EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines

Article abstract of DOI:10.3109/10715762.2010.540242

Superoxide (O₂) has been implicated in the pathogenesis of many human diseases, but detection of the O₂ radicals in biological systems is limited due to inefficiency of O₂ spin trapping and lack of site-specific information. This work studied production of extracellular, intracellular and mitochondrial O₂ in neutrophils, cultured endothelial cells and isolated mitochondria using a new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell permeability. Cyclic hydroxylamines rapidly react with O₂, producing stable nitroxides and allowing site-specific O₂ detection in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium (CAT1-H) detected only extramitochondrial O₂. Inhibition of EPR signal by SOD2 over-expression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O₂ both in isolated mitochondria and intact cells. Both 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CM-H) detected an increase in cytoplasm O₂ stimulated by PMA, but only CM-H and mitoTEMPO-H showed an increase in rotenone-induced mitochondrial O₂. These data show that a new set of hydroxylamine spin probes provide unique information about site-specific production of the O₂ radical in extracellular or intracellular compartments, cytoplasm or mitochondria.

Full text of DOI:10.3109/10715762.2010.540242

Free Radical Research, April 2011; 45(4): 417–430
EPR detection of cellular and mitochondrial superoxide using
cyclic hydroxylamines
SERGEY I. DIKALOV1, IGOR A. KIRILYUK2, MAXIMVOINOV3 & IGOR A. GRIGOR’EV2
1FRIMCORE, Emory University School of Medicine,Atlanta, GA, USA, 2Novosibirsk Institute of Organic Chemistry,
Novosibirsk, Russia, and 3North Carolina State University, Raleigh, NC, USA
(Received date: 2 July 2010; In revised form date: 10 November 2010)
Abstract
•–
Superoxide (O2•–) has been implicated in the pathogenesis of many human diseases, but detection of the O2 radicals in
biological systems is limited due to inefficiency of O2•– spin trapping and lack of site-specific information.This work studied
production of extracellular, intracellular and mitochondrial O2•– in neutrophils, cultured endothelial cells and isolated mito-
chondria using a new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell perme-
ability. Cyclic hydroxylamines rapidly react with O2•–, producing stable nitroxides and allowing site-specific O2 detection
•–
in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,
6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium
(CAT1-H) detected only extramitochondrial O2•–. Inhibition of EPR signal by SOD2 over-expression showed that mitocho-
•–
ndria targeted mitoTEMPO-H detected intramitochondrial O2 both in isolated mitochondria and intact cells. Both
1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrro-
lidine (CM-H) detected an increase in cytoplasm O2•– stimulated by PMA, but only CM-H and mitoTEMPO-H showed
an increase in rotenone-induced mitochondrial O2•–. These data show that a new set of hydroxylamine spin probes pro-
•–
vide unique information about site-specific production of the O2 radical in extracellular or intracellular compartments,
cytoplasm or mitochondria.
Keywords: Superoxide, spin trap, cyclic hydroxylamine, intracellular, extracellular, EPR
•–
Introduction
techniques for O2 detection [6] and the diversity of
the sources of O2•– production [7].
•–
Superoxide radical (O2 ) is one of the most important
free radicals both in chemistry and biology [1]. It is
produced by one-electron reduction of oxygen and fre-
quently a precursor of other reactive oxygen species
(ROS) such as hydrogen peroxide (H2O2), peroxynitrite
(ONOOꢀ), lipid and hydroxyl radicals [2]. In biological
systems O2•– plays a dual role: under physiological con-
The most commonly used chemiluminescence
technique for measurement of O2 is lucigenin-
•–
enhanced chemiluminescence [6].The validity of this
technique, however, has been questioned on the
•–
grounds that O2 production might be artificially
over-estimated because of a phenomenon known as
redox cycling, in which the lucigenin radical can react
with oxygen to generate O2•– [8].
Reduction of ferricytochrome c by O2 to ferrocy-
tochrome c was frequently used in the past for O2
detection [6]. Unfortunately, this spectrophotometric
detection was limited to extracellular O2•– because cyto-
chrome c is not cell permeable and it did lack sensitivity
and specificity due to reduction of ferricytochrome c
•–
ditions O2 is involved in cell signalling [3] and in
pathophysiological states over-production of O2•– leads
to oxidative stress causing cellular dysfunction and cell
death [4]. Pathological conditions such as hypertension,
atherosclerosis and hypercholesterolemia are strongly
•–
•–
•–
associated with increased O2 production [5]. None-
theless, measurements of O2 in biological samples is
still a challenging task due to limitations of existing
•–
Correspondence: Sergey Dikalov, PhD, FRIMCORE Director, Division of Cardiology, Emory University School of Medicine, 1639 Pierce
Drive, Atlanta, GA 30322, USA. Tel: 404-712-9550. Fax: 404-727-3585. Email: dikalov@emory.edu
ISSN 1071-5762 print/ISSN 1029-2470 online © 2011 Informa UK, Ltd.
DOI: 10.3109/10715762.2010.540242

418 S. I. Dikalov et al.
by electrons donated from enzymes and other
molecules.
O2•– radical hydroxylamine, probes can be used at very
low concentrations (0.05–1 mM) compared to spin
traps (10–50 mM), which minimizes side-effects of
the probes on the biological samples. Cyclic hydrox-
One of the most definitive methods of O2•– identi-
fication is electron paramagnetic resonance (EPR)
spectroscopy [9,10]. The EPR spin-trapping tech-
nique has been used to detect short-lived O2•– radicals
by following the formation of persistent radical
adducts. Theoretically, spin trapping should be an
ideal way to detect radicals in biological tissues.
Unfortunately, this turns out not to be the case. First,
•–
ylamine detect the O2 radical in a single chemical
reaction, while other probes like dihydroethidium
and lucigenin require at least two reactions, which
may cause artifacts [17]. In contrast to lucigenin or
2’,7’-dichlorofluorescin, hydroxylamine probes are
not engaged in redox cycling and therefore they allow
•–
•–
nitrone spin traps react with O2 very slowly (k ~
us to avoid problems with artificial O2 production
35–75 Mꢀ1sꢀ1, pH ꢁ 7.4) [11], limiting spin trapping
[21]. It is also important to note that the potential
competition between hydroxylamine and nitroxide
[22] does not affect the O2•–-mediated oxidation of
hydroxylamine because the hydroxylamine/nitroxide
ratio is usually very high [11].
•–
to only extracellular O2 because spin traps cannot
compete with intracellular antioxidants, rapidly react-
•–
ing with O2 (k ~ 105–109 Mꢀ1sꢀ1) [12]. Second,
O2•– radical adducts are unstable and undergo decom-
position to •OH-radical adducts [13–15] or reduction
to EPR-silent products by ascorbate, transition metal
ions or flavin-enzymes [14]. These limitations make
the spin trapping technique useful for identification
Several cyclic hydroxylamines, such as 1-hydroxy-3-
carboxy-2,2,5,5-tetramethylpyrrolidine(CP-H)[23,24],
1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethy-
lpyrrolidine (CM-H) [19,20,23–27], 1-hydroxy-4-
phosphono-oxy-2,2,6,6-tetramethylpiperidine
(PP-H) [11,28,29] and 1-hydroxy-2,2,6,6-tetrameth-
ylpiperidin-4-yl-trimethylammonium chloride (CAT1-H)
[30] (Figure 1) were successfully used for detection
and qualitative analysis but not suitable for quantifi-
•–
cation of intracellular O2
.
Because of the aforementioned problems with
nitrone spin traps, investigators have turned their
attention to other molecules such as dihydroethidium
•–
and quantitative measurements of O2 in cultured
•–
for O2 detection in biological samples [6,16]. The
cells and tissue samples. Some of these cyclic hydrox-
ylamine probes have been successfully applied not
only ex vivo but also in vivo [6,20]. Hydroxylamine
probes can be potentially oxidized by several ROS and,
therefore, to define the contribution of specific ROS
it is necessary to perform control experiments with
supplementation of superoxide dismutase (SOD),
peroxynitrite scavenger urate or other scavengers
[26,31,32]. For example, we have found that in cultured
cells or isolated vessels PEG-SOD lowers the EPR signal
by more than 70% [19].
dihydroethidium method, however, has several prob-
lems. It is a light-sensitive compound which is also
prone to auto-oxidation. Dihydroethidium requires a
two-step reaction in order to detect O2•–, which
involves free radical intermediate [17]. Furthermore,
formation of ‘O2•–-specific’ product can be affected
by peroxidase reactions, which may compromise
specificity of the assay [18].
We have previously reported that cyclic hydroxy-
lamines can be used for measurement of O2•– in cul-
tured cells, tissues and in vivo [19,20]. These
molecules are not spin traps, they do not ‘trap’ radi-
Despite significant progress in applications of
•–
hydroxylamine probes, investigation of O2 produc-
•–
cals, but they are oxidized with O2 to form EPR-
tion in different sub-cellular compartments is still a
challenging problem. The aim of this work was to
detectable stable nitroxides with half-lives of several
hours in cell cultures. Since both spin traps and
cyclic hydroxylamines generate species detectable by
EPR we suggest to name these cyclic hydroxylamines
as ‘hydroxylamine spin probes’ which must not be
confused with nitroxide spin probes. Cyclic hydroxy-
•–
investigate site-specific O2 production in different
cellular compartments using a new set of cationic,
anionic and neutral hydroxylamine spin probes (Fig-
ure 1) with various lipophilicity and cell permeability.
For this purpose we have investigated O2•– production
in a cell-free xanthine oxidase system, mitochondria,
neutrophils and endothelial cells. Efficiency of O2•–
detection was compared with the nitrone spin trap
5-ethoxycarbonyl-5-methyl-1-pyrrolineN-oxide(EMPO).
•–
lamines react with O2 much faster (k ~ 103–104
Mꢀ1sꢀ1, pH ꢁ 7.4) compared to nitrone spin traps
and can compete with cellular antioxidants for intrac-
ellular O2•– [11]. Due to high efficiency in detection of
O
+
O
HN C
Na+
Cl
+
-
O CH3
OH
P(Ph)3
OPO3H
O CH3
N
HN
N
O
O
CO2Et
N
OH
N
N
N+
O
N
OH
N
OH
N
OH
OH
OH
OH
mitoTEMPO-H
CAT1-H
PP-H
TM-H
TMT-H
CM-H
EMPO
CP-H
Figure 1. Chemical structures of hydroxylamine spin probes and the spin trap EMPO.

Superoxide detection by cyclic hydroxylamines 419
Cell permeability and lipophilicity of various cyclic
hydroxylamines were measured.This new set of probes
has demonstrated site-specific measurement of O2
in extracellular or intracellular compartments, cyto-
plasm or mitochondria.
Superoxide measurement
The cell-free xanthine oxidase superoxide-generating
system contained xanthine oxidase (1 mU/ml), xan-
thine (50 μM) and DTPA (0.1 mM) in 50 mM
sodium phosphate buffer (pH ꢁ 7.4) containing 0.9%
NaCl [37].
•–
Materials and methods
Measurement of mitochondrial O2•– was performed
in media containing 125 mM KCl, 10 mM MOPS,
2 mM MgSO4, 2 mM KH2PO4, 10 mM NaCl, 1 mM
EGTA and 0.7 mM CaCl2, 50 μM desferoxamine,
Cyclic hydroxylamines 1-hydroxy-2,2,6,6-tetramethyl-
piperidin-4-yl-trimethylammonium chloride (CAT1-
H), 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine
hydrochloride (CP-H), 1-hydroxy-3-methoxycarbonyl-
2,2,5,5-tetramethylpyrrolidine (CM-H), 1-hydroxy-4-
phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H),
1-hydroxy-4-methoxy-2,2,6,6-tetramethylpiperidine
(TM-H), N-(1-Hydroxy-2,2,6,6-tetramethylpiperidin-
4-yl)-2-methylpropanamide (TMT-H), 1-hydroxy-4-
[2-(triphenylphosphonio)-acetamido]-2,2,6,6-tetra-
methylpiperidine (mitoTEMPO-H,), spin trap 5-
ethoxycarbonyl-5-methyl-1-pyrrolineN-oxide(EMPO)
and 3-morpholinosydnonimine (SIN-1) were purchased
from Enzo Life Sciences (San Diego, CA). Polyethylene
glycol-conjugated superoxide dismutase (PEG-SOD),
phorbol 12-myristate 13-acetate (PMA) and all other
reagents were obtained from Sigma-Aldrich (St. Louis,
MO). Stock solutions of cyclic hydroxylamines (10 mM)
were prepared in argon-purged 0.9% NaCl treated with
25 g/L Chelex-100 and containing 50 μM desferoxamine
or 0.1 mM DTPA. Stock solutions were kept under
argon on ice and were prepared daily.
•–
pH ꢁ 7.2. Mitochondrial O2 production was stud-
ied in the presence of 2 mM malate ꢂ 20 mM gluta-
mate and 20 μg of mitochondrial protein [29]. The
amount of detected O2 was calculated from inhibi-
tion of EPR signal with 50 U/ml Cu,Zn-SOD. All
mitochondria measurements were carried out at 25°C
using a Bruker EMX spectrometer equipped with a
Temperature Controller system.
•–
Neutrophils were incubated with 1 mM of corre-
sponding cyclic hydroxylamine and stimulated by the
addition of 1 μM PMA in Chelex-treated phosphate
buffer, pH ꢁ 7.4 [27].
•–
Production of O2 in bovine aortic endothelial
cells (BAEC) was measured in Krebs–Hepes buffer
(KHB) containing 5.786 g/L NaCl, 0.35 g/L KCl,
0.368 g/L CaCl2,0.296 g/L MgSO4,2.1 g/L NaHCO3,
0.142 g/L K2HPO4, 5.206 g/L Na-Hepes, 2 g/L
D-glucose, pH ꢁ 7.35, in the presence of 25 μM
desferoxamine and 2.5 μM diethyldithiocarbamate.
•–
Cellular production of O2 was stimulated by mito-
chondrial inhibitor antimycin A (5 μM) [16,29] or
pre-treatment of cells with peroxynitrite donor SIN-1
[26]. Confluent endothelial cells were treated with
1 mM SIN-1 for 60 min at 37°C and then washed
with PBS. Superoxide production was analysed in cell
suspensions incubated at room temperature with
hydroxylamine spin probes (1 mM) or the spin trap
Cell culture and mitochondria isolation
Bovine aortic endothelial cells (BAEC, passage 4–8)
were cultured on 100 mm plates in Media 199 con-
taining 10% foetal calf serum supplemented with 2
mM L-glutamine and 1% vitamins. Confluent cells
were used for the experiments [26]. Human aortic
endothelial cells (HAEC) were purchased from
Lonza (Chicago, IL) and cultured in EGM-2
medium supplemented with 2% FBS, but without
antibiotics. On the day before the study, the FBS
concentration was reduced to 1%. HAEC were
transfected with GFP or SOD2 plasmid was
described previously [31]. Rat aortic smooth muscle
cells were purchased from Cell Systems (Kirkland,
Wash). Neutrophils were isolated from whole blood
by Percoll density gradient centrifugation as previ-
ously described [33].
Brain mitochondria were isolated from the male
Lewis rat pooled forebrain. We used the modified
method of Sims [34] to isolate and purify brain mito-
chondria (RBM) in a Percoll gradient. Endothelial
mitochondria were isolated using the digitonin
method as previously described [35].
All animal use complied with National Institutes of
Health guidelines and was approved by the Emory
University Institutional Animal Care and Use
Committee [36].
•–
EMPO (50 mM). The rate of O2 formation was in
xanthine oxidase system, mitochondria, neutrophils
or BAEC was measured by monitoring the amplitude
of the low-field component of the EPR spectrum as
previously described [11]. The concentration of
nitroxide was determined from a calibration curve for
intensity of the EPR signal of 3-carboxyproxyl at
•–
various known concentrations. The rate of O2 pro-
duction was calculated from the accumulation of
nitroxide, obtained from the EPR time scan. For this
purpose, the EPR kinetics were analysed using linear
regression and WinEPR software (Bruker Biospin
Corp, Billerica, MA). EPR settings were as follows:
modulation amplitude, 2 G; microwave power,
20 mW; conversion time, 1.3 s; time constant, 5.2 s.
Samples were scanned immediately after supplemen-
tation of spin probes unless stated otherwise.
•–
Production of intracellular O2 in intact human
aortic endothelial cells (HAEC) was stimulated
with PMA (5 μM, 30 min). Generation of mitochon-
•–
drial O2 was induced by rotenone (5 μM, 30 min).

420 S. I. Dikalov et al.
Following treatment with PMA, rotenone or vehicle,
HAEC were incubated with 0.5 mM spin probes for
20 min at 37°C.Then cells were collected and placed
into a 1 ml syringe with 0.6 ml buffer and the suspen-
sion was snap-frozen in liquid nitrogen. Samples were
analysed in finger Dewar vessel filled with liquid
nitrogen [6]. ESR spectra were recorded using the
following ESR settings: field sweep, 80 G; microwave
frequency, 9.39 GHz; microwave power, 2 mW;
modulation amplitude, 5 G; conversion time, 327.68
ms; time constant, 5242.88 ms; 512 points resolution
and receiver gain, 1 ꢃ 104.
(I0) and after (I) a single extraction with an equal
amount of octanol, Kp ꢁ (I0/I) ꢀ 1. EPR measure-
ments were performed in 50 μL glass capillary tubes
at room temperature using the Bruker EMX spec-
trometer. Spectrometer settings were as follows: field
sweep, 60 G; microwave frequency, 9.82 GHz; micro-
wave power, 20 mW; modulation amplitude, 1 G; con-
version time, 164 ms; time constant 328 ms; sweep
time, 168 s; receiver gain, 1 ꢃ 105; number of scans, 4.
Results
•–
Reaction of hydroxylamine spin probes with O2
Analysis of cell permeability of hydroxylamine probes
All the hydroxylamine spin probes readily reacted with
•–
O2 produced in a cell-free xanthine oxidase system
Cell permeability of hydroxylamine probes was deter-
mined in cellular lysates. For this purpose confluent
rat aortic smooth muscle cells were subjected to
20-min incubation with corresponding hydroxylam-
ine (1 mM) at 37°C.Then cells were centrifuged and
supernatant discarded. Cells were resuspended in
PBS and lysated by ultrasound. Spin probe cellular
content was determined by oxidation of cell lysates
with NaIO4 (50 mM) to convert cyclic hydroxylam-
ines into nitroxides detected by EPR [38].
To determine partition coefficients, solutions of the
hydroxylamines (1 mM) in 50 mM phosphate buffer
saline (pH 7.4) were prepared. The portions of the
solutions were extracted with an equal amount of
octanol.The concentrations of hydroxylamine probes
in water phase were calculated from EPR intensities
of samples treated with NaIO4 (50 mM) to convert
cyclic hydroxylamines into nitroxides. The partition
coefficients were determined from a ratio of hydroxy-
lamine concentrations in the phosphate buffer before
[37]. Figure 2 demonstrates comparison of O2•– detec-
tion using the spin trap EMPO (50 mM) and the spin
•–
probe TMT-H (1 mM). Spin trapping of O2 by
EMPO produced the relatively stable EMPO/•OOH
radical adduct with the characteristic six-line EPR
spectrum (Figure 2A, insert). The accumulation of
EMPO/•OOH radical adduct was monitored by fol-
lowing the intensity of the low-field component of its
EPR spectrum. The rate of accumulation of the radi-
cal adduct (130 nM/min) decreased by more than
60% after 6-min data acquisition (Figure 2A, XOꢂX).
Addition of Cu,Zn-SOD (50 U/ml) to xanthine/xan-
thine oxidase completely blocked the accumulation of
the radical adduct (Figure 2A, XOꢂXꢂSOD).
Reaction of TMT-H with O2•– produced the stable
nitroxide radical 4-isobutyramido-TEMPO (TMT),
which has a three-line EPR spectrum (Figure 2B,
insert). Addition of Cu,Zn-SOD (50 U/ml) to the
reaction mixture blocked nitroxide accumulation
EMPO-OOH, μM
TMT, μM
A
B
1.0
XO+X
XO+X
1.50
1.25
1.0
0.8
0.6
15 G
15 G
EPR sensitivity
100 nM
0.75
0.50
0.25
0
0.4
10 nM
1 nM
0.2
0
XO+X+SOD
XO+X+SOD
0
100 200 300 400
500 600 [sec]
0
100 200 300 400 500 600 [sec]
Figure 2. EPR detection of the nitroxides formed from the spin trap EMPO (A) and the hydroxylamine spin probe TMT-H (B) in a
xanthine oxidase O2•– generating system (XOꢂX, 1 mU/ml xanthine oxidase plus 50 μM xanthine). Graphs show typical time scans of
nitroxide accumulation followed by low field component of EPR spectrum (insert). Analysis of EPR sensitivity shows that Bruker EMX
spectrometer equipped with SHQ microwave cavity has a detection limit of 1 nM nitroxide.

Superoxide detection by cyclic hydroxylamines 421
(Figure 2B, XOꢂXꢂSOD). Interestingly, there was
no significant difference in the initial rates of accumu-
lation of the EMPO/•OOH andTMT, but after 6-min
the difference in the rates of product accumulation
was more than 2-fold (Figure 2). As a result, concen-
tration of TMT was more than 60% higher than con-
centration of EMPO/•OOH after the 11 min scan,
whichislikelyduetothelowerstabilityoftheEMPO/•OOH
radical adduct compared to TMT nitroxide.
Other cyclic hydroxylamine probes (CAT1-H,
PP-H, CP-H and CM-H) showed similar accumula-
tion of nitroxides in the xanthine oxidase system and
supplementation of SOD blocked accumulation of
nitroxides in a dose-dependent manner (Figure 3),
while catalase did not affect kinetics of nitroxide pro-
duction (data not shown). Competition of SOD and
hydroxylamines was used to measure the rate con-
stant of the spin probe reaction with O2•–. Figure 3
shows the linear graphs of V/V0 ꢀ1 (where V and V0
represent the rates of nitroxide accumulation in the
presence or absence of SOD, respectively) as a func-
tion of SOD activity, as was previously described
[23,39]. The slope of the graph reflects the ratio of
reported earlier (3.2 ꢃ 103 M-1s-1) [23] the corre-
sponding rate constants for TM-H, TMT-H, CM-H,
mitoTEMPO-H and CAT1-H were estimated (Table I).
The reactivity of HO2•– is known to be higher than that
•–
of O2 [39]. Moreover, cyclic hydroxylamines are
known to be bases, with pK varying in a broad range
(5–8), depending on electronic and steric effects of the
substituents, and the reactivity of free hydroxylamine
base may differ strongly from that of hydroxylammo-
nium cation [40].Therefore, the apparent reaction rates
•–
•
ofthehydroxylamineprobeswithO2 /HO2 areexpected
to demonstrate complex pH-dependence, which deser-
ves a separate study. Finally, it is important to note that
correct determination of ‘absolute values’ for the reac-
tion rate constants from competition kinetics using
biological sources of superoxide may be complicated.
Therefore, the apparent rate constants presented in
Table I should be taken primarily for the ranking
purpose and considered as a semi-quantitative.
Lipophilicity and cell permeability of spin probes
Cell permeability of the spin probes may affect the
•–
k
SOD/kProbe, therefore, higher slope corresponds to
outcome of the cellular O2 detection. Lipophilicity
lower rate constant. Among the hydroxylamines stud-
ied, the positively charged mitoTEMPO-H and
CAT1-H show the highest rate constants of reaction
with O2•–, while the negatively charged PP-H shows
the smallest rate constant. The ratio of the slopes
equals the ratio of the apparent reaction rate con-
stants of corresponding hydroxylamine spin probes
with superoxide.Taking into account the value of the
is an important factor modulating cellular permeabil-
ity of the cyclic hydroxylamines and the partition
coefficient in a water/octanol mixture is a standard
measure of lipophilicity. The partition coefficients of
the spin probes were determined in octanol/phos-
phate buffer saline at pH ꢁ 7.4 mixture as octanol/
aqueous phase concentration ratio. Relatively high
lipophilicity of CM-H,TMT-H and TM-H (Table I)
is an indication of their ability to cross cell membranes.
•–
rate constant for the reaction of CP-H with O2
y (PP-H) = 4.2 x
10
8
H2O2
O
(<0.2%)
+
2
Vo
— - 1 = —— • ———
kProbe [Probe]
kSOD [SOD]
•
5•105 M-1s-1
O2
2.5 nM/s
V
y (CP-H) = 3.4 x
SOD
6•109 M-1s-1
•
+
H2O2 O2
X+XO
O2
R
y (TM-H) = 2.6 x
y (TMT-H) = 2.2 x
kProbe
N
OH
6
R
y (CAT1-H) = 1.7 x
y (mT-H) = 1.4 x
N
O
4
PP-H
CP-H
TMT-H
TM-H
2
CAT1-H
mT-H
0
0
0.5
1
1.5
2
2.5 SOD, U/ml
Figure 3. Reaction of hydroxylamine spin probe with O2•– and measurement of the rate constants by competition with SOD. Superoxide
radicals were produced by xanthine (50 μM) plus xanthine oxidase (1 mU/ml). The rate constants were calculated from inhibition of
nitroxide formation at various SOD activities using the following equation: (V0/V) ꢀ 1 ꢁ kSOD ꢃ [SOD]/kProbe ꢃ [Probe], where V0 and
V represent the rates of nitroxide formation in the absence or presence of SOD, respectively. The kProbe and kSOD are the second order
•–
rate constants of the O2 reactions with spin probes and SOD. The slope of the graph reflects the ratio of kSOD/kProbe. Higher slope
corresponds to lower rate constant.

422 S. I. Dikalov et al.
Table I. Partition coefficients (Kp) of cyclic hydroxylamines, rate constants of their reactions with O2•– and rates of nitroxide accumulation
in PMA-stimulated neutrophilsa, in BAECb under basal conditions and after treatment with peroxynitrite donor SIN-1.
CAT1-H
PP-H
CP-H
CM-H
TMT-H
TM-H
mT-H
EMPO
Kp
0.001
ꢀ1sꢀ1 6.4 ꢄ 0.4
936 ꢄ 28
0.005
0.84 [28]
929 ꢄ 35
0.05
3.2 [23]
27
12 [6]
1153 ꢄ 52
35
4.9 ꢄ 0.5
43
8
•
k(O2 ), ꢃ 10ꢀ3
M
4.2 ꢄ 0.4
932 ꢄ 31
7.8 ꢄ 0.6
0.074 [9]
562 ꢄ 25
aNeutrophils ꢂ
PMA, nM/min
bBAEC, nM/min
bBAECꢂSIN-1,
nM/min
12 ꢄ 1
22 ꢄ 2
20 ꢄ 3
60 ꢄ 5
95 ꢄ 5
122 ꢄ 6
15 ꢄ 2
41 ꢄ 3
14 ꢄ 2
38 ꢄ 4
12 ꢄ 2
21 ꢄ 2
afor experiment details see Figure 5; bfor experiment details see Figure 6.
Low lipophilicity of CP-H most likely results from
ionization of the carboxylic group (pKa is ca. 5) at
physiological pH values. However, ionization of the
carboxylic group only attenuates the cell permeability
but does not abolish it completely. Ionic PP-H and
CAT1-H showed the lowest lipophilicity.
accumulated in red blood cells which are deficient of
mitochondria (data not shown).
Detection of extracellular O2•– in human neutrophils
Previously, we have shown that human neutrophils
produce predominantly extracellular O2•–, both in
basal and PMA-stimulated conditions [27]. In this
work we compared O2•– detection by spin probes and
the spin trap EMPO in human neutrophil suspen-
sions. Production of O2•– was monitored by accumu-
lation of nitroxides or EMPO radical adducts which
were followed by the increase in the low-field compo-
nent of their EPR spectra.The rate of nitroxide accu-
mulation significantly increased upon stimulation of
We studied cell permeability of the hydroxylamine
spin probes in cultured rat aortic smooth muscle cells.
For this purpose rat aortic smooth muscle cells were
subjected to 20-min incubation with corresponding
hydroxylamine probe. Then cells were centrifuged
and cellular content of the probe was determined by
oxidation of cell lysates with NaIO4 (50 mM) to con-
vert cyclic hydroxylamines into nitroxides detected
by EPR [38]. We found that CM-H, PP-H, TM-H
and TMT-H accumulated in the cellular cytoplasm
(Figures 4A–E). No significant accumulation of
CAT1-H in the cells was observed (Figure 4F).
Lipophylic triphenylphosphonium cation is known
to be cell-permeable and provide mitochondria-
targeted accumulation [41]. Indeed, incubation of
cells with 50 μM mitochondria-targeted spin probe
mitoTEMPO-H resulted in significant cellular accu-
mulation of mitoTEMPO (Figure 4C). The concen-
tration of mitoTEMPO in cellular lysate was close to
nitroxide concentration in lysates of cells incubated
with 1 mM TM-H or TMT-H, thereby supporting
active cellular accumulation of mitoTEMPO-H.
Indeed, mitochondria isolated from mitoTEMPO-H
treated cells showed strong EPR spectra (data not
shown).
•–
neutrophils with PMA. Specificity of O2 detection
by spin probes was confirmed by inhibition of EPR
signal with SOD supplementation (Figure 5).The rate
of CM-H oxidation into nitroxide was 20% higher
than that of spin probes PP-H, TM-H and CAT1-H,
while accumulation of the EMPO radical adduct was
A
CM-H
B
C
PP-H
mitoTEMPO-H
It has been previously been suggested that hydro-
philic nitroxides do not diffuse through cellular lipid
membranes. The absence of cell permeability for
CAT1-H is in line with this assumption. However,
PP-H unexpectedly demonstrates high capacity of cel-
lular accumulation similar to neutral CM-H. Our data
(Figure 4) suggest that cells actively import PP-H
through some unknown mechanism. The mechanism
of PP-H accumulation by cells could be similar to the
active transport of phosphate [42]. This process is
ATP-dependent and mitochondrial uncoupling should
inhibit accumulation of PP-H. Indeed, we found that
accumulation of PP-nitroxide in vascular cells was
inhibited by antimycin A and PP-nitroxide was not
TM-H
TMT-H
D
E
F
CAT1-H
15 G
Figure 4.Cell permeability of hydroxylamine spin probes.Confluent
rat aortic smooth muscle cells were incubated with 1 mM cyclic
hydroxylamines (50 μM mitoTEMPO-H) for 20 min at 37°C. Cell
lysate was treated with 10 mM NaIO4 before EPR measurements.
The figure shows typical EPR spectra observed in at least three
independent experiments.

Superoxide detection by cyclic hydroxylamines 423
Nitroxide, μM
7.0
Cells + PMA + CM-H
6.0
5.0
Cells + PMA + CAT1-H
4.0
3.0
2.0
1.0
Cells + PMA + SOD + CM-H
Cells + CM-H
CM-H
0
0
25 50 75 100 125 150 175 200 225 250 [sec]
Figure 5. Superoxide production was by PMA-stimulated human neutrophils (0.15 mg/ml). Cells were stimulated by 1 μM phorbol
12-myristate 13-acetate (PMA) and incubated with 1 mM hydroxylamine probes or 50 mM EMPO.
•–
2-fold lower than nitroxide formation from the spin
probes (Figure 5, Table I). These differences in O2
detection in PMA-stimulated neutrophils correlated
with the cell-free experiments in a xanthine oxidase
O2•– generating system (Figures 3 and 5).
intra- and extra-cellular O2 production in endothe-
•–
lial cells [25,26]. We have investigated production of
•–
endothelial O2
after eNOS uncoupling using
hydroxylamine probes and the spin trap EMPO.
Both EMPO and the hydroxylamine probes
detected O2•– produced by BAEC (Figure 6,Table I).
Interestingly, CAT1-H and EMPO formed similar
amounts of nitroxides.The nitroxides formation from
CAT1-H and EMPO was completely suppressed by
Cu,Zn-SOD supplementation; thus, it corresponds to
basal extracellular production of O2•–. Reactions with
other spin probes gave stronger EPR signals and sup-
plementation with extracellular SOD did not inhibit
nitroxide accumulation completely. Presumably,
CM-H, PP-H, TM-H and TMT-H react with intrac-
ellular O2•–. CM-H showed highest reactivity among
hydroxylamine spin probes producing the highest
amount of nitroxide (Figure 6, Table I).
•–
Detection of O2 in intact endothelial cells treated with
the peroxynitrite donor SIN-1
It has been previously reported that that exposure of
endothelial cells to peroxynitrite leads to oxidation of
tetrahydrobiopterin (BH4), which is a crucial intracel-
lular cofactor of eNOS (Figure 6, Scheme) [43].
Depletion of cellular BH4 results in uncoupling of
endothelial NO synthase (eNOS), which then pro-
•–
duce O2 instead of NO [25,43]. Nitric oxide syn-
•–
thase inhibitor L-NAME blocks O2 production by
uncoupled eNOS [25,43,44], which is likely due to
inhibition of heme domain of eNOS [45]. Uncoupling
of eNOS by peroxynitrite donor SIN-1 is a well-known
model of endothelial dysfunction which increases both
Uncoupling of eNOS was induced by 1-h incuba-
tion of endothelial cells with 1 mM peroxynitrite
donor SIN-1, the later was then washed out with PBS
CM,μM
EC treated with ONOO¯
SIN-1
2.5
•
•
O2 + NO
EC treated with ONOO¯
plus L-NAME
EC
2.0
1.5
1.0
ONOO¯
EC+SOD
eNOS
BH4
coupled
BH2
eNOS
PBS
uncoupled
L-NAME
•
O2
0
100
200
300
400
500
600 Time, sec
Figure 6. Intra- and extracellular O2•– production in bovine aortic endothelial cells (BAEC) treated with the peroxynitrite donor SIN-1.
Confluent endothelial cells were treated with 1 mM SIN-1 for 60 min at 37°C, then washed with PBS. Superoxide production was analysed
in cell suspensions incubated at room temperature with hydroxylamine spin probes (1 mM) or the spin trap EMPO (50 mM).

424 S. I. Dikalov et al.
Detection of O2•– in intact endothelial cells treated
with antimycin A
300
Buffer
250
200
150
100
50
Cells
•–
Antimycin A stimulates the release of O2 into the
Cells+AA
Cells+AA+SOD
mitochondrial matrix and cellular cytoplasm [16]. It
has been recently reported that nitrone spin traps fail
to detect intracellular O2•– [16]. In this work we have
investigated antimycin A-induced O2 production in
endothelial cells using hydroxylamine spin probes
(Figure 7, Table I).
•–
•–
It was found that all spin probes detected O2 in
BAEC under basal conditions. Incubation of BAEC
with CM-H and PP-H produced the strongest EPR
signal and formation of corresponding nitroxides was
only partially inhibited by extracellularly added Mn-
SOD (50 U/ml). The amount of nitroxides formed
0
CM-H
PP-H
TM-H
TMT-H
CAT1-H
Figure 7. Detection of intra- and extracellular mitochondrial
O2•– in endothelial cells (BAEC) stimulated with the mitochondrial
uncoupling agent antimycin A (AA). Extracellular O2 was
assessed by EPR signal inhibited by supplementation of 50 U/ml
Mn-SOD (SOD).
•–
•–
due to reaction of O2 with TM-H and TMT-H was
3-times less compared to CMH or PP-H.
Treatment of endothelial cells with antimycin A led
to a major increase in CM-H oxidation rate, which
was only partially suppressed by supplementation
with extracellular Mn-SOD. Antimycin A caused a
moderate increase in O2•–-induced oxidation ofTM-H
andTMT-H. In contrast, oxidation of PP-H decreased
in antimycin A-treated cells (Figure 7). Antimycin A
inhibits ATP synthesis, thereby blocking all energy-
dependent processes including active transport. This
may prevent accumulation of PP-H inside antimycin
A-treated cells and decrease the rate of oxidation of
PP-H. A passive cellular diffusion of CM-H, TM-H
and TMT-H was not likely affected by antimycin A
supplementation and, thereby, antimycin A did not
•–
to avoid interference with cellular O2 detection.
•–
Cells were centrifuged and O2 production was
analysed in cell suspension [26].Treatment of endo-
thelial cells with the peroxynitrite donor SIN-1
significantly increased the production of cellular
O2 detected both by hydroxylamine probes and
EMPO. The contribution of the eNOS uncoupling
•–
•–
in O2 production was estimated by decrease of
nitroxide accumulation in the presence of eNOS
inhibitor L-NAME (Figure 6) [26]. Treatment
with SIN-1 resulted in increase of the intensity of
the EPR signal: 2-fold for CAT1-H and EMPO,
2.5-fold for TM-H and TMT-H and 3-fold for
PP-H (Figure 6, Table I). Supplementation of
SIN-1-treated cells with Cu,Zn-SOD significantly
reduced nitroxide accumulation, while addition of
DMSO or uric acid did not affect oxidation of cyclic
hydroxylamines. These data confirm that reaction
•–
decrease the ability to scavenge intracellular O2 by
these probes.
Interestingly, detection of cellular O2•– in BAEC by
cell-impermeable CAT1-H was completely blocked
by supplementation with extracellular Mn-SOD both
in basal conditions and upon stimulation with anti-
mycin A (Figure 7,Table I), indicating that antimycin
•–
of hydroxylamines with O2 is a major source of
accumulated nitroxide in endothelial cells.
A
EMPO
B
TMT-H
Background
RBM
Background
RBM
RBM + Antimycin A
RBM+AA
RBM + Antimycin A + SOD
RBM + AA+SOD
15 G
15 G
•–
Figure 8. Detection of O2 in the suspension of rat brain mitochondria (RBM). Superoxide radical production was measured in the
samples containing RBM (0.5 mg/ml), 2 mM malate ꢂ 20 mM glutamate using spin trap EMPO (A) or cyclic hydroxylamine TMT-H
(B) with or without antimycin A (AA).

Superoxide detection by cyclic hydroxylamines 425
A increased not only intracellular but also extracel-
lular O2•– levels.
No EMPO radical adducts formation was observed
in rat brain mitochondria under basal conditions in
the presence of malateꢂglutamate (Figure 8A).
•–
However, stimulation of mitochondrial O2 pro-
Detection of mitochondrial O2•– in isolated mitochondria
duction with antimycin A resulted in significant
accumulation of EMPO/•OH radical adduct, which
was completely blocked by supplementation with
extra-mitochondrial SOD.
Small fractions of electrons in the mitochondrial respi-
ratory chain can leak out and reduce oxygen, produc-
•–
ing O2 radicals [46]. Superoxide radicals formed on
In contrast, TMT-H has been able to detect basal
production of O2 in rat brain mitochondria with
the inner membrane of the matrix are quickly dismu-
tated by Mn-SOD and, therefore, cannot be detected
outside of mitochondria, but can be estimated indi-
rectly by leakage of H2O2 from mitochondria.
•–
malateꢂglutamate (Figure 8B, RBM vs background).
Antimycin A further stimulated SOD-inhabitable
accumulation of nitroxide (Figure 8B, RBMꢂAA vs
RBMꢂAAꢂSOD). Interestingly, in antimycin A stim-
ulated mitochondriaTMT-H gave a twice higher EPR
signal compared to EMPO (Figures 8A and B).
Superoxide produced on the outer membrane of
the matrix can be released by mitochondria and
detected as extramitochondrial O2•– [29]. Previously,
succinate-dependent mitochondrial production of
•–
•–
We have previously described the detection of O2
O2 has been studied using DMPO and DEPMPO.
in mitochondria using PP-H [29]. Indeed, supplemen-
tation of PP-H to isolated mitochondria resulted in
significant accumulation of nitroxide which was fur-
ther increased by antimycin A (Figure 9A). However,
only a small fraction of the EPR signal was inhibited
by SOD under basal conditions and PP-H showed a
rather high background oxidation (Figure 9A).
In these experiments formation of the radical adducts
was completely prevented by supplementation with
extra-mitochondrial SOD [14,47]. However, identifi-
cation of mitochondrial O2•– using spin trapping was
complicated because of conversion of primary OOH-
adduct into secondary OH-radical adduct by gluta-
thione peroxidase and reduction of radical adducts
[13–15]. Hydroxylamine spin probes can react with
mitochondrial O2•– producing stable nitroxides which
can be resistant to mitochondrial reduction. In this
We have recently found that CAT1-H is a very sta-
ble probe with minimal background oxidation [30].
Supplementation of CAT1-H to mitochondria resulted
in accumulation of CAT1 nitroxide both in basal and
antimycin A stimulated conditions (Figure 9B).These
experiments showed that CAT1-H can be success-
•–
work we have investigated production of O2 in iso-
lated mitochondria under basal conditions and stim-
ulated with antimycin A or rotenone using various
hydroxylamine spin probes and EMPO.
•–
fully used for detection of mitochondrial O2 under
_
_
Mitochondria
OPO3H
OPO3H
N+
N+
+ O2
O2 + mitoTEMPO-H
O2
+
N
N
OH
N
O
N
OH
O
PP-nitroxide, nM
CAT1, nM
mitoTEMPO, nM
1000
750
500
250
0
300
ESR spectrum of PP
100
250
mitoTEMPO-H
15 G
80
60
40
200
A
B
C
PP-H
CAT1-H
150
100
50
20
0
RBM + MG + SOD
mitoTEMPO-H
0
0
[sec]
250
0
100
200
300 [sec]
[sec]
0
50
100 150 200
50
100
150
200
Figure 9. Detection of extramitochondrial O2•– in the suspension of rat brain mitochondria (RBM, 0.5 mg/ml) in the presence of malate
(2 mM) and glutamate (20 mM) (MG); concentration of the hydroxylamine spin probes PP-H (A) or CAT1-H (B) was 0.5 mM.
•–
Mitochondrial matrix O2 reacted with mitoTEMPO-H (C) in mitochondria isolated from bovine aortic endothelial cells (ECM) in the
presence of succinate (10 mM) and glutamate (20 mM).

426 S. I. Dikalov et al.
•–
basal conditions as well as upon stimulation by
antimycin A.The most important difference between
PP-H and CAT1-H was observed in the detection of
Stimulation of mitochondrial O2 by rotenone
increased nitroxide accumulation in cells incubated
with CM-H or mitoTEMPO-H, but not with CP-H
(Figures 10 A–C). Contribution of mitochondrial
O2•– in EPR measurements of cellular O2•– was deter-
mined by a decrease of EPR signal in HAEC trans-
fected with SOD2 plasmid compared to cells
transfected with control GFP plasmid. EPR analysis
of transfected HAEC showed that SOD2 over-expres-
sion did not affect measurements with CP-H (Fig-
ure 10D), but significantly attenuated measurements
with CM-H and mitoTEMPO-H. Over-expression of
mitochondrial SOD2 has significantly decreased
•–
O2 under basal conditions. Supplementation of
SOD decreased the accumulation of CAT1 by a fac-
tor of 4 due to low background oxidation of CAT1-H,
while PP-nitroxide signal was reduced only by 25%.
It is interesting to note that antimycin A did not influ-
ence PP-nitroxide accumulation in the presence of
•–
SOD, indicating that intramitochondrial O2 does
not participate in PP-H oxidation. Thus, similarly
to CAT1-H, PP-H does not penetrate mitochondria
•–
and detects only extramitochondrial O2
.
In order to measure O2•– production in a mitochon-
drial matrix we used mitochondria-targeted probe
mitoTEMPO-H. Addition of mitochondria isolated
from BAEC to mitoTEMPO-H significantly increased
EPR signal (Figure 9C). Both rotenone and antimy-
cin A further increased accumulation of nitroxide. As
expected, supplementation of SOD did not affect
rotenone-induced nitroxide accumulation and only
partially attenuated the EPR signal, indicating that
detection of basal and rotenone-induced O2 by
CM-H or mitoTEMPO-H (Figures 10E and F).
•–
Discussion
In this work we studied production of extracellular,
•–
intracellular and mitochondrial O2 in neutrophils,
cultured endothelial cells and isolated mitochondria
using cyclic hydroxylamines of various lipophilicity
and cell permeability. Despite significant differences
in the chemical structures of cationic (CAT1-H),
anionic (PP-H) and neutral (CM-H,TM-H,TMT-H)
hydroxylamines show comparable rates of oxidation
•–
reaction with O2 proceeds inside of the mitochon-
drial matrix.
Detection of intracellular and mitochondrial O2•– in
intact cells
•–
with O2 in the cell-free xanthine oxidase system,
which are significantly higher than the rate of accu-
mulation of the EMPO/•OOH radical adduct. The
performance of different hydroxylamine spin probes
in cellular systems, however, strongly depend on
differences in lipophylicity, cell permeability and
propensity to specific active transport.
All cationic, anionic and neutral spin probes showed
similar results in detection of O2•– in the cell-free xan-
thine oxidase system (Figure 2) and measurements of
extracellular O2•– produced by PMA-stimulated neu-
trophils (Figure 5, Table I). Meanwhile, detection of
intracellular O2•– was very distinct (Figures 7 and 10).
The above data indicate that that mitoTEMPO-H
and CM-H can penetrate the cells and react with
•–
mitochondrial O2 (Figures 4, 7 and 9). In contrast
to CMH, mitochondria-targeted mitoTEMPO-H is
actively accumulated in the mitochondrial matrix
similar to other triphenylphosphonium cations [40],
while CM-H may only passively diffuse across the
lipid membrane. Another probe, CP-H, can permeate
the cells, but it is mitochondria impermeable [48].
It is conceivable that specific intracellular localization
•–
of these spin probes will allow detection of O2 in
•–
Thus, the efficacy of intracellular O2 detection is
distinct cellular compartments: CP-H in the cyto-
plasm, mitoTEMPO-H in mitochondria, and CM-H
in both compartments. In this work we have stimu-
lated O2•– production in the cytoplasm with NADPH
determined not only by chemical reactivity of the spin
probes, but also by lipophility (Table I, Kp), cell per-
meability (Figure 4) and sub-cellular distribution of
these compounds (Figure 11). The presence of cat-
ionic group (CAT1-H), carrier or targeting groups
such as phosphate or lipophilic triphenylphospho-
nium cation was particularly important for probe sub-
cellular distribution and site-specific O2•– detection.
All the spin probes showed accumulation of nitrox-
ides in the suspension of BAEC both in basal condi-
tions and upon stimulation with peroxynitrite, but in
these experiments cell-impermeable CAT1-H showed
the lowest oxidation rate and only CAT1 nitroxide
accumulation was completely inhibited by extracellular
SOD (Figures 7 and 11). In contrast, formation of
nitroxides from cell-permeable hydroxylamines CM-H,
PP-H, TM-H and TMT-H was only partially sup-
pressed by extracellular SOD (Figures 6, 7 and 11).
•–
oxidases activator PMA while generation of O2 in
the mitochondrial matrix was induced by complex I
inhibitor rotenone [29]. The site-specific production
•–
of O2 in cytoplasm and mitochondria was investi-
gated using 0.5 mM CP-H, 0.5 mM CM-H or 50 μM
mitoTEMPO-H in intact human aortic endothelial
cells (Figure 10) by EPR analysis of frozen samples
[6]. In order to confirm detection of mitochondrial
•–
O2 we used specific over-expression of the mito-
chondrial isoform of superoxide dismutase SOD2,
while transfection of HAEC with GFP plasmid was
used as a control.
Treatment of HAEC with PMA increased nitroxide
accumulation in cells incubated with CP-H or CM-H
but not with mitoTEMPO-H (Figures 10A–C).

Superoxide detection by cyclic hydroxylamines 427
Figure 10. EPR detection of intracellular and mitochondrial O2•– in intact human aortic endothelial cells (HAEC). Production of intracellular
•–
•–
O2 was stimulated with PMA (5 μM, 30 min). Generation of mitochondrial O2 was induced by rotenone (5 μM, 30 min). Following
treatment with PMA, rotenone or DMSO as a vehicle, HAEC were incubated with 0.5 mM spin probes for 20 min at 37°C. Then cells
were collected and into a 1-ml syringe with 0.6 ml buffer and snap-frozen in liquid nitrogen. Samples were analysed in a finger Dewar
•–
filled with liquid nitrogen. Production of O2 in intact non-transfected HAEC was measured in using cell permeable spin probe CPH
•–
(A), mitochondria permeable CMH (B) or mitochondria-targeted mitoTEMPO-H (C). Contribution of mitochondrial O2 in EPR
measurements of cellular O2•– was determined by inhibition of EPR signal in HAEC transfected with SOD2 plasmid compared transfection
with control GFP plasmid. Analysis of transfected HAEC showed that SOD2 over-expression did not affect measurements with CPH (D),
but significantly attenuated measurements with CMH (E) and mitoTEMPO-H (F).
of O2•– by mitochondria. Our data suggest that CAT1-
H is an optimal probe for O2 quantification in
sub-cellular fractions and in cell-free samples.
Differences in cell-permeable spin probes behaviour
in the presence of BAEC and neutrophils coincides
with the previously known facts about extracellular
O2•– production by neutrophils and intracellular loca-
tion of eNOS in BAEC. Thus, supplementation of
cell-permeable hydroxylamines with extracellular
SOD may provide a simple method to differentiate
intracellular and extracellular O2•–. Moreover, quan-
•–
Application of neutral cell permeable hydroxyl-
amines (CM-H, TM-H, TMT-H) showed a 3-fold
increase in intracellular O2•– production by endothelial
cells treated with antimycin A, while cell impermeable
CAT1-H showed only a 30% increase in the extracel-
•–
•–
tification of both extracellular and intracellular O2
production is possible (Figure 11).
lular O2 .These data suggest that only a small fraction
•–
of mitochondrial O2 released into the cytoplasm is
Detection of intracellular O2•– correlates with accu-
mulation of the spin probes inside cells. CM-H
showed very strong cellular accumulation and the
highest rate of intracellular nitroxide formation among
all tested hydroxylamines (Figures 4–7). It is interest-
ing to note that anionic PP-H demonstrated higher
likely to escape from the cell and react with extracel-
lular probes (Figure 7, CM-H vs CAT1-H). Experi-
ments with peroxynitrite-treated BAEC showed that
•–
the extracellular fraction of O2 produced by uncou-
pled eNOS is higher than that from the mitochondrial
source.This difference may result from eNOS localiza-
tion in caveolae associated with extracellular mem-
brane [49]. As a consequence, localization of the
•–
sensitivity in the detection of endothelial O2 than
cationic CAT1-H. The phosphate-containing probe
PP-H was found to accumulate inside cells, presum-
ably via active transport. Recently, we have validated
•–
O2 sources in the proximity to extracellular mem-
branes greatly increases the probability for O2•– release
out of the cell. Therefore, it is likely that extracellular
O2•– detection strongly depends on the sources of cel-
•–
the detection of extracellular O2 in intact tissues
using CAT1-H [30] and found it extremely useful,
particularly due to resistance to auto-oxidation. Here
we successfully applied CAT1-H to study the release
•–
lular O2 , due to differences in their intracellular local-
ization. This may represent an additional challenge in

428 S. I. Dikalov et al.
•
O2
scarcity of spectral information, because the prod-
uct of oxidation has the same three-line EPR signal
regardless of species responsible for oxidation. How-
ever, the simplicity of the EPR spectrum results in
a higher signal-to-noise ratio due to distribution of
EPR intensity between three lines for nitroxides
instead of six lines for radical adducts, and provides
easy analysis both in liquid and frozen samples.The
second potential limitation is the non-specific oxi-
dation by transition metal ions (Cu2ꢂ, Fe3ꢂ) and
some heme proteins. Stability of hydroxylamines
towards background oxidation can be enhanced by
supplementation of chelating agents (DTPA, defer-
oxamine, DETC). In addition, we also found that
six-membered ring hydroxylamines, such as CAT1-
H, TM-H and TMT-H, have lower levels of back-
ground EPR signal compared to five-membered
probes (CP-H, CM-H). The third limitation is the
Extracellular detection
CAT1-H
+
EMPO
Uncoupled eNOS
NADPH oxidase
Mitochondria
CP-H
CM-H
Rot
CM-H
AA
•
O2
•
O2
+
+
PP-H
AA
mitoTEMPO-H
Matrix
TM-H
Xanthine
oxidase
Mitochondrial detection
TMT-H
Figure 11. General scheme of EPR detection of superoxide in
extracellular, intracellular or mitochondrial compartments using
cyclic hydroxylamine spin probes.
•–
the analysis of intracellular O2 using extracellular
detection by spin traps or cytochrome c [6] because a
minor change in extracellular O2•– may correspond to
•–
a several-fold increase in local intracellular O2 . The
•–
results of extracellular O2•– detection, therefore, should
be interpreted with caution. Cell-permeable hydroxy-
lamine spin probes described in this work may provide
a useful tool for investigation of sources of intracellular
O2•– (Figure 11).
lack of specificity in O2 detection. The lack of
specificity of cyclic hydroxylamines can be over-
come by the use of SOD and inhibitors of sources
•–
of O2 production, such as NADPH oxidase, xan-
thine oxidase or mitochondria [33], while reaction
with peroxynitrite can be inhibited by uric acid [26].
We have found that SOD inhibits 70–98% of nitrox-
ide production [19,31], while acute addition of uric
acid did not show a significant effect. These data
A number of mitochondria-targeted nitrone spin
traps have been designed to accumulate in mitochon-
dria and to detect free radicals generated in the matrix
[50]. Spin trapping in mitochondria, however, req-
uired very a high concentration of mito-DEPMPO
(50 mM) and supplementation with SOD completely
abolished the EPR signal to the level of oxygen-
free mitochondria [46], indicating detection of
extra-mitochondrial O2•– only. Our experiments with
SOD2 over-expressed HAEC showed that both
mitoTEMPO-H and CM-H can detect mitochon-
drial O2•–. Mitochondria-targeted accumulation of
•–
suggests that O2 represents a major pull of free
radicals produced in cells and tissue. Overall,
hydroxylamine probes will not replace spin traps,
but they provide a new useful tool for EPR detec-
•–
tion of cellular O2
.
The above-described examples demonstrate that a
new set of hydroxylamine spin probes allows site-
•–
specific detection of cellular and mitochondrial O2
.
•–
mitoTEMPO-H allowed specific O2 detection in
The main advantage of hydroxylamine spin probes is
variability of chemical structure.The use of this vari-
ability permits molecular design of specific probes
‘targeted’ to certain cellular compartment and the
combination of several hydroxylamine spin probes
may provide unique information about location of
radical production, which can hardly be obtained
using other methods.
mitochondria while CM-H measured O2•– both in the
cytoplasm and mitochondria (Figure 11). In contrast,
mitochondria-impermeable CP-H showed PMA-
•–
stimulated O2 production in the cytoplasm but did
•–
not detect rotenone-induced mitochondrial O2
.
Cyclic hydroxylamine probes react with O2•– much
faster (k ~ 103–104 Mꢀ1sꢀ1, pH ꢁ 7.4) than nitrone spin
traps (k ~ 35–75 Mꢀ1sꢀ1, pH ꢁ 7.4) and can compete
with cellular antioxidants for reaction with intracel-
•–
lular O2 [11]. Furthermore, cyclic hydroxylamine
Acknowledgement
probes showed a relatively slow decrease in nitroxide
accumulation rate in the reaction with superoxide while
the nitrone spin trap showed a much higher deviation
from linear accumulation of radical adduct (Figure 2
and Dikalov et al. [51]), presumably due to bimo-
lecular decay of the radical adduct which is typical for
b–proton containing nitroxides. Therefore, hydroxy-
Authors are grateful to Dr Maziar Zafari for his help
in editing the manuscript.
Declaration of interest
This research was supported by NIH grants PO-1
HL058000-05, PO-1 HL075209.
lamine spin probes represent a very useful tool for
•–
quantitative analysis of cellular O2
.
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This paper was first published online on Early Online on
9 December 2010.