study, high concentrations of EPC-K1 (2.5, 25 mM) with 3% H2O2 dissolved
and caused the collapse of dentin blocks. Similar concentrations of EPC-K1 without 3% H2O2, however, dissolved
the dentin blocks without collapse over a 3-week period. In these cases, a ·OH-like signal was detected using an
ESR spin-trapping method. The volume of calcium in solution (including the dentin block) increased on the ad-
dition of EPC-K1 in a concentration-dependent manner. In addition, the calcium : phosphorus ratio changed
from 2 : 1 in sound dentin to 1 : 2 in the collapsed dentin block. High concentrations of EPC-K1 are therefore con-
sidered to have calcium chelating and dentin dissolving activity. The dentin dissolving activity was enhanced
when EPC-K1 was used with H2O2. EPC-K1 had no protective effect when used in tooth whitening with H2O2.
Key words dentin; hydroxyl radical; ESR
In a previous study, it was reported1,2) that hydroxyl radi-
DMSO, which are used as ·OH scavengers, were obtained
from Wako Pure Chemical Industries, Ltd., (Osaka, Japan).
Dentin Blocks The dentin blocks were prepared as de-
scribed by Tsujimoto et al.12) Four blocks were obtained from
the root of an extracted human single cone tooth. The pulp
and smear layers were removed by file, then 15% EDTA was
applied on the dentin wall for 2 min. The sample was washed
with pure water (PW) and blot dried with Kimwipe (Kim-
berly-Clark Co. Ltd., Japan) paper. The sample was then
weighed. Three blocks were used for each examination.
Free Radical Measurement The ESR spin-trapping
method11,13,14) was used to measure free radical generation
from each solution. The final concentration of DMPO in
200 ml of the reaction mixture was adjusted to 89.0 mM for
this experiment. The ESR spectra were measured using a JES
FA 300 (JEOL, Tokyo, Japan). The measurement conditions
were as follows: microwave power, 8 mW; magnetic field,
335.0ꢁ5 mT; sweep time, 2 min; modulation frequency,
100 kHz; and time constant, 0.03 s.
cals (·OH) generated via hydrogen peroxide (H2O2) were re-
sponsible for tooth whitening. However, the detailed mecha-
nism(s) of tooth whitening have not yet been elucidated. It is
well known that ·OH are scavenged by ·OH scavengers such
as methanol, dimethyl sulfoxide (DMSO) and L-ascorbic acid
2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltride-
cyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium
salt (EPC-K1).3—11) EPC-K1, a conjugate of vitamin C and vi-
tamin E that is water-soluble and stable at room temperature,
was developed as a ·OH scavenger and antioxidant agent by
Senju Pharmaceuticals (Osaka, Japan).
In the tooth whitening pilot study, we used EPC-K1 to in-
vestigate the relationship between whitening effect and ·OH.
At that time we accidentally found that the tooth (dentin)
blocks were dissolved by H2O2 with EPC-K1. EPC-K1 has
been developed as an antioxidant for the living body. There is
a huge problem if EPC-K1 is able to dissolve dentin blocks,
because tooth components are almost the same as bone. If
damage to organic and/or inorganic components occurs with
EPC-K1, the components of the human body will become un-
balanced.
Calcium Volume Measurement The calcium volumes
of each solution around the dentin blocks were measured
over time using an inductively coupled plasma atomic emis-
sion spectrometer (SPS 1700R; Seiko Instrument Inc.,
Tokyo, Japan). The measurement conditions were as follows:
modulation frequency, 27.12 MHz; microwave power,
1.30 kW; plasma flow (argon gas), 16.0 l/min.
The aim of the current study is to examine the effects of
EPC-K1 with and without H2O2 on dentin blocks over time.
MATERIALS AND METHODS
Changes in pH The pH of each solution was measured
using an ISFET pH Meter KS723 (Shindengen Electric Man-
ufacturing Co., Ltd., Tokyo, Japan).
Wet Tooth Block Weight The wet tooth block weight
was measured using a R2000D electric scale (Sartorious,
Germany).
Chemicals EPC-K1 was kindly provided by Senju Phar-
maceuticals (Osaka, Japan). 5,5-Dimethyl-1-pyrroline-N-
oxide (DMPO), which is used as a trapping agent for free
radicals, was obtained from Dojin Chemicals (Kumamoto,
Japan). Ethylenediamine-N,N,Nꢀ,Nꢀ-tetraacetic acid (EDTA),
used as an irrigant for the removal of a smear layer, was ob-
tained from Dojin Chemicals (Kumamoto, Japan). H2O2
which is used as a tooth whitening agent, and ethanol and
∗ To whom correspondence should be addressed. e-mail: tsuji@mascat.nihon-u.ac.jp
© 2004 Pharmaceutical Society of Japan

832
Vol. 27, No. 6

June 2004
833
ter radical (·C) was detected when ethanol or DMSO was
added to 3% H2O2 (data not shown). This means that free
·OH was produced followed by conversion to the DMPO-C-
center.15) ·OH was not detected with every concentration of
EPC-K1, however ·OH-like signals were observed at 3 weeks
with high concentrations of EPC-K1 (2.5, 25 mM) (Fig. 4).
·OH-like signals were also detected at 3 weeks in H2O2 solu-
tions with 2.5 and 25 mM EPC-K1. Higher ·OH-like signals
were detected with 2.5 and 25 mM EPC-K1 with H2O2 than
without H2O2.
DISCUSSION
EPC-K1 is composed of vitamin C and vitamin E joined by
a phosphodiester linkage. EPC-K1 has been reported to have
potent ·OH scavenging activity, anti-lipid peroxidation activ-
ity,10) and can chelate iron and copper.11) In addition, Kuriba-
yashi et al.16) suggested that EPC-K1 might be useful in the
treatment of ischemia-reperfusion. It was thought that EPC-
K1 could be used to treat the inflammation of dental pulp and
gingiva, and in tooth replantation and transplantation. It has
not been reported what kind of effect EPC-K1 has on the
tooth component. As mentioned above, we found acciden-
tally that EPC-K1 dissolved dentin during the tooth whitening
experiment. It is therefore very important to clarify the ef-
fects of EPC-K1 before it is applied clinically.
The results of the current study show that high concentra-
tions of EPC-K1 (2.5, 25 mM) with 3% H2O2 dissolved and
caused the collapse of dentin blocks. When the pH of the so-
lution was measured, it was found to be pH 5—6 for 24 h,
then decreased to pH 4—5 in the first week, and then rose
after 3 weeks to pH 5.5—7.
Fig. 4. Typical ESR Signal
(A) ·OH signal from 3% H2O2. (B) ·OH-like signal from 3% H2O2 with 2.5 mM EPC-
K1.
RESULTS
State of the Dentin Blocks The typical state of the
dentin blocks in each solution after 3 weeks is shown in Fig.
1 (results at 1 and 2 weeks not shown). The dentin blocks and
solutions in PW and EPC-K1 showed little change after 3
weeks; however, the color of the solutions in 2.5 and 25 mM
EPC-K1 changed to milk-white. The color of the dentin
blocks became white after 3 weeks in 3% H2O2 and 3% H2O2
with EPC-K1. However, after 3 weeks in 3% H2O2 with 2.5
and 25 mM EPC-K1 the dentin blocks had collapsed. An ex-
ceptional amount of collapse was observed in 3% H2O2 with
25 mM EPC-K1 after 3 weeks.
Calcium Volume of the Solutions The calcium volume
in the solutions is shown in Figs. 2A and B. Calcium volume
increased after the addition of EPC-K1 in a concentration-de-
pendent manner. An exceptional amount of calcium was de-
tected after the addition of 25 mM EPC-K1.
Changes in pH The change in pH of each solution (in-
cluding the dentin block) is shown in Fig. 3. In the case of
EPC-K1, the pH of the solution (approximately pH 6) in-
creased gradually after mixing. After 3 weeks, the pH was
approximately 8 (Fig. 3A). The pH of the solution was ap-
proximately 6 after mixing with 3% H2O2 and differing con-
centrations of EPC-K1. The pH increased to pH 7—8 in 3%
H2O2, 3% H2O2 with 0.025 mM and 0.25 mM EPC-K1. How-
ever, in 3% H2O2 with 2.5 and 25 mM EPC-K1 the pH de-
creased to pH 4—5 at 1 week and then increased to pH 6
after 3 weeks (Fig. 3B).
High concentrations of EPC-K1 (2.5, 25 mM) without 3%
H2O2, on the other hand, dissolved the dentin blocks without
causing a collapse. In this case, the initial pH was approxi-
mately 6 and then increased gradually to pH 8 after 3 weeks.
The ·OH was generated from H2O2 via Fenton reac-
tion17,18) because of H2O2 generally includes a small volume
of metal in the manufacture process. In this experiment, the
EPC-K1 concentrations ranged from 0.025—25 mM. This was
because the ·OH generated from 3% H2O2 was completely
eliminated by the addition of 0.025—25 mM EPC-K1 in a
preliminary study (data not shown). The pH experiment is an
important aspect because teeth are dissolved by acidic solu-
tions such as phosphoric acid, which is used as a tooth etch-
ing agent. The pH was not dependent on dentin being dis-
solved by EPC-K1; the pH decreased to pH 4—5 in the first
week and then rose to pH 5.5—7 after 3 weeks when high
concentrations of EPC-K1 with H2O2 were used. This obser-
vation is difficult to explain, although it is suggested that the
dissolving of dentin by EPC-K1 is enhanced by H2O2. A fur-
ther study to clarify this issue is currently underway.
Wet Dentin Block Weight The wet dentin block weight
showed little change except when 3% H2O2 was used with
25 mM EPC-K1 for 3 weeks. This happened because the
dentin block collapsed (data not shown).
In a previous tooth whitening study,2) the ·OH generated
from H2O2 degraded the amino acids that make up organic
matter, although the structure of hydroxyl apatite, like the in-
organic material in dentin, did not change after application of
H2O2. The weight of the dentin block decreased after applica-
tion of H2O2, but the calcium volume did not change. It has
been suggested that the ·OH generated from H2O2 degraded
the organic matter of dentin, and then destroyed its structure.
In this study, a ·OH-like signal was detected with 2.5 and
Analysis of Collapsed Dentin Blocks The calcium:
phosphorus ratio of the collapsed tooth block was measured
using an inductively coupled plasma atomic emission spec-
trometer (SPS 1700R; Seiko Instrument Inc., Tokyo, Japan).
The calcium : phosphorus ratio was found to be 1 : 2 in 3%
H2O2 with 25 mM EPC-K1.
Free Radical Measurement Generated free radicals
were measured using an ESR spin-trapping method.11,13,14)
·OH were detected from 3% H2O2 (Fig. 4), and a carbon cen-

834
Vol. 27, No. 6
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Acknowledgements We thank Dr. T. Ishizaki (Central
Research Center, Sangi Co. Ltd.) for the calcium volume
measurement work. This study was supported by a Grant
from the Ministry of Education, Culture, Sports, Science and
Technology to promote 2001-Multidisciplinary Research
Projects (in 2001—2005), and from The Tsuchiya Founda-
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