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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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.

Detection of ROS in tissue and blood following in vivo treatment with CPH

Wyche et al. (2004) Hypertension 43(6): 1246-1251.

Detection of extracellular superoxide production by neutrophils using CPH