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Free Radical-Induced Oxidation of Docosahexaenoate Lipids

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Free Radical-Induced Oxidation of Docosahexaenoate Lipids CLINICAL AND ANIMAL STUDIES OF LIPID-DERIVED PROTEIN MODIFICATIONS IN AUTISM, KIDNEY DIALYSIS, KERATITIS AND AGE-RELATED MACULAR DEGENERATION by LIANG LU Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. Robert G. Salomon Department of Chemistry CASE WESTERN RESERVE UNIVERSITY August 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. This thesis is dedicated to my parents, my husband, my daughter, and my sisters. iii TABLE OF CONTENTS Table of Contents iv List of Schemes ix List of Tables xi List of Figures xiv Acknowledgements xxiv List of Abbreviations and Acronyms xxvi Abstract xxxiii CLINICAL AND ANIMAL STUDIES OF LIPID-DERIVED PROTEIN MODIFICATIONS IN AUTISM, KIDNEY DIALYSIS, KERATITIS AND AGE-RELATED MACULAR DEGENERATION Chapter 1. Introduction 1 1.1. Oxidative stress and aging 2 1.2. Lipid oxidation 4 1.3. 4-Hydroxy-2-nonenal and its protein adducts 6 1.4. Levuglandins, isolevuglandins and their protein adducts 7 1.5. Oxidatively truncated phospholipids and carboxyalkylpyrrole modifications of proteins 10 1.6. Carboxyethylpyrroles (CEPs) and their potential clinical applications 11 1.7. References 17 Chapter 2. Syntheses and Characterization of Carboxyethylpyrroles 27 2.1. Background 28 2.2. Results and Discussion 30 2.2.1. Paal-Knoor synthesis using 4,7-dioxoheptanoic acid is ineffective iv for the preparation of CEPs 30 2.2.2. Synthesis of a 9-fluorenylmethyl (Fm) ester of DOHA 31 2.2.3. Syntheses of CEP-peptide and CEP-protein adducts by Paal-Knoor synthesis with DOHA-Fm 31 2.2.4. CEP linked to proteins with an ω-aminohexanoyl tether 34 2.2.5. Syntheses of biotinylated CEP derivatives 36 2.2.6. Syntheses of ethanolamine phospholipid CEP derivatives 39 2.2.7. Synthesis of an active pentafluorophenyl ester of a lysyl CEP 39 2.2.8. Characterization of CEP-modified protein 40 2.2.9. Tertiary protein structures of CEP modified HSA 41 2.3. Conclusions 43 2.4. Experimental Procedures 44 2.5. References 70 Chapter 3. Lipid Oxidative Modifications in Autistic Disease 74 3.1. Background 75 3.2. Materials and Methods 82 3.3. Results and Discussion 84 3.3.1. CEP and iso[4]LGE2 protein adducts immunoreactivity in human plasma 84 3.3.2. Presence of anti-CEP and anti-iso[4]LGE2 autoantibodies in human plasma 87 3.3.3. Protein bound nitrotyrosine, chlorotyrosine and bromotyrosine levels in ASD and healthy controls 90 3.3.4. Correlations among lipid oxidative protein adducts and nitrative proteins 92 v 3.3.5. Correlations between autoantibodies and lipid oxidation immunoreactivities 94 3.3.6. Lipid oxidation products and birth events in ASD patients 96 3.4. Conclusions 99 3.5. Experimental Procedures 101 3.6. References 112 Chapter 4. Serum Vitamin E and Oxidative Protein Modification in Hemodialysis 117 4.1. Background 118 4.2. Results 120 4.3. Discussion 128 4.4. Conclusions 133 4.5. Experimental Procedures 134 4.6. References 150 Chapter 5. Identification of Carboxyethylpyrrole Phosphatidylethanolamine Adducts in vitro and in vivo 161 5.1. Background 162 5.2. Results and Discussion 168 5.2.1. Syntheses of authentic samples 168 5.2.2. Immunoreactivity of authentic CEP-PE 168 5.2.3. Identification of CEP-PE in oxidized lipid extracts 169 5.2.4. Identification of CEP-PE in lipid extracts that had not been oxidized in vitro 170 vi 5.2.5. Quantification of CEP-PE in bovine retina 175 5.2.6. Identification of lysoCEP-PE in light-promoted oxidized lipid extracts 175 5.2.7. Identification of lysoCEP-PE in bovine retina 180 5.2.8. Identification of lysoCEP-PE in human plasma 180 5.2.9. Quantification of lysoCEP-PE in bovine retina and human plasma 183 5.3. Conclusions 185 5.4. Experimental Procedures 187 5.5. References 196 Chapter 6. Pilot Studies towards Identification of Levuglandin Modified Proteins in Cornea 203 6.1. Introduction 204 6.1.1. LPS and inflammation 204 6.1.2. The cyclooxygenase and isoprostane pathways 204 6.1.3. LGE2 and iso[4]LGE2 bind avidly with proteins 207 6.1.4. Pyridoxamine, a potent trap that prevents protein modification by reactive electrophiles 207 6.2. Results and Discussion 209 6.3. Experimental Procedures 212 6.4. References 214 Chapter 7. Syntheses of Isosteric Pyrazole Derivatives 221 7.1. Background 222 7.2. Results and Discussions 225 7.2.1. Synthesis of a 1,3-dione 225 vii 7.2.2. Synthesis and structure identification of 1-alkyl-5-pentylpyrazole-3- carboxylic acid 225 7.2.3. Decarboxylation of a pyrazole carboxylic acid 227 7.2.4. Condensation of an alkylhydrazine with a β-ketoaldehyde 227 7.2.5. Attempted ozonolysis of an alkehylpyrazole 228 7.2.6. A strategy for conjugation of the isostere hapten with peptides 229 7.3. Experimental Procedures 230 7.4. References 236 Appendix 242 Bibliography 303 viii LIST of SCHEMES Chapter 1 Scheme 1.1. Cyclooxygenase oxidation of AA generates PGs and LGs via rearrangement of PGH2. 8 Scheme 1.2. Free radical-induced oxidation of AA-PC produces isoLGs by rearrangement of isoP intermediates. 8 Scheme 1.3. Formation of LG-protein adducts, protein-protein and DNA-protein crosslinks. 10 Scheme 1.4. Oxidation of polyunsaturated fatty acids generates hydroxy-ω- oxoalkenoic acids, that react with proteins and form 2-(ω- carboxyalkyl)pyrroles. 12 Chapter 2 Scheme 2.1. DOHA exists in equilibrium with a spiroacylal hemiacetal. 30 Scheme 2.2. Synthesis of 9H-fluoren-9-ylmethyl ester 4,7-dioxo-heptanoic acid (DOHA-Fm, 2.4). 31 Scheme 2.3. Synthesis of CEP-dipeptide. 31 Scheme 2.4. Syntheses of CEP protein adducts. 33 Scheme 2.5. Syntheses of CEPs bound to proteins through a tether. 35 Scheme 2.6. Synthesis of biotinylated CEP derivative 2.21. 38 Scheme 2.7. Synthesis of biotinylated CEP derivative 2.25. 38 Scheme 2.8. Syntheses of ethanolamine phospholipid CEP derivatives. 39 Scheme 2.9. Synthesis of a lysyl CEP pentafluorophenyl derivative. 40 Chapter 5 Scheme 5.1. 4,5-(E)-Epoxy-2-(E)-heptenal reacts with phosphatidylethanolamine to produce pyrrole derivatives. 166 Scheme 5.2. Synthesis of CEP-PE and lysoCEP-PE. 168 ix Chapter 6 Scheme 6.1. Representative pathways showing enzyme and free radical induced generation of LGE2 and iso[4]LGE2 from AA-PC. 206 Scheme 6.2. Possible mechanism of pyrrole formation between pyridoxamine and 1,4-dicarbonyls. 208 Chapter 7 Scheme 7.1. Synthesis of 1-alkyl-5-pentylpyrazole through a decarboxylation route. 225 Scheme 7.2. General scheme of Barton decarboxylation. 227 Scheme 7.3. An alternative route for synthesis of pyrazole 7.5. 228 Scheme 7.4. Reductive amination of proteins with pyrazole aldehyde 7.6 that is to be generated by ozonolysis of alkene 7.5. 229 x LIST OF TABLES Chapter 2 Table 2.1. Pyrrole concentration (μM) generated in 1 mg/mL CEPH-BSA prepared by coupling various initial protein concentrations with various CEPFmSu/Lys ratios. 36 Table 2.2. ELISA data for CEPH-BSA of Figure 2.1. 69 Table 2.3. ELISA data for CEP-HSA of Figure 2.1. 69 Chapter 3 Table 3.1. ELISA data for iso[4]LGE2-HSA standard for Figure 3.6. 103 Table 3.2. ELISA data for ASD patient (patient code A27) for Figure 3.6. 103 Table 3.3. ELISA data for control (normal code N11) for Figure 3.6. 104 Table 3.4. Data for CEP, iso[4]LGE2 immunoractivites and CEP, iso[4]LGE2 autoantibodies in ASD patients. 105 Table 3.5. Data for CEP, iso[4]LGE2 immunoractivites and CEP, iso[4]LGE2 autoantibodies in normal controls. 106 Table 3.6. Nitrotyrosine, chlorotyrosine and bromotyrosine levels in autistic patients. 109 Table 3.7. Nitrotyrosine, chlorotyrosine and bromotyrosine levels in normal controls. 110 Chapter 4 Table 4.1. Baseline clinical characteristics of patients. 120 Table 4.2. Plasma levels of alpha and gamma tocopherol in placebo and vitamin E treated patients. 122 Table 4.3. Plasma glycoxidation and protein-lipid oxidation products in placebo and vitamin E treated patients. 124 Table 4.4. Linear regression of glycoxidation and protein-lipid oxidation products with alpha or gamma tocopherol at baseline. 126 Table 4.5. Linear regression of iso[4]LGE2 with alpha or gamma tocopherol during placebo treatment at 3 months, 6 months and 9 months. 126 xi Table 4.6. Linear regression of iso[4]LGE2 with alpha or gamma tocopherol during during treatment with α-tocopherol at 3 months, 6 months and 9 months. 127 Table 4.7. Linear regression matrix for glycoxidation and protein-lipid oxidation products at baseline. 127 Table 4.8. α-Tocopherol levels (μg/mL) in plasma from patients treated with vitamin E at baseline, three months, six months, nine months and twelve months. 136 Table 4.9. α-Tocopherol levels (μg/mL) in plasma from HD patients treated with placebo at baseline, three months, six months, nine months and twelve months. 137 Table 4.10. γ-Tocopherol levels (μg/mL) in plasma from patients treated with vitamin E at baseline, three months, six months, nine months and twelve months. 138 Table 4.11. γ-Tocopherol levels (μg/mL) in plasma from HD patients treated with placebo at baseline, three months, six months, nine months and twelve months. 138 Table 4.12.
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