Reevaluation of quantitative ESR spin trapping analysisof hydroxyl radical by applying sonolysis of water as a model system

Article abstract of DOI:10.1246/bcsj.20100078

Electron spin resonance (ESR) spin trapping using 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) is commonly applied for quantitative analysis of hydroxyl radical. For better understanding of the analysis, we investigated kinetics related to formation and decay of hydroxyl radical spin adduct of DMPO compared with that of 3, 3, 5, 5-tetramethyl-1-pyrroline N- oxide (M4PO) and 5-(diphenylphosphinoyl)-5-methyl-1-pyrroline N-oxide (DPPMPO). in our study where hydroxyl radical was generated by sonolysisof water, we found that (1) DMPO-OH formation was saturated even though hydroxyl radical was generated continuously and (2) the concentration of DMPO-OH decreased in inverse proportion to time after cessation of ultrasound irradiation, suggesting that the decay is a second-order reaction. Similar results were obtained in an experiment of DMPO-OH generated by photolysisof H2O2. Other than DMPO, M4PO, and DPPMPO also trapped hydroxyl radical but the spin trap efficiency was less than that of DMPO. Furthermore, M4PO-OH and DPPMPO-OH decayed more quickly than DMPO-OH. From this, we conclude that DMPO is more suitable for quantification of hydroxyl radical than M4PO and DPPMPO but results from the quantitative analysis must be interpreted with consideration of the kinetics related to formation and decay of DMPO-OH, especially in quantification oflarge amount of hydroxyl radical generation.

Full text of DOI:10.1246/bcsj.20100078

© 2010 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 83, No. 9, 1037–1046 (2010) 1037
Reevaluation of Quantitative ESR Spin Trapping Analysis of Hydroxyl
Radical by Applying Sonolysis of Water as a Model System
Keisuke Nakamura,*1,2 Taro Kanno,2 Hiroyo Ikai,2 Emiko Sato,1 Takayuki Mokudai,1
Yoshimi Niwano,1 Toshihiko Ozawa,1,3 and Masahiro Kohno1
1New Industry Creation Hatchery Center, Tohoku University, Sendai 980-8579
2Division of Fixed Prosthodontics, Department of Restorative Dentistry,
Tohoku University Graduate School of Dentistry, Sendai 980-8575
3Yokohama College of Pharmacy, Yokohama 245-0066
Received March 16, 2010; E-mail: keisuke1@mail.tains.tohoku.ac.jp
Electron spin resonance (ESR) spin trapping using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is commonly applied
for quantitative analysis of hydroxyl radical. For better understanding of the analysis, we investigated kinetics related to
formation and decay of hydroxyl radical spin adduct of DMPO compared with that of 3,3,5,5-tetramethyl-1-pyrroline N-
oxide (M4PO) and 5-(diphenylphosphinoyl)-5-methyl-1-pyrroline N-oxide (DPPMPO). In our study where hydroxyl
radical was generated by sonolysis of water, we found that (1) DMPO-OH formation was saturated even though hydroxyl
radical was generated continuously and (2) the concentration of DMPO-OH decreased in inverse proportion to time after
cessation of ultrasound irradiation, suggesting that the decay is a second-order reaction. Similar results were obtained in
an experiment of DMPO-OH generated by photolysis of H2O2. Other than DMPO, M4PO, and DPPMPO also trapped
hydroxyl radical but the spin trap efficiency was less than that of DMPO. Furthermore, M4PO-OH and DPPMPO-OH
decayed more quickly than DMPO-OH. From this, we conclude that DMPO is more suitable for quantification of
hydroxyl radical than M4PO and DPPMPO but results from the quantitative analysis must be interpreted with
consideration of the kinetics related to formation and decay of DMPO-OH, especially in quantification of large amount of
hydroxyl radical generation.
Hydroxyl radical, a free radical as well as reactive oxygen
species (ROS), has been studied in various fields, such as
physics, biochemistry, pharmacology, and medical science
because it reacts non-specifically with most organic molecules
causing various oxidative reactions.1,2 Because of its short
lifetime (less than 10 ns in liquid),3,4 it is difficult to detect
hydroxyl radical directly. Consequently, spin trapping using
electron spin resonance (ESR) spectrometry is commonly used
for measurement of hydroxyl radical.5,6 Among various spin
trap agents, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has
been regarded as the most useful and powerful spin trap used in
determination of hydroxyl radical and other free radicals.7-9
ESR spin trapping can detect very low concentrations of
hydroxyl radical trapped by DMPO (2-hydroxy-5,5-dimethyl-
1-pyrrolidinyloxyl; DMPO-OH) which is regarded as a stable
free radical called a spin adduct. Since the ESR spectrum of
DMPO-OH shows a quartet with 1:2:2:1 signal intensity and
hyperfine coupling constants aN = aH = 1.49 mT, it is possible
to identify and quantify it indirectly.10-12
reaction system. Unless sufficient concentration of DMPO
exists in the reaction system, only a limited amount of
generated hydroxyl radical will be trapped by DMPO. Optimal
concentration of DMPO should be evaluated for each hydroxyl
radical generation system because optimal concentration of
DMPO might vary with the generation constant of hydroxyl
radical of each generation system.
Among several generation systems for hydroxyl radical such
as sonolysis of water,12,15,16 photolysis of hydrogen peroxide
(H2O2),6,17 and Fenton reaction,18 sonolysis of water is the
simplest because it involves only one substance, water
including dissolved oxygen. When the quantitative analysis
of hydroxyl radical formation is considered in the ultrasound
system as an example, formed DMPO-OH will react with other
chemicals and will decay both during ultrasound irradiation
and after cessation of ultrasound irradiation. During the
ultrasound irradiation, there is a possibility that not only
hydroxyl radical but also a slight amount of other ROS such as
H2O2, superoxide anion and other radicals such as hydrogen
trioxide19 are generated and might react with DMPO-OH.
In addition, both during and after cessation of ultrasound
irradiation, dissolved oxygen, remaining substances (water and
DMPO), and product of the reaction may also react with
DMPO-OH. Although these factors should be studied in detail
to quantify hydroxyl radicals precisely, little is known so far.
Regarding the lifetime of DMPO-OH, a few previous studies
It has been reported that the rate constant of the reaction
between hydroxyl radical and DMPO is approximately 2 to
¹113,14
4 © 109 M¹1 s
suggesting that the reaction is quite fast.
However, since the lifetime of hydroxyl radical is very short,
sufficient concentration of DMPO is necessary to trap all of the
generated hydroxyl radical.12 Hence, DMPO-OH formation
will be greatly dependent on the concentration of DMPO in the
Published on the web September 2, 2010; doi:10.1246/bcsj.20100078

1038 Bull. Chem. Soc. Jpn. Vol. 83, No. 9 (2010)
Kinetics of Hydroxyl Radical Spin Adducts
A
B
50
40
30
20
10
0
400 mM
300 mM
200 mM
100 mM
50 mM
25 mM
0
100
200
300
400
330.5
335.5
340.5
DMPO concentration/mM
Magnet field/mT
Figure 1. Saturation curve of DMPO-OH. (A) DMPO-OH increased with the concentration of DMPO and then was saturated. (B)
The representative ESR spectrum of DMPO-OH generated at different concentrations of DMPO. Each value in Figure 1A
represents the mean of triplicate determinations.
reported that the half-life of DMPO-OH was in the range of 20
to 60 min in aqueous solutions.20,21 The lifetime of DMPO-OH
will considerably influence the quantitative analysis of hydrox-
yl radical. If a large amount of DMPO-OH spontaneously
decays in short time, it is difficult to quantify the concentration
of DMPO-OH correctly.
Besides DMPO, other spin trap agents are also used for
quantification of hydroxyl radical. For instance, 3,3,5,5-
tetramethyl-1-pyrroline N-oxide (M4PO) sometimes is used
for quantification of hydroxyl radical in biological samples
because oxidation of M4PO by ferric ions is much slower than
that of DMPO-OH.22 In addition, 5-(diphenylphosphinoyl)-5-
methyl-1-pyrroline N-oxide (DPPMPO) has been newly devel-
oped as a spin trap agent with a higher rate constant for
hydroxyl radical than that of DMPO.23 However, few studies
have been conducted to compare the kinetics of these three spin
trap agents in terms of hydroxyl radical trapping ability, and
stability of the spin adducts.
90
80
70
60
50
40
30
20
10
0
y = 24.026x +1.8382
R2=0.9957
2
0
4
6
8
Irradiation time/min
Figure 2. Saturation curve of DMPO-OH formation versus
ultrasound irradiation time. DMPO-OH increased linearly
up to 2 min with high correlation coefficient of over
0.997. Then, DMPO-OH was gradually saturated with the
increase of irradiation time. Each value represents the
mean of triplicate determinations with standard deviation.
In the present paper, we discuss the influences of kinetics
related to formation and decay of DMPO-OH generated by the
simple generation system of hydroxyl radical, sonolysis of
water, on quantitative analysis by ESR spin trapping. Fur-
thermore, spin trapping efficiency and the decay rate of spin
adduct were compared among DMPO, M4PO, and DPPMPO.
H, which is hydrogen atom (hydrogen radical) spin adduct of
DMPO, was detected (Figure 1B).
Formation of DMPO-OH.
DMPO-OH increased in a
Results
time dependent manner, and a linear relationship with a
correlation coefficient of over 0.99 between the concentration
of DMPO-OH and the irradiation time was observed up to
2 min (Figure 2). Then the DMPO-OH was saturated at
around 80 ¯M (Figure 2). However, ultrasound irradiation to
ultrapure water without DMPO for 8 min in advance did not
affect the DMPO-OH formation after the addition of DMPO
(data not shown). Therefore, it is suggested that hydroxyl
radical is generated in accordance with the linear proportion
(y = 24.026x + 1.8382) as shown in Figure 2 even though the
DMPO-OH is saturated. In other words, it is unlikely that
Optimal Concentration of DMPO for Quantification of
Hydroxyl Radical. When ultrapure water containing different
concentrations of DMPO was irradiated for 2 min by ultrasound
with an output power of 30 W and a frequency of 1650 kHz,
DMPO-OH (aN = aH = 1.49 mT) increased with the concen-
tration of DMPO to a certain extent and then was saturated at
the DMPO concentration of around 300 mM (Figure 1A). The
changes in representative ESR spectra are shown in Figure 1B.
Under the condition used in this study, neither DMPO-OOH,
which is superoxide anion spin adduct of DMPO, nor DMPO-

K. Nakamura et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 9 (2010) 1039
A
B
60
12
10
8
50
40
30
20
10
y = 0.0598x - 0.0451
R2=0.9994
6
y=827.76/(x+15.97)
4
2
0
0
0
50
100
150
200
0
40
80
120
160
200
Time/min
Time/min
Figure 3. Decay curve of 50 ¯M DMPO-OH generated by ultrasound irradiation of ultrapure water. (A) DMPO-OH decreased
inversely with time. The half-life was 16.6 min. (B) The inverse proportion was linearized by the equation I0/Ix ¹ 1 = ax + b,
where I0 is the initial concentration of DMPO-OH and Ix is the concentration of DMPO-OH at each time interval. Each value
represents the mean of triplicate determinations.
Table 1. Actually Measured and Calculated Half-Lives at
Different Initial Concentrations of DMPO-OHa)
DMPO trapped the entire hydroxyl radical generated by
ultrasound for over 2 min.
It was observed that addition of H2O2 to the reaction system
decreased DMPO-OH in a concentration dependent manner
(data not shown). The IC50 of H2O2 against DMPO-OH
formation increased with the concentration of DMPO. The
IC50s against 1, 2, and 4 mM DMPO were 80, 230, and 540 mM
H2O2, respectively. The rate constant was calculated for each
Half-life/min
DMPO-OH/¯M
Actually measured
Calculated
50
40
30
20
16.6
21.0
29.4
41.8
®
20.7
27.6
41.4
concentration of DMPO and then the mean rate constant was
¹1
calculated. The mean rate constant was 3.3 © 107 M¹1 s
,
a) The actually measured half-lives were compared with the
calculated half-lives using the equation of the decay curve
obtained from the 50 ¯M DMPO-OH experiment (y = 827.76/
(x + 15.97)). Each value of the actually measured half-life is
expressed as the mean of duplicate determinations.
which is lower by two orders than that of the reaction between
DMPO and hydroxyl radical (3.4 © 109 M¹1 s¹1).24 However,
addition of SOD, which was used to examine if superoxide
anion and products derived from superoxide anion affect the
decay of DMPO-OH, to the reaction system neither increased
nor decreased the concentration of DMPO-OH.
ed highly to the actually measured values (Table 1). Even when
dissolved oxygen was replaced by Argon (Ar), the decay curve
of DMPO-OH was not affected. The half-life of the 50 ¯M
DMPO-OH in the deoxidized sample was 16.4 min. Addition
of H2O2 to the sample after ultrasound irradiation did not affect
the decay curve of DMPO-OH, either. The half-lives of 40 ¯M
DMPO-OH in samples containing 1 and 2 M of H2O2 were
21.6 and 20.9 min, respectively. Those values corresponded
highly to that of 40 ¯M DMPO-OH from a sample without
H2O2. Furthermore, addition of superoxide dismutase (SOD) to
the sample did not affect the decay curve of DMPO-OH. The
half-lives of the 50 ¯M DMPO-OH in samples containing 10
Decay of DMPO-OH. The decay curve of 50 ¯M DMPO-
OH is shown in Figure 3A. The concentration of DMPO-OH
decreased in inverse relation to time. The values of I0/Ix ¹ 1,
where I0 is the initial concentration of DMPO-OH and Ix is the
concentration of DMPO-OH at each time interval, were plotted
to confirm the inverse relationship between DMPO-OH and
time. The equation of the plot calculated by the least squares is
I0/Ix ¹ 1 = 0.0598 © ¹0.0451 (r = 0.9997) (x is time in the
equation) (Figure 3B). Then, the actually measured initial
concentration of DMPO-OH, 49.5 ¯M, is inserted to I0 of the
equation. The eq 49.5/Ix ¹ 1 = 0.0598 © ¹0.0451 where Ix is
y in Figure 3A is converted to y = 827.76/(x + 15.97) show-
ing inverse proportion between DMPO-OH and time. Hence,
the decay of DMPO-OH proved to be a second-order reaction.
The half-lives of 20, 30, 40, and 50 ¯M DMPO-OH are
calculated using the equation. For instance, to calculate the
half-life of 40 ¯M DMPO-OH, the x values, when y values
are equal to 40 and 20, are calculated to be 4.7 and 25.4,
respectively. Hence, 25.4-4.7 = 20.7 min represents the half-
life of 40 ¯M DMPO-OH. When the calculated half-lives
were compared with the actually measured half-lives of each
concentration of DMPO-OH, the calculated values correspond-
¹1
and 20 U mL SOD were 15.8 and 17.0 min, respectively.
Those values were similar to that of 50 ¯M DMPO-OH from
the sample without SOD.
Effect of Temperature on Decay of DMPO-OH. The
temperature affected the DMPO-OH decay curve (Figure 4).
The half-life of the 50 ¯M DMPO-OH decreased in a temper-
ature dependent manner and the rate constants were calculated
using the half-life of the DMPO-OH (Table 2).
Decay of DMPO-OH Generated by Visible Light Laser
Irradiation to H2O2. When 1 M H2O2 containing different
concentrations of DMPO was irradiated with a laser (an output

1040 Bull. Chem. Soc. Jpn. Vol. 83, No. 9 (2010)
Kinetics of Hydroxyl Radical Spin Adducts
60
50
40
30
20
10
0
60
50
40
30
20
10
0
0 °C
25 °C
50 °C
75 °C
0
10
20
30
0
5
10
15
Time/min
Time/min
Figure 5. Decay curve of 50 ¯M DMPO-OH generated
by laser irradiation of 1 M H2O2. DMPO-OH decreased
inversely with time and the half-life almost corresponded
to that of 50 ¯M DMPO-OH generated by ultrasound
irradiation of ultrapure water. Each value represents the
mean of duplicate determinations.
Figure 4. The influence of temperature on the decay curve
of DMPO-OH. The half-life decreased with the increase of
temperature. Each value represents the mean of triplicate
determinations.
Table 2. Half-Lives of 50 ¯M DMPO-OH and Rate
Constants of the Bimolecular-Reaction of DMPO-OH at
273, 298, 323, and 348 Ka)
80
DMPO-OH
70
60
50
40
30
20
10
0
¹1
M4PO-OH
DPPMPO-OH
Temp/K
Half-life/min
Rate constant/M¹1 s
273
298
323
348
39.8
16.9
6.5
9
20
52
2.8
118
a) Each value is expressed as the mean of triplicate
determinations.
·
power of 300 mW and a wavelength of 405 nm) for 30 s,
DMPO-OH increased with the concentration of DMPO to a
certain extent and then was saturated at the DMPO concen-
tration of around 300 mM which was considered to be an
optimal concentration (data not shown). The saturated level of
DMPO-OH was around 50 ¯M which was coincident with that
of DMPO-OH generated by the sonolysis of water. The 50 ¯M
DMPO-OH generated by photolysis of H2O2 decreased with
time similarly to the 50 ¯M DMPO-OH generated by ultra-
sound irradiation to ultrapure water (Figure 5). The equation
of inverse relation between DMPO-OH and time was found
to be y = 931.86/(x + 18.89). The half-life of the 50 ¯M
DMPO-OH was 18.6 min which was nearly equal to the half-
life (16.6 min) of 50 ¯M DMOP-OH generated by ultrasound
irradiation to ultrapure water.
0
1
2
3
4
5
Irradiation time/min
Figure 6. Relationship between spin adducts formation and
ultrasound irradiation time. Each spin trap agent was used
at their optimal concentration. i.e., 300 mM for DMPO and
M4PO, and 20 mM for DPPMPO. Although spin adduct
of hydroxyl radical increased with irradiation time, the
amount of hydroxyl radical trapped by M4PO and
DPPMPO was less than half of that trapped by DMPO.
Each value represents the mean of duplicate determina-
tions.
Comparison of DMPO, M4PO, and DPPMPO. When
ultrapure water containing M4PO was irradiated for 1 min by
ultrasound, M4PO-OH (aN = 1.53 mT and aH = 1.68 mT)25
was detected. M4PO-OH increased with the concentration of
M4PO and then was saturated at around 16 ¯M. On the other
hand, although DPPMPO-OH (aN = 1.39 mT, aH = 1.39 mT,
and aP = 3.58 mT)23,26 was also detected and saturated around
14 ¯M, then it became undetectable at the DPPMPO concen-
tration of over 40 mM. Since 1.0 M DPPMPO was prepared
in 99% dimethyl sulfoxide (DMSO) and then diluted to the
appropriate concentration with ultrapure water, it was consid-
ered that DMSO in the preparations of DPPMPO at concen-
trations of 40 mM or more scavenged hydroxyl radical,
resulting in disappearance of the ESR spectrum. The optimal
concentrations of M4PO and DPPMPO for this hydroxyl
radical generation system were over 300 mM and between 10
and 30 mM, respectively.
M4PO-OH and DPPMPO-OH increased in an irradiation
time dependent manner, and a linear relationship with a
correlation coefficient of over 0.99 between the spin adducts of
hydroxyl radical and the irradiation time was observed up to
1 min (Figure 6). Then the M4PO-OH was saturated at around
30 ¯M and the DPPMPO-OH was at 25 ¯M when they were
used at their optimal concentrations, 300 mM for M4PO and

K. Nakamura et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 9 (2010) 1041
A
25
DMPO-OH
M4PO-OH
DPPMPO-OH
20
15
10
5
·
0
0
5
10
15
20
25
30
Time/min
B
C
M4PO-OH
DPPMPO-OH
4
5
y = 0.0789x - 0.0385
R2=0.9996
y = -0.1268x + 3.0151
R2=0.9944
3
2
4
3
2
1
0
1
0
-1
0
10
20
30
40
50
60
70
0
5
10
15
20
25
30
35
Time/min
Time/mn
Figure 7. Analysis of decay curve of 20 ¯M spin adduct of hydroxyl radical. (A) DMPO-OH was the most stable followed by
M4PO-OH and DPPMPO-OH. The half-lives of DMPO-OH, M4PO-OH, and DPPMPO-OH were 41.8, 12.7, and 5.5 min,
respectively. (B) M4PO-OH decayed in inverse proportion to time suggesting second-order reaction. The inverse proportion was
linearized by the equation I0/Ix ¹ 1 = ax + b, where I0 is the initial concentration of M4PO-OH and Ix is the concentration of
M4PO-OH at each time interval. (C) On the other hand, since plots of natural logarithm of DPPMPO-OH against time showed a
linear relationship, it was suggested that DPPMPO-OH decayed by first-order reaction. Each value represents the mean of duplicate
determinations.
20 mM for DPPMPO (Figure 6). Thus, it was demonstrated
that hydroxyl radical was trapped more efficiently by DMPO
than M4PO and DPPMPO in this experimental model.
When the decay rates of 20 ¯M spin adducts of hydroxyl
radical were compared at 25 °C, DMPO-OH showed the
longest half-life followed by M4PO-OH and DPPMPO-OH
(Figure 7A). The half-lives of DMPO-OH, M4PO-OH, and
DPPMPO-OH were 41.8, 12.7, and 5.5 min, respectively.
DMPO-OH and M4PO-OH decayed by a second-order reaction
(Figure 7B) while the decay of DPPMPO-OH was first-order
(Figure 7C). The first-order reaction was confirmed by plotting
the natural logarithm of DPPMPO-OH against time. The linear
relationship between them confirmed that the decay was a first-
order reaction.
It was observed that the addition of M4PO or DPPMPO to
DMPO aqueous solution decreased DMPO-OH generated by
the sonolysis in a concentration dependent manner. Since it was
possible to differentiate the ESR spectrum of M4PO-OH and
DPPMPO-OH from that of DMPO-OH in a certain magnet
field, the IC50 for them against DMPO was evaluated. The IC50
of M4PO against 30 mM DMPO was 1.5 mM and that of
DPPMPO was 6.1 mM. The calculated rate constants using
¹1
the IC50s were 6.8 © 1010
M
¹1 s for M4PO and 1.7 © 1010
¹1
M
¹1 s for DPPMPO.
Discussion
In the present study, we demonstrated that (1) the optimal
concentration of DMPO for sonolysis of water and photolysis
of H2O2 under the condition used was 300 mM, (2) DMPO-OH
formation was saturated even though hydroxyl radical was
generated continuously, (3) the concentration of DMPO-OH
decreased in inverse proportion to time after generation of
DMPO-OH, and (4) DMPO was more suitable for the
quantification of hydroxyl radical than M4PO and DPPMPO
in terms of spin trapping efficiency and stability of spin adduct.
We further examined the effects of possible factors on the
kinetics of DMPO-OH. H2O2 and SOD, which was used to
examine if superoxide anion and products derived from
superoxide anion affect the decay of DMPO-OH, did not or
very slightly affected DMPO-OH formation under the con-
dition used in the present study. Although the addition of H2O2
to the reaction system decreased DMPO-OH, the rate constant,

1042 Bull. Chem. Soc. Jpn. Vol. 83, No. 9 (2010)
Kinetics of Hydroxyl Radical Spin Adducts
3.3 © 107 M¹1 s¹1, which is approximately consistent with that
reported in a previous paper,27 was lower by two orders than
that of the reaction between DMPO and hydroxyl radical
(3.4 © 109 M¹1 s¹1). It is considered that H2O2 in the presence
of 300 mM of DMPO scavenged only a small amount of
hydroxyl radical even though H2O2 was generated by ultra-
sound irradiation of water. Furthermore, since the concentra-
tion of H2O2 used for photolysis (1 M) was only 3.4 times
higher than that of DMPO (300 mM), the generation of DMPO-
OH by photolysis of 1 M H2O2 was unlikely affected by the
presence of H2O2. SOD did not or very slightly affected the
decay of DMPO-OH, so that there are two possibilities
considered. One is that superoxide anion did not interact
with DMPO-OH, and the other is that very small amount of
superoxide anion was produced by ultrasound irradiation to
water. Since it was reported that superoxide anion produced
by xanthine-xanthine oxidase reaction induced the depletion
of DMPO-OH,28 it is likely that the decay of DMPO-OH
observed in this study was free from the effect of superoxide
anion. Indeed, in the present study, DMPO-OOH was not
detected as shown in Figure 1B while DMPO-OH was detected
in several dozen ¯M. Similarly, DMPO-H was not detected,
either. It was reported that hydrogen atom as well as hydroxyl
radical were generated by sonolysis of water when low-
frequency ultrasound (several dozen to hundred kHz) was
used.12,29,30 However, the 1650 kHz ultrasound used in this
study generated almost no hydrogen atom as reported in our
previous study.16
The half-life of DMPO-OH depended on its initial concen-
tration and varied from 16 to 42 min in this study. Although we
investigated potential factors affecting lifetime of DMPO-OH,
such as dissolved oxygen and H2O2, those factors did not seem
to influence the DMPO-OH decay curve. In addition, the decay
of DMPO-OH after the cessation of irradiation represents likely
spontaneous decay or mutual interaction with DMPO-OH
molecules themselves because the sample used in this study
contained only the ultrapure water, the remaining DMPO,
and DMPO-OH after ultrasound irradiation. Therefore, one
possibility is that the DMPO-OH decayed by second-order
reaction of two molecules of DMPO-OH so that the half-life
of DMPO-OH was in inverse proportion to the initial con-
centration of DMPO-OH. If the second-order reaction was
attributed to two molecules of DMPO-OH, the activation
energy of the reaction was low (26 kJ mol¹1) according to an
Arrhenius plot using the values in Table 2. Thus, we presumed
that the product from the second-order reaction might be a
dimer of DMPO-OH.
fore, it is suggested that the initial concentration of DMPO-OH
is an important factor affecting quantitative analysis of
hydroxyl radical.
As opposed to the potential factors discussed above
(dissolved oxygen and H2O2), the DMPO-OH decay curve
was affected by temperature. Since higher temperature causes
faster decay of DMPO-OH, it was confirmed that the second-
order reaction was accelerated by temperature. If the second-
order reaction for two molecules of DMPO-OH occurred, the
rate constants were quite low compared to those in the reactions
of hydroxyl radical and DMPO even though the temperature
was 75 °C, suggesting that the reaction was slow. On the other
hand, the influence of the temperature on the quantitative
analysis is not negligible as shown in Figure 4.
When the actually measured half-life of each concentration
of DMPO-OH and the calculated value using the equation
(y = 827.76/(x + 15.97)) of the decay curve of 50 ¯M DMPO-
OH were compared, there is little difference between the
two. This finding suggests that concentration of DMPO-OH
decreases according to the equation. For instance, the calcu-
lated half-life of each concentration of DMPO-OH is as
follows; 8.3 min for 100 ¯M DMPO, 4.1 min for 200 ¯M
DMPO-OH, and 2.1 min for 400 ¯M DMPO. The short
lifetime of several hundred ¯M DMPO-OH might be one of
the reasons for the saturation phenomenon of the actually
measured DMPO-OH. However, the spontaneous decay of
DMPO-OH is too slow to explain the whole reaction of
saturation of the actually measured DMPO-OH. Therefore,
hydroxyl radical generated constantly by ultrasound irradiation
might degrade DMPO-OH, which makes difficulty in measure-
ment of several hundred ¯M DMPO-OH correctly. Indeed,
hydroxyl radical is generated linearly with time (Figure 2)
but the actually measured concentration of DMPO-OH is
saturated at 80 ¯M. This finding is in accordance with previous
reports.12,16 Those studies investigated the relationship between
ultrasound irradiation time and concentration of DMPO-OH.
Iwasawa et al.16 reported that the concentration of DMPO-OH
was saturated at around 15 ¯M. The difference of the concen-
tration of saturation between the previous study and ours might
be due to the concentration of DMPO added to the reaction
system and the power of the ultrasound device. The factors
which cause the saturation of actually measured DMPO-OH
should be studied in the future for better understanding of a
generation system which could produce a large amount of
hydroxyl radical.
Optimal concentration of DMPO for photolysis of 1 M H2O2
was 300 mM under the conditions in which 50 ¯M DMPO-OH
was generated. Although the optimal conditions for photolysis
of H2O2 were coincident with those for sonolysis of water in
this study, optimal conditions should be evaluated for each
experiment because they are probably affected by the amount
and the generation rate of hydroxyl radical in each generation
system. As to decay of DMPO-OH generated by photolysis of
H2O2, a decay curve similar to that generated by sonolysis of
water was observed. Therefore, it is thought that the kinetics
related to the decay of DMPO-OH generated by sonolysis of
water and photolysis of H2O2 is almost the same. However,
kinetics of DMPO-OH may vary in other hydroxyl radical
generation systems, such as Fenton reaction or Haber-Weiss
Considering these findings, the difference of half-life
of DMPO-OH between the previous reports26,20,21 and the
present study was mainly due to the difference of the initial
concentration of DMPO-OH generated from different hydroxyl
radical generation systems. According to the equation
(y = 827.76/(x + 15.97)) used in this study, the 60 min half-
life means that the initial concentration of DMPO-OH was
about 14 ¯M. Similarly, as reported in a previous study on
radiolysis of water, DMPO-OH exhibited second-order radical
termination kinetics at initial concentrations of 13 ¯M or more,
followed by first-order termination kinetics at lower concen-
trations,31 which is in agreement with another study.32 There-

K. Nakamura et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 9 (2010) 1043
Me
Me
H
O
Me
Me
Me
Me
H
H
P
Me
+
+
N
+
N
N
-
-
-
O
O
O
DMPO
M4PO
DPPMPO
Figure 8. Structures of DMPO, M4PO, and DPPMPO.
reaction because they involve more chemicals than in sonolysis
and photolysis used in this study.
analyze hydroxyl radical production as the first step of radical
control. On quantitative analysis using generation systems
which generate a large amount of hydroxyl radical (up to
several hundred ¯M in a short time) as seen in this study, the
following factors for ESR spin trapping using DMPO must be
considered; (1) the optimal concentration of DMPO, (2) the
decay rate of DMPO-OH depending on the initial concen-
tration of DMPO-OH, and (3) temperature effect on the
kinetics of DMPO-OH. If these factors are taken into
consideration, the true amount of hydroxyl radical generation
can be speculated by the linear relationship between the
DMPO-OH level and irradiation time of ultrasound or laser
observed in the short period of time even though the actually
measured DMPO-OH is saturated.
It was confirmed that M4PO and DPPMPO could detect
hydroxyl radical as reported previously. Both of the spin trap
agents also showed the saturation of hydroxyl radical spin
adduct as did DMPO (Figure 6). Since hydroxyl radical was
continuously generated unless ultrasound irradiation was
ceased as discussed above, the saturation of spin adducts was
likely due to the same reason as in the case with DMPO.
Hence, it was suggested that the hydroxyl radical spin adducts
would not reflect the true generation of hydroxyl radical after
certain ultrasound irradiation time irrespective of the types of
spin trap agents used in this study. Although the rate constants
of those two spin trap agents for hydroxyl radical were
relatively higher than that of DMPO, the spin trapping
efficiency of them was less than half (Figure 6). This finding
probably means that M4PO and DPPMPO reacted to hydroxyl
radical but could not trap the spin effectively. In other words,
partial structures such as methyl and diphenylphosphinoyl
moieties interact with hydroxyl radical, which in turn interferes
with trapping. Concerning the kinetics of decay curve, M4PO-
OH and DPPMPO-OH showed shorter half-lives than DMPO-
OH. Interestingly, it was demonstrated that DPPMPO-OH
decayed in the first-order reaction while M4PO-OH and
DMPO-OH decayed in the second-order reaction. DPPMPO-
OH might not react with another molecule of DPPMPO-OH
but decompose by autolysis. As for the chemical structure of
DPPMPO, a major difference from those of DMPO and M4PO
is existence of diphenylphosphinoyl moiety in its structure
(Figure 8). Therefore, one of the possibilities is that the
diphenylphosphinoyl moiety is attributable to fragility of
DPPMPO-OH. These findings suggest that DMPO is more
suitable for quantification of hydroxyl radical than M4PO and
DPPMPO at least in simple reaction systems, such as sonolysis
of water and photolysis of H2O2.
Experimental
Reagents. Reagents were purchased from the following
sources: DMPO, M4PO, and DPPMPO from Labotec (Tokyo,
Japan); H2O2 from Santoku Chemical Industries (Tokyo,
Japan); 4-hydroxy-2,2,6,6-tetramethylpiperidine (TEMPOL)
and SOD from Sigma Aldrich (St. Louis, MO, USA); DMSO
from Wako Pure Chemical Industries (Osaka, Japan). All other
reagents used were of analytical grade.
Optimal Concentration of DMPO for Quantification of
Hydroxyl Radical. An experimental device which generates
1650 kHz ultrasound with an output power of 30 W was made.
A glass tube (15 mm in diameter and 85 mm long) containing
500 ¯L of sample was set into the device. To evaluate an
optimal concentration of DMPO for ESR measurement of
hydroxyl radical generated from this system, the relationship
between the concentration of DMPO and the concentration of
DMPO-OH was investigated. Immediately after mixing of
ultrapure water and given concentration of DMPO, the test tube
was set in the device and was irradiated by ultrasound for
2 min. During the irradiation, the temperature of the water in
the bath was maintained at 22 « 1 °C. The DMPO aqueous
solution was then transferred to a quartz cell for ESR
spectrometry and ESR spectrum was recorded on an X-band
ESR spectrometer (JES-FA-100, JEOL, Tokyo, Japan). The
measurement conditions for ESR were as follows; field sweep,
330.50-340.50 mT; field modulation frequency, 100 kHz; field
modulation width, 0.1 mT; amplitude, 80; sweep time, 2 min;
time constant, 0.03 s; microwave frequency, 9.420 GHz: micro-
wave power, 4 mW. To calculate the concentration of DMPO-
OH, 20 ¯M TEMPOL was used as a standard sample for
quantitative analysis and the ESR spectrum of manganese
(Mn2+) which was equipped in the ESR cavity was used as an
Hydroxyl radical has a bilateral character. The negative
character is thought to be an important factor of oxidative
damage in cells and tissues causing many diseases.33,34 In
addition to host immune defense by polymorphonuclear
leukocytes,35,36 the positive character is applied medically,
such as cancer treatment,37 antibiotics,38 and bactericidal
(fungicidal) treatment.16 For medical application of hydroxyl
radicals, controlling the amount of production is one of the
most important factors. Otherwise, hydroxyl radicals will kill
not only bacteria or undesired cells but also normal cells
causing adverse reactions to the body. We believe that the
present study can give basic knowledge to quantitatively

1044 Bull. Chem. Soc. Jpn. Vol. 83, No. 9 (2010)
Kinetics of Hydroxyl Radical Spin Adducts
internal standard. The concentration of hydroxyl radical was
determined using Digital Data Processing (JEOL, Tokyo,
Japan) and was expressed as that of DMPO-OH in ¯M.
Formation of DMPO-OH. The relationship between the
ultrasound irradiation time and the concentration of DMPO-
OH was investigated. According to the experiment described
above, DMPO was used in the final concentration of 300 mM.
The 300 mM of DMPO aqueous solution was irradiated for 15 s
to 8 min. The ESR measurement and data analysis were
performed in the same way as described above. Then, equations
indicating the linear relationship between DMPO-OH and the
ultrasound irradiation time prepared by least squares fit to
calculate the total amount of hydroxyl radical.
The change in the hydroxyl radical generation from ultrapure
water was evaluated. In brief, ultrapure water was placed in a
glass tube and then set in the ultrasound device without DMPO.
After 8 min of irradiation, DMPO was added to make a final
concentration of 300 mM. Immediately after addition of
DMPO, the sample was irradiated for 2 min again. The amount
of DMPO-OH generated was compared to that from 2 min
ultrasound irradiation to DMPO aqueous solution without prior
ultrasound irradiation. The ESR measurement and data analysis
were performed in the same way as described above.
The effect of H2O2 on DMPO-OH formation in the hydroxyl
radical generation system was investigated. H2O2 and DMPO
were mixed to make a final concentration of 8 mM to 8 M for
H2O2 and 1, 2, and 4 mM for DMPO. The mixture was then
irradiated for 2 min. The inhibition curve of DMPO-OH
against the concentration of H2O2 was made and the half
maximal inhibitory concentration (IC50) of H2O2 was calcu-
lated for each concentration of DMPO. The rate constant of the
reaction between H2O2 and hydroxyl radical was also calcu-
lated on the assumption that the reaction proceeded with the
following equations;
was recorded by ESR using the same conditions as described
above. Fifty ¯M DMPO-OH was generated from 300 mM of
DMPO aqueous solution using the ultrasound device. The
measurement of DMPO-OH was conducted up to 3 h using the
same sample after cessation of irradiation. The mean values of
DMPO-OH at each time interval were plotted. The equation of
the decay curve was calculated to fit to the inverse curve
(y = a/(x ¹ b)). The half-lives of 20, 30, and 40 ¯M DMPO-
OH generated in the same way as described above but shorter
irradiation time than that required for 50 ¯M DMPO-OH were
also evaluated. The actually measured half-lives were com-
pared with the calculated half-lives using the equation of the
decay curve obtained from the result of the 50 ¯M DMPO-OH
experiment.
Since dissolved oxygen of the sample might affect the
DMPO-OH decay curve, the dissolved oxygen was replaced by
Ar gas bubbling. The initial measurement of the DMPO-OH
was performed under the same conditions as described above
but the remaining sample was bubbled by Ar gas in the glass
tube during the initial measurement of the DMPO-OH. Then,
the concentration of DMPO-OH was measured-up to 15 at
3 min intervals.
The influence of H2O2 on the decay curve of DMPO-OH
was evaluated. Fifty ¯M DMPO-OH was generated from
400 ¯L of ultrapure water containing DMPO (final concen-
tration of 300 mM) using the ultrasound device. Immediately
after irradiation, 100 ¯L of ultrapure water or 100 ¯L of H2O2
was added to be 0, 1, and 2 M of the final concentration of
H2O2. Then, the measurement of DMPO-OH was conducted up
to 15 at 3 min intervals.
To investigate the influence of oxidative products which
might be generated through superoxide anion on the decay
curve of DMPO-OH, SOD was added to the reaction system.
SOD and DMPO were dissolved in ultrapure water to make a
¹1
DMPO þ HO• ! DMPO-OH
final concentration of 0, 10, and 20 U mL for SOD and
300 mM for DMPO. Fifty ¯M DMPO-OH was generated from
the sample using the ultrasound device and the measurement of
DMPO-OH was conducted up to 15 min at 3 min intervals. The
half-life of DMPO-OH under each condition was calculated
and compared with that of DMPO-OH without any additional
treatment.
ðrate constant: k1 ¼ 3:4 ꢀ 109 Mꢁ1 sꢁ1 13
Þ
ð1Þ
ð2Þ
H2O2 þ HO• ! X1 ðrate constant: k2Þ
where HO• is hydroxyl radical and X1 is defined as a product
produced by the reaction.
From eq 1 and eq 2, the following equations were derived;
Effect of Temperature on Decay of DMPO-OH. The
influence of temperature on DMPO-OH decay curve was
studied. Fifty ¯M DMPO-OH was generated using the
ultrasound device in the same way as described above. The
samples were kept in crushed ice or in a water bath to maintain
water temperature at 0, 25, 50, and 70 °C, and was used for
subsequent measurement of DMPO-OH. The half-life of
DMPO-OH under each condition was calculated. The rate
constant of the reaction between mutual DMPO-OH was also
calculated on the assumption that the reaction was second-order
as shown in the following equations;
d½DMPO-OHꢂ=dt ¼ k1½HO•ꢂ½DMPOꢂ
ð3Þ
ð4Þ
d½X1ꢂ=dt ¼ k2½HO•ꢂ½H2O2ꢂ
If the concentration of H2O2 corresponded to the IC50, the
following equation was derived;
k2 ¼ k1½DMPOꢂ=½IC50ꢂ
The value of k2 was compared to k1 to evaluate the influence of
the hydroxyl radical scavenging of H2O2 in the radical
generation system.
To investigate the influence of superoxide anion on DMPO-
OH formation, SOD was added to the reaction system to make
a final concentration of 10 and 20 U mL¹1. One unit of SOD
was defined as the amount of SOD required to inhibit reduction
of cytochrome c by 50% in a coupled system with xanthine
oxidase. The concentration of DMPO-OH with or without SOD
was compared.
ð5Þ
DMPO-OH þ DMPO-OH ! X2 ðrate constant: kÞ ð6Þ
where X2 is defined as a product produced by the mutual
DMPO-OH reaction. If the reaction is second-order, the
reaction rate was obtained from the following equation;
2
d½DMPO-OHꢂ=dt ¼ ꢁk½DMPO-OHꢂ
ð7Þ
Decay of DMPO-OH. The concentration of DMPO-OH

K. Nakamura et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 9 (2010) 1045
The eq 7 was converted to eq 8 by indefinite integral.
1=½DMPO-OHꢂ ꢁ 1=½DMPO-OHꢂ0 ¼ kt
where [DMPO-OH]0 means the initial concentration of
DMPO-OH. The eq 9 was delivered from eq 8 using the
half-life (t1/2).
DMPO. The mixture was then irradiated for 1 min. According
to the inhibition curve of DMPO-OH against the concentration
of the spin trap agents, IC50 of each spin trap agent was
determined and then the rate constant was calculated.
ð8Þ
This research was partially supported by the Ministry of
Economy, Trade and Industry, Grant-in-Aid for “Regional
Innovation Creation R&D Programs,” No. 21R2007C, 2010.
t1=2 ¼ 1=ðk½DMPO-OHꢂ0Þ
ð9Þ
Decay of DMPO-OH Generated by Visible Light Laser
Irradiation to H2O2. A commercially available laser device
(RV-1000, RICHO OPTICAL INDUSTRY, Hanamaki, Japan)
was used. DMPO-OH was generated by means of irradiation
of a laser diode with an output power of 300 mW and a
wavelength of 405 « 5 nm to 1 M H2O2. Optimal concentration
of DMPO for photolysis of 1 M H2O2 was investigated under
the conditions in which 50 ¯M DMPO-OH was generated, i.e.,
the irradiation time was 30 s. Then, the kinetic analysis of
decay of 50 ¯M DMPO-OH was performed. In brief, DMPO
and H2O2 were mixed in a 96-well plate to make a final
concentration of 300 mM for DMPO and 1 M for H2O2. Fifty
¯M DMPO-OH was generated by 30 s irradiation and was
measured by ESR along under the same conditions as described
above. The measurement of DMPO-OH was conducted up to
30 min after laser irradiation. The DMPO-OH decay curve and
the half-life of DMPO-OH were compared to those of DMPO-
OH generated by ultrasound irradiation to ultrapure water.
Comparison of DMPO, M4PO, and DPPMPO. Struc-
tures of DMPO, M4PO, and DPPMPO are shown in Figure 8.
M4PO (1.0 M) was prepared in ultrapure water while 1.0 M
DPPMPO was prepared in 99% DMSO. Optimal concentration
of M4PO and DPPMPO for the quantification of hydroxyl
radical was investigated as performed for DMPO. The spin trap
agents were then diluted with ultrapure water and were
irradiated by ultrasound for 1 min. ESR spectrum was recorded
by the same way as described above except that for the ESR
measurement of M4PO, field modulation width was set at
0.05 mT.
The relationship between the ultrasound irradiation time and
the concentration of M4PO-OH and DPPMPO-OH were also
investigated as performed for DMPO. According to the
experiment described above, M4PO and DPPMPO were used
in the final concentration of 300 and 20 mM, respectively. The
aqueous solutions containing the spin trap agents were
irradiated for 15 s to 5 min. The ESR measurement and data
analysis were performed in the same way as described above.
The decay curve of 20 ¯M spin adducts was investigated. To
obtain 20 ¯M DMPO-OH, M4PO-OH, and DPPMPO-OH,
each aqueous solution containing the spin trap agent at the
optimal concentration was irradiated for 45, 90, and 120 s,
respectively, depending on their spin trapping efficiency. The
concentration of each spin adduct of hydroxyl radical was
recorded up to 30 min by ESR in the same way as described
above. Then, the half-lives of each spin adduct of hydroxyl
radical were evaluated.
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