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DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED NITRONES Jeannette V á squez Vivar, Ph.D.

DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED NITRONES Jeannette V á squez Vivar, Ph.D. Medical College of Wisconsin Milwaukee Wisconsin 53226 jvvivar@mcw.edu. Outline. Chemical structures and names of superoxide spin traps Superoxide spin trapping with cyclic nitrones

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DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED NITRONES Jeannette V á squez Vivar, Ph.D.

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  1. DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED NITRONES Jeannette Vásquez Vivar, Ph.D. Medical College of Wisconsin Milwaukee Wisconsin 53226 jvvivar@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 O2O2•– + O2•–H2O2 + O2 2H+ NO GSH/GPx XOD Xanthine Uric Acid ONOO- GSSG CO2 RSH Y •C(O)NH2 Cys-SH Y• RS• CO3•– Cys-SOH

  4. Selection of the Spin Trap • Stable and easy to purify • Radical adduct is persistent • Radical adducts present distinctive EPR spectra • EPR spectra is simple

  5. Nitrones Commonly Used for Detection of Superoxide • DMPO 5,5-Dimethyl-1-pyrroline-N-oxide2,2-Dimethyl-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-pyrrole-1-oxide 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide • BMPO 5-Tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide

  6. 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

  7. g= 2.018 20 G O O OOH OH (Et (Et O O ) ) P P 2 2 H H C C H H N N . . 3 3 O O Application I: Detection of Hydroxyl radical in Superoxide-driven Reactions Aconitase [4Fe-4S]2+ (active) Aconitase [3Fe-4S]1+ (inactive) Vásquez-Vivar et al. J Biol Chem 2000, 275:14064

  8. EPR Spin Trapping Detection of Superoxide with DEPMPO • Characteristics: • Unique EPR spectrum: cis- and trans-DEPMPO-OOH (1:9) and • conformers exchange • Formation of persistent superoxide • DEPMPO-OOH loss of signal • adduct (t1/2~15 min) is not followed by DEPMPO-OH • appearance trans-DEPMPO cis-DEPMPO

  9. EPR Spin Trapping Detection of Superoxide with DEPMPO Limitations: • Substitution with 5-methyl group with 31P (I=1/2 and large hyperfine • coupling constant ~49 G) decreases sensitivity ~0.2 nmol superoxide • Purification is difficult

  10. 10 G 10 G EPR Spin Trapping Detection of Superoxide with 5-Ethoxycarbonyl-5-Methyl-1-Pyrroline N-oxide (EMPO) and 15N-EMPO O2•– O2•– (15N) I=½ (14N) I=1 Olive et al. Free Radical Biol Med 1999, 28: 403 Zhang H et al.FEBS Lett 2000, 473: 58

  11. 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-EMPO<0.05 nmoles superoxide>14N-EMPO • Limitation: • Purification • t½< DEPMPO-OOH

  12. HEME FMN BH4 FAD L-Arg NADPH 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

  13. Ca2+/CaM L-Arg (0.1 mM) L-NAME (1.0 mM) 7,8-BH2 (0.1 mM) L-Arg/BH4 (2 µM) L-Arginine, L-NAME and BH4 Effects on Superoxide Release from eNOS Vásquez-Vivar et al. Circulation 2000, 102: II-63

  14. ) -1 140 14 mg protein 12 120 -1 10 100 •NO nmol min 8 80 6 60 -1 40 4 EMPO-OOH (nmoles min mg protein 2 20 0 0 -1 0.0001 0.001 0.01 0.1 1 10 100 BH ( m M) 4 +L-Arginine -L-Arginine 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, 362:733

  15. O2•– EPR Spin Trapping Detection of Superoxide with BMPO BH4-free nNOS + BH4 (10 nM) Zhang et al Free Radical Biol Med 2001, 31:599 Porter et al. Chem Res Toxicol 2005, 18:864 • Characteristics: • More persistent superoxide radical adducts • BMPO-OOH BMPO-OH • More sensitive measurements ~ 0.01 nmoles superoxide • Solid readily purified by recrystallization in MeOH

  16. O O O O O O H H H H H H 3 3 3 H H H 3 3 3 N N N O O O H H H 5 5 5 H H H 5 5 5 O O O C C C O O O R R R 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,R6=CH3 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

  17. H 3 H 3 H 5 H 5 10 G 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, 1:45

  18. 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-CD96.0 • Protection against reduction (Ascorbate, GSH, GSH/GPx) • EMPO>DEPMPO>DMPO-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, 45:1043

  19. Quantification of Superoxide Using Spin Trapping Methodology • Considerations: • Spin trapping is a kinetic method • Calibration curve • Baseline • Simulation and identification of radical adduct species

  20. 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,

  21. 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. #Scans=100, time<4 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, 35:1149

  22. 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

  23. 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

  24. 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 70:1198-1203 • Clement et al. (2003) Deuterated analogues of the free radical trap DEPMPO: synthesis and EPR studies. Org Biomol Chem 1:1591-1597 • Frejaville et al. (1995) 5-(Diethoxyphosphory)l-5methyl-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 35:1149-1157 • Karoui & Tordo (2004) ESR-spin trapping in the presence of cyclodextrins. Tetrahedron Lett. 45:1043-1045 • 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

  25. References-II • Porter et al. (2005) Reductive activation of Cr(VI) by nitric oxide synthase. Chem Res Toxicol 18:864-843 • Roubaud et al. (1997) Quantitative measurement of superoxide generation using the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide. 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 275:14064-14069 • Vásquez-Vivar et al. (2000) EPR spin trapping of superoxide from nitric oxide synthaseAnalusis (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 362:733-739 • 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 31:599-606 • Public EPR Software and Data Base: http://epr.niehs.nih.gov/pest.html

  26. 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

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