Tetrathiatriarylmethyl radical with a single aromatic hydrogen as a highly sensitive and specific superoxide probe

Article abstract of DOI:10.1016/j.freeradbiomed.2012.09.011

Superoxide (O₂^?-) plays crucial roles in normal physiology and disease; however, its measurement remains challenging because of the limited sensitivity and/or specificity of prior detection methods. We demonstrate that a tetrathiatriarylmethyl (TAM) radical with a single aromatic hydrogen (CT02-H) can serve as a highly sensitive and specific O ₂^?- probe. CT02-H is an analogue of the fully substituted TAM radical CT-03 (Finland trityl) with an electron paramagnetic resonance (EPR) doublet signal due to its aromatic hydrogen. Owing to the neutral nature and negligible steric hindrance of the hydrogen, O ₂^?- preferentially reacts with CT02-H at this site with production of the diamagnetic quinone methide via oxidative dehydrogenation. Upon reaction with O₂^?-, CT02-H loses its EPR signal and this EPR signal decay can be used to quantitatively measure O₂^?-. This is accompanied by a change in color from green to purple, with the quinone methide product exhibiting a unique UV-Vis absorbance (ε =15,900 M^-1 cm^-1) at 540 nm, providing an additional O₂^?- detection method. More than five-fold higher reactivity of CT02-H for O₂^?- relative to CT-03 was demonstrated, with a second-order rate constant of 1.7×10⁴ M^-1 s^-1 compared to 3.1×10³ M^-1 s^-1 for CT-03. CT02-H exhibited high specificity for O₂^?- as evidenced by its inertness to other oxidoreductants. The O₂^?- generation rates detected by CT02-H from xanthine/xanthine oxidase were consistent with those measured by cytochrome c reduction but detection sensitivity was 10- to 100-fold higher. EPR detection of CT02-H enabled measurement of very low O₂^?- flux with a detection limit of 0.34 nM/min over 120 min. HPLC in tandem with electrochemical detection was used to quantitatively detect the stable quinone methide product and is a highly sensitive and specific method for measurement of O₂ ^?-, with a sensitivity limit of ～2×10^-13 mol (10 nM with 20-μl injection volume). Based on the O₂-dependent linewidth broadening of its EPR spectrum, CT02-H also enables simultaneous measurement of O₂ concentration and O₂^?- generation and was shown to provide sensitive detection of extracellular O ₂^?- generation in endothelial cells stimulated either by menadione or with anoxia/reoxygenation. Thus, CT02-H is a unique probe that provides very high sensitivity and specificity for measurement of O ₂^?- by either EPR or HPLC methods.

Full text of DOI:10.1016/j.freeradbiomed.2012.09.011

Free Radical Biology and Medicine 53 (2012) 2081–2091
Contents lists available at SciVerse ScienceDirect
Free Radical Biology and Medicine
journal homepage: www.elsevier.com/locate/freeradbiomed
Original Contribution
Tetrathiatriarylmethyl radical with a single aromatic hydrogen as a highly
sensitive and specific superoxide probe
Yangping Liu a,1, Yuguang Song a,1, Francesco De Pascali a, Xiaoping Liu a, Frederick A. Villamena a,b
,
n
Jay L. Zweier a,
a Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University,
Columbus, OH 43210, USA
b Department of Pharmacology, College of Medicine, The Ohio State University, Columbus, OH 43210, USA
a r t i c l e i n f o
a b s t r a c t
ꢀ
Superoxide (O2ꢁ) plays crucial roles in normal physiology and disease; however, its measurement
remains challenging because of the limited sensitivity and/or specificity of prior detection methods. We
demonstrate that a tetrathiatriarylmethyl (TAM) radical with a single aromatic hydrogen (CT02-H) can
Article history:
Received 27 March 2012
Received in revised form
4 September 2012
ꢀ
ꢁ
serve as a highly sensitive and specific O2 probe. CT02-H is an analogue of the fully substituted TAM
radical CT-03 (Finland trityl) with an electron paramagnetic resonance (EPR) doublet signal due to
its aromatic hydrogen. Owing to the neutral nature and negligible steric hindrance of the hydrogen,
Accepted 13 September 2012
Available online 20 September 2012
ꢀ
ꢁ
Keywords:
O2 preferentially reacts with CT02-H at this site with production of the diamagnetic quinone methide
via oxidative dehydrogenation. Upon reaction with O2ꢁ, CT02-H loses its EPR signal and this EPR
ꢀ
Oxygen free radicals
Reactive oxygen species
Electron paramagnetic resonance
spectroscopy
signal decay can be used to quantitatively measure O2ꢁ. This is accompanied by a change in color from
ꢀ
green to purple, with the quinone methide product exhibiting
a unique UV–Vis absorbance
ꢀ
ꢁ
(e
¼15,900 Mꢁ1 cmꢁ1) at 540 nm, providing an additional O2 detection method. More than five-fold
Spin trapping
ꢀ
ꢁ
higher reactivity of CT02-H for O2 relative to CT-03 was demonstrated, with a second-order rate
Trityl radical
constant of 1.7 ꢂ 104
M
ꢁ1 sꢁ1 compared to 3.1 ꢂ 103 Mꢁ1 sꢁ1 for CT-03. CT02-H exhibited high
Free radicals
ꢀ
ꢁ
ꢀꢁ
specificity for O2 as evidenced by its inertness to other oxidoreductants. The O2 generation rates
detected by CT02-H from xanthine/xanthine oxidase were consistent with those measured by
cytochrome c reduction but detection sensitivity was 10- to 100-fold higher. EPR detection of CT02-H
ꢀ
ꢁ
enabled measurement of very low O2 flux with a detection limit of 0.34 nM/min over 120 min. HPLC
in tandem with electrochemical detection was used to quantitatively detect the stable quinone methide
product and is a highly sensitive and specific method for measurement of O2ꢁ, with a sensitivity limit
ꢀ
of ꢃ2 ꢂ 10ꢁ13 mol (10 nM with 20-
ml injection volume). Based on the O2-dependent linewidth
broadening of its EPR spectrum, CT02-H also enables simultaneous measurement of O2 concentration
ꢀ
ꢁ
ꢀꢁ
and O2 generation and was shown to provide sensitive detection of extracellular O2 generation in
endothelial cells stimulated either by menadione or with anoxia/reoxygenation. Thus, CT02-H is a
ꢀ
ꢁ
unique probe that provides very high sensitivity and specificity for measurement of O2 by either EPR
or HPLC methods.
& 2012 Elsevier Inc. All rights reserved.
Introduction
electron transfer chain [7–10] and through several enzyme-
mediated processes [ꢁ11,12]. It has been estimated that 1–2% of O2
In view of the critical roles of superoxide (O2ꢁ) in both normal
cellular signaling and disease [1–6], as well as the limitations of
prior detection methods, there is a great need for development of
specific high-sensitivity methods for its measurement in biomedical
systems. Superoxide is a one-electron reduction product of mole-
cular oxygen (O2) that is mainly generated from the mitochondrial
ꢀ
ꢀ
is converted into O2 in isolated mitochondria [13]. As a critical
ꢀ
ꢁ
reactive oxygen species (ROS), O2 not only takes part in host
defense in phagocytic cells, but also is involved in signal transduc-
tion and regulation in many tyꢁpes of cells [6]. On the other hand,
ꢀ
oxidative stress induced by O2 and other ROS is also associated
with pathogenesis of many important diseases including cancer,
arteriosclerosis, heart attack, stroke, diabetic vascular disease, and
a variety of inflammatory diseases [3,6]. Thus, sensitive and
ꢀ
ꢁ
selective detection of O2 in in vitro and in vivo systems is of
paramount importance to understand its roles in normal physiology
and disease.
n Corresponding author. Fax:þ(614) 247 7845.
E-mail address: Jay.Zweier@osumc.edu (J. L. Zweier).
1
These authors contributed equally to this study.
0891-5849/$ - see front matter & 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.freeradbiomed.2012.09.011

2082
Y. Liu et al. / Free Radical Biology and Medicine 53 (2012) 2081–2091
ꢀ
ꢁ
of O2 in endothelial cells was also measured. CT02ꢁ-H was found to
ꢀ
provide excellent sensitivity and specificity for O2 detection and
also to enable simultaneous measurement of O2 concentrations.
Materials and methods
Chemicals
Xanthine (X), xanthine oxidase (XO), glutathione (GSH), ascor-
bic acid (Asc), hydrogen peroxide (H2O2), ammonium iron(II)
sulfate hexahydrate ((NH4)2Fe(SO4)2 6H2O), diethylenetriamine-
ꢀ
pentaacetic acid (DTPA), nitrilotriacetic acid disodium salt (NTA),
catalase, cytochrome c, and superoxide dismutase (SOD) from
bovine erythrocytes were purchased from Sigma–Aldrich. TAM
radicals CT-03 and CT02-H were synthesized as previously
described [30,32,39]. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO)
was purchased from Dojindo Molecular Technologies and
5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO)
from Enzo Life Science. The concentration of CT02-H was deter-
mined by EPR using the purified CT-03 as standard. Stock
solutions (1 mM) of CT-03 and CT02-H as carboxylate sodium
forms were prepared in phosphate-buffered saline (PBS) and
stored at ꢁ20 1C. All other chemicals were commercially avail-
able unless otherwise indicated.
Scheme 1. Molecular structure of TAM radicals.
Over the past decades, several techniques have been devel-
ꢀ
oped for the measurement of O2ꢁ, including ferricytochrome c
reduction [14], electron paramagnetic resonance (EPR)–spin trap-
ping [15], electrochemistry [16], and fluorescence and chemi-
luminescence [17,18], but most of these techniques are limited by
low sensitivity and/or specificity. Efforts have been made to
improve these techniques. For example, nꢁew spin traps with
EPR spectroscopy
ꢀ
All EPR spectra were recorded at room temperature using a
Bruker X-band EPR spectrometer. Samples were loaded into EPR
capillary tubes. The following acquisition parameters were used:
microwave power, 0.2–10 mW for TAM radicals and 20 mW for
the spin trapping with nitrones; modulation frequency, 30–
100 kHz; time constant, 21 ms; modulation amplitude, 0.03–1 G.
enhanced stability of the corresponding O2 spin adducts have
been developed [19,20]. In addition, the new HPLC–fluorescence
tandem technique was alsoꢁutilized to compensate for the low
ꢀ
specificity of fluorescent O2 detection [21]. Despite these inno-
vations, theꢁidentification and quantitation of the low biological
ꢀ
fluxes of O2 formation remain challenging.
In recent years a tetrathiatriarylmethyl (TAM) radical (OX063;
Scheme 1) has been proposed as an ideal spin probe for in vivo
EPR spectroscopy and imaging suitable for the measurement of
O2 concentrations based on the O2-dependent linewidth broadening
of its EPR spectrum [22,23]. It was also proposed to serve as a
Stability studies in the presence of biological oxidoreductants
Aqueous solutions of GSH (1 mM), Asc (1 mM), and H2O2
(1 mM) were used. The Fe(II)–NTA complex was prepared by
dissolving (NH4)2Fe(SO4)2 and NTA at a molar ratio of 1:2 in water
under anaerobic conditions. The Fe(III)–NTA solution (Fe:NTA 1:2)
was prepared by slow addition of the appropriate volume of
acidic Fe(III) stock solution into a vigorously stirred solution of
NTA in water. The resulting solution was slowly neutralized to
pH 7.4 using 0.1 M NaOH. Either Fe(II)–NTA or Fe(III)–NTA solution
ꢀ
ꢁ
highly selective and sensitive O2 probe [24,25]. Superoxide was
conveniently measured by following the changes in the EPR signal
intensity or UV–Vis absorbance of OX063. Recently, the reaction
ꢀ
ꢁ
mechanism of the TAM radicals OX063 and CT-03 with O2 was
characterized [26] and the corresponding quinone methide pro-
ducts were identified [26,27]. Owing to unique EPR properties and
high biostability [28], TAM radicals have been so far the most
promising paramagnetic probes for EPR spectroscopy/imaging
applications and have been utilized to measure extracellular
ꢀ
was freshly prepared before use. Hydroxyl radical (HO ) was
continuously generated from the system consisting of Fe(III)–NTA
(0.1 mM) and H2O2 (1 mM). Spin trapping EPR experiments with
the spin trap DMPO were carried out to verify the production of
ꢀ
ꢁ
[29] and intracellular [30,31] O2 levels, O2 generation [24], pH
[32–35], and glutathione levels [36], as well as to assess total
redox status [37,38]. Thus, combined with recent progress in low-
frequency EPR instrumentation, TAM radicals have great potential
ꢀ
HO . Superoxide was generated using the X/XO system using XO
(10 mU/ml) and X (100 mM) in the presence of DTPA (0.1 mM).
ꢀ
ꢁ
for measurement and mapping of O2 in tissues.
Alkylperoxyl radical was generated by thermolysis of 2,20-azobis-
2-methylpropanimidamide, dihydrochloride (AAPH; 1 mM) at
37 1C in the presence or absence of SOD (200 U/ml). Peroxynitrite
(ONOOꢁ) was generated by decomposition of SIN-1 (1 mM) at
37 1C in the presence or absence of SOD (50 U/ml). EPR spectra
were recorded 30 min after mixing the TAM radical solution
In this article, we report a pꢁartially substituted TAM radical,
ꢀ
CT02-H (Scheme 1), as a new O2 probe with a high rate constant
providiꢁng enhanced sensitivity. Using xanthine/xanthine oxidase
ꢀ
ꢀꢁ
as a O2 source, the reactivity of CT02-H with O2 was compared
with that of the fully substituted TAM radical CT-03, and the
ꢀ
ꢁ
reaction rate constants of both TAM radicals with O2 were
determined using O2 self-dismutation as the competition reac-
tion. The reaction mechanism with O2 was determined based on
product analysis. Applications of CT02-H for detection and quan-
titation of high or low O2 fluxes by EPR spectroscopy or HPLC/
electrochemistry techniques were performed. Cellular generation
(20 mM) with various oxidoreductants in PBS. The effects of
ꢀ
ꢁ
various reactive species on CT02-H were expressed as the per-
centage of CT02-H remaining after exposure to the reactive
species for 30 min, which was obtained by comparing the double
integral of the EPR signal in each system with that of CT02-H
ꢀ
ꢁ
ꢀ
ꢁ
(20 mM). Each experiment was conducted three times.

Y. Liu et al. / Free Radical Biology and Medicine 53 (2012) 2081–2091
2083
Cyclic voltammetry
X (100
chrome c (100
m
M), EDTA (100
m
M),ꢁcatalase (200 U/ml), and ferricyto-
ꢀ
mM). The O2 generation rate was determined
Cyclic voltammetry was performed on a potentiostat and
computer-controlled electroanalytical system. Electrochemical
measurements were carried out in a 10-ml cell equipped with a
glassy carbon working electrode (7.07 mm2), a platinum-wire
auxiliary electrode, and a Ag/AgCl reference electrode. Solutions
of TAM radicals (1 mM) were degassed by bubbling with nitrogen
gas before the detection. The redox potentials were calculated
according to the relation E¼(EapþEpc)/2.
from the reduction of ferricytochrome c to ferrocytochrome c by
ꢀ
O2ꢁ. It was measured from the slope of the time-dependent
production of ferrocytochrome c measured at 550 nm using an
extinction coefficient of 2.1 ꢂ 104 Mꢁ1 cmꢁ1 [42,43].
Preparation of QM1 and 18O-labeled QM1
The quinone methide product QM1 was prepared from CT02-H
using the previously reported method [26] with some modifica-
tion. XO (0.05 U/ml) was added to the solution containing CT02-H
(8.6 mg), X (1 mM), and DTPA (0.1 mM) in 100 ml of PBS (pH 7.4).
This reaction mixture was stirred at room temperature for 2 h
with a gentle bubbling of O2 gas. Then, a citric acid solution (30
ml, 8%, w/v) was added and QM1 was extracted with ethyl acetate
(3 ꢂ 50 ml). The combined organic layers were dried on anhy-
drous sodium sulfate and concentrated. The resulting residue was
redissolved in PBS and purified by RP-flash chromatography over
a prepacked C18 column using a gradient from 0/100 to 30/80 of
acetonitrile/water mixture to afford 8.5 mg of pure QM1 as a
purple solid. 1H NMR (600 MHz, acetone-d6) 1.693 (s, 6H), 1.782
Kinetic studies
The apparent second-order rate constants of TAM radicals
ꢀ
ꢁ
ꢀꢁ
(CT02-H and CT-03) with O2 were determined using the O2
self-dismutation as a competition reaction at pH 7.4. XO (10 mU/ml)
was added to the solutions containing various concentrations of
TAM radical, X (100 mM), and DTPA (0.1 mM) in PBS (pH 7.4).
Incremental EPR spectra were recorded 30 s after mixing. The
initial rate of the CT02-H decay was determined within 30–80 s,
during which the earliest linear curve becomes evident. Data
were plotted using Eq. (1),
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
(s, 6H), 1.795 (s, 6H), 1.812 (s, 6H), 1.889 (s, 6H), 1.896 (s, 6H); 13
C
2
ꢁ1þ 1þ4UkdisUF=ðkU½TAMꢄÞ
NMR (600 MHz, acetone-d6) 29.5, 30.0, 31.8, 33.3, 33.7, 35.4, 63.4,
64.0, 64.5, 123.7, 130.8, 131.6, 140.4, 141.3, 141.7, 142.8, 146.1,
153.3, 167.6, 173.3; HRMS (electrospray ionization (ESI), negative)
V ¼
ð1Þ
2
2Ukdis=ðkU½TAMꢄÞ
ꢀ
calcd for C39H37O5S12 ([MꢁH]ꢁ), 968.9290; found, 968.9295.
where V is the initial rate of the TAM radical decay, k and kdis are
ꢀ
ꢁ
the second-order rate constant of the TAM radical with O2 and
A stock solution of QM1 with a concentration of 3 mM was prepared
in PBS (10 mM, pH 7.4) for the subsequent HPLC analysis.
the O2 self-dismutation rate at pH 7.4 (2.4 ꢂ 105 Mꢁ1 sꢁ1
)
ꢀ
ꢁ
ꢀ
ꢁ
For the preparation of the 18O-labeled QM1, isotopically enriched
18O2 gas (99 atom% 18O; Sigma) was used instead of 16O2. Briefly, the
[40,41], respectively, and F is the O2 generation rate in the
X/XO system, which was determined to be 0.115 mM/s by ferri-
cytochrome c kinetic competition (see Supplementary Fig. S6).
solution containing CT02-H (0.2 mM),
X (0.8 mM), and DTPA
The appꢁarent second-order rate constants of CT02-H and CT-03
(0.1 mM) in 3 ml of PBS (pH 7.4) was bubbled with argon for
30 min and then 18O2 for 10 min. Thereafter, XO (0.02 U/ml) was
added and the resulting mixture was stirred at room temperature
for 30 min with gentle bubbling of 18O2 gas. Then, the citric acid
solution (1 ml, 8%, w/v) was added and the 18O-labeled QM1 was
extracted with ethyl acetate. Mass spectrometry (MS) analysis was
carried out on the resulting organic solution.
ꢀ
with O2 were determined to be 1.7 ꢂ 104 and 3.1 ꢂ 103 Mꢁ1 sꢁ1
,
respectively.
Oxygen sensitivity
The O2 sensitivity of CT02-H was evaluated as previously
described [34,36–38]. Briefly, a solution of CT02-H (20 M) in
m
ꢀ
ꢁ
PBS was transferred into a gas-permeable Teflon tube (i.d.
0.8 mm) and then the tube was sealed at both ends. The sealed
sample was placed inside a quartz EPR tube with open ends.
Nitrogen or a N2/O2 gas mixture with varying concentrations of
O2 was allowed to diffuse into the EPR tube. After 20 min of
equilibration, the EPR spectrum was recorded and the peak–peak
linewidth was calculated.
HPLC detection of the product of CT02-H with O2
The solutions (10 l) containing various concentrations of QM1
m
or CT02-H were injected separately into the HPLC system (Coul-
Array System from ESA Analytical, Ltd., Chelmsford, MA, USA) on a
C18 Tosoh Bioscience ODS-80Tm column (250 ꢂ 4.6 mm, 5
mm)
with refrigerated autosampler and ESA software for data collection
and analysis. The mobile phase consisted of 20 mM ammonium
acetate:methanol:acetonitrile 40:20:40 (v/v/v), pH 7.0, at a flow
rate of 1.2 ml/min, which was filtered and degassed before use.
ꢀ
ꢁ
Determination of the generation rate of O2 by EPR and UV–Vis
spectroscopy
ꢀ
ꢁ
CT02-H and its product with O2 (QM1) were detected at þ600
and þ800 mV, respectively, using the electrochemical detector of
the ESI Coulchem 6210 four-sensor electrochemical cell. The reten-
tion time was 4.2 min for the quinone methide QM1 and 9.6 min
for CT02-H. The HPLC peak intensity of QM1 linearly increased with
the concentration, and the detection limit of this HPLC/electro-
chemistry technique was determined to 2 ꢂ 10ꢁ13 mol of QM1,
which was determined from a concentration of 20 nM, with
ꢀ
ꢁ
ꢀꢁ
The O2 generation rates at various O2 fluxes from the X/XO
system were assessed by EPR spectroscopy using CT02-H with
measurement of its signal decay. These results were compared to
values measured by UV–Vis spectroscopy using cytochrome c.
UV–Vis spectra were recorded at room temperature on a Varian
Cary 50 Bio spectrophotometer. First, the enzymatic activity of
the commercial XO was determined by monitoring the produc-
tion of uric acid (12.2 mMꢁ1 cmꢁ1 at 290 nm) in the presence of
excess X. Then, various concentrations of XO (0.1, 0.2, 0.5, 1, 2,
injection volume of 10
ml, or 10 nM with volume of 20
ml.
ꢀ
ꢁ
and 5 mU/ml) were added to the solution containing X (100
m
M),
M), with the O2 generation
rate determined from the decay rate of the EPR signal of CT02-H.
Detection of O2 generation in cells
ꢀ
ꢁ
DTPA (0.1 mM), and CT02-H (50
m
Bovine aortic endothelial cells (BAECs) were grown to con-
fluence in Dulbecco’s modified Eagle’s medium (DMEM) contain-
ꢀ
ꢁ
For the measurements of the O2 generation rate from the
reduction of ferricytochrome c, various concentrations of XO
(2, 5, 10, and 20 mU/ml) were added to solutions containing
ing 10% fetal bovine serum, glutamine (30
mg/ml), penicillin
(100 U/ml), streptomycin (100 g/ml), MEM nonessential amino
m

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Y. Liu et al. / Free Radical Biology and Medicine 53 (2012) 2081–2091
acids (1%), and endothelial cell growth supplement (10 mg/ml) at
37 1C in a humidified atmosphere of 5% CO2 and 95% air. Cells
were used between passages 7 and 10. After removal of the
culture medium, the cells were washed with PBS and treated with
trypsin. The suspended cells were centrifuged at 500g for 5 min
and washed with PBS. The resulting cell pellet was resuspended in
ꢀ
ꢁ
PBS at a concentration of 3.8 ꢂ 106 cells/ml. The O2 generation
was initiated by adding menadione (100 mM) in the presence of
CT02-H (0.2 mM) or DEPMPO (20 mM). Then, the cell sample was
transferred to a glass capillary tube and EPR spectra were
recorded.
ꢀ
ꢁ
The concentration effect of menadione on the cellular O2
generation was investigated by HPLC. The culture medium was
removed from BAECs cultured in a six-well plate. The cells were
then washed three times by PBS and incubated with CT02-H
(50
mM) and various concentrations of menadione (0, 5, 10, 20, 50,
and 100
mM) in PBS for 15 min. The medium containing CT02-H
and QM1 was taken and centrifuged at 11,000g for 10 min, and
the supernatant was analyzed by HPLC. For studies of BAECs
subjected to the pathophysiological stimulus of hypoxia and
reoxygenation, cells were treated in a Billups Rothenberg cham-
ber purged with 95% nitrogen, 5% CO2 gas for 4 h followed by
incubation at 37 1C for 24 h followed by reoxygenation with air.
Fig. 1. EPR spectrum of CT02-H in PBS.
EPR signal intensity was observed for CT-03 in Fig. 2C because of
the decrease in the O2 concentration in the system, which
narrows its EPR signal and offsets the signal intensity decay from
the radical quenching. In fact, the concentration of CT-03 obtained
from the double integration of the EPR signal did gradually
decrease over time (Fig. 2D). The initial decay rates for CT02-H
ꢀ
ꢁ
Simultaneous measurement of O2 generation and O2 consumption
XO (10 mU/ml) was added to a solution containing CT02-H
(100 mM), X (100 mM), DTPA (0.1 mM) in PBS (pH 7.4). The low-
field peak of CT02-H was monitored for 35 min. The concentration
and CT-03 were 5.7 and 1.0
second-order reaction rate constant of CT02-H with O2 was
approximately 5.7 times that for CT-03 because the same con-
m
M/min, respectively, and thꢁus the
of CT02-H at each time point was obtained by comparing the
double integral of its EPR spectrum with that of a 100 mM CT02-H
ꢀ
standard sample, and the linewidth of the probe at each time
point was used to calculate O2 concentration according to the O2
sensitivity of CT02-H as mentioned above. Therefore, the initial
centration (14 mM) was used for both radicals.
To further quantitate the reactivity of CT02-H with Oꢁ2ꢁ, the
ꢀ
ꢀ
ꢀ
ꢁ
apparent second-ordꢁer rate constants of CT02-H with O2 were
O2 generation rate (4.9 mM/min) and O2 consumption rate
ꢀ
determined using O2 self-dismutation as a competition reaction.
(20.7
m
M/min) could be obtained in thꢁis single measurement.
ꢀ
Fig. 3 shows the concentration effect of CT02-H on its initial decay
Based on these data, the efficiency of O2 generation from O2 in
the X/XO system was determined to be 24%, consistent with the
previously reported values (20–30%) [42,44,45].
ꢀ
ꢁ
rates in the presence of O2 (see Supplementary Fig. S7 for the
kinetic decay of CT02-H induced by O2ꢁ). The initial decay rate
ꢀ
increases with the CT02-H concentration and then reaches a
plateau at the concentration of ꢃ45
m
M owing to complete
ꢀ
ꢁ
Results
trapping of O2 by CT02-H. These data were fitted with Eq. (1)
ꢀ
ꢁ
and the apparent second-order rate constant of CT02-H with O2
Fig. 1 shows the typical EPR spectrum of CT02-H under aerobic
conditions, which exhibits a doublet with a hyperfine splitting of
2.35 G at pH 7.4 due to the interaction of the unpaired spin with
the proton on the partially substituted aromatic group. The
magnitude of this hyperfine splitting is pH-dependent, which
was previously proposed to enable pH measurement [32]. In
addition to the doublet small satellite lines are seen due to
natural abundance 13C splittings.
was calculated to bꢁe 1.7 ꢂ 104 Mꢁ1 sꢁ1 using the self-dismutation
ꢀ
rate constant of O2 ꢁ (2.4 ꢂ 105 Mꢁ1 sꢁ1) at pH 7.4 [40,41] and the
ꢀ
predetermined O2 generation rate (0.11570.003
mM/s) (see
Supplementary Fig. S6). For comparison, a similar kinetics assay
was also carried out for CT-03, and its rate constant was
determined to be 3.1 ꢂ 103 Mꢁ1 sꢁ1, which is more than five
times lower than that of CT02-H. Because of the relatively low
ꢀ
reaction rate with O2ꢁ, CT-0ꢁ3 even at 140
mM concentration did
ꢀ
ꢁ
ꢀ
To check if CT02-H can function as a O2 detection probe, its
not fully outcompete the O2 self-dismutation (Fig. 3).
ꢀ
ꢁ
ꢀ
reactivity with O2 was investigated. The X/XO system was used
To explore the reaction mechanism of CT02-H with O2ꢁ, UV–Vis
ꢀ
ꢁ
ꢀꢁ
as a O2 source. As shown in Fig. 2A, O2 induced the rapid decay
of the signal intensity of CT02-H, as detected from the low-field
peak, and the signal almost completely disappeared in 10 min. In
contrast,ꢁthe reaction of the fully substituted TAM radical CT-03
spectroscopic studies were performed. As shown in Fig. 4, CT02-H has
a characteristic peak at 457 nm in the UV–Vis absorption spectrum
with an extinction coefficient of 17,900 Mꢁ1 cmꢁ1. Compared to CT-
03 (467 nm), the maximal absorbance of CT02-H has a blueꢁshift of
ꢀ
ꢀ
with O2 is much slower. The EPR signal of CT-03 gradually
approximately 10 nm. The reaction between CT02-H and O2 led to
decayed over time, with ꢃ45% of the signal remaining after
15 min, and no further reaction was observed after that time,
possibly because xanthine and/or O2 in the system was comple-
the rapid but time-dependent decrease in the absorbance at 457 nm,
consistent with the above EPR experimental results. Concurrently, a
new absorbance at 540 nm (
e
¼15,900 Mꢁ1 cmꢁ1) appeared due to
ꢀ
ꢁ
tely depleted (Fig. 2B). A much higher reactivity of CT02-H to O2
the formation of a product that shared an isosbestic point with CT02-
than of CT-03 was further verified by simultaneous measurement
of the signal intensity decays of both TAM radicals induced by
H at 480 nm. The presence of the isosbestic point indicates that the
ꢀ
ꢁ
reaction of CT02-H with O2 stoichiometrically generates the pro-
ꢀ
ꢁ
ꢀ
ꢁ
ꢀꢁ
O2 (Fig. 2C). Whereas CT02-H was quickly quenched by O2
,
duct, thus enabling quantitation of O2 by measuring the product.
According to previous studies regarding the reaction of CT-03 with
CT-03 was relatively stable. Of note, only minimal change in the

Y. Liu et al. / Free Radical Biology and Medicine 53 (2012) 2081–2091
2085
ꢀ
Fig. 2. (A) O2ꢁ-induced decay of the low-field peak in the EPR signal of CT02-H. XO (0.01 U/ml) was added to the solution containing CT02-H (14
X (100
symbols. (C) Simultaneous measurement of EPR signal decays in both CT02-H and CT-03 after addition of XO (0.01 U/ml) into the solution containing CT02-H (14
CT-03 (14 M), DTPA (100 M), and X (100 M) in PBS (pH 7.4). (D) Plot of TAM radical concentration as a function of time according to the data in (C). TAM radical
m
M), DTPA (100
m
M), and
m
M) in PBS (pH 7.4) and EPR spectra were recorded over time. (B) Plot of TAM radical concentration as a function of time. Error bars are small and within the
mM),
m
m
m
concentration was quantitated by comparing the double integral of the EPR signal of each radical with its predetermined concentration (i.e., 14
mM). All the experiments
were repeated three times.
Fig. 3. Effect of concentration on the O2ꢁ-induced initial decay of CT02-H and
CT-03. XO (10 mU/ml) was added to the solution containing DTPA (100 M),
X (100 M), and various concentrations of CT02-H or CT-03 in PBS (pH 7.4) and
ꢀ
m
ꢀ
Fig. 4. UV–Vis studies of the reaction of CT02-H with O2ꢁ. UV–Vis spectra were
m
continuously recorded after addition of XO (8 mU/ml) into the solution containing
EPR spectra were continuously recorded. The initial rate of the decay of CT02-H or
CT-03 was determined within 30–80 s.
CT02-H (16.9 mM), DTPA (100 mM), and X (200 mM) in PBS (pH 7.4).
ꢀ
ꢁ
O2 [26], the product could be the corresponding quinone methide
QM1 or QM2 (Fig. 5). Subsequent ESI–MS analysis (negative mode)
showed that the product with m/z 968.9295 is QM1 (calculated m/z:
968.9290) instead of QM2 (calculated m/z: 924.9392; see Supple-
mentary Fig. S1). The molecular structure of QM1 was further
characterized by 1H and 13C NMR spectra (see Supplementary Figs.
S2 and S3), which were well consistent with the previously reported
data [46]. To further confirm the origin of the oxygen atom in the
X/XO system. The resulting product has an 18O inserted in the
molecule as determined by the molecular ion of [MþNa]þ in a
high-resolution mass spectrum at 994.9276 (calculated value
994.9308, Supplementary Fig. S14), verifying that the oxygen atom
ꢀ
ꢀꢁ
in QM1 comes from O2ꢁ. Therefore, the reaction of CT02-H with O2
resulted in oxidative dehydrogenation in contrast to the oxidative
decarboxylation in the case of CT-03 [26].
ꢀ
ꢁ
To investigate the specificity of the CT02-H reaction with O2
,
quinone QM1, 18O2 instead of 16O2 was used to generate 18O2 in the
its reactivity toward other reactive species was also studied. As
ꢀ
ꢁ

2086
Y. Liu et al. / Free Radical Biology and Medicine 53 (2012) 2081–2091
ꢀ
ꢁ
.
Fig. 5. Possible mechanisms of CT02-H reaction with O2
Fig. 7. Cyclic voltammograms of CT02-H (1 mM) and CT-03 (1 mM) in PBS (pH 7.4)
containing 0.15 M KCl. Scan rate: 50 mV/s.
Fig. 6. Effects of various reactive species on CT02-H expressed as a percentage of
CT02-H remaining after exposure to reactive species for 30 min. The percentage
was obtained by comparing the double integral of the EPR signal in each system
with that of CT02-H (20 mM). Each experiment was carried out at least three times.
See details under Materials and methods.
TAM cation at E1/2 (ox)¼0.445 V vs Ag/AgCl (
quasi-reversible one-electron reduction to the corresponding anion
E1/2 (red)¼ ꢁ0.642 V vs Ag/AgCl ( Ep¼82 mV). These values are
D
Ep¼79 mV) and
D
shown in Fig. 6, CT02-H was stable toward most reducing agents
such as Asc (1 mM), GSH (1 mM), and Fe(II)–NTA (0.1 mM), as
very similar to those observed for CT-03 under the same conditions,
i.e., E1/2 (ox)¼0.462 V vs Ag/AgCl and E1/2 (red)¼ ꢁ0.623 V vs Ag/
AgCl. Similar redox potentials for CT02-H and CT-03 further verified
their similar stability toward various oxidoreductants. Although the
ester form of CT02-H was previously reported to react with O2 at
high concentration [27], no significant signal decay after CT02-H
ꢀ
well as the oxidants H2O2 (1 mM), Fe(III)–NTA (0.1 mM), HO , and
peroxynitrite generated from decomposition of SIN-1 (1 mM).
Although there were only slight changes in the signal intensity
ꢀ
after 30-min incubation of CT02-H with the highly reactive HO
and peroxynitrite, reactions between CT02-H and these two
species may still exist wherein the paramagnetism of CT02-H is
preserved. Reactions of CT02-H with alkyl peroxyl radical gener-
ated from the thermolysis of AAPH (1 mM) were observed with
ꢃ40% of signal remaining after 30-min incubation. The lack of
effect of SOD (200 U/ml) on the decay of CT02-H induced by
alkylperoxyl radicals confirmed the moderate reactivity of CT02-H
with this species (data not shown). The reaction of CT02-H with
alkylperoxyl radicals ꢁhas the same product (i.e., QM1) as that of
(20 mM) in PBS was observed in room air for 5 h at 37 1C, which
ꢀ
ꢁ
supports its use for the measurement of O2 (see Supplementary
Fig. S10). Additionally, incubation of CT02-H with the serum protein
bovine serum albumin at 37 1C for 5 h resulted in only 4% signal
decay (see Supplementary Fig. S10).
ꢀ
ꢀꢁ
To determine if CT02-H can be used to measure O2ꢁ, various O2
fluxes were generated in the presence of a constant concentration of
X (100 M) and various concentrations of XO (0.1, 0.2, 0.5, 1, 2, and
5 mU/ml). As shown in Fig. 8A, CT02-H had a good response to the
m
ꢀ
ꢀꢁ
the reaction with O2 as verified by UV–Vis spectroscopy (Sup-
O2 fluxes, and the initial decay rate of CT02-H was linearly
correlated with the XO concentration. The slope (0.5670.02 mol/
plementary Fig. S13). Overall, the stability of CT02-H toward
various reactive species is very similar to the results observed
previously for CT03 and OX063 [24,34]. Cyclic voltammetric
studies (Fig. 7) showed that CT02-H, as a neutral radical, can
undergo reversible one-electron oxidization to the corresponding
m
min/U)ꢁof the curve obtained by the linear fitting corresponds tꢁo
ꢀ
ꢀ
the O2 generation activity of XO. Based on this value, the O2
generation activity can be further estimated as 2871% of the
total O2 consumption. Comparatively, the cytochrome c detection

Y. Liu et al. / Free Radical Biology and Medicine 53 (2012) 2081–2091
2087
ꢀ
ꢁ
ꢀꢁ
Fig. 9. Measurement of the low O2 flux by CT02-H (0.1
was generated using a low concentration of XO (0.05 mU/ml), X (2
(100 M) in PBS. The concentration of CT02-H was quantitated by comparing the
double-integrated EPR signal of each radical with its predetermined concentration
(i.e., 0.1 M).
m
M). The low O2 flux
m
M), and DTPA
m
m
ꢀ
ꢁ
Fig. 8. (A) Detection of various O2 fluxes by EPR using CT02-H. The incubation
mixture contained X (100 M), DTPA (100 M), CT02-H (50 M) in PBS (pH 7.4) at
room temperature. Various concentrations of XO (0.1, 0.2, 0.5, 1, 2, and 5 mU/ml)
m
m
m
ꢀ
ꢁ
were added to initiate the reaction. The decay rate of CT02-H, that is, the O2
generation rate, was determined to be 0.5670.02 mol/min/U. (B) Detection of
various O2 fluxes by UV–Vis spectroscopy using ferricytochrome c. The incuba-
tion mixture contained X (100 M), cytochrome c (100 M), EDTA (100 M), and
catalase (200 U/ml) in PBS (pH 7.4) at room temperature. Various concentrations
m
ꢀ
ꢁ
ꢀꢁ
Fig. 10. Cellular generation of O2 detected by CT02-H. The O2 generation was
ꢀ
ꢁ
stimulated by adding menadione (100
m
M) to the cell suspension (3.8 ꢂ 105 (’) or
m
m
m
1.2 ꢂ 105 (K) cells/ml in a 40-
ml EPR capillary tube) containing CT02-H (0.2 mM)
and DTPA (100 mM) in DMEM. EPR spectra were recorded 5 min after the addition
ꢀ
ꢁ
of XO (2, 5, 10, and 20 mU/ml) were added to initiate the reaction. The O2
ꢀ
ꢁ
of menadione. SOD (200 U/ml) (m) was used to verify the O2 generation. The
generation rate was determined to be 0.5870.02 mmol/min/U. See details under
Materials and methods.
concentration of CT02-H was quantitated by comparing the double integral of the
EPR signal of each radical with its predetermined concentration (i.e., 0.2 mM).
ꢀ
ꢁ
technique was also used to determine the O2 generation activity
of XO (Fig. 8B). The obtained value (0.5870.02 mol/min/U) was
m
the decay of CT02-H, verifying that the CT02-H decay is specific to
ꢀ
consistent with that obtained uꢁsing CT02-H, implying that CT02-
O2ꢁ. However, the EPR spin trapping technique using DEPMPO as
ꢀ
H can be used to quantitate O2
.
a spin trap could not detect any signal under the same condition
and required fivefold more XO (i.e., 0.25 mU/ml) to reach a
detectable signal (see Supplementary Fig. S4), indicating that
the new EPR method using CT02-H as a probe can aꢁchieve much
To estimate the detection limit of CT02-H, we monitored the
ꢀ
ꢁ
signal decay of CT02-H (0.1
generated from the mixture containing X (2
and DTPA (100 M). As shown in Fig. 9, CT02-H gradually decayed
m
M) in the presence of low O2 flux
m
M), XO (0.05 mU/ml),
ꢀ
m
higher sensitivity. According to the plot of the O2 generatioꢁn
ꢀ
over time in this system with approximately 25% of CT02-H
rate as a function of the XO concentration in Fig. 8, the O2
remaining after 60-min reaction. The incomplete reaction of
generation rate is estimated to be 25 nM/min, using 0.05 mU/ml
XO and excess X. Assuming that the EPR technique can safely
distinguish 5% of the signal intensity change, the present technique
ꢀ
ꢁ
ꢀꢁ
CT02-H with O2 may result from: (1) less O2 trapped by CT02-H
with time due to a significant decrease in the CT02-H concentra-
ꢀ
ꢁ
ꢀꢁ
tion or (2) decreased O2 generation possibly due to the con-
sumption of xanthine and O2 as well as to decreased enzyme
activity. Addition of Cu/Zn SOD (200 U/ml) completely inhibited
can measure a O2 flux of ꢃ1.7 nM/min over 60 min detection.
ꢀ
ꢁ
The application of CT02-H for measurement of the O2 genera-
ꢀ
ꢁ
tion in cells was also explored. The O2 generation was stimulated

2088
Y. Liu et al. / Free Radical Biology and Medicine 53 (2012) 2081–2091
ꢀ
ꢁ
Fig. 11. (A) HPLC–electrochemistry chromatograms of QM1. (B) HPLC peak intensities of QM1and CT02-H at various concentrations. (C) Reaction of CT02-H with O2
monitored by HPLC–EC at 0, 2, and 10 min. XO (10 mU/ml) was added to the solution containing X (100 M), DTPA (100 M), and CT02-H (14 M) in PBS; then, one aliquot
m
m
m
was taken at 2 or 10 min and SOD (200 U/ml) was added to quench the reaction. For the sample at 0 min, SOD was introduced before addition of XO. The peaks of CT02-H
at 2 and 10 min are shown with 10-fold higher gain because of the weak residual intensity. #These peaks come from the X/XO system (see Supplementary Fig. S11).
ꢀ
ꢁ
(D) (a) HPLC chromatograms of the QM1 peaks centered at a retention time of 4.2 min, measuring the O2 generation in BAECs stimulated by 0, 5, and 100ꢀmM menadione
ꢁ
in the presence of CT02-H (50
m
M) for 15 min at 37 1C. (b) HPLC chromatograms of the QM1 peaks centered at a retention time of 4.2 min, measuring the O2 generation in
BAECs treated with anoxia/reoxygenation. After BAECs were exposed to anoxia for 24 h, CT02-H (50
mM) was added to the medium and then reoxygenation treatment was
ꢀ
ꢁ
conducted for 5 min or 2 h. The medium was analyzed by HPLC. (E) Concentration effect of menadione on the cellular O2 generation in BAECs.
ꢀ
ꢁ
ꢀꢁ
in BAECs by adding menadione [47]. As shown in Fig. 10, the O2
Because the reaction of CT02-H with O2 resulted in the
production of the stable QM1 (Supplementary Fig. S12), HPLC in
tandem with electrochemical detection was used to detect this
specific product. Fig. 11A shows the chromatograms using electro-
chemical detection of QM1 at various concentrations with a reten-
tion time of ꢃ250 s (ꢃ4.2 min). As shown in Fig. 11B, the HPLC peak
intensity of QM1 is in good agreement with the amounts of down to
generation in cells (3.8 ꢂ 105 cells/ml, 40
ml) induced the quick
decay of CT02-H with approximately 60% of signal loss after 20-min
incubation. The decrease in cell density from 3.8 ꢂ 105 to 1.5 ꢂ 105
cells/ml (15,000 to 6000 cells) significantly slowed the decay of
CT02-H, with slightly more than 25% signal loss after 20-min
incubation. Near-complete inhibition of the CT02-H decay by
exogenous addition of SOD (200 U/ml) was ꢁseen, confirming the
2ꢂ 10ꢁ13 mol or 20 nM with an injection volume of 10
ml. With
ꢀ
specificity of the reaction of CT02-H with O2 in this system. The
increasing sample volume from 10 to 20 ml the sensitivity could be
complete inhibition of the CT02-H decay by the cell-impermeative
SOD also implies that CT02-H acts only as an extracellular probe
owing to its negatively charged nature. The sensitivity for detection
of cellular O2 generation with CT02-H was high and O2 could be
detected from as few as 1200 cells over 20 min with a 5% signal
decrease. For comparison, a regular spin trapping experiment was
also performed using DEPMPO (20 mM) but a much higher cell
density of 1.2 ꢂ 106 cells/ml, with corresponding cell number of
48,000, was required to reach a detectable EPR signal (Supplemen-
tary Fig. S5).
extended to ꢃ10 nM QM1. Fig. 11C showsꢁ the HPLC–EC chromato-
ꢀ
grams of the reaction of CT02-H with O2 from the X/XO system.
Similar to the EPR results in Figs. 2A and 2B, CT02-H with the
retention time of 9.6 min was quickly and uniquely transformed into
ꢀ
ꢁ
ꢀꢁ
ꢀ
ꢁ
QM1 upon reaction with O2 with a transformation of ꢃ80% after
2 min incubation, and this reaction was complete at 10 min. Tꢁo
ꢀ
further investigaꢁte the potential of the HPLC technique for O2
ꢀ
detection, the O2 generation from BAECs stimulated by menadione
was also studied by HPLC. As shown in Figs. 11D and 11E, menadione
ꢀ
ꢁ
stimulates O2 generation in a concentration-dependent manner,

Y. Liu et al. / Free Radical Biology and Medicine 53 (2012) 2081–2091
2089
Discussion
ꢀ
ꢁ
The crucial role of O2 in redox cell signaling and in the
development of disease has resulted in an increasing need for
development of new methodologies for the detection and quanti-
fication of this reactive species. However, currently available
ꢀ
ꢁ
techniques for the measurement of O2 are still limited by low
sensitivity and specificity. In this study, we reported that the
partially substitꢁuted TAM CT02-H can serve as a highly sensitive
ꢀ
and specific O2 probe suitable for measurements in biological
systems.
It was shown in our previous study that OX063, a fully
ꢀ
ꢁ
substituted TAM radical, can function as a O2 probe with
relatively high sensitivity and specificity [24]. Similar results were
also observed for CT-03 [34]. Compared to OX063 and CT-03, CT02-H
ꢀ
shows higher sensitivity to O2ꢁ, due to the high reaction rate, but
similarly high specificity (Fig. 6). Upon reaction with O2ꢁ, CT02-H
ꢀ
quickly loses its EPR signal (Figs. 2A and 2B) and is selectively
transformed into the diamagnetic quinone methide QM1 (Fig. 5),
which has a maximal absorbance at 540 nm (
Fig. 4), allowing spectrophotometric measurement of O2
e
¼15,900 Mꢁ1 cmꢁ1
;
.
ꢀ
ꢁ
ꢀ
ꢁ
Interestingly, the reaction of CT02-H with O2 resulted in
oxidative dehydrogenation instead of oxidative decarboxylation
as observeꢁd for CT-03 and OX063 [26]. The rate constant of CT02-
ꢀ
H with O2 was estimated to be 5.7 times higher than that of CT-
03ꢁby concurrently monitoring their EPR signal decays induced by
ꢀ
O2 (Fig. 2D). Kinetic studies (Fig. 3) also showed that CT02-H has
a reaction rate constant of 1.7 ꢂ 104 Mꢁ1 sꢁ1, which is 5.5 times
higher than that of CT-03 (3.1 ꢂ 103 Mꢁ1 sꢁ1), consistent with the
above vꢁalue (5.7 times). The much higher reactivity of CT02-H
ꢀ
with O2 compared to CT-03 and OX063 (3.1 ꢂ 103 Mꢁ1 sꢁ1) [24]
is most likely due to the ease of oxidative dehydrogenation in
CT02-H compared to the oxidative decarboxylation in CT-03 and
OX063 because the hydrogen on the active carbon in CT02-H is
less bulky than the carboxylate group in the other two TAMs, and
the neutral nature of the hydrogen prevents the electrostatic
Fig. 12. (A) Variations in the EPR linewidth of the low-field peak of CT02-H with
ꢀ
ꢁ
O2 concentration. (B) Simultaneous monitoring of O2 and TAM concentrations by
repulsion with the negatively charged O2 upon the reaction.
EPR in the system containing CT02-H (100
DTPA (100 M). The concentration of CT02-H was quantitated by comparing the
double integral of the EPR signal of each radical with its predetermined
concentration (i.e., 100 M).
m
M), X (100
mM), XO (10 mU/ml), and
ꢀ
ꢁ
Therefore, this new EPR probe exhibits higher sensitivity for O2
m
than its fully substituted analogues CT-03 and OX063.
EPR spin trapping is one of the most sensitive and definitive
m
ꢀ
ꢁ
ꢀꢁ
methods for the detection of biological radicals, including O2
.
ꢀ
ꢁ
with more O2 generation at higher concentrations of menadione.
No EPR signal decay of CT02-H (20 M) was observed in the presence
of menadione (500 M; data not shown), proving the specificity of
CT02-H for O2ꢁ. Moreover, reoxygenation of BAECs that underwent
This technique utilizes the reaction of nitrone spin traps with O2
m
resulting in the production of relatively stable spin adducts [15].
Our previous studies showed that the EPR spin trapping techni-
que is more sensitive than the cytochrome c reduction method in
m
ꢀ
ꢀ
ꢁ
ꢀꢁ
anoxia for 24 h immediately induced O2 generation (ꢃ0.08
m
M),
quantifying O2 [44,48]. However, the application of the EPR spin
ꢀ
ꢁ
and extended reoxygenation treatment for 2 h further increased the
trapping method for very low O2 fluxes is still limited because of
slow spin trap reactivity with O2ꢁ, and short adduct half-life, as
ꢀ
ꢁ
ꢀ
O2 production detected (ꢃ0.48
m
M; Fig. 11D).
Like other TAM radicals, CT02-H can also serve as an O2 probe
based on its O2-dependent EPR linewidth broadening. Fig. 12A
shows the EPR line broadening of CT02-H as a function of the O2
concentration. Fig. 12B shows the changes in O2 and CT02-H
concentration over time in the X/XO system. CT02-H gradually
decayed, with the concomitant decrease in O2 concentration.
well as decreased EPR analytical sensitivity due to the compleꢁx
ꢀ
multiline EPR spectra of the spin adduct. In our experiments, O2
generation was not detectable using DEPMPO, the most specific
ꢀ
commonly used spin trap for O2ꢁ, when a very low concentration
of XO (0.05 mU/ml) was utilized. Comparatively, in the same
ꢀ
ꢁ
system, O2 induced a marked decrease in the EPR signal
Because the CT02-H concentration used (100
m
M) is much higher
intensity (ꢃ75%) of CT02-H over 60 min (Fig. 9), indicating that
ꢀ
ꢀꢁ
than the concentration of O2ꢁ, CT02-H largely outcompetes the
CT02-H is well suited to the detection of low O2 flux. Assuming
that EPR spectroscopy can distinguish 5% of the signal decay, the
detection limit of CT02-Hꢁin this experiment is estimated to be
ꢀ
ꢁ
ꢀꢁ
O2 self-dismutation and traps almost all of the O2 generated
during the first several minutes. Therefore, theꢁinitial decay rate
ꢀ
ꢀ
of CT02-H is effectively equal to the initial O2 generation rate.
ꢃ1.7 nM/min of the O2 flux. With the use of an internal
ꢀ
ꢁ
According to Fig. 12B, the rates of initial O2 generation and O2
consumption were calculated to be 4.9 and 20.7 M/min, respec-
nonreactive standard such as the extremely stable dendritic trityl
radical [34] or an external standard such as CT-03 or OX063, one
can precisely measure as little as 2% of the signal iꢁntensity change
m
ꢀ
ꢁ
tively, with the O2 generation efficiency of ꢃ24% from O2 in
accordance with the range of values reported in the literatures
[42,44,45]. Therefore, CT02-H can be used to simultaneously
ꢀ
by EPR, enabling detection of 2.5 times lower O2 flux (i.e., 0.68
nM/min). With longer reaction time the detection limit would be
expected to be further lowered in proportion to the time, with a
value of as low as 0.34 nM/min after 120 min. Furthermore, owing
ꢀ
ꢁ
measure the O2 concentration and O2 generation as well as
the ratio of its generation to the O2 consumed.

2090
Y. Liu et al. / Free Radical Biology and Medicine 53 (2012) 2081–2091
ꢀ
ꢁ
to the high sensitivity, CT02-H can be used to detect O2 from as
few as 600 menadione-treated BAECs (0.04 mlꢂ 1.5 ꢂ 104 cells/ml)
over 20 min using an external or internal standard as mentioned
above, which is 80 times lower than that (48,000 cells) required in
the spin trapping method using DEPMPO as a spin trap. To further
Acknowledgment
This work was supported by NIH Grants HL38324, EB0890,
EB4900 (J.L.Z.), and HL81248 (F.A.V.).
ꢀ
increase the sensitivity of the present EPR method for O2ꢁ, fully
deuterated CT02-H with deuteration of all of the methyl groups
[49] and the aromatic hydrogen can be developed in the future.
On the other hand, HPLC in tandem with the highly sensitivꢁe
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.freeradbiomed.2012.
09.011.
ꢀ
electrochemical detection technique can be used to quantitate O2
with a detection limit of 10 nM equating to measurement of a flux
of only 0.17 nM/min for a 60-min measurement (Figs. 11A anꢁd
ꢀ
11B). Owing to the high sensitivity of this technique, the O2
generation can be detected from BAECs that were stimulated by as
low as 5 mM menadione (Figs. 11D and 11E). In addition, the O2
References
ꢀ
ꢁ
generation in BAECs that were treated with anoxia/reoxygenation
was also determined (Fig. 11D). Therefore, HPLC coupled with the
use of CT02-H allows highly sensitive detection and measurement
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ꢀ
ꢁ
of the O2 generation in various in vitro and ex vivo systems.
The high O2 sensitivity of CT02-H also allowed simultaneous
ꢀ
ꢁ
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ꢀ
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