Reaction targets of antioxidants in azo-initiator or lipid hydroperoxide induced lipid peroxidation

Article abstract of DOI:10.1080/10715762.2020.1761020

Lipid peroxidation (LPO) is reported to be involved in the pathogenesis of several oxidative diseases, and several therapeutic approaches using antioxidants have been proposed. LPO is thought to progress via a complicated series of multistep reactions suggesting that the activity of each antioxidant may be different, and depends on the reacting molecules. Hence, in this study, we evaluated the inhibitory mechanisms of several antioxidants toward arachidonic acid (AA) peroxidation induced by the azo initiator 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) or a lipid hydroperoxide, hydroperoxyoctadecadienoic acid (HpODE)/hemin. Edaravone, ferrostatin-1, TEMPO and trolox effectively inhibited the production of malondialdehyde (MDA) and several oxidised AAs generated in the AAPH-induced LPO because of their scavenging ability toward lipid peroxyl radicals. In contrast, ebselen and ferrostatin-1 showed strong antioxidative activity in the HpODE/hemin-induced peroxidation. Under this condition, ebselen and ferrostatin-1 were thought to reduce HpODE and its derived alkoxyl radicals to the corresponding lipid alcohols. In conclusion, we found that each antioxidant had different antioxidative activities that prevented the progression of LPO. We expect that these findings will contribute to the design of novel therapeutic strategies using an appropriate antioxidant targeted to each step of the development of oxidative stress diseases.

Full text of DOI:10.1080/10715762.2020.1761020

Free Radical Research
ISSN: 1071-5762 (Print) 1029-2470 (Online) Journal homepage: https://www.tandfonline.com/loi/ifra20
Reaction targets of antioxidants in azo-initiator or
lipid hydroperoxide induced lipid peroxidation
Kota Saito, Yuta Matsuoka & Ken-ichi Yamada
To cite this article: Kota Saito, Yuta Matsuoka & Ken-ichi Yamada (2020): Reaction targets
of antioxidants in azo-initiator or lipid hydroperoxide induced lipid peroxidation, Free Radical
Research, DOI: 10.1080/10715762.2020.1761020
To link to this article: https://doi.org/10.1080/10715762.2020.1761020
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Reaction targets of antioxidants in azo-initiator or lipid hydroperoxide induced lipid peroxidation
Kota Saito1, Yuta Matsuoka1,2, Ken-ichi Yamada1,2*
1Physical Chemistry for Life Science Laboratory, Faculty of Pharmaceutical Sciences, Kyushu
University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
2AMED-CREST, Japan Agency for Medical Research and Development, 1-7-1 Ohtemachi, Chiyoda-ku,
Tokyo, 100-0004, Japan
*Correspondence should be addressed to: Ken-ichi Yamada
Physical Chemistry for Life Science Laboratory, Faculty of Pharmaceutical Sciences, Kyushu
University, 3-1-1 Maidashi Higashi-ku, Fukuoka 812-8582, Japan
Tel: +81-92-642-6624
Fax: +81-92-642-6626
E-mail: kenyamada@phar.kyushu-u.ac.jp
Co-Authors’ e-mail address
Kota Saito : saitokoge31101061@gmail.com
Yuta Matsuoka : ymatsu@phar.kyushu-u.ac.jp

ABSTRACT
Lipid peroxidation (LPO) is reported to be involved in the pathogenesis of several oxidative diseases,
and several therapeutic approaches using antioxidants have been proposed. LPO is thought to
progress via a complicated series of multistep reactions suggesting that the activity of each
antioxidant may be different, and depends on the reacting molecules. Hence, in this study, we
evaluated the inhibitory mechanisms of several antioxidants toward arachidonic acid (AA)
peroxidation induced by the azo initiator 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) or
a lipid hydroperoxide, hydroperoxyoctadecadienoic acid (HpODE)/hemin. Edaravone, ferrostain-1,
TEMPO and trolox effectively inhibited the production of malondialdehyde (MDA) and several
oxidized AAs generated in the AAPH-induced LPO because of their scavenging ability toward lipid
peroxyl radicals. In contrast, ebselen and ferrostatin-1 showed strong antioxidative activity in the
HpODE/hemin-induced peroxidation. Under this condition, ebselen and ferrostatin-1 were thought to
reduce HpODE and its derived alkoxyl radicals to the corresponding lipid alcohols. In conclusion,
we found that each antioxidant had different antioxidative activities that prevented the progression of
LPO. We expect that these findings will contribute to the design of novel therapeutic strategies using
an appropriate antioxidant targeted to each step of the development of oxidative stress diseases.
KEYWORDS: lipid peroxidation, antioxidant, lipid radical, lipid hydroperoxide.

INTRODUCTION
Lipid peroxidation (LPO) is recognized as an important contributor to the pathological events of
oxidative diseases [1]. A polyunsaturated fatty acid (PUFA), which contains several carbon-carbon
double bonds, is easily oxidized by reactive oxygen species (ROS), causing chain peroxidation [2].
The primary step of this chain reaction is hydrogen atom abstraction from the bis-allylic methylene
groups of the PUFA and addition of molecular oxygen, resulting in the formation of lipid peroxyl
radicals [3]. Then, these radicals promote the excessive generation of lipid hydroperoxides or
electrophiles, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) [4]. Additionally,
lipid hydroperoxides easily decompose to highly reactive lipid alkoxyl radicals via the Haber-Weiss
reaction in the presence of metal ions, and further accelerate the propagation step in the chain
reaction [5]. Each oxidized lipid and electrophile contributes to the onset or progression of several
oxidative diseases. For example, accumulation of lipid hydroperoxide leads to lipid
peroxidation-dependent cell death or ferroptosis [6,7]. Lipid-derived electrophiles cause the
chemical modification of proteins [8,9] and induce cardiovascular and neurodegenerative diseases
[10,11].
Hence, antioxidants that specifically inhibit lipid peroxidation may be effective therapeutic agents
[12]. To date, there have been many studies on the protective effects of antioxidants toward several
diseases. α-Tocopherol, which is a lipophilic antioxidant, has been reported to improve the
pathological condition of non-alcoholic steatohepatitis [13]. Edaravone has been clinically applied
for the treatment of acute cerebral infarction and amyotrophic lateral sclerosis [14,15]. Additionally,
ferrostatin-1 and liproxstatin-1, which are potential inhibitors of ferroptosis, reduced the
pathogenesis in various disease model animals, including those for acute renal failure, myocardial
disease and ischemic stroke [16-18]. Thus, LPO is a promising therapeutic target in oxidative stress
diseases, and is the subject of novel antioxidant therapeutic strategies.

However, it is unclear as to which peroxidation step each antioxidant targets. As described above,
LPO involves a complex multistep reaction and a number of oxidized lipids and oxidative factors are
involved in the reaction processes [2,5]. Thus, the types and amounts of oxidized lipids vary greatly
depending on the reaction conditions [19]. Therefore, it is also assumed that antioxidants show
different inhibition effects toward LPO depending on where in the multistep reaction process they
act. However, commonly used antioxidant assays [20], such as oxygen radical absorbance capacity
and ferric reducing antioxidant power (FRAP) [21] [22], only enable a limited evaluation of the
capabilities of antioxidants, such as their ROS scavenging and reducing capacities. Accordingly, if
each reaction target of an antioxidant could be comprehensively investigated and clarified, it will
contribute to the design of appropriate antioxidants for each LPO condition.
In this study, we investigated the inhibitory mechanisms of several antioxidants toward LPO. For
this purpose, we comprehensively analyzed the oxidized lipid species generated in the presence of
antioxidants using the thiobarbituric acid reactive substances (TBARS) assay and LC/MS/MS
measurements. As a result, the employed antioxidants showed different removal activities toward
oxidized lipids, such as lipid radicals and lipid hydroperoxides.

MATERIALS AND METHODS
Reagents
Arachidonic acid (AA, from porcine liver, ≥99%), linoleic acid (LA, ≥99%) and soybean lipoxidase
from Glycine max (s15LOX) were purchased from Sigma-Aldrich (St. Louis, MO), 2,2’-azobis
(2-amidinopropane) dihydrochloride (AAPH) was obtained from Wako Pure Chemical Industries,
Ltd. (Osaka, Japan), and hemin was from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The
antioxidants edaravone and ebselen were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo,
Japan), trolox was from Merck Millipore (Burlington, MA) and ferrostatin-1 was from Focus
Biomolecules
(Plymouth
Meeting,
PA).
4-(4-Nitrobenzo[1,2,5]
oxadiazol-7-ylamino)-6-pentyl-2,2,6-trimethylpiperidine-1-oxyl (NBD-Pen) was synthesized
according to a previously reported method [23]. For high-performance liquid chromatography
(HPLC), acetonitrile (LCMS grade, ≥99.9%) was purchased from Nacalai Tesque (Kyoto, Japan),
and ammonium acetate (Wako 1st grade) was from Wako Pure Chemical Industries, Ltd.
Preparation of hydroperoxyoctadecadienoic acid
LA (500 µM, 0.5% ethanol) and s15LOX (10 µg/mL) were mixed in PBS (pH 7.4) and incubated 15
min at 37 °C. After the incubation, the reaction mixture was extracted by the Bligh and Dyer method
[24]. The organic layer was dried with nitrogen gas and dissolved in 100 µL of ethanol. The solution
of hydroperoxyoctadecadienoic acid (HpODE) was stored at -80 °C until use.
AA peroxidation systems
AA (500 µM) and each antioxidant (ebselen, edaravone, ferrostatin-1, TEMPO and trolox; 10 or 100
µM) were mixed in PBS containing 0.5% ethanol (pH 7.4). Radical initiators (system-1; 50 mM
AAPH, system-2; 50 µM HpODE and 5.0 µM hemin) were added to the reaction mixture. After
incubation for 15 min at 37 °C, the reaction mixture was extracted by the Bligh and Dyer method
[24]. The organic layer was dried with nitrogen gas and dissolved in 200 µL of methanol. The

solution was stored at -80 °C until use.
Thiobarbituric acid reactive substances assay
A mixture of 10% sodium dodecyl sulfate, 20% acetic acid and 1.3% thiobarbituric acid were added
to oxidized AA solutions and incubated at 60 °C for 1 h. Samples were cooled and an n-butanol/
pyridine (15:1) solution was added. After centrifugation at 720 g for 10 min, samples were plated
onto a 96-well microplate and the fluorescence was measured (λex 512 nm, λem 553 nm) using an
EnSpire Multimode Plate Reader (PerkinElmer Japan Co., Ltd., Kanagawa, Japan).
Measurement of oxidized arachidonic acids by liquid chromatography–tandem mass spectrometry
Liquid chromatography–tandem mass spectrometry (LC/MS/MS) was carried out using a
LCMS-8060 (Shimadzu Co., Kyoto, Japan). The LC/MS/MS analyses were carried out using
LabSolutions version 5.80 (Shimadzu Co.). The mass spectrometer was equipped with an electron
spray ionization source. The extracted solution was measured by LC/MS/MS with multiple reaction
monitoring (MRM) and a LC/MS/MS Method Package for Lipid Mediators (Shimadzu Co.).
Detection of lipid-derived radicals
AA (500 μM) and antioxidants at the indicated concentration (50, 100, 200 and 400 μM) were mixed
in PBS (pH 7.4). A radical initiator (system-1; 50 mM AAPH, system-2; 50 µM HpODE and 5.0 µM
hemin) was added to the reaction mixture and incubated at 37 °C for 60 min. Then NBD-Pen (10
μM) was added to the mixture, and further incubated at 37 °C for 15 min. After the reaction, the
fluorescence intensity (λex 470 nm, λem 530 nm) of the reaction solution was measured using an
Enspire Multimode Plate Reader.
Statistical analysis
Statistical analyses were carried out using StatView 5.0 (SAS Institute Inc.), and the data were
analyzed by the Tukey-Kramer test. The results are expressed as mean ± standard deviation, and P
<0.05 was considered statistically significant.

RESULTS
In this study, LPO was induced using two radical initiators (Fig. 1a). In system-1, the azo-initiator
AAPH was used, which pyrolytically decomposes to radical species and promotes the production of
peroxyl radicals [25]. In system-2, a combination of HpODE and hemin was applied, which mimics
LPO induced by the decomposition of lipid hydroperoxide [5]. The chemical structures of the
antioxidants used in this study, ebselen, edaravone, ferrostatin-1, TEMPO and trolox, are shown in
Fig. 1b.
Lipid peroxide inhibitory effects of antioxidants in an AAPH-induced lipid peroxidation system.
First, we examined whether antioxidants inhibit MDA production using system-1 (Fig. 2). After
adding AAPH to the AA solution, most of the tested antioxidants inhibited MDA production except
for ebselen. Next, we investigated the effect of the antioxidants on the production of AA-derived
oxidants, including hydroperoxyeicosatetraenoic acids (HpETEs; 15-HpETE, 12-HpETE and
5-HpETE; Fig. 3a–c), hydroxyeicosatetraenoic acid (HETEs; 15-HETE, 12-HETE and 5-HETE; Fig.
3d–f) and prostaglandins (PGs; 8-iso-PGE2, PGE2 and PGD2; Fig. 3g–i) using LC/MS/MS. The
formation of oxidized AAs was enhanced by addition of AAPH, and decreased by addition of all the
tested antioxidants except ebselen. Interestingly, ebselen inhibited HpETEs formation, but not the
production of HETEs and PGs. In addition, ebselen enhanced the formation of 15-HETE, 5-HETE
and 8-iso-PGE2. The formation of MDA and PGs are reported to occur via the cyclization of lipid
peroxyl radicals through a reaction process that does not involve the formation of lipid
hydroperoxides [26,27] (Fig. 4a). Therefore, we hypothesized that the small inhibitory effect
observed for ebselen toward MDA and PGs formation is attributed to its low reactivity toward
lipid-derived radicals. To investigate whether ebselen could react with the AA-derived radicals
generated in system-1, we used NBD-Pen, which is a probe developed in-house for the detection of
lipid radicals [23]. As expected, ebselen did not inhibit the enhanced fluorescence intensity of

NBD-Pen, but TEMPO suppressed it in a concentration-dependent manner (Fig. 4b). These results
clearly suggest that most of the antioxidants used in this study inhibited the generation of
AA-derived radicals, while ebselen only suppressed the accumulation of lipid hydroperoxide but not
AA-derived radicals and their secondary products, such as MDA and PGs.
LPO inhibitory effects of antioxidants in an HpODE/hemin-induced lipid peroxidation system.
Next, we examined the inhibitory effect of antioxidants on MDA production in a
HpODE/hemin-induced peroxidation system (system-2) (Fig. 5). In system-2, enhanced MDA
production was inhibited by all tested antioxidants, even ebselen although it did not suppress the
MDA level in system-1. Next, the amounts of oxidized lipids derived from AA and 13-HpODE were
measured (Fig. 6). 13-HpODE was completely consumed by adding hemin (Fig. 6a). AA-derived
oxidized lipids increased in the presence of the HpODE/hemin peroxidation system and decreased in
the presence of antioxidants (Fig. 6b-j). Interestingly, ebselen and ferrostatin-1 increased the level of
13-hydroxyoctadecadienoic acid (13-HODE) (Fig. 6k). Ebselen is known to reduce lipid
hydroperoxides to lipid alcohols [28]. Hence, ferrostatin-1 was also assumed to react with
13-HpODE-derived active species to prevent the propagation of LPO.
Reducing capacities of ferrostain-1 toward HpODE-derived alkoxyl radicals
In the presence of metal ions, lipid hydroperoxides immediately decompose to lipid-derived alkoxyl
radicals via the Haber-Weiss reaction. Lipid alkoxyl radicals are usually reduced to lipid alcohols by
a one-electron reduction. In comparison, lipid alkoxyl radicals may also be converted to epoxyaryl
radicals via a radical rearrangement [29], which are highly reactive carbon-centered radicals that act
to accelerate LPO as chain carriers (Fig. 7a) [30]. Hence, we investigated whether ferrostatin-1
eliminates lipid alkoxyl radicals by promoting the one electron reduction to produce 13-HODE. As a
result, 13-HODE increased with the addition of ferrostatin-1 in a concentration-dependent manner in
the presence of hemin, but not in the absence of hemin (Fig. 7b). Furthermore, under this condition,

lipid-derived radical generation was suppressed by ferrostatin-1 (Fig. 7c). These results suggest that
ferrostatin-1 reduced 13-HpODE derived radicals, and then effectively inhibited the subsequent LPO
process.
DISCUSSION
As shown in this study, the antioxidants exhibited different inhibitory effects toward LPO and
regulated the production of oxidized lipids depending on their mechanism of action. Scheme 1
demonstrates the reaction points of the antioxidants using in this study. Edaravone, TEMPO and
trolox inhibited the generation of a wide variety of oxidized lipids using both the system-1 and
system-2 peroxidation mixtures (Fig. 2, 3, 5, 6). The inhibition was attributed to the scavenging
effect of the above antioxidants toward the lipid-derived radicals, which are the upstream active
species in LPO. Moreover, TEMPO showed much higher antioxidative activity than edaravone or
trolox. TEMPO has been reported to catalytically inactivate lipid-derived radicals via the electron
transfer or radical-radical coupling reaction [31,32]. Indeed, TEMPO had a higher anti-oxidative
effect even at a lower concentration (10 µM) (Fig. 2, 5). In contrast, chromanol derivatives, such as
trolox, react with peroxyl radicals to produce unstable semiquinone radical intermediates resulting in
loss of their antioxidative capacities [33]. Edaravone forms an enolate anion in aqueous solution, and
is inactivated after reaction with the radical species [34]. For these reasons, despite the fact that the
above antioxidants react with the same reaction target (lipid-derived radicals), TEMPO in particular
showed strong antioxidative effects because of its catalytic scavenging capacity against radical
species.
Although ebselen completely inhibited the production of lipid hydroperoxides (Fig. 3a–c), it did
not suppress the generation of MDA, PGs and lipid-derived radicals (Fig. 2, 3g–i, 4b). Organic
selenium is known to reduce hydroperoxides to their corresponding alcohols [28]. Indeed ebselen, a

synthetic organoselenium compound, exhibited a large cytoprotective effect against ferroptosis
characterized by the accumulation of lipid hydroperoxide [6]. In contrast, there are few studies that
show ebselen has radical scavenging ability. In agreement with these observations, ebselen
sufficiently inhibited LPO induced by lipid hydroperoxides (system-2), but not by a radical reaction
(system-1). On the other hand, ebselen enhanced the production of some HETEs and PGs, including
15-HETE, 5-HETE and 8-iso-PGE2 in system-1 (Fig. 3). These oxidized AAs were thought to
generate through the reduction of hydroperoxides (Scheme S1), and ebselen can promote this
reaction [35]. Therefore, ebselen could increase the formation of 15-HETE, 5-HETE and 8-iso-PGE2
in AAPH induced LPO system.
The ferroptosis inhibitor ferrostatin-1 showed excellent antioxidative effects in both systems (Fig. 2,
5). It is thought that ferrostatin-1 exhibits scavenging activity against not only lipid peroxyl radicals,
but also lipid alkoxyl radicals. In fact, ferroptosis requires both the accumulation of lipid
hydroperoxide and iron [6,7], which may indicate that a large amount of lipid alkoxyl radicals are
generated from this mechanism of cell death. These observations are consistent with the result
described herein that ferrostatin-1 showed a high scavenging ability toward lipid alkoxyl radicals
(Fig. 7). In addition, other anitoxidants, including edaravone, TEMPO, and trolox, also enhanced the
production of 13-HODE from HpODEs in the presence of hemin, but these capabilities were weaker
than that of ferrostatin-1 (Fig. S1).
Furthermore, the strong LPO inhibitory effect of ferrostatin-1 was observed even at a lower
concentration (10 µM) in both peroxidation systems (Fig. 2, 5). In recent years, aniline-type
antioxidants including ferrostatin-1 have been reported to exhibit excellent protective effects against
oxidative stress because of their catalytic antioxidative activities [36,37]. Additionally, the high
hydrophobicity of ferrostatin-1 allows its successful accumulation in lipid vesicles [38]. Taken
together, these properties of ferrostatin-1 support why it efficiently inhibits LPO in both systems.

Since all the experiments in this study were performed in test tubes, it is unclear whether these
antioxidants show similar activities in vivo. Therefore, future work should include investigation of
the antioxidative activity and the reaction target of each antioxidant in biological systems using
disease model animals.
To date, many researchers have worked on treatments using antioxidants, but their effects have been
controversial [39]. In this study, we demonstrated that each tested antioxidant has a different reaction
target in LPO, including lipid peroxyl radical, lipid hydroperoxide and lipid alkoxyl radical. These
observations suggest that antioxidants may not be broadly applicable to conventional treatment
methods because of a lack of understanding of their reaction points in LPO. Based on these findings,
we expect that novel antioxidant therapeutic strategies must be developed with knowledge of the
specific LPO reaction targets responsible for the pathology of each oxidative stress disease.

ACKNOWLEDGMENTS
This work was supported in part by the AMED under AMED-CREST Grant [JP19gm0910013 to
K.Y.] and JSPS under KAKENHI Grants [18K14884 to Y.M., 18K19405 and 17H03977 to K.Y.],
Japan. This work was supported in part by the Platform Project for Supporting Drug Discovery and
Life Science Research from the AMED. We appreciate the technical support provided by the
Research Support Center of the Graduate School of Medical Sciences, Kyushu University. We thank
Renee Mosi, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this
manuscript.
DISCLOSURE OF INTEREST
The authors report no conflict of interest.

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Figure Legends
Figure 1. Arachidonic acid (AA) peroxidation systems (a) and antioxidants (b) employed in this
study.

Figure 2. MDA production in system-1. AAPH (50 mM) was added to the reaction mixture
containing AA (500 µM) and antioxidants (white columns; 10 µM, black columns; 100 µM). After
incubation, the level of MDA was measured by the TBARS assay. Data are presented as the mean ±
s.d. of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, as compared to AAPH
(-) group without adding antioxidants.

Figure 3. LC/MS/MS analyses of oxidized AAs generated in system-1. After AAPH-induced AA
peroxidation in the presence of several antioxidants (100 µM), oxidized AAs were extracted by the
Bligh and Dyer method. The extracted solution was measured by LC/MS/MS with multiple reaction
monitoring (MRM). (a) 15-HpETE, (b) 12-HpETE, (c) 5-HpETE. (d) 15-HETE, (e) 12-HETE, (f)
5-HETE. (g) PGD2, (h) PGE2, (i) 8-iso-PGE2. Data are presented as the mean ± s.d. of three
independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, as compared to AAPH (-) group
without adding antioxidants.

Figure 4. Generation of AA-derived radicals in system-1. (a) Plausible mechanism of MDA/PGs
formation initiated by an AA-derived radical. (b) Detection of lipid radicals using NBD-Pen (λex;
470 nm, λem: 530 nm). NBD-Pen (10 µM) was mixed with an AA solution 60 min after adding
antioxidants at the indicated concentrations (TEMPO, ebselen; 50, 100, 200 and 400 µM) and AAPH
(50 mM). Data are presented as the mean ± s.d. of three independent experiments. ***P <
0.001, **P < 0.01, *P < 0.05, as compared to AAPH group without adding antioxidants.

Figure 5. MDA production in system-2. HpODE (50 µM) and hemin (10 µM) were added to the
reaction mixture containing AA (500 µM) and antioxidants (white columns; 10 µM, black columns;
100 µM). After incubation, the level of MDA was measured by the TBARS assay. Data are presented
as the mean ± s.d. of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, as
compared to HpODE/hemin (-) group without adding antioxidants.

Figure 6. LC/MS/MS analyses of oxidized AAs generated in system-2. After HpODE/hemin was
used to induce AA peroxidation in the presence of several antioxidants (100 µM), AA or
HpODE-derived oxidized lipids were extracted by the Bligh and Dyer method. The extracted
solution was measured by LC/MS/MS with MRM and the MS peak area determined (a) 13-HpODE,
(b) 15-HpETE, (c) 12-HpETE, (d) 5-HpETE. (e) 15-HETE, (f) 12-HETE, (g) 5-HETE. (h) PGD2, (i)

PGE2, (j) 8-iso-PGE2, (k) 13-HODE. Data are presented as the mean ± s.d. of three independent
experiments. ***P < 0.001, **P < 0.01, *P < 0.05, as compared to HpODE/hemin (-) group without
adding antioxidants.
Figure 7. Conversion of 13-HpODE-derived radicals to 13-HODE or carbon centered radicals. (a) A
plausible mechanism to form 13-HODE and epoxyaryl radicals from 13-HpODE-derived alkoxyl
radicals. (b) Measurement of 13-HODE generated in the presence of ferrostatin-1 at the indicated
concentrations (50, 100, 200 and 400 µM) with (white columns) or without (meshed columns) added
hemin (10 µM). (c) Detection of lipid radicals generated in the HpODE/hemin system using
NBD-Pen (λex 470 nm, λem 530 nm). NBD-Pen (10 µM) was mixed with HpODE (50 µM) and
hemin (10 µM) in the presence of ferrostatin-1 at the indicated concentrations (50, 100, 200, 400

µM). Data are presented as the mean ± s.d. of three independent experiments. ***P < 0.001, **P <
0.01, *P < 0.05, as compared to HpODE/hemin group without adding ferrostatin-1.
Scheme 1. Proposed mechanism of how each antioxidant suppresses LPO induced by two radical
initiator systems. System-1 consists of AAPH and system-2 consists of HpODE/hemin.
Supporting information Figure S1. Measurement of 13-HODE generated in system-2 with
antioxidants ((a)edaravone (b) TEMPO and (c) trolox) at the indicated concentrations (50, 100, 200
and 400 μM) with added HpODE/hemin. Data are presented as the mean +s.d. of three independent
experiments. ***P < 0.001, **P < 0.01, *P < 0.05, as compared to control group without adding
antioxidant.
Supporting information Scheme S1. Proposed mechanism of formation of (a) 15-HETE and (b)
PGs.