Sergey Dikalov

1
Detection of Superoxide with Cyclic Hydroxylamines
Sergey Dikalov Director of Free Radicals in
Medicine CORE Division of Cardiology, Emory
University School of Medicine
2
Detection of O2? _ with EPR spectroscopy
1. Direct detection
SOD

1 e _
O2? _
O2
H2O2
2. Spin trapping (DMPO, EMPO, DEPMPO)
DMPO
DMPO/?OOH
35 M-1s-1

O2? _
N
N
O-
O?
3. Spin probes (cyclic hydroxylamines)
CP-H
CP

H2O2
3.2x103 M-1s-1

O2? _
3
Problems with direct O2? detection
1. O2? has extremely short life-time ( 1
msec). 2. It is present at very low steady-state
concentration ( 1 nM). 3. No EPR spectrum at
room temperature.
Superoxide cannot be directly detected in
biological samples.
4
Problems with spin trapping of O2?
EMPO/ ? OOH
EMPO
EMPO/ ? OH
EMPO/ OH2
GPx
E
t
O
C
2

N
Reduction
O
74 M-1s-1
slow
105 109 M-1s-1
O2? _
SOD, Ascorbate, GSH
fast
1. Slow kinetics of O2?- trapping and obstruction
by antioxidants EMPO ? OOH ?? EMPO/
? OOH (74 M-1s-1)
2. Decomposition to OH-radical adduct (GSH
peroxidase) EMPO/ ? OOH ?? EMPO/ ?
OH
GPx
3. Reduction to EPR silent hydroxylamine
(ascorbate, metals, enzymes) EMPO/ ? OOH
Fe2 ?? EMPO/OH2 Fe3
Spin trapping is limited by slow kinetics and
biodegradation of the radical adducts.
5
Advantages of O2? detection with cyclic
hydroxylamines
1) High reactivity with O2? _. The reactions of
cyclic hydroxylamines with O2?- are hundred
times faster than those with nitrone spin traps,
thereby enabling the hydroxylamines to compete
with cellular antioxidants and react with
intracellular O2? _.
CM-H
CM
1.2X104 M-1s-1


O2? _
H2O2
2) Stability of the reaction product. Cyclic
hydroxylamines produce stable nitroxides with a
much longer life time than radical adducts.
Hydroxylamines allow quantitative O2?- detection
with higher sensitivity than spin traps.
6
Nitroxide stability
1)  Absence of b-protons, which is a major site
for oxidative decay of the radical adducts.
2)  Reduction into EPR silent hydroxylamines is
a major pathway for decay of the
nitroxides I. Reduction in electron transport
chain depends on oxygen concentration and
permeability II. Reduction by flavin-enzymes
depends on oxygen concentration and permeability
III. Reduction by thiols (RSH) depends on the
presence of the metals IV. Reduction by
ascorbate (AH _) direct reaction and major
pathway in plasma. V. Reduction via formation of
oxoammonium cation and its reaction with NADH or
AH _.
Comparison of the nitroxide reduction (mM/min)
Dikalov et al. Biophys. Res. Comm. 231, 701-704
1997.
7
Spin probe stability
CP-H
CP ?

Fe3

1.
Fe2
Inhibited by Desferal
CP-H
CP ?

Cu2

2.
Cu1
Inhibited by DTPA or DETC
X
CP-H
CP ?

H2O2
3.
There is no direct reaction
Formation of ferryl species
H2O2
Fe4O

Fe2
4.
CP-H

CP ?
Fe4O

5.
Inhibited by DTPA or DETC
Fe3
Stabilization Ice, metal chelators (DF, DTPA or
DETC), Argon, fresh buffers (no H2O2)
8
Relative specificity of cyclic hydroxylamines
SOD
2x102 M-1s-1

ONOO_

NO2_
Urate


?NO2
NO2_
RSH
9
Detection of superoxide with cyclic hydroxylamines
Dikalov S.I., Dikalova A.E., Mason R.P. Arch.
Biochem. Biophys. 402, 218-226 2002.
10
Comparison of superoxide detection by spin trap
DEPMPO and spin probe PP-H
O2? 200 nM/min, 50mM DEPMPO
A
O2? 20 nM/min, 50mM DEPMPO
B
C
No O2?, 0.5 mM PP-H
D
O2? 20 nM/min, 0.5 mM PP-H
40 G
PP ?, nM
E
O2? 20 nM/min, 0.5 mM PP-H
100
0
100
200
300
400
500
600
Time, sec
Dikalov S. I., Dikalova A.E., Mason R.P. Arch.
Biochem. Biophys. 402, 218-226 2002.
11
Summary
1)  Advantages of cyclic hydroxylamines over
nitrone spin traps are I. High reactivity with
O2? rate constants are 103-104 M-1s-1 vs 30 of
DMPO II. Reaction product nitroxide has superior
life-time over radical adducts. III. Cyclic
hydroxylamines can be used for intracellular
superoxide detection.
2)  The major limitations of cyclic
hydroxylamines are I. Nitroxide radical as a
product of the reaction does not have specific
EPR spectrum II. Nitroxide can be formed by
non-specific oxidation of cyclic hydroxylamines.
3)  The lack of specificity of cyclic
hydroxylamines can be overcome by I. Superoxide
dismutase II. Inhibitors of sources of O2?
production, such as NADPH oxidase, xanthine
oxidase or mitochondria.
4)  Stability of cyclic hydroxylamines can be
increased by metal chelating agents (DTPA,
deferoxamine, DETC) and use of 6-membered ring
structures.
12
Applications of cyclic hydroxylamines
1. Quantitative O2? detection in blood plasma,
membrane fraction and purified enzymes.
2. Extra- and intracellular superoxide
measurements.
3. Detection of O2? in tissue samples.
4. In vivo O2? detection.
13
Measurements of xanthine oxidase activity in the
human blood plasma using CPH
Figure 2. A, Endothelium-bound xanthine-oxidase
activity as determined by EPR spectroscopy in
patients with chronic heart failure (CHF) and
control subjects. B, Representative EPR spectra
of CP demonstrating a greater increase of
xanthine-oxidase activity in plasma after heparin
injection (5000 U) in a patient with CHF compared
with a control subject. (The background signal
from plasma without xanthine was
subtracted.) Landmesser U. et al. Circulation.
2002106(24) 3073-3078.
14
Quantification of O2? in the membrane fractions
CP, mM
1.00
MNADPH
EPR spectrum of CP
0.5 mM O2
0.75
15 G
M
0.50
MNADPHSOD
PBS
0.25
sec
0
100
200
300
400
500
600
SOD-inhibitable CP-nitroxide formation reflects
the amount of O2?- detected by CPH in the
membrane fraction (M) in the presence of NADPH.
Sorescu D et al. (2001) Free Radic Biol Med
30603-612 Dikalov et al. 2003 Hanna IR,
Hilenski LL, Dikalova A, et al. (2004) Free
Radic. Biol. Med. 37(10) 1542-1549 Khatri et
al. (2004) Circulation 109 520-525 Dudley et
al. (2005) Circulation 1121266-73.
15
Calculation of the rate constant of superoxide
reaction with antioxidant by competition with
spin probe CMH
CM
EPR signal
CMH
O2
O2? _
e-
e-
e-
NADPH
Cyt P-450 reductase
MQ
MQ? _
Antioxidant
CM, mM
0 mM
3.0
10 mM
20 mM
2.0
50 mM
1.0
50 U/ml SOD
Background
0.0
sec
0
50
100
150
200
250
300
(A0/A) 1 kSCAV/kCPH x cSCAV/cCPH, where
A0 is the EPR amplitude in absence of antioxidant
and A the EPR amplitude in presence scavenger, k
is reaction rate constant and C is
concentration. (V0/V) - 1 kSCAV/kCPH x
cSCAV/cCPH, where V0 is the rate of nitroxide
accumulation in absence of antioxidant and V is
the rate in presence of scavenger. Kuzkaya et al.
(2003) J Biol Chem 278(25) 22546-22554.
16
Lipophilicity
KpOctanol/Water, PBS pH 7.4
17
Cell permeability
CM-H
PP-H
TM-H
TMT-H
CAT1-H
15 G
RASMCs were incubated with hydroxylamines 20 min
at 37 C. Cell lysate was treated with 10mM NaIO4.
18
Detection of extracellular O2? production by
PMA-stimulated neutrophills
Nitroxide, mM
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
sec
0
25
50
75
100
125
150
175
200
225
250
Wyche et al. (2004) Hypertension 43(6)
1246-1251.
19
Intra- and extracellular O2? in endothelial cell
(EC) treated with peroxynitrite
SIN-1
O2? NO?
ONOO
CM, mM
EC treated with ONOO-
2.5
eNOS uncoupled
EC
eNOS
EC treated with ONOO- plus L-NAME
ONOO
BH4
2.0
EC
BH2
ECSOD
EC
eNOS uncoupled
1.5
PBS
1.0
O2?
0
Time, sec
100
200
300
400
500
600
20
Detection of O2? production by endothelial
cells. Basal production and stimulation of O2?
release by mitochondria.
AA Antinamycin A, mitochondrial uncoupling
agent SOD extracellular superoxide dismutase
(50 U/ml Mn-SOD)
21
Detection of O2? by DEPMPO, EMPO and CMH in
cultured Lymphocytes
DEPMPO-OOH, mM
EMPO-OOH, mM
CM, mM
0.9
Cells PMA
2.4
Cells PMA
Cells PMA
2.0
0.6
1.6
1.2
0.3
0.8
Cells SOD PMA
Cells SOD PMA
Cells
0.4
CellsSOD
0
0
0
sec
0
100
200
300
400
500
600
sec
0
100
200
300
400
500
600
sec
0
100
200
300
400
500
600
Table 1. Detection of superoxide with cytochrome
C, DEPMPO, EMPO, CMH (pmol/mln/min).
Dikalov S., Wei L., Zafari M. 2005
22
Detection of extramitochondrial O2? by PP-H in
brain mitochondria (RBM) with glutamatemalate
Mitochondria

O2?
1000
RBMGMAA
750
EPR spectrum of PP
Antimycin A induced O2? production
PP-nitroxide, nM
15 G
500
RBMGM
Basal O2?
RBMGMAASOD
RBMGMSOD
250
PPH
0
0
50
100
150
200
250
300
350
sec
Panov A., Dikalov S., Shalbueva N. et al. J Biol
Chem. 2005 Oct 21
23
Measurements of PMA-stimulated superoxide
production in rat aorta segments using CMH spin
probe
37 C
21 C
CM, mM
100
Control
1
2.5
50 ml capillary tube (Fisher)
2.0
Control Apocynin
2
90
3
4
1.5
1.0
50 ml label
133
PMA
5
0.5
0
100
200
300
400
500
600
sec
PMA Apocynin
Tissue sample
71
1 Aorta PMA
2 Aorta (control)
14 mm
3 Aorta Apocycin PMA
Sealing compound
EPR spectra of tissue incubated 60 min with CMH
at 37 C.
4 Aorta Apocycin (Apocycin control)
5 CMH only, no aorta (background)
Apocynin inhibited 52 in PMA vs 10 in control.
24
Preparation of the frozen samples for ROS
measurements
1. Cut the top of the syringe. 2. Fill 200 ml
buffer. 3A. Insert tissue to position of 300 ml
from the bottom or 3B. Put 200 ml cell
suspension on the top of the buffer. 4A. Fill
the rest with the buffer 4B. Freeze and then
fill the rest of the syringe with buffer. 5.
Freeze whole sample.
0.0
0.1
0.2
0.3
Tissue or Cell suspension
0.4
0.5
300 ml
0.6
0.7
1 ml syringe
P-s buffer must have chelating agent DF-DETC or
DTPA.
25
Atrial fibrillation increased production of O2?
in left atrium measured using intracellular spin
probe CMH and frozen samples (liquid nitrogen)
CMH
CM
COOH
COOH
1.2.104 M-1s-1

O2? _

H2O2
N
N
O ?
OH
EPR signal
EPR silent
Left atrium
Right atrium
A
Control
D
Control
B
E
AF
AF
F
C
AF S178
AF S178
15 G
15 G
Dudley et al. (2005) Circulation 1121266-73.
26
Detection of superoxide in aorta of Tg SM nox1
mice using CMH
Dikalova A. et al. Circulation Circulation.
2005 112(17) 2668-76.
27
Measurements of ROS in blood using spin probe
PPH, CPH or CAT1H
Dikalov S.I., Dikalova A.E., Mason R.P. (2002)
New non-invasive diagnostic tool for
inflammation-induced oxidative stress using
electron spin resonance spectroscopy and cyclic
hydroxylamine. Arch. Biochem. Biophys. 2,
218-226.
28
In vivo measurements of superoxide production
induced by nitroglycerin
In vivo formation of 3-carboxy-proxyl nitroxide
in control rabbit (A), after injection of 130
µg/kg GTN (B), after injection of 1mg/ml SOD and
130 µg/kg GTN (C), after injection of 30 µg/kg
vitamin C and 130 µg/kg GTN (D). Superoxide
radical formation was determined from the
oxidation of CP-H to 3-carboxy-proxyl nitroxide.
Concentration of CP-H in blood was maintained
constant by continuos infusion of CP-H (2.5
mg/min).
Dikalov et al. (1999) Free Radical Biology
Medicine 27 (1-2), 170-176.
29
Increase in the O2? _ production or decrease in
antioxidant activity (SOD)
30
Conclusion

• Hydroxylamine spin probe should be selected based
  on its lipophilicity, cell permeability,
  stability and reactivity.
• Selective inhibitors and antioxidants must be
  used to identify ROS.
• Probes can be scanned immediately or analyzed in
  the frozen state.
• Frozen samples should be analyzed with caution
  due to overlapping with the EPR signals of
  bioradicals.
• Cyclic hydroxylamines can be used in vivo or ex
  vivo for tissue analysis.
• Cyclic hydroxylamines have been successfully used
  to assay O2? production by mitochondria,
  neutrophils, endothelial, and smooth muscle
  cells.
• Cyclic hydroxylamines are capable to detect both
  intra- and extracellular O2?-.

31
Acknowledgments
32
Free Radicals in Medicine CORE Division of
Cardiology, Emory University School of
Medicine, Atlanta, Georgia
33
Literature
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34
Detection of ROS in tissue and blood following in
vivo treatment with CPH
ROS generation in control rats and LPS-treated
rats.
Generation of NO and ROS in blood of control
animals and animals receiving LPS.
Detection of CP -radicals and NO-Hb complexes in
blood.
Kozlov A.V. et al. Free Radic Biol Med. 2003
34(12) 1555-62.
35
Detection of extracellular superoxide production
by neutrophils using CPH
Wyche et al. (2004) Hypertension 43(6)
1246-1251.