Kinetic study of the α-tocopherol-regeneration reaction of ubiquinol-10 in methanol and acetonitrile solutions: Notable effect of the alkali and alkaline earth metal salts on the reaction rates

Article abstract of DOI:10.1021/jp2118086

A kinetic study of regeneration reaction of α-tocopherol (α-TocH) by ubiquinol-10 has been performed in the presence of four kinds of alkali and alkaline earth metal salts (LiClO₄, NaClO₄, NaI, and Mg(ClO₄)₂) in methanol and acetonitrile solutions, using double-mixing stopped-flow spectrophotometry. The second-order rate constants (k_r's) for the reaction of α-tocopheroxyl (α-Toc?) radical with ubiquinol-10 increased and decreased notably with increasing concentrations of metal salts in methanol and acetonitrile, respectively. The k_r values increased in the order of no metal salt < NaClO₄ ～ NaI < LiClO₄ < Mg(ClO ₄)₂ at the same concentration of metal salts in methanol. On the other hand, in acetonitrile, the k_r values decreased in the order of no metal salt > NaClO₄ ～ NaI > LiClO₄ > Mg(ClO₄)₂ at the same concentration of metal salts. The metal salts having a smaller ionic radius of cation and a larger charge of cation gave a larger k_r value in methanol, and a smaller k _r value in acetonitrile. The effect of anion was almost negligible in both the solvents. Notable effects of metal cations on the UV-vis absorption spectrum of α-Toc? radical were observed in aprotic acetonitrile solution, suggesting complex formation between α-Toc? and metal cations. On the other hand, effects of metal cations were negligible in protic methanol, suggesting that the complex formation between α-Toc? and metal cations is hindered by the hydrogen bond between α-Toc? and methanol molecules. The difference between the reaction mechanisms in methanol and acetonitrile solutions was discussed on the basis of the results obtained. High concentrations of alkali and alkaline earth metal salts coexist with α-TocH and ubiquinol-10 in plasma, blood, and many tissues, suggesting the contribution of the metal salts to the above regeneration reaction in biological systems.

Full text of DOI:10.1021/jp2118086

Article
pubs.acs.org/JPCB
Kinetic Study of the α-Tocopherol-Regeneration Reaction of
Ubiquinol-10 in Methanol and Acetonitrile Solutions: Notable Effect
of the Alkali and Alkaline Earth Metal Salts on the Reaction Rates
Kazuo Mukai,* Masanori Oi, Aya Ouchi, and Shin-ichi Nagaoka
Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790-8577, Japan
ABSTRACT: A kinetic study of regeneration reaction of α-
tocopherol (α-TocH) by ubiquinol-10 has been performed in
the presence of four kinds of alkali and alkaline earth metal
salts (LiClO4, NaClO4, NaI, and Mg(ClO4)2) in methanol and
acetonitrile solutions, using double-mixing stopped-flow
spectrophotometry. The second-order rate constants (kr's)
for the reaction of α-tocopheroxyl (α-Toc•) radical with
ubiquinol-10 increased and decreased notably with increasing
concentrations of metal salts in methanol and acetonitrile,
respectively. The kr values increased in the order of no metal
salt < NaClO4 ∼ NaI < LiClO4 < Mg(ClO4)2 at the same
concentration of metal salts in methanol. On the other hand, in acetonitrile, the kr values decreased in the order of no metal salt >
NaClO4 ∼ NaI > LiClO4 > Mg(ClO4)2 at the same concentration of metal salts. The metal salts having a smaller ionic radius of
cation and a larger charge of cation gave a larger kr value in methanol, and a smaller kr value in acetonitrile. The effect of anion
was almost negligible in both the solvents. Notable effects of metal cations on the UV−vis absorption spectrum of α-Toc• radical
were observed in aprotic acetonitrile solution, suggesting complex formation between α-Toc• and metal cations. On the other
hand, effects of metal cations were negligible in protic methanol, suggesting that the complex formation between α-Toc• and
metal cations is hindered by the hydrogen bond between α-Toc• and methanol molecules. The difference between the reaction
mechanisms in methanol and acetonitrile solutions was discussed on the basis of the results obtained. High concentrations of
alkali and alkaline earth metal salts coexist with α-TocH and ubiquinol-10 in plasma, blood, and many tissues, suggesting the
contribution of the metal salts to the above regeneration reaction in biological systems.
−
α ‐ Toc • + UQ H (and/or AsH )
1. INTRODUCTION
2
10
It is well-known that vitamin E (α-tocopherol, α-TocH) is
localized in biomembranes, plasma, and LDL and functions as
an efficient inhibitor of lipid peroxidation.1−5 The antioxidant
properties of the α-TocH have been ascribed to the initial
oxidation of the phenolic hydroxyl group by a lipid peroxyl
radical (LOO•), producing corresponding α-tocopheroxyl (α-
Toc•) radical (reaction 1).1,2,6 If α-TocH exists in biomem-
branes and edible oils, the α-Toc• radicals may react with
unsaturated lipids (LH) (reaction 2). Reaction 2 is known as
prooxidant reaction, which induces the degradation of
unsaturated lipids.7−11 Ubiquinol-10 (UQ10H2)1,12−14 and/or
vitamin C (ascorbate anion, AsH−)1,15−17 enhance the anti-
oxidant activity of α-TocH by regenerating α-Toc• to α-TocH
(reaction 3), to protect the above prooxidant effects. Further,
α-Toc• radicals disappear by bimolecular self-reaction with another
α-Toc• to give nonradical products (NRP) (reaction 4),6,18−20 if
the above ubiquinol-10 and vitamin C do not coexist in the
system.
k
r
−
→ α ‐ TocH + UQ H • (and/or As• )
10
(3)
(4)
2k
d
α ‐ Toc • + α ‐ Toc• ⎯⎯⎯→ non ‐ radical products (NRP)
It is also well-known that alkali and alkaline earth metal ions
(Na+, K+, and Mg2+) are included in blood and plasma at rela-
tively high concentrations.21 These metal ions play important
roles in many kinds of enzymatic reactions.22 α-TocH and
UQ10H2 coexist with these metal ions in human blood, plasma,
and various tissues.12,23,24 However, the examples of the kinetic
studies of the effect of these metal ions on the free radical-
scavenging reaction of the natural antioxidants, including α-
TocH and UQ10H2, are very limited.25−28
In previous works,29,30 in order to investigate the effect of alkali
and alkaline earth metal ions (Li+, Na+, K+, and Mg2+) on the
scavenging reaction of free radical by α-TocH, we measured the
second-order rate constants (ks's) for the reaction of α-TocH
k
inh
(1)
(2)
LOO • + α ‐ TocH ⎯⎯⎯⎯→ LOOH + α ‐ Toc •
Received: December 7, 2011
Revised: February 6, 2012
Published: February 8, 2012
k
p
α ‐ Toc • + LH → α ‐ TocH + L •
© 2012 American Chemical Society
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with aroxyl radical (ArO•, see Figure 1) in protic methanol
in a refrigerator at −20 °C. The purity of the UQ10H2 was
checked spectrophotometrically before the use, using the above
molar extinction coefficient (ε) in ethanol. Experiments were
always performed using pure UQ10H2.
and aprotic acetonitrile solutions including various metal salts
2.2. Measurements. Measurement of the UV−vis absorp-
tion spectrum of α-Toc• radical (see Figure 2a) was performed
Figure 1. Molecular structures of α-tocopherol (α-TocH), α-
tocopheroxyl (α-Toc•) radical, aroxyl (ArO•) radical, and ubiquinol-
10 (UQ10H2).
(LiI, LiClO4, NaI, NaClO4, KI, and Mg(ClO4)2) (reaction 5),
using stopped-flow spectrophotometry. The effect of these
metal ions on the bimolecular self-reaction (reaction 4) was
also studied.29,30 From the results, the mechanisms for the above
reactions 4 and 5 were discussed.
k
s
(5)
ArO • + α ‐ TocH → ArOH + α ‐ Toc •
On the other hand, recently, kinetic study of the regeneration
reaction of α-TocH by UQ10H2 (reaction 3) was carried out in
ethanol (C2H5OH) and ethanol-d1 (C2H5OD) solutions by
means of double-mixing stopped-flow spectrophotometry.14
A
substantial deuterium kinetic isotope effect was observed on the
second-order rate constant (kr) and the activation energy. From
the result, it was considered that proton tunneling plays an
important role in the regeneration reaction of α-TocH by
UQ10H2.
In the present work, in order to clarify the effect of alkali and
alkaline earth metal ions (Li+, Na+, and Mg2+) on the rege-
neration reaction of α-TocH by UQ10H2, we measured the rate
constants (kr's) for the reaction of α-Toc• with UQ10H2 in
methanol and acetonitrile solutions including various metal
salts (LiClO4, NaClO4, NaI, and Mg(ClO4)2), using double-
mixing stopped-flow spectrophotometry. The effect of these
metal ions on the UV−vis absorption spectrum of α-Toc• was
also studied.
2. EXPERIMENTAL METHODS
2.1. Materials. LiClO4, NaClO4, NaI, and Mg(ClO4)2 are
commercially available. Optical grades of methanol and
acetonitrile were used for the measurement. α-Tocopherol
(α-TocH) was kindly supplied from Eisai Co. Ltd. Ubiquinone-
10 (UQ10) was kindly supplied by Kaneka Corporation. Aroxyl
radical (ArO•, 2,6-di-tert-butyl-4(4′-methoxyphenyl)phenoxyl)
was prepared according to the method of Rieker et al.31
Ubiquinol-10 (UQ10H2) was prepared by the reduction of
UQ10 with sodium hydrosulfite in n-hexane under a nitrogen
atmosphere.32,33 It was recrystallized from ethanol/petroleum
ether solution: mp 46−47 °C. UV spectrum (ethanol):
λmax = 289 nm (ε = 4340 M−1 cm−1) and λmax = 290 nm (ε =
4010 M−1 cm−1). The UQ10H2 prepared was kept under vacuum
Figure 2. (a) Change in electronic absorption spectrum of ArO• and
α-Toc• radicals during reaction of ArO• with α-TocH in methanol
solution at 25.0 °C. The spectra were recorded at 20 ms intervals. The
arrow indicates a decrease (ArO•) and an increase (α-Toc•) in
absorbance with time. Initial concentrations of [α-TocH] in cell A and
[ArO•] in cell B used for the reaction are shown in part a. (b) Change
in absorption spectrum (at 60 ms intervals) during reaction 3 of
UQ10H2 and α-Toc• in methanol at 25.0 °C. The initial concentration
of [UQ10H2] in cell C was 9.70 × 10−5 M. The arrow indicates a
decrease in absorbance of α-Toc•. (c) Absorbance decay of α-Toc• at
428.9 nm during reaction 3 in methanol at 25.0 °C.
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by mixing equal volumes of methanol (or acetonitrile) solutions
of ArO• and α-TocH (reaction 5) under a nitrogen atmo-
sphere, using a Unisoku single-mixing stopped-flow spectro-
photometer (model RSP-1000).
Measurement of the kr value (reaction 3) was performed with
a Unisoku double-mixing stopped-flow spectrophotometer
(model RSP-1000-03F).14 By the first mixing of equal volumes
of methanol (or acetonitrile) solutions of α-TocH (cell A) and
ArO• (cell B), α-Toc• radical was prepared (reaction 5), and
after 2 s the second-mixing of equal volumes of α-Toc• solu-
tion and UQ10H2 solution (cell C) (reaction 3) was performed
(see Figure 2b,c), using double-mixing unit of the RSP-1000.
The rate constants (kr's) were determined by analyzing the
decay curve of α-Toc• as described later. The time between
mixing two solutions and recording the first data point (that is,
dead time) was 10−20 ms. The reaction was monitored with
either single wavelength detection (Figure 2c) or photodiode
array detector (Figure 2b) attached to the stopped-flow spectro-
photometer. All the measurements were performed at 25.0
0.5 °C. Experimental errors in the rate constants (kr's) were
estimated to be about 5% in methanol and acetonitrile solutions.
Figure 3. Plots of the peak wavelength (λmax) of α-Toc• radical versus
the concentration of metal salts: (a) LiClO4, (b) NaClO4, and (c)
Mg(ClO4)2. Measurements were performed in acetonitrile and
methanol solutions at 25.0 °C.
complex formation ([α-Toc•···M+ (or M2+)]) between α-Toc•
radical and metal cations (Li+, Na+, and Mg2+) in acetonitrile.30
Furthermore, the effects of metal salts on the UV−vis
absorption spectra of α-TocH and UQ10H2 included in reaction
3 were studied in methanol and acetonitrile solutions. However,
any shifts of the absorption peaks of these molecules were not
observed in the presence of high concentrations of metal salts
(4.00 × 10−1 M) in both the solvents. The interaction between
each metal cation and neutral α-TocH (or UQ10H2) molecule is
considered to be negligible in methanol and acetonitrile. UQ10H•
radical may form a complex with metal cations. However, UQ10H•
is very unstable (lifetime τ = 1.17 μs),34 and we were unsuccessful
in observing its UV−vis spectrum in methanol and acetonitrile
solutions.
3.2. Effect of Metal Salts on the Regeneration Rate (kr)
of α-Tocopherol by Ubiquinol-10 in Methanol. As
described in section 2.2, measurements of the rate constant
(kr) for the reaction of α-Toc• radical with UQ10H2 (reaction 3)
were performed in methanol solution, using a double-mixing
stopped-flow spectrophotometer. For example, α-Toc• radical
was prepared by the first-mixing of equal volumes of α-TocH
(5.56 × 10−3 M) (cell A) and ArO• (2.42 × 10−4 M) (cell B)
solutions (reaction 5), and after 2 s the second-mixing of equal
volumes of α-Toc• solution and UQ10H2 solution (0, 9.70 ×
10−5, 1.94 × 10−4, 2.92 × 10−4, or 4.86 × 10−4 M) (cell C)
(reaction 3) was performed (see Figure 2b). The decay curves of
the absorbance of α-Toc• at 428.9 nm in methanol were shown in
Figure 2c, indicating that the decay rates increase with increas-
ing concentrations of UQ10H2. The decay of the absorbance of
α-Toc• at 428.9 nm without UQ10H2 (see Figure 2c; line of
[UQ10H2] = 0 M) is due to a bimolecular reaction of α-Toc•
radicals (reaction 4). It is slow (2kd = 9.2 × 102 M−1 s−1)29 and
negligible, because the reaction between α-Toc• and UQ10H2 is
very fast (kr = 1.48 × 105 M−1 s−1) (see Table 1).14
3. RESULTS
3.1. Effect of Metal Salts on UV−Vis Absorption
Spectra of α-Tocopheroxyl Radical in Methanol and
Acetonitrile Solutions. ArO• radical is stable in the absence
of α-TocH, and shows absorption peaks at λmax = 375.2 and
579.8 nm in methanol solution, as shown in Figure 2a.29 By
adding a methanol solution of α-TocH (2.78 × 10−3 M) to the
solution of ArO• (8.36 × 10−4 M) (1:1 in volume) at 25.0 °C,
the absorption peak of ArO• disappeared quickly, and the
spectrum was changed to that of α-Toc• with three absorption
peaks at λmax = 428.9, 408.4, and 379.1 nm (reaction 5) (see
Figure 2a,b), where the measurements were performed by using
a single-mixing stopped-flow spectrophotometer. The absorp-
tion of ArO• radical disappeared in ∼0.5 s, and changed to that
of α-Toc•. α-Toc• is unstable at 25.0 °C, and its absorption
decreased gradually, after passing though the maximum at ∼0.5 s
(data are not shown).29
In the present work, we studied the effect of four kinds of
metal salts (LiClO4, NaClO4, NaI, and Mg(ClO4)2) on the
UV−vis absorption spectra of α-Toc• radical in methanol and
acetonitrile solutions. The measurements were performed by
varying the concentrations of metal salts. In methanol solution,
the shift of the absorption peak of α-Toc• at λmax = 428.9 nm
was negligible in the presence of each metal salt used (see
Figure 3), as reported in a previous work.29 The hydrogen bond
formation between α-Toc• radical and OH group of methanol
may hinder the approach of the metal cations.
On the other hand, in acetonitrile solution, the shift of the
absorption peak of α-Toc• was observed in the presence of
metal salts.30 The λmax value (424.3 nm) of α-Toc• in the
absence of metal salts increased with increasing concentrations
of metal salts, and approached different constant values,
depending on the kinds of metal salts, as shown in Figure 3.
The λmax value of α-Toc• increased in the following order at
the same concentration of metal salts.
The reaction rate was measured by following the decrease in
absorbance at 428.9 nm of the α-Toc• radical. The pseudo-
first-order rate constants (kobsd's) observed at 428.9 nm were
linearly dependent on the concentration of UQ10H2, and thus
the rate law is expressed as follows
without metal salt < NaClO ∼ NaI < LiClO < Mg(ClO )
4
4
4 2
(6)
−d[α ‐ Toc•]/dt = k
[α ‐ Toc•]
obsd
The effect of anion of the metal salts was negligible, because
NaClO4 and NaI show a similar shift. The result suggests the
= k [UQ H ][α ‐ Toc•]
(7)
r
2
10
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value (krav = 1.48 × 105 M−1 s−1) obtained is listed in Table 1.
Experimental errors are estimated to be less than 5%.
Similar measurements were performed for the reaction of α-
Toc• with UQ10H2 in methanol solution, including various
concentrations (0−6.00 × 10−1 M) of LiClO4, NaClO4, NaI,
and Mg(ClO4)2 salts. The results obtained for Mg(ClO4)2 salt
are shown in Figure 4a. The rate constants (kr's) obtained
for LiClO4, NaClO4, NaI, and Mg(ClO4)2 salts are listed in
Table 1. The kr values increased with increasing concentrations
of metal salts, as shown in Figure 4b. Further, the kr values
increased in the following order at the same concentration of
metal salts:
Table 1. Second-Order Rate Constants (kr's) for the
Reaction of Ubiquinol-10 with α-Toc• Radical in Methanol
Solution Including Various Concentrations of Alkali and
Alkaline Earth Metal Salts at 25.0 °C
LiClO4 kr/
NaClO4 kr/ NaI kr/M−1 Mg(ClO4)2
[salt]/M
M−1 s−1
M−1 s−1
s−1
kr/M−1 s−1
a
a
a
a
0
1.48 × 105
1.75 × 105
1.94 × 105
2.13 × 105
0.76
1.48 × 105
1.53 × 105
1.72 × 105
1.93 × 105
1.02
1.48 × 105
1.57 × 105
1.76 × 105
2.00 × 105
1.02
1.48 × 105
1.79 × 105
2.03 × 105
2.48 × 105
0.72
0.200
0.400
0.600
ionic radii of
cations r/Å
(six
coordination)
without metal salt < NaClO ∼ NaI < LiClO < Mg(ClO )
4
4
4 2
a
Measurement of the kr value for [salt] = 0 M was repeated three
times (1.46 × 105, 1.53 × 105, and 1.45 × 105 M−1s−1). An average
value (av 1.48 × 105 M−1 −1) was shown in the table.
(8)
s
The rate constants (kr's) obtained in the presence of 6.00 ×
10−1 M of LiClO4 and Mg(ClO4)2 are 1.44 and 1.68 times
larger than that in the absence of metal salts, respectively. On
the other hand, the effects of NaClO4 and NaI are less than
those of LiClO4 and Mg(ClO4)2. The reason will be discussed
in the Discussion section.
where kr is the second-order rate constant for the reaction of
α-Toc• radical with UQ10H2. The kr value was obtained by
plotting kobsd against [UQ10H2], as shown in Figure 4a. The
3.3. Effect of Metal Salts on the Regeneration Rate (kr)
of α-Tocopherol by Ubiquinol-10 in Acetonitrile.
Similarly, measurements of the second-order rate constants
(kr's) for the reaction of α-Toc• radical with UQ10H2 were per-
formed in aprotic acetonitrile solution at 25.0 °C, in the pre-
sence and absence of four kinds of metal salts (LiClO4, NaClO4,
NaI, and Mg(ClO4)2). The rate constants (kr's) were obtained by
plotting kobsd against [UQ10H2]. As an example, the results
obtained for Mg(ClO4)2 salt are shown in Figure 5a. Notable
effects of metal salts on the reaction rates (kr) were observed, as
listed in Table 2. As shown in Figure 5b, the kr values decreased
remarkably with increasing concentrations of metal salts, differing
from the results obtained in methanol (see Figure 4b). Further-
more, the kr values decreased in the following order at the same
concentration of metal salts:
without metal salt > NaClO ∼ NaI > LiClO > Mg(ClO )
4
4
4 2
(9)
The rate constants (kr's) obtained in the presence of 5.00 ×
10−1 M of LiClO4 and NaClO4 are 29% and 83% of that in the
absence of metal salts, respectively. Furthermore, the rate
constant (kr) obtained in the presence of 2.00 × 10−1 M of
Mg(ClO4)2 is 0.091% of that in the absence of metal salts. The
reason for the notable decrease in the rate constants (kr's) will
be discussed in the Discussion.
4. DISCUSSION
4.1. Mechanism of the Regeneration Reaction of α-
Tocopherol by Ubiquinol-10 in Methanol Solution
Including Metal Salts. Measurements of the reaction rates
of galvinoxyl• (and DPPH•) radicals with α-tocopherol model
without a phytyl side-chain were performed by Nakanishi et al.25,26
in methanol solution including Mg(ClO4)2 salt. They found that
the rate constants (ks's) increase with increasing concentration
of Mg(ClO4)2. Ouchi (one of the present authors) et al.29
measured the rate constant (ks's) for the reaction of ArO• with
α-TocH (reaction 5) in methanol solutions including six kinds
of metal salts (LiI, LiClO4, NaI, NaClO4, KI, and Mg(ClO4)2).
The rate constants (ks's) increased with increasing concentrations
Figure 4. (a) Dependence of pseudo-first-order rate constant (kobsd
)
on concentration of ubiquinol-10 in methanol including 0.000, 0.200,
0.400, and 0.600 M Mg(ClO4)2 salt. (b) Plots of the second-order rate
constant (kr) for the reaction of ubiquinol-10 with α-Toc• radical versus the
concentration of metal salts: (a) LiClO4, (b) NaClO4, and (c) Mg(ClO4)2.
Measurements were performed in methanol solution at 25.0 °C.
measurements of the kr values (=1.46 × 105, 1.53 × 105, and
1.45 × 105 M−1 s−1) were repeated three times, and the average
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From the results, it has been suggested that reaction 5 proceeds
via an electron-transfer intermediate [ArO:−···α-TocH•+] in
methanol (see Figure 7 in ref 29).
In the present work, measurement of the rate constant (kr)
for the reaction of α-Toc• with UQ10H2 was also performed in
protic methanol solution including LiClO4, NaClO4, NaI, and
Mg(ClO4)2 salts. As described in the Results section, the
changes of UV−vis absorption spectra of α-Toc•, α-TocH, and
UQ10H2 were negligible in methanol in the presence of four
metal salts (4.00 × 10−1 M), showing that the interactions
between these neutral molecules and the cations (and anions)
of metal salts are negligibly weak. Similarly, the interaction
between UQ10H• and the cations (and anions) of metal salts is
thought to be negligible in methanol, although we could not
observe the absorption spectrum of UQ10H• radical because of
its instability.34 The results suggest that both the total energy
levels of the reactants (α-Toc• + UQ10H2) and the reaction
products (α-TocH + UQ10H•) in reaction 3 do not vary in the
presence of metal salts, as shown in Figure 6a.
Figure 5. (a) Dependence of pseudo-first-order rate constant (kobsd) on
concentration of ubiquinol-10 in acetonitrile including 0.000, 0.050, 0.100,
0.150, and 0.200 M Mg(ClO4)2 salt. (b) Plots of the second-order rate
constant (kr) for the reaction of ubiquinol-10 with the α-Toc• radical versus
the concentration of metal salts ((a) LiClO4, (b) NaClO4, and (c) Mg-
(ClO4)2). Measurements were performed in acetonitrile solution at 25.0 °C.
Table 2. Second-Order Rate Constants (kr's) for the
Reaction of Ubiquinol-10 with α-Toc• Radical in
Acetonitrile Solution Including Various Concentrations of
Alkali and Alkaline Earth Metal Salts at 25.0 °C
Figure 6. (a) Mechanism of the α-tocopherol-regeneration reaction of
UQ10H2 in methanol. The figure shows that the transition state for the
reaction of α-Toc• with UQ10H2 has the property of an electron-
transfer intermediate [α-Toc:−···UQ10H2•+]. The effects of metal salts
on the reaction rates (kr's) may be explained by considering two kinds
of equilibrium between (i) α-Toc:− anion and M+ cation and (ii)
[metal
salt]/M
LiClO4 kr/
NaClO4 kr/
NaI kr/
Mg(ClO4)2 kr/
M−1s−1
M−1s−1
M−1s−1
M−1s−1
a
a
a
a
0
1.20 × 105
7.01 × 104
1.20 × 105
1.20 × 105
1.20 × 105
0.050
0.100
0.150
0.200
0.300
0.400
0.500
0.600
5.27 × 104
1.80 × 104
1.44 × 104
1.07 × 104
−
UQ10H2•+ cation and ClO4 anion at the transition state (see text).
1.17 × 105
(b) Mechanism of the α-tocopherol-regeneration reaction of UQ10H2
in acetonitrile. The energy level of the reactants (α-Toc• + UQ10H2) is
lowered by the complex formation between α-Toc• and metal cations
(M+ (or M2+)) (see text).
1.15 × 105
1.08 × 105
1.06 × 105
1.02 × 105
1.18 × 105
1.13 × 105
1.07 × 105
6.05 × 104
5.52 × 104
3.55 × 104
As shown in Figure 4b, the kr values increased in the order of
eq 8 in methanol. The order of eq 8 is the same as that of eq 10,
suggesting that reaction 3 in methanol also proceeds via an
electron-transfer intermediate. As shown in Figure 6a, if the
transition state of the α-Toc•-scavenging reaction by UQ10H2
has the property of an electron-transfer intermediate [α-
Toc:−···UQ10H2•+], the metal cations (Li+, Na+, and Mg2+) will
interact with α-Toc:− anion by Coulomb interaction and form a
complex with a α-Toc:− anion [α-Toc:−···M+ (or M2+)].29
a
Measurement of the kr value for [salt] = 0 M was repeated three
times (1.23 × 105, 1.21 × 105, and 1.15 × 105 M−1 s−1). An average
value (av 1.20 × 105 M−1 s−1) was shown in the table.
of metal salts. Furthermore, the ks values increased in the following
order at the same concentration of metal salts:
without metal salt < KI ∼ NaClO ∼ NaI ≤ LiClO
4
4
−
Similarly, the ClO4 anion of the salts will also interact with
UQ10H2•+ cation and form a complex with an UQ10H2•+ cation
< Mg(ClO ) < LiI
(10)
4 2
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[UQ10H2•+···ClO4 ]. As a result, the energy level of an
the following way: as the stability constant (K) increases in the
order NaClO4 < LiClO4 < Mg(ClO4)2, the stabilization energy
of the reactants (α-Toc• + UQ10H2) will increase in the order
NaClO4 < LiClO4 < Mg(ClO4)2. Consequently, the activation
energy for reaction 3 increases in the order NaClO4 < LiClO4 <
Mg(ClO4)2, resulting in the decrease in the kr value in the order
of NaClO4 > LiClO4 > Mg(ClO4)2 (see Figure 6b).
As reaction 3 indicates, the products for reaction 3 are
α-TocH + UQ10H•. We may expect the complex formation
[UQ10H•···M+ (or M2+)] between UQ10H• radical and metal
cations in acetonitrile solution. However, we could not observe
the UV−vis absorption spectrum of UQ10H• radical, because
the UQ10H• radical is unstable (half-life τ = 1.17 μs)34 and
immediately changes to UQ10 and UQ10H2 by disproportiona-
tion reaction. Therefore, we could not ascertain a lowering of
the total energy level of the reaction products (α-TocH +
UQ10H•). The result of the present kinetic study indicates that
a lowering of the energy level of the α-Toc• due to the complex
formation mainly contributes to the decrease of the reaction
rates (kr's) in acetonitrile.
4.3. Alkali and Alkaline Earth Metal Salts May Affect
the Regeneration Reaction of α-Tocopherol by Ubiq-
uinol-10 in Biological Systems and the Prooxidant
Reaction of α-Tocopherol in Edible Oils. As described in
the Introduction, α-TocH, UQ10H2, and alkali and alkaline
earth metal ions (Na+, K+, Mg2+, and Ca2+) coexist in blood,
plasma, plasma lipoproteins, and cellular membranes.21−24 For
instance, high concentrations of Na+ and K+ ions are included
in human blood ([Na+] = 81.7 mM and [K+] = 44.6 mM) and
plasma ([Na+] = 136 mM and [K+] = 3.6 mM).21 The concen-
trations of metal cations found in mammals are [Na+] = 140
mM, [K+] = 4 mM, [Mg2+] = 1.5 mM in extracellular fluid and
[Na+] = 10 mM, [K+] = 140 mM, [Mg2+] = 30 mM in intra-
cellular fluids.37 The concentrations of α-TocH and UQ10H2 in
human plasma were reported to be av 22.0 (12.0−36.0) μM23,24
and av 0.71 (0.36−1.10) μM,12 respectively. Therefore, the con-
centrations of Na+, K+, and Mg2+ ions are much higher than those
of α-TocH and UQ10H2 in human plasma.
−
electron-transfer intermediate [α-Toc:−···UQ10H2•+] will be
stabilized (that is, the activation energy (Eact) will decrease),
and thus, the kr values increase. The Coulomb interaction
energy will increase with decreasing the ionic radius of cations
(Na+, Li+, and Mg2+) and with increasing the cation charge
from +1e to +2e (see Table 1). In fact, the rate constants (kr's)
increase in the order of eq 8. The kr value observed for NaI
showed a little bit larger value than that for NaClO4, although
−
the difference is very small. As the ionic radii of anions (ClO4
and I−) are larger than those of cations, the effect of the anion
may be smaller than that of the cation. The details of the effect
of the anion are not clear at present.
4.2. Mechanism of the Regeneration Reaction of α-
Tocopherol by Ubiquinol-10 in Acetonitrile Solution
Including Metal Salts. In the present work, measurement of
the rate constant (kr) for the reaction of α-Toc• with UQ10H2
was performed in aprotic acetonitrile solution including LiClO4,
NaClO4, NaI, and Mg(ClO4)2 salts. As shown in Figure 5 and
listed in Table 2, a notable effect of metal salts on the rate
constants (kr) was observed in acetonitrile. The rate constants
(kr's) decreased rapidly with increasing concentrations of metal
salts. Furthermore, the kr values decreased in the order of eq 9
at the same concentration of metal salts, differing from the
increase of the kr values in methanol.
As described in section 3.1, a notable effect of the metal salts
(LiClO4, NaClO4, NaI, and Mg(ClO4)2) on the UV−vis absorp-
tion spectrum of α-Toc• was observed in acetonitrile. As shown in
Figure 3, the λmax value (424.3 nm) of α-Toc• without metal salts
increased with increasing concentrations of metal salts in
acetonitrile, suggesting the [α-Toc•···M+ (or M2+)] complex
formation between α-Toc• radical and metal cations.25,30 The
effect of anion of the metal salts is negligible, because NaClO4 and
NaI show a similar shift (data are not shown). If M+ ion forms a
(1:1) complex with α-Toc•, the following equilibrium between
α-Toc• and M+ ion may exist in acetonitrile solution.
K
+
+
α ‐ Toc • + M ⇌ [α ‐ Toc •···M ]
(11)
Na+ ion has the highest concentration ([Na+] = 136 mM)
among alkali and alkaline earth metal ions in plasma. As listed
in Tables 1 and 2, the kr values in methanol and acetonitrile
with 136 mM of Na+ ion are by ∼3% larger and by ∼3% smaller
than those without Na+ ion, respectively. Similarly, the kr values
in methanol and acetonitrile with 30 mM of Mg2+ ion are
by ∼3% larger and by ∼34% smaller than those without Mg2+
ion, respectively. The effects of Na+ ion on the α-tocopherol-
regenerating rate constants (kr's) are small and negligible in
methanol and acetonitrile solutions. On the other hand, the effect
of Mg2+ ion on the reaction rate (kr) is notable in acetonitrile.
As described in the Introduction, α-Toc• radical is an im-
portant key radical, which appears in the process of the
antioxidant and prooxidant actions of α-TocH (see reactions
1−4). In the present work, a detailed kinetic study has been
performed for regeneration reaction 3, and the mechanisms
involved and the effect of metal salts on the reaction rates (kr)
in protic methanol and aprotic acetonitrile solutions have been
studied.
Here, K is a stability constant for eq 11. With increasing
concentrations of the metal salts, the concentrations of the
complex ([α-Toc•···M+]) will increase gradually, and thus, the
shifts of the absorption maximum (λmax) proceed. Recently, the
stability constant (K) was determined to be 9.2, 2.8, and
45 M−1 for LiClO4, NaClO4, and Mg(ClO4)2, respectively.30 The
value increases in the order NaClO4 < LiClO4 < Mg(ClO4)2.
The result indicates that the metal cation having smaller ionic
radius and larger positive charge of +2e gives a larger shift of
the λmax value and a larger K value.
As described above, a notable red shift of the λmax value of the
α-Toc• was observed in the presence of the metal salts in
acetonitrile, indicating the complex formation between α-Toc•
and metal cations. On the other hand, the shift of the λmax value
of UQ10H2 (and α-TocH) was negligible in acetonitrile in the
presence of metal salts, showing that the interactions between
UQ10H2 (and α-TocH) and metal cations are negligibly weak.
The results suggest that the total energy level of the reactants
(α-Toc• + UQ10H2) in reaction 3 will be lowered by the
complex formation (see Figure 6b). In such a case, the dec-
rease in the reaction rate constant (kr) will be observed in the
presence of metal salts, as expected from the Evans−Polanyi
theory.35,36 Further, the difference between the rate constants
(kr's) for NaClO4, LiClO4, and Mg(ClO4)2 will be explained in
As described in section 4.2, α-Toc• radical molecule forms a
1:1 complex with metal cations in acetonitrile. When α-Toc•
forms a complex with metal cations (such as Na+, K+, and Mg2+
ions), the total energy levels of the reactants (α-Toc• + LH) in
reaction 2 will decrease,30 and thus, the kp value will decrease.11
That is, the prooxidant effect of α-TocH, which is observed in
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Article
(13) Mukai, K.; Itoh, S.; Morimoto, H. J. Biol. Chem. 1992, 267,
22277−22281.
aprotic edible oils including high concentration of α-TocH, may
be suppressed by the addition of the metal salts (NaCl, KCl,
MgCl2, and CaCl2). We can expect notable effects of metal salts
on the above biologically important reactions 2 and 3. However,
the rate constant (kr) increased and decreased with increas-
ing concentrations of metal salts, depending on the kinds of the
solvents used for the reaction (that is, the character of the
reaction field). It will be interesting to investigate the effects of
metal salts on the reaction rates (kp and kr) in model membrane
systems, such as micellar solution or liposome.
(14) Ouchi, A.; Nagaoka, S.; Mukai, K. J. Phys. Chem. B 2010, 114,
6601−6607.
(15) Packer, J. E.; Slater, T. F.; Willson, R. L. Nature 1979, 278, 737−
738.
(16) Bisby, R. H.; Parker, A. W. Arch. Biochem. Biophys. 1995, 317,
170−178.
(17) Nagaoka, S.; Kakiuchi, T.; Ohara, K.; Mukai, K. Chem. Phys.
Lipids 2007, 146, 26−32.
(18) Lucarini, M.; Pedulli, G. F.; Cipollone, M. J. Org. Chem. 1994,
59, 5063−5070.
(19) Gregor, W.; Grabner, G.; Adelwhrer, C.; Rosenau, T.; Gille, L.
J. Org. Chem. 2005, 70, 3472−3483.
5. SUMMARY
A kinetic study of regeneration reaction of α-TocH by UQ10H2
has been performed in the presence of four kinds of alkali and
alkaline earth metal salts in methanol and acetonitrile solutions,
using double-mixing stopped-flow spectrophotometry. In protic
methanol solution, the second-order rate constant (kr) for the
reaction of α-Toc• radical with UQ10H2 increased, with increas-
ing concentrations of metal salts, and it increased in the follow-
ing order at the same concentration of metal salts: no metal salt
< NaClO4 ∼ NaI < LiClO4 < Mg(ClO4)2. On the other hand,
in aprotic acetonitrile solution, the rate constant (kr) decreased
notably, with increasing concentrations of metal salts, and it
decreased in the following order at the same concentration of
(20) Mukai, K.; Ouchi, A.; Mitarai, A.; Ohara, K.; Matsuoka, C. Bull.
Chem. Soc. Jpn. 2009, 82, 494−503.
(21) Takeda, R.; Nakamura, T.; Saito, Y.; Takeda, A.; Yamashita, C.;
Shigetomi, H.; Takeda, T.; Kimura, M. Biomed. Res. Trace Elem. 2007,
18, 277−280.
(22) Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman and Company:
New York, 1988.
(23) Colome, C.; Artuch, R.; Vilaseca, M.-A.; Sierra, C.; Brandi, N.;
Lambruschini, N.; Cambra, F. J.; Campistol, J. Am. J. Clin. Nutr. 2003,
77, 185−188.
(24) Machlin, L. J. Vitamin E. In Handbook of Vitamins; Machlin, L.
J., Ed.; Marcel Dekker Inc.: New York, 1991; pp 99−144.
(25) Nakanishi, I.; Fukuhara, K.; Shimada, T.; Ohkubo, K.; Iizuka, Y.;
Inami, K.; Mochizuki, M.; Urano, S.; Itoh, S.; Miyata, N.; Fukuzumi, S.
J. Chem. Soc., Perkin Trans. 2 2002, 1520−1524.
(26) Nakanishi, I.; Kawashima, T.; Ohkubo, K.; Kanazawa, H.; Inami,
K.; Mochizuki, M.; Fukuhara, K.; Okuda, H.; Ozawa, T.; Itoh, S.;
Fukuzumi, S.; Ikota, N. Org. Biomol. Chem. 2005, 3, 626−629.
(27) Nakanishi, I.; Miyazaki, K.; Shimada, T.; Ohkubo, K.; Urano, S.;
Ikota, N.; Ozawa, T.; Fukuzumi, S.; Fukuhara, K. J. Phys. Chem. A
2002, 106, 11123−11126.
(28) Nakanishi, I.; Shimada, T.; Ohkubo, K.; Manda, S.; Shimizu, T.;
Urano, S.; Okuda, H.; Miyata, N.; Ozawa, T.; Anzai, K.; Fukuzumi, S.;
Ikota, N.; Fukuhara, K. Chem. Lett. 2007, 36, 1276−1277.
(29) Ouchi, A.; Nagaoka, S.; Abe, K.; Mukai, K. J. Phys. Chem. B
2009, 113, 13322−13331.
(30) Kohno, Y.; Fujii, M.; Matsuoka, C.; Hashimoto, H.; Ouchi, A.;
Nagaoka, S.; Mukai, K. J. Phys. Chem. B 2011, 115, 9880−9888.
(31) Rieker, A.; Scheffler, K. Liebigs Ann. Chem. 1965, 689, 78−92.
(32) Podda, M.; Weber, C.; Traber, M. G.; Packer, L. J. Lipid Res.
1996, 37, 893−901.
(33) Mukai, K.; Tokunaga, A.; Itoh, S.; Kanesaki, Y.; Ohara, K.;
Nagaoka, S.; Abe, K. J. Phys. Chem. B 2007, 111, 652−662.
(34) Parker, A. W.; Hester, R. E.; Phillips, D.; Umapathy, S. J. Chem.
Soc., Faraday Trans. 1992, 88, 2649−2653.
(35) Evans, M. G.; Polanyi, M. Trans. Faraday Soc. 1938, 34, 11−24.
(36) Kagiya, T.; Sumida, Y.; Inoue, T. Bull. Chem. Soc. Jpn. 1969, 42,
2422−2425.
metal salts: no metal salt > NaClO4 ∼ NaI > LiClO4
>
Mg(ClO4)2. The decrease of the kr value in acetonitrile will be
due to the complex formation between α-Toc• and metal
cations. The alkali and alkaline earth metal salts having smaller
ionic radius of cation and larger cation charge gave a larger kr
value in methanol solution, and a smaller kr value in acetonitrile
solution. From the results, the mechanisms for the above two
different types of reactions observed in methanol and acetonitrile
solutions were discussed.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 81-89-927-9588. Fax: 81-89-927-9590. E-mail: mukai-k@
dpc.ehime-u.ac.jp.
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Niki, E. Chem. Phys. Lipids 1987, 44, 227−253.
(2) Barclay, L. R. C. Can. J. Chem. 1993, 71, 1−16.
(3) Traber, M. G.; Atkinson, J. Free Radical Biol. Med. 2007, 43, 4−
15.
(37) Murray, R. K.; Granner, D. K.; Mayes, P. A.; Rodwell, V. W.
Membranes: Structure, Assembly, and Function. Harper’s Biochemistry,
25th ed.; McGraw-Hill: New York, 2000; Chapter 43, pp 505−533.
(4) Esterbauer, H.; Dieber-Rotheneder, M.; Striegel, G.; Waeg, G.
Am. J. Clin. Nutr. 1991, 53, 314S−321S.
(5) Neuzil, J.; Thomas, S. R.; Stocker, R. Free Radical Biol. Med. 1997,
22, 57−71.
(6) Burton, G. W.; Doba, T.; Gabe, E. J.; Hughes, L.; Lee, F. L.;
Prasad, L.; Ingold, K. U. J. Am. Chem. Soc. 1985, 107, 7053−7065.
(7) Cillard, J.; Cillard, P.; Cormier, M.; Girre, L. J. Am. Oil Chem. Soc.
1980, 57, 252−255.
(8) Terao, J.; Matsushita, S. Lipids 1986, 21, 255−260.
(9) Bowry, V. W.; Stocker, R. J. Am. Chem. Soc. 1993, 115, 6029−
6044.
(10) Mukai, K.; Noborio, S.; Nagaoka, S. Int. J. Chem. Kinet. 2005, 37,
605−610.
(11) Ouchi, A.; Ishikura, M.; Konishi, K.; Nagaoka, S.; Mukai, K.
Lipids 2009, 44, 935−943.
(12) Coenzyme Q: Molecular Mechanisms in Health and Disease;
Kagan, V. E., Quinn, P. J., Eds.; CRC Press: Boca Raton, FL, 2001.
2621
dx.doi.org/10.1021/jp2118086 | J. Phys. Chem. B 2012, 116, 2615−2621