DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED NITRONES

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DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED
NITRONES Jeannette Vásquez Vivar,
Ph.D. Medical College of Wisconsin Milwaukee,
Wisconsin 53226 jvvivar_at_mcw.edu
2
Outline

• Chemical structures and names of superoxide spin
  traps
• Superoxide spin trapping with cyclic nitrones
• Experimental considerations and applications
• Quantification of superoxide from radical adduct
  data

3
Sources of Superoxide and other Reactive Species
OH
NO2
HOCl HOBr
Fe2
BH4-deficient NOS NADPH Oxidase Mitochondria
NO2
Cl-/Br-
Aconitase
MPO
Drug metabolism
O2 O2 O2 H2O2 O2
2H
NO
GSH/GPx
XOD
Xanthine
ONOO-
Uric Acid
GSSG
CO2
RSH
Y
C(O)NH2
Cys-SH
CO3
Y RS
Cys-SOH
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Selection of the Spin Trap

• Stable and easy to purify
• Radical adduct is persistent
• Radical adducts present distinctive EPR spectra
• EPR spectra is simple

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Nitrones Commonly Used for Detection of Superoxide

• DMPO
• 5,5-Dimethyl-1-pyrroline-N-oxide2,2-Dimethy
  l-3,4-dihydro-2H-pyrrole 1-oxide
• DEPMPO
• 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-
  N-oxide2-Diethylphosphono-2-methyl-3,4-dihydro-2H
  -pyrrole 1-oxide(2-Methyl-3,4-dihydro-1-oxide-2H-
  pyrrol-2-yl) diethylphosphonate
• EMPO
• 2-Ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyr
  role-1-oxide
• 5-ethoxycarbonyl-5-methyl-1-pyrroline
  N-oxide
• BMPO
• 5-Tert-butoxycarbonyl-5-methyl-1-pyrroline
  N-oxide

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EPR Spin Trapping Detection of Superoxide with
5-Diethoxyphosphoryl-5-Methyl-1-Pyrroline
N-oxide (DEPMPO)
2 min 15 min 20 min
O2
DEPMPO-OOH
SOD
20 G
DEPMPO-OH
Frejaville et al. J Med Chem 1995, 38 258
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Application I Detection of Hydroxyl radical in
Superoxide-driven Reactions
Aconitase 4Fe-4S2 (active)
Aconitase 3Fe-4S1 (inactive)
Vásquez-Vivar et al. J Biol Chem 2000, 27514064
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EPR Spin Trapping Detection of Superoxide with
DEPMPO

• Characteristics
• Unique EPR spectrum cis- and trans-DEPMPO-OOH
  (19) and
  
• conformers exchange
• Formation of persistent superoxide
  DEPMPO-OOH loss of signal
• adduct (t1/215 min)
  is not followed by DEPMPO-OH
• 
  appearance

trans-DEPMPO
cis-DEPMPO
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EPR Spin Trapping Detection of Superoxide with
DEPMPO
Limitations

• Substitution with 5-methyl group with 31P (I1/2
  and large hyperfine
• coupling constant 49 G) decreases
  sensitivity 0.2 nmol superoxide
• Purification is difficult

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EPR Spin Trapping Detection of Superoxide with
5-Ethoxycarbonyl-5-Methyl-1-Pyrroline N-oxide
(EMPO) and 15N-EMPO
O2
O2
(15N) I½
(14N) I1
Olive et al. Free Radical Biol Med 1999, 28
403 Zhang H et al.FEBS Lett 2000, 473 58
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EPR Spin Trapping Detection of Superoxide with
EMPO

• Characteristics
• Distinctive EPR spectra EMPO-OOH composite of
  two conformers
• EMPO-OOH is more persistent than DMPO-OOH
• EMPO-OOH EMPO-OH
• Sensitivity 15N-EMPOlt0.05 nmoles
  superoxidegt14N-EMPO

• Limitation
• Purification
• t½lt DEPMPO-OOH

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Application II. Quantification of Superoxide from
Nitric Oxide Synthase
O2-
Electron acceptor Reduced

• Electron acceptors such as
• cytochrome c, lucigenin and NBT are
• directly reduced by NOS
• In the case of redox-active compounds,
• this reaction increases superoxide
• generation
• BH4 reduces cytochrome c
• Spin trapping is the ideal technique to detect
  superoxide from NOS

Oxygenase Domain
Reductase Domain
Vásquez-Vivar et al. Methods in Enzymology 1999,
301 169
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L-Arginine, L-NAME and BH4 Effects on Superoxide
Release from eNOS
Vásquez-Vivar et al. Circulation 2000, 102 II-63
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Tetrahydrobiopterin Coordinates the Inhibition of
Superoxide and the Stimulation of NO Formation
from eNOS
97.7 nmoles O2 min-1 mg protein-1 BH4 IC50?
0.15 µM
Vásquez-Vivar et al. Biochem J 2002, 362733
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EPR Spin Trapping Detection of Superoxide with
BMPO
BH4-free nNOS
BH4 (10 nM)
Zhang et al Free Radical Biol Med 2001, 31599
Porter et al. Chem Res Toxicol 2005, 18864

• Characteristics
• More persistent superoxide radical adducts
• BMPO-OOH BMPO-OH
• More sensitive measurements 0.01 nmoles
  superoxide
• Solid readily purified by recrystallization in
  MeOH

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Superoxide Spin Trapping in the Presence of
ß-Cyclodextrins
Ramdom-ß-cyclodextrin (RM-ß-CD) R2, R3, R6 H
and CH3
Dimethyl-ß-cyclodextrin (DM-ß-CD) R2,R6CH3 R3
H
Bardelang et al. J Phys Chem B 2005, 109 10521
Karoui et al. Chem Commun 2002, 24 3030
Hydrophobic core
6.5 A
6 A
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Superoxide Spin Trapping with BMPO in
DM-ß-Cyclodextrin Containing Solutions
Control
6 mM
12 mM
KNITROXIDE 660 M-1 KNITRONE 230 M-1
25 mM
100 mM
BMPO-OOH / DM-b-CD
Karoui et al. EPR-2005 Abstracts 2005, 145
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Properties of the Superoxide Radical Adduct and
in ß-Cyclodextrin Inclusion Complex

• Characteristics
• Enhanced persistence
• Superoxide Radical Adduct t½
  (min) Inclusion complex t½ (min)
• DMPO-OOH 0.8
  DMPO-OOH/RM-ß-CD 5.9
• EMPO-OOH 4.6
  EMPO-OOH/ RM-ß-CD 38.0
• DEPMPO-OOH 14.0
  DEPMPO-OOH/ RM-ß-CD 96.0
• Protection against reduction (Ascorbate, GSH,
  GSH/GPx)
• EMPOgtDEPMPOgtDMPO-OOH
• Limitations
• Changes in hyperfine coupling constant of the
  radical adduct in inclusion complex

Bardelang et al. J Phys Chem B 2005, 109 10521
Karoui Tordo Tetrahedron Lett. 2004, 451043
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Quantification of Superoxide Using Spin Trapping
Methodology

• Considerations
• Spin trapping is a kinetic method
• Calibration curve
• Baseline
• Simulation and identification of radical adduct
  species

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Superoxide Spin Trapping Kinetic Analysis
BIOLOGICAL PROCESS (E)
P
kd
k1
k2
k3
Nitrone O2 Nitroxide-OOH Other
Under steady-state concentrations of superoxide
and saturating concentrations of the nitrone, then
thus,
and,
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Quantification of Superoxide Using Spin Trapping
Methodology

• Data acquisition
• i. Static scanning of spectra- 2D data set
• ii. Rapid scan of spectra- 3D data set

Kinetics of DMPO-OOH formation in
incubations containing DMPO (10 mM), Xanthine
(0.5 mM), Xanthine Oxidase (50 mU/ml) in
phosphate buffer 50 mM, pH 7.4 and DTPA 0.1
mM. Scans100, timelt4 s EPR Spectra after
SVD (identification total components and
isolation of the main component) and Spectral
Analysis
Keszler et al. Free Radical Biol Med 2003, 351149
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Calculating Initial Rates of Superoxide Radical
Adduct Formation
B. Standard reactions- known rates of superoxide
flux (µM/min) - Xanthine Oxidase
and hypoxanthine, xanthine or acetaldehyde -
Spin trap concentration (10-100 mM), buffers
(concentration, pH) C. Simulation and
integration - Corrects baseline -
Identify major component of analysis D.
Calculating initial rates of superoxide radical
formation - Use results with standard reaction
to calculate superoxide concentration
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Superoxide Radical Adduct Data Analysis
Simulation
- Simulation rationale correction baseline
drifting and analysis of one species only.
Public EPR Software (WinSim)
Table I. EPR Parameters of Superoxide Radical
Adducts
Radical Adduct Conformers
Hyperfine coupling constant (G)
()
aN aHß aH? aP
aH DMPO-OOH 67
14.15 11.34 1.58 -
- 33
14.09 11.78 0.17 14N
EMPO-OOH 54 12.8
12.1 0.15 - -
46
12.8 8.6 - 15N EMPO-OOH
55 17.9 12.0 0.3
- -
45 17.8 8.7
- BMPO-OOH 55
13.4 12.1 - -
- 45
13.37 9.42
DEPMPO-OOH 50 13.4
11.9 0.8 52.5 0.4
50 13.2
10.3 0.9 48.5 0.43
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References-I

• Bardelang et al. (2005) Inclusion complexes of
  PBN-type nitrones spin traps and their superoxide
  spin adducts with cyclodextrin derivatives
  parallel determination of the association
  constants by NMR-titrations and 2D-EPR
  simulations. J Phys Chem B 109 10521-10530
• Clement et al. (2005) Assignment of the EPR
  spectrum of 5,5-dimethyl-1-pyrroline N-oxide
  (DMPO) superoxide spin adduct. J Org Chem
  701198-1203
• Clement et al. (2003) Deuterated analogues of the
  free radical trap DEPMPO synthesis and EPR
  studies. Org Biomol Chem 11591-1597
• Frejaville et al. (1995) 5-(Diethoxyphosphory)l-5m
  ethyl-1-pyrroline N-oxide A new efficient
  phosphorylated nitrone for the in vitro and in
  vivo spin trapping of oxygen centered radicals. J
  Med Chem 38 258-265
• Keszler et al. (2003) Comparative investigation
  of superoxide trapping by cyclic nitrone spin
  traps the use of singular value decomposition
  and multiple linear regression analysis. Free
  Radical Biol Med 351149-1157
• Karoui Tordo (2004) ESR-spin trapping in the
  presence of cyclodextrins. Tetrahedron Lett.
  451043-1045
• Karoui et al. (2002) Spin trapping of superoxide
  in the presence of ß- cyclodextrins. Chem Commun
  24 3030-3031
• Olive et al. (1999) 2-Ethoxycarbonyl-2-methyl-3,4
  -dihydro-2H-pyrrole-1-oxide evaluation of the
  spin trapping properties Free Radical Biol Med
  28 403-408

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References-II

• Porter et al. (2005) Reductive activation of
  Cr(VI) by nitric oxide synthase. Chem Res Toxicol
  18864-843
• Roubaud et al. (1997) Quantitative measurement of
  superoxide generation using the spin trap
  5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxid
  e. Anal Biochem 247 404-411
• Vásquez-Vivar et al. (1999) ESR Spin-trapping
  detection of superoxide generated by neuronal
  nitric oxide synthase. In Methods in Enzymology
  301 169-177.
• Vásquez-Vivar et al. (2000) Mitochondrial
  aconitase is a source of hydroxyl radical. J Biol
  Chem 27514064-14069
• Vásquez-Vivar et al. (2000) EPR spin trapping of
  superoxide from nitric oxide synthase Analusis
  (Eur J Anal Chem) 28 487-492
• Vásquez-Vivar et al. (2000) BH4/BH2 ratio but not
  ascorbate controls superoxide and nitric oxide
  generation by eNOS. Circulation 102 II-63
• Vasquez-Vivar et al. (2002) The ratio between
  tetrahydrobiopterin and oxidized
  tetrahydrobiopterin analogues controls superoxide
  release from endothelial nitric oxide synthase
  an EPR spin trapping study. Biochem J
  362733-739
• Zhang H et al. (2000) Detection of superoxide
  anion using an isotopically labeled nitrone spin
  trap potential biological applications. FEBS
  Lett 473 58-62
• Zhao et al. (2001) Synthesis and biochemical
  applications of a solid cyclic nitrone spin trap
  a relatively superior spin trap for detecting
  superoxide anions and glutathiyl radicals. Free
  Radical Biol Med 31599-606
• Public EPR Software and Data Base
  http//epr.niehs.nih.gov/pest.html

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Acknowledgements

• B. Kalyanaraman
• Joy Joseph
• Hakim Karoui
• Neil Hogg
• Hao Zhang
• Hongtao Zhao
• Medical College of Wisconsin
• Free Radical Research Center
• National Biomedical EPR Center