Transcript No Slide Title

Detection of Superoxide with Cyclic Hydroxylamines
Sergey Dikalov
Director of Free Radicals in Medicine CORE
Division of Cardiology, Emory University School of Medicine
+
Na OPO 3H
-
+
Cl
O
HN C
N
O CH3
N
OH
N
OH
N
OH
N
OH
PP-H
CAT1-H
TM-H
TMT-H
O
O CH3
N
OH
CM-H
OH
O
N
OH
CP-H
Detection of O2
1. Direct detection
+ 1e
O2

_
with EPR spectroscopy
_
O2

_
SOD
H2O2
2. Spin trapping (DMPO, EMPO, DEPMPO)
DMPO/OOH
DMPO
+
+
N
O2

_
35 M-1s-1
N
O
-
O
3. Spin probes (cyclic hydroxylamines)
CP-H
CP
CO2H
N
OH
+
O2

_
3.2x103 M-1s-1
N
O
CO2H
+ H2O2
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.
Problems with spin trapping of O2¯
EMPO/  OOH
EMPO
OOH EtO 2C
EtO 2 C
H
+
N
N
O
slow
74
O2
GPx
EtO 2C
EMPO/ OH2
EtO 2C
H
N
OOH
OH
O
O
M-1s-1

EMPO/  OH
H
N
Reduction
OH
OH
_ 105 – 109 M-1s-1
fast
SOD, Ascorbate, GSH
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
GPx

EMPO/  OH
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.
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
CO2CH3
CO2CH3

+ O2
_
4
1.2X10 M-1s-1
+ H2O2
N
N
OH
O
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.
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;
_
Reduction by ascorbate (AH ): direct reaction and major pathway in plasma.
IV.
V.
_
Reduction via formation of oxoammonium cation and its reaction with NADH or AH .
Comparison of the nitroxide reduction (mM/min)
Nitroxide
(40 mM)
Cysteine
1 mM
GSH
1 mM
RASMC
4000 per mL
Ascorbate
1 mM
Rate constant
M-1s-1
3-Carboxyproxyl
0.012
0.12
0.06
0.23
0.11
TEMPONE
0.013
0.21
0.13
15.7
7.2
Dikalov et al. Biophys. Res. Comm. 231, 701-704: 1997.
Spin probe stability
1. CP-H +
Fe3+
CP  + Fe2+
Inhibited by Desferal
2. CP-H +
Cu2+
CP  + Cu1+
Inhibited by DTPA or DETC
CP 
There is no direct reaction
Fe4+=O
Formation of ferryl species
CP  + Fe3+
Inhibited by DTPA or DETC
3. CP-H + H2O2
4. H2O2 +
Fe2+
5. CP-H + Fe4+=O
X
Stabilization: Ice, metal chelators (DF, DTPA or DETC), Argon, fresh buffers (no H2O2)
Relative specificity of cyclic hydroxylamines
CO2H
CO2H
+

O2
_
3.2x103 M-1s-1
N
N
SOD
OH
CO2H
O
CO2H
+ ONOO
_ ~ 2x102 M-1s-1
+ NO2
_
N
N
Urate
OH
CO2H
+
N
OH
+ H2 O2
O
CO2H
NO
+ NO2
2
RSH
N
O
_
Detection of superoxide with cyclic hydroxylamines
+
+
Na OPO 3H
75
50
25
0
-25
-50
-75
-
I
EPR Spectra
A
N
OH
PP-H + xanthine
100
N
O
D
PP-H + xanthine
0
-100
-200
0
50
100
150
200
250
300 [sec]
PP-H + xanthine + xanthine oxidase
I
B
400
300
E
200
PP-H + xanthine + xanthine oxidase
100
0
3485 3490 3495 3500 3505 3510 3515 3520 3525 3530 3535
75
50
25
0
-25
-50
-75
Time scans
200
3485 3490 3495 3500 3505 3510 3515 3520 3525 3530 3535
75
50
25
0
-25
-50
-75
Na OPO 3H -
0
50
100
150
200
250
300 [sec]
200
C
PP-H + xanthine + SOD + xanthine oxidase
F
100
PP-H + xanthine + SOD + xanthine oxidase
0
-100
-200
3485 3490 3495 3500 3505 3510 3515 3520 3525 3530 3535
Magnetic field, G
0
50
100
150
200
Time, sec
Dikalov S.I., Dikalova A.E., Mason R.P. Arch. Biochem. Biophys. 402, 218-226: 2002.
250
300 [sec]
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
100
0
O2 20 nM/min, 0.5 mM PP-H
100
200
300
400
500
600 Time, sec
Dikalov S. I., Dikalova A.E., Mason R.P. Arch. Biochem. Biophys. 402, 218-226: 2002.
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.
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.
+
Cl
N
+
Na OPO 3H -
N
OH
N
OH
CAT1-H
PP-H
OH
O
N
OH
CP-H
O CH3
O
HN C
N
OH
N
OH
TM-H
TMT-H
O
O CH3
N
OH
CM-H
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. 2002;106(24): 3073-3078.

Quantification of O2 in the membrane fractions
CP, mM
1.00
0.75
0.5 mM O2
EPR spectrum of CP
•
15 G
0.50
0.25
0
100
200
300
400
500
600 [sec]
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 30:603-612; 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 112:1266-73.
Calculation of the rate constant of superoxide reaction with antioxidant
by competition with spin probe CMH
CM
NADPH
e-
Cyt P-450 reductase
e-
MQ
e-
MQ
_
O2
O2
EPR signal
_
CM, mM
3.0
2.0
1.0
50 U/ml SOD
Background
0.0
0
50
100
150
200
250
300 [sec]
(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.
Lipophilicity
PP-H
+
Na OPO 3H -
CAT1-H
CP-H
+
O H
N
Cl
O
CM-H
O
TM-H
O CH3
O
HN C
N
OH
N
OH
35
43
N
OH
N
OH
N
OH
N
OH
0.005
0.01
0.05
27
Kp=[Octanol]/[Water], PBS pH 7.4
TMT-H
O CH3
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 NaIO 4.
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
0
nM/min
PMN-PMA
25
50
CM-H
1123
75
100
125
PP-H
929
150
175
200
225
TM-H
932
250 [sec]
CAT1-H
936
Wyche et al. (2004) Hypertension 43(6): 1246-1251.
EMPO
562
Intra- and extracellular O2 in endothelial cell (EC) treated with peroxynitrite
nM/min CM-H
Control
95
ONOO¯ 122
SIN-1
O2 + NO
PP-H
20
60
TM-H
14
38
TMT-H CAT1-H EMPO
15
12
12
41
22
21
CM, mM
ONOO¯
EC treated with ONOO-
2.5
eNOS
uncoupled
EC
BH4
ONOO¯
eNOS
EC treated with ONOOplus L-NAME
EC
2.0
BH2
EC+SOD
EC
eNOS
uncoupled
1.5
O2
1.0
PBS
0
100
200
300
400
500
600
Time, sec

Detection of O2 production by endothelial cells.

Basal production and stimulation of O2 release by mitochondria.
nM/min
Buffer
BAECs
BAECs+AA
EC+AA+SOD
CM-H
35
112
272
222
PP-H
28
102
49
34
TM-H
7.4
36.6
59
44.6
TMT-H
8.8
26.7
35.8
20.1
AA – Antinamycin A, mitochondrial uncoupling agent
SOD – extracellular superoxide dismutase (50 U/ml Mn-SOD)
CAT1-H
2.7
8.7
15.1
3.6
Detection of

O2
by DEPMPO, EMPO and CMH in cultured Lymphocytes
DEPMPO-OOH, mM
EMPO-OOH, mM
0.9
0.5
Cells +
PMA
0.4
CM, mM
Cells +
PMA
2.4
Cells+
PMA
2.0
0.6
1.6
0.3
1.2
0.2
0.3
0.8
Cells + SOD + PMA
0.1
Cells
Cells+SOD
0
0
100 200 300 400 500 600 [sec]
0
0
100 200
300 400 500
600 [sec]
Cells +SOD+PMA
0.4
0
0
100 200 300 400 500 600 [sec]
Table 1. Detection of superoxide with cytochrome C, DEPMPO, EMPO, CMH (pmol/mln/min).
Cytochrome
DEPMPO
EMPO
CMH
Cells
2.4±0.18
2.3±0.3
2.7±0.4
5.5±0.5
Cells+PMA
11.2±0.87
9.4±0.9
16±2.1
48.6±8.2
Dikalov S., Wei L., Zafari M. 2005

Detection of extramitochondrial O2 by PP-H in
brain mitochondria (RBM) with glutamate+malate
Mitochondria
O2

+
O
O P OH
O
O
O P OH
O
N
OH
N
O
Antimycin A induced
750
EPR spectrum of PP
O2

15 G
production
500
Basal O2
PP-nitroxide, nM
1000

250
0
0
50
100
150
200
250
300
350 [sec]
Panov A., Dikalov S., Shalbueva N. et al. J Biol Chem. 2005 Oct 21
Measurements of PMA-stimulated superoxide production in rat aorta segments
using CMH spin probe
100%
Control
2
3
4
90%
Control+
Apocynin
5
133%
1
2.5
2.0
1.5
1.0
0.5
0
100
200
300
400
500
1 – Aorta + PMA
600 [sec]
71%
PMA
PMA+
Apocynin
50 ml label
Tissue
sample
2 – Aorta (control)
3 – Aorta + Apocycin + PMA
4 – Aorta + Apocycin (Apocycin control)
EPR spectra of tissue incubated 60 min with CMH at 37 C.
5 – CMH only, no aorta (background)
Apocynin inhibited 52% in PMA vs 10% in control.
Sealing
compound
14 mm
CM, mM
50 ml capillary tube (Fisher)
37 °C
21 °C
Preparation of the frozen samples for ROS measurements
0.0
1. Cut the top of the syringe.
0.1
2. Fill 200 ml buffer.
0.2
0.3
Tissue or
Cell suspension
0.4
0.5
300 ml
0.6
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
0.7
4B. Freeze and then fill the rest of
the syringe with buffer.
1 ml syringe
5. Freeze whole sample.
P-s: buffer must have chelating agent DF-DETC or DTPA.

Atrial fibrillation increased production of O2 in left atrium measured using
intracellular spin probe CMH and frozen samples (liquid nitrogen)
CMH
CM COOH
COOH
+ O 2
N
1.2.104 M-1s-1
_
OH
O
EPR silent
EPR signal
Left atrium
A
B
C
+ H2O2
N
Right atrium
Control
D
AF
E
AF + S178
F
15 G
Dudley et al. (2005) Circulation 112:1266-73.
15 G
Control
AF
AF + S178
Detection of superoxide in aorta of Tg SM nox1 mice using CMH
Dikalova A. et al. Circulation Circulation. 2005; 112(17): 2668-76.
Measurements of ROS in blood using spin probe PPH, CPH or CAT1H
Shaking, 37 ° C
Blood, 2ml
CPH
30 min
30 min
Heparin
0.7 ml
0.7 ml
0.7 ml
Store at –80 C,
Ship in dry ice,
EPR analysis in
liquid nitrogen
1 ml syringe in
liquid nitrogen
0 min
30 min
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.
60 min
15 G
IEPR
In vivo measurements of superoxide production induced by nitroglycerin
ESR amplitude, mm
160
C
160
A
SOD GTN
140
140
120
120
Control experiment
SOD+GTN experiment
100
100
ESR amplitude, mm
0
20
40
60
80
100
B
160
0
20
40
60
80
160
100
D
GTN
140
140
120
120
Vitamin C
GTN experiment
GTN
Vitamin C experiment
100
100
0
20
40
60
80
Time after CP-H infusion, minutes
100
0
20
40
60
80
100
Time after CP-H infusion, minutes
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.
Increase in the O2

_
production or decrease in antioxidant activity (SOD)
Antioxidant
system
ROS formation
Conclusion
1.
Hydroxylamine spin probe should be selected based on its lipophilicity, cell
permeability, stability and reactivity.
2.
Selective inhibitors and antioxidants must be used to identify ROS.
3.
Probes can be scanned immediately or analyzed in the frozen state.
4.
5.
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.

6.
Cyclic hydroxylamines have been successfully used to assay O2 production by
mitochondria, neutrophils, endothelial, and smooth muscle cells.
7.
Cyclic hydroxylamines are capable to detect both intra- and extracellular O2-.
Acknowledgments
Emory University School of Medicine
Division of Cardiology, Atlanta, GA
Institute of Organic Chemistry
Novosibirsk, Russia
Prof. David G. Harrison
Prof. Kathy Griendling
Dr. Maziar Zafari
Dr. Anna Dikalova
Prof. Igor A. Grigor’ev
Dr. Igor Kiriluk
Dr. Maxim Voinov
National Institute of Environmental Health Sciences
Free Radical Metabolite Section, RTP, NC
Dr. Ronald P. Mason
Free Radicals in Medicine CORE
Division of Cardiology, Emory University School of Medicine,
Atlanta, Georgia
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in blood of control animals
and animals receiving LPS.
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rats and LPS-treated rats.
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