Kinetics of the reaction by which natural vitamin E is regenerated by vitamin C

Article abstract of DOI:10.1016/j.chemphyslip.2006.12.001

The rate constant and activation energy of the regeneration reaction of natural vitamin E by vitamin C were determined with a double-mixing stopped-flow spectrophotometer. The formation of vitamin C radical was observed in the absorption spectrum. The kinetic effect of methyl substitution on the aromatic ring of vitamin E radical indicates that partial charge-transfer plays a role in the reaction. Since a substantial deuterium kinetic isotope effect was not found, the tunneling effect may not play an important role under the present experimental conditions.

Full text of DOI:10.1016/j.chemphyslip.2006.12.001

Chemistry and Physics of Lipids 146 (2007) 26–32
Kinetics of the reaction by which natural
vitamin E is regenerated by vitamin C
Shin-ichi Nagaoka∗, Takuhiro Kakiuchi, Keishi Ohara, Kazuo Mukai∗∗
Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790-8577, Japan
Received 9 June 2006; received in revised form 4 December 2006; accepted 22 December 2006
Available online 30 December 2006
Abstract
The rate constant and activation energy of the regeneration reaction of natural vitamin E by vitamin C were determined with a
double-mixing stopped-flow spectrophotometer. The formation of vitamin C radical was observed in the absorption spectrum. The
kinetic effect of methyl substitution on the aromatic ring of vitamin E radical indicates that partial charge-transfer plays a role in
the reaction. Since a substantial deuterium kinetic isotope effect was not found, the tunneling effect may not play an important role
under the present experimental conditions.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Vitamin E regeneration; Vitamin C; Reaction rate; Activation energy; Tocopherol; Ascorbate
1. Introduction
Fig. 1 shows the scheme of LOO• production and of
a part of the antioxidant reaction of TocH (Burton and
Ingold, 1986; Niki, 1987, 1989; Diplock et al., 1989). A
lipid radical (L•) is first formed from lipid (LH), usually
because of radicals, light, heat, metal (Fukuzawa et al.,
1991) or irradiation. The L• radical reacts with oxygen
to produce LOO•, and the reaction of LOO• and TocH
results in production of lipid hydroperoxide (LOOH) and
a vitamin E radical (tocopheroxyl radical, Toc•). Fig. 2
shows the molecular structures of natural TocH’s (␣-, ␤-,
␥- and ␦-TocH’s), each of which has a hydroxychroman
ring and a phytyl side chain that stabilizes TocH in the
cell membrane. These TocH’s differ from one another
only in the number and positions of the methyl groups
on the aromatic ring. The most biologically active of
these four TocH’s is the fully methylated ␣-TocH.
Although no theory advanced to account for aging is
generally accepted, one of the promising is the free rad-
ical theory (Harman, 2001) postulating that aging is in
part caused by lipid peroxyl radicals (LOO•’s) formed by
the reactions of lipids and oxygen (Cutler, 1984; Burton
andIngold, 1986;Niki, 1987, 1989;Diplocketal., 1989).
The living body, however, has a way to scavenge LOO•
and thus to help prevent aging: it is the so-called antiox-
idant reaction of vitamin E (d-tocopherols, TocH’s).
Evidence for this is that the lifetimes of mammalian
species are proportional to their plasma levels of a TocH
(Cutler, 1984).
The Toc• produced in the above-mentioned reaction
reacts with LH and LOOH and again produces L• and
LOO• (Fig. 1). This prooxidant action of TocH (Cillard
et al., 1980; Terao and Matsushita, 1986) is suppressed
when TocH is regenerated by the reaction of Toc• with
∗
Corresponding author. Tel.: +81 89 927 9592; fax: +81 89 927
9590.
∗∗
Corresponding author. Fax: +81 89 927 9590.
E-mail addresses: nagaoka@ehimegw.dpc.ehime-u.ac.jp
(S.-i. Nagaoka), mukai@chem.sci.ehime-u.ac.jp (K. Mukai).
0009-3084/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2006.12.001

S.-i. Nagaoka et al. / Chemistry and Physics of Lipids 146 (2007) 26–32
27
vitamin C (ascorbate monoanion, AsH−) at the interface
of the cell membrane and the water phase (Packer et al.,
1979; Scarpa et al., 1984; Niki et al., 1984; Mukai et al.,
1987, 1989, 1991; Sato et al., 1990; Bisby and Parker,
1995; Watanabe et al., 1999) (Fig. 1).
All of the above-mentioned reactions of TocH are
essentially proton- or hydrogen-transfer reactions:
TocH + LOO• → Toc• + LOOH,
Toc• + LH → TocH + L•,
(1)
(2)
(3)
(4)
Toc• + LOOH → TocH + LOO•,
Fig. 1. Scheme of LOO• production and of part of the antioxidant,
prooxidant and regeneration reactions of TocH in the cell membrane.
Toc• + AsH− → TocH + As−
,
•
where As−• denotes the dehydroascorbate monoanion
radical. Reaction (3) is the reversal of reaction (1). It
would be interesting to study the tunneling effect in the
proton or hydrogen transfer of these reactions.
In previous studies using a conventional stopped-
flow spectrophotometer (Mukai et al., 1987, 1989, 1991;
Nagaoka et al., 2000; Nagaoka, 2004), we investigated
the kinetics of reaction (4) by using 5,7-diisopropyl-
Toc• or 7-t-butyl-5-isopropyl-Toc• (Fig. 2) because the
Toc•’s originating from natural ␣-, ␤-, ␥- and ␦-TocH’s
are so short-lived that the kinetics of their reaction (4)
cannot be examined with the conventional method. The
results obtained using 5,7-diisopropyl-Toc• showed that
the tunneling effect plays an important role in reaction
(4) (Nagaoka et al., 2000; Nagaoka, 2004). Bisby and
Parker (1995) studied the kinetics of reaction (4) using
␣-Toc• and laser flash photolysis, but the formation of
As−• has not yet been observed nor has the effect of
methyl substitution on the aromatic ring of Toc• been
examined. In the work presented here we have observed
the decays of ␣-, ␤- and ␥-Toc•’s and the formation
of As−• by using a double-mixing stopped-flow spec-
trophotometer, which allows us to examine the kinetics
of the reactions of short-lived radicals. We have deter-
mined the rate constants and activation energies for
reaction (4) and examined the deuterium kinetic isotope
effect.
2. Experimental procedures
2.1. Sample preparation
The structures of the molecules studied in this work
are shown in Fig. 2. The d-␣-, d-␤-, d-␥-, and d-␦-
TocH’s kindly supplied by Eisai Co., Ltd. were used
without further purification. We prepared 2,6-di-t-butyl-
4-(4-methoxyphenyl)phenoxyl (ArO•) as reported in a
previous paper (Rieker and Scheffler, 1965). In aqueous
Fig. 2. Structures of molecules used in the present work.

28
S.-i. Nagaoka et al. / Chemistry and Physics of Lipids 146 (2007) 26–32
solutions around pH 7, vitamin C exists mostly as the
monoanion (AsH−) in which the proton at the 3-position
is dissociated (Fig. 2) (Mukai et al., 1991). Accordingly,
sodium ascorbate (Na+AsH−) was used in the present
work. Na+AsH− (guaranteedreagent)wasobtainedfrom
Nacalai Tesque and was used without further purifica-
tion. Ethanol-d0 (EtOH) was obtained from Wako, dried
and purified by distillation. Water-d0 (H2O) was purified
by ion exchange. Ethanol-d1 (C2H5OD, EtOD) of 99%
purity and water-d2 (D2O) of 99% purity were purchased
from Cambridge Isotope Laboratories and used without
further purification.
When TocH is dissolved in EtOD, the hydrogen atom
of the OH group is easily replaced by a deuteron to
yield the deuterated molecule TocD. This replacement
was confirmed by proton NMR. When Na+AsH− is dis-
solved in D2O, the hydrogen atoms of the OH groups
are easily replaced by deuterons to yield the deuterated
molecule Na+AsD−. This replacement was confirmed
by the proton NMR spectrum of Na+AsH− in DMSO
showing peaks due to the hydrogen atoms of the OH
groups and by the disappearance of those peaks when a
small amount of D2O was added.
In the experiments evaluating the kinetics of reac-
tion (4), EtOH (EtOD) and H2O (D2O) were mixed in a
5:1 volume ratio to obtain a mixed solvent EtOH/H2O
(EtOD/D2O) because Toc• is alcohol-soluble and
Na+AsH− is water-soluble. An EtOH/H2O (EtOD/D2O)
solution of Na+AsH− (Na+AsD−) was produced by first
dissolving Na+AsH− in H2O (D2O) and then adding
EtOH (EtOD), and an EtOH/H2O (EtOD/D2O) solution
of TocH (TocD) was produced by first dissolving TocH
in EtOH (EtOD) and then adding H2O (D2O).
Fig. 3. Change in absorption spectrum (at 20-ms intervals) during
the reaction of ␣-TocH and ArO• in EtOH/H2O at 25 ◦C (reaction
(1ꢀ)). [␣-TocH]t=0 = 0.839 mM. The arrows indicate the decrease in
the absorbance of ArO• and the increase in the absorbance of ␣-Toc•.
umes of EtOH/H2O (EtOD/D2O) solutions of TocH
(TocD) and ArO• were mixed under a nitrogen atmo-
sphere (reaction (1ꢀ)). The change in the absorption
spectrum measured during reaction (1ꢀ) is shown in
Fig. 3. Although ArO• was stable in the absence of
TocH (TocD), when an EtOH/H2O (EtOD/D2O) solu-
tion with excess TocH (TocD) was added to the ArO•
solution, the ArO• absorption peak disappeared imme-
diately, the Toc• peak appeared, and the isosbestic points
were observed. Since ␦-Toc• is very unstable, its peak
was not seen in the absorption spectra measured during
reaction (1ꢀ).
Reaction (1ꢀ) yielded the maximum concentration
of Toc• after about 1 s in EtOH/H2O (about 10 s in
EtOD/D2O), andtheresultantmixtureandanEtOH/H2O
(EtOD/D2O) solution of Na+AsH− (Na+AsD−) were
mixed under a nitrogen atmosphere (reaction (4ꢀ)). The
change in absorption spectrum during reaction (4ꢀ) is
shown in Fig. 4: the Toc• absorption peak disappeared,
2.2. Measurements
The reactions studied in the present work were the
following ones:
TocH (TocD) + ArO• → Toc• + ArOH (ArOD),
(1ꢀ)
Toc• + Na+AsH− (Na+AsD−)
−→ TocH (TocD) + Na+As−
k
•
,
(4ꢀ)
where k denotes the second-order rate constant for reac-
tion (4ꢀ).
The kinetic data of reaction (4ꢀ) were obtained using a
Unisoku double-mixing stopped-flow spectrophotome-
ter Model RSP-1000-03F. The experimental error in the
temperature was less than 0.5 ◦C. First, equal vol-
Fig. 4. Change in absorption spectrum (at 2-ms intervals) during the
reaction of ␣-Toc• and Na+AsH− in EtOH/H2O at 25 ◦C (reaction
(4ꢀ)). [Na+AsH−]t=0 = 38.5 ␮M. Thearrowsindicatethedecreaseinthe
absorbance of ␣-Toc• and the increase in the absorbance of Na+As−•
.

S.-i. Nagaoka et al. / Chemistry and Physics of Lipids 146 (2007) 26–32
29
Fig. 5. Dependence of kobsd on [Na+AsH−] in the reaction of ␣-Toc•
and Na+AsH− in EtOH/H2O at 25 ◦C (Eq. (5)), where [Na+AsH−]
stands for the concentration of Na+AsH− in the solution. The value
of kobsd was obtained by monitoring the decrease in the absorbance of
␣-Toc• at 430 nm (Fig. 4).
Fig. 6. Arrhenius plots of the k’s for the reactions of ␣-Toc• with
Na+AsH− in EtOH/H2O (open circles), ␣-Toc• with Na+AsD− in
EtOD/D2O (closed circles), ␤-Toc• with Na+AsH− in EtOH/H2O
(open squares), and ␤-Toc• with Na+AsD− in EtOD/D2O (closed
squares).
the Na+As−• (Fig. 2) peak appeared, and the isosbestic
points were observed.
against the reciprocal of temperature (1/T) (an Arrhenius
plot) is a straight line (Atkins and de Paula, 2002). This
behavior is normally expressed mathematically in the
following way:
Pseudo-first-order conditions were assumed in the
analyses (Mukai et al., 1987, 1989, 1991; Nagaoka et
al., 2000; Nagaoka, 2004), and the absorption decay
of Toc• in reaction (4ꢀ) was well characterized by a
single-exponential decay. The pseudo-first-order rate
constant (kobsd) was determined by evaluating the
decrease in the absorbance of Toc•. As shown in
Fig. 5, kobsd was linearly dependent on the concen-
tration of Na+AsH− (Na+AsD−). The location of the
intercept depends on the rate of the dimerization reac-
tion (Toc• + Toc• → Toc − Toc) (Watanabe et al., 1999,
2000). The rate equation is thus expressed as
E
log k = log A −
,
(6)
2.303RT
where E, A and R, respectively, denote the activation
energy, frequency factor and gas constant. Linear rela-
tionships between log k and 1/T can be seen in the
Arrhenius plots of the k’s for the reaction of Toc•’s with
Na+AsH− and Na+AsD− (Fig. 6). Let the k’s for the
reactions of a Toc• with Na+AsH− and of the Toc• with
Na+AsD−, respectively, be kH and kD. The values of k at
25 ◦C and the average ratio of kH to kD (kH/kD) at tem-
peratures from 15 to 35 ◦C are listed in Table 1 along
with the values of E and log A.
d[Toc•]
−
= kobsd [Toc•]
dt
For Na+AsH− at 25 ◦C the order of the k values is as
follows: ␣-Toc• < ␤-Toc• ≈ ␥-Toc•. Thus the k value for
␣-Toc•, having three electron-donating methyl groups,
is less than the k values for ␤-Toc• and ␥-Toc•, each
having two electron-donating methyl groups, and the k
values for ␤-Toc• and ␥-Toc• are close to each other. The
weaker the electron-donating property of the substituent
in Toc•, the larger the k value. These results suggest
that the following partial charge-transfer plays a role in
reaction (4):
= k[Na+AsH− (Na+AsD−)][Toc•],
(5)
where [X] (X = Toc•, Na+AsH− or Na+AsD−) stands
for the concentration of X in the solution. The k
value was obtained by plotting kobsd against [Na+AsH−
(Na+AsD−)] (Fig. 5) and using a standard least-squares
analysis.
The temperature dependences of the k’s for ␣-Toc•
and ␤-Toc• were measured (Fig. 6), but the temperature
dependence of the k for ␥-Toc• could not be examined
with our apparatus because ␥-Toc• is unstable. Only its k
value in EtOH/H2O at 25 ◦C was obtained in the present
study.
Toc• + AsH− → [(Toc•)δ−· · ·(AsH−)δ+
]
→ TocH + As−
.
(7)
•
3. Results and discussion
A similar charge-transfer in reaction (1) was suggested
by the results of stopped-flow spectroscopy (Nagaoka et
al., 1992, 2000; Nagaoka, 2004) and femtosecond spec-
troscopy (Nagaoka and Ishihara, 1996) as well as by
It is found experimentally that for many reactions a
plot of the logarithm of the reaction rate constant (log k)

30
S.-i. Nagaoka et al. / Chemistry and Physics of Lipids 146 (2007) 26–32
Table 1
E, log A, k at 25 ◦C and kH/kD values for the reactions of various tocopherol radicals with ascorbic acid and ascorbate monoanions
Reaction
k at 25 ◦C (M−1 s−1
)
kH/kD
E (kJ/mol)
log Aa
␣-Toc• + Na+AsH−
␣-Toc• + Na+AsD−
␤-Toc• + Na+AsH−
␤-Toc• + Na+AsD−
␥-Toc• + Na+AsH−
␣-Toc• + ascorbic acidb
2.73 × 106
4.87 × 105
3.65 × 106
1.00 × 106
3.81 × 106
4.97 × 104
5.6
–
3.9
–
–
7.97
4.0 0.4
6.4 0.1
27.2 1.3
40.5 3.5
–
7.1 0.1
6.8 0.1
11.3 0.2
13.1 0.6
–
18.9 0.5
–
a
Because log A was obtained by extrapolating the linear log k vs. 1/T plot in a limited 1/T range around room temperature to the intercept, the
log A values thus obtained have a large uncertainty.
b
In SDS micelles (Bisby and Parker, 1995).
time-dependent density functional theory (Duan et al.,
2004).
The absence of the tunneling effect in ␣- and ␤-Toc•’s
is not consistent with the important role the tunneling
effect plays in reaction (4ꢀꢀ-1) (Nagaoka et al., 2000;
Nagaoka, 2004). This inconsistency seems to be a result
of steric hindrance due to the two isopropyl groups at
the 5- and 7-positions of the Toc•. This steric hindrance
prevents access of Na+AsH− (Na+AsD−) to the O• at
the 6-position of 5,7-diisopropyl-Toc• (Fig. 2) in solu-
tions and gives large E’s in reaction (4ꢀꢀ-1) [11.1 kJ/mol
for Na+AsH− and 24.2 kJ/mol for Na+AsD− (Nagaoka
et al., 2000)]. Tunneling allows the proton to cut a
corner on the potential surface with the high energy
barrier, and the proton tunneling takes place below
the transition state in reaction (4ꢀꢀ-1). In reaction (4ꢀ)
for natural TocH’s, in contrast, since the steric hin-
drance giving large E’s is absent, the proton prefers
to jump semiclassically over the low energy barrier
(E = 4.0 kJ/mol for ␣-Toc• + Na+AsH− and 6.4 kJ/mol
for ␣-Toc• + Na+AsD−) rather than to tunnel through
it. As a result, the tunneling effect does not play an
important role in reaction (4ꢀ) in vitro. In vivo, how-
ever, the energy barrier of reaction (4) would be higher
than that in a uniform solution because the collisions
between Toc• in the cell membrane and AsH− in the
water phase (Fig. 1) would be less frequent than the
collisions between the free Toc• and AsH− in a uni-
form solution. Accordingly, owing to the high energy
barrier in vivo, the proton tunneling might take place
below the transition state in reaction (4). In fact, the
E value of reaction (4ꢀꢀ-1) for Na+AsH− in a micellar
dispersion (29.7 kJ/mol), which is a model of the cell
membrane, is much larger than that in a uniform solution
(11.1 kJ/mol) (Nagaoka et al., 2000). Bisby and Parker
(1995) also reported high E’s for reaction (4) in micelles
and bilayers (17.3–28.8 kJ/mol) and low E’s for reac-
tion (4) in uniform solutions (2.2–2.3 kJ/mol). Miyazaki
and Kumagai (2004) also concluded that the tunneling
effect plays an important role in mutation-suppression by
vitamin C.
It is interesting that ␤- and ␥-TocH’s are more
effective in antioxidant regeneration (reaction (4)) than
␣-TocH, which is the most biologically active of the four
natural TocH’s shown in Fig. 2. In fact, Willett suggested
that ␥-TocH has important antioxidant and other proper-
ties not shared by ␣-TocH (Willett, 2003), and Jiang et
al. (2001) noted that ␥-TocH is the major form of vitamin
E in the United States diet and deserves more attention.
Substantial deuterium kinetic isotope effects on the
k and E for reaction (4ꢀ) were not found under the
present experimental conditions. The values of kH/kD do
not exceed the maximum semiclassical ratio [6–8 (Bell,
1980; Kwart, 1982)], nor does the E difference between
the reaction of ␣-Toc• with Na+AsH− and the reaction
of ␣-Toc• with Na+AsD− exceed the maximum semi-
classical difference [1.3–4.2 kJ/mol (Bell, 1980; Kwart,
1982)]. These results show that although the tunneling
effect plays an important role in reaction (1ꢀ) (Nagaoka
et al., 1992, 2000; Nagaoka, 2004), it does not play an
important role in reaction (4ꢀ) under the present experi-
mental conditions. The E difference between the reaction
of ␤-Toc• with Na+AsH− and that the reaction of ␤-Toc•
with Na+AsD− is discussed in the paragraph after the
following one.
In the work reported in previous papers (Mukai et al.,
1987, 1989, 1991; Nagaoka et al., 2000; Nagaoka, 2004),
we used 5,7-diisopropyl-Toc• or 7-t-butyl-5-isopropyl-
Toc• (Fig. 2) in the studies of reaction (4) (reaction (4ꢀꢀ)):
5, 7-diisopropyl-Toc• + Na+AsH− (Na+AsD−)
→ 5, 7-diisopropyl-TocH (TocD) + Na+As−
,
•
(4ꢀꢀ-1)
7-t-butyl-5-isopropyl-Toc• + Na+AsH− (Na+AsD−)
→ 7-t-butyl-5-isopropyl-TocH (TocD) + Na+As−
.
•
(4ꢀꢀ-2)

S.-i. Nagaoka et al. / Chemistry and Physics of Lipids 146 (2007) 26–32
31
The E and A values of ␤-Toc• are much larger than
those of ␣-Toc• (Table 1), and there seems to be a large
deuterium kinetic isotope effect on E for ␤-Toc•. At
present we do not have an unambiguous explanation for
this, but a possible reason is the isokinetic relationship
(compensation effect) (Leffler, 1955; Nagaoka, 1987), in
which the correlation between the enthalpy and entropy
changes in a series of related reactions results in E being
proportional to log A.
Diplock, A.T., Machlin, L.J., Packer, L., Pryor, W.A. (Eds.), 1989.
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4. Conclusions
The k and E values of reaction (4ꢀ) for Toc•’s
originating from natural TocH’s were determined with
a double-mixing stopped-flow spectrophotometer. The
formation of Na+As−• was observed in the absorption
spectrum. The kinetic effect of methyl substitutions on
the aromatic ring of Toc• indicates that partial charge-
transfer plays a role in reaction (4ꢀ). Since a substantial
deuterium kinetic isotope effect was not found, the tun-
neling effect may not play an important role under the
present experimental conditions.
Jiang, Q., Christen, S., Shigenaga, M.K., Ames, B.N., 2001. ␥-
Tocopherol, the major form of vitamin E in the US diet, deserves
more attention. Am. J. Clin. Nutr. 74, 714–722.
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Acknowledgments
We thank Eisai Co., Ltd. for the generous gifts of d-
␣-, d-␤-, d-␥ and d-␦-TocH’s. We also thank Dr. Umpei
Nagashima of National Institute of Advanced Industrial
Science and Technology for his valuable discussion and
to Mr. Hiroyuki Shiode of Ehime University for his kind
help in our experiments. This work was partly supported
by a Grant-in-Aid for Scientific Research on Priority
Areas “Applications of Molecular Spins” (Area No. 769
and Proposal No. 15087104) from the Japanese Ministry
of Education, Culture, Sports, Science and Technology
(MEXT), Japan.
Nagaoka, S., Kuranaka, A., Tsuboi, H., Nagashima, U., Mukai, K.,
1992. Mechanism of antioxidant reaction of vitamin E. Charge
transfer and tunneling effect in proton-transfer reaction. J. Phys.
Chem. 96, 2754–2761.
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