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Sergey Dikalov Director of Free Radicals in Medicine CORE

Detection of Superoxide with Cyclic Hydroxylamines. Sergey Dikalov Director of Free Radicals in Medicine CORE Division of Cardiology, Emory University School of Medicine. CO 2 H. CO 2 H. N. N. O . OH. Detection of O 2  _ with EPR spectroscopy. 1. Direct detection. SOD. +.

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Sergey Dikalov Director of Free Radicals in Medicine CORE

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  1. Detection of Superoxide with Cyclic Hydroxylamines Sergey Dikalov Director of Free Radicals in Medicine CORE Division of Cardiology, Emory University School of Medicine

  2. CO2H CO2H N N O OH Detection of O2_ with EPR spectroscopy 1. Direct detection SOD + 1 e_ O2 O2_ H2O2 2. Spin trapping (DMPO, EMPO, DEPMPO) DMPO DMPO/OOH 35 M-1s-1 + O2_ N N+ O O- 3. Spin probes (cyclic hydroxylamines) CP-H CP + H2O2 3.2x103 M-1s-1 + O2_

  3. 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.

  4. E t O C H E t O C 2 H 2 OOH OH N OOH N O O E t O C H 2 OH N OH Problems with spin trapping of O2¯ EMPO EMPO/  OOH EMPO/  OH EMPO/ OH2 GPx E t O C 2 + N Reduction O 74 M-1s-1 slow 105 – 109 M-1s-1 O2_ SOD, Ascorbate, GSH fast 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 EMPO/  OH GPx 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.

  5. CO2CH3 CO2CH3 N N O OH 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 enablingthe hydroxylamines to compete with cellular antioxidants and react with intracellular O2_. CM-H CM 1.2X104 M-1s-1 + + O2_ H2O2 2) Stability of the reaction product. Cyclichydroxylaminesproducestablenitroxideswithamuchlongerlifetimethanradicaladducts. Hydroxylamines allow quantitative O2- detection with higher sensitivity than spin traps.

  6. 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; 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 (mM/min) Dikalov et al. Biophys.Res. Comm. 231, 701-704: 1997.

  7. Spin probe stability CP-H + 1. Fe3+ + CP  Fe2+ Inhibited by Desferal CP-H + 2. Cu2+ + CP  Cu1+ Inhibited by DTPA or DETC X CP-H + 3. H2O2 CP  There is no direct reaction Formation of ferryl species H2O2 + 4. Fe2+ Fe4+=O CP-H + 5. Fe4+=O + CP  Inhibited by DTPA or DETC Fe3+ Stabilization: Ice, metal chelators (DF, DTPA or DETC), Argon, fresh buffers (no H2O2)

  8. CO2H CO2H 3.2x103 M-1s-1 + + O2_ H2O2 CO2H CO2H N N O OH N N OH OH CO2H CO2H N N O O Relative specificity of cyclic hydroxylamines SOD ~ 2x102 M-1s-1 + + ONOO_ NO2_ Urate + + NO2 NO2_ RSH

  9. I EPR Spectra Time scans 200 75 PP-H + xanthine 50 PP-H + xanthine 100 A D 25 0 0 -25 -100 -50 -200 -75 3485 3490 3495 3500 3505 3510 3515 3520 3525 3530 3535 0 50 100 150 200 250 300 [sec] PP-H + xanthine + xanthine oxidase I 75 400 50 B 300 25 0 E 200 -25 PP-H + xanthine + xanthine oxidase 100 -50 -75 0 3485 3490 3495 3500 3505 3510 3515 3520 3525 3530 3535 [sec] 0 50 100 150 200 250 300 200 75 F 50 PP-H + xanthine + SOD + xanthine oxidase 100 C PP-H + xanthine + SOD + xanthine oxidase 25 0 0 -25 -100 -50 -200 -75 [sec] 3485 3490 3495 3500 3505 3510 3515 3520 3525 3530 3535 0 50 100 150 200 250 300 Magnetic field, G Time, sec Detection of superoxide with cyclic hydroxylamines Dikalov S.I., Dikalova A.E., Mason R.P.Arch.Biochem. Biophys.402, 218-226: 2002.

  10. 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 O2 20 nM/min, 0.5 mM PP-H 100 0 100 200 300 400 500 600 Time, sec Dikalov S. I., Dikalova A.E., Mason R.P. Arch.Biochem. Biophys.402, 218-226: 2002.

  11. Summary 1)  Advantages of cyclic hydroxylamines over nitrone spin traps are: I. High reactivity with O2 : rate constants are 103-104 M-1s-1vs 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.

  12. 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 vivoO2 detection.

  13. 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.

  14. Quantification of O2 in the membrane fractions CP,mM 1.00 M+NADPH EPR spectrum of CP 0.5mM O2• 0.75 15G M 0.50 M+NADPH+SOD PBS 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.

  15. Calculation of the rate constant of superoxide reaction with antioxidant by competition with spin probe CMH CM EPR signal CMH O2 O2 _ e- e- e- NADPH Cyt P-450 reductase MQ MQ _ Antioxidant CM, mM 0 mM 3.0 10 mM 20 mM 2.0 50 mM 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.

  16. Lipophilicity Kp=[Octanol]/[Water], PBS pH 7.4

  17. 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 NaIO4.

  18. 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 25 50 75 100 125 150 175 200 225 250 [sec] Wyche et al.(2004)Hypertension 43(6): 1246-1251.

  19. Intra- and extracellular O2 in endothelial cell (EC) treated with peroxynitrite SIN-1 O2+ NO ONOO¯ CM, mM EC treated with ONOO- eNOS uncoupled 2.5 EC eNOS EC treated with ONOO- plus L-NAME ONOO¯ BH4 2.0 EC BH2 EC+SOD EC eNOS uncoupled 1.5 PBS O2 1.0 0 Time, sec 100 200 300 400 500 600

  20. Detection of O2 production by endothelial cells. Basal production and stimulation of O2 release by mitochondria. AA – Antinamycin A, mitochondrial uncoupling agent SOD – extracellular superoxide dismutase (50 U/ml Mn-SOD)

  21. Cytochrome DEPMPO EMPO CMH 0.5 Cells 2.4±0.18 2.3±0.3 2.7±0.4 5.5±0.5 0.4 0.3 Cells+PMA 11.2±0.87 9.4±0.9 16±2.1 48.6±8.2 0.2 0.1 Detection of O2 by DEPMPO, EMPO and CMH in cultured Lymphocytes DEPMPO-OOH, mM EMPO-OOH, mM CM, mM 0.9 Cells + PMA 2.4 Cells + PMA Cells+PMA 2.0 0.6 1.6 1.2 0.3 0.8 Cells +SOD+PMA Cells + SOD + PMA Cells 0.4 Cells+SOD 0 0 0 0 100 200 300 400 500 600 [sec] 0 100 200 300 400 500 600 [sec] [sec] 0 100 200 300 400 500 600 Table 1. Detection of superoxide with cytochrome C, DEPMPO, EMPO, CMH (pmol/mln/min). Dikalov S., Wei L., Zafari M. 2005

  22. Detection of extramitochondrial O2 by PP-H in brain mitochondria (RBM) with glutamate+malate Mitochondria + O2 1000 RBM+GM+AA 750 EPR spectrum of PP Antimycin A induced O2 production PP-nitroxide, nM 15G 500 RBM+GM Basal O2 RBM+GM+AA+SOD RBM+GM+SOD 250 PPH 0 0 50 100 150 200 250 300 350 [sec] Panov A., Dikalov S., Shalbueva N. et al. J Biol Chem. 2005 Oct 21

  23. Measurements of PMA-stimulated superoxide production in rat aorta segments using CMH spin probe 37 °C 21 °C CM, mM 100% Control 1 2.5 50 ml capillary tube (Fisher) 2.0 Control+ Apocynin 2 90% 3 4 1.5 1.0 50 ml label 133% PMA 5 0.5 0 100 200 300 400 500 600 [sec] PMA+ Apocynin Tissue sample 71% 1 – Aorta + PMA 2 – Aorta (control) 14 mm 3 – Aorta + Apocycin + PMA Sealing compound EPR spectra of tissue incubated 60 min with CMH at 37 C. 4 – Aorta + Apocycin (Apocycin control) 5 – CMH only, no aorta (background) Apocynin inhibited 52% in PMA vs 10% in control.

  24. Preparation of the frozen samples for ROS measurements 1. Cut the top of the syringe. 2. Fill 200 ml buffer. 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 4B. Freeze and then fill the rest of the syringe with buffer. 5. Freeze whole sample. 0.0 0.1 0.2 0.3 Tissue or Cell suspension 0.4 0.5 300 ml 0.6 0.7 1 ml syringe P-s: buffer must have chelating agent DF-DETC or DTPA.

  25. Atrial fibrillation increased production of O2in left atrium measured using intracellular spin probe CMH and frozen samples (liquid nitrogen) CMH CM COOH COOH 1.2.104 M-1s-1 + + O2_ H2O2 N N O OH EPR signal EPR silent Left atrium Right atrium A Control D Control B E AF AF F C AF + S178 AF + S178 15 G 15 G Dudley et al. (2005) Circulation 112:1266-73.

  26. Detection of superoxide in aorta of Tg SM nox1 mice using CMH Dikalova A. et al. Circulation Circulation. 2005; 112(17): 2668-76.

  27. 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 60 min 0 min 30 min IEPR 15 G 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.

  28. 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.

  29. Antioxidant system ROS formation Increase in the O2_ production or decrease in antioxidant activity (SOD)

  30. Conclusion • Hydroxylamine spin probe should be selected based on its lipophilicity, cell permeability, stability and reactivity. • Selective inhibitors and antioxidants must be used to identify ROS. • Probes can be scanned immediately or analyzed in the frozen state. • Frozen samples should be analyzed with caution due to overlapping with the EPR signals of bioradicals. • Cyclic hydroxylamines can be usedin vivo or ex vivo for tissue analysis. • Cyclic hydroxylamines have been successfully used to assay O2 production by mitochondria, neutrophils, endothelial, and smooth muscle cells. • Cyclic hydroxylamines are capable to detect both intra- and extracellular O2-.

  31. Acknowledgments

  32. Free Radicals in Medicine CORE Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia

  33. Literature Rosen GM, Finkelstein E, Rauckman EJ. Arch Biochem Biophys. 1982:215(2):367-378. Dikalov S, Skatchkov M, Bassenge E. Biochem. Biophys. Res. Comm. 1997:231, 701-704. DikalovS, Grigor'evIA, VoinovM, Bassenge E. Biochem Biophys Res Commun 1988:248,211-215. Valgimigli L, Pedulli GF, Paolini M. FreeRadicBiolMed. 2001:31, 708-716. Dikalov S, Fink B, Skatchkov M, Bassenge E. Free Radic Biol Med. 1999:27, 170-176. Saito K, Takeshita K, Anzai K, Ozawa T. Free Radic Biol Med. 2004: 36, 517-525. Dikalov SI, Dikalova AE, Mason RP. Arch Biochem Biophys 2002:2, 218-226. Kozlov AV, Szalay L, Umar F, Fink B, Kropik K, Nohl H, Redl H, Bahrami S. Free Radic Biol Med. 2003: 34,1555-1562. Kuzkaya N, Weissmann N, Harrison DG, Dikalov S. Biochem Pharmacol. 2005:70,343-354. Dudley SC, Hoch NE, McCann LA, Honeycutt C, Diamandopoulos L, Fukai T, Harrison DG, Dikalov SI, Langberg J. Circulation 2005:112(9),1266-1273.

  34. Detection of ROS in tissue and blood following in vivo treatment with CPH • Detection of CP-radicals and NO-Hb complexes in blood. • Generation of NO and ROS in blood of control animals and animals receiving LPS. • ROS generation in control rats and LPS-treated rats. • Kozlov A.V. et al. Free Radic Biol Med. 2003; 34(12): 1555-62.

  35. Detection of extracellular superoxide production by neutrophils using CPH Wyche et al.(2004)Hypertension 43(6): 1246-1251.

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