Proposed mechanisms for HOOOH formation in two typical enzyme reactions responsible for superoxide anion production in biological systems

Article abstract of DOI:10.1246/cl.2009.302

We investigated the hypoxanthine (HPX)-xanthine oxidase (XOD) reaction by examining the chemiluminescence (CL) response mediated by a luminol analog, 8-amino-5-chloro-7-phen-y]pyrido[3,4-d]pyridazine-1,4-(2H, 3H)-dione sodium salt (1-012). It was found th

Full text of DOI:10.1246/cl.2009.302

3
02
Chemistry Letters Vol.38, No.4 (2009)
Highlight Review
Proposed Mechanisms for HOOOH Formation
in Two Typical Enzyme Reactions Responsible
for Superoxide Anion Production in Biological Systems
ꢀ
Masahiro Kohno, Emiko Sato, Noriko Yaekashiwa, Takayuki Mokudai, and Yoshimi Niwano
(Received December 17, 2008; CL-088013)
Abstract
The authors of that study postulated that antibodies carry the re-
action through HOOOH as a key intermediate, as in the follow-
11
ing reaction 1.
We investigated the hypoxanthine (HPX)–xanthine oxidase
XOD) reaction by examining the chemiluminescence (CL) re-
sponse mediated by a luminol analog, 8-amino-5-chloro-7-phen-
(
1
2 O2 þ 2H2O ! 2HOOOH
! H2O4 þ H2O2 ! O2 þ 2H2O2
Although HOOOH has not yet been detected in biological
ylpyrido[3,4-d]pyridazine-1,4-(2H,3H)-dione sodium salt (L-
012). It was found that addition of a high concentration of di-
3
ð1Þ
ꢁ
methyl sulfoxide (DMSO), a potent OH scavenger, could not
completely reduce the CL response. This result suggests the ex-
istence of an unknown reactive oxygen intermediate other than
systems in vivo, its in vitro production has been confirmed ex-
12,13
ꢂ
ꢁ
ꢁ
perimentally.
generated reductantly from ozone decomposes into H2O and O2
For instance, it has been shown that HOOOH
1
O2 and OH. We further examined the HPX–XOD reaction
and the nicotinamide adenine dinucleotide phosphate (NADPH)
oxidation reaction by applying an electron spin resonance (ESR)
spin-trapping method. In both reaction systems, similar re-
sponses were observed. That is, addition of DMSO increased
the formation of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)
1
4
in a process catalyzed by a water molecule. The reverse reac-
tion is surmised to be catalyzed by one or more molecules of
1
1
water, as described above.
Recently, we postulated the existence of HOOOH as a new
reactive intermediate oxygen species found in hypoxanthine
–OOH in a concentration-dependent manner. This indicates that
scavenging of OH increases the detected O2 level, further sug-
ꢁ
ꢂ
ꢁ
1
5
(
HPX) and xanthine oxidase (XOD) reactions and in nicotina-
gesting the existence of an intermediate oxygen species derived
from O2 and OH. One candidate for this species is HOOOH,
presumably formed in the following way.
ꢂ
ꢁ
ꢁ
mide adenine dinucleotide phosphate (NADPH) oxidation reac-
tions (unpublished data). Our interest is in whether HOOOH
plays an important role in biological systems. Therefore, this
Highlight Review focuses on the possible mechanisms for
HOOOH formation in these two typical enzyme reactions of
XOD and NADPH oxidase.
ꢂ
ꢁ
þ
ꢁ
O2 þ H þ OH ! HOOOH
Ç
Introduction
Ç
Xanthine Oxidase Reaction
ꢂ
ꢁ
Oxygen is one of the most important biomediators for aero-
XOD is known to generate O2 when acting on its sub-
1
6–18
bic organisms, since these organisms take oxygen into the body
during respiration, and the consumed oxygen is used for oxida-
tion–reduction reactions. These reactions generate four kinds
of active intermediates called strict reactive oxygen species
strates in the presence of oxygen. The consumption of oxy-
gen by the XOD system has been postulated as the following
pathway.
1
7
þ
ꢂ
ꢁ
HPX þ XOD þ 2O2 ! 2H þ 2O2
ð2Þ
ð3Þ
ꢂ
ꢁ
ꢁ
(
ROSs): superoxide anion (O2 ), hydroxyl radical ( OH), hy-
1
ꢂ
ꢁ
þ
2O2 þ 2H ! O2 þ H2O2
drogen peroxide (H2O2), and singlet oxygen ( O2). A dismuta-
ꢂ
ꢁ
tion reaction of O2 with water results in the formation of H2O2
and O2. H2O2 is then converted to OH by a Fenton-like reac-
tion or a metal-ion-catalyzed Haber–Weiss reaction.
also generated by a peroxidase, H2O2, and halide system, such
Furthermore, it has been reported that superoxide dismutase
(SOD) inhibits the lipid peroxidation caused by XOD, as does
catalase, indicating that both O2 and H2O2 are essential inter-
1
1
ꢁ
2
,3
1
ꢂ
ꢁ
O2 is
1
8
mediates.
4
–7
as myeloperoxidase-catalyzed bactericidal action.
Electron spin resonance (ESR) spectrometry using the spin-
trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has
been applied to characterize the different radical species gener-
ated by XOD, along with the mechanisms of their generation.19
In those authors’ study, the reaction of xanthine with XOD equi-
librated with air resulted in DMPO–OOH (an adduct from
DMPO and O2 ) and DMPO–OH (an adduct from DMPO
and OH). Since ꢀ-hydroxylethyl or methyl radicals are gener-
ated in the presence of ethanol or dimethyl sulfoxide (DMSO),
Beside these ROSs, attention has gradually been paid to hy-
drogen trioxide (HOOOH) in the past decade. Several reports
have suggested that RO3H (R = H and alkyl) species are key
intermediates in both natural and polluted atmospheric environ-
ments and in low-temperature ozonization of various organic
substances.10 In the case of biological systems, it has been re-
ported that all antibodies have the ability to catalyze the oxida-
8,9
ꢂ
ꢁ
ꢁ
1
tion of water by O2 to generate H2O2 and probably O3 as well.
ꢀ
Prof. Masahiro Kohno, Emiko Sato, Noriko Yaekashiwa, Takayuki Mokudai, and Yoshimi Niwano
New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai 980-8579
E-mail: mkohno@niche.tohoku.ac.jp
Copyright Ó 2009 The Chemical Society of Japan

Chemistry Letters Vol.38, No.4 (2009)
303
ꢁ
respectively, both of which are potent scavengers of OH, it has
been shown that DMPO–OH is generated directly from OH
rather than from the breakdown of DMPO–OOH.
Taking the reported results together, the following reactions
(
a)
ꢁ
5
4
0,000
0,000
Solvent (50 mM Tirs-HCl)
2
–4 are proposed for the HPX–XOD system.
þ
ꢂ
ꢁ
HPX þ XOD þ 2O2 ! 2H þ 2O2
ð2Þ
ð3Þ
ð4Þ
30,000
0
.4 mM
ꢂ
ꢁ
þ
2
O2 þ 2H ! O2 þ H2O2
1.9 mM
2
1
0,000
0,000
0
ꢂ
ꢁ
ꢂ
ꢁ
O2 þ H2O2 ! O2 þ OH þ OH
7.5 mM DMSO
0 mM
20 mM
480 mM
3
As shown by these reactions 2–4, the enzyme system gener-
1
ꢁ
ꢂ
ꢁ
ates OH as well as O2 , so it has been widely used for the
screening and development of novel antioxidants as functional
active ingredients for neutraceuticals, cosmeceuticals, and phar-
maceuticals.2
0
50
100
150
200
250
300
Time/s
0–26
ESR spin-trapping and chemiluminescence (CL) methods
(b)
ꢂ
ꢁ
ꢁ
are widely used for analyses of ROSs such as O2 and OH.
The ROS selectivity of CL methods, however, is poor. In addi-
tion, the luminol CL reaction is well documented, whereas the
reaction processes in ROS generation systems, such as the
HPX–XOD system, are not clear. It has been reported that a
luminol-mediated CL response occurs in the HPX–XOD system,
100
8
6
4
2
0
0
0
0
0
2
7
according to the following reactions 2–7.
þ
ꢂ
ꢁ
HPX þ XOD þ 2O2 ! 2H þ 2O2
2
ð2Þ
ð3Þ
ð4Þ
ð5Þ
ð6Þ
ð7Þ
ꢂ
ꢁ
þ
O2 þ 2H ! O2 þ H2O2
ꢂ
ꢁ
ꢂ
ꢁ
O2 þ H2O2 ! O2 þ OH þ OH
0
100
200
DMSO/mM
300
400
500
ꢁ
ꢂ
ꢂ
þ
ꢂ
ꢁ
OH þ LH ! OH þ H þ L
ꢂ
ꢁ
ꢂ
ꢁ
2ꢂ
L
þ O2 ! LO2
ꢁ
Figure 1. Effect of DMSO, a potent OH scavenger, on the L-
0
LO22 ! N2 þ AP
ꢂ
ꢀ2ꢂ
! AP2ꢂ þ hꢁ
12-mediated CL response in the HPX–XOD system. (a) Repre-
sentative CL responses, and (b) inhibition rate of the CL re-
sponse through the addition of different concentrations of
DMSO. Each CL response curve or inhibition rate value repre-
sents the mean of duplicate trials.
ꢂ
ꢂ
ꢁ
2ꢂ
LH : luminol monoanion, L : luminol radical, AP : amino-
ꢀ
2ꢂ
2ꢂ
phthalate dianion, AP : electronically excited state of AP .
ꢂ
ꢁ
ꢂ
ꢁ
The reactions show that LH reacts with OH to form L ,
ꢂ
ꢁ
ꢂ
ꢁ
and the resultant L reacts with O2 . According to the reac-
tions, if OH is completely scavenged, no CL response is as-
sumed to be observed. Thus, we previously examined the effect
ꢁ
100
80
ꢁ
of DMSO, a potent OH scavenger, on the CL response in the
HPX–XOD system.15 In that study, we used a luminol analog, 8-
amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)-
dione sodium salt (L-012), as a CL probe. Figure 1a shows the
effect of DMSO on the L-012-mediated CL response in the
HPX–XOD system. When HPX was added to the mixture, a
rapid CL response was observed, and the CL response was re-
duced by the presence of DMSO in a concentration-dependent
manner. Figure 1b, which shows the DMSO-induced inhibition
rate, demonstrates that DMSO at a concentration of 240 mM
maximized the reduction of the CL response, and an even high-
er concentration of DMSO reduced the CL response by up to
6
4
0
0
In the presence of DMSO
In the absence of DMSO
20
1
10
100
1000
10000
100000
−
1
Concentration/µg mL
Figure 2. Inhibition curves for DMPO–OOH formation, ob-
tained by the addition of different concentrations of L-012 in
the presence or absence of DMSO in the HPX–XOD system.
Each value represents the mean of duplicate trials.
9
in the presence of either SOD or DMPO (data not shown). As
0%. In contrast, the CL response was reduced by 99% or more
5
1
ꢂ
ꢁ
ꢂ
ꢁ
described above, LH reacts with OH to form L , and then the
resultant L reacts with O
ping method. In either the presence or absence of DMSO, dif-
ferent concentrations of L-012 were added to the HPX–XOD
system, and the DMPO–OOH adduct was monitored by ESR.
As shown in Figure 2, even in the presence of DMSO, the
DMPO–OOH formation was inhibited by L-012 in a concentra-
tion-dependent manner, although the inhibition was weaker
than in the absence of DMSO. Our preliminary study showed
ꢂ
ꢁ
ꢂ
ꢁ
22
2
.
Thus, maximal scavenging of
ꢁ
OH is assumed to cause the complete lack of CL response.
The addition of DMSO, however, could not reach a level near
00% reduction of the CL response, so we speculate the exis-
tence of an intermediate, other than OH, that is reactive to
1
ꢁ
ꢂ
ꢂ
ꢁ
LH . Since one candidate intermediate is O
ꢂ
2
, we examined
by applying an ESR spin-trap-
ꢁ
ꢂ
ꢁ
whether L-012 reacts with O
2
that the rate constant for LH with OH is smaller than that
Published on the web (Advance View) February 28, 2009; doi:10.1246/cl.2009.302

3
04
Chemistry Letters Vol.38, No.4 (2009)
ꢁ
8 28,29
for mannitol with OH (order of 10 ), and the rate constant
for DMSO with OH has been reported to be on the order of
1
DMSO
1.92 M
ꢁ
9
27
ꢁ
0 . In other words, the rate constant for DMSO with OH
ꢂ
ꢁ
is much higher than that for LH with OH, so that the high con-
centration of DMSO used in the study was sufficient to com-
pletely scavenge OH. Thus, under the condition of a lack of
0
.96 M
ꢁ
0
.48 M
ꢁ
ꢂ
ꢁ
OH, L-012 likely competes with DMPO to react with O
From these considerations, the reactions 2–7 given above
2
.
ꢂ
0.24 M
should likely be amended as follows, in which LH reacts di-
30
ꢂ ꢂ
ꢁ
ꢁ
rectly with O
2
to form L , as reported in a previous paper.
DMPO-OH
! 2Hþ þ 2O2ꢂ
ꢁ
In the absence of DMSO
DMPO-OOH
HPX þ XOD þ 2O
2
ð2Þ
ð3Þ
ð4Þ
ð5Þ
ð8Þ
ð6Þ
ꢂ
ꢁ
þ
2
O
2
þ 2H ! O
2
þ H
þ OH þ OH
2 2
O
2ꢂ
ꢁ
2
2
2
ꢂ
ꢁ
O
þ H
O
! O
330.5
340.5
3
35.5
Magnetic field/mT
ꢁ
ꢂ
ꢂ
þ
ꢂ
ꢁ
OH þ LH ! OH þ H þ L
ꢂ
ꢂ
ꢁ
þ
ꢂ
Figure 3. Representative ESR spectra showing the effect of
DMSO, a potent OH scavenger, on DMPO–OOH and
DMPO–OH formation in the HPX–XOD system.
LH þ O
2
þ H ! L þ H
2
O
2
ꢁ
ꢂ
ꢁ
ꢂ
ꢁ
2ꢂ
L
þ O
2
! LO
2
We found an interesting phenomenon in another experi-
ment, in which the effect of different concentrations of DMSO
on DMPO–OOH and DMPO–OH formation was examined by
the ESR spin-trapping method, as shown in Figure 3. DMPO–
OOH formation was increased two- to threefold by the addition
of DMSO, whereas DMPO–OH formation was almost complete-
ꢂ
Together with the possibilities of direct reaction of LH
with O2 to form L and interaction of O2 and OH to gen-
erate HOOOH, we finally postulate the following reactions 2–
ꢂ
ꢁ
ꢂ
ꢁ
ꢂ
ꢁ
ꢁ
1
1 for the HPX–XOD system, as summarized schematically in
Figure 4.
þ
ꢂ
ꢁ
ꢂ
ꢁ
HPX þ XOD þ 2O ! 2H þ 2O
2
2
ð2Þ
ð3Þ
ly reduced. These results suggest an interaction between O2
and OH. We postulate a possible interaction via the following
reaction.
ꢁ
ꢂ
ꢁ
þ
2O þ 2H ! O þ H O
2
2
2
2
ꢂ
ꢁ
ꢂ
ꢁ
O2 þ H2O2 ! O2 þ OH þ OH
ð4Þ
ꢂ
ꢁ
þ
ꢁ
ꢁ
ꢂ
ꢂ
þ
ꢂ
ꢁ
O2 þ H þ OH ! HOOOH
ð9Þ
OH þ LH ! OH þ H þ L
ð5Þ
ꢂ
ꢂ
ꢁ
þ
ꢂ
Although this should be confirmed in a future study,
HOOOH may contribute to the luminol-mediated CL response
in the following way.
LH þ O2 þ H ! L þ H2O2
ð8Þ
ꢂ
ꢁ
þ
ꢁ
O2 þ H þ OH ! HOOOH
ð9Þ
ꢁ
ꢁ
HOOOH þ OH ! HOOO þ H2O
ð10Þ
ð11Þ
ð6Þ
ꢁ
ꢁ
ꢁ
ꢂ
ꢂ
ꢁ
HOOOH þ OH ! HOOO þ H2O
ð10Þ
ð11Þ
ð6Þ
HOOO þ LH ! L þ HOOOH
ꢁ
ꢂ
ꢂ
ꢁ
ꢁ
ꢁ
ꢂ
ꢂ
2ꢂ
HOOO þ LH ! L þ HOOOH
L
þ O ! LO2
2
ꢂ
ꢁ
ꢂ
ꢁ
2ꢂ
2ꢂ
ꢀ2ꢂ
! AP2ꢂ þ hꢁ
L
þ O2 ! LO2
LO2 ! N2 þ AP
ð7Þ
O
O
O
XOD
XOD
N
H
N
N
HN
HN
HN
O
N
H
N
H
O
N
H
N
H
N
O
N
H
HPX
XA
UA
O2
O2
O -
.
-.
H O
O2
2
2
2
H+
•OH
•OH
H O
2
HOOO.
HOOOH
-.
O2
NH2
O
C
NH2
O
C
NH2
O
C
O-
O-
NH
N-
N
+
hv
N-
C
O
C
O
C
O
LH-
O2
-.
OH , H O
-
L
-.
AP2-
2
Figure 4. Schematic illustration of the proposed mechanism inducing the luminol-mediated CL response.

Chemistry Letters Vol.38, No.4 (2009)
305
DMPO-OH
DMPO-OOH
DMSO
.92 M
1
0
.96 M
0
.48 M
0
.24 M
330.5
335.5
340.5
In the absence of DMSO
Magnetic field/mT
DMPO-OOH
DMPO-OH
Figure 5. Representative ESR spectrum obtained from the
NADPH–NADH oxidase system.
3
30.5
335.5
340.5
Magnetic field/mT
Ç
NADPH Oxidase Reaction
Figure 6. Representative ESR spectra demonstrating the effect
of DMSO, a potent OH scavenger, on DMPO–OOH and
DMPO–OH formation in the NADPH–NADH oxidase system.
Phagocytic cells such as neutrophils and macrophages play
ꢁ
an important role in host defense against microbial infections.
The microbicidal mechanism consists of phagocytosis of patho-
gens and production and subsequent release of ROSs and bac-
tericidal proteins to phagosomes. During the microbicidal re-
1
ꢂ
ꢁ
2
sponse, professional phagocytic cells produce O2 , a precursor
3
1–33
of microbicidal oxidants. The process involves activation of
NADPH oxidase, which catalyzes the reduction of molecular
0.5
DMPO-OOH
DMPO-OH
y = -1.0339x + 0.8929
R = -0.883 (p<0.01)
ꢂ
ꢁ
1
.5
1
oxygen to O2 at the expense of NADPH in the phagocytes.
0
NADPH þ 2O2 ! NADP þ 2O2ꢂꢁ þ Hþ
þ
ð12Þ
0
0.2
0.4
DMPO-OH
The necessity of NADPH oxidase in host defense is demon-
strated by the recurrent, life-threatening infections occurring in
patients with chronic granulomatous disease, a disorder in which
NADPH oxidase is nonfunctional because of a deficiency in its
0.5
0
3
4–36
oxidase components.
Besides the essential role of NADPH
oxidase in the innate immune system, it is also a prominent con-
tributor to a variety of inflammatory disorders. A vast amount of
circumstantial evidence implicates ROSs produced uncontrolla-
bly by phagocytic cells as mediators of inflammation, shock, and
0.06 0.12 0.24 0.48 0.96 1.92
.1 M PB
0
DMSO/M
3
7
ischemia/reperfusion injury.
The NADPH oxidases are a group of plasma-membrane-
Figure 7. Effect of different concentrations of DMSO on the
relative signal intensities of DMPO–OOH and DMPO–OH.
The relative signal intensities of DMPO–OOH and DMPO–
OH were obtained by dividing each by the signal intensity of
associated multicomponent enzymes consisting of at least two
phox
components bound to the plasma membrane (gp91
phox
and
p22 , which together form the flavocytochrome b558), three
2þ
Mn as an internal standard. Each value represents the mean
of duplicate trials.
phox
phox
phox
cytosolic components (p47 , p67 , and p40 ), and a
small GTPase Rac.38 In resting phagocytic cells, NADPH oxi-
dases are dormant, and their components exist separately in
the membrane and in the cytosol. Once phagocytic cells are
primed by appropriate stimuli, NADPH oxidases are activated
DMPO–OH. The inset figure in Figure 7 shows the negative cor-
relation between DMPO–OOH and DMPO–OH formation.
Addition of different concentrations of DMSO augmented
the DMPO–OOH level and reduced the DMPO–OH level in a
concentration-dependent manner. In other words, scavenging
ꢂ
ꢁ
to produce O2 by association of these cytosolic components
3
9–42
with the plasma or phagosome membrane components.
In our study using the cell-free NADH oxidase system with
ꢁ
ꢂ
ꢁ
of OH increases the detected O2 level, suggesting the exis-
ꢂ
ꢁ
ꢁ
the ESR spin-trapping method, OH was detected as shown in
Figure 5. It was presumably formed according to the following
tence of an intermediate oxygen species derived from O2
and OH. We thus postulate that this species might be HOOOH,
14
as reported in a previous study on the HPX–XOD system.
ꢁ
2
7
reaction 4 given by Hodgson and Fridovich.
ꢂ
ꢁ
ꢂ
ꢁ
ꢂ
ꢁ
þ
ꢁ
O2 þ H2O2 ! O2 þ OH þ OH
ð4Þ
O2 þ H þ OH ! HOOOH
ð9Þ
Figure 6 shows representative ESR spectra demonstrating
the effect of DMSO on DMPO–OOH and DMPO–OH formation
in the NADPH oxidation reaction, and Figure 7 summarizes the
effect of DMSO on the signal intensities of DMPO–OOH and
As with Figure 4 summarizing the proposed mechanism for
HOOOH formation in the HPX–XOD system, Figure 8 schemat-
ically illustrates the proposed mechanism for HOOOH formation
in the NADPH–NADH oxidase system.

3
06
Chemistry Letters Vol.38, No.4 (2009)
H
O
O
H
H
NH2
NH2
+
O
P
N
O
P
N
O
NAD(P)H oxidase
O
HO
HO
O
HO
HO
O
H
H
NH2
H
H
NH2
O
P
H
O
O
P
H
O
OH OH
OH OH
N
N
N
N
O
O
N
H
O
N
H
O
N
N
O2
H
H
H
H
H
H
OH
O
OH
O
O
O
P
OH
P
OH
OH
OH
H+
O2-
.
H O
2 2
.
OH
HOOOH
Figure 8. Schematic illustration of the proposed mechanism by which HOOOH is formed in the NADPH oxidation reaction.
1
0 J. Cerkovnik, E. Er zˇ en, J. Koller, B. Plesni cˇ ar, J. Am. Chem.
Soc. 2002, 124, 404.
11 P. Wentworth Jr., L. H. Jones, A. D. Wentworth, X. Zhu,
N. A. Larsen, I. A. Wilson, X. Xu, W. A. Goddard, III,
K. D. Janda, A. Eschenmoser, R. A. Lerner, Science 2001,
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12 J. Koller, B. Plesni cˇ ar, J. Am. Chem. Soc. 1996, 118, 2470.
13 F. Cacace, G. de Petris, F. Pepi, A. Troiani, Science 1999,
285, 81.
14 J. M. McCord, I. Fridovich, J. Biol. Chem. 1968, 243, 5753.
15 E. Sato, T. Mokudai, Y. Niwano, M. Kamibayashi, M.
Kohno, Chem. Pharm. Bull. 2008, 56, 1194.
Ç
Conclusion
XOD, which catalyzes the oxidation of HPX to xanthine and
further catalyzes the oxidation of xanthine to uric acid, plays an
important role in catabolism of purines. In addition, XOD is
thought to be a primary source of ROSs that mediate ische-
mic-reperfusion-related injuries. NADPH oxidase is essential
in host defense through ROS production of primed phagocytic
cells, while ROSs produced uncontrollably by phagocytic cells
act as mediators of inflammation. In these important biological
systems, O2 , OH, and H2O2 have been considered the major
ROSs. Our recent studies propose both the existence of HOOOH
as an additional ROS and mechanisms for its formation. As a
next step, detection of HOOOH and the biological implications
of HOOOH should be addressed.
4
3
ꢂ
ꢁ
ꢁ
16 J. M. McCord, I. Fridovich, J. Biol. Chem. 1969, 244, 6049.
17 I. Fridovich, J. Biol. Chem. 1970, 245, 4053.
18 E. W. Kellogg, III, I. Fridovich, J. Biol. Chem. 1975, 250,
8
812.
We thank the Pharmaceutical Society of Japan for giving us
permission to reprint Figures 1, 2, 3, and 4 of this review from
Chem. Pharm. Bull. 2008, 56, 1194. All rights, including copy-
right for the figures, belong to the Pharmaceutical Society of
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