sergey dikalov director of free radicals in medicine core division of cardiology, emory university...
TRANSCRIPT
Sergey Dikalov
Director of Free Radicals in Medicine CORE
Division of Cardiology, Emory University School of Medicine
Detection of Superoxide with Cyclic Hydroxylamines
NOH
OPO3H
NOH
N+
NOH
O CH3Na
+ -
NOH
NH CO
N
OH
OO CH3
Cl
N
OH
OOH
PP-H CAT1-H CM-HTMT-HTM-H CP-H
Detection of O2
_ with EPR spectroscopy
CP-H CP
+ O2
_ + H2O23.2x103 M-1s-1
+ 1 e_
O2
_O2
3. Spin probes (cyclic hydroxylamines)
2. Spin trapping (DMPO, EMPO, DEPMPO)
N+
O-
DMPO DMPO/OOH
+ O2
_ 35 M-1s-1
N
CO2H
OH
N
CO2H
O
N
O
1. Direct detection
H2O2
SOD
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¯
N
O
H
OOH
EtO 2C
N+
O
EtO2C OOH
2. Decomposition to OH-radical adduct (GSH peroxidase)
EMPO/ OOH EMPO/ OH
3. Reduction to EPR silent hydroxylamine (ascorbate, metals, enzymes)
EMPO/ OOH + Fe2+ EMPO/OH2 + Fe3+
N
OH
H
OH
EtO 2C
N
O
H
OH
EtO 2CGPx
EMPO/ OOHEMPO EMPO/ OH EMPO/ OH2
1. Slow kinetics of O2- trapping and obstruction by antioxidants
EMPO + OOH EMPO/ OOH (74 M-1s-1)
O2
_SOD, Ascorbate, GSH
74 M-1s-1
105 – 109 M-1s-1
slow
fast
Spin trapping is limited by slow kinetics and biodegradation of the radical adducts.
GPx
Reduction
Advantages of O2¯ detection with cyclic hydroxylamines
Hydroxylamines allow quantitative O2- detection with higher sensitivity than spin traps.
1) High reactivity with O2
_.
The reactions of cyclic hydroxyl amines 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
_.
2) Stability of the reaction product.
Cyclic hydroxylamines produce stable nitroxides with a much longer life time than radical adducts.
N
CO2CH3
OH
CM-H CM
+ O2
_ + H2O2
N
CO2CH3
O
1.2X104 M-1s-1
1) Absence of -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 (M/min)
Nitroxide (40 M)
Cysteine1 mM
GSH1 mM
RASMC4000 per L
Ascorbate1 mM
Rate constantM-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.
Nitroxide stability
Spin probe stability
CP-H CP + Fe3+ + Fe2+1.
CP-H CP + Cu2+ + Cu1+2.
Inhibited by Desferal
Inhibited by DTPA or DETC
CP-H CP + H2O23.
H2O2 Fe4+=O+ Fe2+4.
CP-H CP + Fe4+=O + Fe3+5.
X There is no direct reaction
Formation of ferryl species
Inhibited by DTPA or DETC
Stabilization: Ice, metal chelators (DF, DTPA or DETC), Argon, fresh buffers (no H2O2)
Relative specificity of cyclic hydroxylamines
N
CO2H
OH
+ O2
_ + H2O2
N
CO2H
O
3.2x103 M-1s-1
N
CO2H
OH
+ ONOO_ + NO2
_
N
CO2H
O
~ 2x102 M-1s-1
N
CO2H
OH
+ NO2 + NO2
_
N
CO2H
O RSH
SOD
Urate
Detection of superoxide with cyclic hydroxylamines
Dikalov S.I., Dikalova A.E., Mason R.P. Arch. Biochem. Biophys. 402, 218-226: 2002.
NOH
OPO3HNa+ -
NO
OPO3HNa+ -
-75
-50
-25
0
25
50
75
-75
-50
-25
0
25
50
75
-75
-50
-25
0
25
50
75
I
PP-H + xanthine
3485 3490 3495 3500 3505 3510 3515 3520 3525 3530 3535
PP-H + xanthine + xanthine oxidase
3485 3490 3495 3500 3505 3510 3515 3520 3525 3530 3535
PP-H + xanthine + SOD + xanthine oxidase
3485 3490 3495 3500 3505 3510 3515 3520 3525 3530 3535
Time scans
PP-H + xanthine
0 50 100 150 200 250 300
-200
-100
0
100
200
PP-H + xanthine + xanthine oxidase
0 50 100 150 200 250 300
200
100
0
300
400
PP-H + xanthine + SOD + xanthine oxidase
0 50 100 150 200 250 300
-200
-100
0
100
200
EPR Spectra
Time, secMagnetic field, G
I
A D
E
FC
B
[sec]
[sec]
[sec]
Comparison of superoxide detection by spin trap DEPMPO and spin probe PP-H
40 G
A
B
C
D
O2 200 nM/min, 50mM DEPMPO
O2 20 nM/min, 50mM DEPMPO
No O2, 0.5 mM PP-H
O2 20 nM/min, 0.5 mM PP-H
Time, sec 0 100 200 300 400 500 600
100
PP , nM
E O2 20 nM/min, 0.5 mM PP-H
Dikalov S. I., Dikalova A.E., Mason R.P. Arch. Biochem. Biophys. 402, 218-226: 2002.
4) Stability of cyclic hydroxylamines can be increased by metal chelating agents (DTPA,
deferoxamine, DETC) and use of 6-membered ring structures.
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.
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.
Summary
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.
Applications of cyclic hydroxylamines
NOH
OPO3H
NOH
N+
NOH
O CH3Na
+ -
NOH
NH CO
N
OH
OO CH3
Cl
N
OH
OOH
PP-HCAT1-H CM-HTMT-HTM-HCP-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
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.
[sec] 0 100 200 300 400 500 600
0.25
1.00
0.75
0.50
CP, M
M+NADPH+SOD
M+NADPH
M15 G
EPR spectrum of CP 0.5
M O
2 •
PBS
NADPH e-
Cyt P-450 reductase e-
MQ e-
MQ_
O2
O2
_CMH
Antioxidant
CM EPR signal
[sec] 0 50 100 150 200 250 300
2.0
3.0
1.0
0.0
CM, M
50 M
20 M
10 M
0 M
50 U/ml SOD
Background
Calculation of the rate constant of superoxide reaction with antioxidant by competition with spin probe CMH
(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.
PP-H CAT1-H CP-H CM-H TMT-H TM-H
0.005 0.01 0.05 27 35 43
NOH
OPO3HNa+ -
NOH
N+
Cl
NOH
O CH3
NOH
NH CO
N
OH
OO CH3
N
OH
OO H
Lipophilicity
Kp=[Octanol]/[Water], PBS pH 7.4
15 G
Cell permeability
PP-H
TM-H
TMT-H
CAT1-H
CM-H
RASMCs were incubated with hydroxylamines 20 min at 37 C. Cell lysate was treated with 10mM NaIO4.
nM/min CM-H PP-H TM-H CAT1-H EMPOPMN-PMA 1123 929 932 936 562
[sec] 0 25 50 75 100 125 150 175 200 225 250
1.0
4.0
5.0
6.0
7.0
2.0
3.0
0
Nitroxide, M
Cells + PMA + CM-H
CM-H
Cells + PMA + SOD + CM-HCells + CM-H
Cells + PMA + CAT1-H
Detection of extracellular O2 production by PMA-stimulated neutrophills
Wyche et al. (2004) Hypertension 43(6): 1246-1251.
CM, M EC treated with ONOO-
EC
PBS
Time, sec 0 100 200 300 400 500 600
1.0
1.5
2.0
2.5
EC treated with ONOO-
plus L-NAME
EC+SOD
nM/min CM-H PP-H TM-H TMT-H CAT1-H EMPOControl 95 20 14 15 12 12ONOO¯ 122 60 38 41 22 21
SIN-1
O2 + NO
ONOO¯
BH4
eNOS
eNOSuncoupled
BH2
O2
Intra- and extracellular O2 in endothelial cell (EC) treated with peroxynitrite
eNOSuncoupledEC
ON
OO
¯
EC
nM/min CM-H PP-H TM-H TMT-H CAT1-HBuffer 35 28 7.4 8.8 2.7
BAECs 112 102 36.6 26.7 8.7BAECs+AA 272 49 59 35.8 15.1EC+AA+SOD 222 34 44.6 20.1 3.6
AA – Antinamycin A, mitochondrial uncoupling agentSOD – extracellular superoxide dismutase (50 U/ml Mn-SOD)
Detection of O2
production by endothelial cells.
Basal production and stimulation of O2
release by mitochondria.
Cells +
PMA
[sec]0 100 200 300 400 500 6000
0.4
0.8
1.2
1.6
2.0
2.4
Cells + SOD + PMA
CM, M
[sec]0 100 200 300 400 500 6000
Cells + PMA
DEPMPO-OOH, M
0.1
0.2
0.3
0.4
0.5
Cells + SOD + PMA
Detection of O2
by DEPMPO, EMPO and CMH in cultured Lymphocytes
Dikalov S., Wei L., Zafari M. 2005
[sec] 0 100 200 300 400 500 600
0.9
0.6
0.3
0
EMPO-OOH, M
Cells
Cells+SOD
Cells + PMA
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
Detection of extramitochondrial O2
by PP-H in
brain mitochondria (RBM) with glutamate+malate
[sec] 0 50 100 150 200 250 300 3500
250
500
750
1000
PP
-nit
roxi
de,
nM
PPH
RBM+GM
RBM+GM+SODRBM+GM+AA+SOD
RBM+GM+AAB
asal O2
An
timycin
A in
du
ced O
2 pro
du
ction
15 G
EPR spectrum of PP
Panov A., Dikalov S., Shalbueva N. et al. J Biol Chem. 2005 Oct 21
Mitochondria
O2
NOH
OP OHOO
+NO
OP OHOO
[sec] 0 100 200 300 400 500 600
2.0
1.0
1.5
0.5
2.5
CM, M
1
234
5
1 – Aorta + PMA
2 – Aorta (control)
3 – Aorta + Apocycin + PMA
4 – Aorta + Apocycin (Apocycin control)
5 – CMH only, no aorta (background)
Measurements of PMA-stimulated superoxide production in rat aorta segments using CMH spin probe
100% Control
90%
133%
71%
Apocynin inhibited 52% in PMA vs 10% in control.
Control+Apocynin
PMA
PMA+Apocynin
EPR spectra of tissue incubated 60 min with CMH at 37 C.
37 °C21 °C
Tissuesample
50
l cap
illa
ry t
ub
e (F
ish
er)
Sealing
compound
50 l label
14 m
m
Preparation of the frozen samples for ROS measurements
Tissue orCell suspension
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1 ml syringe
1. Cut the top of the syringe.
2. Fill 200 l buffer.
3A. Insert tissue to position of 300 l from the bottom or
3B. Put 200 l 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.
300 l
P-s: buffer must have chelating agent DF-DETC or DTPA.
ControlControl
AF
AF + S178
AF
Left atrium Right atrium
AF + S178
Atrial fibrillation increased production of O2
in left atrium measured using intracellular spin probe CMH and frozen samples (liquid nitrogen)
A
C
B
F
E
D
15 G 15 G
N
COOH
OH
CMH CM
+ O2
_ + H2O2N
COOH
O
1.2.104 M-1s-1
EPR silent EPR signal
Dudley et al. (2005) Circulation 112:1266-73.
Dikalova A. et al. Circulation Circulation. 2005; 112(17): 2668-76.
Detection of superoxide in aorta of Tg SM nox1 mice using CMH
Blood, 2ml CPH
Heparin
1 ml syringe inliquid nitrogen
Shaking, 37 ° C
30 min 30 min
0.7 ml 0.7 ml0.7 ml
Store at –80 C,Ship in dry ice,EPR analysis inliquid nitrogen
15 GIEPR
30 min 60 min0 min
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.
Time after CP-H infusion, minutes
0 20 40 60 80 100100
120
140
160
Vitamin C experiment
GTNVitamin C
D
0 20 40 60 80 100100
120
140
160
SOD+GTN experiment
SOD
C
0 20 40 60 80 100
ES
R a
mp
litu
de,
mm
100
120
140
160
GTN experiment
GTN
B
0 20 40 60 80 100
ES
R a
mp
litu
de,
mm
100
120
140
160
Control experiment
A
Time after CP-H infusion, minutes
GTN
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.
ROS formation
Antioxidant system
Increase in the O2
_ production or decrease in antioxidant activity (SOD)
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. Frozen samples should be analyzed with caution due to overlapping with the
EPR signals of bioradicals.
5. 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
Prof. David G. Harrison
Prof. Kathy Griendling
Dr. Maziar Zafari
Dr. Anna Dikalova
Institute of Organic Chemistry
Novosibirsk, Russia
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 COREDivision of Cardiology, Emory University School of Medicine,
Atlanta, Georgia
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Wyche et al. (2004) Hypertension 43(6): 1246-1251.
Detection of extracellular superoxide production by neutrophils using CPH