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Reaction of Peroxynitrite and Uric Acid Studied by ESR Spin Trapping and Mass Spectrometry

Permanent Link: http://ufdc.ufl.edu/UFE0022880/00001

Material Information

Title: Reaction of Peroxynitrite and Uric Acid Studied by ESR Spin Trapping and Mass Spectrometry Free Radical Formation and Product Identification
Physical Description: 1 online resource (120 p.)
Language: english
Creator: Imaram, Witcha
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: acid, adduct, electron, esr, free, hydrogen, mass, peroxynitrite, radical, resonance, spectrometry, spin, trapping, uric
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Uric acid is the most abundant antioxidant in plasma. However, under conditions of elevated uric acid levels and oxidative stress, it becomes a pro-oxidant and causes endothelial dysfunction. It is hypothesized that the generation of reactive intermediates such as free radicals from the reaction of peroxynitrite and other oxidants mediate the pathological pro-oxidant properties of uric acid. Peroxynitrite is a reactive oxidant produced in vivo in response to oxidative and other stress by the diffusion-limited reaction of nitric oxide and superoxide. Our research is focused on the identification of free radical metabolites of uric acid formed from its reaction with peroxynitrite. Our experimental approach included the electron spin resonance (ESR) spin trapping of the radical generated from the reaction between uric acid and peroxynitrite at pH 7.4. Using PBN (N-tert-butyl-alpha-phenylnitrone) as the spin trapping agent, a six-line ESR spectrum was obtained and its hyperfine coupling constants, a(N) = 15.6 G; and a(H) = 3.6 G, corresponded to two carbon-based radicals. Further structural identifications of the PBN-radical adducts were carried out using liquid chromatography-mass spectrometry (LC-MS). After comparison with the control reactions, we could identify two molecules, corresponding to the fragment ions of m/z 352 and 223, respectively. The PBN-triuretcarbonyl radical adduct was characterized for m/z 352 and the latter was identified as a PBN-aminocarbonyl radical adduct. The pH dependence study of the reaction between uric acid and peroxynitrite revealed the formation of hydrogen adduct at high pH and could be observed even without urate. Its formation was proposed to undergo the inverted spin trapping mechanism, in which the spin trap was initially oxidized rather than the antioxidant substrate, and followed by electron transfer. We extended our studies to investigate the effect of methyl substitution at various nitrogen positions on product and radical formation. No ESR signal was observed when conducting the reactions with N-7 methylated uric acids in phosphate buffer pH 7.4. Moreover, the reactions were purposely conducted in methanol to trap the reaction intermediate. Various products have been identified by LC-MS, and those products indicated that a common intermediate in various urate oxidation conditions, dehydroisouric acid, was formed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Witcha Imaram.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Angerhofer, Alexander.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022880:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022880/00001

Material Information

Title: Reaction of Peroxynitrite and Uric Acid Studied by ESR Spin Trapping and Mass Spectrometry Free Radical Formation and Product Identification
Physical Description: 1 online resource (120 p.)
Language: english
Creator: Imaram, Witcha
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: acid, adduct, electron, esr, free, hydrogen, mass, peroxynitrite, radical, resonance, spectrometry, spin, trapping, uric
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Uric acid is the most abundant antioxidant in plasma. However, under conditions of elevated uric acid levels and oxidative stress, it becomes a pro-oxidant and causes endothelial dysfunction. It is hypothesized that the generation of reactive intermediates such as free radicals from the reaction of peroxynitrite and other oxidants mediate the pathological pro-oxidant properties of uric acid. Peroxynitrite is a reactive oxidant produced in vivo in response to oxidative and other stress by the diffusion-limited reaction of nitric oxide and superoxide. Our research is focused on the identification of free radical metabolites of uric acid formed from its reaction with peroxynitrite. Our experimental approach included the electron spin resonance (ESR) spin trapping of the radical generated from the reaction between uric acid and peroxynitrite at pH 7.4. Using PBN (N-tert-butyl-alpha-phenylnitrone) as the spin trapping agent, a six-line ESR spectrum was obtained and its hyperfine coupling constants, a(N) = 15.6 G; and a(H) = 3.6 G, corresponded to two carbon-based radicals. Further structural identifications of the PBN-radical adducts were carried out using liquid chromatography-mass spectrometry (LC-MS). After comparison with the control reactions, we could identify two molecules, corresponding to the fragment ions of m/z 352 and 223, respectively. The PBN-triuretcarbonyl radical adduct was characterized for m/z 352 and the latter was identified as a PBN-aminocarbonyl radical adduct. The pH dependence study of the reaction between uric acid and peroxynitrite revealed the formation of hydrogen adduct at high pH and could be observed even without urate. Its formation was proposed to undergo the inverted spin trapping mechanism, in which the spin trap was initially oxidized rather than the antioxidant substrate, and followed by electron transfer. We extended our studies to investigate the effect of methyl substitution at various nitrogen positions on product and radical formation. No ESR signal was observed when conducting the reactions with N-7 methylated uric acids in phosphate buffer pH 7.4. Moreover, the reactions were purposely conducted in methanol to trap the reaction intermediate. Various products have been identified by LC-MS, and those products indicated that a common intermediate in various urate oxidation conditions, dehydroisouric acid, was formed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Witcha Imaram.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Angerhofer, Alexander.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022880:00001


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1 REACTION OF PEROXYNITRITE AND URIC ACID STUDIED BY ESR SPIN TRAPPING AND MASS SPECTROMETRY: FREE RADICAL FORMATION AND PRODUCT IDENTIFICATION By WITCHA IMARAM A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSI TY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Witcha Imaram

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3 To my parents, to my brother, and to my grandparents

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4 AC KNOWLEDGMENTS I particularly owe my deepest gratitude to my advisor Dr. Alexander Angerhofer for his encouragement, valuable guidance and support throughout this study. I would like to express my sincere gratitude to Dr. Richard J. Johnson for inspiring me to be involved in the exciting field of biochemistry, in uric acid study. I am pleased to thank Dr. George N. Henderson for making the MS work a reality, as well as his invaluable suggestions and discussion. I would like to thank my doctoral dissertation committee members (Dr. William R. Dolbier, Dr. Lisa McElwee White, and Dr. Kirk S. Schanze) for their input and constructive comments. This study was supported by grants from Gatorade and the University of Florida through an opportunity grant from its Divi sion of Sponsored Research, and by the University of Florida Chemistry Department I am grateful for the graduate stipend and tuition sponsored by the Development and Promotion of Science and Technology Talent Project (DPST) scholarship from t he Institute for the Promotion of Teaching Science and Technology, Thailand I would also like to take this opportunity to further express my appreciation to my uric acid project collaborators Dr. Christine Gersch, Dr. Sergiu P. Palii, and Dr. Kyung Mee Kim for their i nvaluable training and guidance. I am also thankful to Dr. Yuri Y. Sautin for his helpful suggestion. In addition to Dr. George N. Henderson and Dr. Kyung Mee Kim, I owe a debt of gratitude to my MS collaborators Dr. David H. Powell and Dr. Jodie V. Johnson of the Mass Spectrometry Services at the University of Florida Chemistry Department. I am grateful to my colleagues and friends Dimitri Dascier Fabrizio Guzzetta, Dooho Park Vijay B. Krishna, Jirapat Jangjamras Yun Wang, Suwussa Bamrungsap and Pattara porn Vanachayangkul for their help, suggestion, and friendship.

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5 I thank Dr. Ben Smith, Graduate Coordinator and chemistry departmental staff members Lori Clark, Beth Douglas, and Vivian Thompson for their help and advice. I gratefully acknowledge Rich Athe y for his friendship and critical reading of this dissertation. Finally, I would like to acknowledge my parents who have always supported, encouraged, and inspired me to complete my dream.

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6 TABLE OF CONTENTS page ACKNOWLE DGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 9 LIST OF FIGURES ............................................................................................................................ 10 ABSTRACT ........................................................................................................................................ 14 CHAPTER 1 INTRODUCTION ....................................................................................................................... 16 General Overview of Electron Spin Resonance Spin Trapping ............................................... 16 Uric Acid ...................................................................................................................................... 18 Generation and Degradation ............................................................................................... 18 Uric Acid: The Oxidant -Antioxidant Paradox ................................................................... 20 Peroxynitrite ................................................................................................................................ 22 Peroxynitrite vs. Uric Acid ......................................................................................................... 25 Research Objectives .................................................................................................................... 26 2 RADICAL FORMATION FROM THE REACTION BETWEEN URIC ACID AND PEROXYNITRITE ..................................................................................................................... 28 Introduction ................................................................................................................................. 28 Materials and Methods ................................................................................................................ 29 Chemicals ............................................................................................................................. 29 ESR Experiments ................................................................................................................. 29 Sample Preparation for Liquid Chromatography -Mass Spectrometry ............................. 29 Liquid Chromatography -Mass Spectrometry Analysis ..................................................... 29 Ful lscan liquid chromatography -mass spectrometry. ................................................ 30 Liquid chromatography-mass spectrometry tandem mass spectrometry analysis. ..................................................................................................................... 30 Results .......................................................................................................................................... 30 Electron Spin Resonance Spin Trapping ............................................................................ 30 Product Identification of the Radical Adducts by Liquid Chromatography -Mass Spectrometry Analysis ..................................................................................................... 35 Discussion .................................................................................................................................... 36 3 REACTIONS OF PEROXYNITRITE WITH MONO DI AND TRI METHYLURIC ACIDS STUDIED BY LIQUID CHROMATOGRAPHY MASS SPECTROMETRY AND ELECTRON SPIN RESONANCE SPECTROSCOPY ................................................. 46 Introduction ................................................................................................................................. 46 Materials and Methods ................................................................................................................ 47

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7 Chemicals. ............................................................................................................................ 47 ESR Spin Trapping Experiments ........................................................................................ 48 Product Identification of t he Reactions Conducted in Phosphate Buffer ......................... 49 Product Identification of the Reactions Conducted in Methanol. ..................................... 50 Results .......................................................................................................................................... 52 Electron Spin Resonance Spin Trapping. ........................................................................... 52 Liquid Chromatography -Mass Spectrometry Analysis of the Reactions Conducted in Phosphate Bu ffer. ........................................................................................................ 54 Liquid Chromatography -Mass Spectrometry Analysis of the Reactions Conducted in Methanol. ...................................................................................................................... 55 Discussion .................................................................................................................................... 62 Reactions in Phosphate Buffer. ........................................................................................... 62 Reactions in Methanol. ........................................................................................................ 63 4 ESR SPIN TRAPPI NG OF THE REACTION BETWEEN URIC ACID AND PEROXYNITRITE: THE HYDROGEN ADDUCT ................................................................ 70 Introduction ................................................................................................................................. 70 Materials and Methods ................................................................................................................ 70 Chemicals. ............................................................................................................................ 70 pH Dependence Experiments on Urate Peroxynitrite Reactions. ..................................... 70 Effect of Spin Trapping Agents on the Hydrogen Adduct Formation ............................. 71 Electron Spin Resonance Parameters ................................................................................. 71 Results .......................................................................................................................................... 71 pH Dependence Studies on Urate-Peroxynitrite Reactions .............................................. 71 Electron Spin Resonance Spin Trapping by PBN and POBN .......................................... 72 Discussion .................................................................................................................................... 76 5 CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK ......................................... 81 Conclusions ................................................................................................................................. 81 Suggestions for Future Work ...................................................................................................... 82 APPENDIX A LC CHROMATOGRAMS AND ELECTROSPRAY MASS SPECTRA OBTAINED FROM THE REACTIONS OF VARIOUS METHYLATED URIC ACIDS WITH PEROXYNITRITE CONDUCTED IN PHOSPHATE BUFFER ........................................... 84 B LC CHROMATOGRAMS OBTAINED FROM THE REACTIONS OF VARIOUS METHYLATED URIC ACIDS WITH PEROXYNITRITE CONDUCTED IN METHANOL ............................................................................................................................... 91 C FRAGMENTATION PATTERNS OF DIMETHOXYDEHYDROURIC ACIDS ................ 98

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8 D EFFECT OF DIVALENT METAL IONS ON THE REACTION BETWEEN URIC ACID AN D PEROXYNITRITE .............................................................................................. 107 Introduction ............................................................................................................................... 107 Materials and Methods .............................................................................................................. 107 Chemicals ........................................................................................................................... 107 Electron Paramagnetic Resonance Parameters ................................................................ 107 Reaction Mixtures .............................................................................................................. 107 Results and Discussion ............................................................................................................. 108 LIST OF REFERENCES ................................................................................................................. 112 BIOGRAPHICAL SKETCH ........................................................................................................... 120

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9 LI ST OF TABLES Table page 3 1 Hyperfine coupling constants a (Gauss) of PBN radical adducts from the reaction between uric acid and methylated uric acids with peroxynitrite at pH 7.4, and their relative ESR intensities compared to uric acid. .................................................................... 53 3 2 Reaction percent conversions and product yields of uric acids with one equivalent of peroxynitrite based on LC -MS analysis of the reaction products. ...................................... 55

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10 LIST OF FIGURES Figure page 1 1 Examples of nitrone spin trapping agents. ............................................................................ 18 1 2 O xidative reaction of urate by xanthine oxidase (XO) ....................................................... 21 1 3 Scheme summarized the chemistry of peroxynitrite ............................................................ 23 1 4 Geometrical isomers of peroxynitrite. .................................................................................. 23 2 1 ESR spectra of PBN radical adducts ..................................................................................... 31 2 2 Effect of urate concentration o n the production yield of the PBN radical adduct. ............ 32 2 3 Effect of peroxynitrite concentration on the production yield of the PBN -radical adduct ...................................................................................................................................... 33 2 4 Effect of pH on the production yield of the PBN radical adduct ........................................ 34 2 5 Effect of CO2 on the production yield of the PBN radical adduct ..................................... 35 2 6 LC MS study of the reaction between urate and peroxynitrite in phosphate buffer pH 7.4. ........................................................................................................................................... 37 2 7 MS/MS spectrum of the PBN aminocarbonyl radical adduct. ............................................ 38 2 8 MS/MS spectrum of the PBN -triuretcarbonyl radical adduct. ............................................ 38 2 9 Fragmentation pattern of the hydroxylamine form of PBN amin ocarbonyl radical adduct. ..................................................................................................................................... 40 2 10 Fragmentation pattern of the hydroxylamine form of PBN triuretcarbonyl radical adduct. ..................................................................................................................................... 41 2 11 S tructures of PBN -radical adducts of aminocarbonyl radical 2 and triuretcarbonyl radical 3 .................................................................................................................................. 42 2 12 Proposed radical formation mechanism of the reaction between urate and peroxynitrite. ........................................................................................................................... 45 3 1 Structures of uric acid, monomethyluric acid, dimethyluric acid, and trimethyluric acid .......................................................................................................................................... 47 3 2 ESR spectra of the PBN radical adducts from the reaction between uric acid and its methyl derivatives with peroxynitrite. .................................................................................. 52

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11 3 3 LC MS study of the reaction between uric acid and peroxynitrite in phosphate buffer pH 7.4.. .................................................................................................................................... 54 3 4 Proposed structures of the reaction products obtained from the reaction between uric acid (or various methylated uric acid analogues) and peroxynitrite. .................................. 56 3 5 LC MS study of the reaction between uric acid and peroxynitrite in the presence of methanol. ................................................................................................................................. 58 3 6 LC MS study of the reaction between uric acid and per oxynitrite in the presence of d4-methanol. ............................................................................................................................ 59 3 7 F ragmentation pattern of uric acid glycol dimethyl ether 3a. ............................................. 60 3 8 F ragmentat ion pattern of uric acid glycol di -d3-methyl ether 3a. ....................................... 61 3 10 S teric hindrance between the methyl group of triuret moiety and the phenyl group of PBN prevents the PBN radical adduct formation. ............................................................... 63 3 11 P roposed mechanism of the reaction between urate or methylurate derivatives with no methyl group at postion 3 and peroxynitrite, leading to the formation of various products. .................................................................................................................................. 66 3 12 P roposed mechanism of the reaction between methylurate derivatives with a methyl group at position 3 and peroxynitrite, leading to the formation of various products.. ....... 67 3 13 P roposed mechanism of the formation of 9 -methylurate -peroxynitrite adduct. ................ 68 4 1 pH dependence study of the urate -peroxynitrite reaction in Tri s -buffer. ........................... 72 4 2 ESR spectrum of the PBN radical adduct at pH 7.4. ........................................................... 73 4 3 ESR spectrum of the POBN radical adduct at pH 7.4. ........................................................ 74 4 4 ESR spectrum of the PBN radical adduct at pH 12 ............................................................. 74 4 5 ESR spectrum of the POBN radical adduct at pH 12. ......................................................... 75 4 6 ESR spectra of the control reactions (without uric acid). .................................................... 75 4 7 Mechanism scheme shows the possible pathway of the PBN H adduct formation. .......... 79 4 8 Resonance stabilization of POBN and PBN radical cations. .............................................. 80 A 1 LC MS study of the reaction between 1 -methyluric acid and peroxynitrite in phosphate buffer pH 7.4. ........................................................................................................ 84 A 2 LC MS study of the reaction between 9 -methyluric acid and peroxynitrite in phosphate buffer pH 7.4.. ...................................................................................................... 85

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12 A 3 LC MS study of the reaction between 1,3 dimethyluric acid and peroxynitrite in phosphate buffer pH 7.4. ........................................................................................................ 86 A 4 LC MS study of the reaction between 1,7 dimethyluri c acid and peroxynitrite in phosphate buffer pH 7.4. ........................................................................................................ 87 A 5 LC MS study of the reaction between 1,9 dimethyluric acid and peroxynitrite in phosphate buffer pH 7.4. ........................................................................................................ 88 A 6 LC MS study of the reaction between 3,7 dimethyluric acid and peroxynitrite in phosphate buffer pH 7.4. ........................................................................................................ 89 A 7 LC MS study of the reaction between 1, 3,7 trimethyluric acid and peroxynitrite in phosphate buffer pH 7.4. ........................................................................................................ 90 B1 LC chromatogram of 10 mM 1 -methylurate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate bu ffer solution at pH 7.4. ...................................... 91 B2 LC chromatogram of 10 mM 9 -methylurate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4. ...................................... 92 B3 LC chromatogram of 10 mM 1,3 -dimethylurate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4. ............................. 93 B4 LC chromatogram of 10 mM 1,7 -dimethylurate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4. ............................. 94 B5 LC chromatogram of 10 mM 1,9 -dimethylurate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4. ............................. 95 B6 LC chromatogram of 10 mM 3,7 -dimethylurate treated with 10 mM peroxy nitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4. ............................. 96 B7 LC chromatogram of 10 mM 1,3,7 trimethylurate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueo us phosphate buffer solution at pH 7.4. ......................... 97 C1 Fragmentation pattern of 1-methyluric acid glycol dimethyl ether 3b .............................. 98 C2 Fragm entation pattern of 1-methyluric acid glycol di d3-methyl ether 3b ........................ 99 C3 Fragmentation pattern of 9-methyluric acid glycol dimethyl ether 3c ............................ 100 C4 Fragmentation pattern of 9-methyluric acid glycol di d3-methyl ether 3c ...................... 101 C5 Fragmentation pattern of 1,3-dimethyluric acid glycol di -d3-methyl ether 3d ............... 102 C6 Fragmentation pattern of 1,7-dimethyluric acid glycol dimethyl ether 3e ...................... 103 C7 Fragmentation pattern of 1,7-dimethyluric acid gl ycol di -d3-methyl ether 3e ................ 104

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13 C8 Fragmentation pattern of 1,9-dimethyluric acid glycol dimethyl ether 3f ....................... 105 C9 Fragmentati on pattern of 1,9-dimethyluric acid glycol di -d3-methyl ether 3f ................ 106 D 1 EPR spectra of the urate Cd(II) complex ........................................................................... 109 D 2 EPR sp ectra urate Zn(II) complex ...................................................................................... 110 D 3 Effect of ascorbate on the urate -Zn(II) complex formation. ............................................. 111

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14 Abstract of Dissertation Presented to the Graduate School o f the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy REACTION OF PEROXYNITRITE AND URIC ACID STUDIED BY ESR SPIN TRAPPING AND MASS SPECTROMETRY: FREE RADICAL FORMATION AND PRODUCT ID ENTIFICATION By Witcha Imaram December 2008 Chair: Name Alexander Angerhofer Major: Chemistry Uric acid is the most abundant antioxidant in plasma. However, u nder conditions of elevated uric acid levels and oxidative stress, it becomes a pro -oxidant and causes endothelial dysfunction. It is hypothesized that the generation of reactive intermediates such as free radicals from the reaction of peroxynitrite and other oxidants mediate the pathological pro -oxidant properties of uric acid. Peroxynitrite is a r eactive oxidant produced in vivo in response to oxidative and other stress by the diffusionlimited reaction of nitric oxide and superoxide. Our research is focused on the identification of free radical metabolites of uric acid formed from its reaction wit h peroxynitrite. Our experimental approach included the electron spin resonance (ESR) spin trapping of the radical generated from the reaction between uric acid and peroxynitrite at pH 7.4. Using PBN ( N tert -butyl alpha -phenylnitrone ) as the spin trapping agent, a six line ESR spectrum was obtained and its hyperfine coupling constants, a(N) = 15. 6 G; and a(H) = 3.6 G, corresponded to two carbon-based radicals. Further structural identifications of the PBN radical adducts were carried out using liquid chroma tography -mass spectrometry (LC MS) After comparison with the control reactions, we could identify two molecules, corresponding to the fragment ions of m/z 352 and 223, respectively. The PBN -triuretcarbonyl

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15 radical adduct was characterized for m/z 352 and the latter was identified as a PBN aminocarbonyl radical adduct The pH dependence study of the reaction between uric acid and peroxynitrite revealed the formation of hydrogen adduct at high pH and could be observed even without urate. Its formation was p roposed to undergo the inverted spin trapping mechanism, in which the spin trap was initially oxidized rather than the antioxidant substrate, and followed by electron transfer. We extended our studies to investigate the effect of methyl substitution at var ious nitrogen positions on product and radical formation. No ESR signal was observed when conducting the reactions with N 7 methylated uric acids in phosphate buffer pH 7.4 Moreover, the reactions were purposely conducted in methanol to trap the reaction intermediate. Various products have been identified by LC MS, and those products indicated that a common intermediate in various urate oxidation conditions, dehydroisouric acid, was formed.

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16 CHAPTER 1 INTRODUCTION General Overview of E lectron Spin Resona nce Spin Trapping Free radicals are any chemical species capable of independent existence possessing one or more unpaired electrons (1 ). Free radicals, and especially reactive oxygen species, play an important role in living systems and they are widely bel ieved to contribute to the development of several age related diseases and implicated in the pathology of a range of diseases including ischemic and post ischemic reperfusion damage, inflammation processes, cancers, and neurodegenerative diseases (1 -5 ). In these pathologies, oxidative damage initiated by free radicals, such as lipid peroxidation process (6 ), DNA damage (7 ), and proteins and enzy me inactivation, is occurring. Thus, the development of methods capable of detecting free radicals in biological s ystems is very important and has become an active field in free radical research. Electron spin resonance ( ESR ), known by many synonyms such as, electron paramagnetic resonance (EPR), or electron magnetic resonance (EMR), has emerged as a powerful method for direct detection and characterization of free radicals ( 2 ). Because of its high selectivity to detect paramagnetic species such as free radicals, ESR is one of the most widely chosen methods for studying the free radi c al mediated process in a complex b iological system ( 8 ). However, ESR alone has some disadvantages, one of which is a short time -period for free rad ical detection (half life t1/2 109 to 10 s). In many occasions, some radical processes may not be ESR detectable under various physiological c onditions because some free radicals are very short -lived. These short lived free radical species may not have sufficient enough time to accumulate their concentration to reach a steady state level above the detection limit of ESR (~108 M) ( 8 ). Moreover, if free radicals have very short spin relaxation time, this will make their line width too broad to be

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17 observed by ESR ( 9 ). To circumvent this drawback, the spin trapping technique was introduced in late 1960s ( 10). Named by Janzen and Blackburn in 1969 ( 1 1 ), spin trapping is the chemical reaction (Equation 1 1) in which a reactive free radical (R) adds to a diamagnetic compound and spin trapping agent (ST) to form a more stable radical, the spin adduct ([ST -R]). R + ST [ST R] (1 1) The spin adduct (usually a nitroxide) that is paramagnetic gives the ESR spectrum which contains useful information called hyperfine parameters. In general, one can characterize the original free radical, from which the spin adduct was derived, by analyzing t he number of hyperfine parameters and the magnitude of the hyperfine coupling constants in the ESR spectrum (8 ). Practically, such a full characterization is rarely obtained; however, it is still possible to extract some information about the type of the r adical (i.e., whether it is carbon -centered, oxygen -centered, or nitrogen -centered) ( 9 ). Nitrone and nitroso compounds are the most popular spin trapping agents. Although the spin adducts of nitroso compounds are relatively less stable, more information i n the hyperfine splitting parameters can be obtained from nitroso compounds, such as 2 -methyl 2 -nitrosopropane (MNP ), than nitrone because the radical adds directly to the nitroso nitrogen (Equation 1 2), (1 2) The spin adducts from nitrones are qui te stable, but some information is lost because the nitroxyl radical is far away from the original radical by one carbon atom. Nevertheless, useful information can still be obtained from a -hydrogen that is present in many widely used nitrone spin traps: DMPO, PBN, and POBN ( Figure 1 1).

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18 Figure 1 1 Examples of nitrone spin trapping agents The spin trapping technique of short lived free radicals, especially reactive oxygen species (ROS), coupled with E S R has become a valuable tool in the study of the fate of the radicals in the biological milieu. However, the use of the spintrapping technique has been limited when applied to the biological system due to the short persistence of the spin adducts, in which the aminoxyls are reduced to E S R silent product s by the biological antioxidants such as ascorbate anion and glutathione peroxidase (12). In an effort to develop more efficient spin traps, in the past decade, a number of different nitrone spin traps, such as DEPMPO (13), EMPO (14), and BMPO (15) ( Figure 1 1 ) have been synthesized. The challenge of designing a new spin trap is not only getting more persistent spin adducts, but also improving the spectral resolution between the different spin adducts. Moreover, the development of spin traps that can accumu late in relevant sites and cell compartments is an important issue as well. Uric Acid Generation and Degradation Uric acid is a purine degradation product from RNA, DNA and adenine nucleotides (16). Its immediate precursor, xanthine, is converted to uric a cid via the enzymatic reaction with xanthine oxidase or xanthine dehydrogenase (Equation 1 3)

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19 (1 3) The former enzyme generates oxidants (O2 and H2O2) during this reaction. In most mammals, uric acid is degraded to allantoin by the enzyme urate oxida se (uricase) which is present in the liver. However, during primate evolution, 5 to 20 million years ago, two parallel but distinct mutations occurred in early humanoids that rendered the uricase gene nonfunctional (17). As a result, humans and the great a pes have higher uric acid levels (range 3 14 mg/d L ) compare with most mammals (1mg/d L ) (18). In plasma, uric acid is largely present as monoanion urate (pKa = 5.4) ( 19). Serum uric acid can be generated from endogenous and exogenous sources. Exogenous sources include foods rich in purines, such as fatty meats, beer, and organ meats. Interestingly, fructose can increase the level of uric acid in serum ( 20). Fructose is different from other sugars in that it is degraded by a specific enzyme pathway (fructoki nase aldolase B) that results in the rapid generation of uric acid The mechanism is due to the unregulated rapid phosphorylation of fructose to fructose 1 -phosphate, resulting in ATP depletion, phosphate depletion, AMP deaminase synthesis, and generation of urate (21). Because of the lack of uricase and the ability to regulate uric acid tightly, dietary intake of purines or fructose can lead to a rapid rise in serum uric acid In addition to exogenous sources, uric acid can be generated under conditions of high cell turnover or in the setting of ischemia (22). Being an antioxidant, uric acid can react with a variety of substances that can lead to its stepwise degradation. It can react with O2 -, H2O2, and peroxynitrite (OONO) (23,24). These

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20 reactions lead to the complete degradation of uric acid and result in the generation of a number of stable end products, including allantoin, alloxan, parabanic acid, and triuret (25,26). Uric Acid: The Oxidant Antioxidant Paradox Because of the lack of uricase in humans after the primate evolution, u ric acid has been theorized to replace vitamin C and become a major antioxidant in human (23 ). Urate has been proposed to inhibit the formation of nitrotyrosine resulted from peroxynitrite -mediated damage (27) by scavenging t he radical intermediates, the decomposition products of peroxynitrite (28), which are responsible for nitration of tyrosine Moreover, uric acid can chelate transition metal ions and scavenge many reactive oxygen and nitrogen species; for example, superoxi de, the hydroxyl radical, singlet oxygen, and peroxynitrite (19,29).The antioxidant properties of uric acid have been thought to be initiated by the donation of an electron by uric acid to generate the urate radical (with a redox potential of 0.59V) follo wed by its nonreversible degradation to a variety of products (25,26). In this regard the urate reaction is distinct from ascorbate, for although ascorbate will also generate the ascorbyl radical, this latter reaction is reversible (30). Despite the role o f antioxidant in plasma, the growing evidence of uric acid being a true risk factor to develop obesity, hypertension, and cardiovascular disease, conditions associated with oxidative stress, has recently been reported (18,31). Uric acid can be a pro -oxidant by forming free radials in various reactions Maples and Mason detected the urate radical in a flow cell ES R experiment with both permanganate and the peroxidase/H2O2 system (32). The data delocalized radical that resided on the 5 -membered ring. Kahn et al. studied the oxidative reaction of urate (anion form of uric acid) by xanthine oxidase (XO) and found that hydroperoxide and dehydrourate w ere two distinct intermediates (Figure 1 2) (33,34). 5 hydroxyisourate was the primary product of the enzymatic oxidation and through

PAGE 21

21 subsequent non-enzymatic ring opening leads to allantoin (33,35). This is an effective 2 e/2H+ oxidation mechanism which c an also be observed by electrochemical oxidation (26,35). Figure 1 2. The oxidative reaction of urate by xanthine oxidase (XO). Attacked by peroxynitrite, urate was decomposed to form urate -derived radicals, which are responsible for the amplification of lipid oxidation products found in liposomes and LDL when treated with peroxynitrite (36). The fact that urate can be oxidized via a radical mechanism is significant since this opens up the possibility for an explanation of the pro oxidant effect of uric acid ( 20,37-39). If the oxidation of uric acid in vivo processes via radical mechanism it is possible that radical chain reactions may be started and damage the cell (16). Whether uric acid functions as an antioxidant or pro -oxidant remains ambiguous. Sa utin et al. suspected that uric acid may have a protective effect only in the hydrophilic environment, like in plasma ( 16). On the other hand, the pro -oxidative effects of uric acid, which are usually associated with lipid, may take place only in the hydro phobic environment created by lipid within the cell ( 16). Thus, one may determine the true function of uric acid by analyzing the effect of uric acid in various circumstances.

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22 Peroxynitrite Peroxynitrite (OONO) or oxoperoxynitrate(1 ) has been receiving great interest in several fields. Beside earth, peroxynitrite was observed by the Mars Viking biology experiments to be generated by photolysis on Mars ( 40). It can be formed by the fast reaction (k = 5 19 109 M1 s1) between nitric oxide (NO) and supe roxide (O2 ) (41-43) (Equation 1 4 ). NO + O2 ONOO (1 4) In vivo t his reaction frequently occurs in the vasculature due to the presence of superoxide generated by NADPH oxidase or by xanthine oxidase, and nitric oxide generated by en dothelial nitric oxide synthase (eNOS) (44). The formation of peroxynitrite from relatively unreactive radicals, NO and O2 is regulated by superoxide dismutase (SOD), an enzyme capable of lowering superoxide (O2 ) (Figure 1 3 ). From thermodynamic calculations, the oxidation potential of peroxynitrite was calculated. It indicated that peroxynitrite is a strong oxidant, with a one -electron reduction potential, E (OONO, 2H/ NO2 H2O), around 1.6 V at pH 7, and that it is unstable with respect to dispr oportionation to nitrogen dioxide and the nitrosyldioxyl radical, ONOO (45). Moreover, the product, nitrogen dioxide, is also a strong oxidant, with a one -electron reduction potential, E (NO2 /NO2 ) = 1.04 V ( 46). Peroxynitrite is somewhat stable, thou gh decomposes slowly to nitrite and dioxygen at and above pKa, and at high concentration (exceed 0.1 mM) (Equation 1 5) (47). 2ONOO O2 + 2NO2 (1 5) Peroxynitrite anion can exist in two geometries, the cis and trans isomers (Figure 1 4 ). In solution, it is present in the cis -form ( 48), which is 3 4 kcal/mol more stable than the trans -form

PAGE 23

23 (49). The energy barrier between the two conformers is approximately 24 kcal/mol for the anion, and 10 kcal/mol less for the protonated form ( 49). Fi gure 1 3. Scheme summarized the chemistry of peroxynitrite, including the postulated formation of peroxynitrite in vivo the reaction between peroxynitrite and CO2, the decomposition of peroxynitrous acid, and the reaction pathways leading to the oxidatio n products. Figure 1 4. Geometrical isomers of peroxynitrite.

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24 Known to react as a nucleophile, peroxynitrite anion (OONO) can undergo nucleophilic addition with CO2 (50,51), aldehydes ( 52), and ketones ( 53), while peroxynitrous acid (ONOOH) mediates eb selen oxidaton (52,54). Peroxynitrous acid (ONOOH), a conjugate acid of peroxynitrite, is a strong both one and two -electron oxidizing agent (55), with a pKa of 6.5 7.5, depending on the ionic strength of the medium ( 47). Unlike peroxynitrite anion, peroxynitrous acid isomerizes to nitrate (70%) with a rate of 1.2 s1 at 25 C. In addition to isomerisation pathway, peroxynitrous acid has been proposed to undergo homolysis (30%) to form the hydroxyl radical and nitrogen dioxide (Equation 1 6 ) (56). However, Kissner et al. suggested that homolysis of the O -O bond in peroxynitrous acid is unlikely( 55), and concluded that peroxynitrous acid is not the source of hydroxyl radicals ( 46). ONOOH HO + NO2 (1 6 ) The actual nature of the decomposition of peroxynitrite is still debated. However, whether peroxynitrous acid undergoes homolysis is irrelevant in a biological system, where the carbon dioxide (CO2) exists in a high concentration (approximately 1 mM). The reaction of peroxynitrite with CO2 is one of the main pathways of peroxynitrite chemistry in physiology. The rate constant for reaction of CO2 with OONO is large (approximately 5.8 104 M1 s1 depending on pH), and faster than the rate of direct reaction between peroxynitrite and biologi cal target molecules such as ascorbate, glutathione, and urate ( 57). As a result, only a limited number of biomolecules can compete with CO2 to scavenge peroxynitrite. Nitrosoperoxycarbonate (ONOOCO2 ) is the first intermediate formed by the reaction of ONOO and CO2 (Equation 1 7 ). This intermediate is very short -lived and decomposes to regenerate CO2 and NO3 in ca. 70% yield (Equation 1 8 ), and produce the free radicals (in ca.

PAGE 25

25 30% yield ) CO3 and NO2 (Equation 1 9 ) that are responsible for the oxida tions and nitrations of many biological species ( 58). ONOO + CO2 ONOOCO2 (1 7 ) ONOOCO2 CO2 + NO3 (1 8 ) ONOOCO2 CO3 + NO2 (1 9 ) The decomposition processes of nitrosoperoxycarbonate (ONOOCO2 ) describ ed above is postulated to undergo via a second intermediate, which is proposed to be the caged radicals (Figures 1 3 ). These caged radicals can break apart to give free radicals and react further with scavengers, or reform and give a new cage product ( 57). Peroxynitrite vs. Uric A cid It was proposed in 1990 that peroxynitrite exists in vivo (59). Under oxidative stress, peroxynitrite ( OONO-) can react with various biomolecules ( 60). It has been shown that peroxynitrite reacts with tyrosine residue to produc e nitrotyrosine; this is major eviden ce for peroxynitrite -mediated cell damage ( 61). Furthermore, peroxynitrite can induce DNA base damage ( 62) and lipid peroxidation ( 63). The finding of the potential scavengers of peroxynitrite is important, and has beco me one of the most interesting topics in the field ( 60). Among scavengers, uric acid is one of the most abundant in human. In many cases, its reaction with peroxynitrite has been reported to benefit the cells from peroxynitrite -mediated damage ( 16). In th e mice model of experimental allergic encephalomyelitis ( EAE), uric acid can block peroxynitrite activity by preventing the nitration of neuronal proteins ( 64). Interestingly, the protective properties of uric acid in EAE against peroxynitrite related chem ical reaction is even more superior than ascorbic acid ( 65). Recently, uric acid was found to have a high potential to scavenge peroxynitrite (24). Peroxynitrite reacts with uric acid 16 times faster than with ascorbate, and 3 times quicker than with cyste ine; however, to achieve the maximum

PAGE 26

26 protection, ascorbate and cysteine must be present along with urate in scavenging of peroxynitrite ( 24). As described in the previous topic, uric acid can become a pro -oxidant under a certain condition, by forming radi cals after reacting with oxidants including peroxynitrite. Santos et al. observed a carbon -centered radical by ESR spin -trapping with the spin trap DMPO in reaction mixtures of uric acid and peroxynitrite (36). It was identified as the aminocarbonyl radica l and its presence explained due to follow up reactions between peroxynitrite and the primary reaction products such as all oxan and parabanic acid ( 36). The generation of the aminocarbonyl radical from urate was proposed to be responsible for amplified oxi dation of LDL and liposomes. Research Objectives In recent years, Johnson et al. (31,66-71) demonstrated in a series of cell culture, animal, and human studies that uric acid may be a true risk factor for hypertension, kidney disease, and metabolic syndrome A key issue is the upstream mechanism of how urate mediates these biological effects. It is thus important to know that when uric acid gives up an electron and proton as an anti -oxidant, it then becomes a urate radical (32) that can act as a pro -oxidant. A major question is whether the effects of uric acid to activate cells is due to the uric acid itself or to the urate radical, which is easily generated from uric acid in the presence of mild oxidative stress (32,36). This has led us to hypothesize that uric acid, particularly in the presence of oxidative stress, may convert to a urate radical or to an oxidative radical product of urate which could then activate cells contributing to the pathogenesis of hypertension, the metabolic syndrome, renal disease and cardiovascular disease. Although the presence of the urate radical has been shown previously (32,36), as well as its ability to act as a pro oxidant (72-75), to date there has been minimal study of the major urate radicals that are generated under ph ysiological

PAGE 27

27 conditions following reactions with oxidants, particularly as it relates to their structure, kinetics and possible mechanisms of generation. Here we examined the reaction between urate and peroxynitrite using electron spin resonance (ESR) coupled with spin trapping as a primary tool to identify the radicals generated from this reaction. Moreover, the structure of the radical adduct was also characterized by LC MS.

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28 CHAPTER 2 RADICAL FORMATION FROM THE REACTION BETWEEN URIC ACID AND PEROXYNITRITE Introduction A comprehensive study of uric acid oxidation by peroxynitrite has been done by Santos et al (36). An apparent second order rate constant of this reaction has been determined (k = 4.8 102 M1 s1), and oxygen consumption has been observe d (36). The reaction was proposed to involve multiple interactions between uric acid and peroxynitrite, and generated various products including radicals. The radical formation has been claimed to be produced by the addition of peroxynitrite anion to the oxidation products that contain a carbonyl vicinal to aminocarbonyl group such as alloxan and parabanic acid. This assumption is unlikely because no radical was detected by ESR in incubations of peroxynitrite with those urate oxidation products in their stu dy (36). Due to the complexity of the peroxynitrite -mediated oxidation of uric acid, its mechanism has not been well established. During our investigation to unfold the chemistry of uric acid when treated with peroxynitrite at pH 7.4, we discovered a novel urate -derived radical triuretcarbonyl radical which could be an intermediate for the production of the aminocarbonyl radical. The radicals were studied by electron spin resonance spectroscopy using spin trapping method, and PBN was used as a spin trappi ng agent. The structure of the PBN radical adducts were characterized by liquid chromatography -mass spectrometry (LC -MS)

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29 Materials and Methods Chemicals Uric acid was purchased from Sigma. Diethylenetriaminepentaacetic acid (DTPA) was purchased from Fluk a. N tert -butyl -phenylnitrone (PBN) was obtained from Alexis Biochemicals. Peroxynitrite was synthesized following the method reported by Uppu and Pryor (76). The peroxynitrite concentration was measured spectrophotometrically at 302 nm ( = 1670 M1 cm1). ESR Experiments 100 mM stock solutions of uric acid (1) was prepared in 0.3 M potassium hydroxide. The reaction mixtures, typically conducted in 0.3 0.5 M potassium phosphate buffer at pH 7.4, contained the final concentration of 3 mM urate, 30 mM N t ert butyl -phenylnitrone (PBN), 0.1 mM DTPA, and 9 mM peroxynitrite. Then, the reaction mixture was transferred into a quartz capillary of approximately 1 2 mm ID OD for ESR measurement. After two minutes, the ESR spectrum was recorded at room temperature, using a commercial Bruker Elexsys E580 spectrometer, employing Brukers highQ cavity (ER 4123SHQE). Spectral parameters were typically: 100 kHz modulation frequency, 1 G or 0.1 G modulation amplitude, 20 mW microwave power, 9.87 GHz microwave frequency, 82 ms ti me constant, and 164 ms conversion time/point. Sample P reparation for L iquid C hromatography -M ass S pectrometry The reaction mixtures were prepared at room temperature in 0.3 M potassium phosphate buffer pH 7.4 and contained a final concentration of 10 mM u ric acid, 30 mM PBN, 0.1 mM DTPA, and 30 mM peroxynitrite with the final volume of 10 mL. Then, the reaction mixtures were subsequently extracted by 2 20 ml of CH2Cl2, dried under nitrogen gas, and re -suspended in 1 mL CH3CN. Liquid C hromatography -M ass S pe ctrometry Analysis The LC MS analyses were carried out with an Agilent 1100 liquid chromatography system (Agilent Technologies, Palo

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30 Alto, CA, USA) and an TSQ 7000 triple -quadruple mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with APCI i nterface operated in positive -ion mode detection. In a TSQ 7000 instrument, nitrogen was used as both the sheath and the auxiliary gases. The second quadrupole was used as a collision chamber, with argon as a collision gas, at a pressure in vicinity of 2. 5 103 Torr. The operation of the LC -MS and data analyses was performed using the ThermoFinnigan Xcalibur 1.4 software. Fullscan liquid chromatography mass spectrometry Liquid chromatography analyses were performed in a gradient elution mode using Pheno menex Luna 5 C18(2) 100 (150 mm 4.6 mm) column (Phenomenex, Torrance, CA, USA) coupled with a Phenomenex Luna C18 (2), 5 m particle size guard column. The mobile phase used included 5 mM ammonium acetate / 0.1 % acetic acid (A) and methanol (B) as a gradient. The mobile phase flow was 0.6 mL min1, and the i to 95% B over the next 10 min, remained constant for 3 min, then reversed to the original composition of 90% A over 1 min, after which it was kept constant for 1 min to re -equilibrate the column. The extracted reaction products, control samples and standard samples, were analyzed in the fullscan mode at a mass range of m/z 90450. Liquid chromatography -mass spectrometry tandem mass spectrometry analysis LC analysis was performed as outlined above. The MS/MS analysis performed for M+1 ion 223 and 352 in the positive mode at a collision energy of 20V, with a mass scan range of m/z 40230 for M+1 ion 223, and a mass scan range of m/z 45360 for the latter. Results Electron Spin R esonance S pin Trapping To probe the generation of the PBN radical adducts at pH 7.4, the reaction between urate with peroxynitrite was monitored by ESR using spin trapping method. The reaction between urate and peroxynitrite resulted in a six-line ESR

PAGE 31

31 spectrum (Figure 2 1A). The trapped radical adducts displayed the average hyperfine coupling constants a(N) = 15.6 G, and a(H) = 3.6 G. No trapped radicals were observed when the reactions were conducted without urate or peroxynitrite. PBN alone, or mi xed with urate or peroxynitrite, did not yield any ESR signal (Figure 2 1C). Figure 2 1 ESR spectra of PBN radical adducts obtained from the incubation of 3 mM urate, 30 mM N tert -butyl phenylnitrone (PBN), 0.1 mM DTPA, and 9 mM peroxynitrite in phos phate buffer pH 7.4. At room temperature, the ESR spectrum was recorded at 2 minutes after adding peroxynitrite. (A) The ESR spectrum of the PBN radical adducts using spectral parameters at 100 kHz modulation frequency, 1 G modulation amplitude, 20 mW micr owave power, 9.87 GHz microwave frequency, 82 ms time constant, and 164 ms conversion time/point. (B) The high resolution ESR spectrum of the PBN radical adducts using spectral parameters at 100 kHz modulation frequency, 0.1 G modulation amplitude, 20 mW m icrowave power, 9.87 GHz microwave frequency, 82 ms time constant, and 164 ms conversion time/point. (C) The control reaction conducted in the same condition as described in (A) but without urate.

PAGE 32

32 Furthermore, the ESR intensities increased when the urate concentration was increased (Figure 2 2). These experiments confirmed that the observed radicals were derived from urate, not artifacts. Moreover, when the experiment was performed at higher resolution (lower modulation amplitude), we found that PBN could trap at least two different carbon based radicals (Figure 2 1B). The radical formation increased with the concentration of peroxynitrite, but yielded maximum at a four -fold molar excess of peroxynitrite over urate (Figure 2 3). Figure 2 2 Effect of ura te concentration on the production yield of the PBN radical adduct derived from urate obtained from the oxidation of urate by peroxynitrite. The ESR spectra were recorded after 2 min of incubation of various urate concentraions 19 mM N tert -butyl phenylnitrone (PBN), 0.1 mM DTPA, and 23 mM peroxynitrite in 0.3 M phosphate buffer pH 7.4. The instrumental parameters were 100 kHz modulation frequency, 0.5 G modulation amplitude, 20 mW microwave power, 9.87 GHz microwave frequency, 82 ms time consta nt, and 164 ms conversion time/point.

PAGE 33

33 Figure 2 3 Effect of peroxynitrite concentration on the production yield of the PBN radical adduct derived from urate obtained from the oxidation of urate by peroxynitrite. The ESR spectra were recorded after 2 m in of incubation of 3 mM urate, 30 mM N -tert butyl -phenylnitrone (PBN), 0.1 mM DTPA, and various concentrations of peroxynitrite in 0.5 M phosphate buffer pH 7.4 The instrumental parameters were 100 kHz modulation frequency, 1 G modulation amplitude, 20 mW microwave power, 9.87 GHz microwave frequency, 20 ms time constant, and 82 ms conversion time/point. Depending on pH, both uric acid and peroxynitrite can exist in either neutral or anion form. Therefore, the radical formation should be affected by pH as well. Indeed, the pH profiles show that the ura te -peroxynitrite reaction yielded more trapped radicals when pH increased (Figure 24). This indicates that the radicals may not be produced by the direct reaction between peroxynitrous acid or its decomposition products with uric acid The simulated ESR t itration curve obtained a better fitting when the initial pHs of the buffers were used. Although the final pHs of the solutions were preferred, we have not been able to find proper parameters to fit the

PAGE 34

34 data obtained from the shifted pHs. Nevertheless, bot h pH profiles exhibited the same results; the yield of radical adducts increased with pH. The obtained pH sigmoidal curve shows an inflection point of 8.1, which is near the pKa of peroxynitrous acid determined previously by Kissner et al. (47). Figure 2 4. Effect of pH on the production yield of the PBN radical adduct derived from urate obtained from the oxidation of urate by peroxynitrite. The ESR spectra were recorded after 2 min of incubation of 3 mM urate, 30 mM N tert -butyl -phenylnitrone (PBN), 0.1 mM DTPA, and 9 mM peroxynitrite in 0.5 M phosphate buffer at various pH s. The instrumental parameters were 100 kHz modulation frequency, 1 G modulation amplitude, 20 mW microwave power, 9.87 GHz microwave frequency, 20 ms time constant, and 82 ms conve rsion time/point. The measured ESR intensities corresponded to the first PBN radical adduct peak to peak heigthts. In addition to the effect of pH, we have investigated the effect of CO2 on the production of radical adducts. The administration of CO2 decreased the observed ESR signals (Figure 2 5). Interestingly, a four -fold excess of bicarbonate over peroxynitrite was required to completely prevent the formation of the radical adducts. This is probably because the reactions were not

PAGE 35

35 Figure 2 5. Effect o f CO2 on the production yield of the PBN radical adduct derived from urate obtained from the oxidation of urate by peroxynitrite. The ESR spectra were recorded after 2 min of incubation of 3 mM urate, 30 mM N tert -butyl -phenylnitrone (PBN), 0.1 mM DTPA, and 9 mM peroxynitrite in 0.5 M phosphate buffer at pH 7.4 in the present of various concentrations of bicarbonate (HCO3 ). The instrumental parameters were 100 kHz modulation frequency, 1 G modulation amplitude, 20 mW microwave power, 9.87 GHz microwave frequency, 82 ms time constant, and 164 ms conversion time/point. conducted in the gas tight system. Therefore, some carbon dioxide gas could escape the reaction mixtures. As a result, the excess amount of bicarbonate is required in order to completely quench peroxynitrite. Product Identification o f t he Radical Adducts b y Liquid Chromatography -Mass Spectrometry Analysis From the reaction between urate and peroxynitrite in phosphate buffer pH 7.4, the extracted radical ad ducts were separated and characterized by liquid chromatography coupled with mass spectrometry (LC -MS). After comparison with the control reaction (Figure 2 6B)the reaction without urate the fullscan LC MS showed two products, at the retention time

PAGE 36

36 of 10: 56 minute s corresponding to the quasi ions at m/z 223; and at 13:13 minutes corresponding to the quasi ion at m/z 352 (Figure 2 6A). As displayed in Figure 2 6A and 2 6B, the large area of the LC traces at the retention time stating from 11.12 minutes to 15.00 minutes belonged to the mass derived from PBN (M+1 = 178), and the small peak at 9 minutes was consistent with benzaldehyde oxime (M+1 = 122), which is the decomposition product of PBN. In addition to the fullscan, the ions at m/z 223 and 352 were s elected and analyzed by tandem mass spectrometry (MS/MS). At m/z 223, the following ions were identified: m/z (% intensity): 223 (4%; M+1), 167 (30), 149 (29), 134 (12), 132 (5), 122 (24) and 104 (100%), see Figure 2 7, and the fragment of m/z 352 exhibite d the following ions m/z (% intensity): 352 (2%; M+1), 335 (13%), 296(33), 279(25), 263 (100), 246 (83), 218 (7), 193(12), 177(24), 175 (22), 167 (9), 147(24), 122(8), 118 (6), 104 (9); and 61 (1), see Figure 2 8. Discussion Spin trapping allows the trapp ing of short lived radicals with a more stable radical adduct, thereby allowing analysis. It is the method of choice to study short lived radical intermediates (77). The identification of the radical adducts and their proposed formation mechanism from the peroxynitrite urate reaction will be discussed. The first spin trapping studies on urate -derived radicals were reported by Santos et al. on the peroxynitrite urate system (36). A carbon -centered radical was trapped with the spin trap

PAGE 37

37 DMPO (Figure 1 1) and analyzed by CW -ESR. It was tentatively identified as an aminocarbonyl radical. Figure 2 6. LC -MS study of the reaction between urate and peroxynitrite in phosphate buffer pH 7.4. Before submitting to LC, the reaction products were extracted by methy lene chloride (CH2Cl2), dry under nitrogen gas, and resuspended in acetronitrile. (A ) LC chromatogram of 10 mM urate treated with 30 mM peroxynitrite in the presence of 30 mM PBN and 0.1 mM DTPA. (B ) LC chromatogram of the control reaction, which every rea gent contained the same concentration as described in a) but without urate.

PAGE 38

38 Figure 2 7. MS/MS spectrum of the PBN aminocarbonyl radical adduct. Figure 2 8. MS/MS spectrum of the PBN -triuretcarbonyl radical adduct.

PAGE 39

39 Unlike reported by Santos et al (i.e ., only spin trapping agent DMPO gave stable radical adducts) (36), our experiment showed that PBN could trap at least two radicals. After a comparison with data reported in a review by Buettner (9 ), the PBN adducts were found to belong to carbon -centered radicals. The information from ESR spectra only gave a rough estimation of the type of trapped radicals. Thus, the LC MS analysis was performed to acquire more structural information of the radical adducts. The fullscan LC -MS study showed two distinctive p roducts with m/z 223 (M+1) and 352 (M+1), which were absent in the control reaction (Figure 2 6). The analysis of the LC MS/MS spectra (Figure 2 7 and 2 8) revealed that both ions showed the same initial fragment loss of a neutral compound with a molecular weight (MW) of 56, corresponding to the loss of 2-methylpropene ([M+H -C4H8]+). This fragment loss could be originated from the cleavage of tert -butyl group from the PBN moiety. Moreover, the fragmentation pattern of both parent ions contained the same fra gment daughter ion at m/z 122. This ion was identified as benzaldehyde oxime (Figure 2 9 and 210), which was converted from PBN as well. These results indicated that both parent ions were derived from PBN. Furthermore, the loss of formamide (MW 45), in bo th 223 and 352 ions, and isocyanic acid (HNCO, MW 43), in the case of 352 ion, indicated that at least one amide group was present in both compounds. This hypothesis is further supported by the loss of ammonia (MW 17) in both 223 and 352 ions, which reflec ts that the parent compounds have a functional group that contained a primary amine such as amide. Based on the observations described above, the MS/MS fragmentation analysis confirmed that the structure of the product corresponding to m/z 223 was consiste nt with the hydroxylamine form of the PBN aminocarbonyl radical adduct 2 (Figure 2 -11), and the structure corresponding to m/z 352 was proposed to be the hydroxylamine form of the PBN -

PAGE 40

40 triuretcarbonyl radical adduct 3 (Figure 2 1 1 ). The existence of triuret moiety in compound 3 is confirmed by the fragment ions at m/z 147, 104, and 61(Figure 2 10) which is a signature fragmentation pattern of triuret (M+1 = 147) (25). Figure 2 9. Fragmentation pattern of the hydroxylamine form of PBN aminocarbonyl radi cal adduct.

PAGE 41

41 Figure 2 10. Fragmentation pattern of the hydroxylamine form of PBN -triuretcarbonyl radical adduct.

PAGE 42

42 Figure 2 11. The s tructures of PBN -radical adducts of aminocarbonyl radical 2 and triuretcarbonyl radical 3 Although the aminocarbonyl radical has been previously characterized, little is known about its formational mechanism. Many possible pathways have been proposed for the generation of aminocarbonyl radical. Santos et al. suggested that the aminocarbonyl radical was produced from one of the products of a urate -peroxynitrite reaction, such as alloxan, parabanic acid, or allantoin (36). Robinson et al. later suggested that triuret, a novel product from peroxynitrite induced oxidation of urate, could be accounted for the detection of aminocarbonyl radical (25). Th e s e hypothes e s, however, is unlikely because we have tested the reaction of these products with peroxynitrite under the same condition as the oxidation of urate and we did not observe any radical adduct formation Our observation indicated that aminocarbonyl radical was not derived from these known urate -derived products. In our study, the trapping of radical adduct 3 led us to postulate that triuretcarbonyl radical 12 can be an intermediate for the production of aminocarbonyl rad ical 13, and their formations are proposed as depicted in (Figure 212). At pH 7.4, peroxynitrite, which its pKa is about 6.8, can exist as a mixture of peroxynitrite anion, peroxynitrous acid, or its decomposition products (25,46). We propose that urate i s oxidized via one electron transfer by peroxynitrous acid forming urate radical 4 (Equation 2 1 to 2 2 ). Because we did not observe the PBN OH radical adduct, so the electron is likely transferred to oxygen of the hydroxyl moiety of peroxynitrous acid, f orming a hydroxide (OH) and a nitrogen dioxide radical (NO2) (Equation 2 2).

PAGE 43

43 (2 1) (2 2) Then diimine 5 an oxidative intermediate of uric acid described in many urate oxidation studies (2 6,36), can be produced by one electron and one proton transfer from urate radical 4 to peroxynitrous acid. This second electron oxidation can be initiated by nitrogen dioxide radical (NO2), which is a decomposition product of peroxynitrous acid (Equation 2 2), as well b ecause of its strong oxidizing potentia l (46). In contrast to s ome studies (25,33), the dehydrourate ( with its structure depicted in Figure 1 2 ) is unlikely to be formed because it is antiaromatic molecule. Therefore, we propose that the intermediate of the second electron transfer from urate i s diimine 5 Th e diimine 5 is susceptible to nucleophilic addition and can rapidly react with nucleophile such as water or ammonia (26). Peroxynitrite anion has been reported to undergo nucleophilic addition in the reaction with diacetyl (78) and cyclicqui none (79). Moreover, our pH dependence experiments show that the radical formation was increased when pH was raised (Figure 2 4), and decreased when CO2 was present (Figure 2 5). This result indicates that peroxynitrite anion is responsible for the product ion of radicals by reacting with an intermediate, initially formed from the oxidation of original urate. Taken together, we thus propose that peroxynitrite anion can react with diimine 5 to produce peroxo adduct 6 After undergoing homolysis of the O O bon d (78), the peroxo adduct 6 decomposes to yield nitrite and radical intermediate 7 which then rearrange s to form carbonyl radical 8 The carbonyl radical 8 can be converted to radial 9 by either losing carbon monoxide (CO) or reacting with O2 and releasin g carbon dioxide (CO2). Another molecule of peroxynitrite anion may react with intermediate 10, which is produced by reduction

PAGE 44

44 of compound 9, to generate allantoin peroxo adduct 1 1 The multi -molar equivalent consumption of peroxynitrite leading to the rad ical formation was supported by our observation that the yield of radical formation was at maximum when a four -fold excess peroxynitrite over urate was used (Figure 2 3). By analogy similar to the degradation of 6 triuretcarbonyl radical 1 3 is obtained. T he -cleavage of triuretcarbonyl radical 1 3 yields isocyanic acids and aminocarbonyl radical 1 4 which is subsequently trapped by PBN (Figure 2 12). Aside from being the intermediate for aminocarbonyl radical, triuretcarbonyl radical could be an intermedia te candidate for the generation of triuret 1 5 as well (Figure 2 11). This hypothesis was supported by our observation that no triuret was observed when the reaction mixtures contained PBN. This indicates that PBN could quantitatively trapped the triuretami nocarbonyl radical, preventing the formation of triuret.

PAGE 45

45 Figure 2 1 2 Proposed radical formation mechanism of the reaction between urate and peroxynitrite.

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46 CHAPTER 3 REACTIONS OF PEROXYNITRITE WITH MONO DI AND TRI METHYLURIC ACIDS STUDIED BY LIQUID CHROMATOGRAPHY -MASS SPECTROMETRY AND ELECTRON SPIN RESONANCE SPECTROSCOPY Introduction Uric acid is a purine degradation product from RNA, DNA and adenine nucleotides (16). Several isomers of methyluric acid are also commonly present in vivo and are deri ved from the metabolism of methylated purines such as caffeine, theobromine, theophyl l ine and plant methylxanthines (80). Methyluric acids can attain significant concentrations in vivo, and have even been reported to cause kidney stones in subjects who ar e heavy coffee consumers (81). Indeed, Safranow and Machoy reported that methylated uric acids are found in all uric acid stones (82). Nevertheless, unlike the role of uric acid in quenching peroxynitrite mediated reactions, which is well established, less is known about the reaction of peroxynitrite with methyluric acids. In previous studies, depending on the reaction condition and the sources of the oxidants, the methylation at certain nitrogen position s in uric acid has been reported to affect the reacti vity of uric acid (83,84). 1,3 Dimethyluric acid and 1,3,7 trimethyluric acid were found to have higher potency than uric acid to prevent lipid peroxidation in human erythrocyte membranes (85-87). However, when N 7 was methylated, the reactivity of uric ac id toward the oxidation by 1,1 diphenyl 2 picrylhydrazyl (DPPH) radical was markedly decreased (84). The radicals derived from uric acid and its methyl derivatives upon photolysis in the presence of mild oxidants (not including peroxynitrite) have been investigated by ESR spectroscopy and calculation study (88). The radical s were proposed to be formed by hydrogen abstraction mechanism ( 88). The pKa values of the corresponding N -methyluric acid radical anions were determined to be in the range of 9.7 -

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47 11.2, a nd the intrinsic acidity of the N H protons in both neutral and radical -anion forms was calculated to follow the order N1H < N9H < N3H (88). We therefore examined the reactivity of various methyluric acids with peroxynitrite using liquid chromatography-ma ss spectrometry (LC MS) and electron sp in resonance (ESR) spectroscopy, a) to understand the reactivity of these biologically significant molecules with peroxynitrite and b) to test the hypothesis that methyl ation at N 7 might interfere with the oxidative reaction. This report shows the radical formations and reaction products generated from seven commercially available methyluric acids ( Figure 3 1) upon reaction with peroxynitrite in phosphate buffer pH 7.4. The radicals formed from these reactions were in vestigated by E S R spin trapping method, using PBN as the spin trapping agent, and the reaction products were identified by LC -MS. In an attempt to probe the intermediates in these reactions we have incorporated methanol into the reaction mixtures to invest igate if methanol could trap any intermediates generated during the course of the reactions, which were monitored by LC -MS. Figure 3 1 S tructures of uric acid, monomethyluric acid, dimethyluric acid, and trimethyluric acid Materials and Methods Chemicals Shown in Figure 3 1 are the different methylated uric acid compounds used. Uric acid ( UA, 1a ), 1,3 -dimethyluric acid ( 1,3 -DiMeUA, 1d ) and 9 -methyluric acid ( 9 -MeUA,

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48 1c ) were purchased from Sigma. 1 -Methyluric acid ( 1 -MeUA, 1b ), 1,7 -dimethyluric acid ( 1 ,7 DiMeUA, 1e ), 1,9 dimethyluric acid ( 1,9 DiMeUA, 1f ), 3,7 dimethyluric acid ( 3,7 DiMeUA, 1g), and 1,3,7 -trimethyluric acid ( 1,3,7 TriMeUA, 1h ) were purchased from Fluka. The d4m ethanol w as purchased from Cambridge Isotopes. Distilled, deionized water (m etal ion free) and EDTA (500 mM) were purchased from Gibco. Peroxynitrite, used in the experiments containing methanol was purchased from Cayman Chemical For the experiments conducted in phosphate buffer, peroxynitrite was synthesized following the method reported by Uppu and Pryor (76). The peroxynitrite concentration was measured spectrophotometrically at 302 nm ( = 1670 M1 cm1). To ensure the same reactivity, both sources of peroxynitrite reacted with uric acid in the same reaction conditions; the s ame reaction products, determined by LC MS, were obtained. ESR Spin Trapping Experiments 100 mM stock solutions of uric acid ( 1 a ) and selected methyl derivatives of uric acids; namely, 1 -methyluric acid ( 1b ), 9 -methyluric acid ( 1c ), 1,3 dimethyluric acid (1d ), 1,7 -dimethyluric acid ( 1e ), 1,9 -dimethyluric acid ( 1f ), 3,7 dimethyluric acid ( 1g), and 1,3,7 trimethyluric acid ( 1h ) were prepared in 0.3 M potassium hydroxide. The reaction mixtures, conducted in 0.3 M potassium phosphate buffer at pH 7.4, containe d the final concentration of 3 mM urate (or its analogues), 30 mM N -tert Butyl phenylnitrone (PBN), 0.1 mM DTPA, and 9 mM peroxynitrite. Then, the reaction mixture was transferred into a quartz capillary of approximately 1 2 mm ID OD for ESR measurement. After two minutes, the ESR spectrum was recorded at room temperature using a commercial Bruker Elexsys E580 spectrometer, employing Brukers highQ cavity (ER 4123SHQE). Spectral parameters were typically: 100 kHz modulation frequency, 1 G modulation ampl itude, 20 mW microwave power, 9.87 GHz microwave frequency, 82 ms time constant, and 164 ms conversion time/point.

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49 Product Identification of the Reactions Conducted in Phosphate Buffer Each reaction sample conducted in 0.5 M phosphate buffer pH 7.4, cont ained the final concentration of 10 mM uric acid (or its methyl analogues) 0.1 M DTPA and 30 mM peroxynitrite. Then, the reaction mixtures were placed on ice, transferred to LC -MS vials on ice, and stored at 4 C to await LC MS analysis The reaction mixtu res were analyzed by LC -MS in the positive fullscan (both negative and positive) mode using electrospray ionization (ESI) method. This enabled us to detect the positive ions (mass range m/z : 50.0 900.0) as well as the fragmentation pattern of each intermediate, or product. The TSQ Quantum Discovery mass spectrometer (ThermoFinnigan, San Jose, CA, USA) and a ThermoFinnigan Surveyor liquid chromatography system (ThermoFinnigan, San Jose, CA, USA) was equipped with a Phenominex Luna C18 column (250x4.6 mm). To minimize the decomposition of labile intermediates, samples were kept cold until analysis. The mobile phases included 5mM NH4OAc/0.1% AcOH and methanol (as gradient). Typical HPLC analyses were carried out in a gradient elution mode, using an aqueous mobi le phase A (5 mmol/L ammonium acetate and 0.1% by volume of AcOH in water) and MeOH as organic mobile phase B. Mobile phase flow was 0.6 mL/min The HPLC run time was 16 min and the gradient used was as follows: the gradi ent began at 85% A. The composition was linearly ramped to 10% A over the next 9 min and remained constant for 3 min, and reversed to the original composition of 85% A over 2 min, after which it was kept constant for 2 min to reequilibrate the column. In the Quantum Discovery mass spectrometer, nitrogen was used as both the sheath (40 psi) and the auxiliary (15 units) gas. The spray voltage was 4000 V. The heated capillary temperature was maintained at 300 C. The collision pressure was 1.5 103 Torr. Th e operation

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50 of the LC MS and data analyses was performed using the ThermoFinnigan Xcalibur 1.4 software. Product Identification of the Reactions Conducted in Methanol Each reaction sample contained the final concentration of 10 mM uric acid (or its methyl analogues) and 10 mM peroxynitrite in a mixed solvent of 50% alcohol (methanol or d4-methanol) and 50% aqueous phosphate buffer solution at pH 7.4. U ric acid, or its methyluric acid powder, was added to alcohol/aqueous solution. Under this condition, uric acid only partially dissolved. However, after one equivalent of peroxynitrite was added to the reaction mixture, agitated, and allowed to incubate at room temperature for 3 min, uric acid dissolved completely as the reaction proceeded. Then, the reaction mixtures were placed on ice, transferred to LC -MS vials on ice, and stored at 80C to await LC -MS analysis The reaction mixtures were analyzed by LC -MS in the fullscan (both positive and negative), tandem mass spectrometry (MS/MS), and single reaction mo nitoring (SRM) modes, using the atmospheric pressure chemical ionization (APCI) method. In addition, some samples were further analyzed using the electrospray ionization (ESI) method. This enabled us to detect both positive and negative ions, as well as the fragmentation pattern of each intermediate or product. The LC MS analyses were performed using a Finnigan triple quadrupole mass spectrometer model TSQ 7000 and an Agilent quaternary pump HPLC model 1100, equipped with a Phenominex Luna C18 column (either 150 or 250x4.6 mm). To minimize the decomposition of labile intermediates, samples were kept cold until analysis. The mobile phases, used in the LC separation of the uric acid reaction products, included NH4OAc/AcOH and methanol (as gradient), formic ac id/methanol gradient, or NH4OAc/AcOH/acetonitrile gradient. Typical HPLC analyses were carried out in a gradient elution mode using an aqueous mobile

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51 phase A (5 mmol/L ammonium acetate and 0.1% by volume of AcOH in water) and MeOH as organic mobile phase B Mobile phase flow was 0.6 mL/min. Two typical HPLC gradients used are as follows: Gradient 1 (10 min HPLC run): the gradient began at 90% A. The composition was linearly ramped to 75% A over the next 9 min, then remained constant for 0.5 min, and then reversed to the original composition of 90% A over 0.5 min. Gradient 2 (16 min HPLC run): the gradient began at 95% A. The composition was linearly ramped to 80% A over the next 9 min and to 10% A over the next 3 min, then remained constant for 2 min, and reversed to the original composition of 95% A over 1 min, after which it was kept constant for 1 min to reequilibrate the column. Evaluation of the molecular weight and fragmentation patterns of the intermediates and products was performed using the mass spectrometer after the compounds had been separated on the LC colu Uric acid concentrations were determined in SRM mode using an APCI source in the negative mode. For uric acid, reactions were monitored at the m/z 166.9 95.9 and 166.9 123.9 usi ng 25V. In the TSQ 7000 instrument, nitrogen was used as both the sheath (80 psi) and auxiliary (10 units) gases. In the APCI mode, the vaporizer was kept at 500C, and the heated capillary temperature was maintained at 200C. The corona current was set to 3 kA by applying approximately 4 kV to the corona needle. The second quadrupole was used as a collision chamber with argon as a collision gas at a pressure around 2.5 103 Torr. The LC -MS/MS ran on Xcalibur software, which is a flexible Windows NT PC -ba sed data acquisition system that allowed complete instrument control. The operations of the LC -MS and data analyses were performed using the ThermoFinnigan Xcalibur software.

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52 In addition to the fullscan LC -MS analyses, MS/MS experiments, at 15 and 25 V, we re also conducted for M+1 ions (m/z 230.9, 245, 259), the mass corresponding to the uric acid (and its mono di -, and tri -methyl isomers) glycol dimethyl ethers. Results Electron Spin Resonance Spin Trapping. At pH 7.4, t he ESR spectra obtained from the reactions of u rate and its analogues with peroxynitrite are shown in Figure 3 2. Figure 3 2 ESR spectra of the PBN radical adducts from the reaction between uric acid and its methyl derivatives with peroxynitrite. The reaction mixture contained 3 mM ur ic acid or its analogues, 0.1 mM DTPA, 30 mM PBN and 9 mM peroxynitrite in 0.3 M phosphate buffer pH 7.4. (a) uric acid 1a, (b) 1 -methyluric acid 1b (c) 9 -methyluric acid 1c (d) 1,3 -dimethyluric acid 1d (e) 1,7 -dimethyluric acid 1e (f) 1,9 dimethyluric acid 1f (g) 3,7 -dimethyluric acid 1g, (h) 1,3,7 -trimethyluric acid 1h The reaction between urate 1a and peroxynitrite resulted in a sixline ESR spectrum with the hyperfine coupling constants: a (N) = 15.7 G, and a (H) = 4.3 G (Table 3 -1 ). All methylated uric acids that gave ESR signals displayed similar hyperfine coupling constants, ranging from 4.2 4.4 G for a (H) and 15.716.0 G for a (N). The s e are close to the observed value from the uric

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53 acid reaction, indicating that methylurates were converted to radicals, which are similar to the radicals produced by urate. In addition, the ESR spectrum obtained from 9 -methyluric acid 1c was composed of a mixture of two spectra. These spectra were characterized by a six line and a nine -line ESR spectrum, with the hyp erfine coupling constants: a (N) = 15.9 G, a (H) = 4.2 G and a (N) = 16.8 G, a (H) = 10.6 G, respectively. The latter showed a characteristic hyperfine structure of the hydrogen-PBN radical adduct. Table 3 1 Hyperfine coupling constants a (G auss ) of PBN -radi cal adducts from the reaction between uric acid and methylated uric acids with peroxynitrite at pH 7.4, and their relative ESR intensities compared to uric acid. Radicals from compound a (H) a (N) ESR Intensity (arb. unit.) Relative to Uric acida UA ( 1a ) 4. 3 15.7 1.00 1 MeUA ( 1b ) 4.4 15.9 0.61 9 MeUA ( 1c ) 4.2 15.9 0.09 9 MeUA ( 1c ) ( H adduct) 10.6 16.8 N/A 1,3 DiMeUA ( 1d ) 4.4 16.0 0.18 1,7 DiMeUA ( 1e ) N/A N/A N/A 1,9 DiMeUA ( 1f ) 4.3 15.9 0.14 3,7 DiMeUA ( 1g ) N/A N/A N/A 1,3,7 TriMeUA ( 1h ) N/A N/A N/A a Carbon based radical adducts To compare the relative radical formation quantity, the measured ESR intensities corresponded to the first PBN radical adduct peak. Methyluric acid 1b formed radical adducts that were approximately 60% of that observed wit h uric acid (Table 3 1 ). The radicals derived from urate 1c, 1d, and 1f exhibited much lower ESR intensity than 1a but still showed a noticeable number of radical adducts formed. However, the generations of radical adducts from 1e 1g and 1h (R7 = CH3) we re barely detectable, and their ESR intensities were too low to be accurately measured.

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54 Figure 3 3. LC -MS study of the reaction between uric acid and peroxynitrite in phosphate buffer pH 7.4 (A) LC chromatogram obtained from the incubation of 10 mM ur ate 0.1 mM DTPA anf 3 0 mM peroxynitrite in 0.5 M phosphate buffer pH 7.4. (B) Electrospray mass spectrum of triuret conducted in positive mode. Liquid Chromatography -Mass Spectrometry Analysis of the Reactions Conducted in Phosphate Buffer. Under positive mode, t he fullscan LC -MS analysis of the reaction between urate and peroxynitrite in phosphate buffer pH 7.4 showed a trace of mass peak of m/z 147 (Figure 3 3), which was consistent with triuret 2 a (Figure 3 4). We did not find other common urate oxidati on products such as allantoin or alloxan in our reactions. With other methylated uric acid analogues, we observed the mass peaks at 161, 175, and 189 (Figure A -1 to A 7 ), which were attributed to monomethyltriurets 2 b ; 2 c dimethyltriurets 2 d ; 2 e ; 2 f; 2 g and trimethyltriurets 2 h respectively. This indicates that the reactions of methylated uric acid derivatives upon oxidation by peroxynitrite underwent similar oxidation mechanism like urate

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55 Liquid Chromatography -Mass Spectrometry Analysis of the Reactions Co nducted in Methanol. Analyzed by LC -MS, e ach of the urate and seven methylurates individually reacted with peroxynitrite producing various products, which are s ummarized in Table 3 2, and their structures are displayed in Figure 3 4 E ach reaction procee ded to various extents (Table 3 2 ), which depended on the methylation position. The percent conversion of each reaction was obtained by measuring the peak area of the untreated starting material and comparing with the peak area of the standard solution of the starting material. When N 3 was methylated, a low percent completion was obtained (22 35%), while the reaction of urate and 1 -methylurate proceeded to 100 % completion. In addition to methylation at N 3, having methyl groups on N 7 or N 9 also decreased the percent co nversion. The LC trace and mass spectra obtained from the interaction between uric acid ( 1 a ) and peroxynitrite in methanol are shown as examples in Figure 3 5 (see Appendix B for full data set of other methylated uric acids).The fullscan MS spectra exhibited two products corresponding to the fragments of m/z 231, which is attributed to uric acid glycol dimethyl ether 3 a and m/z 173, which is consistent with allantoin derivative 5 a (Figure 3 4). Table 3 2 Reaction percent conversions and p roduct yields of uric acids with one equivalent of peroxynitrite based on LC -MS analysis of the reaction products Compound Conversion (%) Products (% Yield) 3 4 5 6 7 8 9 UA ( 1a ) 100 53.1 44.4 1 MeUA ( 1b ) 100 74.7 25.1 9 MeUA ( 1c ) 62 32.8 8.5 6.3 (i) 10.6 1,3 DiMeUA ( 1d ) 30.4 2.3 10.4 4.7 (ii) 1,7 DiMeUA ( 1e ) 72.4 37.4 19.9 11.9 1,9 DiMeUA ( 1f ) 52 36.0 0.5 9.3 6.2 (i) 3,7 DiMeUA ( 1g ) 35 3.0 10.6 10.5 1,3,7 TriMeUA ( 1h ) 22.8 2 .4 13.3

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56 Figure 3 4. Proposed structures of the reaction products obtained from the reaction between uric acid (or various methylated uric acid analogues) and peroxynitrite. The presence of methoxy groups in both products 3a and 5a is evidenced b y using d4methanol to substitute nonlabeled methanol. The ions at m/z 176 corresponding to d3-methoxy

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57 compound 5 a and m/z 237 corresponding to di -d3-methoxy adduct 3 a were observed (Figure 3 6). The losses of methanol molecules (MW 32) exhibited in MS/M S fragmentation analysis (Figure 3 7) further supported the proposed structure of the uric acid glycol dimethyl ether 3 a Similar fragmentation patterns were also found in the MS/MS experiment of the corresponding uric acid glycol di d3-methyl ether 3 a (Fi gure 3 8). Similar products found in the urate peroxynitrite reaction were observed in the reactions of other methylated uric acid analogues treated with peroxynitrite as well. The fullscan MS spectra obtained from the reaction mixtures of monomethylurate 1b and 1c displayed t he quasi ions at m/z 245, which were attributed to the methyluric acid glycol dimethyl ether 3 b and 3 c (Figure 3 4) respectively. In the case of dimethylurate 1d 1e and 1f the fragments of m/z 259 were observed and identified as th e dimethyluric acid glycol dimethyl ether; 3 d 3 e and 3 f ( Figure 3 4 ), respectively. Using the same analogy as conducted in urate reaction, t he identification s of these dimethyl ethers 3 w ere confirmed by conducting isotope -labeled experiments using d4me thanol and performing MS/MS analysis. In the presence of d4-methanol, t he fragments of m/z 251; and 265 were found in the reactions of monomethylated urates 1b 1c and dimethylated urates 1d 1e 1f respectively. Furthermore, the fragmentation analys es f rom MS/MS studies of ions m/z 251 and 265 exhibited a similar pattern ( APPENDIX C) as observed in the MS/MS spectrum of dimethyl ether 3 a (Figure 3 7 and 38) These results indicate that two methoxy groups are present in compounds with fragment ions of m/ z 245 and 259, which were consistent with dimethyl ether adducts 3 We did not observe the dimeth yl ether adducts 3 when dimethylurate 1g and trimethylurate 1h were used as reactants. However, in the reaction mixture of dimethylurate 1g, we detected a mono -methoxy adduct 4 g (m/z 229) as a minor product. A small quantity (0.5%) of 4 f was found in the reaction mixture of dimethylurate 1f

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58 Figure 3 5. LC -MS study of the reaction between uric acid and peroxynitrite in the presence of methanol. (A) The fullsc an LC chromatogram obtained from the reaction of 10 mM urate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4. The first peak was monitored at m/z 172.9 (B), corresponding to allantoin derivative 5 a The second peak was monitored at m/z 230.9 (C), corresponding to uric acid glycol dimethyl ether 3 a

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59 Figure 3 6. LC -MS study of the reaction between uric acid and peroxynitrite in the presence of d4-methanol. (A) The fullscan LC chromatogram obtained from t he reaction of 10 mM urate treated with 10 mM peroxynitrite in 50% d4-methanol and 50% aqueous phosphate buffer solution at pH 7.4. The first peak was monitored at m/z 17 6.0 (B), corresponding to d3-methoxyallantoin derivative 5 a The second peak was moni tored at m/z 237 (C) corresponding to uric acid glycol di -d3-methyl ether 3 a

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60 Figure 3 7. The fragmentation pattern of uric acid glycol dimethyl ether 3 a In addition to dimethyl ethers 3 allantoin derivatives 5 (Figure 3 4 ) were observed in most reac tion mixtures as well (except 1e ). We identified the structures of methoxy allantoin like products 5 based on the quasi ions at m/z 187 from methylurate 1b and 1c m/z 201 from dimethylurate 1d 1e 1f and 1g, and m/z 215 from trimethylurate 1h The prese nce of the methoxy group, derived from solvent methanol, was confirmed by using d4methanol. Indeed, the expected ions at m/z 190, 204, and 218 were detected. In addition to these allantoin derivatives 5 methylallantoin derivatives 6 ( Figure 3 4 ), interpr eted from m/z 187, and m/z 201, were observed when R7 = CH3; however, when R7 = H (except 1a and 1b ), the dehydroallantoins 7 corresponding to m/z 171 from methylurate 1c and m/z 185 from dimethylurate 1d and 1f were obtained.

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61 Figure 3 8. The fragment ation pattern of uric acid glycol di d3-methyl ether 3 a In addition to diadduct 3 e and allantoin derivatives 5 e and 6 e LC MS analysis of 1,7 dimethyl ur ic acid 1e exhibited m/z 245, which was tentatively identified as methyl ether 8 e We also found a trac e of an ion at m/z 246, which was identified as adduct 9 c the addition product of peroxinitrous acid to 9 -methylurate 1c We did not find the same intermediate in other reactions. Other minor products were found in some of the reactions, and their proposed structures are illustrated in Appendix B. One of the puzzling aspects of our study was the identification of multiple isomers of some of the compounds in the LC -MS analysis. For example, four isomers of dimethyl ether 3 b were identified (Figure B 1) and the MS/MS data of the four isomers were identical. These isomers comprised tautomers including an amide form and an imidic acid form. In solution, where

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62 tautomerization is possible, the chemical equilibrium of the tautomers is reached quickly (Figure 3 9 ). As long as the hydrogen exchange is relatively slow, the tautomers may be separated by HPLC and LC MS. Several examples of such separations have been reported (89). Formation of such tautomers could explain other compounds in this study exhibiting isomer formation. Figure 3 9. Example of Tautomerization of compound 2b. Discussion These studies were initiated to determine the significance of the N H group at various positions, and to determine the reactive intermediates and products formed from their reac tions with peroxynitrite. In addition, we wanted to understand the mechanistic interaction of methyluric acids with peroxynitrite as these molecules are ubiquitous in the body. Reactions in Phosphate Buffer. The methyl groups, methylated on various nitroge n position, were hypothesized to alter the course of radical adduct formation. The results indeed showed a difference when a certain hydrogen of ureide nitrogen wa s replaced by a methyl group. As shown in Figure 3 2, the intense six line ESR spectra were o bserved only if N 7 was not methylated. With methylation at N 7, the production of radical adduct was greatly diminished (Figure 3 2). Moreover, not only methylation at N 7, but methylation at N 9 ( 1c 1f ) and N 3 (1d ) also reduced the radical formations. This data indicated that the methyl group on N 7 and either on N 3 or N 9 positions of uric acid has a great impact on the reactiv ity of urate with peroxynitrite.

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63 The LC MS analysis suggested that all methylated uric acid analogues reacted with peroxynitri te and formed methylated triuret derivatives as major products. As described in Chapter 2, we hypothesized that triuret, a product generated from the oxidation of urate by peroxynitrite, could be produced from the triuretcarbonyl radical. It is possible th at methylated triurets 2 derived from N 7 methylated uric acids could be produced from their corresponding methylated triuretcarbonyl radicals as well. But these radicals may not be trapped by PBN because of the steric hindrance between the methyl group of triuret moiety and the phenyl group of PBN (Figure 3 10). As a result, there was no detected ESR signal in the reactions of N 7 methyl derivatives. Figure 3 10. The steric hindrance between the methyl group of triuret moiety and the phenyl group of PBN prevents the PBN -radical adduct formation. Reactions in Methanol. In the presence of methanol, our study documents that all methyluric acids react with peroxynitrite as determined by mass spectrometry, with different degree of conversions, depending on the position and extent of substitution (UA=MeUA > DiMeUA > TriMeUA). Replacing hydrogen with a methyl group at N 3, N -7, or N 9 retarded the reactions, giving relatively lower percent conversion. These studies suggest that the C4 C5 bond is the reaction cen ter and w e speculate that methylation at N 3, N 7, or N 9 that are adjacent to C4 C5 bond may control the stability of the intermediates, resulting to different degree of conversion s The addition of alcohols to the C4 and C5 position forming dimethyl e ther 3 and the formation of alkylated allantoin analogues (compound 5 and 6 ) suggested that an intermediate

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64 that can rapidly react with methanol and water may be produced. We propose that the reactive intermediate 12 is formed (Figure 3 11 and 3 12), pres umably via the electron transfer mechanism similar to the mechanism proposed for the oxidation of urate by peroxynitrite as described in Figure 2 12. Moreover we proposed that there are two possible structures of intermediate 12, 12(i) and 12(ii) dependi ng upon the methylation position. As reported in the study of the intrinsic acidity of uric acid and its derivatives by Telo ( 88), the N H proton at N 3 is the most acidic one followed by the N H proton at N 9. When N 3 is not methylated (i.e., in the ca se of 1a, 1b 1c 1e and 1f ), the N3H proton is deprotonated to produce anion form of compound 1a 1b 1c 1e and 1f ( Figure 3 11). The oxidation of these anion compounds by peroxynitrous acid will give intermediates 12(i) (Figure 3 11). While, in the c ase of 1d 1g, and 1h where N 3 is methylated, the deprotonation occurs at N9H (Figure 3 12). After oxidized by peroxynitrous acid, the anion forms of 1d 1g, and 1h are converted to intermediates 12(ii) The formation of 12(ii) is however highly unfavorab le because the compound 12(ii) is antiaromatic Consequently, t his could be the reason why we observed very low percent conversion in the oxidation of 1d 1g, and 1h which contain a methyl group at N 3. We speculated that intermediate 12 can undergo nucle ophilic addition with methanol to form product 3 and 5 and with water to generate methylated allantoin derivatives 6 (Figure 3 11 and 3 12). It is possible that allantoin derivatives 6 could be oxidized by peroxynitrous acid to produce the oxidized form o f the corresponding allantoins 7 However, we had a LC MS result showing that no product was formed from the reaction between allantoin 6a and peroxynitrite.

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65 Nevertheless, this result may not be the case with these methylated allantoins 6 In addition to t he oxidation of compound 6 product 7 can be generated from the process initiated by the nucleophilic addition of peroxynitrite to intermediate 12 as well (Figure 3 11 and 3 12). By the same analogy described in Chapter 2 (Figure 2 12), one would expect the formation of methylated triurets in these reactions. Although we did not observe any methylated triuret products in these reactions that contained methanol, this argument may still be valid because based on our proposed mechanism in Figure 2 12, four mol e of peroxynitrite is required to form triuret. However, in the reaction condition that we performed, we have used only one equivalent of peroxynitrite. This may be the factor that controls the progress of the reaction. Furthermore, we have shown that the oxidation of uric acid 1a and 1 -methyluric acid 1b with one equivalent of peroxynitrite went to completion Table 3 2. These results suggested that the second electron transfer from urate radical 10 could be initiated by nitrogen dioxide radical ( NO2) (Fig ure 3 11 and 3 12). Moreover, the existence of urate radical 10 is evidenced by the formation of product 4 because the only way that C 5 could form a covalent bond with hydrogen would be by a radical process. Beside the products that derived from intermed iates 12, we also detected compound 9c which is the adduct of 9-methylurate and peroxynitrous acid. A similar product to compound 9 has been proposed in the reaction between peroxynitrite and deoxyguanosine and characterized as 4,5 Dihyrdo5 hydroxy4 ( nitrosooxy) deoxyguanosine (90,91).

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66 Figure 3 11. The proposed mechanism of the reaction between urate 1a or methyl urate derivatives 1b 1c 1e 1f and peroxynitrite leading to the formation of various products. *I ntermediates 12(i) contain a positiv e charge at N 7 if N 7 is methylated.

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67 Figure 3 12. The proposed mechanism of the reaction between methylurate derivatives 1d 1g, 1h and peroxynitrite, leading to the formation of various products. *Intermediates 12(i i ) contain a positive charge at N 7 if N 7 is methylated.

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68 In our study, among the eight tested reactions, only the reaction of 1c yielded the proposed adduct 9 c Its proposed formation was initiated by a single electron transfer from uric acid to peroxynitrite. Peroxynitrite then dissociate s to the nitrogen dioxide (NO2) and the hydroxide anion (O H ) which add s to the urate free radical, generating adduct 9 c (Figure 3 13, route A ). Figure 3 13. The proposed mechanism of the formation of 9 -methylurate -peroxynitrite adduct 8c Alternat iv ely, the formation of intermedi ate 9c could also be obtained by the addition of hydroxyl (HO) and nitrogen dioxide (NO2) free radicals, which are the decomposition products from the homolytic cleavage of peroxynitrous acid ( Equation 3 2), across the C4 C5 bond (9294) (Figure 3 13, ro ute B) ONOO+ H+ ONOOH (3 1 ) ONOOH [O NO OH] OH + NO2 (3 2 )

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69 However, the formation of the two radicals according to equation 3 2 is still debated ( 46). Moreover, our ESR spin trapping experiments did not show any formation of the hydroxyl radical. Therefore we believe that the formation of adduct 9 c by the addition of hydroxyl (HO) and nitrogen dioxide (NO2) free radicals pathway (Figure 3 13, route B) is less feasible than the proposed mechanism depicted in Figure 3 13, route A Nevertheless, the reason why we did not observe this adduct in other methyl urate derivatives is unknown. Because of the complexity of these reactions, the mechanistic study of these reactions deserves further consideration.

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70 CHAPTER 4 ESR SPIN TRAPPI NG OF THE REACTION B ETWEEN URIC ACID AND PEROXYNITRITE: THE H YDROGEN ADDUCT Introduction Previously, we reported the trapping of the carbon-centered radicals from the reaction between uric acid and peroxynitrite under phosphate buffer pH 7.4. In an attempt to understand the in depth chemistry of the radical formation, we have employed the ESR spin trapping experiment to investigate the generation of the free radicals from the reaction between uric acid and peroxynitrite at various chemical conditions. Under a certain circumstance, we surprisingly found a nine line ESR spectrum, corresponding to the trapping of a hydrogen atom when PBN was used as a spin trapping agent. This intriguing finding led us to study, in more detail, the production of the hydrogen adduct (H adduct) In this report, the factors that could affect the forming of H adduct and the mechanism of its formation will be discussed. Materials and Methods Chemicals. Uric acid was purchased from Sigma. D iethylenetriaminepentaacetic acid (DTPA) was purchased from Fluka. N tert -butyl -phenylnitrone (PBN) and (4 -Pyridyl 1 oxide) N -tert -butylnitrone (POBN) w ere obtained from Alexis Biochemicals. Peroxynitrite was synthesized following the method reported by Uppu and Pryor (76). The peroxynitrite con centration was measured spectrophotometrically at 302 nm ( = 1670 M1 cm1). pH Dependence Experiments on Urate -Peroxynitrite Reactions. The ESR spin trapping of the reaction between uric acid and peroxynitrite at various pHs was performed by using 0.1 M Tris buffer with a range of pH 7.4 9.0. PBN was used as a spin trapping agent. The reaction mixtures contained: 1.5 mM uric acid, 15 mM PBN, 0.1 mM DTPA, and 6 mM peroxynitrite in 0.1 M Tris buffer.

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71 Effect of Spin Trapping Agents on the Hydrogen Adduct For mation The effect of the spin trapping agent on the formation of the hydrogen adduct was performed in 0.3 M potassium phosphate buffer pH 7.4 or 0.3 M KOH. The reaction mixtures contained: 3 mM urate, 30 mM PBN or POBN 0.1 mM DTPA, and 9 mM peroxynitrite Electron Spin Resonance Parameters T he reaction mixture was transferred into a quartz capillary of approximately 1 2 mm ID OD for ESR measurement. After two minutes, the ESR spectrum was recorded at room temperature, using a commercial Bruker Elexsys E580 spectrometer, employing Brukers highQ cavity (ER 4123SHQE). Spectral parameters were typically: 100 kHz modulation frequency, 1 G or 0. 2 G modulation amplitude, 20 mW microwave power, 9.87 GHz microwave frequency, 82 ms time constant, and 164 conversi on time/point. Results pH Dependence Studies on Urate -Peroxynitrite Reactions By using Tris -buffer, the six line ESR spectrum was the only observed ESR signal at pH 7.48.0 The ESR intensity of this six -line spectrum was increased when we raised the pH f orm 7.4 to 8.0, and started to decrease when the pH was raised to pH 8.5. At pH 9.0 the ESR spectrum exhibited an overlapping between a six line and a nine -line ESR (Figure 4 1). On the other hand, the ESR spectrum obtained from pH 9 was derived from a mi xture of a two trapped radical species The six line ESR spectrum corresponded to the trapping of carboncentered radical by PBN, while the nine -line ESR spectra with relative intensities (1:2:1:1:2:1:1:2:1) displayed a characteristic spectrum of the hydrogen PBN adduct with the hyperfine splitting a(H) = 10.68 G and a(N) = 16.64 G.

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72 Figure 4 1 The pH dependence study of the urate -peroxynitrite reaction in Tris buffer The ESR spectra obtained from the spin trapping exp eriment of the reaction between 1. 5 mM urate and 6 mM peroxynitrite in the presence of 15 mM PBN and 0.1 mM DTPA in 0.1 M Tris buffer at pH ranging from 7.4 9.0. Electron Spin Resonance Spin Trapping b y PBN and POBN The hydrogen adduct formation has been detected recently when POBN, anoth er nitrone spin trapping agent, was used (95). This prompted us to investigate whether we would obtained the same result when POBN was used as a spin trapping agent under the same reaction condition as we performed with PBN. In phosphate buffer at pH 7.4, the ESR spin trapping by PBN exhibited a six line ESR spectrum corresponding to the trapping of the carbonbased radical, which is the only observed product from the oxidation of urate by peroxynitrite (Figure 4 2), while spin trapping by POBN displayed a mixture of two radical species, a six line and a nine line spectrum (Figure 4 3). The nine -line ESR spectrum belonged to the H POBN adduct and has a much higher ESR intensity than the six line ESR spectrum which is consistent with the carbon -based radical adduct.

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73 Fi gure 4 2. E SR spectr um of the PBN radical adduct at pH 7.4 obtained from the spin trapping exp eriment of the reaction between 3 mM urate and 9 mM peroxynitrite in the presence of 30 mM P BN and 0.1 mM DTPA in 0. 3 M phosphate buffer pH 7.4 As described earlier, the pH study exhibited the formation of H adduct at basic condition (pH 9.0). We thus performed the spin trapping experiment under very basic condition, by replacing phosphate buffer with 0.3 M KOH, to investigate whether basic condition could promote the formation of the H adduct. The final pH of the solution mixture averaged around pH 12. Indeed, we observed the H adduct from both spin trapping agents, PBN and POBN at this pH. The ESR spectrum obtained from PBN exhibited a six-line ESR spectrum, corresponding to the carbon-centered radical adduct, overlapped with a nine line ESR spectrum, corresponding to the H adduct (Figure 4 4). With POBN, only the nine line ESR spectrum, which was consistent with the H adduct, was observed, and its ESR intensity was relatively much higher than PBN (Figure 4 5).

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74 Fi gure 4 3. ESR spectrum of the POBN radical adduct at pH 7.4 obtained from the spin trapping exp eriment of the reaction between 3 mM urate and 9 mM peroxynitrite in the presence of 30 mM P OBN and 0.1 mM DTPA in 0. 3 M phosphate buffer pH 7.4. Fi gure 4 4. ESR spectr um of the PBN radical adduct at pH 12 obtained from the spin trapping exp eriment of the reaction between 3 mM urate and 9 mM peroxynitrite in the presence of 30 mM P BN and 0.1 mM DTPA in 0. 3 M KOH

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75 Figure 4 5. ESR spectrum of the P O BN radical adduct at pH 12 obtained from the spin trapping experiment of the reaction between 3 mM urate and 9 mM peroxynitrite in the presence of 30 mM P O BN and 0.1 mM DTPA in 0.3 M KOH. Figure 4 6. ESR spectra of the control reactions (without uric acid). (A) the ESR spectrum obtained from the spin trapping experiment of the reaction contained 30 mM PBN, 0.1 mM DTPA, and 9 mM peroxynitrite in 0.3 M phosphate buffer pH 7.4. (B) the ESR spectrum obtained from the spin trapping experiment of the reaction contained 30 mM PBN, 0.1 mM DTPA, and 9 mM peroxynitrite in 0.3 M KOH. (C) the ESR spectrum obtained from the spin trapping experiment of the reaction contained 30 mM POBN, 0.1 mM DTPA, and 9 mM p eroxynitrite in 0.3 M KOH.

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76 Interestingly, in the control reaction without uric acid, conducted in 0.3 M KOH, the PBN H adduct was also observed with less intensity than the reaction containing uric acid (Figure 4 6). This indicates that the PBN H adduct wa s not only derived from uric acid; however, the presence of uric acid amplified the formation of H adduct. Beside PBN, POBN H adduct was detected in the control reaction without uric acid as well, and its ESR intensity was even higher than PBN H. These res ults indicate that the formation of the H adduct depends on the type of the spin trapping agent. Discussion Recently, hydrogen adducts were identified in the peroxynitrite -mediated oxidation of tetrahydrobiopterin by using spin trap, POBN (95). The auth ors reported a mixture of two spin trapped components: a six-line spectrum corresponding to a carbon -centered radical and a nine-line spectrum resulting from an H atom trapped by POBN. It was proposed that peroxynitrite promoted a homolytic cleavage of a N H bond of tetrahydrobiopterin, yielding a hydrogen radical, which could further react with POBN. We speculated that e ven though a hydrogen atom can be generated, it is unlikely that the hydrogen atom would survive long enough t o be captured by the spin tr ap POBN We postulated that hydrogen adducts may be formed by an electron transfer mechanism. Under basic condition, the electron transfer between PBN and a reducing agent could be favored as depicted in Equation 4 1.

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77 (4 1) But what could be the source of the electron? In our study, we discovered that peroxynitrite must be present in the reaction mixture in order to produce the H adduct. Without it, the reaction mixture of urate and PBN did not yield any ESR signal. This indicates that the reaction between urate and peroxynitrite must take place and it may be one of the urate -derived intermediates that provide an electron to the spin trapping agent (Equation 4 2) (4 2) This proposed mechanism could not however explain the formation of the H adduc t in the control reaction that contained only the spin trapping agents and peroxynitrite. From this reaction (the reaction without urate) we observed the H adduct formation (in both PBN and POBN) but its ESR intensity is much lower than the reaction that contained urate. Nevertheless, this observation indicates that uric acid is not necessary to be the only specie that can give up one electron to the spin trapping agents However, it is unlikely that peroxynitrite can directly reduce PBN, due to the strong oxidant property of peroxynitrite ( 45). This led us to postulate that other specie s that could transfer an electron to spin trapping agents may be formed by a different mechanism called inverted spin trapping ( 96-98).

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78 Inverted spin trapping is a process where the oxidation initially occurs at the spin trapping agent ([ST]) to form its radical cation ([ST]+ followed by the reaction with nucleophile ([Nu ]) such as water or hydroxide and thus the radical adduct is formed (Equation 4 3) (4 3 ) In basic condition without uric acid, peroxynitrite could potentially oxidize PBN (or POBN) to PBN radical cation, which is susceptible to a nucleophilic attack by hydroxide, forming a PBN OH radical adduct (Figure 4 7). However, we did not detect the existence of this PBN OH radical in our experiment. This is probably due to the instability of this radical adduct which has been reported to be unstable by hydrolysis reaction with its half -life less than one second at a pH higher than 9 .0 (99). The decomposit ion products of this process have been identified as N -tert -butylhydroaminoxyl and benzaldehye (Equation 4 4) (99 ). (4 4) We hypothesize that it could be either PBN -OH radical adduct or N tert butylhydroaminoxyl that transfers an electron to PBN giving rise to the generation of the H adduct (Figure 4 7). The existence of inverted spin trapping in our condition is further supported by the observation that POBN yielded more H adduct than PBN both in the presence or absence of uric acid. Comparing to PBN, one would predict that POBN may have less potential for oxidation than PBN because of the presence of another nitroxide (N O) group, which can increase the electron withdrawing ability of POBN.

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79 However, the electrochemical study of spin traps by McIntir e et al. ( 100) discovered that in fact, PBN is harder to oxidize than POBN. This can be rationalized by the fact that the conjugation between the two nitroxide functions of POBN helps to stabilize the radical cation that is formed after oxidation. On the other hand, POBN radical cation exhibits the number of resonance structures more than PBN (Figure 4 8). As a consequence, POBN is more susceptible toward oxidation by peroxynitrite than PBN, causing more pronounced hydrogen adduct formation according to the inverted spin trapping scheme. Figure 4 7. Mechanism scheme shows the possible pathway of the PBN -H adduct formation.

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80 Figure 4 8. The resonance stabilization of POBN and PBN radical cations.

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81 CHAPTER 5 CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK Conclusions By using ESR spin trapping coupled with LC MS, we have discovered two urate derived radicals trapped by a spin trap, PBN, from the peroxynitrite -mediated urate oxidation. The structures of the trapped radicals were characterized as the aminoca rbonyl radical, which has previously been described ( 36), and the new finding radical triuretcarbonyl radical, which we proposed to be the intermediate for the formations of aminocarbonyl radical and triuret. Observed by ESR, the radical formation was pH d ependent and exhibited lower yield when CO2 is present. These results indicate that peroxynitrite anion is responsible for the production of the urate derived radicals. The generation of this radical intermediate may be responsible for the deleterious effe ct of urate in a biological system. In addition to uric acid, we have extended our study to investigate the peroxynitrite mediated oxidation with various methylated uric acid isomers. We have reported the first mass spectrometric analysis of these reactio ns In phosphate buffer pH 7.4, t he major products from these reactions were methyl triuret dervatives, which were proposed to be produced by radical processes as evidenced by ESR spin trapping results. Methylation at N 7 did not yield any ESR signal; howe ver, the radicals may still be produced, but the formation of the radical adducts are not favored due to the steric effect (Figure 3 10). To trap the reaction intermediate, the reactions were purposely conducted in methanol. The extent of the reactions depended on the position and extent of the substitution (urate = methylurate > dimethylurate > trimethylurate). The administration of methanol resulting in the formation of d imeth yl ether s 3 and allantoin derivatives 5 (Figure 3 4 ) indicated that diimine a c ommon intermediate found in various urate oxidation conditions, was formed (Figure 3 11 and 3 12) Methylation at N -3, N 7, and N 9

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82 could all reduce the extent of the reaction as compared to the reaction with uric acid, suggesting that the C4 -C5 bond of th e uric acid scaffold is the initial reaction site The ability of these methylated uric acid analogues, which exist in the human body, to quench peroxynitrite may prove to be important in the ir metabolism Under a very basic condition, the hydrogen adduct was formed as a major product from the reaction between uric acid and peroxynitrite studied by ESR spin trapping, and it could be observed even without urate. Without uric acid, its formation is proposed to undergo a nontraditional spin trapping process c alled inverted spin trapping mechanism, followed by the electron transfer between spin traps and the PBN -OH (or POBN OH) adduct or its decomposition product. The H adduct formation is amplified when uric acid and peroxynitrite are present. However, the exa ct nature of this amplification by urate is still unknown. Different yields of H adducts obtained from various spin traps supported the inverted spin trapping mechanism and can be rationalized by the redox potential of the spin traps. Suggestions for Futur e Work It would be of interest to determine whether a urate radical resulting from one electron oxidation by peroxynitrite is actually formed. Although, the direct detection of a urate radical has been reported in various oxidizing conditions ( 32,100,101), the peroxynitrite -mediated oxidation of urate is still not known. To detect a very short l ived radical, one can use a rapid mixing/continuous flow set up coupled with ESR. This would allow enough accumulation of the free radicals to reach the ESR detectio n limit. It would also be of interest in terms of urate peroxynitrite mechanism to determine if one of the oxygen of triuret is derived from peroxynitrite as proposed in Figure 2 12. One can synthesize peroxynitrite with 18O isotope labeled. The oxygen att ached to C 2 of triuret should be labeled (Equation 51).

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83 (5 1) Based on the finding of the hydrogen adduct, it would be worthwhile to investigate the H adduct formation in the presence of 1,1,1,3,3,3 -hexafluoropropan 2 ol (HFP), which is a solvent rep orted to suppress the effect on the inverted spin trapping mechanism (101). If the H adduct formation is indeed generated by inverted spin trapping mechanism, its yield should be decreased when HFP is present Another interesting topic for uric acid chemist ry is the complex formation between uric acid and transition metals. It has been shown that uric acid can form complexes with transition metal ions such as Cd(II) and Pb(II) (102). Little is known if transion metal ions can stabilize urate derived radicals Our preliminary results showed that an EPR signal was observed from the incubation of uric acid with peroxynitrite, in the presence of Cd(II) or Zn(II) (Appendix D).Unfortunately, we could not identify the structures of these complexes because of their p oor solubility. It is necessary to investigate the method to purify these complexes for further structural characterization. The oxidant antioxidant paradox of uric acid is still a major interest among researchers. Therefore, the chemistry of uric acid, w ith oxidant like peroxynitrite, deserves further investigation to complete the jigsaw that is still missing from its mechanism scheme.

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84 APPENDIX A LC CHROMATOGRAMS AND ELECTROSPRAY MASS SPECTRA OBTAINED FROM THE REACTIONS OF VARIOUS METHYLATED URIC ACIDS WITH PEROXYNITRITE CONDUCTED IN PHOSPHATE BUFFER Figure A 1 LC MS study of the reaction between 1 -methyl uric acid and peroxynitrite in phosphate buffer pH 7.4 (A) LC chromatogram obtained from the incubation of 10 mM 1 -methyl urate 0.1 mM DTPA anf 3 0 mM peroxynitrite in 0.5 M phosphate buffer pH 7.4 (B) Electrospray mass spectrum of triuret conducted in positive mode.

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85 Figure A 2 LC MS study of the reaction between 9 -methyl uric acid and peroxynitrite in phosphate buffer pH 7.4 (A) LC chromatogram obtained from the incubation of 10 mM 9 -methyl urate 0.1 mM DTPA anf 3 0 mM peroxynitrite in 0.5 M phosphate buffer pH 7.4 (B) Electrospray mass spectrum of triuret conducted in positive mode.

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86 Figure A-3. LC MS study of the reaction between 1,3 -dimeth yl uric acid and peroxynitrite in phosphate buffer pH 7.4 (A) LC chromatogram obtained from the incubation of 10 mM 1,3 dimethylurate 0.1 mM DTPA anf 3 0 mM peroxynitrite in 0.5 M phosphate buffer pH 7.4 (B) Electrospray mass spectrum of triuret conducted in positive mode.

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87 Figure A-4. LC MS study of the reaction between 1,7 -dimethyl uric acid and peroxynitrite in phosphate buffer pH 7.4 (A) LC chromatogram obtained from the incubation of 10 mM 1,7 dimethylurate 0.1 mM DTPA anf 3 0 mM peroxynitrite in 0 .5 M phosphate buffer pH 7.4 (B) Electrospray mass spectrum of triuret conducted in positive mode.

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88 Figure A 5 LC MS study of the reaction between 1,9 -dimethyl uric acid and peroxynitrite in phosphate buffer pH 7.4 (A) LC chromatogram obtained from the incubation of 10 mM 1,9 dimethylurate 0.1 mM DTPA anf 3 0 mM peroxynitrite in 0.5 M phosphate buffer pH 7.4 (B) Electrospray mass spectrum of triuret conducted in positive mode.

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89 Figure A 6 LC MS study of the reaction between 3,7 -dimethyl uric acid a nd peroxynitrite in phosphate buffer pH 7.4 (A) LC chromatogram obtained from the incubation of 10 mM 3,7 dimethylurate 0.1 mM DTPA anf 3 0 mM peroxynitrite in 0.5 M phosphate buffer pH 7.4 (B) Electrospray mass spectrum of triuret conducted in positive mode.

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90 Figure A 7 LC MS study of the reaction between 1,3,7 trimethyl uric acid and peroxynitrite in phosphate buffer pH 7.4 (A) LC chromatogram obtained from the incubation of 10 mM 1,3,7 trimethyl urate 0.1 mM DTPA anf 3 0 mM peroxynitrite in 0.5 M ph osphate buffer pH 7.4 (B) Electrospray mass spectrum of triuret conducted in positive mode.

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91 APPENDIX B LC CHROMATOGRAMS OBTAINED FROM THE REACTIONS OF VARIOUS METHYLATED URIC ACIDS WITH PEROXYNITRITE CONDUCTED IN METHANOL Figure B-1. LC chromatogram of 10 mM 1-methylurate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4.

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92 Figure B-2. LC chromatogram of 10 mM 9-methylurate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4.

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93 Figure B-3. LC chromatogram of 10 mM 1,3-dimethylurate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4.

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94 Figure B-4. LC chromatogram of 10 mM 1,7-dimethylurate treated wi th 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4.

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95 Figure B-5. LC chromatogram of 10 mM 1,9-dimethylurate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4.

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96 Figure B-6. LC chromatogram of 10 mM 3,7-dimethylurate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4.

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97 Figure B-7. LC chromatogram of 10 mM 1,3,7-trimethylurate treated with 10 mM peroxynitrite in 50% methanol and 50% aqueous phosphate buffer solution at pH 7.4.

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98 APPENDIX C FRAGMENTATION PATTER NS OF DIMETHOXYDEHYDROURIC ACIDS Figure C 1 Fragmentation pattern of 1 -methyluric acid glycol dimethyl ether 3 b

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99 Figure C 2 Fragmentation pattern o f 1 -methyluric acid glycol di -d3-methyl ether 3 b

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100 Figure C 3 Fragmentation pattern of 9 -methyluric acid glycol dimethyl ether 3 c

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101 Figure C 4 Fragmentation pattern of 9 -methyluric acid glycol di -d3-methyl ether 3 c

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102 Figure C 5 Fragmentation pattern of 1,3 dimethyluric acid glycol di -d3-methyl ether 3 d

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103 Figure C-6. Fragmentation pattern of 1,7 dimethyluric acid glycol dimethyl ether 3e

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104 Figure C 7 Fragmentation pattern of 1,7 dimethyluric acid glycol di -d3-methyl ether 3 e

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105 Figure C-8. Fragmentation pattern of 1,9 dimethyluric acid glycol dimethyl ether 3f.

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106 Figure C 9 Fragmentation pattern of 1,9 dimethyluric acid glycol di -d3-methyl ether 3 f.

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107 APPENDIX D EFFECT OF DIVALENT METAL IONS ON THE REACTION BETWEEN URIC ACID AND P EROXYNITRITE Introduction We extended our study to investigate the effect of divalent metal ions on the stability of the urate derived radicals generated from the reaction between ur ic acid and peroxynitrite at pH 12 The nine divalent metal ions, Ca (II), Zn (II), Mg (II), Cu (II), Ni (II) Co (II) Fe (II) Cd (II) and Mn (II) were tested and monitored by X band EPR. Materials and Methods Chemicals Uric aid was purchased from Sigma. L (+) -Ascorbic Acid was purchased from Fisher and isoamyl nitrite was purchased from Acros Organics. All of the divalent metals used were in the form of chloride salts and purchased from Fisher Urate stock solutions were prepared with 0.3 M potassium hydroxide (100 mM urate). Peroxynitrite was synthesized according to Uppu and Pryor (76). The peroxynitrite stock solution concentration was measured spectrophotometrically at 302 nm ( = 1670 M1 cm1) Electron Paramagnetic Resonance Parameter s EPR spectra were recorded at room temperature using a Bruker Elexsys E580 spectrometer employ ing Brukers high Q cavity (model) and using quartz capillaries of approximately 1 mm ID. Spectral parameters were typically: 100 kHz modulation frequency, 1 G modulation amplitude, 2 mW microwave power, 9.87 GHz microwave frequency, 20.48 ms time constan t and 81.92 conversion time/point. All spectra were recorded after the addition of peroxynitrite. Reaction Mixtures All reaction Mixtures contained a solution of 5 mM of urate and 25 mM of metal ions. Peroxynitrite was added to these solutions to 15 mM fi nal concentration.

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108 Results and Discussion Of all the metal ions we tested. Only the reactions in the presence of Zn (II) and Cd(II) gave EPR signals. The precipitates were formed from both reactions. After filtration, it was clearly shown that the EPR sign als came from the precipitates. As shown in Figure D 1, the Cd(II) spectrum consists of a single intense line at g = 2.0149, while the Zn (II) spectrum displays a similar line shape with g = 2.0124 (Figure D 2). Both g -values are consistent with carbon cent ered radical s Interestingly, when ascorbate was added to the reaction mixture, the EPR s ignal was diminished completely (Figure D 3) and the doublet EPR spectrum, corresponding to the ascorbyl radical, was formed. We postulated that the EPR spectra arose from the formation of the complexes between urate derived radicals, generated from the reaction between uric acid and peroxynitrite, and metal ions, Zn(II) and Cd(II) However, we still need to do additional experiments to identify the structures of these complexes.

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109 Figure D 1 EPR spectra of the urate Cd(II) complex obtained by incubating uric acid (5 mM), CdCl2 (25 mM) and peroxynitrite ( PN ) (15 mM) at room temperature at pH 12. The reaction mixture was filtered to separate the precipitate from the fi ltrate and monitored by EPR separately. Spectrometer settings: microwave power = 2 mW; modulation frequency = 100 kHz; modulation amplitude = 1 GHz; time constant = 20.48 ms; sweep time = 83.89 s; sweep width = 100 G; and receiver gain = 60 db.

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110 Figure D 2 EPR spectra urate Zn(II) complex obtained by incubating uric acid (UA) (5 mM), ZnCl2 (25 mM) and peroxynitrite ( PN ) (15 mM) at room temperature at pH 12. The reaction mixture was filtered to separate the precipitate from the filtrate and monitored b y EPR separately. Spectrometer settings: microwave power = 2 mW; modulation frequency = 100 kHz; modulation amplitude = 1 GHz; time constant = 20.48 ms; sweep time = 83.89 s; sweep width = 100 G; and receiver gain = 60 db.

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111 Figure D 3 Effect of ascorb ate on the urate -Zn(II) complex formation. EPR spectra obtained by incubating urate ( 5 mM), ZnCl2 (25 mM) and peroxynitrite ( PN ) (15 mM) at room temperature at pH 12. An aliquot amount of the reaction mixture was withdrew to examine the stability of the ra dical complexes in the presence of ascorbate ( 1 mM) and monitored by EPR ( ). Spectrometer settings: microwave power = 19.97 mW; modulation frequency = 100 kHz; modulation amplitude = 1 GHz; time constant = 20.48 ms; sweep time = 83.89 s; sweep width = 54 G; and receiver gain = 60 db.

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120 BIOGRAPHICAL SKETCH Wit cha Imaram was born in Ratchaburi, a province in the western part of Thailand. Awarded by t he Institute for the Promotion of Teaching Science and Technology, he received the Development and Promotion of Science and Technology Talent Project (DPST) scholars hip to complete his high school education at Sriboonyanon School, Nonthaburi, Thailand in 1999, and a Bachelor of Science (1st class honours) degree in c hemistry from Kasetsart University, Bangkok, Thailand in 2003. After graduation, he received a Thai sc holarship to pursue his Ph.D. degree at the University of Florida in Fall 2003. In his graduate research work, he joined Dr. Alexander Angerhofer s research group to elucidate the mechanisms involved in the biological effects of uric acid, using E S R spin t rapping methods to probe the formation of reactive radical species derived from uric acid