LIPID-BASED OXIDATIVE PROTEIN MODIFICATIONS IN ...

LIPID-BASED OXIDATIVE PROTEIN MODIFICATIONS IN GLAUCOMA

by

ANNANGUDI PALANI SURESH BABU

Submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Thesis Advisor: Dr. Robert G. Salomon

Co-Advisors: Dr. John W. Crabb

Dr. Sanjoy K. Bhattacharya

Department of Chemistry

CASE WESTERN RESERVE UNIVERSITY

January 2006

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.

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This thesis is dedicated to my Mom and Dad

Banumathi & Palani

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TABLE OF CONTENTS

Table of Contents v

List of Schemes xii

List of Tables xvi

List of Figures xvii

Index of Appendix xxiii

Acknowledgements xxvii

List of Abbreviations and Acronyms xxviii

Abstract xxxii

Lipid-Based Oxidative Protein Modification in Glaucoma

Chapter 1. Introduction 1

1.1 Background 2

1.2 HNE as an important lipid peroxidation product 3

1.3 Levuglandins – discovery, formation and pathology 8

1.4 Anatomy of anterior section of the eye 13

1.5 Glaucoma 15

1.5.1 Primary open angle glaucoma (POAG) 16

1.5.2 Secondary glaucomas 16

1.6 Animal models for glaucoma. 17

1.7 Glaucoma and oxidative stress 18

1.8 References 22

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Chapter 2. A Short and Efficient Synthesis of 4-Oxo-2-alkenoic

Acids from 2-Alkylfurans 25

2.1. Background 26

2.1.1. Previous synthesis of γ-keto α,β-unsaturated alkenoates 27

2.2. Results and discussion 33

2.3. Conclusions 40

2.4. Experimental procedures 41

2.5. References 51

Chapter 3. Detection and Characterization of Multiple 4-

Hydroxynonenal Adducted Amino acids Using

Deuterium Labeled HNE and Mass Spectrometry 54

3.1. Background 55

3.1.1. Previous syntheses of deuterated HNE 58

3.2. Results 62

3.2.1. Synthesis of [8,8,9,9-2H4]-HNE 62

3.2.2. Reactions of N-acetyl amino acids with HNE 64

3.2.3. Mass spectrometric analysis of the HNE-amino acid adducts 64

3.3. Discussion 85

3.4. Conclusion 101

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3.5.1. General methods 103

3.6. References 115

Chapter 4. Oxidative Protein Modifications in the Pathogenesis

of Primary Open Angle Glaucoma 118

4.1 Background 119

4.1.1. Oxidative stress and glaucoma 119

4.1.2. Levuglandins and isolevuglandins: formation and pathobiology 122

4.1.3. 4-Hydroxynonenal and its role in pathobiology 124

4.1.4. Advanced glycation end products and their pathobiology 125

4.1.5. Oxidative products of tryptophan and α-hydroxykynurenine 127

4.2 Results 129

4.2.1. Immunoblot analysis of TM tissues for analyzing the levels of

oxidative protein modifications in glaucomatous TM compared to

the controls 129

4.2.2. Localization of modified proteins by immunohistochemical

analysis 132

4.2.3. Immunoprecipitation of modified proteins 133

4.2.4. Identification of immunoprecipitated proteins 134

4.2.6. Identification of modified proteins by two-dimensional gel

electrophoresis 136

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4.3 Discussion 139

4.4 Experimental procedures 148


4.4.2. Tissue procurement 148

4.4.3. Protein extraction 149

4.4.4. Western analysis 149

4.4.5. Histochemical analysis 150

4.4.6. Immunoprecipitation 151

4.4.7. 2D Gel electrophoresis 151

4.4.8. LC MS/MS analysis and protein identification 152

4.5 References 154

Chapter 5. Iso[4]LGE2 Modified Proteins in Trabecular

Meshwork of Glaucomatous DBA/2J mice 159

5.1. Background 160

5.1.1 Oxidative stress and inflammation pathways 160

5.1.2. Different forms of secondary glaucoma 162

5.1.3. DBA/2J, mouse model for pigmentary glaucoma 163

5.1.4. Iso[4]Levuglandin E2 – formation and pathology 164

5.2. Results 167

5.2.1. Clinical examination of DBA/2J mice 167

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5.2.2. Increase in levels of iso[4]LGE2 modified proteins in DBA/2J

with age 169

5.2.3. Immunohistochemical localization of iso[4]LGE2 modified

proteins 171

5.2.4. Immunoprecipitation of iso[4]LGE2-modified proteins 172

5.2.5. Identification of modified proteins using LC-MS/MS 174

5.3. Discussion 176


5.4.1. Tissue procurement 180



5.4.4. Immunohistochemistry 181

5.4.5. Immunoprecipitation 182

5.4.6. LC MS/MS analysis 182

5.5. References 184

Chapter 6.

Part A: Pilot Studies Towards Identification of Levuglandin

Modified Proteins in Macrophages

Part B: Initial Studies Towards Developing a Model System

to Differentiate Enzyme Mediated and Free- Radical

Mediated Formation of Levuglandins 187

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6.1. Background 188

6.1.1. Macrophages, inflammation and atherosclerosis 188

6.1.2. Macrophages and COX-2 188

6.1.3. Lipopolysaccharides (LPS) and cyclooxygenase (COX)

expression 190

6.1.4. Distinguishing levuglandins and isolevuglandins 191

6.2. Results 194

PART A

6.2.1. LPS stimulates formation of LGE2 and iso[4]LGE2 modified

proteins in mouse peritoneal macrophage cell cultures

194

6.2.2. Separation and identification of modified protein by 2D PAGE

and mass spectrometry 196

6.2.3. Immunohistochemical analysis of macrophages 197

PART B

6.2.4. Protein modifications in LPS treated cornea – 1D SDS PAGE 200

6.2.5. Immunohistochemical analysis 201

6.3. Discussion PART A 203

6.3. Discussion PART B 205

6.4. Experimental Procedures 206




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6.4.4. Histochemical analysis 207

6.4.5. 2D gel electrophoresis 208

6.4.6. LC MS/MS analysis and protein identification 208

6.5. References 210

Appendix (See p. xxiii for Index of Appendix) 213

Thesis conclusion 243

Bibliography 245

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LIST OF SCHEMES

Chapter 1

Scheme 1.1: Proposed mechanisms for the formation of HNE from LA. 4Scheme 1.2: Adducts formed by HNE with amino acid side chain

residues. 5Scheme 1.3: Proposed mechanism for the formation of Michael adduct,

Schiff base, amine-HNE (2:1) crosslink adduct and pyrrole adduct. 6

Scheme 1.4: The free radical mediated oxidation of AA-PC and LA-PC leading to the formation of HOOA-PC and HODA-PC that are analogous to HNE and their subsequent reaction with lysine side chain residues to form the corresponding pyrrole adducts, 2-(ω-carboxypropyl) pyrrole (CPP) and 2-(ω-carboxyheptyl) pyrrole (CHP). 7

Scheme 1.5: Formation of prostaglandin endoperoxides from arachidonic acid. 9

Scheme 1.6: Formation of levuglandins from prostaglandin endoperoxides. 9

Scheme 1.7: Representative pathways showing a difference in enzyme and free radical mediated pathways generating LGs from AA-PC. 10

Scheme 1.8: Formation of levuglandins from arachidonyl phosphatidylcholine esters by free radical mediated processes. 11

Scheme 1.9: Formation of isolevuglandin and levuglandin based protein adducts. 12

Scheme 1.10: Daughter ion generated by LG-lysine lactam in the mass spectrometric ionization process. This feature was used in tandem MS/MS to identify the LG modified proteins. 13

Chapter 2

Scheme 2.1: Suggested mechanism for generation of KOdiA-PC and

KDdiA-PC. 26Scheme 2.2: Natural products incorporating 4-oxo-2-alkenoate

functionality. 27Scheme 2.3: 2-Alkoxyfuran as precursor for 4-oxo-2-alkenoate. 28Scheme 2.4: 2-Acylfuran as precursor for 4-oxo-2-alkenoate. 28Scheme 2.5: 2-Siloxyfuran as precursor for 4-oxo-2-alkenoate. 29Scheme 2.6: 2-Silylfuran and 2-siloxyfuran oxidation using dimethyl

dioxirane. 29

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Scheme 2.7: Oxidation of a 2-alkylfuran using pyridinium chlorochromate. 30

Scheme 2.8: Oxidation of 2-alkylfuran using N-bromo succinimide. 30Scheme 2.9: Methods reported for synthesis of a pyrenophorin

precursor: a) using Jones reagent with furyl precursor; b) using (3+2) cyclo-addition of primary nitro group and methyl acrylate, c) using NBS to oxidize a furyl precursor. 31

Scheme 2.10: A patented process for generation of 4-oxo-2-alkenoic acids by Takeya. 32

Scheme 2.11: Synthetic method for generating 2-alkylfurans from furan. 33

Scheme 2.12: Synthesis of the 4-oxo-2-alkenoic acid functional array. 34Scheme 2.13: Ring-chain tautomers of 4-oxo-2-pentenoic acid. 34Scheme 2.14: Comparison of synthetic methods used to generate 4-oxo-

2(E)-alkenoic acids from 2-alkylfurans. 35Scheme 2.15: Synthesis of 4-oxo-2-alkenoic acids from 2-alkylfurans. 36 Scheme 2.16: Synthesis of pyrenophorin precursor from 2-alkylfuran

2.20. 37Scheme 2.17: Synthesis of KOdiA-PC and KDdiA-PC by furan

oxidation protocols employing two different solvent conditions. 38

Scheme 2.18: Synthesis of 1,4-enediones from 2,5-dialkylfurans. 39

Chapter 3

Scheme 3.1. Synthesis of [2H1]-HNE reported by Sugamoto’s group. 59Scheme 3.2. Synthesis of [2H3]-HNE and [2H11]-HNE from

fumaraldehyde dimethylacetal. 60Scheme 3.3. Synthesis of [8,8,9,9-2H4]-HNE. 63Scheme 3.4. Synthesis of HNE. 63Scheme 3.5. Proposed mass spectrometric fragmentation of a 1:1 N-

acetyl histidine - HNE Michael adduct parent ion at m/z 354. See Scheme 3.2S in appendix for fragment structures. 66

Scheme 3.6: Possible Michael adduct HNE - (N-acetyl-Gly-Lys-OMe) fragmentation. See appendix Scheme 3.4S for fragment structures. 72

Scheme 3.7: Possible Schiff base HNE- (gly-lys) Schiff base fragmentation. See appendix Scheme 3.5S for fragment structures. 73

Scheme 3.8: Possible fragmentation of the molecular ion m/z 380 corresponding to an HNE-(N-acetyl-Gly-Lys-OMe) pyrrole adduct. See appendix Scheme 3.6S for fragment structures. 75

Scheme 3.9: Structure of (N-acetyl-Gly-Lys-OMe)–HNE (2:1) 76

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crosslink proposed by Xu et. al. Scheme 3.10: Suggested mode of fragmentations for N-acetyl cysteine

dimer m/z 324. See Scheme 3.7S in appendix for fragment structures. 80

Scheme 3.11: Fragmentation of the molecular ion m/z 302 arising from the dehydration of N-acetyl-Cys-HNE Michael adduct. See Scheme 3.8S in appendix for fragment structures. 81

Scheme 3.12: Possible isomers of the m/z 510 (2:1) HNE-N-acetyl-Cys adduct. 89

Scheme 3.13: Possible fragmentation of the putative parent ion m/z 510 to generate a unique daughter ion at m/z 310 and 408. 90

Scheme 3.14: Possible mass spectrometric cleavage sites of adduct at 510 m/z with a structure 3.19. See Scheme 3.9S in appendix for fragment structures. 91

Scheme 3.15a: Possible fragmentations of the m/z 666 3:1 (HNE/N-acetyl-His) adduct. Inset (tandem MS/MS of the parent ions m/z A) 510, B) 674, C) 670 and D) 666 in the range m/z 394-402). See Scheme 3.10S in appendix for fragment structures. 93

Scheme 3.15b: Possible structures of daughter ions formed by retro-Michael cleavage of the 3:1 parent ion m/z 666. 93

Scheme 3.16: One of the possible structures and the mass spectrometric fragmentation for the molecular ion at m/z 728 (3:1, HNE/(N-acetyl-Gly-Lys-OMe) adduct). See Figure 3.10 for mass spectra. 96

Scheme 3.17: Daughter ions formed from the tandem MS/MS of 2:1 N-acetyl-Cys: HNE adduct A) d0-HNE-2N-acetyl-Cys adduct B) d4-HNE-2N-acetyl-Cys adduct. 98

Scheme 3.18: Daughter ions formed from the tandem MS/MS of 2:2 (N-acetyl cysteine: HNE) adduct m/z 603, 607 and 611.

99Chapter 4

Scheme 4.1: Enzymatic (cyclooxygenase) and non enzymatic (free

radical) routes for the formation of LGE2 and iso[4]LGE2 and their protein adducts. 123

Scheme 4.2: Some of the commonly reported adducts formed by 4-hydroxynonenal reaction with histidine, lysine and cysteine residues. 124

Scheme 4.3: Mechanistic pathway for the formation of AGE’s and argpyrimidine. 126

Scheme 4.4: Catabolic pathway of tryptophan under oxidative conditions. 128

Chapter 5

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Scheme 5.1: Diagrammatic representation of activation and expression of proteins involved in inflammation pathway by oxidative stress. Details of the scheme are discussed in the text. (ROS – reactive oxygen species, COX-2 – cyclooxygenase-2, LO – lipooxygenase, NF-кB – nuclear factor- kappa B, IL-18 – interleukin 18, TNF – tumor necrosis factor). 161

Scheme 5.2: Generation of levuglandins from arachidonic acid by enzyme mediated and free radical mechanisms (Detailed scheme presented in Scheme 4.1.2, page 4-4). 165

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LIST OF TABLES

Chapter 2

Table 2.1: Yield and reaction time for alcohol protected alkyl furans in Scheme

2.15. 36Table 2.2: Synthesis of 2-ene-1,4-diones from 2,5-dialkyl furans (Scheme 2.16) 39

Chapter 3

Table 3.1: Nucleophilic functional groups in LDL 57Table 3.2: Isotopic mass abundances of some common elements 61Table 3.3: Optimized parameters for mass spectrometer. 111

Chapter 4

Table 4.1: Total number of tissue samples used for Western blots for each of

the antibodies probed. Some of the tissues were used in more than one Western blot analysis. 129

Table 4.2: Identification of TM proteins immunoprecipitated using iso[4]LGE2 and HNE pAbs. A. Proteins identified by in-gel digestion of bands from lane A-2 (Figure 4.9) using mass spectrometry (LC-MS/MS). B. Proteins identified by in-gel digestion of bands from lane B-2 (Figure 4.9) using mass spectrometry (LC-MS/MS). 136

Table 4.3: Iso[4]LGE2 and HNE immunoreactive proteins identified by 2D PAGE and mass spectrometry. Calculated MW and pI refers to value derived from Swissprot protein database; Measured MW and pI refers to value from the 2D PAGE. 138

Table 4.4: List of crosslinked proteins identified by 2D PAGE and mass spectrometry of glaucomatous TM. 145

Chapter 5

Table 5.1: Proteins immunoprecipitated from TM extract of DBA/2J mice

using iso[4]LGE2-pAb. Gel slices from top to the bottom of lane 4 of the gel in Figure 5.6 were digested using trypsin and the proteins identified using LC-MS/MS and MassLynx™ software with the Swissprot database. 174

Chapter 6

Table 6.1: List of LGE2 – modified proteins electrophoretically separated by 2D gel electrophoresis and identified by mass spectrometry. 197

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LIST OF FIGURES

Chapter 1

Figure 1.1: Anatomy of human eye showing the anterior and posterior regions (Copyright© www.nie.nih.gov). 14

Figure 1.2: Artistic rendering of anterior section of the eye, showing pathway of aqueous flow through the trabecular meshwork (arrow). (Copyright© www.nie.nih.gov) 14

Figure 1.3: Flow chart illustration of the components involved in the pathogenesis of POAG induced by decrease of vitamin E in the ciliary body. MMP – matrix metalloproteinases, TIMP – tissue inhibitor of metalloproteinases, ON – optic nerve, ECM – extracellular matrix. (Copyright obtained from Br. J. Nutrition, CABI Publishing)62 20

Chapter 2

Figure 2.1: UV-spectrograph of 4-oxo-2-pentenoic acid at varing pH. 35

Chapter 3

Figure 3.1: ESI-TOF-MS of reaction mixture containing HNE (d4:d0, 1:1) with amino acids A) N-acetyl-gly-lys-OMe, B) N-acetyl-cysteine and C) N-acetyl-histidine. 65

Figure 3.2: ESI-TOF-MS/MS analysis of Michael adducts parent ions at m/z 354 and 358 from the infusion of reaction mixture from HNE and N-acetyl-His (1:1). 66

Figure 3.3: ESI-MS/MS of molecular ions at A) m/z 518, B) m/z 514 and C) m/z 510, from the infusion of reaction mixture containing HNE and N-acetyl-His. Peaks labeled with an asterix (*) or a solid rectangle (■) denote fragments with or without the C5-C9 alkyl chain of HNE. 67

Figure 3.4: ESI-MS/MS of molecular ions A) m/z 674, B) m/z 670 and C) m/z 666 from infusion of reaction mixture HNE and N-acetyl-His (1:1). 68

Figure 3.5: ESI-LC-SIR for the reaction mixture from N-acetyl-His and HNE with 13 channels of which 7 channels are shown here. A) m/z 670, B) m/z 666, C) m/z 518, D) m/z 514, E) m/z 510, F) m/z 358 G) m/z 354 and H) TIC (for 13 channels).(for chromatograms of channels not shown here, see appendix figure 3.1S) 70

Figure 3.6: ESI-MS/MS analysis of m/z 260 molecular ion. Tandem MS/MS analysis of a solution of N-acetyl-glycine-lysine-OMe was injected into a Q-TOF by infusion at a flow rate of 0.5 µL/min. 71

Figure 3.7: ESI-MS/MS of molecular ions at A) m/z 416 and B) m/z 420 corresponding to a 1:1 HNE-(N-acetyl-Gly-Lys-OMe) Michael adduct. 72

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Figure 3.8: ESI-MS/MS analysis of molecular ions A) m/z 402 and B) m/z 398 corresponding to an HNE- (N-acetyl-Gly-Lys-OMe) Schiff base adduct. 73

Figure 3.9: ESI-MS/MS analysis of molecular ions at A) m/z 384 and B) m/z 380 corresponding to the HNE- (N-acetyl-Gly-Lys-OMe) pyrrole adduct 74

Figure 3.10: ESI-MS/MS analysis of molecular ions A) m/z 656 and B) m/z 652 corresponding to the 1:2 (HNE:(N-acetyl-Gly-Lys-OMe)) crosslink. 76

Figure 3.11: ESI-MS/MS analysis of molecular ions corresponding to the 2:1 (HNE:(N-acetyl-Gly-Lys-OMe)) adducts A) m/z 580, B) m/z 576, C) m/z 572 and the 3:1 (HNE:(N-acetyl-Gly-Lys-OMe)) adducts D) m/z 736, E) m/z 732 and F) m/z 728 G) m/z 740. 77

Figure 3.12: ESI-LC-SIR for the (N-acetyl-Gly-Lys-OMe) and HNE reaction mixture monitored through 17 channels of which the spectrum of 7 channels are shown here (Chromatograms for other channels in presented in appendix). LC-SIR chromatograms for molecular ions at A) m/z 732 (M+4 of 3:1 adduct), B) m/z 728 (M of 3:1 adduct). C) m/z 576 (M+4 of 2:1 adduct), D) m/z 572(M of 2:1 adduct); E) m/z 416 (M of 1:1 Michael adduct); F) m/z 398 (Schiff base adduct); G) m/z 380 (M of 1:1 pyrrole adduct); and H) total ion chromatogram (TIC). (See appendix figure 3.3S for M+8 and M+12 of 3:1 adducts and M+4 of Michael, Schiff base and pyrrole 1:1 adducts) 78

Figure 3.13: ESI-MS/MS analysis for N-acetyl cysteine. 79Figure 3.14: ESI-MS/MS analysis for N-acetyl cysteine dimer at m/z 325. 79Figure 3.15: ESI-MS/MS analysis of the dehydrated N-acetyl-Cys–HNE Michael

adduct. 81Figure 3.16: ESI-MS/MS analysis for molecular ion at m/z A) 469 and B) 465

corresponding to HNE-N-acetyl-Cys (1:2) adduct. 82Figure 3.17: ESI-MS/MS analysis of the ions with A) m/z 611, B) m/z 607 and C)

m/z 603, which correspond to 2:2 N-acetyl-Cys-HNE adducts. 83Figure 3.18: ESI-LC-SIR analysis of the molecular ions observed in N-acetyl-Cys-

HNE reaction mixture through 8 channels. (Chromatograms shown for 8 channels: A) m/z 611, B) m/z 607, C) m/z 603, D) m/z 469, E) m/z 465, F) m/z 346, G) m/z 342 H) m/z 324 m/z and I) TIC. 84

Figure 3.19: Tandem MS/MS of the parent ions A) m/z 358, B) m/z 354, C) m/z 518, D) m/z 514 and E) m/z 510 in the m/z range 207-212 and 238-244. Only the (2:1) HNE/N-acetyl-His adducts C, D and E shows unique daughter ions at m/z 209, 213 and m/z 239, 243. 90

Figure 3.20: Tandem MS/MS of the parent ions at A) m/z 358, B) m/z 354, C) m/z 518, D) m/z 514 and E) m/z 510 in the mass range m/z 309-316. 91

Figure 3.21: Fragmentation of parent ions at A) m/z 518, B) m/z 514 and C) m/z 510 showing the presence of daughter ions m/z 408 and/or m/z 412. 92

Figure 3.22: Relative amounts of adducts formed by N-acetyl-His/HNE reaction, calculated using LC-SIR. The amounts reflect the relative amount (of the adducts monitored) of each of the adduct present in the reaction mixture. Values are average of 2 independent experiments and the

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error bars indicate half the difference between the experimental values. 94

Figure 3.23: Relative amounts of adducts formed by (N-acetyl-Gly-Lys-OMe)/HNE reaction, calculated using LC-SIR. The ‘y’ scale reflects percentage of the each adduct present in the reaction mixture (with respect to the adducts monitored). Values are average of 2 independent experiments and the error bars indicate half the difference between the experimental values. 97

Figure 3.24: Relative amounts of adducts formed by N-acetyl-Cys and HNE reaction, calculated using LC-SIR. The ‘y’ scale reflects percentage of the each adduct present in the reaction mixture (with respect to all the adducts monitored). Values are average of 2 independent experiments and the error bars indicate half the difference between the experimental values. 100

Figure 3.25: A) ESI-MS/MS of CHD derived HNE B) ESI-MS/MS of CHD derived d4-HNE. 110

Chapter 4

Figure 4.1: Diagrammatic representation of the cross section of an eye showing the two forms of glaucoma defined according to the difference in the angle of the anterior chamber formed by lines drawn parallel to the iris and cornea (iridial angle). A. normal open angle (~40o) associated with POAG and B. a closed angle (~15o) associated with angle closure glaucoma. 120

Figure 4.2: Diagrammatic representation of the cross section of the human eye showing the aqueous humor outflow pathway. 121

Figure 4.3: Western analysis of POAG and normal trabecular meshwork using anti-iso[4]LGE2 pAb antibodies. TM extracts (5 µg) were subjected to SDS-PAGE, blotted to PVDF membrane and probed with anti-iso[4]LGE2 antibodies. A,C,E . Coomassie blue stained gels. B,D,E. Western blot; Age, race and gender of the tissue samples are indicated (M-Male; F-Female; W-Caucasian; B-African American). 130

Figure 4.4: Western analysis of POAG and normal trabecular meshwork using anti-HNE pAb antibodies. TM extracts (5 µg) were subjected to SDS-PAGE, blotted to PVDF membrane and probed with anti- anti-HNE antibodies. A,C,E. Coomassie blue stained gels. B,D,E. Western blot. M-Male; F-Female; W-Caucasian; B-African American). 130

Figure 4.5: Western analysis of POAG and normal trabecular Meshwork using anti-argpyrimidine mAb. TM extracts (5 µg) were subjected to SDS-PAGE, blotted to PVDF membrane and probed with anti-argpyrimidine antibodies. A,C,E. Coomassie blue stained gels. B,D,E. Western blot. Age, race and gender of the tissue samples are indicated (M-Male; F-Female; W-Caucasian; B-African American). 131

Figure 4.6: Western Analysis of POAG and Normal Trabecular Meshwork using

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anti-OHKYN mAb. TM extracts (5 µg) were subjected to SDS-PAGE, blotted to PVDF membrane and probed with anti-OHKYN antibodies. Age, race and gender of the tissue samples are indicated (M-Male; F-Female; W-Caucasian; B-African American). 131

Figure 4.7: Localization of iso[4]LGE2 modified proteins in glaucomatous TM. Anterior segment histochemical sections through the trabecular meshwork are shown. A, B, C – Probed with pre-immune serum; D, E, F,G, H, I – Probed with anti-iso[4]LGE2 antibody; Red Channel – iso[4]LGE2 specific immunofluorescence ; Green Channel – autofluorescence 132

Figure 4.8: Localization of HNE modified proteins in glaucomatous TM. Anterior segment histochemical sections through the trabecular meshwork are shown. A, B, C – Probed with pre-immune serum; D, E, F,G, H, I – Probed with anti-HNE antibody; Red Channel – HNE specific immuno-fluorescence; Green Channel – auto-fluorescence 133

Figure 4.9: Immunoprecipitation of antibody modified protein from TM extract. 10µg of antibody coupled beads and 30µg of TM proteins were used for immunoprecipitations. A. Anti-iso[4]LGE2 antibody B. Anti-HNE antibody .Lane 1 – Protein extract, Lane 2 – IP, Lane 3 – Ab coupled beads, Lane 4 – control-protein A beads, Lane 5 – Western blot of protein extract. 134

Figure 4.10: 2D PAGE analysis of TM extract. ~80 µg of extracted TM proteins was used for the 2D PAGE. The gel was partially transferred onto a PVDF membrane for immunochemical analysis. A. Coomassie stained gel B. Western analysis using Iso[4]LGE2 pAb. C. Western analysis using HNE pAb. D. Merged image showing Coomassie stain in black and Western blots probed with iso[4]LGE2 in blue; HNE in orange. 137

Figure 4.11: Quantification of immunoreactive bands in 1D Western blots. Intensity for each of the lanes detected (all the bands in the lane included) in the Western blots was normalized with respect to the total of all the bands detected in that blot (details of calculations in the experimental section). The minimum intensity for band detection was fixed for each of the blots. ◊ – Relative intensity of a control sample on the blot; □ – OD of a POAG tissue on the blot. A. Levels of iso[4]LGE2 modified proteins (tissue donors – 25 POAGs, 25 controls), B. Levels of HNE modified proteins (tissue donors – 24 POAGs, 25 controls). ‘p’ values calculated by students t-test using Microsoft ® Excel 2003. 141

Figure 4.12: Quantification of immunoreactive bands in 1D Western blots. Relative intensity of optical density (OD) for each of the lanes (all the bands in each lane included) in the Western blots was normalized for each of the blots (8 lanes each). The minimum intensity for band detection was fixed for each of the blots. ◊ – Relative intensity of a control sample on the blot; □ – relative intensity of a POAG tissue on the blot. A. Levels of argpyrimidine modified proteins (tissue donors – 25 POAGs, 25 controls). B. Levels of OHKYN modified proteins (tissue donors – 8

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POAGs, 8 controls). ‘p’ values calculated by students t-test using Microsoft ® Excel 2003. 142

Chapter 5 Figure 5.1: Diagrammatic illustration of sagital and transverse sections of the eye

(not drawn to scale). The sagital section passes through the longitudinal axis of the eye from the anterior to the posterior region. The transverse section passes perpendicularly axis to the sagital axis. 167

Figure 5.2. Anterior segment assessment of DBA/2J mice. The anterior segment pictures were taken with a Leica MZ stereomicroscope (Leica Microsystems Inc., Bannockburn, IL) equipped with a SpotCam RT KE digital camera (Sterling Heights, MI). A. 14-month-old, B. 8-month-old and C. 6-month-old DBA/2J mouse. 168

Figure 5.3. Histochemical assessment of mouse optic nerve. The mouse eye sections (10 µm) passing though optic nerve were stained with hematoxylin. A. 8 months old DBA/2J mouse (x 20) and C. a section through the optic nerve (x 100) B. age matched C57BL6J control mice (x 20) and D. a section through the optic nerve (x 100). A,B – Sagital sections; C,D – Transverse sections. 169

Figure 5.4. Western analysis of TM proteins. TM proteins (~5 µg/lane) were subjected to 1D SDS/PAGE, electroblotted onto a poly(vinylidene difluoride) membrane, and probed with anti-iso[4]LGE2 antibodies. Bovine serum albumin modified with iso[4]LGE2 was used as a positive control. A. Immunoblot of DBA/2J and C57BL6J TM proteins B. Coomassie stained immunoblot.C. Densitometric quantification of the Western blot. 170

Figure 5.5. Histochemical localization of isoLGE2 modified proteins in TM. Representative immunohistochemical analyses with a rabbit polyclonal anti-isoLGE2 are shown. Rhodamine conjugated secondary antibody was used for detection. Immunofluoresence. A. DBA/2J mice TM, 8 months (green channel) B. DBA/2J mice TM, 8 months(red channel) C. C57BL6J mice TM, 8months (green channel). D. C57BL6J mice TM, 8 months (red channel). 172

Figure 5.6: Immunoprecipitation of oxidatively modified TM proteins from 8 month old DBA/2J mice. Immunoprecipitations (Ips) utilized antibody coupled beads (10 µg) and TM protein extracts (10 µg). They were analyzed by SDS-PAGE and mass spectrometry. Coomassie blue stained gels are shown: lane 1, low molecular weight marker; lane 2, antibody coupled beads without TM; lane 3, TM protein extract (5 µg); lane 4, IP products; lane 5, wash containing proteins post IP. 173

Figure 5.7. Proteasome crosslinked in 8-month-old DBA/2J mice. A. Coomassie stained SDS PAGE (truncated Figure 5.4). B. Immunoblot probed with anti-rabbit proteasome polyclonal antibody (arrow shows the position of high molecular weight band immunoreactive for the proteasome antibody). 179

xxii

Chapter 6

Figure 6.1: Schematic description of one of the factors contributing to foam cell f ormation through endocytosis of oxLDL by macrophages. 189

Figure 6.2: LGE2-modified proteins in LPS stimulated macrophages. A. Coomassie stained SDS PAGE proteins from macrophages. B. Western blot analysis for LGE2 immunoreactivity in the electroblotted PVDF membrane. C. Densitometric quantification of the total immunoreactive bands in each of the lanes (as relative intensity). 1- control macrophages, 2 – LPS (10 µg/mL) treated macrophages. 194

Figure 6.3: Iso[4]LGE2-modified proteins in LPS stimulated macrophages. A. Coomassie stained SDS PAGE of proteins from macrophages. B. Iso[4]LGE2 immunoreactivity of the electro-blotted PVDF membrane. C. Densitometric quantification of the total immunoreactive bands in each of the lanes (as relative intensity). 195

Figure 6.4: Separation of proteins extracted from macrophages treated with LPS. A. Western blot using anti-LGE2-KLH polyclonal antibody. B. Coomassie blue stained 2D PAGE. 196

Figure 6.5: Immunohistochemical analysis of macrophages for LGE2 modified proteins. macrophages grown to confluency were plated on to a glass slide and treated with or without LPS. A. Cells treated with LPS and stained with pre-immune serum. B. Controls cells stained with anti-LGE2-KLH pAb. C. Cells treated with LPS stained with anti-LGE2-KLH pAb D. Cells treated with LPS along with indomethacin for 24 h and stained with anti-LGE2-KLH pAb. 198

Figure 6.6: Western blot using anti-LGE2 pAb of C57BL/6 mouse corneal protein extract (2 µg) treated with PBS or LPS. Lane 1. Naïve cornea. Lane 2. PBS treated cornea. Lane 3. LPS (1 µg/mL) treated cornea. 200

Figure 6.7: Relative amount of LGE2 immunoreactivity. Densitometric quantification of immunoreactive bands of the Western blots probed with LGE2-pAb. Naïve – corneal proteins of untreated C57BL/6 mouse, PBS – corneal proteins of mice treated with PBS, LPS - corneal proteins of mice treated with LPS (10 µg/mL). Error bars indicate standard deviation for three experiments. 201

Figure 6.8: Cornea of C57BL/6 mouse was treated with LPS or PBS in vivo and preserved after 6 h and 24 h. Sections (5 µm) of cornea was stained with LGE2 and iso[4]LGE2 pAb. Naïve corneal sections were used as additional controls. A,B,C – Naïve cornea; D,E,F – PBS treated (24 h); G,H,I – LPS treated (6 h); J,K,L – LPS treated (24 h). Panels treated with preimmune, LGE2 and iso[4]LGE2 pAb’s are indicated in the figure

202

xxiii

Index of Appendix

List of Figures

Figure 2.1S: a) 13C NMR and b) 1H NMR of 2.14 214Figure 2.2S: 1H NMR of 2.38 (crude) 215Figure 2.3S: a) 13C NMR and b) 1H NMR of 2.37 215Figure 2.4S: a) 13C NMR and b) 1H NMR of 2.17 216Figure 2.5S: a) 13C NMR and b) 1H NMR of 2.38 217Figure 2.6S: 1H NMR of 2.21 218Figure 2.7S: a) 13C NMR and b) 1H NMR of 2.22 219Figure 2.8S: 1H NMR of 2.39 (crude) 220Figure 2.9S: a) 13C (APT) NMR and b) 1H NMR of sec-butyl ester of 2.40 221Figure 2.10S: 1H NMR of 2.27 (crude) 222Figure 2.11S : 1H NMR of 2.26 223 Figure 3.1S : a) 1H NMR and b) 13C NMR of 3.3 224Figure 3.2S : 2H NMR of 3.4 226Figure 3.3S : a)1H NMR and b) 13C NMR of 3.6 225Figure 3.4S: 2H NMR of 3.7 227Figure 3.5S : a) 1H NMR and b) 13C NMR of 3.7 228Figure 3.6S: How to read a mass spectra? 229Figure 3.7S: ESI-TOF-SIR of N-acetyl-His:HNE reaction mixture.

Channels shown here m/z 696, m/z 692, m/z 688, m/z 684, m/z 678 and m/z 674. 232

Figure 3.8S: ESI-TOF-SIR of N-acetyl-Gly-Lys-OMe:HNE reaction mixture. Channels shown here are from m/z 384, 402, 420, 580, 640, 644, 652, 656, 736 and 740. 233

Figure 3.9S: ESI-TOF-SIR of N-acetyl-His:HNE reaction mixture. Channels shown here are from channels monitored at m/z 320, m/z 326, m/z 352, m/z 356 and m/z 360. 235

Figure 4.1S: Western blot analyses of normal and POAG trabecular

meshwork using anti-iso[4]LGE2 antibodies as described in Section 4.4.4. 238

Figure 4.2S: Western blot analyses of normal and POAG trabecular meshwork using anti-HNE antibodies as described in Section 4.4.4. 238

xxiv

Figure 4.3S: Western blot analyses of normal and POAG trabecular meshwork using anti-argpyrimidine antibodies as described in Section 4.4.4. 239

Figure 4.3S: Immunoprecipitation of POAG trabecular meshwork using anti-iso[4]LGE2 pAb and preimmune serum antibodies as described in Section 4.4.6 239

Figure 5.1S: Western blot of TM proteins from DBA/2J mouse of different

age groups. 5 µg of protein was electrophoretically separated on a SDS PAGE and electroblotted on a PVDF membrane and probed with iso[4]LGE2 pAb. Time course of DBA/2J mice. A. Western blot. B. Coomassie stained SDS PAGE. 240

Figure 5.2S: Immunoprecipitation of trabecular meshwork proteins from a 8 month old DBA/2J mouse using anti-iso[4]LGE2 pAb and preimmune serum antibodies as described in Section 5.4.6. 241

Figure 6.1S: LGE2-modified proteins in LPS stimulated macrophages.

Western blot analysis for LGE2 immunoreactivity in the electroblotted PVDF membrane. 242

xxv

List of Tables

Table 3.1: Summary of tandem MS/MS fragments incorporating the C5

alkyl chain of HNE derived from the precursor ions 354, 358, 510, 514, and 510 thus showing the 4 Da difference. 237

xxvi

List of Schemes

Scheme 3.1S: Fragmentation of N-acetyl-Histidine 229Scheme 3.2S: Fragmentation of (N-acetyl-His)-HNE Michael adduct 230Scheme 3.3S: Suggested fragmentation of (N-acetyl-His)-HNE Michael

(1:1) adduct. 230Scheme 3.4S: Suggested fragmentation of (N-acetyl-Gly-Lys-OMe)-HNE

Schiff base adduct 231Scheme 3.5S: Fragmentation of (N-acetyl-Gly-Lys-OMe)-HNE pyrrole

adduct 231Scheme 3.6S: Fragmentation of (N-acetyl-Cys)-(N-acetyl-Cys) disulphide

adduct 234Scheme 3.7S: Fragmentation of (N-acetyl-Cys)-HNE Michael adduct 234Scheme 3.8S: Suggested fragmentation of (N-acetyl-His)-HNE 1:2 adduct 236Scheme 3.9S: Suggested fragmentation of (N-acetyl-His)-HNE 1:2 adduct 236

xxvii

Acknowledgements

I wish to express my deepest sense of respect and gratitude to Dr. Robert G.

Salomon for his expert guidance, constant encouragement, and advice that helped me to

become a professional researcher over the years.

I also wish to express my deepest thanks to Dr. John W. Crabb, Cole Eye

Institute, Cleveland Clinic Foundation, for giving me a chance to work in his lab and

helped me in learning basic protein chemistry and mass spectrometry. I am indebted to

Dr. Sanjoy K. Bhattacharya for his expert guidance, many insightful discussions and

importantly friendship. He taught me basic molecular biology and related biological

techniques that has enormously helped in completing two projects that are in chapter 4 &

5. Special thanks also to Dr. Podrez (CCF) and Dr. Eric Carlson for their collaboration as

well as friendship.

I thank all my people in the lab, Nathan (my buddy), Xiaorong Gu, Jaiyin Gu,

Bharathi, Jack, Karen and Bogdan for for their friendship and enormous help.

I would like to thank my labmates at CASE - Jim laird, Wujuan, Liang Lu, Xi

Chen, Wei Li, Xiaodong Gu and Jaewoo for their help on many situations and for their

friendship.

I would like to dedicate this thesis to my parents as this could be virtually

impossible without their hard work and sacrifices.

xxviii

LIST OF ABBREVIATIONS AND ACRONYMS

Abbreviations and Acronyms Equivalent AA arachidonic acid AA-PC arachidonyl phosphatidylcholine AcOH acetic acid AD Alzheimer’s disease AGE advanced glycoxidation end products ALS amyotrophic lateral sclerosis AMD age-related macular degeneration Apo B apolipoprotein b APT attached proton test AS atherosclerosis BHT butylated hydroxytoluene BSA bovine serum albumin CapLC capillary liquid chromatography CCD charge coupled device CDCl3 deutrated chloroform CHCA α-cyano-4-hydroxy-cinnamic acid CHCl3 chloroform CHD cyclohexa-1,4-dione CHP 2-(ω-carboxyheptyl) pyrrole Cys cysteine d4-HNE [8,8,9,9-2h4]-4-hydroxynonenal DAPI 4', 6-diaminidophenylindole DCC dicyclohexylcarbodiimide DHA-PC 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-

phosphatidylcholine DMAP 4-dimethylaminopyridine DMF N,N-dimethyl formamide DMP dimethyl pimelimidate DTPA diethylenetriaminepentaacetate DTT dithiotheoritol EDTA ethylenediaminetetraacetate EI electrospray ionization ESI-MS electrospray ionization mass EtOAc ethyl acetate

xxix

FAB fast atom bombardment GFAP glial fibrillary acidic protein GSH glutathione HHE 4-hydroxy-2-hexenal HNE (E)-4-hydroxy-2-nonenal HPLC high performance liquid chromatography HRMS high resolution mass spectrometry HSA human serum albumin HSP heat shock protein Hz hertz IACUC institutional animal care and use committees IEF isoelectric focusing IgG immunoglobin g TGF- βIGH3 transforming growth factor β inducing gene h3 IL-18 interleukin-18 IOP intraocular pressure IP immunoprecipitation IPD iris pigmentary dispersion IPG iris pigmentary glaucoma ISA iris stromal atrophy iso[4]LGE2 iso[4]levuglandin E2

iso[n]LGs iso[n]levuglandin(s) isoLG(s) isolevuglandin(s) isoLGE2 isolevuglandin E2

ITIC integrated total ion current J hyperfine coupling constant kDa kilo Dalton KHdiA-PC 1-palmitoyl-2-(7-carboxy-4-oxohex-5-enoyl)-sn-

glycero-3-phosphatidylcholine KLH keyhole limpet hemocyanin KODA-PC 1-palmitoyl-2-(9-oxo-12-oxododec-10-enoyl)-sn-

glycero-3-phosphatidylcholine KYN kyneurinin LA linoleic acid LC liquid chromatography LC-ESI-SIR liquid choromatograpy electrospray ionization selected

ion recording LC-MS liquid choromatograpy mass LDL low-density lipoprotein LG levuglandins MALDI matrix assisted laser desorbtion ionization MALDI-TOF matrix assisted laser desorption ionization-time of flight

xxx

MDA malondialdehyde Me methyl MeOH methanol MG methylglyoxal MHz megahertz MMP-1 matrix metalloproteinases MRM multiple reaction monitoring MS/MS tandem mass spectrometry MW molecular weight NAC N-acetyl cysteine NAH N-acetyl histidine NAL N-acetyl lysine NBS N-bromosuccinimide NDRI National Disease Research Interchange NF-kB necrosis factor –kb NGL N-acetyl glycine-lysine methyl ester NL nonlinear NMR nuclear magnetic resonance OCT optimal cutting temperature OHKYN 3-hydroxykynurenine ON optic nerve oxLDL oxidized low density lipoprotein oxPC oxidized phosphatidylcholines p probability pAb polyclonal antibody PAF platelet activation factor PBS phosphate buffered saline PC phosphatidylcholine PCC pyridinium chlorochromate PDC pyridinium dichromate PDI-A3 protein disulphide isomerase a3 PG prostaglandins PGH2 prostaglandin H2

PI4K phosphoinisitol-4-kinase POAG primary open angle glaucoma ppm parts per million PPTS pyridinium p-toluenesulfonate PUFAs polyunsaturated fatty acid(s) PVDF polyvinylidene fluoride Q-TOF quadrapole time-of-flight Rf retention factor

xxxi

ROS reactive oxygen species RP-HPLC reverse phase high performance liquid SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel SIR selected ion recording SOD superoxide dismutase SPE solid phase extraction TBAF tetrabutylammonium fluoride TBARS thiobarbituric acid-reacting substance TBDMS tert-butyldimethylsilyl TBDMSCl tert-butyldimethylsilyl chloride t-BHT tert-butylated hydroxytoluene TBS tris buffered saline TBST 20 mM tris, 150 mm NaCl, ph 7.5, 0.5% tween 20 t-Bu t-butyl TGF transforming growth factor THF tetrahydrofuran TIC total ion chromatogram TLC thin layer chromatography TM trabecular meshwork TNF tumor necrosis factor UV ultraviolet

xxxii

Lipid-Based Oxidative Protein Modifications in Glaucoma

Abstract

by

Annangudi Palani Suresh Babu

We and others have postulated that oxidative protein modifications, including

covalent crosslinks, may accumulate in trabecular meshwork (TM) and contribute to

impaired aqueous outflow and primary open angle glaucoma (POAG). To test this

hypothesis, human TM from normal and POAG donors was probed with antibodies for

oxidative protein modifications. Studies using SDS PAGE and Western blotting showed

elevated levels of covalent protein modifications derived from lipid oxidation products

(iso[4]Levuglandin E2 (isoLGE2) and 4-hydroxy-nonenal (HNE)), an advanced glycation

end product (methylglyoxal), and a tryptophan oxidation product (3-hydroxy-

kynurenine), in POAG compared to age-and-gender matched controls.

Immunoprecipitation (IP) using iso[4]LGE2 and HNE antibodies showed the presence of

several apparently crosslinked proteins in POAG donor TM. 2D PAGE Western analyses,

also showed proteins with altered molecular weights and pIs, implying modified and/or

crosslinked proteins. Immunohistochemistry showed that iso[4]LGE2 and HNE

modifications were localized to the TM. Thus, the study provides direct evidence for

lipid-based oxidative modification of TM proteins in POAG. Additionally, increased

levels of iso[4]LGE2 in the TM of the DBA/2J mouse model of glaucoma was also

xxxiii

established and several putative crosslinked proteins were identified using IP and mass

spectrometry. These results support a role for levuglandins (LGs) in POAG pathology.

LGs are known to avidly bind with and crosslink proteins.

The inability of macrophages to processes modified proteins contributes to “foam

cell” formation and atherosclerosis (AS). To identify the modified proteins in inflamed

macrophages, lipopolysaccharide (LPS) stimulated macrophages were studied. LPS

stimulation increased the levels of LGE2 modified proteins in macrophages.The modified

proteins were identified by 2D PAGE and mass spectrometry. Many of the modified

proteins are involved in cholesterol trafficking, gene expression, and/or lipid efflux. This

suggests a role for LGE2 in AS. These observations inspired a pilot study to explore the

possible role of LGs in LPS-induced inflammation of cornea in vivo. We found that LPS

promotes the generation of LGs in cornea.

An efficient synthesis of the γ-keto-α,β-unsaturated alkenoic acid functional array

present in some of the oxidatively truncated phospholipids, was achieved by oxidation of

furyl precursors using NaClO2. Additionally, detection and structural characterization of

multiple HNE adducts onto lysine and histidine side-chain residues were accomplished

using deuterated HNE and mass spectrometry.

Chapter 1

Introduction

1

1.1. Background

Aerobic organisms use oxygen in processes involving energy metabolism, which

makes them prone to radical induced in vivo oxidative damage. This problem can be

averted by antioxidants and antioxidant enzymes in addition to systems that recycle the

oxidatively damaged molecules (proteins). In conditions that compromise one or more

factors of the antioxidant machinery, the free radical mediated damage may lead to a vast

array of diseased conditions. Aging is one of the important phenomenona that have been

attributed to damage by these radicals in situ.1

Biological membranes consist of lipids that provide structural integrity to cells

and cellular structures. These lipids are also involved in signaling processes across the

membrane, through lipid mediators.2 For example, enzyme mediated peroxidation of

PUFAs is involved in signaling normal physiological functions by generating

prostaglandins and thromboxanes.3,4 Additionally, analogous free radical induced

peroxidation can also generate prostaglandin isomers (isoprostanes) and highly reactive

lipid by-products (levuglandins). These highly reactive lipid by-products are known to

form adducts with side chains of proteins and DNA bases, that are involved in various

pathological processes viz, atherosclerosis,5,6 age related macular degeneration,7

rheumatoid arthritis,8 multiple sclerosis,9 Alzheimer’s disease,10 Parkinson’s disease11

and Alexander’s disease.12

2

Ancient scriptures in Sanskrit have identified the potential for pathological

involvements of lipids in the body.

“Those who eat heavy, cold, and excessively oily foods in excessive quantity and do excessive mental work, suffer from disease of the vessels that nourish the heart”

- The Charaka Samhita (400-200 BC)13 (Translated from Sanskrit)

1.2. HNE as an important lipid peroxidation product. 4-Hydroxy-2-nonenal is one of

the most highly investigated lipid peroxidation products. It has been implicated in a vast

array of disease conditions.14 HNE is considered as one of the established markers for

oxidative stress.15 The ready availability of HNE synthetically and its relatively stable

nature has made the handling and processing easier than other lipid oxidation products,

e.g., LGs and oxidized phospholipids.

HNE is a product of oxidative fragmentation of arachidonyl (AA) or linolenyl

(LA) phospholipid esters. Dr. Herman Esterbauer, who discovered HNE, proposed a

mechanism for the formation of HNE from LA through a dioxetane intermediate from

13-HPODE (Scheme 1.1).16 Fragmentation through the intermediacy of 9-PODE can also

be explained based on the peroxy dioxetane intermediate that is formed as depicted in

Scheme 1.1.17,18 A similar mechanism may be proposed for the formation of HNE from

AA-PC, a ω-6 fatty acid. However, there is no evidence to support the actual

involvement of such a mechanism in the generation of HNE through auto-oxidation of

PUFAs. Numerous other mechanisms, also untested, have been proposed for the

formation of HNE in vivo.19-22

3

HOOCC5H11

C5H11HOOC HOOC C5H11

HOOC C5H11

OHO

13-HPODE

HOOC C5H11

OHO

O O

HOOCO

C5H11

OHO

O

C5H11

OH

O

HOOCC5H11

911

13

LA

H

+

L

LH

O2

O2

dioxetene fragmentation

Oxononanoic acid HPNE

HNE

C5H11HOOCO O

9-PODE

C5H11HOOCO O

9,10-dioxetane

O2

O2

LLH

Scheme 1.1: Proposed mechanisms for the formation of HNE from LA.

The pathological activities of HNE are believed to stem from its bifunctional

nature, i.e., the α,β-unsaturated aldehyde and the alcohol. The β carbon of the α,β-

unsaturated aldehyde group acts as an electrophilic center and thus a potential target for

the nucleophilic amino acid residues of proteins. Nucleophilic side chain residues of

4

lysine, histidine and cysteine are known to form Michael adducts with HNE (Scheme

1.2).23 Apart from the Michael adducts, the ε-amino group of lysine can react with the

aldehyde of HNE to form a Schiff base adduct. Both the Michael adduct and the Schiff

base adduct are formed reversibly with lysyl residues.

OOH

4-HydroxynonenalN

OOH

NH

OOH

N

Lysine Michael adduct

Histidine Michael adduct

Histidine

Lysine

S

OOH

Cysteine Michael adduct

Cysteine

OHN

Lysine

NLysine

LysineN

OHLysine N

Lysine-Lysine crosslink

Lysine Schiff base adduct

Lysine pyrrole adduct

Scheme 1.2: Adducts formed by HNE with amino acid side chain residues.

In the case of the Schiff base, the adduct can undergo enol-keto tautomerization

followed by nucleophilic attack of the imine on the carbonyl carbon, resulting in a

cyclized product that undergoes dehydration and concomitant aromatization to form a

pyrrole (Scheme 1.3, next page).24 The HNE-pyrrole adduct is a relatively stable “end

product” of oxidative protein modification mediated by HNE. Antibodies developed

5

against HNE-pyrrole have been useful for analyzing the levels of proteins modified by

HNE in human plasma.25

RNH2

-H2O

RNH2

n-C5H11O

OH

+ n-C5H11NR

OH

n-C5H11NHR

OH

n-C5H11NHR

O

n-C5H11O

OH

NHR

O OHn-C5H11

NHR

NR

n-C5H11

RN

OHn-C5H11

[O]

n-C5H11NHR

O

n-C5H11NHR

O

NHR

n-C5H11NHR

OH

NHR Pyrrole adduct

Michael Adduct

Schiff base Adduct

[O]n-C5H11

NHRO

NH2R

N

NHR

OHR

R

N

NH2R

OHR

R

Amine-HNE (2:1) crosslink

Scheme 1.3: Proposed mechanism for the formation of Michael adduct, Schiff base,

amine-HNE (2:1) crosslink adduct and pyrrole adduct.24

Lipid oxidation products incorporating a phospholipid group, that are analogous

to HNE, with a γ-hydroxy α,β-unsaturated aldehyde core structure, can be formed from

AA-PC and LA-PC. Oxidative fragmentation of AA-PC generates HOOA-PC and LA-

PC generates HODA-PC (Scheme 1.4). These phospholipids occur intact in vivo.

HOOA-PC and HODA-PC can react with lysine residues as described for HNE and form

2-(ω-carboxypropyl)pyrrole (CPP) and 2-(ω-carboxyheptyl)pyrrole (CHP) respectively

(Scheme 1.4) which have lost the PC ester functionality.

6

ROOCC5H11

ROOC

C5H11OO

9-PODE-PC

HOOCOH

ROOCC5H11

9 1113

LA-PC

H

O2

R = 3-lysophosphatidylcholine

O

HODA-PC

PLA2

ROOC

C5H11

9

AA-PC

ROOCO

O

HOOC OOH

HOOA-PC

PLA2

COOH

NH2ProteinNH2Protein

NProteinNProtein

COOH

Carboxypropylpyrrole (CPP) Carboxyheptylpyrrole (CHP)

ROOC

O2

5-PETE-PC

Scheme 1.4: Free radical mediated oxidation of AA-PC and LA-PC leads to the

formation of HOOA-PC and HODA-PC. These lipids are analogous to HNE. They

subsequently react with lysine side chain residues to form the corresponding pyrrole

adducts, 2-(ω-carboxypropyl) pyrrole (CPP) and 2-(ω-carboxyheptyl) pyrrole (CHP).

Antibodies developed against the CPP modification were useful for

demonstrating a considerable increase in the amounts of CPP in patients with renal

failure and atherosclerosis.26 Analogous oxidative cleavage of docosohexanoic acid

7

(DHA) generates the 2-(ω-carboxyethyl)pyrrole (CEP) that has been implicated in age

related macular degeneration.27,28

Since lysine residues can reversibly form a Schiff base with the aldehyde group

of HNE or a Michael adduct at the β-carbon of HNE, HNE can mediate the formation of

lysine-lysine crosslinks. Such crosslinks were identified and characterized through model

studies.24,29,30 Additionally, monoclonal antibodies were generated against the crosslinks

by Dr. Uchida’s group. These antibodies were used to detect the presence of the adducts

in atherosclerotic lesions from human aorta.31 A suggested mechanism for the formation

of the crosslink adducts is depicted in Scheme 1.3 (see page 5).24

1.3. Levuglandins – discovery, formation and pathology. Arachidonyl phospholipids

(AA-PC), ω-6 fatty acid esters, are precursors to a vast array of biologically active

oxidized by-products through enzyme (cyclooxygenase (COX) or lipoxygenase) or free

radical mediated processes. The enzyme-mediated peroxidation requires the free fatty

acid form, while the free radical pathway operates on esters as well.32 Spontaneous

rearrangements of the prostaglandin endoperoxide, PGH2, generate PGD2 and PGE2.

8

COOH COOH

C5H11

OO

OOH

COX

AA-PC Prostaglandin endoperoxide,PGG2

COOH

C5H11

OO

OHPGH2

hydroperoxidase

Scheme 1.5: Formation of prostaglandin endoperoxides from arachidonic acid.

Dr. Salomon discovered an alternative pathway through which these highly

reactive endoperoxides could also rearrange to levulinaldehydes (γ-ketoaldehydes) with

prostanoid side chains. The rearrangement is triggered by abstraction of the electron rich

bridgehead hydrogen of the endoperoxide by the incipient electron deficient methylene

group and a subsequent rearrangement of bonds (as shown in Scheme 1.6) results in

levuglandins (LGs).

OOO

O

OO

H HOH

H

R1

R2

R1

R2

H

R1

R2δ−

δ+

Scheme 1.6: Formation of levuglandins from prostaglandin endoperoxides.

LGs isomers that are generated by free radical mediated pathways are referred to as

isolevuglandins.

9

C5H11

(CH2)3COOH

C5H11

(CH2)3COOPC

OO

(CH2)3COOPC

OH

C5H11

OHC

(CH2)3COOH

OH

C5H11

O

AA-PC

C5H11

(CH2)3COOHOO

OH

C5H11

(CH2)3COOH

OHCOH

O

Levuglandin E2 Isolevuglandin E2

Cyclooxygenase

Phospholipase A2

Free radical-induced

Scheme 1.7: Representative pathways showing a difference in enzyme and free radical

mediated pathways generating LGs from AA-PC.

OO C15H31

O

OP

ON(CH3)3

O OOC5H11

710

13

OR

OC5H11

710

13

OR

OC5H11

710

13

OR

OC5H11

710

13

(CH2)3COOR

C5H11

710

13

OO

(CH2)3COOR

C5H11

710

13

OO

OO

(CH2)3COORC5H11

OO

10 128

(CH2)3COOR8O

O1012 (CH2)3COOR7

10

C5H11O

O 13

(CH2)3COOR710

C5H11OO

(CH2)3COORC5H11

OO

108

OO

(CH2)3COOROO C5H11

OO

OO

(CH2)3COOR

C5H11

OH

OO

8

9

[H][H]

(CH2)3COOR

OH

OO

OO

OH

(CH2)3COORC5H11

iso[4]-PGH2-PC iso[10]-PGH2-PCiso[7]-PGH2-PC

OO

(CH2)3COOR

OH

C5H11

8-epi-LGE2-PC

[H][H]

Arachidonyl phosphatidylcholine ester

(CH2)3COOR

OHOHC

OHCOHC

OH

(CH2)3COOR(CH2)3COOR

OH

C5H11

OOO

C5H11

iso[4]-LGE2-PC iso[10]-LGE2-PCiso[7]-LGE2-PC

OHC

(CH2)3COOR

C5H11

OH

O

12

8-epi-LGE2-PC

8

9

Scheme 1.8: Formation of levuglandins from arachidonyl phosphatidylcholine esters by

free radical mediated processes.

10

The γ-ketoaldehyde functionality of the LGs makes them highly reactive towards

nucleophiles present in the biological system. Crosslinking of proteins is one of the

important features that have been implicated in pathological conditions, for e.g.,

Alzheimer’s disease (AD), which is also associated with increase in the expression of

COX enzymes. LGs are also known to crosslink proteins orders of magnitude greater

than other products of AA-PC oxidation (MDA and HNE) and additionally, the possible

generation of LGs through the COX pathway makes them a possible suspect in AD. A

possible mechanism of crosslink formation by LGs with proteins is depicted in Scheme

1.9. The initial Schiff base adduct formed by the LGs can cyclize and undergo

dehydration and form pyrroles. These pyrrole can also be oxidized to electrophilic

intermediates33 which can undergo crosslinking with other nucleophilic side chain

residues of the protein or the pyrrole itself to form bis-pyrrole adducts.34 Alternatively, an

aminal crosslinking may be generated.

11

O

OHC C5H11

(CH2)3COOH

OH

NH

H2O

Protein

NH2Protein

Protein

NProtein

NR2

R1O

R1

R2

ProteinNH

R2

R1O

NR1

R2

OH

Protein

Protein

NH2Protein

Iso[4]levuglandin E2

H2O

IsoLGE - pyrrole adductProtein-protein aminal crosslink

NH

NProteinR1

R2

N

R1

R2Protein

Pyrrole-pyrrole crosslink

NProteinR1

R2

N

R2

R1Protein

OR

Scheme 1.9: Formation of isolevuglandin and levuglandin based protein adducts.

LGs react with DNA and form DNA-protein crosslinks. These repair-resistant

DNA-protein crosslinks were shown to cause cell death in Chinese hamster fibroblasts.35

Recently, mass spectrometric identification of adducts formed by LGs that were present

12

in brain tissues of patients with AD was reported.36 In this study proteins from brain

tissue were digested to individual amino acids and analyzed by tandem LC-MS/MS and

selective reaction monitoring (479.3 332.1) (Scheme 1.10) as demonstrated earlier in

cell culture studies.37

C5H11

(CH2)3COOH

OH

H3N

OHO

N

O

(CH2)3COOHH2N

O

m/z 479.4

m/z 332.1

(CH2)3COOHHN

HO m/z 332.1

Scheme 1.10: Daughter ion generated by LG-lysine lactam in the mass spectrometric

ionization process. This feature was used in tandem MS/MS to identify the LG modified

proteins.36,37

1.4. Anatomy of anterior section of the eye. A portion of this thesis involves lipid-

derived oxidative modifications of the trabecular meshwork (TM) of the eye. The

anatomy of the eye can be best described by dividing transversely through the lens into

anterior and posterior segments. The anterior segment consists of the cornea, iris and the

anterior chamber. The posterior segment contains the retina, optic nerve and the vitreous

chamber (Figure 1.1, see next page). As background for our study involving the TM, the

anterior chamber is described here in detail.

13

Figure 1.1: Anatomy of human eye showing the anterior and posterior regions

(Copyright© permission obtained from www.nei.nih.gov).

Figure 1.2: Artistic rendering of anterior section of the eye, showing pathway of

aqueous flow through the trabecular meshwork (arrow). (Copyright© permission

obtained from www.nei.nih.gov)

The aqueous humor is actively secreted by the ciliary body and flows into the

anterior chamber through the pupil. The aqueous humor leaves the anterior chamber

14

through an extensive meshwork of tissues leading to the episcleral veins. Aqueous humor

serves two important functions viz. nourishing the cornea and maintaining its shape. The

amount of aqueous humor entering the eye should be equal to the amount flowing out in

order to maintain the pressure in the eye (intraocular pressure). Any decrease in the

outflow will cause an increase in the intraocular pressure (IOP) leading to a serious

blinding disease called glaucoma (vide infra). There are two main pathways of aqueous

drainage. The major pathway is through the trabecular meshwork into the episcleral veins

through the canal of Schlemm. The minor pathway is the uveoscleral pathway, in which

the aqueous passes through the ciliary muscles into the spaces between the cilary body

and sclera leading to the veins. Another pathway through the iris is described in rabbits,

which lack the canal of Schlemm. This pathway is absent in humans.

1.5. Glaucoma. Glaucoma refers to a group of eye diseases that often occur with increase

in the intraocular pressure leading to irreversible blindness and optic neuropathy.38

Glaucoma is a leading cause of blindness.39 A common system of classification and

definition of glaucomas has not yet been defined. But broadly, glaucomas can be divided

into two broad categories, primary and secondary. Primary glaucoma is a diagnosis of

exclusion, which refers to those conditions where no known potential cause(s) can be

attributed to the disease. Secondary glaucoma are often associated with an injury,

inflammation or a previous illness. These two classes can be further classified as open

angle (OAG) and angle closure glaucoma. This classification is based on the iridial angle,

defined by the angle formed by the tangents of iris and cornea, i.e., around 40o (normal

angle) for open angle glaucoma and around 15o for angle closure glaucoma.

15

1.5.1. Primary open angle glaucoma (POAG). Affecting around 3 million Americans,

this form of glaucoma has no symptoms or early warning signs. POAG is often, but not

always, associated with an increase in the fluid pressure inside the eye (intraocular

pressure, IOP) and involves optic nerve damage leading to irreversible blindness. As the

iridial angle is normal, the aqueous pathway is clear up to the trabecular meshwork (TM),

and it is thought that there could be a clogging of the aqueous drainage in the TM region

or beyond. This type of glaucoma develops slowly and sometimes without noticeable

sight loss for many years. It usually responds well to medication, especially if caught

early and treated.40

1.5.2. Secondary glaucomas. There are several forms of secondary glaucoma, classified

either as open angle or closed angle as described above.

Pseudoexfoliative glaucoma. In this condition, thin flaky dandruff-like material peels off

the outer layer of the lens within the eye and collects in the iridial angle and can clog the

drainage system of the eye, causing eye pressure (IOP) to rise. Treatments include

medication or surgery.

Pigmentary glaucoma. This form of open angle glaucoma has midperipheral iris

transillumination defects and is characterized by heavy pigmentation of the TM. The TM

is pigmented due to the release of pigment granules from the heavily pigmented iris into

the aqueous humor, which then can clog the outflow pathway causing the pressure (IOP)

to rise. The etiology of the disease is poorly understood and the primary causes are still

unknown.41

Traumatic glaucoma. Injury to the eye may cause this type of glaucoma known as

secondary glaucoma, which may also be caused by post injury trauma years later.

16

Neovascular glaucoma. This form of glaucoma is caused by formation of abnormal blood

vessels in the iris or in the drainage canals of the anterior chamber resulting in increased

IOP. Neovascular glaucoma is often associated with other abnormalities, e.g., diabetes.

This form of glaucoma is difficult to treat.38

Iridocorneal endothelial syndrome. This rare form of glaucoma usually appears in only

one eye, rather than both. Cells on the back surface of the cornea spread over the eye’s

drainage tissue and across the surface of the iris, increasing IOP and damaging the optic

nerve. These corneal cells also form adhesions that bind the iris to the cornea, further

blocking the drainage channels.38

1.6. Animal models for glaucoma. Use of animal models to study the molecular and

genetic basis of disease processes provides economical and functional feasibility.42 For

example, differences in the anatomical and physiological features across different species

of animals can broaden the understanding of an experimental outcome, which is not

possible using cell culture or in vitro experiments. A part of this thesis involves use of the

DBA/2J mouse, model of glaucoma, to assess the oxidative damage to the TM in

glaucoma. The pathological mechanisms causing glaucoma in the DBA/2J mouse are

incompletely understood but may be associated with pigmentary dispersion.

In glaucomas, increase in IOP is often implicated in optic neuropathy and

progression of vision loss. The biochemical changes that follow the increase in IOP have

been studied by inducing an increase in the IOP by several mechanisms.42 One of the

commonly used procedures is treating rodent models with hypertonic saline injections

into episcleral veins. This causes an increase in the IOP and subsequent damage to the

17

optic nerve head.43 Use of adrenalin, which induces β-adrenoreceptors, in the

development of experimental glaucoma in rabbits has been reported.44 Adrenalin triggers

a cascade of cellular signaling that causes dystrophic changes in ocular blood vessels and

tissues (including those in the drainage zone), mainly due to excessive entry of Ca2+ into

cells, which are typical symptoms of primary open angle glaucoma in humans.44 IOP

increases have been induced in Rhesus monkeys by cauterization, using an argon laser, in

the anterior chamber angle leading to clogging of the aqueous flow.45,46 α-Chymotrypsin

induced ocular hypertension models in various animals viz, monkey, rabbit and dog, have

been described.42,47 The effects in this model are ascribed to TM blockage by

inflammatory exudates and by debris resulting from enzymatic hydrolysis of the zonules

(sensory ligaments holding the lens).

1.7. Glaucoma and oxidative stress. Increase in oxidative stress has been suggested to

be an important factor in glaucoma pathogenesis. Increased levels of lipid peroxidation

products in the TM of primary open angle glaucoma (POAG) were noted as early as 1989

by Babizhayev et al., through experiments that spectrophotmetrically quantitate the

amount of dienes present in TM lipid extracts.48 Aging, an important factor that has been

long associated with decrease in antioxidant capacity and increase in aggregation of

protein due to oxidation, has also been associated with glaucoma.49 Clinical evidence

supporting this view was substantiated by reports that show the loss of cellularity of TM

cells50 and loss of superoxide dismutase (SOD) activity,51 an antioxidant enzyme, in TM

tissues with aging. Additional evidence obtained through TM cell culture studies, showed

18

decreased amounts of reduced glutathione (GSH)52 and lowered cell adhesion

characteristics53 when the cells were treated with H2O2.

Protein expression is altered in diseased conditions as a homeostatic mechanism

to prevent any damage to the tissues. In the cases of human and monkey TM tissue, there

is an over expression of alpha B-crystallin in response to stress, to prevent any cellular

damage.54 Extracellular matrix (ECM) remodeling in the TM tissue of glaucoma patients

causes an increase in the levels of collagen deposition, which can in turn cause an

increase in the outflow resistance.55

Reactive oxygen species (ROS) are generated in the anterior chamber

photochemically giving rise to reactive substances, e.g., lipid peroxides.56 A decrease in

antioxidant levels of the lacrimal secretions in patients with POAG have also been

reported as the disease progresses.57 A decrease in the levels of plasma GSH58 and an

increase in the lipid peroxidation product, malonaldehyde59 have been observed in

plasma of POAG. These reactive substances have been known to play a role in cataract, a

disease associated with oxidation of lens proteins.60 The aqueous humor is suggested to

be one of the important routes through which ROS may reach the TM, which can act on

the ECM proteins and the membranes.61 TGF-β, a fibrogenic cytokine involved in

atherosclerotic and pulmonary fibrogenesis, is also fibrogenic in TM cells.62 Increase in

the TGF-β levels in the aqueous humor may influence the cytoskeletal structures and

cellular signaling cascades.63 A review62 of the effects of vitamin E deficiency describes

various components that are involved in the pathology in POAG and how they influence

each other (Figure 1.3, next page) .

19

Figure 1.3: Flow chart illustration of the components involved in the pathogenesis of

POAG induced by decrease of vitamin E in the ciliary body. MMP – matrix

metalloproteinases, TIMP – tissue inhibitor of metalloproteinases, ON – optic nerve,

ECM – extracellular matrix. (Copyright permission obtained from Br. J. Nutrition, CABI

Publishing)62

Elevated levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), a marker for

oxidative DNA damage, have been found in TM tissues of POAG patients.64 This study

also revealed that there is an increase in 8-OH-dG levels in glutathione S-transferase

20

(GSTM) null subjects (GSTM is a gene corresponding to the enzyme involved in

antioxidant activity of GSH).64 Increase in oxidative stress in the retinal ganglion cell65

and stress related enzymes in the optic nerve head62,66 was observed in animal glaucoma

models. Rescue of retinal ganglion cells in rats having increased IOP has been successful

by using tropic factors and antioxidants.67

“M: Too many free radicals. That's your problem. James Bond: "Free radicals," sir? M: Yes. They're toxins that destroy the body and the brain, caused by eating too much red meat and white bread and too many dry martinis! James Bond: Then I shall cut out the white bread, sir. M: Oh, you'll do more than THAT, 007. From now on you will suffer a strict regimen of diet and exercise; we shall PURGE those toxins from you! James Bond: Shrublands? M: You got it!”

- From “Never say never again” (1983).

21

1.8. References.

(1) Finkel, T.; Holbrook, N. J. Nature 2000, 408, 239-47. (2) Molecular and Cellular Basis of Inflammation; Serhan, C. N.; Ward, P. A., Eds.;

Humana Press: Totowa, New Jersey, 1999. (3) Chakraborti, T.; Ghosh, S. K.; Michael, J. R.; Batabyal, S. K.; Chakraborti, S. Mol

Cell Biochem 1998, 187, 1-10. (4) Tapiero, H.; Ba, G. N.; Couvreur, P.; Tew, K. D. Biomed Pharmacother 2002, 56,

215-22. (5) Brown, M. S.; Goldstein, J. L. Annu Rev Biochem 1983, 52, 223-61. (6) Arlt, S.; Kontush, A.; Muller-Thomsen, T.; Beisiegel, U. Z Gerontol Geriatr

2001, 34, 461-5. (7) Kopitz, J.; Holz, F. G.; Kaemmerer, E.; Schutt, F. Biochimie 2004, 86, 825-31. (8) Winyard, P. G.; Tatzber, F.; Esterbauer, H.; Kus, M. L.; Blake, D. R.; Morris, C.

J. Ann Rheum Dis 1993, 52, 677-80. (9) Newcombe, J.; Li, H.; Cuzner, M. L. Neuropathol Appl Neurobiol 1994, 20, 152-

62. (10) Sayre, L. M.; Zelasko, D. A.; Harris, P. L.; Perry, G.; Salomon, R. G.; Smith, M.

A. J Neurochem 1997, 68, 2092-7. (11) Yoritaka, A.; Hattori, N.; Uchida, K.; Tanaka, M.; Stadtman, E. R.; Mizuno, Y.

Proc Natl Acad Sci U S A 1996, 93, 2696-701. (12) Castellani, R. J.; Perry, G.; Harris, P. L.; Cohen, M. L.; Sayre, L. M.; Salomon, R.

G.; Smith, M. A. Brain Res 1998, 787, 15-8. (13) http://www.ayurveda-ayurvedic.com/e-zine/health-ezine-404.html, Volume 1,

Issue 4. April 2004. (14) Zarkovic, K. Mol Aspects Med 2003, 24, 293-303. (15) Zarkovic, N. Mol Aspects Med 2003, 24, 281-91. (16) Esterbauer, H.; Schaur, R. J.; Zollner, H. Free Radic Biol Med 1991, 11, 81-128. (17) Gardner, H. W.; Deighton, N. Lipids 2001, 36, 623-8. (18) Schneider, C.; Tallman, K. A.; Porter, N. A.; Brash, A. R. J Biol Chem 2001, 276,

20831-8. (19) Schneider, C.; Tallman, K. A.; Porter, N. A.; Brash, A. R. J. Biol. Chem. 2001,

276, 20831-20838. (20) Lee, S. H.; Blair, I. A. Chem Res Toxicol 2000, 13, 698-702. (21) Noordermeer, M. A.; Feussner, I.; Kolbe, A.; Veldink, G. A.; Vliegenthart, J. F.

Biochem Biophys Res Commun 2000, 277, 112-6. (22) Pryor, W. A.; Porter, N. A. Free Radic Biol Med 1990, 8, 541-3. (23) Uchida, K. Amino Acids 2003, 25, 249-57. (24) Xu, G.; Sayre, L. M. Chem Res Toxicol 1998, 11, 247-51. (25) Kaur, K.; Salomon, R. G.; O'Neil, J.; Hoff, H. F. Chem Res Toxicol 1997, 10,

1387-96. (26) Salomon, R. G.; Kaur, K.; Podrez, E.; Hoff, H. F.; Krushinsky, A. V.; Sayre, L.

M. Chem Res Toxicol 2000, 13, 557-64. (27) Crabb, J. W.; Miyagi, M.; Gu, X.; Shadrach, K.; West, K. A.; Sakaguchi, H.;

Kamei, M.; Hasan, A.; Yan, L.; Rayborn, M. E.; Salomon, R. G.; Hollyfield, J. G. Proc Natl Acad Sci U S A 2002, 99, 14682-7.

22

http://www.ayurveda-ayurvedic.com/e-zine/health-ezine-404.html

(28) Gu, X.; Meer, S. G.; Miyagi, M.; Rayborn, M. E.; Hollyfield, J. G.; Crabb, J. W.; Salomon, R. G. J Biol Chem 2003, 278, 42027-35.

(29) Xu, G.; Liu, Y.; Sayre, L. M. Chem Res Toxicol 2000, 13, 406-13. (30) Itakura, K.; Osawa, T.; Uchida, K. J Org Chem 1998, 63, 185-187. (31) Itakura, K.; Oya-Ito, T.; Osawa, T.; Yamada, S.; Toyokuni, S.; Shibata, N.;

Kobayashi, M.; Uchida, K. FEBS Lett 2000, 473, 249-253. (32) Salomon, R. G. Chem Phys Lipids 2005, 134, 1-20. (33) Amarnath, V.; Valentine, W. M.; Amarnath, K.; Eng, M. A.; Graham, D. G. Chem

Res Toxicol 1994, 7, 56-61. (34) DiFranco, E.; Subbanagounder, G.; Kim, S.; Murthi, K.; Taneda, S.; Monnier, V.

M.; Salomon, R. G. Chem Res Toxicol 1995, 8, 61-7. (35) Murthi, K. K.; Friedman, L. R.; Oleinick, N. L.; Salomon, R. G. Biochemistry

1993, 32, 4090-7. (36) Zagol-Ikapitte, I.; Masterson, T. S.; Amarnath, V.; Montine, T. J.; Andreasson, K.

I.; Boutaud, O.; Oates, J. A. J Neurochem 2005, 94, 1140-5. (37) Boutaud, O.; Li, J.; Zagol, I.; Shipp, E. A.; Davies, S. S.; Roberts, L. J., 2nd;

Oates, J. A. J Biol Chem 2003, 278, 16926-8. (38) http://www.glaucoma.org/learn/; Vol. 2005. (39) Quigley, H. A. N Engl J Med 1998, 338, 1063-4. (40) Epstein, D. L. Chandler and Grant's glaucoma; 4th ed.; Williams & Wilkins, co.,:

Baltimore, Md, 1997. (41) Sowka, J. Optometry 2004, 75, 115-22. (42) Ritch, R.; Shields, M.; Krupin, T. The Glaucomas; 2 ed.; Mosby: St. Louis, MO,

1996. (43) Morrison, J. C.; Moore, C. G.; Deppmeier, L. M.; Gold, B. G.; Meshul, C. K.;

Johnson, E. C. Exp Eye Res 1997, 64, 85-96. (44) Mikheytseva, I. N.; Kashintseva, L. T.; Krizhanovsky, G. N.; Kopp, O. P.;

Lipovetskaya, E. M. Int Ophthalmol 2004, 25, 75-9. (45) Gaasterland, D.; Kupfer, C. Invest Ophthalmol 1974, 13, 455-7. (46) Gross, R. L.; Ji, J.; Chang, P.; Pennesi, M. E.; Yang, Z.; Zhang, J.; Wu, S. M.

Trans Am Ophthalmol Soc 2003, 101, 163-9; discussion 169-71. (47) Bucolo, C.; Campana, G.; Di Toro, R.; Cacciaguerra, S.; Spampinato, S. J

Pharmacol Exp Ther 1999, 289, 1362-9. (48) Babizhayev, M. A.; Bunin, A. Acta Ophthalmol (Copenh) 1989, 67, 371-7. (49) Babizhayev, M. A.; Brodskaya, M. W. Mech Ageing Dev 1989, 47, 145-57. (50) Alvarado, J.; Murphy, C.; Polansky, J.; Juster, R. Invest Ophthalmol Vis Sci 1981,

21, 714-27. (51) De La Paz, M. A.; Epstein, D. L. Invest Ophthalmol Vis Sci 1996, 37, 1849-53. (52) Kahn, M. G.; Giblin, F. J.; Epstein, D. L. Invest Ophthalmol Vis Sci 1983, 24,

1283-7. (53) Zhou, L.; Li, Y.; Yue, B. Y. J Cell Physiol 1999, 180, 182-9. (54) Tamm, E.; Russell, P.; Johnson, D.; Piatigorsky, J. Invest Ophthalmol Vis Sci

1996, 37, 2402-13. (55) Gonzalez-Avila, G.; Ginebra, M.; Hayakawa, T.; Vadillo-Ortega, F.; Teran, L.;

Selman, M. Arch Ophthalmol 1995, 113, 1319-23.

23

http://www.glaucoma.org/learn/;

(56) Kurysheva, N. I.; Vinetskaia, M. I.; Erichev, V. P.; Demchuk, M. L.; Kuryshev, S. I. Vestn Oftalmol 1996, 112, 3-5.

(57) Bunin, A.; Filina, A. A.; Erichev, V. P. Vestn Oftalmol 1992, 108, 13-5. (58) Gherghel, D.; Griffiths, H. R.; Hilton, E. J.; Cunliffe, I. A.; Hosking, S. L. Invest

Ophthalmol Vis Sci 2005, 46, 877-83. (59) Yildirim, O.; Ates, N. A.; Ercan, B.; Muslu, N.; Unlu, A.; Tamer, L.; Atik, U.;

Kanik, A. Eye 2004, 1-4. (60) Srivastata, S. K.; Awasthi, S.; Wang, L.; Bhatnagar, A.; Awasthi, Y. C.; Ansari,

N. H. Curr Eye Res 1996, 15, 749-54. (61) Babizhayev, M. A.; Costa, E. B. Biochim Biophys Acta 1994, 1225, 326-37. (62) Veach, J. Br J Nutr 2004, 91, 809-29. (63) Tripathi, R. C.; Li, J.; Chan, W. F.; Tripathi, B. J. Exp Eye Res 1994, 59, 723-7. (64) Izzotti, A.; Sacca, S. C.; Cartiglia, C.; De Flora, S. Am J Med 2003, 114, 638-46. (65) Tezel, G.; Yang, X.; Cai, J. Invest Ophthalmol Vis Sci 2005, 46, 3177-87. (66) Yan, X.; Tezel, G.; Wax, M. B.; Edward, D. P. Arch Ophthalmol 2000, 118, 666-

73. (67) Ko, M. L.; Hu, D. N.; Ritch, R.; Sharma, S. C. Invest Ophthalmol Vis Sci 2000,

41, 2967-71.

24

Chapter 2

A Short and Efficient Synthesis of 4-Oxo-2-alkenoic Acids from 2-Alkylfurans

25

2.1. Background

Polyunsaturated fatty acids (PUFAs) are especially prone to damage by free

radicals owing to the presence of homoconjugated C=C double bonds. The chemistry of

PUFA peroxidation is particularly harmful because the immediate damage may be easily

amplified by the additional release of reactive substances, radicals or fatty acid-derived

aldehydes that can initiate further modification of cellular structures.1-5 However, they

can also be detoxified.6,7

OO C15H31

O

OP

ON(CH3)3

O OO

C4H9 m n

O

On PC

OO

PA-PC, n=3, m=4 (2.1) LA-PC, n=7, m=2 (2.2)

HPOOA-PC, n=3HPODA-PC, n=7

O

On PC

OHO

HO

HOOA-PC, n=3HODA-PC, n=7

O

On PC

OHO

HOdiA-PC, n=3HDdiA-PC, n=7

OH

O

On PC

OO

KOOA-PC, n=3KODA-PC, n=7

O

On PC

OO

KOdiA-PC, n=3 (2.3)KDdiA-PC, n=7 (2.4)

OH

[H]

O2O2

O2

O2

O2

-H2O

O2

Scheme 2.1: Suggested mechanism for generation of KOdiA-PC and KDdiA-PC.8

26

Oxidative modification of low-density lipoprotein and subsequent uptake by

macrophages is an early event in the formation of atherosclerotic plaques.9 Among the

products that are formed by oxidative fragmentation of arachidonyl 2.1 or linoleyl 2.2

phosphatidylcholine (PC), the γ-keto-α,β-unsaturated alkanoate phosphatidylcholine

esters, KOdiA-PC 2.3 and KDdiA-PC 2.4 (Scheme 2.1), are potent ligands responsible

for recognition and uptake of oxidized low-density lipoprotein.10,11

The functional array containing 4-oxo-2-alkenoic acid is also present in many

pharmaceutically important natural products, e.g., pyrenophorin, patulolide, and

grahamimycin that have antifungal, anti-bacterial and/or anti-inflammatory properties.12-

18 To facilitate studies on the biological properties of these compounds, a short and

efficient synthesis was needed to make them readily available.

OO

OO

OOO

OO

O

O

O

O

OH

O

GrahamimycinPyrenophorin Patulolide

Scheme 2.2: Natural products incorporating 4-oxo-2-alkenoate functionality.

2.1.1. Previous syntheses of γ-keto α,β-unsaturated alkanoates

Most of the previous syntheses of these functional arrays either used elaborately

functionalized precursors19-23 or multiple step procedures involving ring opening and

subsequent oxidation (vide infra).

Synthesis using functionalized furans as precursors. Synthesis of 4-oxo-alkenoates

using 2-alkoxy-5-alkylfurans as precursors was achieved previously by using oxidizing

27

agents like bipyridinium chlorochromate,24 followed by isomerization using I2 of the Z-

to E- isomer (Scheme 2.3). Another method, using the hydrogen peroxide adduct of urea

(UHP) in the presence of a catalytic amount of methyltrioxorhenium (MTO) generated

this functional array in good yields.25

OO

1. PCC,CH2Cl2, 2.5h2. I2, 4h

OO

O< 40%

OOO

OO

UHP -Urea-H2O2 adductMTO -Methyltrioxorhenium

UHP5 mol % MTO

95%

ReO

OO

MTO

Scheme 2.3: 2-Alkoxyfuran as precursor for 4-oxo-2-alkenoate.

Photooxidation of 2-acyl-5-alkylfurans using singlet oxygen followed by

reduction was found to generate such products in fair to poor yields (Scheme 2.4).26

OO

O1.1O2, Rose Bengal2.K2CO3, Me2SO4

< 60%, 2 stepsOOHC

O OO O

Scheme 2.4: 2-Acylfuran as precursor for 4-oxo-2-alkenoate.

Lead(IV) acetate mediated oxidation of 2-siloxy-5-alkylfurans and subsequent

acidification yielded 2-alkyl-2-hydroxybutenolides, ring-form tautomers of cis-4-oxo-2-

alkenoates,27 in good yields (Scheme 2.5).28

28

O OSi

OO

Si

O OHOO

H3O+

76% 86%

Pb(OAc)4

Scheme 2.5: 2-Siloxyfuran as precursor for 4-oxo-2-alkenoate.

Based on an earlier method, that used dimethyl dioxirane to oxidatively convert a

4,5-disubstituted furans to γ-keto-enals,29 the use of 2-siloxy-5-alkylfurans30 or 2-silyl-

5-alkylfurans31 as precursors generates hydroxybutenolides in good to excellent yields

(Scheme 2.6).

O OSi

OO

Si

OOHO

O

O OH3O+

100% 77%

OR Si OHOO

R

O O

H3O+

95%OR SiO

Scheme 2.6: 2-Silylfuran and 2-siloxyfuran oxidation using dimethyl dioxirane.

Synthesis using simple furans. Though the use of simple furans to synthesize the 4-oxo-

2-alkenoic acids have been reported, these procedures involved multi-step reactions. For

example, synthesis of (E)-4-oxonon-2-enoic acid (2.6), a natural antibiotic produced by

Streptomyces olivaceus, was achieved Ballini et al.32 by oxidation of 2-pentylfuran (2.5)

using pyridinium chlorochromate followed by Jones reagent gave the product in good

29

yields. However, the procedure is harsh and has limitations in the kind of substrates that

can be used (Scheme 2.7).

OC5H11 C5H11O

OC5H11

OO

OH

PCCJones

reagent70% 85%

2.5 2.6

Scheme 2.7: Oxidation of a 2-alkylfuran using pyridinium chlorochromate.

Use of bromine to oxidize the furan ring has been reported earlier, but gives very

low yields.33-35 Kobayashi et al. modified this procedure and used N-bromosuccinimide

(NBS) to oxidize the furan ring (Scheme 2.8).36,37 This methodology was used by our

group to synthesize some oxidized phospholipids.38

OC5H11 C5H11O

OC5H11

OO

OH

PCCJones

reagent70% 85%

2.5 2.6

Scheme 2.8: Oxidation of 2-alkylfuran using N-bromosuccinimide.

Synthesis of pyrenophorin precursor. Pyrenophorin, a macrocyclic natural product, is

a lactone a of γ-keto-α,β-unsaturated ester of an alkan-7-ol-1-oic acid (Scheme 1.2).

There are many reported methods to synthesize this precursor.15,20,28,36,39-41 Some of

methods are described in Scheme 2.9.

30

a.

OAc

Oa) Jones reagentb) 2-mercaptobenzimidazole

c) HOCH2CH2OH, p-TsOH

O

OHOAc

OO

< 36%

b.

NO2

OAcN

OAc

O

COOMe

COOMeOAc

O OH

COOMeOAc

O

COOMe

PhNCOEt3N80%

H2, Ni-Raneyacetic acid: H2O.

MeSO2ClEt3N

70%

70%

c.

OAc

O

CHOOAc

O

COOHOAc

O

NBS

pyridine

NaClO2

64%

83%

Scheme 2.9: Methods reported for synthesis of a pyrenophorin precursor: a) using Jones

reagent with furyl precursor; b) using (3+2) cycloaddition of primary nitro group and

methyl acrylate;42 c) using NBS to oxidize a furyl precursor.36

31

A patent application reported a one-pot synthesis of 4-oxo-5-phthalimido-2-

pentenoic acid from N-furfurylphthalimide through singlet oxygenation.43 Though this is

a one-step method, the methodology was not substantially exemplified.

O

HO

O

O

NO

O

O

NO O2

hν76%

Scheme 2.10: A patented process for generation of 4-oxo-2-alkenoic acids by Takeya.43

32

2.2. Results and discussion

4-Oxo-2-alkenoic acids, and the closely related α,β-unsaturated-γ-diketones, are

versatile intermediates for organic synthesis.44-52 An important shortcoming of previous

methods for preparing 4-oxo-2-alkenoic acids is the requirement for more elaborately

functionalized furan intermediates such as 2-alkoxy-5-alkyl,24,25 2-acyl-5-alkyl,53 2-

siloxy-5-alkyl28,30 or 2-silyl-5-alkyl31 as precursors (see section 2.1.1). Generation of 4-

oxo-2-alkenoic acids from 2-alkylfurans has also been accomplished previously through a

variety of multi-step procedures involving treatment with reagents such as bromine41 or

NBS36 followed by oxidation with sodium chlorite; PCC followed by Jones reagent;32 or

nitration followed by acidic oxidation (see section 2.1.2).54

Alkylfurans are easily accessible,44-46,55 robust precursors that are compatible with

a wide variety of reagents and reaction conditions making them higly desirable

substrates. The 2-alkylfuran precursors can be prepared by alkylation of 2-furyllithium

using the corresponding alkyl halide as reported earlier (Scheme 2.11).38

O RO t-BuLi O Li RX

Scheme 2.11: Synthetic method for generating 2-alkylfurans from furan.

This study focuses on a efficient stereocontrolled methodology, that expliots

sodium chlorite as an oxidizing agent, for the conversion of 2-alkylfurans 2.7 into 4-oxo-

2-alkenoic acids 2.10 and for the conversion of 2,5-dialkylfurans into 1,4-enediones. The

oxidation cleanly generates cis isomers 2.9 (butenolides 2.8) stereospecifically and in

33

high yield. If desired, these can be cleanly and quantitatively converted to the

corresponding trans isomers by treatment with pyridine.56

O R

2.7 HO RO O

OR

O

OH2.10

OR

OOH

NaClO2 Pyridine2.8

2.9

Scheme 2.12: Synthesis of the 4-oxo-2-alkenoic acid functional array.

Synthesis of 4-oxo-2-alkenoic acids. Oxidation of 2-alkylfurans 2.7 in the presence of

sodium chlorite in a slightly acidic aqueous solution (~ pH 3.5) at room temperature

generates 2-alkyl-2-hydroxy-butenolides 2.8. The 2-alkyl-2-hydroxybutenolides 2.8 are

the ring tautomers of 4-oxo-2(Z)-alkenoic acids 2.9 (Scheme 2.12).27 To confirm this, 2-

pentyl-2-hydroxybutenolide (2.5, R = C5H11) was synthesized (vide infra) and subjected

to various pH conditions and the UV spectrum recorded (Scheme 2.13). The spectrum

shows the appearance of a peak around 240 nm under basic conditions indicating the

predominance of a keto functionality (Figure 2.1).

OC5H11HO

O C5H11 OHO OpH >7

pH <7

Scheme 2.13: Ring-chain tautomers of 4-oxo-2-pentenoic acid.

34

Figure 2.1: UV-spectra of 4-oxo-2-pentenoic acid at varying pH.

Using this furan oxidation protocol, simple 2-alkyl substituted furans were

converted to 4-oxo-alkenoic acids in excellent yields. Compared to the earlier synthetic

methods,32,57 synthesis of 4-oxo-2-nonenoic acid, a natural antibiotic produced by

Streptomyces olivaceus, was prepared conveniently in excellent yields (Scheme 2.14).

C6H13

COOEt COOEt

C5H11

O

COOH

C5H11

O

OR OR

OHO

OC5H11CHO

C5H11

O

CrO3

PCC

NaClO2

Pyridine

K2CO356%

68%

95%100%

70% 85%Jones

R = -CH3, -C5H11

Scheme 2.14: Comparison of synthetic methods used to generate 4-oxo-2(E)-alkenoic

acids from 2-alkylfurans.

35

The generality of the protocol for substituents with commonly used alcohol

protecting groups, as depicted in Scheme 2.15, was established and the results are shown

in Table 2.1.

OPHOOC

O

O OP OOP

OHOa b

a - NaClO2 (3 eq.), NaH2PO4 (1.5 eq.), tBuOH:H20 (5:1), rtb - Pyridine, THF/Acetone/H2O (5:4:1), rt

2.11 2.12 2.13

Scheme 2.15: Synthesis of 4-oxo-2-alkenoic acids from 2-alkylfurans.

Table 2.1: Yield and reaction time for alcohol protected alkylfurans in Scheme 2.15.

Substrate P Yield 2.12

(%)

Reaction time

2.11 2.12 (h)

Yield 2.13

(%)

Reaction time

2.12 2.13 (h) Product

2.14 Ts 90 4 96 2 2.17

2.15 THP 78 2 90 2 2.18

2.16 TBDMS 77 2 95 2 2.19

36

Pyrenophorin, a natural product having antibiotic and antifungal properties, can

be assembled by lactonization of 2.22 as reported earlier.36 This precursor 2.22 was

synthesized using our new furan oxidation protocol (Scheme 2.16) in good yield,

compared to earlier protocols (Scheme 2.9).36

OOTBDMS

OOTBDMS

OHO

OO

OTBDMS

NaClO2

Pyridine

OH

OO

O

OO

O

77%

95%

2.20 2.21

2.222.23

Scheme 2.16: Synthesis of pyrenophorin precursor from 2-alkylfuran 2.20.

Synthesis of oxidatively truncated phospholipids, KOdiA-PC (2.3) and KDdiA-

PC (2.4), for studying their biological properties,10,11 was our primary motivation to

devise an efficient method for the synthesis of 4-oxo-2-alkenoic acid. Our previous

syntheses of 2.3 and 2.4 from 2-substituted furan phospholipid precursors 2.24 and 2.25,

using oxidation with NBS followed by further oxidation with sodium chlorite, gave 46%

and 36% yields, respectively.38 For these phospholipids, the sodium chlorite oxidation

method using t-BuOH-water (5:1) as solvent afforded poor yields, even after long

reaction times. Total syntheses involving phospholipids present special challenges owing

to their unique solubility properties. The direct oxidation of 2.24 or 2.25 with sodium

chlorite can be accomplished by employing a biphasic solvent system containing

37

phosphate buffer (10 mM, pH 3.5), chloroform and water in the ratio 2:1:1. The biphasic

aqueous sodium chlorite protocol decreased the reaction time from 9 h to 1 h, and

consistently gave 40-80% higher yields than the NBS-NaClO2 procedure.

OO

OPC

n

NaClO2 (3 eq.) ,NaH2PO4 (1.5 eq.),tBuOH:H2O (5:1), rt

NaClO2 (3 eq.) PBS (pH 3.5):CHCl3:H2O(2:1:1), rt

< 10%

>75%

O O

OPC

n

HO

O

OOO

OOHPC

Pyridine, THF/Acetone/H2O (5:4:1), rt

> 90%

n

n=2 (2.24)n=6 (2.25)

n=2, KOdiA-PC (2.3)n=6, KDdiA-PC (2.4)

n=2 (2.26)n=6 (2.27)

Scheme 2.17: Synthesis of KOdiA-PC and KDdiA-PC by furan oxidation protocols

employing two different solvent conditions.

Synthesis of 1,4-enediones. Oxidation of 2,5-dialkylfurans 2.28 using the new furan

oxidation protocol generated the 1,4-alk-2-enediones 2.29 (Scheme 2.18).8

R R'O OOR R'

a. NaClO2 ( 3 equiv.), NaH2PO4 ( 1.5 equiv.), tBuOH:H2O (5:1), 93%

a

2.28 2.29

Scheme 2.18: Synthesis of 1,4-enediones from 2,5-dialkylfurans.

38

Substrates incorporating a vinyl substituent present a special challenge. For

example, under the usual reaction conditions, 2.31 did not give any of the corresponding

enedione 2.34. However, in the presence of 2-methylbut-2-ene, a chlorine scavenger,58

the sodium chlorite oxidation protocol gave enedione 2.34 in good yield (Table 2.2).

Table 2.2: Synthesis of 2-ene-1,4-diones from 2,5-dialkylfurans (Scheme 2.16)

Substrate R R’ Product Yield(%)

2.30 -CH3 -CH3 2.33 93b

2.31 -(CH2)7CHO -CH=CH(E)-C4H9 2.34d 76c

2.32 -CH3 -CH=CH(E)-C4H9 2.35 78 c

a NaClO2 (3 equiv.), NaH2PO4 (1.5 equiv.), t-BuOH:H2O (5:1) b Reaction time 1 h c 2-Methyl-2-butene (0.1 mol equiv.) added to the reaction, reaction time ~20

min, yield based on starting material consumed. d The aldehyde is completely oxidized to the acid.

39

2.3. Conclusions

In summary, this new methodology provides ready access to either cis- or trans-

isomers of 4-oxo-2-alkenoic acids from simple 2-alkylfuran precursors in high yields,

using readily available reagents.59 Compared to our previous syntheses, the new

procedure enables a major improvement in the overall yield and ease of preparing

biologically active phospholipids that contain this functional array, thus providing a ready

access for further studies. This protocol is also efficient for converting 2,5-dialkylfurans

to the corresponding 1,4-enediones.

40

2.4. Experimental Procedures

General Methods. Proton magnetic resonance (1H NMR) spectra were recorded on a

Varian Gemini spectrometer operating at 300 or 200 MHz. Proton chemical shifts are

reported in parts per million (ppm) on the δ scale relative to CDCl3 (δ 7.24). Carbon

magnetic resonance (13C NMR) spectra were recorded on a Varian Gemini spectrometer

operating at 50 MHz. Carbon chemical shifts are reported in parts per million (ppm) on

the δ scale relative to CDCl3 (δ 77.0). Thin layer chromatography (TLC) was performed

on glass plates precoated with silica gel (Kiesegel 60 F254, E. Merck, Darmstadt,

Germany). The plates were visualized by viewing under short wavelength UV light or by

treatment with iodine. Flash column chromatography was performed on silica gel (60 Å,

32-63 µm) supplied by Sorbent Technology (Atlanta, GA). UV-visible spectra were

recorded on a Perkin Elmer Lambda 35 UV-visible spectrometer using Fisher Scientific

10 mm cuvette. MALDI-ToF was performed on a Voyager – DE PRO from Applied

Biosystems®, using α-cyano-4-hydroxycinnamic acid (CHCA) as matrix. Chemical were

purchased from Aldrich Chemical Co. (Milwaukee, WI) unless otherwise stated. Solid

phase extraction columns (SPE) were purchased from Whatman Inc, Alabama.

Typical procedure for furan oxidation. To a magnetically stirred solution of NaH2PO4

(1.5 equiv.) and NaClO2 (3 equiv.) in t-BuOH-H2O (5:1, v/v) was added the 2-alkylfuran.

The resulting mixture was stirred at room temperature until the yellow color disappeared

or until the starting material was consumed as determined by TLC. Then the solvent was

removed on a rotary evaporator. The residue can be directly used for the isomerization.

To purify the intermediate formed, the residue is extracted with ethyl acetate, washed

41

with brine, dried over MgSO4 and passed through a short silica gel column using ethyl

acetate as eluent.

Typical procedure for cis/trans isomerization. A catalytic amount of freshly distilled

pyridine was added to a solution (THF/acetone/water 5:4:1) of hydroxybutenolide. The

mixture was stirred for 2 hours at room temperature. Solvents were removed on a rotary

evaporator and a high vacuum pump.

4-Furan-2-ylbutyl-4-toluenesulphate (2.14).

O OTsO OP TsCl, Et3N, DMAP

CH2Cl2, 0 oC

91% 2.142.11, P = H

To a suspension of 2-furylbutan-4-ol38 (33 mg, 0.23 mmol) in CH2Cl2 (3 mL) was

added triethylamine (100 µL, 0.7 mmol), p-toluenesulfonyl chloride (50 mg, 0.26 mmol),

DMAP (3 mg, 0.023 mmol) with stirring at room temperature. When TLC showed no

starting material, 5 mL of dil. HCl (pH 3) was added and the resulting mixture was

extracted with CH2Cl2 (3 x 20 mL). The combined extract was washed with brine, dried

over MgSO4, solvent was removed in vacuo and the residue was purified by column

chromatography with 15% ethyl acetate in hexanes (Rf = 0.4) to yield an oily liquid (62

mg, 91%).60 1H NMR (CDCl3, 300 MHz) δ 7.77 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz,

2H), 7.25 (dd, J1 = 1.8 Hz, J2 = 0.9 Hz, 1H), 6.24 (dd, J1 = 3.0 Hz, J2 = 1.8 Hz, 1H), 5.92

(dd, J1 = 3.0 Hz, J2 = 0.9 Hz, 1H), 4.01 (t, J = 6.3 Hz, 2H), 2.56 (t, J = 6.6 Hz, 2H), 2.43

(s, 3H), 1.63-1.64 (4H); 13C NMR (CDCl3, 75 MHz) δ 155.1, 144.6, 140.8, 132.9, 129.7,

127.8, 110.0, 105.0, 70.1, 28.1, 27.0, 23.8, 21.5; HRMS (EI) m/z calcd for

C15H18O4S+(M+) 294.0926 found 294.0924.

42

4-(2-Hydroxy-5-oxo-2,5-dihydro-furan-2-yl)-butyl-4-toluenesulphate (2.36).

O

OTs

OHOO OTs NaClO2, NaH2PO4

tBuOH:H2O(5:1), rt

90%2.14 2.36

To a magnetically stirred solution of NaH2PO4 (12.4 mg, 0.09 mmol) and NaClO2

(16 mg, 0.18 mmol) in 1mL t-BuOH-H2O (5:1, v/v) was added alkylfuran 2.14 (20 mg,

0.06 mmol). The resulting mixture was stirred at room temperature until the starting

material was consumed as determined by TLC. Then the solvent was removed on a rotary

evaporator. The residue was extracted with ethyl acetate, washed with brine, dried over

MgSO4 and passed through a short silica-gel column using ethyl acetate to afford 2.36

(17mg, 90%). 1H NMR (CDCl3, 300 MHz) δ 7.75 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz,

2H), 7.19 (d, J = 5.5 Hz, 1H), 6.09 (d, J = 5.5 Hz, 1H), 4.03 (m, 2H), 2.43 (bs, 3H), 1.4-

1.8 (6H). This was converted to the trans isomer that was further characterized by 1H and

13C NMR as well as HRMS.

4-Oxo-8-(toluene-4-sulphonyloxy)-oct-2-enoic acid (2e).

O

OTs

OHO

HO

O

O

OTsPyridine

THF:Acetone:H2O(5:4:2) 2h, rt2.36 2.17

To a solution of 2.36 (17 mg, 0.05 mmol) in 1 mL THF/acetone/water (5:4:1)

was added a catalytic amount (10 µL) of freshly distilled pyridine. The mixture was

stirred for 2 h at room temperature. Solvents were removed on a rotary evaporator and a

vacuum pump to yield the product 2.17 quantitatively. (TLC: 30% ethyl acetate in

43

hexanes, Rf = 0.32). 1H NMR (CDCl3, 200 MHz) δ 7.77 (d, J = 8.2 Hz, 2H), 7.33 (d, J =

7.9 Hz, 2H), 7.12 (d, J = 15.9 Hz, 1H), 6.63 (d, J = 16.1 Hz, 1H), 4.03 (m, 2H), 2.62 (m,

2H), 2.43 (bs, 3H), 1.6-1.8 (4H); 13C NMR (CDCl3, 50 MHz) δ 198.1, 168.1, 144.9,

144.8, 140.6, 132.9, 129.8, 127.8, 69.9, 40.5, 28.0, 21.6, 19.4; HRMS (FAB) m/z calcd

for C15H19O6S+(M+H+) 327.0902 found 327.0903.

5-Hydroxy-5-pentyl-5H-furan-2-one (2.37).

OC5H11

OHOO C5H11 NaClO2, NaH2PO4

tBuOH:H2O(5:1), rt95%2.5 2.37

Oxidation of 2-pentylfuran (50 mg, 0.36 mmol) was carried out as mentioned in

the general procedure for oxidation using NaH2PO4 (74 mg, 0.54 mmol) and NaClO2 (98

mg, 1.08 mmol) in 3 mL t-BuOH-H2O (5:1, v/v). 1H NMR (300 MHz, CDCl3) δ 7.17 (d,

J = 5.7 Hz, 1H), 6.11 (d, J = 5.7 Hz, 1H), 1.98 (m, 2H), 1.28-1.41 (7H), 0.87 (t, J = 6.3

Hz, 3H). This compound was further characterized by conversion to the trans isomer.

OC5H11

OHO

Pyridine

THF:Acetone:H2O(5:4:2) 2h, rt

HOC5H11

O

O2.37 2.6

The butenolide 2.37 was converted to 4-oxo-2-nonenoic acid (2.6) by the general

isomerization procedure mentioned above in quantitative yields. The spectral data

matched the earlier report.32

44

8-(tert-Butyldimethylsilanyloxy)-4-oxo-oct-2(Z)-enoic acid (2.38).8

O

OTBDMS

OHOO OTBDMS NaClO2, NaH2PO4

tBuOH:H2O(5:1), rt77%2.16 2.38

The oxidation of 2.16 by the general procedure yielded the corresponding

butenolide 2.38. (TLC: 20% ethyl acetate in hexanes, Rf = 0.37) Yield: 77%. 1H NMR

(200 MHz, CDCl3) δ 7.21 (d, J = 5.5 Hz, 1H), 6.10 (d, J = 5.5 Hz, 1H), 3.64 (t, J = 5.9

Hz, 2H), 2.1-1.9 (2H), 1.7-1.4 (4H), 0.88 (s, 9H), 0.05 (s, 6H); 13C NMR (75 MHz,

CDCl3) δ 170.3, 154.2, 123.1, 108.0, 62.9, 36.9, 31.8, 25.9, 20.2, 18.3, -5.3. HRMS

(FAB) m/z calcd for C14H27O4Si+ (M+H+) 287.1673, found 287.1679.

5-[3-(tert-Butyldimethylsilanyloxy)butyl]-5-hydroxy-5H-furan-2-one (2.21).

HO

OH

I

OTBDMS

1.TsCl, Et3N, DMAP2.TBDMSCl, 3.NaI, K2CO3

OTBDMSO

O1.tBuLi2.2.43

2.42 2.43

2.20

O OHOO NaClO2, NaH2PO4

tBuOH:H2O(5:1), rt

TBDMSOOTBDMS

2.20 2.21

The furyl precursor 2.20 was prepared in 4 steps. The diol 2.42 was selectively

tosylated using p-toluenesulfonyl chloride as reported earlier,61 followed by TBDMS

protection of the secondary alcohol using TBDMSCl 62 and iodination63 to yield the iodo

compound 2.43. Alkylation of furan to generate the 2-alkyl derivative 2.20 using tBuLi

45

and 2.43 was performed as reported earlier.64 To a magnetically stirred solution of furan

2.20 (35 mg, 0.05 mmol) in 2 mL of tBuOH:H2O (5:1) was added NaClO2 (0.15 mmol)

and NaH2PO4 (0.75 mmol). The reaction was continued until the reactant was completely

consumed. The solvent was removed by rotary evaporation. The residue, resuspended in

ethyl acetate, was passed through a short plug of Celite to give the butenolide 2.21 in

70% yield. (TLC: 20% ethyl acetate in hexanes, Rf = 0.3) Yield: 75%. 1H NMR (CDCl3,

300 MHz) δ 7.19 (dd, J1 = 12.9 Hz, J2 = 6 Hz, 1H), 6.05 (m, 1H), 4.01 (m, 1/2H), 3.95

(m, 1/2H), 1.6-2.2 (6H), 1.1-1.2 (6H), 0.8-0.9 (9H), (-0.01)-0.16 (6H). This compound

was further characterized by conversion to the trans isomer.

O OHO

HO

O

O

Pyridine

THF:Acetone:H2O(5:4:2) 2h, rt

OTBDMS OTBDMS

2.222.21

The butenolide 2.21 was isomerized to the trans form using the general procedure

described above to yield 2.22 in 95% yield. The proton and carbon spectra were

comparable to the earlier reports.36

5-Hydroxy-5-[4-(tetrahydropyran-2-yloxy)-butyl]-5H-furan-2-one (2.39).

O

OTHP

OHOO OTHP NaClO2, NaH2PO4

tBuOH:H2O(5:1), rt

2.15 2.39

A magnetically stirred suspension of sodium chlorite (24 mg, 0.27 mmol) and

NaH2PO4 (18 mg, 0.13 mmol) in 1mL of tBuOH/H2O (5:1) was added to 2-(4-furan-2-yl-

butoxy)tetrahydropyran (2.39) (20 mg, 0.09 mmol). The reaction mixture was stirred at

46

room temperature and monitored by TLC. After 2 h, solvent was removed by rotary

evaporation, the NMR of the crude residue showed characteristic peaks of the butenolide

2.39. The residue was used for next step without purification.

O

OTHP

OHO

HO

O

O

OTHPPyridine

THF:Acetone:H2O(5:4:2) 2h, rt2.39 2.18

The crude butenolide 2.39, was converted to the trans derivative 2.18 using the

general procedure. The compound 2.18 is not stable on a silica gel column. The sec-butyl

ester derivative of the acid was characterized as described below.

4-Oxo-8-(tetrahydro-pyran-2-yloxy)-oct-2-enoic acid, sec-butyl ester (2.41).

HO

O

O

OTHPO

O

O

OTHPsec-BuOH, DCC,DMAP

CH2Cl2, rt70%, 3 steps2.18 2.40

To a solution of 2.18 (27 mg, 0.1 mmol) in CH2Cl2 (5 mL) was added DCC (31

mg, 0.15 mmol), DMAP (2.4 mg, 0.02 mmol) and sec-butanol (10 µL, 0.11 mmol). The

solution was stirred over night. The reaction mixture was diluted with CH2Cl2, washed

with water, then brine and dried on MgSO4. The solvent was then removed and the

residue was purified using column chromatography (40% ethyl acetate in hexanes Rf =

0.4) to obtain an oil 2.40 (27 mg, 70%). 1H NMR (300 MHz, CDCl3) δ 7.03 (d, J = 16

Hz, 1H), 6.66 (d, J = 16 Hz, 1H), 4.93 (m, 1H), 4.55 (m, 1H), 3.6-3.8 (m, 2H), 3.3-3.9

(2H), 2.68 (t, J = 6.9 Hz, 2H), 1.4-1.8 (14H), 1.25 (d, J = 6.3 Hz, 2H), 1.18 (d, J = 6.3 Hz,

1H), 13C NMR (APT, 50 MHz) (+)199.7, (+)165.2, (-) 139.1, (-) 131.3, (-) 98.9, (-) 73.5,

47

(+) 67.0, (+) 62.4, (+) 41.0, (+) 30.7, (+) 29.0, (+) 28.7, (+) 25.4, (+) 20.6, (+) 19.6, (-)

19.3, (-) 9.6. HRMS (FAB) m/z calcd for C17H29O5 (M+H+) 313.2015 found 313.2017.

1-Palmityl-2-(1-carboxy-4-(2-hydroxy-5-oxo-2,5-dihydro-furan-2-yl)-butanoyl)-sn-

glycero-3-phosphatidylcholine (2.26).

O O

OPC

2

HO

O

OO

OPC

2


>75%

2.20

2.24 2.26

To a magnetically stirred solution of 8-furyl butanoyl ester derivative of the

phosphatidylcholine 2.2438 (5 mg, 8 µmol) in a solution of phosphate buffer pH 3.5,

chloroform and water (2:1:1) (1 mL) was added sodium chlorite (3.5 mg, 39 µmol). After

20 min, the solution was concentrated and purified using a 1 mL solid phase extraction

(SPE) column, eluting with a methanol:water gradient (50 –100% methanol) to yield 2.26

(3.5 mg, 66%). 1H NMR (CD3OD, 300 MHz) δ 7.41 (bs, 1H), 6.15 (d, J = 6 Hz, 1H),

5.25 (m, 2H), 4.39 (m, 1H), 4.28 (m, 2H), 4.21 (m, 1H), 4.01 (m, 2H), 3.66 (m, 2H), 3.23

(s, 9H), 2.3-2.4 (4H), 1.5-2.1 (6H), 1.2-1.3 (24H), 0.88 ( t, J = 6 Hz, 3H); HRMS

(MALDI-TOF): m/z calcd for C32H59NO11P+ (MH+) 664.3826 found 664.3820

1-Palmityl-2-(7-carboxyl-5-oxohept-6-enoate)-sn-glycero-3-phosphatidylcholine

(KOdiA-PC, 2.3).

OOO

OOHPC

2

O O

OPC

2

HO

O

Pyridine

THF:Acetone:H2O(5:4:2) 2h, rt 2.32.26

48

The butenolide 2.26 was isomerized by the general method mentioned above to

the trans form 2.3. The 1H NMR spectral data of the product 2.3 is in agreement with that

previously reported.38

1-Palmityl-2-(1-carboxy-8-(2-hydroxy-5-oxo-2,5-dihydro-furan-2-yl)-octanoyl)-sn-

glycero-3-phosphatidylcholine (2.27).

O O

OPC

6

HO

O

OO

OPC

6


>75%2.25 2.27

To a magnetically stirred solution of the 8-furyl octanoyl ester derivative of

phosphatidylcholine 2.2538 (9 mg, 13 µmol) in a solution of phosphate buffer pH 3.5,

chloroform and water (2:1:1) (1 mL) was added sodium chlorite (3.5 mg, 39 µmol). After

1 h the solution was concentrated, and purified using an SPE column, eluting with a

methanol:water gradient (50 –100% methanol) to yield 2.27 (6 mg, 64%). 1H NMR

(CD3OD, 300 MHz) δ 7.41 (bs, 1H), 6.12 (d, J = 6 Hz, 1H), 5.25 (m, 2H), 4.4 (m, 1H),

4.27 (m, 2H), 4.16 (dd, J1 = 12.6 Hz, J2 = 6.3 Hz, 1H), 4.01 (t, J = 5.1 Hz, 2H), 3.66 (m,

2H), 3.23 (s, 9H), 2.3- 2.4 (4H), 1.5-2.1 (5H), 1.2-1.3 (30H), 0.88 ( t, J = 6 Hz, 3H);

HRMS (MALDI-ToF): m/z calcd for C36H67NO11P+ (MH+) 720.4457 found 720.4424.

1-Palmityl-2-(11-carboxyl-9-oxoundec-6-enoate)-sn-glycero-3-phosphatidylcholine

(KDdiA-PC, 2.4).

OOO

OOHPC

6

O O

OPC

6

HO

O

Pyridine

THF:Acetone:H2O(5:4:2) 2h, rt 2.42.27

49

The butenolide 2.27 was isomerized by the general method described above. The

spectral data of the product 2.4 is in agreement with the previously reported data.38

Ring-Chain Tautomerism:

The product of 2-alkylfuran oxidation by sodium chlorite was confirmed to be a

butenolide under acidic conditions and the corresponding chain isomer at higher pH

through monitoring the λmax. The UV data was collected by dissolving compound 2.37 in

water of varying pH. The graph shows the increase of the peak intensity at 240-250 nm at

higher pH.

50

2.5. References: (1) Witztum, J. L.; Berliner, J. A. Curr Opin Lipidol 1998, 9, 441-8. (2) Girotti, A. W. J. Lipid Res. 1998, 39, 1529-1542. (3) Farooqui, A. A.; Horrocks, L. A. Cellular Mol Neurobiol 1998, 18, 599-608. (4) Balamraju, Y. N.; Sun, M.; Salomon, R. G. J Am Chem Soc 2004, 126, 11522-8. (5) Bergamini, C.; Gambetti, S.; Dondi, A.; Cervellati, C. Curr Pharm Design 2004,

10, 1611-26. (6) McCall, M. R.; Frei, B. Free Radic Biol Med 1999, 26, 1034-53. (7) Finkel, T.; Holbrook, N. J. Nature 2000, 408, 239-47. (8) Sun, M. Ph. D, Case Western Reserve University, 2004. (9) Parthasarathy, S.; Quinn, M. T.; Schwenke, D. C.; Carew, T. E.; Steinberg, D.

Arteriosclerosis 1989, 9, 398-404. (10) Podrez, E. A.; Poliakov, E.; Shen, Z.; Zhang, R.; Deng, Y.; Sun, M.; Finton, P. J.;

Shan, L.; Febbraio, M.; Hajjar, D. P.; Silverstein, R. L.; Hoff, H. F.; Salomon, R. G.; Hazen, S. L. J Biol Chem 2002, 277, 38517-23.

(11) Podrez, E. A.; Poliakov, E.; Shen, Z.; Zhang, R.; Deng, Y.; Sun, M.; Finton, P. J.; Shan, L.; Gugiu, B.; Fox, P. L.; Hoff, H. F.; Salomon, R. G.; Hazen, S. L. J Biol Chem 2002, 277, 38503-16.

(12) Walton, H. M. J Org Chem 1957, 22, 308-12. (13) Sekiguchi, J.; Kuroda, H.; Yamada, Y.; Okada, H. Tetrahedron Lett 1985, 26,

2341-2. (14) Pfefferle, C.; Kempter, C.; Metzger, J. W.; Fiedler, H. P. J Antibiot (Tokyo) 1996,

49, 826-8. (15) Nozoe, S.; Hirai, K.; Tsuda, K.; Ishibashi, K.; Shirasaka, M.; Grove, J. F.

Tetrahedron Lett 1965, 4675-7. (16) Kalita, D.; Khan, A. T.; Barua, N. C.; Bez, G. Tetrahedron 1999, 55, 5177-5184. (17) Kobayashi, Y.; Kumar, G. B.; Kurachi, T.; Acharya, H. P.; Yamazaki, T.;

Kitazume, T. J Org Chem 2001, 66, 2011-8. (18) Ronsheim, M. D.; Zercher, C. K. J Org Chem 2003, 68, 1878-1885. (19) Appleton, D.; Duguid, A. B.; Lee, S.-K.; Ha, Y.-J.; Ha, H.-J.; Leeper, F. J. J

Chem Soc, Perkin Trans 1: Org Bio-Org Chem 1998, 89-101. (20) Obrecht, D.; Weiss, B. Helvetica Chim Acta 1989, 72, 117-22. (21) Kawashima, M.; Sato, T.; Fujisawa, T. Bull Chem Soc Jpn 1988, 61, 3255-64. (22) Pappalardo, P.; Ehlinger, E.; Magnus, P. Tetrahedron Lett 1982, 23, 309-12. (23) D'Auria, M.; Piancatelli, G.; Scettri, A. Tetrahedron 1980, 36, 3071-4. (24) Gunn, B. P.; Brooks, D. W. J Org Chem 1985, 50, 4417-18. (25) Finlay, J. M., M. Anthony; Gunaratne, H. Q. Nimal Tetrahedron Lett 1998, 39,

5651. (26) Cottier, L.; Descotes, G.; Eymard, L.; Rapp, K. Synthesis 1995, 303-6. (27) Franzen, R.; Tanabe, K.; Morita, M. Chemosphere 1998, 38, 973-980. (28) Asaoka, M. Y., N.; Sugimura, N.; Takei, H. Bull Chem Soc Jpn 1980, 53, 1061-4. (29) Oishi, S.; Nelson, S. D. J Org Chem 1992, 57, 2744-2747. (30) Boukouvalas, J.; Lachance, N. Synlett 1998, 31-32. (31) Adger, B. M. B., C.; Brennan, J.; McKervey, M. A.; Murray, R. W. J Chem Soc,

Chem Comm 1991, 21, 1553-4.

51

(32) Ballini, R.; Bosica, G. J Nat Prod 1998, 61, 673-4. (33) Hirsch, J. A.; Szur, A. J. Tetrahedron 1972, 28, 2961-7. (34) Gunn, B. P. Tetrahedron Lett 1985, 26, 2869-72. (35) Hase, T. A.; Nylund, E. L. Tetrahedron Lett 1979, 2633-6. (36) Kobayashi, Y.; Kishihara, K.; Watatani, K. Tetrahedron Lett 1996, 37, 4385-

4388. (37) Kobayashi, Y.; Nakano, M.; Kumar, G. B.; Kishihara, K. J Org Chem 1998, 63,

7505-7515. (38) Sun, M.; Deng, Y.; Batyreva, E.; Sha, W.; Salomon, R. G. J Org Chem 2002, 67,

3575-84. (39) Barco, A.; Benetti, S.; De Risi, C.; Pollini, G.; Zanirato, V. Tetrahedron 1995, 51,

7721. (40) Ngooi, T. J Org Chem 1989, 54, 911. (41) Rao, A. V. R.; Reddy, K. B.; Dhar, T. G. M. Indian J Chem, Section B: Org Chem

Incl Med Chem 1986, 25B, 1014-16. (42) Barco, A.; Benetti, S.; De Risi, C.; Pollini, G. P.; Zanirato, V. Tetrahedron 1995,

51, 7721-7726. (43) Takeya, H. In Eur. Pat. Appl.; (Cosmo Research Institute, Japan; Cosmo Oil Co.,

Ltd.). Ep, 1995. (44) Lukevics, E. Y.; Ignatovich, L. M.; Gol'dberg, Y. S.; Shymanskaya, M. V.

Khimiya Geterotsiklicheskikh Soedinenii 1986, 853-4. (45) Nakamura, I.; Saito, S.; Yamamoto, Y. J Am Chem Soc 2000, 122, 2661-2662. (46) Ochiai, M.; Arimoto, M.; Fujita, E. Tetrahedron Lett 1981, 22, 4491-4. (47) Ballini, R.; Bosica, G.; Fiorini, D.; Gil, M. V.; Petrini, M. Org Lett 2001, 3, 1265-

1267. (48) Yu, J.-Q.; Corey, E. J. J Am Chem Soc 2003, 125, 3232-3233. (49) Carles, L.; Narkunan, K.; Penlou, S.; Rousset, L.; Bouchu, D.; Ciufolini, M. A. J

Org Chem 2002, 67, 4304-4308. (50) Dittami, J. P.; Xu, F.; Qi, H.; Martin, M. W.; Bordner, J.; Decosta, D. L.;

Kiplinger, J.; Reiche, P.; Ware, R. Tetrahedron Lett 1995, 36, 4197-200. (51) Halland, N.; Hazell, R. G.; Jorgensen, K. A. J Org Chem 2002, 67, 8331-8338. (52) Halland, N.; Aburel, P. S.; Jorgensen, K. A. Angwante Chem, Int Edn 2003, 42,

661-665. (53) Gollnick, K.; Griesbeck, A. Tetrahedron 1985, 41, 2057-68. (54) Saldabol, N. O.; Popelis, J.; Slavinska, V. Chem Heterocyclic Compds (New York,

NY, United States)(Translation of Khimiya Geterotsiklicheskikh Soedinenii) 2002, 38, 873-881.

(55) Noyori, R.; Sato, T.; Kobayashi, H. Bull Chem Soc Jpn 1983, 56, 2661-79. (56) Williams, P.; Eugene, L. J Org Chem. 1981, 46(21), 4143-4147. (57) Shet, J.; Desai, V.; Tilve, S. Synthesis 2004, 1859-1863. (58) Bouchain, G.; Leit, S.; Frechette, S.; Khalil, E.; Lavoie, R.; Moradei, O.; Woo, S.;

Fournel, M.; Yan, P.; Kalita, A.; Trachy-Bourget, M.; Beaulieu, C.; Li, Z.; Robert, M.; MacLeod, A.; Besterman, J.; Delorme, D. J Med Chem 2003, 46, 820-30.

(59) Annangudi, S. P.; Sun, M.; Salomon, R. G. Synlett 2005, 2005, 1468-1470.

52

(60) O'Neill, J. A.; Lindell, S. D.; Simpson, T. J.; Willis, Christine L. Tetrahedron: Asymm 1994, 5, 117-118.

(61) O'Neil, J. A.; Lindell, S. D.; Simpson, T. J.; Willis, C. L. Tetrahedron: Asymm 1994, 5, 117-18.

(62) Bonini, C.; Chiummiento, L.; Evidente, A.; Funicello, M. Tetrahedron Lett 1995, 36, 7285-6.

(63) Allingham, M. T.; Howard-Jones, A.; Murphy, P. J.; Thomas, D. A.; Caulkett, P. W. R. Tetrahedron Lett 2003, 44, 8677-8680.

(64) Bracher, F.; Schulte, B. Nat Prod Res 2003, 17, 293-299.

53

Chapter 3

Detection and Characterization of Multiple 4-Hydroxynonenal Adducted Amino

acids Using Deuterium Labeled HNE and Mass Spectrometry

54

3.1. Background

Aerobic organisms use oxygen extensively for anabolic as well as catabolic

processes, which makes them prone to radical induced oxidative damage in vivo.1 These

organisms have developed mechanisms to circumvent this problem primarily by using

antioxidants that can quench the toxic radicals2-8 as well as by evolving efficient

recycling pathways to of dispose of the damaged biomolecules.1 Polyunsaturated fatty

acids 9 (PUFAs), proteins10 and DNA11 are some of the targets of radical induced

damage. Enzyme mediated lipid peroxidation products are known to be involved in

normal physiological functions, e.g., generating prostaglandins and thromboxanes.12,13

Analogous radical induced peroxidation of PUFAs generates prostaglandin isomers

(isoprostanes) and in addition it can form highly reactive levulinaldehyde derivatives

with prostaglandin side chains (isolevuglandins).14 Isolevuglandins form adducts with

side chains of proteins and DNA bases. The adducts so formed are implicated in various

pathological processes such as atherosclerosis,15 age related macular degeneration 16-18

and neurodegeneration.19,20

Arachadonic and linoleic acids phospholipids have four and two conjugated

double bonds respectively, making them highly susceptible to free radical attack followed

by rearrangements and/or peroxidation leading to an array of reactive oxidation

products.21 The staggering number of molecular targets in the intracellular as well as

intercellular environment for these highly reactive lipobolites (products of lipid

degradation) form a complicated mixture of products, making it very difficult to identify

them and assign molecular structures.22 Our group is involved in a systematic and

55

mechanistic approach to predict structures for lipobolites and the stable adducts that they

form with macromolecules.

Hydroxynonenal (HNE), a lipid peroxidation product of arachidonyl and linoleyl

lipids, has been very well studied mainly due to its proclivity to form pathological

adducts23-26 with nucleophilic amino acid residues of proteins. Modification by HNE has

been shown to alter the structure27 and function28,29 of proteins. The adducts are

immunogenic and can elicit an immune response, thus demonstrating that these are not

native to the human body.30,31 The HNE- Michael adduct has been studied extensively.

Lysine, cysteine and histidine residues form Michael adducts readily.24 Besides the

reversible formation of Michael adduct, the ε-amino group of lysine residues can

reversibly form Schiff bases, that may rearrange followed by dehydration to form more

stable pyrroles.32 In contrast, histidine and cysteine amino acid residues only form stable

Michael adducts.33 In vitro, a histidine-lysine crosslink was observed for model

compounds, but there are no reports of their detection in vivo.34

NMR studies in collaboration with Dr. Sayre’s group detected the formation of

adducts that have 2 or more aldehydes per amine group, from the reaction of equimolar

amounts of HNE and primary amines.32 The detection and characterization of these

adducts involved derivatization using strong reducing and/or acidic conditions.

Therefore, only adducts that are stable under these conditions were detected. There is a

need for mild techniques that are compatible with the products, which are stable under

physiological conditions, but readily decompose under harsher conditions. To address

this problem, we are applying isotope labeling and mild conditions to probe adduction

chemistry that occurs under physiological conditions.

56

Studies on HNE modification of LDL by Esterbauer’s group,35 have shown that

there is a steady increase in the amount of HNE bound per mol of LDL with increasing

amount of HNE. The number of nucleophilic amino acid residues in LDL proteins

modified by 13C labeled HNE was estimated to be 0.71 - 1.3 mol/mol of HNE. This

suggests that a single amino acid residue might be modified by more than one HNE.

Furthermore, a high concentration of the HNE (> 10 mM) caused the LDL to precipitate.

This may be because HNE neutralizes the positively charged residues (lysine, histidine,

arginine) on the LDL and/or cause protein aggregation.

Deng et al. performed similar studies with tritium labeled [9-3H]-HNE.36

Incubation of [9-3H]-HNE with LDL, revealed that the number of HNE’s that can

become covalently linked to an LDL particle i.e., 486 molecules per particle, was far

greater than the number of “reactive” lysine, ethanolamine phospholipids, histidine and

cysteine residues i.e., 365 present in each particle (Table 2.1). This indicated that 1:1

Table 3.1. Nucleophilic functional groups in LDLa

Residue Total Reactive

lysine ε-NH2 350 225*

PEs 80 80

histidine 115 115 (?)

cysteine 25 25 (?)

Total 570 365

*TNBS reactive amino groups are 317±29 that would include all reactive lysines and all EPs: which is 225 + 80 = 305. "?" means the amount of actually reactive histidine and cysteine remains unclear. (Adapted from Dr. Deng’s Thesis)

57

adducts e.g. Michael adducts, Schiff bases and pyrroles alone cannot account for the

level of adduction observed. Rather adducts incorporating two or more HNE per

nucleophilic residue must be generated.

To characterize the type of adducts that might be generated in an LDL particle, the

present study focused on qualitative analysis of these adducts formed by HNE with

amino acids (N-acetylated lysine, glycine-lysine methyl histidine and cysteine) using

deuterium labeled HNE and mass spectrometric analyses.

3.1.1. Previous syntheses of deuterated HNE. HNE being one of the most investigated

molecules of the lipid oxidation products, a wide range of synthetic methods have been

reported.36 Synthesis incorporating carbon and hydrogen isotopes have been developed

for use as a probe for investigating metabolic pathways,4 as mass spectrometric

standards37 or to study adducts formed by HNE.38 Among the many procedures

described previously to synthesize deuterium labeled HNE, only two were used

extensively. One involved cobalt (II) porphyrin-catalyzed reductive oxygenation of

commercially available α,β,γ,δ-unsaturated aldehydes. Sugamoto’s group used this

method to synthesize a deuterium labeled HNE from 2(E),4(E)-nonadienal in two steps

(Scheme 3.1). 39,40

58

O

Co II (tdcpp)(0.001 eq.)O2 (1 atm)Et3SiD (1.2 equiv.)

PriOD-DCM (1:1)

OOOH

D

PPh3

OOH

D

54%

Scheme 3.1: Synthesis of [2H1]-HNE reported by Sugamoto’s group.40

Another method involved the use of fumaraldehyde dimethylacetal that is

commercially available, as a precursor for synthesis of HNE. Selective deprotection and

alkylation, of the formaldehyde dimethylacetal using readily available deuterated

alkylating reagents for the deuterium incorporation, followed by deprotection of the

dimethylacetal, generated the required HNE (Scheme 3.2).41,42

59

H3CO

OCH3

OCH3

OCH3H3CO

OCH3

O

Amberlyst 15catalyst

RMgBr

H3COR

OCH3

OH

OR

OH

Amberlyst 15catalyst

R = -(CH2)4-CD3, -(CD2)4-CD3

Scheme 3.2: Synthesis of [2H3]-HNE and [2H11]-HNE from fumaraldehyde

dimethylacetal.

[8,8,9,9-2H4]-HNE is optimal for detecting multiple adducts. Choosing the right kind

of deuterated HNE is important for many practical reasons. Firstly, the deuterium atoms

should be in a position that precludes it from loss or any chemical reactivity. The carbons

1-4 are involved in the adduction chemistry and the protons at carbon 5-7 may be

involved in the McLafferty type rearrangements43 that could occur in the mass

spectrometric ionization process as shown below.

BA

D

HBA D

H

αβ

γ

δ

Secondly, the number of deuteria present should be sufficient to readily

differentiate adducts of labeled and unlabeled HNE from the isotopic peaks that arise

from the various isotopic abundances of some of the elements. Since our study was to be

60

focused on the elements C, H, O, N and S, the highest isotopic peak (> 1%) is at M+1 for

carbon and M+2 for sulphur Table 2.2. Thirdly, the number of deuteria incorporated

should be easily achieved synthetically. The use of multiples of two is readily achieved

by hydrogenation of alkenes. All these considerations indicated that the deuterium atoms

should be located at C8 or C9 of the HNE molecule and that there should be > 2

deuterium atoms. We, therefore, chose to incorporate four deuterium atoms at the C8 and

C9 carbons by reduction of an alkyne bond. Consequently, we prepared a deuterated

alkylating reagent by deuteration of an alkyne triple bond.

Table 3.2. Isotopic mass abundances of some common elements 44

Element Mass No. Isotopic Composition (%) Isotopic mass

1H 2H

1

2

100

0.0115

1.007

2.014 12C 13C

12

13

100

1.08

12.000

13.003 14N 15N

14

15

100

0.369

14.003

15.000 16O 17O 18O

16

17

18

100

0.038

0.205

15.995

16.999

17.999 32S 33S 34S 36S

32

33

34

36

100

0.08

4.52

0.02

31.972

32.971

33.967

35.967

61

3.2. Results

3.2.1. Synthesis of [8,8,9,9-2H4]-HNE. 4-Pentynol (3.1) was used as a precursor for

carbons 5-9 and the protected aldehyde 3.5 as the precursor for carbons 1-4 of the d4-

HNE. 4-Pentynol was converted to its tosyl-derivative 3.2 for two reasons; (i) to decrease

the loss of the alcohol due to its low boiling point and (ii) to be easily converted to the

iodopentane 3.4 for the subsequent alkylation step. The d4-pentanol tosyl ester 3.3 was

obtained in 74% yield by deuteration of pent-4-ynyl tosyl ester (3.2)45 using Wilkinson’s

catalyst, RhCl(PPh3)4, and deuterium.46 The d4-pentyl iodide (3.4) was generated by

refluxing the tosylate 3.3 with NaI and K2CO3 in acetone (Scheme 3.3).36

d4-Pentyllithium was generated by metal halogen exchange of 3.4 using t-BuLi.

Alkylation of aldehyde 3.5 by pentyllithium yielded the stable precursor 3.6 of d4-HNE in

84 % overall yield. Preparation of HNE from this precursor was performed by a one-step

procedure, using NaIO4 and aqueous acetic acid, to give d4-HNE (3.7) in 89 % yield

(Scheme 3.3). An alternative two-step procedure using acetic acid and lead acetate gave

HNE in 78 % yield (Scheme 3.3).47 Commercially available pentylmagnesium bromide

(3.8) was used as alkylating agent to synthesize unlabeled HNE (Scheme 3.4).

62

OR

TsCl

RhCl(PPh3)3, D2HD2C

D2C O

SO

O

HD2C

D2C R

3.3, R=OTs3.4, R=I

NaI,Acetone

+ OOO

OOHD2C

CD2 OH

BuLi

Ethyl ether

NaIO4

Acetic acid:H2O (2:1)

OOHD2C

CD2 OH

OHD2C

CD2 OH

3.3

3.6

3.7

3.1, R= H3.2, R=Ts, 86%

C6H6

3.5

89%

84%

74%

Scheme 3.3: Synthesis of [8,8,9,9-2H4]-HNE.

MgBr + OOO

OO

OH

BuLi

Ethyl ether

NaIO4

Acetic acid:H2O (2:1)

OO

OHO

OH

3.9

3.10

3.5

90%

87%3.8

Scheme 3.4: Synthesis of HNE.

63

3.2.2. Reactions of N-acetyl amino acids with HNE. A solution of HNE and d4-HNE

(1:1) (10 µM) was incubated with various amino acids (N-acetyl glycine-lysine-methyl

ester (N-acetyl-Gly-Lys-OMe), N-acetyl lysine, N-acetyl histidine (N-acetyl-His) and N-

acetyl-cysteine (N-Acetyl-Cys) individually in PBS at 37 ºC for 24 h. The resulting

mixtures were dried on a speed vacuum, extracted with methanol and dried using a

stream of nitrogen. The resulting residue was dissolved in 50% methanol/water with a

trace of formic acid (0.01%) and analyzed by mass spectrometry.

3.2.3. Mass spectrometric analysis of the HNE-amino acid adducts. A preliminary

analysis of the reaction mixtures using MALDI-TOF mass spectrometry revealed the

presence of numerous peaks that appear as unique sets of multiplets with a mass

difference of 4 Da as expected. Further analysis was performed using a Q-TOF mass

spectrometer by ESI-MS (Figure 3.1) and ESI-MS/MS to identify the products formed.

Also, semi-quantitative analyses of the molecular ions identified were done by liquid

chromatography and selected ion recording (LC-SIR) using a Quattro Ultima ™

(Micromass) mass spectrometer.

64

Figure 3.1: ESI-TOF-MS of reaction mixture containing HNE (d4:d0, 1:1) with amino

acids A) N-acetyl-gly-lys-OMe, B) N-acetyl-cysteine and C) N-acetyl-histidine.

(i) N-Acetyl-histidine-HNE adducts. The parent ion for the Michael adduct of N-acetyl-

His with HNE (d4:d0, 1:1) appears as a doublet at m/z 354 and 358 (Figure 3.1, panel C).

Tandem MS/MS analysis of these ions resulted in a specific signature of fragments

corresponding to the labeled or unlabeled HNE. Thus, MS/MS of the m/z 354 ion yields

daughter ions at m/z 336, 312, 308, 266, 198, 180, 177, 156, 152, 139, 110 and 95, while

tandem MS/MS of the ion at m/z 358 yields daughter ions at m/z 340, 316, 312, 270, 198,

180, 177, 156, 152, 143, 110 and 95 (Figure 3.2). Based on mass differences between the

ions from both of the spectra, daughter ions containing the C5-C9 alkyl chain of the HNE

were identified (vide infra). The structures of the ions in the mass spectrum can be

rationalized based on the patterns observed in the fragmentation of N-acetyl-His. Similar

to the fragmentation pattern reported for N-acetyl-His,48 fragmentation of the Michael

65

adduct exhibits a base peak formed by consecutive loss of the acetyl and the carboxyl

group at m/z 266 (d0) and 270 (d4) respectively. The daughter ion at m/z 139 corresponds

to the HNE moiety derived from retro Michael cleavage of the adduct after loss of a

molecule of water (Scheme 3.5).42,49,50

Figure 3.2: ESI-TOF-MS/MS analysis of Michael adducts parent ions at m/z 354 and

358 from the infusion of reaction mixture from HNE and N-acetyl-His (1:1).

HN OHO

N

N

O

O

3.11, m/z 354 (M + H+)

HO

311 (M-CH3CO + H+)

307 (M-COOH + H+)266 (M-CH3CO-COOH + H+)

251

198157-H2O = 139

Scheme 3.5: Proposed mass spectrometric fragmentation of a 1:1 (N-acetyl His)-HNE

Michael adduct parent ion at m/z 354. See Scheme 3.2S in Appendix for fragment

structures.

66

The total ion chromatogram of the reaction mixture (Figure 3.1) showed ions with

m/z much greater than 400. These molecular ion peaks can only be formed by reaction of

two or more of the reaction components. A triplet was observed at m/z 510, 514 and 518

(1:2:1) corresponding to an adduct with 2 HNE’s and one N-acetyl-His (Figure 3.1). The

tandem MS/MS of the m/z 510 precursor ion generates some of the prominent fragments

*

*

*

*

*****

*

**

* * * *

***

■■

■

■

■■

■■■

■

■

■

■

■

■

A

B

C

*

*

*

*

*****

*

**

* * * *

***

■■

■

■

■■

■■■

■

■

■

■

■

■

*

*

*

*

*****

*

**

* * * *

***

■■

■

■

■■

■■■

■

■

■

■

■

■

A

B

C

Figure 3.3: ESI-MS/MS of molecular ions at A) m/z 518, B) m/z 514 and C) m/z 510,

from the infusion of reaction mixture containing HNE and N-acetyl-His. Peaks labeled

with an asterix (*) or a solid rectangle (■) denote fragments with or without the C5-C9

alkyl chain of HNE, respectively.

67

corresponding to the loss of carboxyl, acetyl and HNE-d0 at m/z 464, 468 and 354

respectively (Figure 3.3), including ions observed for the fragmentation of the m/z 354

ion (Figure 3.2). The fragmentation of the parent ions at m/z 514 and 518 generated

daughter ions with masses 4 or 8 Da more than those observed for the m/z 510 ion

(Figure 3.3).

A quadruplet of ions (Figure 3.1C) at m/z 666, 670, 674 and 678 is present in the

total ion chromatogram of the reaction mixture representing adduction of three HNE’s

with one N-acetyl-His in an approximate 1:3:3:1 distribution. Tandem MS/MS analysis

of the m/z 666 ion yielded the peaks at m/z 624, 626, 578, 510 and peaks that were

observed in the fragmentation of m/z 510 ions (Figure 3.3). The fragmentation of the m/z

670 and 674 parent ions gave daughter ions that correspond to 4 or 8 Da more compared

to those observed for the m/z 666 ion (Figure 3.4).

Figure 3.4: ESI-MS/MS of molecular ions A) m/z 674, B) m/z 670 and C) m/z 666 from

infusion of reaction mixture HNE and N-acetyl-His (1:1) in Figure 3.1C.

68

HPLC separation of the (N-acetyl-His)-HNE reaction mixture followed by

selected ion recording (LC-ESI-SIR) of the prominent ions mentioned above in 13

channels was monitored on a Quattro ultima® electrospray mass spectrometer

(Micromass). As evident from the chromatograms in Figure 3.5, the peaks derived from

each adduct and its d4 labeled isomer have similar signatures. The 1:1 Michael adduct is

expected to be comprised of four diastereomers, that emerge at 8.6 – 9.4 min. In the case

of 2:1 and 3:1 adducts, much more complex mixtures of diastereomers are anticipated.

The total ion current for 1:1 adducts is greater than for 2:1 adducts, and that for 3:1

adducts is relatively minor. Also, with the increase in the number of HNE molecules

adducted on to N-acetyl-His, the polarity of the adducts change dramatically (1:1 > 2:1 >

3:1), as is evident from the selected ion recording (SIR) chromatograms (Figure 3.5).

69

Figure 3.5: LC-ESI for the reaction mixture from N-acetyl-His and HNE with selected

ion recording for 13 channels of which 7 channels are shown here. A) m/z 670, B) m/z

666, C) m/z 518, D) m/z 514, E) m/z 510, F) m/z 358 G) m/z 354 and H) TIC (for 13

channels).(for chromatograms of channels not shown here, see Appendix Figure 3.1S)

(ii) Lysine HNE adducts. N-acetyl-glycine-lysine methyl ester (N-acetyl-Gly-Lys-OMe)

was used as a lysine residue surrogate for the analysis of the adduction pattern with HNE.

Preliminary analysis using MALDI-TOF indicated that both N-acetyl lysine (NAL) and

N-acetyl-Gly-Lys-OMe showed similar patterns of adduction. Only the (N-acetyl-Gly-

70

Lys-OMe)-HNE reaction mixture was analyzed by electrospray MS to determine the

nature of these adducts. Fragmentation of the parent ion at m/z 260 (Figure 3.1A) from

this dipeptide generates daughter ions at m/z 242, 218, 201, 144 (base peak), 161, 129 and

84 (Figure 3.6).

Figure 3.6: ESI-MS/MS analysis of m/z 260 molecular ion. Tandem MS/MS analysis of

a solution of N-acetyl-Gly-Lys-OMe was injected into a Q-TOF mass spectrometer by

infusion at a flow rate of 0.5 µL/min.

The ESI- MS/MS spectrum of the molecular ion at m/z 416 and the isotope

labeled peak at m/z 420 (Figure 3.1A) corresponds to the Michael (1:1) adduct based on

the molecular weight and pattern of fragmentation. A carbinolamine formed by reaction

of the ε-amino group of N-acetyl-Gly-Lys and the aldehyde group of HNE has the same

molecular weight as the Michael adduct (m/z 354). The possibility of such structural

variations in the molecular ions cannot be ruled out. The major peaks in the spectrum are

formed from the loss of water (-18), an acetyl group (-44) and by retro-Michael

elimination (-156) (Figure 3.7, Scheme 3.6).

71

Figure 3.7: ESI-MS/MS of molecular ions at A) m/z 416 and B) m/z 420) (Figure 3.1A)

corresponding to a 1:1 HNE-(N-acetyl-Gly-Lys-OMe) Michael adduct.

NH O

HN

O

HNO

O

OHO

372 (M - CH3CO)

260 (MH+ - HNE)

139 (M - NGL -H2O)

398(M - H2O + H+)

3.12, m/z 416 (M + H+)

Scheme 3.6: Possible Michael adduct HNE - (N-acetyl-Gly-Lys-OMe) fragmentation.

See Appendix Scheme 3.4S for fragment structures.

The molecular ions corresponding to a Schiff base 3.13, formed from N-acetyl-Gly-Lys-

OMe and HNE, were observed at m/z 398 and 402 (Figure 3.7). Tandem MS analyses of

these ions are shown in Figure 3.8 and possible fragmentations proposed in Scheme 3.7.

72

Figure 3.8: ESI-MS/MS analysis of molecular ions A) m/z 402 and B) m/z 398 (Figure

3.7) corresponding to an HNE- (N-acetyl-Gly-Lys-OMe) Schiff base adduct.

NHO

N

O

NH

O

O

HO

222 (α−cleavage)204 (222 - H2O)

281 (M -H2O - amide cleavage)

354 (M - CH3CHO)

3.13, m/z 398 (M+H+)

Scheme 3.7: Possible Schiff base HNE- (N-acetyl-Gly-Lys-OMe) Schiff base

fragmentation. See Appendix Scheme 3.5S for fragment structures.

73

The tandem MS/MS of molecular ions at m/z 380 and m/z 384, corresponding to

the pyrrole adduct of the HNE with lysine, show a fragmentation consistent with the

structure of these adducts. One prominent feature among these spectra is the absence of

the retro-Michael adduct fragment at m/z 139/143 in the MS/MS of both the Schiff base

adduct 3.13 and the pyrrole adduct 3.14.

Figure 3.9: ESI-MS/MS analysis of molecular ions at A) m/z 384 and B) m/z 380

corresponding to the HNE-(N-acetyl-Gly-Lys-OMe) pyrrole adduct

74

NHO

N

O

NHO

O

338 (M -CH3CO + H+)

281 (amide cleavage)

204 (α -cleavage)

3.14, m/z 380 (MH+)

138

Scheme 3.8: Possible fragmentation of the molecular ion m/z 380 corresponding to an

HNE-(N-acetyl-Gly-Lys-OMe) pyrrole adduct. See Appendix Scheme 3.6S for fragment

structures.

A 1:2 ((N-acetyl-Gly-Lys-OMe):HNE) Schiff base-Michael adduct 3.15 has a

mass of m/z 656 which is same as that of the d4-HNE-(N-acetyl-Gly-Lys-OMe)-(N-

acetyl-Gly-Lys-OMe) crosslink 3.16.51 Since the amounts were low and peaks not

apparent from the TIC (Figure 3.1A), the tandem mass spectra was recorded based on the

structure proposed by Xu et al. This makes ESI-MS/MS of the molecular ions that

correspond to 3.16 at m/z 652 and m/z 656 difficult to analyze (Scheme 3.9). The

fragmentation shows peaks with M /M+ 4 patterns, nevertheless the assignment of

structures to the fragments was not possible due to interferences from 3.15 (Figure 3.10).

75

Figure 3.10: ESI-MS/MS analysis of molecular ions A) m/z 656 and B) m/z 652

corresponding to the 1:2 (HNE:(N-acetyl-Gly-Lys-OMe)) crosslink proposed by Xu et

al.51

NH

O

N

O

HNO

O

OH

HN

O

N

OHN

OO

N

HNO

HN

O

HN

O

O

OH

HN

OO

NH

O

O

Lysine-Lysine Crosslink

3.15, m/z 656 3.16, m/z 652

Scheme 3.9: Structure of (N-acetyl-Gly-Lys-OMe)–HNE (2:1) crosslink proposed by

Xu et. al.51

Ions that correspond to more than one HNE adducted onto N-acetyl-Gly-Lys-OMe

were observed analogous to the (N-acetyl-His)-HNE adducts. The total ion

76

chromatogram showed ions at m/z 572,576,580 and m/z 728,732,736,740 along with the

fragments derived from dehydration of these adducts (Figure 3.1, panel A). The ESI-

MS/MS of these molecular ions generated prominent daughter ions from the loss of

water, an acetyl group, and HNE. A set of MS/MS spectra corresponding to 2:1 adducts

with m/z 572, 576 and 580 (corresponding to M, M+4 and M+8) and those of 3:1

(HNE:(N-acetyl-Gly-Lys-OMe)) adducts at with m/z 728, 732,736 and 740

(corresponding to M, M+4, M+8 and M+12) are shown in Figure 3.11.

Figure 3.11: ESI-MS/MS analysis of molecular ions corresponding to the 2:1 (HNE:(N-

acetyl-Gly-Lys-OMe)) adducts A) m/z 580, B) m/z 576, C) m/z 572 and the 3:1

(HNE:(N-acetyl-Gly-Lys-OMe)) adducts D) m/z 740, E) m/z 736 and F) m/z 732 G) m/z

728.

77

The (N-acetyl-Gly-Lys-OMe)-HNE reaction mixture was separated by HPLC

using a methanol/water gradient and the molecular ions (discussed above) were

monitoring by LC-ESI through SIR of 17 channels (Figure 3.12). The chromatograms of

the adducts which differ by the type of HNE (d0 or d4) adducted, had similar features

(retention times of peaks). Also the complexity of the chromatogram peaks increases with

increasing complexity of the adducts formed.

Michael adduct

Schiff base adduct

Pyrrole adduct

Michael adduct

Schiff base adduct

Pyrrole adduct

Figure 3.12: LC-ESI for the N-acetyl-Gly-Lys-OMe and HNE reaction mixture

monitored through SIR of 17 channels of which the spectrum of 7 channels are shown

here (Chromatograms for other channels are presented in Appendix). LC-SIR

chromatograms for molecular ions at A) m/z 732 (M+4 of 3:1 adduct), B) m/z 728 (M of

3:1 adduct). C) m/z 576 (M+4 of 2:1 adduct), D) m/z 572(M of 2:1 adduct); E) m/z 416

(M of 1:1 Michael adduct); F) m/z 398 (M of 1:1 Schiff base adduct); G) m/z 380 (M of

1:1 pyrrole adduct); and H) total ion chromatogram (TIC). (See Appendix Figure 3.3S for

M+8 and M+12 of 3:1 adducts and M+4 of Michael, Schiff base and pyrrole 1:1 adducts)

78

(iii) Cysteine HNE adducts. ESI-MS analysis of a reaction between N-acetyl-Cys and

HNE (d0 : d4) in PBS buffer at 37 ºC for 24 h, revealed the presence of several multiple

ion sets as observed in the reactions of lysyl residues in N-acetyl-Gly-Lys-OMe and the

histidine derivative, N-acetyl-His. The fragmentation of N-acetyl-Cys was obtained as

reported earlier (Figure 3.13).52

Figure 3.13: ESI-MS/MS analysis for N-acetyl-cysteine.

Cysteine forms a dimer under oxidative conditions through a disulphide bond.53

The molecular ion at m/z 325, corresponding to the dimer of N-acetyl-Cys, generated

daughter ions at m/z 307, 285 and 208 (Figure 3.14 and Scheme 3.10).

Figure 3.14: ESI-MS/MS analysis for N-acetyl-cysteine dimer at m/z 325.

79

HN

HO

O

S

O

NH

OHO

S

O307(M+ - OH)

281 (M+ - CH3CO)

m/z 324 (M+)

208 (α-cleavage)

Scheme 3.10: Suggested mode of fragmentations for N-acetyl-cysteine dimer m/z 324.

See Scheme 3.7S in Appendix for fragment structures.

The Michael adducts of N-acetyl-Cys with HNE (d0:d4) have calculated

molecular masses of m/z 319 and m/z 324. Since both the N-acetyl-Cys dimer and the

isotope labeled (N-acetyl-Cys)-HNE Michael adduct have the same m/z value, the

MS/MS spectra of (N-acetyl-Cys)-HNE adduct ions are complicated. However, the ions

that correspond to the loss of water, i.e., m/z 302 and m/z 306, exhibit fragmentations

without any interference as shown in Figure 3.15. The ions at m/z 139 and 143

correspond to the daughter ions generated by retro-Michael cleavage (vide supra).

Similar fragmentation of a cysteine-HNE Michael adduct was reported earlier using a

deuterated cysteine.54 The molecular ions at m/z 342 and 346 correspond to the M + Na+

ion of the Michael adduct.

80

Figure 3.15: ESI-MS/MS analysis of the dehydrated (N-acetyl-Cys)–HNE Michael

adduct.

NH

OHO

S

O

O

OH

260(M - CH3CO - H2O)

139(M - NAC - H2O)

164 (M -HNE)

m/z 302 (MH+ - H2O)

242 (M - CH3CO - 2H2O)

Scheme 3.11: Fragmentation of the molecular ion m/z 302 arising from the dehydration

of (N-acetyl-Cys)-HNE Michael adduct. See Scheme 3.8S in Appendix for fragment

structures.

The total ion chromatogram of the ESI-TOF-MS (Figure 3.1B) of the (N-acetyl-

Cys)-HNE reaction product mixture shows many peaks that have the signature multiple

ions with m/z incremented by 4 Da. The tandem MS/MS of these ions was recorded.

81

Here, we present some of the tandem MS/MS data of molecular ions that were observed,

but a definite structure could not be assigned based only on the fragmentation.

In the case of the doublet at m/z 465 and 469 ions (Figure 3.1B), may correspond

to an adduct formed by N-acetyl-Cys and HNE in a ratio of 2:1. Tandem MS/MS

analysis of these ions generated daughter ions at m/z 327,302,164 and 327,304,164

respectively (Figure 3.16) (see section 3.3 for discussion, page 45).

Figure 3.16: ESI-MS/MS analysis for molecular ion at A) m/z 469 and B) m/z 465

corresponding to HNE-(N-acetyl-Cys) (1:2) adduct.

A triplet in the TIC at m/z 603, 607 and 611 (Figure 3.1B) correspond to 2HNE+

2(N-acetyl-Cys) - 2H2O (Figure 3.17). ESI-MS/MS spectra of these adducts showed a

fragmentation with 4 set of peaks (m/z [465,327,302,164]; [465,469, 327, 306,302,164];

[469,327,306,164]) (Figure 3.17, see section 3.3 for discussion, page 45). These values

include the daughter ions obtained from the tandem MS/MS 2:1 (N-acetyl-Cys)/HNE

adduct m/z 465 shown above. Other multiplets that had mass difference of 4 Da (see

82

Figure 3.1, panel B) were too complicated to be analyzed based on the mass

fragmentation alone.

Figure 3.17: ESI-MS/MS analysis of the ions with A) m/z 611, B) m/z 607 and C) m/z

603, which correspond to 2:2 (N-acetyl-Cys)-HNE adducts.

LC-ESI analysis of the N-acetyl-Cys and HNE reaction mixture selectively monitored 13

channels accounting for most of the peaks that appear in the total ion chromatogram. ESI

chromatograms shown in Figure 3.18 represent the channels that have similar peak

features for molecular ion sets corresponding to an adduct (M) and its deuterium labeled

isomer(s) (M+4 and/or M+8). For example, molecular ions at m/z 603, 607 and 611 have

peaks with the same features, however, for the parent ions m/z 320 and 324 ((N-acetyl-

Cys)-HNE Michael adduct), the peak features are different. This difference may be due to

interference from molecular ions corresponding to (N-acetyl-Cys)-(N-acetyl-Cys) dimer

83

that also have m/z 324. So, the Michael adduct is monitored as the M + Na+ ion at m/z

342 and 346 (Figure 3.18, for discussions see section 3.3, page 45).

Figure 3.18: LC-ESI-SIR analysis of the molecular ions observed in (N-acetyl-Cys)-

HNE reaction mixture through 8 channels. (Chromatograms shown for 8 channels: A)

m/z 611, B) m/z 607, C) m/z 603, D) m/z 469, E) m/z 465, F) m/z 346, G) m/z 342 H) m/z

324 and I) TIC.

84

3.3. Discussion

HNE is a ubiquitous product of lipid peroxidation.23,55 In the present study, a new

approach to identify and characterize adducts formed by HNE with surrogate amino acid

residues is described. The methodology developed involves mass spectrometric analysis

of the adducts formed using d4-HNE : HNE (1:1) and amino acids, which confers the

spectrum with multiplets characteristic of the number of HNE present in the adduct.

Unlike previous methods, this approach provides an analytical tool for identification of

adducts that are labile under harsh conditions and/or present in low abundance. Adducts

previously cited, i.e., (see section 1.2) were characterized as reference standards for

solving the structures of previously unknown adducts. This study primarily focused on

the adducts formed by the nucleophilic side chains of lysine, histidine and cysteine with

HNE, because earlier reports suggested that other nucleophilic amino acids (arginine,

serine, methionine and tyrosine) required non-physiological conditions (high alkalinity

and temperatures >>37 oC) for adduct formation.56,57

Methodology. Incubation of amino acids individually with a mixture of deuterium

labeled (d4) HNE and unlabeled (d0) HNE in the ratio 1:1 can potentially yield adducts

that can be formed from either the d0 or d4 HNE. Analysis of these reaction mixtures by

mass spectrometry revealed that unique signatures are conferred upon the spectra. Sets of

ions are found with mass differences in multiples of 4 Da owing to the presence of

various combinations of d0 and d4 HNE. Thus, HNE with 4 deuterium atoms will have a

molecular weight of 156 + 4 Da. Therefore, an ion with ‘n’ HNE will have ‘n + 1’ peaks

with successive mass differences of 4 Da. Accordingly, adducts containing more than

85

one molar equivalent of HNE per molar equivalent of N-acetyl-His should appear as

triplets (1:2:1) or multiplets of n + 1 ions, where ‘n’ corresponds to the number of

equivalents of HNE present. Based on this premise, the following conclusions can be

drawn from the pattern of daughter ions derived from tandem MS/MS each of the

molecular ion (P) and their corresponding isotopic isomers ( P + 4 and/or P + 8 ):

a. If the daughter ion appears as M ( from P ) with no M+4 but M ( from P + 4 )

also, then the fragment has no C5-C9 alkyl chain of HNE.

P

M

M

Inte

nsity

m/z

MS/MS of P+ 4

MS/MS of P

P

M

M

Inte

nsity

m/z

MS/MS of P+ 4

MS/MS of P

Parent ion

Daughter ions

b. If the daughter ion appears as both M ( from P ) and M+4 ( from P + 4 ), then the

daughter ion has one C5-C9 alkyl chain from HNE.

P

M+4

M

m/z

Inte

nsity

MS/MS of P+ 4

MS/MS of P

P

M+4

M

m/z

Inte

nsity

MS/MS of P+ 4

MS/MS of P

Parent ion

Daughter ions

c. If the daughter ion appears as M ( from P ), M+4 ( from P + 4 ), and M+8 ( from

P + 8 ), then the daughter ion has two C5-C9 alkyl chains from HNE.

86

Inte

nsity

P

M

M+4

M+8

m/z

MS/MS of P+ 8

MS/MS of P

MS/MS of P+ 4

Parent ion

Daughter ions

Inte

nsity

P

M

M+4

M+8

m/z

MS/MS of P+ 8

MS/MS of P

MS/MS of P+ 4

Inte

nsity

P

M

M+4

M+8

m/z

MS/MS of P+ 8

MS/MS of P

MS/MS of P+ 4

Parent ion

Daughter ions

d. If the daughter ion of a parent ion with two C5-C9 alkyl chains from HNE

appears as [ M ( from P )] , [ M & M+4 ( from P + 4 )] and [ M+4 ( from P + 4 )],

then the daughter ion has one C5-C9 alkyl chain of HNE.

Inte

nsity

P

M

M+4

M+4M

m/z

MS/MS of P+ 8

MS/MS of P

MS/MS of P+ 4

Parent ion

Daughter ions

Inte

nsity

P

M

M+4

M+4M

m/z

MS/MS of P+ 8

MS/MS of P

MS/MS of P+ 4

Parent ion

Daughter ions

(N-acetyl-His)-HNE Adducts

Mass spectrometric analysis of an (N-acetyl-His)/HNE reaction mixture (Figure

3.1C, page 12) showed the presence of a unique triplet at m/z 510, 514 and 518 (1:2:1)

corresponding to an adduct with two HNE’s and one N-acetyl-His, in addition to the

known Michael adduct at m/z 354 and 358. Though, HNE has been suggested to undergo

aldol condensation32 and form adducts with the nucleophilic side chains of amino acids,

87

peaks corresponding to the HNE-HNE aldol (m/z 312) or dehydrated aldol product (m/z

298 ) were not detected under these reaction conditions. The molecular ion m/z 510 (156

+ 156 + 197 + H+) correspond to a molecule formed from two HNEs and one N-acetyl-

His without loss of any neutral molecules. The fragmentation pattern suggests that there

is minimal change in the basic structure of the reactants. The MS/MS of the m/z 510 ion

(Figure 3.1C) shows a strong signal for a m/z 354 daughter ion, which suggests that the

ion at m/z 510 is a simple adduct of d0-HNE with Michael adduct 3.11 (Figure 3.2).

Daughter ions of the 2:1 adduct that are unique compared to the daughter ions

from the Michael 1:1 adduct 3.11 (in the range of m/z 350-150), may be useful for

suggesting a core structure for the m/z 510 adduct. Some of these are shown in Figures

3.19 and 3.20. There are three sets of daughter ions, at a) m/z 209, 213; b) m/z 239, 243;

and c) m/z 310, 314 that are derived from the fragmentation of m/z 510, 514, 518, parent

ions of 2:1 (HNE:(N-acetyl-His)) adducts that are absent in the 1:1 adduct 3.11 (Figures

3.19, 3.20 and 3.21). As discussed earlier (previous page, rule d), these daughter ions are

derived from a parent ion with two HNEs and they are present in an M, (M & M+4) and

M+4 pattern. This implies that these peaks correspond to a fragment that contains one

C5-C9 alkyl chain. Some of the possible structures that have a molecular mass of 510

amu are shown in Scheme 3.12. Also, analysis of the set of daughter ions at m/z 408, 412

from the 2:1 adduct parent ions at m/z 510, 514, 518 are informative (vide infra).

88

C5H11

O OH

HNOH

O

N

N

O

HO

C5H11

OC5H11

OHOH

NH

HOO

N

N

O

OC5H11

O

m/z 510

C5H11

OH

O

HNHO

ON

N

O

O

C5H11

HO

3.21 3.19

3.20

O

OHHO

C5H11

OH

C5H11

HNHO

ON

N

O

3.22

Scheme 3.12: Possible isomers of the m/z 510 (2:1) HNE-(N-acetyl-Cys) adduct.

Among the structures in Scheme 3.12 that may correspond to the parent ion m/z

510, the daughter ions 310, 314 and 408, 412 are expected by α-cleavage in the case of

the acyclic 3.19 or 3.22 or 3.21 (Scheme 3.13, Figures 3.30 and 3.21) but not the bicyclic

3.20. Other daughter ions from the 2:1 adduct that are unique (Figures 3.19 and 3.20)

were also observed, but their structural significance could not be deduced.

89

OH

HNOH

O

N

N

O

O

O

OH

HNOH

O

N

N

O

m/z 408

OHOH

HNOH

O

N

N

O

O

O

m/z 510 m/z 310

Scheme 3.13: Possible fragmentation of the putative parent ion m/z 510 to generate a

unique daughter ion at m/z 310 and 408.

Figure 3.19: Tandem MS/MS in the m/z range 207-212 and 238-244 from the (N-acetyl-

His)-HNE (Figure 3.1C) of the parent ions A) m/z 358, B) m/z 354, C) m/z 518, D) m/z

514 and E) m/z 510. Only the (2:1) HNE/(N-acetyl-His) adducts C, D and E shows

unique daughter ions at m/z 209, 213 and m/z 239, 243.

90

OHOH

HNOH

O

N

N

O

O

O

310 (α−cleavage)

3.21, m/z 510

466 (MH+ - COOH)468 (M - CH3CO-H+)

408 (α−cleavage)

Scheme 3.14: Possible mass spectrometric cleavage sites of adduct at 510 m/z with a

structure 3.19. See Scheme 3.9S in Appendix for fragment structures.

Figure 3.20: Tandem MS/MS in the mass range m/z 309-316 of the parent ions at A) m/z

358, B) m/z 354, C) m/z 518, D) m/z 514 and E) m/z 510 from (N-acetyl-His)-HNE

(Figure 3.1C).

91

Figure 3.21: Fragmentation of parent ions from (N-acetyl-His)-HNE (Figure 3.1C) at A)

m/z 518, B) m/z 514 and C) m/z 510 showing the presence of daughter ions m/z 408

and/or m/z 412.

Each of the molecular ions in the quadruplet m/z 666, 670, 674 and 678 have a

definite fragmentation pattern making them unique molecular ions in contrast to being

simple aggregated molecules or clusters in the MS analyzer, which would have yielded

the corresponding molecular ion peaks. In spite of very low abundance of these adducts

the tandem MS/MS of their molecular ions made the structure determination possible.

The unique fragments from the 3:1 HNE-(N-acetyl-His) adduct, viz, m/z 395, 399 which

were absent in the fragmentation of the 2:1 HNE-(N-acetyl-His) adduct, m/z 510, greatly

facilitated assignment of structure (Scheme 3.15) (additional information in Appendix,

see Scheme 3.9S). In the tandem MS/MS of the diastereomeric 3:1 HNE-(N-acetyl-His)

adducts, daughter ions that correspond to two consecutive retro-Michael cleavages,

92

leading to the formation of the 2:1 and 1:1 adducts is observed (Figure 3.4). Scheme

3.15b (see below) illustrates this fragmentation.

OHOH

HNOH

O

N

N

O

O

HO

O

O

466 (M-HNE+H+)

395

3.22, m/z 666

467

Scheme 3.15a: Possible fragmentations of the m/z 666 3:1 (HNE/(N-acetyl-His)) adduct.

Inset (tandem MS/MS of the parent ions m/z A) 510, B) 674, C) 670 and D) 666 in the

range m/z 394-402). See Scheme 3.10S in Appendix for fragment structures.

OHOH

HNOH

O

N

N

O

O

O

O

HO

m/z 665

O OH

HNOH

O

N

N

O

OH

O

m/z 509 O OH

HNOH

O

N

N

O

m/z 354

Retro-Michael

Retro-Michael

3:1 HNE:NAH

2:1 HNE:NAH 1:1 HNE:NAH

Scheme 3.15b: Possible structures of daughter ions formed by retro-Michael cleavage of

the 3:1 parent ion m/z 666. (NAH = N-acetyl-His)

93

To establish semi-quantitatively (i.e., without internal standard) the amounts of

adducts formed between (N-acetyl-His) and HNE, each of the molecular ion adducts in

the reaction mixture as shown in Figure 3.5 was selectively monitored by LC-ESI.

Multiple peaks are observed for each parent ion m/z. This indicates that the reaction

product contains a complex mixture of isomers, presumably, diastereomers.

The relative amounts of each of the adducts were calculated based on the area

under the curve for each of the peaks in LC-SIR chromatogram (Figure 3.5). The amount

of Michael adduct accounts for about 70% of the adducts formed, while the 1:2 and 1:3

((N-acetyl-Gly-Lys-OMe):HNE) adducts are present in 25% and 6% abundances,

respectively.

0

10

20

30

40

50

60

70

80

Michaeladduct

(1:2)adduct

(1:3)adduct

Rel

ative

amou

nt (%

)

Figure 3.22: Relative amounts of adducts formed by (N-acetyl-His)/HNE reaction,

calculated using LC-ESI. The amounts reflect the relative amount (of the adducts

selectively monitored) of each of the adduct present in the reaction mixture. Values are

average of 2 independent experiments and the error bars indicate the range.

94

Multiple reaction monitoring (MRM) is a widely used technique to quantify

amounts of molecular ion using LC-MS/MS. MRM measures the amount of the

molecular ion of interest, based on its most abundant daughter ion formed during tandem

MS/MS. MRM analyses were not effective compared to selected ion monitoring (SIR)

analyses of molecular ions, probably due to the low abundance of their fragments. LC-

SIR measures the amount of the ion of interest irrespective of its daughter ions.

Lysine HNE adducts

The Michael adduct (Figure 3.7 and Scheme 3.6), Schiff base (Figure 3.8 and

Scheme 3.7), pyrrole (Figure 3.9 and Scheme 3.8) and crosslink (2:1, Lysine:HNE)

(Figure 3.7 and Scheme 3.6) type adducts formed by lysine with HNE were detected. The

fragmentation patterns of the corresponding molecular ions for the unmodified N-acetyl-

glycine-lysine methyl ester (N-acetyl-Gly-Lys-OMe) and the various adducts support the

structures suggested earlier.32,33,51

ESI-MS/MS of the (N-acetyl-Gly-Lys-OMe)-HNE (1:2) molecular ion generated

fragments with loss of H2O, acetyl and HNE’s (see Figure 3.10, panels A, B and C),

which were similar to fragments generated from the (N-acetyl-His)-HNE (1:2) adduct

(see Figure 3.3, panels A, B and C). This suggests that the molecular ions of the

corresponding multiple adducts may share similar structural characteristics. For example,

in the set of of tandem MS/MS spectra of molecular ions at m/z 572, 576, 580, that

correspond to the 1:2 ((N-acetyl-Gly-Lys-OMe):HNE) adducts, as well as in the set of

tandem MS/MS spectra of molecular ions at m/z 728, 732 and 736, that correspond to the

95

1:3 ((N-acetyl-Gly-Lys-OMe):HNE) adducts (Figure 3.11), the formation of multiple

fragments with a difference of 4 Da is a prominent feature.

As reasoned in the case of the histidine adducts, the doublets and triplets of

daughter ions produced from the 1:2 and 1:3 adducts reflect the presence or absence of

d0-HNE or d4-HNE. The tandem MS/MS of the m/z 728 (1:3 adduct) ion gives daughter

ions at m/z 572 and 416 that are formed by retro-Michael elimination of 1 and 2 HNE

molecules respectively (Scheme 3.16). This fragmentation pattern involving sequential

loss of 1 and 2 HNE molecules was also seen for the 1:3 (N-acetyl-His)-HNE adducts

(Scheme 3.15b).

H2N

O NH

O

NH

O

O

OOH

OHO

HO

O

NH2O

HNHN

OO

OH

O

m/z 372 (M+H)+

HN

O

HN

ONH

O

O

HO

m/z 372 (M+H)+

m/z 728 (M+H)+ m/z 572 (M+H)+3.333.34

3.35 3.36

retro-Michael adduct

Retro-Michael adduct1: 3 (NGL:HNE) Adduct 1: 2 (NGL:HNE) Adduct

NH2O

NH

O

NHO

O O

OHm/z 416 (M+H)+

1: 1 (NGL:HNE) Adduct

3.12a

ba b

H2N

O NH

O

NH

O

O

OOH

OHO

Scheme 3.16: One of the possible structures and the mass spectrometric fragmentation

for the molecular ion at m/z 728 (3:1, HNE/(N-acetyl-Gly-Lys-OMe) adduct). See Figure

3.10 for mass spectra (NGL = N-acetyl-Gly-Lys-OMe).

96

A semi-quantitative analysis of the chromatogram of the adducts detected by LC-

ESI-SIR shows virtually identical mass chromatograms for each of the isotopic molecular

ions (M, M+4, …, M + 4n) of the 3:1 and the 2:1 adducts (Figure 3.12, page 24). In

addition to the fragmentation pattern discussed earlier, the similarity in the SIR

chromatograms suggests that the structure corresponding to the isotopic molecular ions

(d0 and d4) of an adduct are identical.

In addition, the relative amounts of each of the adduct based on the area under the

curve of LC-SIR spectra (Figure 3.12) showed the predominance of the 1:1 adducts,

Schiff base (49%) and Michael adduct (29%), over the multiple HNE-(N-acetyl-Gly-Lys-

OMe) adducts (2:1 (~9%) and 3:1 (9%) adducts) (Figure 3.23). The amounts of

relatively stable adducts, pyrrole and crosslink ((N-acetyl-Gly-Lys-OMe):HNE (2:1)),

were 10% and 1% respectively.

0

5

10

15

20

25

30

35

40

45

50

Pyrrole SchiffBase

Michael Crosslink (1:2)adduct

(1:3)adduct

Rela

tive

amou

nts (

%)

Figure 3.23: Relative amounts of adducts formed by (N-acetyl-Gly-Lys-OMe)/HNE

reaction, calculated using LC-ESI. The ‘y’ scale reflects percentage of the each adduct

present in the reaction mixture (with respect to the adducts selectively monitored). Values

are average of 2 independent experiments and the error bars indicate the range.

97

HNE- Cysteine adducts

Cysteine forms a Michael adduct with HNE and to the best of our knowledge

there have been no reports of any other adducts that can be formed. Here, HNE adducts

with the cysteine were analyzed using N-acetylated cysteine (N-acetyl-Cys). The

molecular ion corresponding Michael adduct with HNE and the (N-acetyl-Cys)-(N-

acetyl-Cys) (disulphide) were analyzed, and the fragmentation pattern was in agreement

with the structure.54

A prominent doublet in the total ion chromatogram of the (N-acetyl-Cys)-HNE

reaction mixture occurs at m/z 465 and 469 (Figure 3.1, panel B). This corresponds to a

2:1 adduct of N-acetyl-Cys and HNE. As shown in Scheme 3.17, the tandem MS/MS of

these ions (Scheme 3.16) results in daughter ions at m/z 164 (corresponding to (N-acetyl-

Cys)+H+) and 302 or 306 (corresponding to (N-acetyl-Cys)+HNE(or d4-HNE)-H2O +H)+.

Another prominent daughter ion from both the parents has m/z 327 that indicates loss of

the d4 labeled portion of the adduct. However, the structure of this daughter ion is not

obvious, and was not assigned.

m/z 465 (2NAC + HNE - H2O+ H+)

m/z 302(NAC + HNE - H2O+ H+)

m/z 327?

m/z 164 (NAC + H+)

m/z 469 (2NAC + (d4-HNE) - H2O+ H+)

m/z 306

(NAC + (d4-HNE) - H2O+ H+)

m/z 327?

m/z 164 (NAC + H+)

A B

Scheme 3.17: Daughter ions formed from the tandem MS/MS of 2:1 (N-acetyl-Cys):

HNE adduct A) d0-HNE-2(N-acetyl-Cys) adduct B) d4-HNE-2(N-acetyl-Cys) adduct,

(NAC = N-acetyl-cysteine).

98

The 2:2 adducts that correspond to the molecular ions at m/z 603, 607 and 611

(Figure 3.1, panel B) may be composed of 2HNE+ 2 (N-acetyl-Cys) - 2H2O. ESI-

MS/MS spectra of these adducts showed a fragmentation with 4 prominent peaks (Figure

3.17) as depicted in Scheme 3.18. A structure was not assigned to this 2:2 adduct. Unlike

(N-acetyl-His) and N-acetyl-Gly-Lys-OMe adducts, peaks corresponding 2:1 or 3:1

adducts of HNE with N-acetyl-Cys were not observed.

302 302 306

327327

164164

302

327

164

(2NAC + 2(HNE) - H2O+ H+) (2NAC + HNE + d4-HNE - H2O+ H+) (2NAC + 2(d4-HNE) - H2O+ H+)

603 (M) 607(M+4) 611(M+8)

465 (M) 465(M) 469(M+4) 469(M+4)

2:2 adducts

2:1 adducts

Scheme 3.18: Daughter ions formed from the tandem MS/MS of 2:2 (N-acetyl-cysteine:

HNE) adduct m/z 603, 607 and 611. (NAC = N-acetyl-cysteine)

99

The relative amounts of each of the adducts with respect to the total number of

adducts monitored showed that the Michael adduct (~50%) was the predominant product

compared to the other adducts (Figure 3.24). Surprisingly, 2:1 and 2:2 adducts were

present in comparatively high levels i.e. ~30 % and ~5% respectively.

0

10

20

30

40

50

60

70

MichaelAdduct

NAC-NACdimer

2:1 adduct 2:2 adduct

Rel

ativ

e am

ount

s (%

)

Figure 3.24: Relative amounts of adducts formed by N-acetyl-Cys and HNE reaction,

calculated using LC-ESI. The ‘y’ scale reflects percentage of the each adduct present in

the reaction mixture (with respect to all the adducts selectively monitored). Values are

average of 2 independent experiments and the error bars indicate the range, (NAC = N-

acetyl-cysteine).

100

3.4. Conclusions

Using a mixture of deuterium labeled HNE and unlabeled HNE in the ratio (1:1)

to study the adduction pattern of HNE on lysine, histidine and cysteine is a robust method

for the characterization of unique adducts. Adducts formed with HNE appear as apparent

multiplets that are indicative of the number of HNE moieties present in each adduct. The

tandem mass spectra of each of the adducts gave sets of unique daughter ions. This

method is valuable for detecting and characterizing different types of adducts that are

formed under mild conditions, including some present in low abundances.

Apart from adduct types reported earlier from the reactions of lysine or histidine

with HNE, the present studies with isotope labeled HNE revealed adducts that

incorporate multiple HNE molecules adducted with a single lysine or histidine residue.

Possible structures for some of the multiple HNE adducts were proposed based on the

unique fragments that were identified. The relative amount of 1:2 plus 1:3 adducts

formed from N-acetyl-Gly-Lys-OMe was about 19%. The amount of 1:1 adducts

(Michael, Schiff base and pyrrole adduct) was about 80% of the total adducts and the 2:1

(N-acetyl-Gly-Lys-OMe)/HNE crosslink comprised about 1% of the total adducts

monitored.

Apart from forming predominantly Michael adduct, incubation of N-acetyl-

cysteine with HNE also generated relatively high amounts of unique adducts with (N-

acetyl-Cys):HNE in the ratio 2:1(~30%) and 2:2 (~5%).

Though high concentrations of HNE might only be possible in certain micro-

domains in situ, e.g. oxidized LDL particles, identification of such multiple HNE adducts

may help in attaining a complete understanding of HNE’s role in physiological processes.

101

The multiple HNE-amine adducts discussed here may constitute an initial response of

proteins towards high levels of HNE. These multiple HNE adducts may rearrange to form

other toxic products or by corollary, this could be one of the in vivo homeostatic

mechanisms to detoxify lipid oxidation products by effectively quenching the HNE’s

reactivity.

102

3.5. Experimental Methods and Procedures

General methods. Proton magnetic resonance (1H NMR) spectra were recorded on a

Varian Gemini spectrometer operating at 200, 300, 400 or 600 MHz. Proton chemical

shifts are reported in parts per million (ppm) on the δ scale relative to CDCl3 (δ 7.25) or

CD3OD (δ 3.30). 1H NMR spectral data are tabulated in terms of multiplicity of proton

absorption (s, singlet; d, doublet; t, triplet; m, multiplet; br, broad), coupling constants

(Hz), number of protons. Carbon magnetic resonance (13C NMR) spectra were recorded

on a Varian Gemini spectrometer operating at 50 MHz and chemical shifts are reported

relative to solvents. Deuterium magnetic resonance (2H NMR) spectra were recorded on a

Varian Gemini spectrometer (400 MHz) and chemical shifts are reported relative to

solvents. All high resolution mass spectra were recorded on a Kratos AEI MS25 RFA

high resolution mass spectrometer at 20 eV. All solvents were distilled under a nitrogen

atmosphere prior to use. Diethyl ether was freshly distilled over lithium aluminum

hydride. Pentane was dried over phosphorus pentoxide. Tetrahydrofuran (THF) was

freshly distilled over potassium and benzophenone. All reaction flasks were flame dried

under argon before use. All materials were obtained from Aldrich (Milwaukee, WI)

unless otherwise specified.

Chromatography was performed with ACS grade solvent (ethyl acetate and

hexanes). Thin layer chromatography (TLC) was performed on glass plates precoated

with silica gel (Kieselgel 60 F254, E. Merck, Darmstadt, West Germany). Rf values are

quoted for plates of thickness 0.25 mm. The plates were visualized with iodine or

phosphomolybdic acid reagent.58 Flash column chromatography was performed on 230 -

400 mesh silica gel supplied by Merck. All other chemicals were obtained from Aldrich

103

(Milwaukee, WI) and Fisher (Chicago, IL). For all reactions performed in an inert

atmosphere, argon was used unless specified.

Synthesis of [8,8,9,9-2H4]-4-hydroxy-2-nonenal.

OSO

OOH TsCl+

3.2Pent-4-yn-1-ol

Et3N

CH2Cl2, 0oC

86%3.1

Toluene-4-sulfonic acid, pent-4-ynyl ester (3.2). A suspension of p-toluenesulfonyl

chloride ( 3.4 g, 17.9 mmol) and triethylamine (6 mL) in CH2Cl2 (25 mL) was added

dropwise to pent-4-yn-1-ol (1 g, 11.9 mmol) at 0 ºC and the reaction mixured was stirred

at 0 ºC. When TLC showed no starting material, the reaction mixture was extracted with

ethyl ether (3 x 20 mL). The extract was washed with saturated sodium bicarbonate,

water, and brine. After drying over MgSO4, the solvent was removed in vacuo and

purified by column chromatography with 5% ethyl acetate in petroleum ether (Rf = 0.7)

to yield an oily liquid 3.2 (2.43 g, 86%).45

OSO

OHD2C

D2C O

SO

O

3.3

RhCl(PPh3)3, D2

C6H6

74%3.2

104

Toluene-4-sulfonic acid, [4,4,5,5-2H4] pent-4-nyl ester (3.3). To a flask containing

benzene (2 mL) flushed with argon was added freshly prepared the Wilkinson catalyst

RhCl(PPh3)3 (12 mg, 12.9 µmol).59 The flask was flushed with argon for 5 min and the

valve turned to deuterium gas. The color of the solution turned ice-tea brown. After 5

min, a solution of 3.2 (336 mg, 1.41 mmol) in degassed benzene (1 mL) was added

dropwise. The reaction was allowed to proceed under a positive pressure of D2 until the

reaction ceased to consume D2. The solvent was removed on a rotatory evaporator. The

product was extracted by trituration with hexanes (3 x 20 mL) and purified by column

chromatography with 5% ethyl acetate in hexanes (Rf = 0.5) to yield a oily liquid 3.3

(260 mg, 74%). 1H NMR (CDCl3, 300 MHz) δ 7.77 (d, J1 = 6.2 Hz, J2 = 1.6 Hz, 2H),

7.33 (d, J = 8.4 Hz, 2H), 4.02 (t, J = 6.5 Hz, 2H), 2.45 (s, 3H), 1.63-1.64 (2H), 1.26 (t, J

= 7.2 Hz, 2H), 0.8031 (bs, 1H); 13C NMR (CDCl3, 75 MHz) δ 144.8, 133.4, 129.9, 128.0,

70.9, 28.6, 27.3, 21.8, 21.1(t), 13.3(q); HRMS (EI) m/z calcd for C12H14D4O3S+(M+)

246.1224 found 247.1292 (M+H)+.46

105

HD2C

D2C IHD2C

D2C O

SO

O

3.3

NaI,Acetone

3.4

HD2C

D2C I

3.4

+O

OOO

OHD2CCD2 OH

tBuLi

Ethyl ether

3.5-78 oC

84% 3.6

[7,7,8,8-2H4]-1-(2,2-Dimethyl-[1,3]dioxolan-4-yl)-oct-1-en-3-ol (3.6). To a stirred

solution of sodium iodide (750mg, 5 mmol) in freshly distilled acetone (15 mL) was

added potassium carbonate (550 mg, 4 mmol) and tosylate 3.3 (500 mg, 2 mmol).36 The

solution was refluxed for 3 h or until the reaction is complete. The solvent was removed

on a rotatory evaporator and the crude product dried on a vacuum pump. This product

was used for the next step without purification. 2H NMR (CDCl3, 90MHz) δ 1.19, 1.12,

0.88, 0.74 1H NMR (CDCl3, 300 MHz) δ 3.19 (t, J = 7 Hz, 2H), 1.78-1.83 (m, 2H), 1.36

(t, J = 7.7 Hz, 2H), 0.86 (s, 1H).

A solution of t-butyllithium (6 mmol) in hexanes was added dropwise to a stirred

solution of 3.4 (~150 mg, 0.74 mmol) in freshly distilled ethyl ether:pentane (2:1, 15 mL)

at -78 ºC under argon. After 10 min at -78 ºC, the reaction mixture was allowed to warm

to room temperature to generate the alkyllithium. The reaction mixture was then cooled

to -78 ºC and 3-(2,2-dimethyl-[1,3]dioxolan-4-yl)-propenal (3.5) 60 (108 mg, 0.7 mmol)

was added dropwise with stirring over 5 min. After stirring the resulting mixture for 30

106

min, saturated NH4Cl was added. The resulting mixture was extracted with ethyl ether (3

x 15 mL). The combined organic extract was washed with brine and solvent removed on

a rotatory evaporator. The crude product was purified on a flash column with 25% ethyl

acetate in hexanes to yield 3.6 (136 mg, 84%). 1H NMR (CDCl3, 300 MHz) δ 5.82 (dd,

J = 15.4, 6.1 Hz, 1H), 5.64 (ddd, J = 16.5, 7.4, 2.3 Hz, 1H), 4.5 (dd, J = 13.7, 7.1 Hz, 1H),

4.06-4.13 (m, 2H), 3.57 and 3.58 (2t, J = 7.9 Hz, J = 7.7 Hz, 1H), 1.25-1.60 (8H), 1.40 (s,

3H), 1.37 (s, 3H), 0.82 (s, 1H); 13C NMR (CDCl3, 300 MHz, some resonances appear as

1:1 apparent doublets owing to the presence of two diastereomers) δ 137.46 and 137.39,

127.81 and 127.76, 109.40 and 109.38, 76.57, 72.04 and 71.98, 69.49 and 69.47, 37.15

and 37.05, 31.48, 26.71, 25.92, 25.05 and 24.99 , 21.62(q), 13.23 (q) HRMS m/z calcd

C13H20D4O3 232.1972 (M)+, found 233.2035 (M+H)+, 231.1872 (M-H)+

[8,8,9,9-2H4]-4-Hydroxy-2(Z)-nonenal (3.7). This product was accessed by two

different methods.

NaIO4

acetic acid:H2O (2:1)

OOHD2C

CD2 OH

OHD2C

CD2 OH

3.6 3.789%40 oC

Method A: A suspension of sodium periodate (24 mg, 0.117 mmol) in acetic acid/water

(2:1, v/v, 2 mL) was added to compound 3.6 (13 mg, 0.056 mmol) and the mixture was

stirred magnetically for 4 h at 40 °C or until the reaction was complete. Then, the solvent

was removed by rotary evaporation under reduced pressure. The last traces of HOAc

were removed by azeotropic distillation with n-heptane (3:1 mL) under high vacuum.

107

The resulting residue was flash chromatographed on a silica gel column (30% ethyl

actetate in hexanes) to give 3.7 (8 mg, 89%).

1. acetic acid:H2O (2:1), 40 o C2. Pb(OAc)4, K2CO3, -78 oC

OOHD2C

CD2 OH

OHD2C

CD2 OH

3.6 3.7

78%

Method B: A solution of the compound 3.6 (13 mg, 0.056 mmol) in acetic acid/water

(2:1, v/v, 1.2 mL) was stirred magnetically for 4 h at 40 °C. Then the solvent was

removed by rotary evaporation under reduced pressure. The last traces of HOAc were

removed by azeotropic distillation with n-heptane (3:1 mL) under high vacuum. Dry

methylene chloride (2 mL) and Na2CO3 (5.7 mg, 0.054 mmol) were added to the residue.

The solution was stirred magnetically at -78 °C under an argon atmosphere, and

Pb(OAc)4 (31 mg, 0.07 mmol) was added. The resulting solution was stirred for 30 min.

The solvent was then removed. The residue was flash chromatographed on a silica gel

column (30% ethyl actetate in hexanes) to give 3.7 (7 mg, 78%). 1H NMR (CD3Cl, 300

MHz) δ 9.57 (d, J = 7.7 Hz, 1H), 6.80 (dd, J1 = 15.7 Hz, J2 = 4.5 Hz, 1H), 6.29 (dd, J1 =

15.7 Hz, J2 = 7.7 Hz, 1H), 4.02 (t, J = 6.5 Hz, 2H), 2.45 (s, 3H), 1.63-1.64 (2H), 1.26 (t,

J = 7.2 Hz, 2H), 0.8031 (bs, 1H); 13C NMR (CD3Cl, 75 MHz) δ 193.1, 158.7, 130.6,

92.3, 71.1, 36.5, 31.3, 29.2, 24.8; HRMS (EI) m/z calcd for (M+) 246.1224 found

247.1292 (M+H)+.

108

Preparation of cyclohexanedione (CHD) reagent. Cyclohexanedione (CHD) was

freshly recrystallized from ethyl acetate. CHD (125 mg), ammonium acetate (5g), and

acetic acid (2.5 mL) were dissolved in deionized water and diluted to 50 mL. The pale

yellow solution was heated at 60 °C for 1 h, cooled with an ice bath, and purified by

passing through a C18 SPE cartridge (1 mL), which was preconditioned with water (5

mL) and methanol (5 mL). The elute was collected and stored at 4 °C.42

Derivatization of 4-hydroxy-2-nonenal by CHD reagent. Stock solutions of HNE, d4-

HNE and benzaldehyde (100 ng/µL) were prepared. Benzaldehyde solution (10 µL) was

mixed individually with 10 µL and 50 µL of HNE and d4-HNE solutions. The solvent

was removed with a gentle steam of dry nitrogen, and the residue was redissolved in 100

µL of methanol. CHD reagent (1 mL) was added, and the mixture was incubated at 60 oC

for 1 h. The resulting solution was cooled, and then passed through a C18 SPE cartridge,

which had been washed sequentially with methanol (5 mL x 2) and water (5 mL x 2). The

cartridge was washed with 5% acetonitrile in water (9 mL) and the product was eluted

with acetonitrile containing 5% water (9 mL). The solvent in the eluant was removed

with a stream of dry nitrogen. Before ESI-MS/MS analysis, the residue was dissolved in

methanol containing 25% water, and diluted to 0.01 ng/µL (final concentration) was

injected into the LC-MS for analysis. ESI-MS/MS analysis of the HNE-CHD derivative

produced characteristic fragments: m/z 326 ([M+H-18]+); m/z 308 ([M+H-18-18]+); m/z

216. ESI-MS/MS analysis of the d4-HNE-CHD derivative produced characteristic

fragments: m/z 330 ([MH-18]+); m/z 312 ([MH-18-18]+); m/z 216. ESI-MS/MS analysis

109

of benzaldehyde-CHD derivative produce characteristic fragments: m/z 294 ([MH]+); m/z

216. (Figure 3.25).

HN

OHC5H7D4

O O

HN

OHC5H11

O O

HN

OHC5H7D4

O O

HN

OHC5H11

O O

A B

Figure 3.25: A) ESI-MS/MS of CHD derived HNE B) ESI-MS/MS of CHD derived d4-

HNE.

Synthesis of HNE adducts of amino acids: A deuterium labeled [8,8,9,9-2H4]-HNE

solution (5 mM in ethyl acetate) was prepared freshly and added to a solution of

unlabeled HNE (5 mM in ethyl acetate) to make a 1:1 stock solution. The concentration

of the stock HNE solution was determined by measurement of UV absorbance at 224 nm

presuming a molar extinction coefficient of 13,750 M-1 for HNE in MeOH.61 To a 1.5

mL glass vial was added 1 mL stock solution of HNE (d4:d0) and the solvent evaporated

using a gentle stream of N2 gas.

To a solution of N-acetyl-lysine (20 µM) in PBS (10 mM, pH 7.4) was added d4-

HNE and HNE in the ratio (1:1) (10 µM). The mixture was incubated at 37 ºC for 24 h.

110

In rare cases longer reaction times were required for the formation of adducts that are

present in low abundances but are of interest for this study, e.g., 36-48 h at room

temperature for the 3:1 (HNE:(N-acetyl-Gly-Lys-OMe)) adduct. Following the reaction,

the reaction mixture is quickly dried using a high-speed vacuum evaporator and stored at

-20 ºC until used. The isolated aliquot was analyzed by either MALDI-TOF or QTOF2

mass spectrometer.

Mass spectrometry. ESI-MS/MS analysis of CHD-d4-HNE derivative was performed

on API-3000 triple quadrapole electrospray mass spectrometer (Applied Biosystems

Inc.). The source temperature was maintained at 100 ºC, and the desolvation temperature

at 200 °C. The drying gas (N2) was maintained at ca. 450 L/Hr, and the cone flow gas at

ca. 50 L/Hr. The multiplier was set at an absolute value of 500. MS scan at m/z 50-800

were obtained for standard compounds.

Table 3.3. Optimized parameters for mass spectrometer.

Ion Mode positive Capillary (KV) 3 Cone (V) 30 Hex 1 (V) 10 Aperture (V) 0 Hex 2 (V) 0.5 LM 1 Resolution 19 HM 1 Resolution 19 Ion Energy 1 1 LM 2 Resolution 19 HM 2 Resolution 19 Ion Energy 2 2

111

Argon was used as collision gas at a pressure of 5 psi for MS/MS analysis. For MS/MS

analysis, the collision energies were optimized for each compound. The instrument was

operated in the positive ion mode.

Analysis of adducts using MALDI-TOF. Initial diagnostic analysis of the samples was

performed by MALDI-TOF mass spectrometry using a PE Biosystems Voyager DE Pro

instrument equipped with a nitrogen laser (337 nm) and operated in the delayed

extraction and reflector mode with a matrix of α-cyano-4-hydroxycinnamic acid (CHCA,

5 mg/ml in acetonitrile/water/3% trifluoroacetic acid, 5:4:1, v/v/v). Internal standards

used for calibration included two synthetic peptides G9I (50 fmol/µL, MH+ 1015.579)

plus L20R (200 fmol/µL, MH+ 2474.6300)29. Each of the reaction aliquot was diluted in

50% aqueous acetonitrile solution to give a final concentration of 100 fmol/µL of the

total amino acid. Each of the solution is properly vortexed before used. One microliter of

sample was mixed with 1 µL of matrix and 0.5 µL of internal standard mix then 1.5 µL

applied to the target and allowed to dry. Each spectrum was accumulated for ~ 50-200

laser shots. The data was processed on Data Explorer 4.0 (Applied Biosystems) software.

Additional sample applications were utilized as needed.

Analysis of adducts using Q-TOF mass spectrometer. The samples were analyzed by a

quadrupole-time of flight (Q-TOF-2) mass spectrometer (Micromass) equipped with

MassLynx™ acquisition and processing software. The samples were infused using a 100

µL Hamilton syringe onto a capillary column (PicoFrit™ 0.050 x 50mm, 5µ tip ID; New

Objective Inc., Woburn, MA) at 0.5 µL/min using a syringe pump (Harvard apparatus,

112

Pump 11) and ionized immediately using an electrosprayer designed in-house. The mass

spectrometer was operated in standard MS and MS/MS switching mode. The MS data

was collected for the mass range of 50-1500 m/z and MS/MS data analyses utilized

Micromass software (MassLynx®) Version 3.5.

The machine was calibrated using a solution of 2 fmol of [Glu1 ]-Fibrinopeptide

B (Sigma) in 50% aqueous acetonitrile (+ 0.1% formic acid) infused using glass

micropipette with 5 µ tip. The intensity of the peak from the MS/MS spectrum at 785.4

m/z was used as a reference for calibration. The final mass measurement accuracy of ≤ 10

ppm was achieved before the use of the machine.

Chromatography. The samples analyzed for selected ion recording were

chromatographically separated on a reverse phase column and recorded in positive mode.

The chromatographic separation was obtained using a 150 × 2.0 mm i.d. Prodigy ODS-2,

5 µ column (Phenomenex, UK), with a binary solvent (water and methanol) gradient.

The solvents were supplemented with 0.2% formic acid. The separation was effected

through a gradient started with 100% water and rose to 100% methanol linearly in 15

min, and elution was continued for 5 min with 100% methanol. Then the gradient was

reversed to 100% water in 2 min, and then held for 5 min at 100% water. The solvents

were delivered at 200 µl/min.

Method to quantify the relative amounts of adducts formed. The selective

monitoring of LC-ESI chromatograms for each of the adduct was integrated using the

Masslynx® software ver 3.5. The total peak area of the chromatogram for each of the

113

adduct (A) was determined and using the formulae below the values of relative amounts

in percentage was calculated. The error bars reflect the results of two independent

experiments.

n – the total number of adducts

A(n) – Total area of the chromatogram for the adduct A(n) in A(n) channel of LC-ESI-

SIR.

LC-ESI. LC-ESI analysis was performed on Quattro Ultima® mass spectrometer

(Micromass,UK). The source temperature was maintained at 100 ºC, and the desolvation

temperature at 200 °C. The drying gas (N2) was maintained at ca. 450 L/Hr, and the cone

flow gas at ca. 50 L/Hr. The multiplier was set at an absolute value of 500. MS scans at

m/z corresponding to selected singly charged molecular ions were obtained for the

adducts. Optimized parameters can be found in Table 3.3.

114

3.6. References.

(1) Hazen, S. L.; Chisolm, G. M. Proc Natl Acad Sci U S A 2002, 99, 12515-7. (2) Hrbac, J.; Kohen, R. Drug Dev Res 2000, 50, 516 - 527. (3) Grandjean, E. M.; Berthet, P.; Ruffmann, R.; Leuenberger, P. Clin Ther 2000, 22,

209-21. (4) Boon, P. J. M.; Marinho, H. S.; Oosting, R.; Mulder, G. J. Toxicol Appl

Pharmacol 1999, 159, 214-223. (5) Rodriguez-Martinez, E.; Rugerio-Vargas, C.; Rodriguez, A. I.; Borgonio-Perez,

G.; Rivas-Arancibia, S. Int J Neurosci 2004, 114, 1133-45. (6) Kondo, Y.; Murakami, S.; Oda, H.; Nagate, T. Adv Exp Med Biol 2000, 483, 193-

202. (7) De Mattia, G.; Bravi, M. C.; Laurenti, O.; Cassone-Faldetta, M.; Proietti, A.; De

Luca, O.; Armiento, A.; Ferri, C. Diabetologia 1998, 41, 1392-6. (8) Wagberg, M.; Jansson, A. H.; Westerlund, C.; Ostlund-Lindqvist, A. M.;

Sarnstrand, B.; Bergstrand, H.; Pettersson, K. J Pharmacol Exp Ther 2001, 299, 76-82.

(9) Marnett, L. J.; Hurd, H. K.; Hollstein, M. C.; Levin, D. E.; Esterbauer, H.; Ames, B. N. Mutat Res 1985, 148, 25-34.

(10) Stadtman, E. R. Science 1992, 257, 1220-4. (11) Fraga, C. G.; Shigenaga, M. K.; Park, J. W.; Degan, P.; Ames, B. N. Proc Natl

Acad Sci U S A 1990, 87, 4533-7. (12) Chakraborti, T.; Ghosh, S. K.; Michael, J. R.; Batabyal, S. K.; Chakraborti, S. Mol

Cell Biochem 1998, 187, 1-10. (13) Tapiero, H.; Ba, G. N.; Couvreur, P.; Tew, K. D. Biomed Pharmacother 2002, 56,

215-22. (14) Salomon, R. G. Antioxid Redox Signal 2005, 7, 185-201. (15) Arlt, S.; Kontush, A.; Muller-Thomsen, T.; Beisiegel, U. Z Gerontol Geriatr

2001, 34, 461-5. (16) Kopitz, J.; Holz, F. G.; Kaemmerer, E.; Schutt, F. Biochimie 2004, 86, 825-31. (17) Crabb, J. W.; Miyagi, M.; Gu, X.; Shadrach, K.; West, K. A.; Sakaguchi, H.;


(18) Gu, X.; Meer, S. G.; Miyagi, M.; Rayborn, M. E.; Hollyfield, J. G.; Crabb, J. W.; Salomon, R. G. J Biol Chem 2003, 278, 42027-35.

(19) Gotz, M. E.; Kunig, G.; Riederer, P.; Youdim, M. B. Pharmacol Ther 1994, 63, 37-122.

(20) Sun, A. Y.; Chen, Y. M. J Biomed Sci 1998, 5, 401-14. (21) Mlakar, A.; Spiteller, G. Chem Phys Lipids 1996, 79, 47-53. (22) Harrison, K. A.; Murphy, R. C. J Biol Chem 1995, 270, 17273-8. (23) Poli, G.; Schaur, R. J. IUBMB Life 2000, 50, 315-21. (24) Uchida, K. Prog Lipid Res 2003, 42, 318-43. (25) Salomon, R. G.; Kaur, K.; Podrez, E.; Hoff, H. F.; Krushinsky, A. V.; Sayre, L.

M. Chem Res Toxicol 2000, 13, 557-64. (26) Sayre, L. M.; Zelasko, D. A.; Harris, P. L.; Perry, G.; Salomon, R. G.; Smith, M.

A. J Neurochem 1997, 68, 2092-7.

115

(27) Macdonald, S.; Dowle, M.; Harrison, L.; Clarke, G.; Inglis, G.; Johnson, M.; Shah, P.; Smith, R.; Amour, A.; Fleetwood, G.; Humphreys, D.; Molloy, C.; Dixon, M.; Godward, R.; Wonacott, A.; Singh, O.; Hodgson, S.; Hardy, G. J Med Chem 2002, 45, 3878-90.

(28) Szweda, L. I.; Uchida, K.; Tsai, L.; Stadtman, E. R. J Biol Chem 1993, 268, 3342-7.

(29) Crabb, J. W.; O'Neil, J.; Miyagi, M.; West, K.; Hoff, H. F. Protein Sci 2002, 11, 831-40.

(30) Sayre, L. M.; Sha, W.; Xu, G.; Kaur, K.; Nadkarni, D.; Subbanagounder, G.; Salomon, R. G. Chem Res Toxicol 1996, 9, 1194-201.

(31) Xu, G.; Liu, Y.; Sayre, L. M. Chem Res Toxicol 2000, 13, 406-13. (32) Sayre, L. M.; Arora, P. K.; Iyer, R. S.; Salomon, R. G. Chem Res Toxicol 1993, 6,

19-22. (33) Nadkarni, D. V.; Sayre, L. M. Chem Res Toxicol 1995, 8, 284-91. (34) Liu, Y.; Sun, G.; David, A.; Sayre, L. M. Chem Res Toxicol 2004, 17, 110-8. (35) Jurgens, G.; Lang, J.; Esterbauer, H. Biochim Biophys Acta 1986, 875, 103-14. (36) Deng, Y. Ph.D, Case Western Reserve University, 2000. (37) van Kuijk, F. J.; Siakotos, A. N.; Fong, L. G.; Stephens, R. J.; Thomas, D. W.

Anal Biochem 1995, 224, 420-4. (38) Amarnath, V.; Valentine, W. M.; Montine, T. J.; Patterson, W. H.; Amarnath, K.;

Bassett, C. N.; Graham, D. G. Chem Res Toxicol 1998, 11, 317-328. (39) Matsushita, Y.-i.; Sugamoto, K.; Matsui, T. Tennen Yuki Kagobutsu Toronkai

Koen Yoshishu 1998, 40th, 613-618. (40) Sugamoto, K.; Matsushita, Y.-i.; Matsui, T. J Chem Soc, Perkin Trans 1: Org

Bio-Org Chem 1998, 3989-3998. (41) Selley, M. L.; Bartlett, M. R.; McGuiness, J. A.; Hapel, A. J.; Ardlie, N. G. J

Chromatogr 1989, 488, 329-40. (42) Gioacchini, A. M.; Calonghi, N.; Boga, C.; Cappadone, C.; Masotti, L.; Roda, A.;

Traldi, P. Rapid Commun Mass Spectrom 1999, 13, 1573-9. (43) Kingston, D. Chem Rev 1974, 74, 215. (44) Gross, J. H. Mass Spectrometry- A Textbook; Springer-Verlag: Heidelberg, 2004. (45) Edwards, G. L.; Muldoon, C. A.; Sinclair, D. J. Tetrahedron 1996, 52, 7779-7788. (46) Felder, S.; Rowan, D. D. J Labelled Compd Radiopharm 1999, 42, 83 - 92. (47) Deng, Y.; Salomon, R. G. J Org Chem 1998, 63, 3504-3507. (48) SDBSWeb : http://www.aist.go.jp/RIODB/SDBS/ (National Institute of

Advanced Industrial Science and Technology; Vol. 2005. (49) Enoiu, M.; Herber, R.; Wennig, R.; Marson, C.; Bodaud, H.; Leroy, P.; Mitrea,

N.; Siest, G.; Wellman, M. Arch Biochem Biophys 2002, 397, 18-27. (50) Aldini, G.; Granata, P.; Carini, M. J Mass Spectrom 2002, 37, 1219-28. (51) Xu, G.; Sayre, L. M. Chem Res Toxicol 1998, 11, 247-51. (52) Steen, H.; Mann, M. J Am Soc Mass Spectrom 2001, 12, 228-32. (53) Doreleijers, J. F., Universiteit Utrecht, 1999. (54) Rathahao, E.; Peiro, G.; Martins, N.; Alary, J.; Gueraud, F.; Debrauwer, L. Anal

Bioanal Chem 2005, 381, 1532-9. (55) Liu, Y.; Jinno, H.; Kurihara, M.; Miyata, N.; Toyo'oka, T. Biomed chromatogr

BMC. 1999, 13, 75-80.

116

http://www.aist.go.jp/RIODB/SDBS/

(56) Isom, A. L.; Barnes, S.; Wilson, L.; Kirk, M.; Coward, L.; Darley-Usmar, V. J Am Soc Mass Spectrom 2004, 15, 1136-47.

(57) Doorn, J. A.; Petersen, D. R. Chem Biol Interact 2003, 143-144, 93-100. (58) Kates, M. Techniques of lipidology : isolation, analysis, and identification of

lipids; 2nd rev. ed. ed.; Elsevier: Amsterdam,The Netherlands, 1986. (59) Birch, A. J.; Walker, K. A. M. J. Chem. Soc. (C) 1966, 1894. (60) Deng, Y.; Salomon, R. G. J Org Chem 2002, 63, 3504-07. (61) Esterbauer, H.; Schaur, R. J.; Zollner, H. Free Radic Biol Med 1991, 11, 81-128.

117

Chapter 4

Oxidative Protein Modifications in the Pathogenesis of Primary Open Angle

Glaucoma

118

4.1. Background

Free radical induced oxidative damage of polyunsaturated fatty acids generates

highly reactive molecules, which may have deleterious effects on cellular processes by

modifying protein and DNA.1-3 Additionally, these reactive molecules may act as ligands

for receptors 4,5 or they can form adducts on the cellular macromolecules that may be

immunogenic.6,7 Since these modifications are involved in a large variety of disease

processes, a method for early detection or prevention of such damage is important. Our

group has been involved in identification and characterization of the products of lipid

oxidation and their protein adducts.7,8 Antibodies generated against these modifications

and their subsequent use in their immunodetection in vivo were instrumental in

establishing a correlation between their levels in normal and diseased states viz,

atherosclerosis, renal disease6 and age related macular degeneration (AMD).9

4.1.1. Oxidative stress and glaucoma. The lipids present in the eye are prone to light

induced oxidative damage owing to the presence of light in conjunction with high oxygen

concentration.10,11 The products of docosahexaenoate (an ω-3 fatty acid) phospholipid

oxidation have been implicated in AMD.12 Unlike these phospholipids that are present in

high levels only in the posterior region of the eye, the ω-6 fatty acids (arachidonyl and

linolenyl phospholipids) are present in the anterior region, and are implicated in the

pathogenesis of glaucoma.13

Glaucoma, a group of late onset and progressive eye diseases that lead to

irreversible blindness often without any initial symptoms, affects about 70 million people

worldwide.14 The glaucomas are classified as primary, when no known contributory

119

factor is linked to the disease, and as secondary, when a previous disease or injury could

be attributed as a cause. Further, they can be classified as open angle glaucoma, when the

angle between the cornea and iris is normal or open for free flow of aqueous humor and

as angle closure glaucoma, when the angle is more acute than the normal (Figure 4.1).

The angle closure form glaucoma is due to an anatomical condition. However, the open

angle form of glaucoma remains poorly understood. Primary open angle glaucoma

(POAG) occurs most commonly but not always with an increase in the intraocular

pressure (IOP), and leads to progressive optic nerve damage.15

Figure 4.1: Diagrammatic representation of the cross section of an eye showing the two

forms of glaucoma defined according to the difference in the angle of the anterior

chamber formed by lines drawn parallel to the iris and cornea (iridial angle). A. normal

open angle (~40o) associated with POAG and B. a closed angle (~15o) associated with

angle closure glaucoma.

120

The aqueous humor, actively secreted by the ciliary epithelium, flows through the

pupil into the anterior segment of the eye and then passes through the trabecular

meshwork (TM), and drains into the Schlemm’s canal (Figure 4.2). The amount of

aqueous humor flowing into the anterior chamber in comparison with the amount drained

out is important in maintaining the IOP. Also, the TM is the site of maximum resistance

to the flow of aqueous humor, and any change in the TM can adversely affect the

aqueous flow, thus changing the IOP.15

Figure 4.2: Diagrammatic representation of the cross section of the human eye showing

the aqueous humor outflow pathway (Illustration by N. Aravindhan © 2005).

Tissues in the anterior chamber are subjected to chronic oxidative stress due to the

formation of reactive oxygen species (ROS),16,17 and increased ROS may be responsible

for damage to the trabecular meshwork and the Schlemm’s canal.18,19 POAG is

considered as a multifactorial disease and the role of oxidative damage in causing

changes in the trabecular meshwork and blockage of aqueous outflow cannot be

121

ignored.17,20-22 One of the factors contributing to increased oxidative stress may be a

decrease in the “antioxidant potential” of the TM, e.g., owing to inactivation of an

enzyme involved in detoxifying peroxides17,21 or decrease in levels of antioxidants.23-25

Furthermore, obstruction in the flow has also been attributed to the aggregation of

proteins under oxidative stress conditions.26

4.1.2. Levuglandins and isolevuglandins: formation and pathobiology. Levuglandins

(LGs) and isolevuglandins (isoLGs) are arachidonyl phospholipid (AA-PC) ester

metabolites that are formed by cyclooxygenase (COX) or free radical mediated pathways

respectively (Scheme 4.1, next page).27,28 The substrate for COX mediated

peroxidation requires the presence of free arachidonic acid, while the free radical

mediated pathway occurs predominantly in the phospholipid ester form.29 As

depicted in Scheme 4.1, LGE2 can be formed by either of these pathways, while

iso[4]LGE2 can only be formed through the free radical mediated pathway. These

γ-ketoaldehydes are highly reactive and are known to form adducts with lysine residues

followed by dehydration to form pyrroles (Scheme 4.1). LGs have also been shown to

form protein cross links rapidly compared to other lipid peroxidation products viz, 4-

hydroxynonenal (HNE) and malondialdehyde (MDA).30 LG modified LDL was shown

to be recognized and processed by macrophages through a scavenger receptor that

recognizes oxidized low density lipoproteins (oxLDL).31 The LGs and isoLGs-derived

protein adducts as well as free LGs have been shown to hinder the activity of the 20S

proteasome, a component of the protein degradation machinery, in a dose dependent

manner.32 Iso[4]LGE2-protein adducts are generated in blood and vasculature 33

122

AA

(CH2)3COOR

C5H11

OH

OHC

(CH2)3COOR

C5H11

OH

O

OO

O

OO C (CH2)nCH3

OP

ON(CH3)3

O OC5H11

(CH2)3COOR

OH

OO

(CH2)3COOR

OHOHC

O

C5H11

C5H11

(CH2)3COOH

C5H11

OH

N

H3C(CH2)3COOH

OH

C5H11N

H3C

(CH2)3COOR

C5H11

(CH2)3COOR

C5H11

O

(CH2)3COOH

C5H11

OH

OHC

(CH2)3COOH

C5H11

OH

O

OO

(CH2)3COOH

C5H11

NH2

2O2

protein

iso[4]PGH2-PC

protein

12

isoLGE2

AA-PC

iso[4]LGE2

iso[4]LGE2-pyrrole

isoPGH2-PC

LGE2-pyrrole

-2 H2O

-2 H2O

8

13

8

7

710

13

10

7

13

10

2O2

9

1 15

6912

LGE2

PGH2

8

8

7

13

10

2O2

9

1

-2 H2O

PLA2

cyclooxygenase

free radicals

-2-lysoPC-2-lysoPCprotein NH2protein

NH2protein

Scheme 4.1: Enzymatic (cyclooxygenase) and non enzymatic (free radical) routes for the

formation of LGE2 and iso[4]LGE2 and their protein adducts.

and are present in oxidized LDL.34 The presence of these adducts was detected with

antibodies raised against these epitopes using immunodetection techniques viz, ELISA,

Western blots and immunohistochemical analysis.33,34 The detection of isoLGs is

probably indicative of localized oxidative stress within tissues as they form adducts

123

rapidly.30 Accumulation of modified proteins has been identified in various pathological

conditions such as muscular dystrophy,35 rheumatoid arthritis36 and atherosclerosis.37

4.1.3. 4-Hydroxynonenal and its role in pathobiology. HNE is by far the most

investigated of the lipid peroxidation products.38-40 HNE is generated from AA or linoleyl

phospholipids through free radical mediated pathways. Because HNE is a small

bifunctional molecule, it easily transverses the cell membrane and reacts with a wide

variety of substrates.39 HNE is known to form Michael adducts with nucleophilic amino

acid residues viz, lysine, cysteine and histidine (Scheme 4.2).41

OOH

4-Hydroxynonenal

N

OOH

NH

OOH

N

Lysine Michael adduct

Histidine Michael adduct

Histidine

Lysine

S

OOH

Cysteine Michael adduct

Cysteine

OHN

Lysine

NLysine

LysineN

HO

LysineN

Lysine-Lysine crosslink

Lysine Schiff base adduct

Lysine pyrrole adduct

Scheme 4.2: Some of the commonly reported adducts formed by 4-hydroxynonenal

reaction with histidine, lysine and cysteine residues.

124

The presence of three functional groups, viz. aldehyde, C=C double bond and a

hydroxyl group, predisposes HNE to react with more than one residue.42 Consequently,

HNE formation of protein-protein crosslinks has been observed in both in vitro as well as

in vivo model studies.43,44 The HNE derived modifications on proteins have been

implicated in a variety of disease processes including atherosclerosis,45

neurodegeneration46 and proliferative vitreoretinopathies.47 See section 2.1 for a detailed

background.

4.1.4. Advanced glycation end products and their pathobiology. Modification of

proteins by the Maillard reaction is thought to play a role in chemical aging of

proteins.48,49 In the Maillard reaction, the amino side chain residues of a protein are non-

enzymatically glycosylated by reducing sugars to form aminoketoses, also called as

Amadori products.50 This initial adduct can undergo rearrangements, cyclizations and

dehydrations to form products, mostly with fluorescent and chromophoric properties,

collectively called advanced glycation end products (AGEs). At present, there is evidence

that protein glycation is involved in the pathogenesis of several amyloid diseases, such as

Alzheimer's disease51-53 and dialysis-related amyloidosis.54 Some of the cellular effects of

AGEs are mediated by interactions with specific cellular receptors, such as the receptor

for AGEs (RAGE).55

In addition to glucose, methylglyoxal (MG) a product formed by non-enzymatic,

amine catalyzed sugar fragmentation reactions and by spontaneous decomposition of

triose phosphate intermediates of glycolysis, can also modify proteins and contribute to

AGE’s (Scheme 4.3).56 MG may also be formed from lipid oxidation products.57 MG is

125

one of the most powerful glycating reagents that can irreversibly modify lysine and

arginine residues in proteins.

NOH

HOOH

Protein

OHHOH2C

Protein-NH2

OH

O O

N N

OH

HN (CH2)3

NH2

COOH

+ Glucose

Arginine

Argpyrimidine

H

O

O

OH

O

OH

H+

HCOOH

HNOH

HOOH

Protein

OHHOH2C

HNO

HOOH

Protein

OHHOH2C

"Classical"Amadori

rearrangement

O

OHNH

OH

OH

OH

Methylglyoxal

Protein

Intermediates of Glycolysis

Amadori product

Scheme 4.3: Mechanistic pathway for the formation of AGE’s and argpyrimidine.

MG is known to form carboxyethyl lysine (CEL) by adduction with lysine

residues and argpyrimidine [Nd-(5-hydroxy-4,6-dimethylpyrimidin-2-yl)-L-ornithine], a

fluorescent product, by adduction with arginine.56 Recently, argpyrimidine was found in

spinal cord of familial sporadic amyotrophic lateral sclerosis (ALS) patients and mutant

126

SOD-1 (superoxide dismutase 1) mice. Methylglyoxal modification of arginine may

contribute to the pathophysiology associated with aging and other diseases.57,58

4.1.5. Oxidative products of tryptophan and α-hydroxykynurenine. Oxidative

damage of tryptophan by both enzymatic and non-enzymatic pathways generates

products that can modify proteins to form yellow-brown pigments.59 The metabolism of

tryptophan proceeds through the kynurenine pathway.60 The products of this pathway as

well as toxic intermediates are implicated in a range of neurodegenerative, inflammatory,

and immunologic phenomena diseases/disorders, including Huntington’s disease,61

Parkinson’s disease,62 and HIV encephalopathy.63

As outlined in Scheme 4.4, the products formed from the N-formyl kynurenines

include kynurenine (KYN), 3-hydroxykynurenine (OHKYN), 3-hydroxyanthranilic acid,

quinolinic acid, and nicotinic acid.60 OHKYN can form various reactive catabolites,

which can modify proteins and cause them to aggregate.64 One of the products of the

reaction of OHKYN with lens glutathione (GSH) is glutathionyl-3-hydroxykynurenine

glucoside,65 which accumulates during lens aging and accumulates to relatively high

levels in cataractous lenses.66 In addition to the modifications formed by kynurenine and

its auto oxidation products, it can also form adducts by non-oxidative pathways, e.g., α,

β- unsaturated OHKN, 67 formed by deamination of KYN, reacts with nucleophilic amino

acids, such as cysteine, histidine, and lysine in lens proteins.68

Recently, antibodies against the protein modifications formed by OHKYN were

generated and characterized.59 These antibodies were used in this study to assess the

levels of these modifications in TM tissue of POAG subjects.

127

NH2

HOO

NH

NH2

HOO

ONH

O

NH2

OHO

ONH2

NH2

OHO

ONH2HO

O2

Kynurenineformidase

Kynureninehydroxylase

INF-γIndoleamine

2,3-dioxygenase

Proteins

α−Hydroxykynurenine

Protein Adducts

TryptophanN-formyl Kynurenine

Kynurenine

Scheme 4.4: Catabolic pathway of tryptophan under oxidative conditions.

Accumulation of oxidative protein modifications in TM from lipids and sugars69

may contribute to the onset and/or progression of glaucoma. However, their occurrence

in TM tissue in vivo in glaucoma has yet to be demonstrated. In this chapter, a

multifaceted approach was used to assess the levels of oxidative injury/insult in

glaucomatous TM compared to that of a control TM by using antibodies against protein

modifications derived from oxidized lipids, glucose and tryptophan.

128

4.2. Results.

4.2.1. Immunoblot analysis of TM tissues for analyzing the levels of oxidative

protein modifications in glaucomatous TM compared to the controls. To compare the

levels of protein modifications in POAG and normal TM, a western blot analysis of TM

extracts of POAG and age-matched controls was performed. The Western blot analysis

using the anti-iso[4]LGE2 (Figures 4.3 and 4.1S (appendix)), HNE (Figures 4.4 and 4.2S

(appendix)), argpyrimidine (Figures 4.4 and 4.3S (appendix)) and OHKYN antibodies

showed increased immunoreactivity in protein extracts of the POAG TM compared to

that of the control TM extracts. Quantification of this difference is presented in Figures

4.11 and 4.12, see discussion in section 4.3 for details. Totally, 52 POAG samples were

compared with 52 age-matched control samples (Table 4.1). Bovine or human serum

albumin modified by these oxidized lipids was used as a positive control.

Table 4.1: Total number of tissue samples used for Western blots for each of the

antibodies probed. Some of the tissues were used in more than one Western blot analysis.

Number of POAG samples analyzed

Number of Control samples analyzed

Immunoreactivity* Immunoreactivity*

Western Blots

Total + - Total + -

Iso[4]LGE2 23 1 22 22 21 1

HNE 23 2 21 22 12 10

Argpyrimidine 16 2 14 16 16 9

OHKYN 8 1 7 8 4 4

*Immunoreactivity (+) = values above 1 standard deviation (SD) and below mean SD of POAG

*Immunoreactivity (-) = values below 1 SD and below mean SD of POAG

129

88M

W

74 M

W

87 M

W

84 M

W

80 F

W

72 M

W

87 M

W

87 M

W

LMW

c

11488

50

35

28

20

11488

50

35

28

20

Normal TM Glaucomatous TM

D

74 M

W

6 1 F

W

74 M

W

7 0 M

W

78 M

W

64 F

W

7 6 M

W

69 M

W


B

E

42 F

B

44 M

B

74 F

B

79 F

B

42 F

B

42 M

B

74 F

B

81 F

B


C

F

Coo

mas

sie

Wes

tern

Blo

t

A

88M

W

74 M

W

87 M

W

84 M

W

80 F

W

72 M

W

87 M

W

87 M

W

LMW

c

11488

50

35

28

20

11488

50

35

28

20

11488

50

35

28

20


D

74 M

W

6 1 F

W

74 M

W

7 0 M

W

78 M

W

64 F

W

7 6 M

W

69 M

W


B

E

42 F

B

44 M

B

74 F

B

79 F

B

42 F

B

42 M

B

74 F

B

81 F

B


C

F

Coo

mas

sie

Wes

tern

Blo

t

A

Figure 4.3: Western analysis of POAG and normal trabecular meshwork using anti-

iso[4]LGE2 pAb antibodies. TM extracts (5 µg) were subjected to SDS-PAGE, blotted to

PVDF membrane and probed with anti-iso[4]LGE2 antibodies. A,B,C . Coomassie blue

stained gels. D,E,F. Western blot: age, race and gender of the tissue samples are

indicated (M-Male; F-Female; W-Caucasian; B-African American).

6645

2631

14

97

6645

2631

14

97

74 M

W

77 M

W

80 F

W

87 M

W

74 M

W

78M

W

80 F

W

86 M

W

77 M

W

60 F

W

80 F

W

74 M

W

76 M

W

75 F

W

94 F

W

77 M

B

43 M

W

50 M

W

58 M

W

72 M

W

72 M

W

58 F

A

50 M

W

43 M

W

Normal TM Glaucomatous TMNormal TM Glaucomatous TM Normal TM Glaucomatous TM

Coo

mas

sie

Wes

tern

Blo

t

D

B

E

C

F

A

6645

2631

14

97

6645

2631

14

97

74 M

W

77 M

W

80 F

W

87 M

W

74 M

W

78M

W

80 F

W

86 M

W

77 M

W

60 F

W

80 F

W

74 M

W

76 M

W

75 F

W

94 F

W

77 M

B

43 M

W

50 M

W

58 M

W

72 M

W

72 M

W

58 F

A

50 M

W

43 M

W


Coo

mas

sie

Wes

tern

Blo

t

6645

2631

14

976645

2631

14

97

6645

2631

14

976645

2631

14

97

74 M

W

77 M

W

80 F

W

87 M

W

74 M

W

78M

W

80 F

W

86 M

W

77 M

W

60 F

W

80 F

W

74 M

W

76 M

W

75 F

W

94 F

W

77 M

B

43 M

W

50 M

W

58 M

W

72 M

W

72 M

W

58 F

A

50 M

W

43 M

W

74 M

W

77 M

W

80 F

W

87 M

W

74 M

W

78M

W

80 F

W

86 M

W

77 M

W

60 F

W

80 F

W

74 M

W

76 M

W

75 F

W

94 F

W

77 M

B

43 M

W

50 M

W

58 M

W

72 M

W

72 M

W

58 F

A

50 M

W

43 M

W


Coo

mas

sie

Wes

tern

Blo

t

D

B

E

C

F

A

Figure 4.4: Western analysis of POAG and normal trabecular meshwork using anti-HNE

pAb antibodies. TM extracts (5 µg) were subjected to SDS-PAGE, blotted to PVDF

membrane and probed with anti- anti-HNE antibodies. A,B,C. Coomassie blue stained

gels. D,E,F. Western blot. M-Male; F-Female; W-Caucasian; B-African American).

130

Normal TM

250150

75

3750

25

100

20

1015

7 4 M

W

61 F

W

7 4 M

W

7 0 M

W

78 M

W

6 4 F

W

7 6 M

W

69 M

W

250150

75

37

50

25

100

20

1015

Glaucomatous TM Normal TM

85 F

W

59 F

W

6 7 F

W

6 1 M

W

81 F

W

5 9 F

W

6 7 F

W

61 M

W


82 F

W

5 9 F

W

8 6 F

W

61 M

W

82 F

W

59 F

W

8 6 F

W

61 M

W

Glaucomatous TM

250150100

Coo

mas

sie

Wes

tern

Blo

t

D

B

E

C

F

A

Normal TM

250150

75

3750

25

100

20

1015

7 4 M

W

61 F

W

7 4 M

W

7 0 M

W

78 M

W

6 4 F

W

7 6 M

W

69 M

W

250150

75

37

50

25

100

20

1015


85 F

W

59 F

W

6 7 F

W

6 1 M

W

81 F

W

5 9 F

W

6 7 F

W

61 M

W


82 F

W

5 9 F

W

8 6 F

W

61 M

W

82 F

W

59 F

W

8 6 F

W

61 M

W

Glaucomatous TM

250150100

Coo

mas

sie

Wes

tern

Blo

t

D

B

E

C

F

A

Figure 4.5: Western analysis of POAG and normal trabecular Meshwork using anti-

argpyrimidine mAb. TM extracts (5 µg) were subjected to SDS-PAGE, blotted to PVDF

membrane and probed with anti-argpyrimidine antibodies. A,B,C. Coomassie blue

stained gels. D,E,F. Western blot: age, race and gender of the tissue samples are

indicated (M-Male; F-Female; W-Caucasian; B-African American).

Coo

mas

sie

Wes

tern

Blo

t

11488

50

35

28

20

Normal TM

85 F

W

5 9 F

W

67 F

W

61 M

W

81 F

W

5 9 F

W

6 7 F

W

61 M

W

Glaucomatous TM

11488

50

35

28

20


82 F

W

59 F

W

8 6 F

W

61 M

W

82 F

W

59 F

W

8 6 F

W

61 M

W

Coo

mas

sie

Wes

tern

Blo

t

11488

50

35

28

20

11488

50

35

28

20

Normal TM

85 F

W

5 9 F

W

67 F

W

61 M

W

81 F

W

5 9 F

W

6 7 F

W

61 M

W

Glaucomatous TM

11488

50

35

28

20

11488

50

35

28

20


82 F

W

59 F

W

8 6 F

W

61 M

W

82 F

W

59 F

W

8 6 F

W

61 M

W

Figure 4.6: Western Analysis of POAG and Normal Trabecular Meshwork using anti-

OHKYN mAb. TM extracts (5 µg) were subjected to SDS-PAGE, blotted to PVDF

membrane and probed with anti-OHKYN antibodies. Age, race and gender of the tissue

samples are indicated (M-Male; F-Female; W-Caucasian; B-African American).

131

4.2.2. Localization of modified proteins by immunohistochemical analysis. To

visualize the localization of modified proteins in the trabecular meshwork, we probed the

tissue sections from POAG and age-matched cadaver tissue with antibodies to these

modifications. Anti-iso[4]LGE2 and anti-HNE antibodies (red fluorescence of the

secondary antibody with Alexa 594 fluorescent tag) showed a strong immunoreactivity in

the TM of POAG compared to the normal TM or the pre-immune stained POAG TM

sections. Adjacent sections were used for immunohistochemical analysis using the

antibody and corresponding pre-immune serum to compare the relative

immunoreactivity.

Figure 4.7: Localization of iso[4]LGE2 modified proteins in glaucomatous TM. Anterior

segment histochemical sections through the trabecular meshwork are shown. A, B, C –

Probed with pre-immune serum; D, E, F,G, H, I - Probed with anti-iso[4]LGE2 antibody;

Red Channel – iso[4]LGE2 specific immunofluorescence ; Green Channel –

autofluorescence ; DAPI - nuclear stain (blue fluorescence)

132

A

B

C

D

E

G

H

I

Glaucomatous TM Glaucomatous TM Normal TM

DAP

IG

reen

Cha

nnel

Red

Cha

nnel

F

A

B

C

D

E

G

H

I

Glaucomatous TM Glaucomatous TM Normal TM

DAP

IG

reen

Cha

nnel

Red

Cha

nnel

F

Figure 4.8: Localization of HNE modified proteins in glaucomatous TM. Anterior

segment histochemical sections through the trabecular meshwork are shown. A, B, C –

Probed with pre-immune serum; D, E, F,G, H, I - Probed with anti-HNE antibody; Red

Channel – HNE specific immuno-fluorescence; Green Channel – auto-fluorescence ;

DAPI – nuclear stain (blue fluorescence)

4.2.3. Immunoprecipitation of modified proteins. To identify the modified proteins

from the TM extracts, we immunoprecipitated the iso[4]LGE2 and HNE modified

proteins using the corresponding antibody-coupled protein A sepharose beads. The

immunoprecipitated proteins were subjected to SDS-PAGE and partially electroblotted

onto a PVDF membrane. The Coomassie stained IP gels clearly show the enrichment of

modified proteins compared to the total protein extracts (Figures 4.9 and 4.3S

(Appendix)). The immunoblot of the IP exhibited protein bands corresponding to those in

133

the total extract (Figure 4.9 and 4.4S (Appendix)). The bands detected at ~55 kDa that is

common to both the lane 2 and 3 (shown with dotted lines in Figure 4.9A & 4.9B)

corresponds to the heavy chain of IgG originating from the cleavage of IgG chains from

the antibody-coupled beads because the extraction was done with Laemmli’s buffers that

contains DTT as reported previously.70 DTT cleaves the disulphide bond that anchor the

IgG heavy chain to the light chain.

114

88

50

35

28

20

250

1 2 3 4 5A

345

2

1

1 2 3 4 5

114

88

50

35

28

20

250

B

IgG(long chain)

12

456

3

114

88

50

35

28

20

250

1 2 3 4 5A

345

2

1

114

88

50

35

28

20

250

1 2 3 4 5A

345

2

1

1 2 3 4 5

114

88

50

35

28

20

250

1 2 3 4 5

114

88

50

35

28

20

250

114

88

50

35

28

20

250

B

IgG(long chain)

12

456

3

Figure 4.9: Immunoprecipitation of antibody modified protein from TM extract. 10µg of

antibody coupled beads and 30µg of TM protein extract were used for

immunoprecipitations. A. Anti-iso[4]LGE2 IP B. Anti-HNE IP .Lane 1 – Protein extract,

Lane 2 – IP products, Lane 3 – Ab coupled beads, Lane 4 – control-protein A beads

without antibody, Lane 5 – Western blot of protein extract. Bands numbered here

corresponds to proteins identified and listed in Table 4.2. Detection was performed by

Coomassie blue staining. Preimmune probed IP is presented in the Appendix Figure 4.4S.

134

4.2.4. Identification of immunoprecipitated proteins. The Coomassie stained 1D gel

bands of the immunoprecipitated proteins in Figure 4.9 were excised and digested in situ

with trypsin. The tryptic peptides were analyzed by liquid chromatography-coupled

electrospray tandem mass spectrometry 71 and the proteins were identified by well

established bioinformatics methods. Two independent immunoprecipitations from two

different POAG TM samples were performed and proteins that are common in both the

samples are listed in Table 4.2. Serum albumin, transforming growth factor β-IGH3,

decorin and HSP-70 are identified to be immunoprecipitated by both anti-Iso[4]LGE2 as

well as anti-HNE antibody. Lumican, prolargin, PDI-A3, pyruvate kinase and vitronectin

were uniquely immunoprecipitated by HNE antibody-coupled beads, while complement -

C3, and GFAP were immunoprecipitated by iso[4]LGE2 antibody-coupled beads.

135

Table 4.2: Identification of TM proteins immunoprecipitated using iso[4]LGE2 and HNE

pAbs. A. Proteins identified by in-gel digestion of bands from lane A-2 (Figure 4.9) using

mass spectrometry (LC-MS/MS). B. Proteins identified by in-gel digestion of bands

from lane B-2 (Figure 4.9) using mass spectrometry (LC-MS/MS). Proteins listed here

were present in three IP experiments.

27

40

42

187

70

74

69

Calculated* MW (kDa)

2,5

1,3

3,5

2

4,5

3,5

3,4,5

BandNo.

70,3301P07585Decorin

ObservedMW (kDa)

Peptide matche

s

Accession NumberProteins Identified

65-701P48677Glial fibrillary acidic protein

70, 2651P48741Heat shock 70 kDa protein 7

2651P01024Complement C3 precursor

65-702P08107Heat shock 70 kDa protein 1

65-703Q15582TGF-β induced protein IG-H3

65-707P02768Serum albumin precursor

27

40

42

187

70

74

69

Calculated* MW (kDa)

2,5

1,3

3,5

2

4,5

3,5

3,4,5

BandNo.

70,3301P07585Decorin

ObservedMW (kDa)

Peptide matche

s


65-701P48677Glial fibrillary acidic protein

70, 2651P48741Heat shock 70 kDa protein 7

2651P01024Complement C3 precursor

65-702P08107Heat shock 70 kDa protein 1

65-703Q15582TGF-β induced protein IG-H3

65-707P02768Serum albumin precursor

54

75

69

24

58

57

44

38

40

50

Calculated*MW (kDa)

6

3,6

3,5,6

6

2

6

3,6

6

1,4

4,6

BandNo.

63, >2002Q15582TGF-β induced protein IG-H3

ObservedMW (kDa)

Peptide matches


631Q8TZS0RNase P component 3

63-2001P49822Serum albumin

631P30101Protein disulphide-isomeraseA3

631P04004Vitronectin precursor

2431P14618Pyruvate kinase isozymes

63, >2003P51888Prolargin

632P51884Lumican

67, >2981P07585Decorin

67, 632P30838Aldehyde dehydrogenase

54

75

69

24

58

57

44

38

40

50

Calculated*MW (kDa)

6

3,6

3,5,6

6

2

6

3,6

6

1,4

4,6

BandNo.

63, >2002Q15582TGF-β induced protein IG-H3

ObservedMW (kDa)

Peptide matches


631Q8TZS0RNase P component 3

63-2001P49822Serum albumin

631P30101Protein disulphide-isomeraseA3

631P04004Vitronectin precursor

2431P14618Pyruvate kinase isozymes

63, >2003P51888Prolargin

632P51884Lumican

67, >2981P07585Decorin

67, 632P30838Aldehyde dehydrogenase

A

B

* for monomeric protein

4.2.6. Identification of modified proteins by two-dimensional gel electrophoresis.

We also identified the iso[4]LGE2- and HNE-modified proteins from glaucomatous

trabecular meshwork by mass spectrometry following two-dimensional immunoblot

analysis. Trabecular meshwork from a 79-year-old caucasian male with primary open

angle glaucoma was used for the 2D SDS PAGE. The mass spectrometry analysis

136

pH

20

28

35

50

88

114

kDa345678910

12

3

54

7

6

8 9 10

15

11 12 13

14-19

20-2425-27

52

42-46 48 49

51

50

37-41

31-35

28-30

54

47

53

1

36

pH

20

28

35

50

88

114

kDa345678910 345678910

12

3

54

7

6

8 9 10

15

11 12 13

14-19

20-2425-27

52

42-46 48 49

51

50

37-41

31-35

28-30

54

47

53

1

36

pH345678910

pH345678910 345678910

345678910pH

20

28

35

50

88114

kDaHNE

345678910 345678910pH

20

28

35

50

88114

20

28

35

50

88114

kDaHNE

A B

C

pH345678910

pH345678910 3456789 345678910

D

pH

20

28

35

50

88

114

kDa345678910

12

3

54

7

6

8 9 10

15

11 12 13

14-19

20-2425-27

52

42-46 48 49

51

50

37-41

31-35

28-30

54

47

53

1

36

pH

20

28

35

50

88

114

kDa345678910 345678910

12

3

54

7

6

8 9 10

15

11 12 13

14-19

20-2425-27

52

42-46 48 49

51

50

37-41

31-35

28-30

54

47

53

1

36

pH345678910

pH345678910 345678910

345678910pH

20

28

35

50

88114

kDaHNE

345678910 345678910pH

20

28

35

50

88114

20

28

35

50

88114

kDaHNE

A B

C

pH345678910

pH345678910 3456789 345678910

D

Figure 4.10: 2D PAGE analysis of TM extract. ~80 µg of extracted TM proteins was

used for the 2D PAGE. The gel was partially transferred onto a PVDF membrane for

immunochemical analysis. A. Coomassie stained gel B. Western analysis using

Iso[4]LGE2 pAb. C. Western analysis using HNE pAb. D. Merged image showing

Coomassie stain in black and Western blots probed with iso[4]LGE2 in blue; HNE in

orange.

revealed the presence of many small proteins present in higher molecular weight region

suggesting an aggregation and a change in their polarity, In corroboration with the 1D

immunoblots.

137

Table 4.3: Iso[4]LGE2 and HNE immunoreactive proteins identified by 2D PAGE and

mass spectrometry. Calculated MW and pI refers to value derived from Swissprot protein

database; Observed MW and pI refers to values from the 2D PAGE.1031

1 homodimer ; 2 multimer * for monomeric protein except where indicated otherwise 95.6 - 4.667, 505.15327,47Vimentin

24.866,1997.96835Transketolase

23--6.269

2,3,5,9,15,16,19-22,24-31,35,36, 38,52,54

Serum albumin precursor

56.1 - 8.4100, 67, 559.643

7-11,16-24,26, 29,30,34,35,38,54

Prolargin

26.1 - 8.492, 676.5387,26-31,33,39, 41,43,53

Lumican

56.47077420,22Lamin A/C

13.3488.64050Trace amine receptor 15

14.5467.84748beta-enolase

14.75064947alpha-enolase

26.3707.27721Transferrin

18.2525124213Transcription factor 8

25.052 - 7287430,43TGF-bIG H3

36.5557.75817,19Pyruvate kinase

16.7556.75218,19Prolineaminopeptidase

28.4927.2407Keratocan

45 - 75586117,18Glutamate dehydrogenase

17.3978.8378,18Cathepsin K

25 - 750 -536.5,5.26017,46ATP synthase

27 - 85585412,17, 18Annexin A11

56.7557.5,6.75418,19, 52Aldehyde dehydrogenase

19.2535.3103111,36a-actinin

pIMWpIMWPeptide match

ObservedCalculated*Spot No.Protein Identifiedz

95.6 - 4.667, 505.15327,47Vimentin


23--6.269

2,3,5,9,15,16,19-22,24-31,35,36, 38,52,54


56.1 - 8.4100, 67, 559.643

7-11,16-24,26, 29,30,34,35,38,54

Prolargin

26.1 - 8.492, 676.5387,26-31,33,39, 41,43,53

Lumican

56.47077420,22Lamin A/C






25.052 - 7287430,43TGF-bIG H3





17.3978.8378,18Cathepsin K

25 - 750 -536.5,5.26017,46ATP synthase

27 - 85585412,17, 18Annexin A11


19.2535.3103111,36a-actinin


ObservedSpot No.Protein Identifiedz

A. Iso[4]LGE2 immunoreactive proteins

B. HNEimmunoreactive proteins

C. Iso[4]LGE2 HNEimmunoreactive proteins

95.6 - 4.667, 505.15327,47Vimentin


23--6.269

2,3,5,9,15,16,19-22,24-31,35,36, 38,52,54


56.1 - 8.4100, 67, 559.643

7-11,16-24,26, 29,30,34,35,38,54

Prolargin

26.1 - 8.492, 676.5387,26-31,33,39, 41,43,53

Lumican

56.47077420,22Lamin A/C






25.052 - 7287430,43TGF-bIG H3





17.3978.8378,18Cathepsin K

25 - 750 -536.5,5.26017,46ATP synthase

27 - 85585412,17, 18Annexin A11


19.2535.3103111,36a-actinin


ObservedCalculated*Spot No.Protein Identifiedz

95.6 - 4.667, 505.15327,47Vimentin


23--6.269

2,3,5,9,15,16,19-22,24-31,35,36, 38,52,54


56.1 - 8.4100, 67, 559.643

7-11,16-24,26, 29,30,34,35,38,54

Prolargin

26.1 - 8.492, 676.5387,26-31,33,39, 41,43,53

Lumican

56.47077420,22Lamin A/C






25.052 - 7287430,43TGF-bIG H3





17.3978.8378,18Cathepsin K

25 - 750 -536.5,5.26017,46ATP synthase

27 - 85585412,17, 18Annexin A11


19.2535.3103111,36a-actinin


ObservedSpot No.Protein Identifiedz

A. Iso[4]LGE2 immunoreactive proteins

B. HNEimmunoreactive proteins

C. Iso[4]LGE2 HNEimmunoreactive proteins

138

4.3. Discussion Oxidative stress is often the final common pathway of cell damage that has been

implicated in pathogenesis in many neurodegenerative diseases such as Alzheimer’s

disease, Parkinson’s disease and glaucoma.72,73 In the eye, oxidative damage has been

implicated in degenerations of the posterior segment, the retina and the optic nerve.74,75 In

glaucoma the increase in the intraocular pressure may be associated with the oxidative

damage in the TM tissue. Increase in oxidative stress and decrease in the total reactive

antioxidant potential have been detected in aqueous humor of glaucoma patients.76

Oxidative DNA damage is increased in the trabecular meshwork of patients with

glaucoma.77,78 Most of the oxidative protein modifications6,7,12,79,80 have been linked to

tissue damage in aging 11 and may be an important component in onset and progression

of POAG.

The present study provided the first direct evidence establishing a correlation

between oxidative stress in the TM and glaucoma. Western blot analysis of protein

extracts from the TM tissue of glaucoma and control subjects was performed using

multiple antibodies against oxidative stress markers derived from lipid and protein

oxidation as well as AGEs. The proteins that may be targets of increased oxidative stress

were isolated either by immunoprecipitation or by 2D PAGE and subsequently identified

by mass spectrometry. The modified proteins were also localized at the tissue level by

immunohistochemical analysis.

Iso[4]LGE2 derived protein modifications are distinctly increased in the

glaucomatous TM compared to that of the controls (p < 2E-11) (Figure 4.11).

Iso[4]LGE2’s are known to irreversibly form adducts that accumulate, and thus reflect the

139

total oxidative stress undergone by the tissue. Thus, the adduct level may be considered

as a dosimeter of oxidative stress. Protein modifications derived from HNE, another lipid

oxidation product, are also elevated in glaucomatous TM compared to that of the controls

(p < 0.0003) (Figure 4.11). Thus, through immunoblots, a strong correlation between

oxidative stress and POAG was established. The vast difference in the ‘p’ values of these

two different lipobolites may suggest that adducts formed from HNE may be formed

reversibly or slowly compared to the iso[4]LGE2 adducts. This is supportive of earlier

reports suggesting that levuglandins bind far more avidly to LDL compared to HNE or

MDA.31,81 So the latter may be cleared before they have an opportunity to form adducts.

140

-1

1

3

5

7

9

11

13

15

17

0

2

4

6

8

10

12

14

Rel

ativ

e in

tens

ity (O

D)

(HN

E-im

mun

orea

ctiv

e ba

nds)

Controls POAG

p≤0.0003

BR

elat

ive

inte

nsity

(OD

) (is

o[4]

LGE 2

imm

unor

eact

ive

band

s)

p≤2E-11

Controls POAG

A

-1

1

3

5

7

9

11

13

15

17

0

2

4

6

8

10

12

14

Rel

ativ

e in

tens

ity (O

D)

(HN

E-im

mun

orea

ctiv

e ba

nds)

Controls POAG

p≤0.0003

BR

elat

ive

inte

nsity

(OD

) (is

o[4]

LGE 2

imm

unor

eact

ive

band

s)

p≤2E-11p≤2E-11

Controls POAG

A

Figure 4.11: Quantification of iso[4]LGE2 and HNE immunoreactive bands in 1D

Western blots. Intensity for each of the lanes detected (all the bands in the lane included)

in the Western blots was normalized with respect to the total of all the bands detected in

that blot (details of calculations in the experimental section). The minimum intensity for

band detection was fixed for each of the blots. ◊ – Relative intensity of a control sample

on the blot; □ – OD of a POAG tissue on the blot. A. Levels of iso[4]LGE2 modified

proteins (tissue donors – 23 POAGs, 22 controls, shown in Figures 4.3 and 4.1S

(appendix)), B. Levels of HNE modified proteins (tissue donors – 23 POAGs, 22

controls, shown in Figures 4.4 and 4.2S (appendix)). ‘p’ values calculated by students t-

test using Microsoft ® Excel 2003.

Additionally, a moderate increase in the levels of argpyrimidine modification was

evident through Western blot analysis using antibodies against argpyrimidine in

glaucomatous TM compared to the controls (p < 0.0035). Though the analysis did not

take into account whether the subjects are diabetic or not, the difference in these levels

along with the evidence for increased lipid oxidation, may point to an increase in the

141

contribution of oxidative lipid fragmentations to the production of methyl glyoxal. This is

additional evidence to previous reports that suggested a contribution of oxidative stress to

the formation of methyl glyoxal in addition to fragmentation of sugars and glycolysis

products.69 There is also increase in the amount of oxidized tryptophan as measured by

the amount of OHKYN using the anti-OHKYN antibodies.

2

3

4

5

0

5

10

15

20

25

30

35

40

Rel

ativ

e in

tens

ity (O

D)

(Arg

pyrim

idin

e im

mun

orea

ctiv

e ba

nds)

Rel

ativ

e in

tens

ity (O

D)

(OH

KYN

imm

unor

eact

ive

band

s)

p≤0.0015p≤0.001

Controls POAGControls POAG

BA

2

3

4

5

0

5

10

15

20

25

30

35

40

Rel

ativ

e in

tens

ity (O

D)

(Arg

pyrim

idin

e im

mun

orea

ctiv

e ba

nds)

Rel

ativ

e in

tens

ity (O

D)

(OH

KYN

imm

unor

eact

ive

band

s)

p≤0.0015p≤0.0015p≤0.001p≤0.001

Controls POAGControls POAG

BA

Figure 4.12: Quantification of argpyrimidine and OHKYN immunoreactive bands in 1D

Western blots. Relative intensity of optical density (OD) for each of the lanes (all the

bands in each lane included) in the Western blots was normalized for each of the blots (8

lanes each). The minimum intensity for band detection was fixed for each of the blots. ◊ –

Relative intensity of a control sample on the blot; □ – relative intensity of a POAG tissue

on the blot. A. Levels of argpyrimidine modified proteins (tissue donors – 12 POAGs, 12

controls, shown in Figures 4.5 and 4.3S (appendix)). B. Levels of OHKYN modified

proteins (tissue donors – 8 POAGs, 8 controls, shown in Figures 4.6). ‘p’ values

calculated by students t-test using Microsoft ® Excel 2003.

Based on our data from the immunoblots, we performed the immunoprecipitation

(IP) of the modified proteins from the glaucomatous TM. IP was done using antibody

142

(Iso[4]LGE2 and HNE) covalently coupled with sepharose A beads. The

immunoprecipitated proteins were analyzed for their immunoreactivity by immunoblot

analysis (Figure 5, Lane 1’). Following 1D SDS PAGE of the immunoprecipitated

proteins, the gel bands were excised, and the proteins analyzed by mass spectrometry. In

both of the IPs a majority of the proteins identified are the extracellular matrix (ECM)

proteins, suggesting that these proteins are especially vulnerable to oxidative protein

modifications. The proteins identified by immunoprecipitation to be oxidatively modified

were also reported in our earlier proteomic analysis of the TM.82

Keratoepithelin (TGF-βig-h3) one of the proteins identified, is found in many

connective tissues and considered to have a binding function of these tissues.83 Elevated

levels and/or abnormal aggregation of keratoepithelin has been associated with type 2

diabetic nephropathy and corneal dystrophies.84 We also found β-IGH3, a protein

localized in the corneal epithelial tissue that is apparently modified by lipid oxidation

products. However, the possibility that these deposits in the TM originate by detachment

from the corneal epithelium, dispersal into the aqueous humor, and deposition in the TM

cannot be ruled out. Protein disulphide isomerase (PDI),85 an enzyme involved in

formation of disulphide bonds in native proteins that also acts as a chaperone, is

immunoprecipitated by HNE antibodies. Modification of this enzyme could cause protein

misfolding and aggregation. Other prominent proteins that immunoprecipitated are the

extracellular matrix associated proteins (viz, decorin, lumican, prolargin and vitronectin)

and those involved in energy metabolism (aldehyde dehydrogenase and pyruvate kinase).

This observation suggests that increase in oxidative stress can potentially compromise the

morphology of the cells lining the TM of POAG as reported earlier.86

143

In addition to IP, 2D SDS PAGE analysis of the TM extract was also performed

for identification of the oxidatively modified proteins. Owing to better separation of

proteins, this analysis resulted in more protein hits compared to the IP. The mass

spectrometric analysis of immunoreactive spots revealed several ECM matrix proteins,

corroborating the results from the IP. Oxidative stress conditions have been shown to

lessen the adhesion of cultured TM cells to ECM proteins.86 This could induce changes in

the cell cycle 87 or apoptosis.88 Identification of transferrin, a iron regulating protein

shown to be elevated in retinal tissue of experimental glaucoma in monkey,89 shows the

presence of antioxidant systems.

The changes observed in the molecular weight of the proteins identified by 2D

PAGE may reflect aggregation by crosslinking (increased MW) caused by the oxidized

lipids or oxidative protein degradation (decreased MW), while the changes in the pI

reflect charge changes due to modifications and/or transformation. Some of the protein

spots identified on the 2D gel had molecular weight that corresponds to a dimer of the

protein identified. Table 4.4 shows a list of the proteins that were identified by mass

spectrometry of the anti-iso[4]LGE2 pAbs and the anti-HNE pAbs immunoreactive spots

on the 2D PAGE (Figure 4.10).

α-Actinin is a homodimer involved in the crosslinking of actins, which is

important in the formation of actin filaments.90 Actinin appears at ~52 kDa (spots 11, 36,

Figure 4.10) that corresponds to the monomer. The list includes a cysteine protease

(cathepsin k) that cleaves collagens I and II in bone and has been implicated in diseases

such as osteoporosis91 and amyloidosis.92 Several of the ECM proteins were also

144

identified to be crosslinked by these lipid oxidation products. Crosslinking of proteins

can affect the mobility and functions of the proteins.

Table 4.4: List of putatively crosslinked proteins identified by 2D PAGE and mass

spectrometric analyses of glaucomatous TM.

Actual Observed ImmunoreactiveProtein Identified MW

(kDa) pI MW (kDa) pI isoLGE HNE

Peptide match

Accession No.

cathepsin K 37 8.8 98 7.3 √ 1 P43235

Keratocan 40 7.2 93 8.4 √ 2 O62702

Lumican 38 6.5 92, 67 - √ √ 2 P51884

prolargin 44 9.6 100-45 - √ √ 5 P51888

Lumican is a kertansulphate proteoglycan found mostly in the sclera and some

connective tissues involved in modulating collagen fibril formation. The 2D shows the

presence of lumican crosslinked as a high molecular weight species (putative dimer and

trimer) in the 2D PAGE. Crosslinking of this protein could result in malformed collagen,

a key structural protein in the TM.93 Prolargin, a proline-arginine-rich end leucine-rich

repeat protein, anchors basement membranes to the connective tissue.94 Prolargin also

appears as a high molecular weight species (putative dimer) (Table 4.4). Earlier reports of

the protein crosslinking ability of iso[4]LGE2 and HNE,30,95 are in accord with these

results.

As the TM isolated from the corneal rim region or by trabeculectomy is likely to

have some of the surrounding corneal, scleral or iris tissue, the proteins identified cannot

145

be unambiguously attributed to the TM region. To localize the modified proteins,

immunohistochemical analysis was performed in the TM region using the iso[4]LGE2

and HNE antibodies. There is an increased amount of staining in the TM region of the

glaucomatous TM sections for the lipid specific antibodies compared to that of the

preimmune IgG treated controls (Figure 4.7, Figure 4.8). Preimmune staining of the

sections eliminated the presence of any non-specific binding artifacts.

We postulate that oxidative modifications, including covalent crosslinks,

accumulate in TM and contribute to aqueous outflow blockage and onset of POAG. The

trabecular meshwork environment is conducive to oxidative damage especially with

age.16,17 The risk of glaucoma increases with age, with POAG at age 80 being 5 to 10

times more prevalent than at age 40.96,97 The body combats free radicals and other

reactive oxidants through a highly efficient and adaptive antioxidant system (enzymatic

and non-enzymatic). It has been shown that there is a decrease in the levels of GSH 98,

while no difference in the activity of MPO and catalase in plasma, whilst a big difference

in the MDA derived protein modifications72 is present in POAG compared to that of

controls. Decrease in the activity of proteasome function and GSH levels, and increase in

lipid peroxides 99 and a stress response mediated by ELAM-1100 have been observed in

cultured TM cells under stress. TM cells have been shown to be able to synthesize a

specific set of proteins that may act as molecular chaperones to prevent oxidative or heat

shock damage.16 In addition to these reports, our findings suggest an oxidative damage

component in glaucoma pathogenesis, and raise many additional questions about the role

of lipid peroxidation in glaucoma. Is it the increase of lipid oxidation-regulating machine

146

that is affected or is it the cellular degradation machinery that is impaired during the

process of aging?

Our findings might thus represent identification of an oxidative stress response

component in glaucoma. It is important, now, to address this question directly by testing

the efficacy of glaucoma therapies that target antioxidant systems. Potentially

encouraging is the finding that antioxidant therapy can be beneficial in certain forms of

ARMD and cataract.11 However, enthusiasm for this possibility must be tempered by the

report that antioxidant therapy is not effective in age-related cataract, a disease also

linked to oxidative stress and iron abnormalities.101

147

4.4. Experimental Procedures.

4.4.1. General methods. All chemicals used were of high purity analytical grade. The

following commercially available materials were used as received: bovine serum albumin

(BSA, fraction V, 96-99%) and human serum albumin (HSA, fraction V) were from

Sigma (St. Louis, MO); goat anti-rabbit IgG-alkaline phos¬phatase (Boehringer-

Mannheim); p-(N,N-dimethylamino) benzaldehyde (DMAB, Aldrich, WI). Spectrapor

membrane tubing (Mr cutoff 14,000 No. 2) for dialysis was obtained from Fisher

Scientific Co. Phosphate buffered saline (PBS) was prepared from a pH 7.4 stock

solution containing 0.2 M NaH2PO4/Na2HPO4, 3.0 M NaCl, and 0.02% NaN3 (w/w).

This solution was diluted to 10 mM or as needed. ON-KLH antibodies102 and

iso[4]LGE2-KLH antibodies6 were prepared as described previously. Iso[4]LGE2 was

prepared by Jim Laird and ON-BSA was prepared by Liang Lu in our laboratory. The

iso[4]LGE2 –HSA, iso[4]LGE2-BSA103 and ON/PPC-HSA104 were prepared as described

previously. The monoclonal anti-argpyrimidine antibody was obtained as gift from Dr.

Kochi Uchida (Nagoya University, Japan) and MG-BSA56 was obtained from Dr. Ram H.

Nagaraj (Case Western Reserve University, USA).

4.4.2. Tissue procurement. Human eyes from 52 normal donors and 52 POAG donors,

all between the ages of 43 and 95 years of age, used in this study were obtained through

the Cleveland Eye Bank and the Cleveland Clinic Foundation (Clinical practices of Drs.

Edward Rockwood and Scott Smith). Eyes were enucleated within 10 h of death and

stored at -80 ºC until TM tissue was isolated by dissection. Control eyes were from

donors with no visual field defects, no evidence of glaucoma and without any disease of

148

the central nervous system. Fixed human TM tissues used for immunohistochemistry

were obtained from the National Disease Research Interchange (NDRI, Philadelphia,

PA). Glaucomatous eyes and tissues were from clinically documented POAG donors.

Glaucomatous TM tissues (~1-2 mm3) were obtained by trabeculectomy of POAG

patients in the Cole Eye Institute, of the Cleveland Clinic Foundation with institutional

review board approval. Human tissues obtained by trabeculectomy were predominantly

TM but possible contamination with small amounts of surrounding tissue (e.g., sclera)

cannot be excluded.

4.4.3. Protein extraction. TM from cadaver and trabeculectomy samples was extracted

by homogenization in 100 mM Tris-Cl buffer pH 7.8 containing 5 mM dithiothreitol,

1mM SnCl2, 50 mM NaHPO4, 1mM diethylenetriaminepentaacetic acid, 100 mM

butylated hydroxy toluene and 0.5% SDS. Insoluble material was removed by

centrifugation (8000g for 5 min). Soluble protein was quantified by the Bradford assay 105

using amino acid quantified BSA as standard, yielding ~15-20 µg total soluble protein

per trabeculectomy tissue sample (~1-2 mm3). Protein extracts were subjected to SDS-

PAGE on 10% gels (Bio-Rad Laboratories, Hercules, CA) and the gels were used either

for Western analyses or for mass spectrometric proteomic analyses.12,82 The soluble

protein was subjected to SDS-PAGE or kept at -80 ºC until analyzed.

4.4.4. Western analysis. Electrophoresis and Western blotting were carried out

according to published protocols.12,82 Protein extracts (~ 10-15 µg) from TM tissue were

suspended in Laemmli sample buffer,82 fractionated on a 10% SDS PAGE, electro blotted

149

on to polyvinyl difluoride (PVDF) membrane (Millipore, Bedford, MA) and probed using

various antibodies: rabbit polyclonal anti-HNE,41,102 rabbit polyclonal anti-iso[4]LGE2 ,33

mouse monoclonal anti-methylglyoxal (argpyrimidine)56 and anti-hydroxykynurenine

mAb.59 As the amount of protein extracted from TM was very low, equal loading was

verified by Coomassie blue stained gels concomitantly with Bradford assay.105 The

immunoreactive bands were quantified using GS-710 scanner and QuantityOne ®

software from Biorad™ (Hercules, CA).Quantification of the Western blot were

performed in two steps. Firstly, the Western blots were normalized based on the

Coomassie blue stained gels to ensure the incorporation of variations in the protein

amounts. Secondly, normalization was performed between Western blots to exclude any

variations between the blots.

4.4.5. Histochemical analysis. Immunohistochemical analyses to localize modified

proteins in ocular tissue were performed with cadaver eyes enucleated within six hours of

death and fixed immediately with calcium acetate buffered 4% para-formaldehyde.

Paraffin embedded tissue was blocked and sectioned (10 µm). The sections were serially

incubated in 1% BSA in phosphate buffered saline pH 7.5, then with 10-100 ng antibody

(rabbit polyclonal anti-(HNE) 41 or rabbit polyclonal anti-Iso[4]LGE2 33) overnight at 4

°C. The sections were washed with a solution of 1% BSA in PBS (3 x 5 min) and

subsequently incubated with 10 ng secondary antibody conjugated with Alexa 594 or

Alexa 488 (Molecular Probes Inc, Eugene, OR) for one hour at room temperature.

Sections were washed with 1% BSA in PBS as mentioned above and sealed with

Vectashield® and analyzed with a Nikon EFD-3 fluorescence microscope attached to a

150

CCD camera. For localization of TM, slides were stained with hematoxylin by serially

incubating in staining solution for 3 min, tap water for 5 min and finally in distilled water

for 5 min. The slides were then dried in air and visualized by light microscopy. The

microscopic images were assembled in Adobe® Photoshop® CS.

4.4.6. Immunoprecipitation. Immunoprecipitation required protein extracts without any

SDS as this might hinder the antibody antigen interaction. Protein was extracted using

100 mM Tris-Cl buffer pH 7.5, 50 mM NaCl and 0.01% genapol unlike the methods

mentioned above. Antibody-coupled protein A beads were used for all

immunoprecipitations. About 100 µg of swelled protein A sepharose CL-4B beads

(Amersham Pharmacia Biotech, CA) was coupled with 100 µg antibody using 4 x 25 mg

dimethylpimelimidate (DMP), each with 2 h incubation at room temperature in 50 mM

sodium borate buffer pH 8.3. During each of the washes, the beads were centrifuged at

2000 rpm for 2 min and the supernatant carefully drained. Following incubation, the

beads were washed with 200 mM ethanolamine pH 8.0 and incubated in the same

solution for 2 h. Beads (100 µg antibody-coupled beads) were finally washed in

phosphate buffered saline pH 7.4 and incubated with protein extracts (~30 µg) prepared

in 100 mM Tris-Cl buffer pH 7.5, 50 mM NaCl and 0.01% genapol. After incubation, the

beads were recovered by centrifugation at 2500 g for 5 minutes and washed 3 x 500 µL

of 100 mM Tris-Cl buffer pH 7.5, 100 mM NaCl and 0.02% genapol. The beads were

boiled with 5 µL Laemmli buffer82 for 2 minutes and separated on a 10% SDS-PAGE.

The gels were either subjected to Coomassie staining and/or subsequent LC MS/MS of

gel bands or Western analyses.

151

4.4.7. 2D Gel electrophoresis. Protein extract from a dissected TM of a 79 year old

Caucasian male, extracted using solution B (7 M urea, 4% Chaps, 2 M thiourea, and 0.5%

Triton-X), was used for the two dimensional electrophoresis. Protein extract (~80 µg)

was transferred to 7 cm IPGphore coffins (Amersham Pharmacia Biosciences,

Piscataway, NJ) and the volume adjusted to 300 µL with additional detergent solution. A

7 cm precast ProteomIQ™ immobilized non-linear pH gradient strip gel (IPG, pH 3-10

NL) was placed in the coffin and overlaid with 600 µL of mineral oil. First dimension

IEF was performed using the Pharmacia IPGphor and the following programmed voltage

gradient; IPG strips were rehydrated with sample at 30 V/6 h; then 300V/1 min; followed

by a linear increase to 3500 V/8 h, held at 3500 V/1 h; then at 8000 V/21 h to reach

approximately 80 kVh. For the second dimension, the IPG strip was reduced in a buffer

(50 mM TrisCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS) containing 1% dithiothreitol

(DTT) for 12 min and followed by alkylation using 0.25% iodoacetamide with a trace of

bromophenol as described earlier.71 The equilibrated IPG strip was embedded in 0.7%

w/v agarose on the top of a 10% acrylamide gel containing a 4% stacking gel. Second

dimension SDS-PAGE (2h at 80 V constant voltage) was performed with Bio-rad

electrophoresis equipment. The gel was stained using GelCode Blue® (Pierce) and

scanned with a GS-710 Imaging Densitometer™ (BioRad). The coordinates of spots on

the 2D image was digitalized using Origin® 7.5 software assuming a linear scale between

the least measurable units shown (both pI and molecular weight).

152

4.4.8. LC MS/MS analysis and protein identification. Following the SDS PAGE, the

Gelcode Blue® stained gel bands were excised in 1mm3 slices and destained according to

standard protocol for in-gel digestion.12,82 The tryptic digested proteins were analyzed by

LC-MS/MS using a quadrupole-time of flight (QTof2) mass spectrometer equipped with

a CapLC system (Waters, Millford, MA), ProteinLynx Global Server acquisition and

processing software. Peptide digests were trapped on a precolumn (0.3 x 1mm, 5µC18,

LC Packing) with 0.1% formic acid in 2% acetonitrile as loading solvent then eluted onto

a capillary column (PicoFrit 0.050 x 50mm, 5µ tip ID; New Objective Inc., Woburn,

MA). Chromatography was performed with a flow of 250 nL/min with aqueous

acetonitrile/formic acid solvents and 100% of the eluant was directed towards the mass

spectrometry. The mass spectrometer was operated in standard MS/MS switching mode

with the three most intense ions in each survey scan subjected to MS/MS analysis.

Protein identifications from MS/MS data utilized ProteinLynxTM Global Server (Waters

Corporation) and Mascot (Matrix Science) search engines and the Swiss-Protein and

NCBI protein sequence databases.12,82

153

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Chapter 5

Iso[4]LGE2 Modified Proteins in Trabecular Meshwork of Glaucomatous DBA/2J

Mice

159

5.1. Background

Glaucoma, affects 70 million people worldwide,1 and in one of the leading causes

of irreversible blindness. Glaucoma refers to a group of eye diseases that progressively

steal sight, mostly without any initial symptoms. Primary open angle glaucoma (POAG)

is a form of glaucoma in which there usually is an increase in the intraocular pressure

(IOP), except normal pressure glaucoma, leading to progressive optic nerve damage and

vision loss. Secondary glaucomas may result from an injury, inflammation or tumor2 and

may also occur in advanced cases of cataract, diabetes or as a result of the use of

steroids.3

Increased IOP is a major risk factor for glaucoma. The insidious nature of

pressure-induced changes in the glaucomatous eye makes it difficult to study in humans.

Also, as the disease condition occurs in elderly patients, it is additionally confounded by

other medical complications.4 Several animal models of glaucomas have been developed

that have broadened our understanding of the disease. The diversity from species to

species with regard to differences in the anatomical features, especially, the outflow

angle, has substantially broadened the view on our understanding of anterior segment

diseases, e.g., glaucoma and cataract.4 A detailed discussion of these animal models for

glaucoma is presented in Chapter 1.

5.1.1 Oxidative stress and inflammation pathways. The anterior segment is constantly

exposed to oxidative stress due to the formation of reactive oxygen species (ROS) by

light catalyzed reactions. The increase in ROS can trigger formation of lipid and

160

carbohydrate-derived reactive fragments, leading to oxidative protein modifications,5 and

accumulation of these modified proteins may be pathological.

Lipid Peroxides

NF-кB

IL-18

ROS TNF-α

COX-2LO

Oxidative Stress

Prostaglandins, levuglandins and other lipid peroxidation products

Inflammation

Free radical pathway

Enzyme pathway

Lipid Peroxides

NF-кB

IL-18

ROS TNF-α

COX-2LO

Oxidative Stress

Prostaglandins, levuglandins and other lipid peroxidation products

Inflammation

Free radical pathway

Enzyme pathway

Scheme 5.1. Diagrammatic representation of activation and expression of proteins

involved in inflammation pathway by oxidative stress. Details of the scheme are

discussed in the text. (ROS – reactive oxygen species, COX-2 – cyclooxygenase-2, LO –

lipooxygenase, NF-кB – nuclear factor- kappa B, IL-18 – interleukin 18, TNF – tumor

necrosis factor).

Formation of ROS participate in a positive feedback loop that activates and/or

promotes expression of proteins that are involved inflammatory processes, and which

inturn may be involved in generation of reactive oxygen species (Scheme 5.1). NF-κB, an

161

oxidative stress-responsive transcription factor, is known to be activated, by several

factors including oxidative stress (H2O26,7 or by lipid peroxidation8), to express

proinflammatory cytokines.6 For example, the proinflammatory cytokine TNF-α, may be

induced by oxidative stress directly or through NF-κB.7,9 TNF-α can, in turn, induce

several cascades of activation or inhibition of pathways that are involved in

inflammation, immunity, and cell death.10-12 Interleukin-18 (IL-18), a regulator of innate

and acquired immunity, is involved in a variety of pathways including the release of

cytokines, activation of NF-κB and upregulation of cyclooxygenase-2.13 There are reports

of NF-κB regulating the expression of IL-18.14

5.1.2. Different forms of secondary glaucoma. There are several forms of secondary

glaucoma viz, pigmentary dispersion, traumatic, pseudoexfoliation and neovascular

glaucoma. Pigmentary glaucoma is caused by release of pigments present in the inner

portion of the iris, which get deposited near the trabecular meshwork (TM) region

causing increased resistance to aqueous outflow. Traumatic glaucoma, as the name

suggests, is caused by an injury that has traumatic impact on the eye, causing an increase

in the IOP and causing optic nerve damage. In the case of the pseudoexfoliation

syndrome, thin flaky dandruff-like material, peeling off from the lens, clogs the outflow

facility and causes the IOP to increase. Neovascular glaucoma occurs due to the

formation of blood vessels on the iris and over the drainage canals of the anterior

chamber of the eye (www.glaucoma.org/ learn). There are a number of factors that are

attributed to be bases for the optic nerve degeneration in glaucoma viz, genetic

predisposition,15-19 involvement of receptors and transporters for glutamate,20,21 nitric

162

oxide19 or γ-synuclein22 and of heat shock proteins23 and retinal autoantibodies.24,25

However, detailed molecular mechanisms involved in ganglion cell death are still poorly

understood.26

Pigmentary glaucoma, caused by pigment dispersion syndrome, is one of the

common forms of secondary glaucoma affecting about 0.22 million Americans.13 This is

an open angle form of secondary glaucoma that presents a heavily pigmented TM

towards the end stages. Although use of drugs that lower the IOP of the eye help in

slowing the progression of the disease, but the molecular and biochemical mechanisms

underlying the disease have yet to be elucidated.27

5.1.3. DBA/2J, mouse model for glaucoma. The DBA/2J mouse model for glaucoma

presents several hallmarks of the human pigmentary glaucoma including iris stromal

atrophy (ISA), pigment dispersion (IPD), retinal ganglion loss and optic nerve

cupping.28,29 Iridial pigment dispersal in DBA/2J mice is first clinically evident at around

6-9 months. By 9–10 months iris depigmentation is pronounced and occurs with

increased IOP, resulting in anterior chamber enlargement and glaucoma.28 The

depigmenting iris disease of DBA/2J mice is genetically separable into two distinct

phenotypes, IPD and ISA, which appear to be caused by mutations in the Gpnmb and

Tyrp1 genes respectively. The two missense mutations (Cys110Tyr, Arg326His) in the

tyrosinase related protein-1(TYRP1) gene cause disrupted melanosomes and clumping of

pigments, and a mutation on the stop codon of Gpnmb (Arg150X) causes iris pigmentary

dispersion.30,31 These mutations are suggested to be involved in the leakage of toxic

intermediates of pigment production from the melanosomes causing ISA and IPD.

163

However, the hypopigmented DBA/2J mouse strain does not exhibit iris pigment

dispersion, iris stromal atrophy or glaucoma despite homozygosity for the Gpnmb and

Tyrp1 mutations. Hence, all the factors contributing to elevated IOP in DBA/2J mice

remain to be identified. Nevertheless, DBA/2J mice provide a valuable resource for

defining factors that may contribute to human glaucoma.

Recently, Zhou et. al.13 showed that there is a considerable increase in the

expression of IL-18 mRNA in the iris/ciliary body, and a consequent increase in IL-18

levels in the aqueous humor, of DBA/2J mice with age. This study further showed that

there are increases in the amounts of activated NF-κB and caspase-3. Additionally, they

noted elevation in the expression of matrix metallo-proteinase-1 (MMP-1) and apoptosis

related genes. MMP’s are known to be involved in the degradation of extracellular matrix

proteins in response to cytokines and inflammatory mediators.32,33 Thus, this study

suggests an involvement of inflammation, degradation and apoptosis in the iris/ciliary

body of the DBA/2J mouse model of glaucoma.

Since, oxidative stress plays a role in the regulation of factors involved in the

inflammation process (Scheme 5.1), the study of their levels in the TM of DBA/2J mice

may be important for understanding the changes occurring in the outflow pathway. In this

chapter, the levels of oxidative stress in the TM of DBA/2J mice will be assessed based

on the levels of isolevuglandin-modified proteins.

5.1.4. Iso[4]Levuglandin E2 – formation and pathology. Previously, Dr. Salomon’s lab

discovered that spontaneous rearrangements of prostaglandin endoperoxides generate

levulinaldehyde derivatives, with prostaglandin side chains appended at the carbons α

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and β to the aldehyde group.34,35 Levuglandins (Scheme 4.1, page 163),36,37 seco

prostanoic acids, are co-generated with prostaglandins (PGs)38,39 by rearrangements of

the endoperoxide PGH2, which occur readily under the free radical-induced and/or

cyclooxygenase -promoted biosynthetic pathway from arachidonic acid (AA).36,40

C5H11

(CH2)3COOH

C5H11

(CH2)3COOHOO

OH

C5H11

(CH2)3COOH

OHCOH

O

Levuglandin E2

Cyclooxygenase

Phospholipase A2

NProtein

NProtein

X

O

Pyrrole Adduct

Lactam (X = H) Hydroxylactam (X = OH)

C5H11

(CH2)3COOPC

OO

(CH2)3COOPC

OH

C5H11

OHC

(CH2)3COOR

OH

C5H11

O

AA-PC

Iso[4] levuglandin E2

Free radical induced

R= H or PC

Other PG's

Scheme 5.2: Generation of levuglandins from arachidonic acid by enzyme mediated and

free radical mechanisms (Detailed scheme presented in Scheme 4.1, page 163).

Iso[4]LGE2 is formed by a free-radical pathway unlike its structural isomer LGE2,

which can be formed through either a free radical or enzyme mediated pathway. These

165

reactive aldehydes bind avidly with proteins forming a protein-bound pyrrole, e.g.,

iso[4]LGE2-pyrrole, as well as protein-protein41,42 and DNA-protein crosslinks.43 We

have also reported mass spectral characterization of several lysine-based modifications

that are generated by covalent adduction of levuglandins with proteins.41 Levels of

levuglandin-protein adducts are markedly elevated in the blood of atherosclerosis and end

stage renal disease patients versus healthy controls.44,45

These highly reactive oxidized lipids, the LGs and isoLGs, form protein

aggregates by cross linking proteins at a very high rate compared to other lipid

peroxidation products viz, HNE and MDA.46 This suggests that they might play a

prominent role in causing oxidative injury. These adducts as well as free LGs have been

shown to hinder the activity of the 20S proteasome, a component of cytosolic protein

degradation machinery, in a dose dependent manner.47 The formation of stable adducts

by LGs and their localization in the TM reflects the localized oxidative stress at TM.

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5.2. Results

5.2.1. Clinical examination of DBA/2J mice. Examinations of the DBA/2J mice were

performed, as described earlier using a slit lamp, visually as well as by sectioning the eye

post-mortem.28 The sectioning of the eyes was performed transversely for assessing the

TM. The optic nerve was assessed by analyzing both sagital and transverse sections.

Sagital sections pass through the longitudinal axis of the eye from anterior to the

posterior regions, while the transverse sections are perpendicular to the sagital sections

(Figure 5.1). Haematoxylin stained sections were used as reference to locate the TM

region.

Figure 5.1: Diagrammatic illustration of sagittal and transverse sections of the eye. The

sagital section passes through the longitudinal axis of the eye from the anterior to the

posterior region. The transverse section passes perpendicularly axis to the sagittal axis.

Pigment dispersion. DBA/2J mice develop a form of glaucoma with similarities to human

pigmentary dispersion syndrome. A visual inspection of the ~ 6-month-old DBA/2J

mouse eye clearly showed the signs of degenerating iris with pigment dispersed across

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the pupil (Figure 5.2A). By 8-9 months, iris depigmentation is pronouncedly increased

(Figure 5.2C) and elevated IOP has developed, resulting in anterior chamber enlargement

and glaucoma.

A B CA B C

Figure 5.2. Anterior segment assessment of DBA/2J mice. The anterior segment pictures

were taken with a Leica MZ stereomicroscope (Leica Microsystems Inc., Bannockburn,

IL) equipped with a SpotCam RT KE digital camera (Sterling Heights, MI). A. 6-month-

old DBA/2J mouse, B. 8-month-old and C. 14-month-old. Arrows show the iris

degeneration (A), heavy prigmentation (B) and complete iris degeneration and

depigmentation (C).

Optic nerve damage. The optic nerve sections of 8-month DBA/2J and C57BL6 mice

were performed using both sagital as well as transverse sections. The sections were

stained with haematoxylin stain. The longitudinal section shows the optic cupping

feature, reflecting the optic nerve excavations (arrow in Figure 5.3A), which is a feature

of the optic nerve degeneration compared with the normal optic nerve in Figure 5.3B.

The cross sections across the optic nerve show a distinct difference in the myelination of

the nerve fibres, a characteristic of optic neuropathy. Figure 5.3C shows disorganized

nerve fibres (arrows) compared to the normal control in Figure 5.3D.

168

DBA/2J C57BL6

A B

C D

DBA/2J C57BL6

A B

C D

DBA/2J C57BL6

A B

C D

Figure 5.3. Histochemical assessment of mouse optic nerve. The mouse eye sections (10

µm) passing though optic nerve were stained with hematoxylin. A. Sagittal section of 8

months old DBA/2J optic nerve (x 20) and C. a transverse section of DBA/2J optic nerve

(x 100) B. Sagittal section of age matched C57BL6 control optic nerve (x 20) and D. a

transverse section of age matched C57BL6 control optic nerve (x 100). Arrows denote

the optic nerve excavations (A) and demylenation of the nerve fibres (C).

5.2.2. Increase in levels of iso[4]LGE2 modified proteins in DBA/2J with age. At age

8-10 weeks, elevated IOP is typically exhibited by DBA/2J mice in contrast to C57BL6

controls. The TM of DBA/2J mice was isolated surgically and the proteins were extracted

immediately.

169

A

B

2.5

mon

ths

8 m

onth

sD

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2J

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2

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mnt

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h

8 m

nth

Rel

ativ

e in

tens

ity (%

)

C57BL6

DBA/2J

CA

B

2.5

mon

ths

8 m

onth

sD

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2J

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BL6

2.5

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0

10

20

30

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50

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mnt

h

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nth

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h

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nth

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DBA/2J

C

Figure 5.4. Western analysis of DBA/2J TM proteins with iso[4]LGE2 antibodies. TM

proteins (~5 µg/lane) were subjected to 1D SDS/PAGE, electroblotted onto a

poly(vinylidene difluoride) membrane, and probed with anti-iso[4]LGE2 antibodies.

Bovine serum albumin modified with iso[4]LGE2 was used as a positive control. A.

Immunoblot of DBA/2J and C57BL6 TM proteins. B. Coomassie stained immunoblot.C.

Densitometric quantification of the Western blot (n=1), see experimental section p. 182

for details.

170

As described previously for our studies of oxidative stress in human TM (chapter

4), we used iso[4]LGE2-protein adduct as a marker for oxidative stress. We probed the

TM extract of DBA/2J mice for iso[4]LGE2-derived protein modifications by Western

analyses using anti-iso[4]LGE2 polyclonal antibodies (pAb). The immunoblots showed

the presence of background levels of some modified proteins at 3 weeks to 5 months old

DBA/2J mice and in control mice of comparable ages. Nevertheless, there is a significant

difference in the number and intensity of immunoreactive bands at 8 months in TM of

DBA/2J compared to controls (Figure 5.4). Subsequent time course analyses in DBA/2J

revealed the presence of iso[4]LGE2 modification prominently at 8 weeks of age (see

Appendix). There is intense staining for iso[4]LGE2 modification in the high molecular

weight region of the SDS-PAGE.

5.2.3. Immunohistochemical localization of iso[4]LGE2 modified proteins. To

visualize the localization of iso[4]LGE2 modified proteins in the limbus region of the

DBA/2J mice, a transverse section of the anterior segment was analyzed. The anti-

iso[4]LGE2 antibody stained section showed the presence of immunoreactivity in the TM

in 6 out of 8 slides analyzed (4 DBA/2J and 4 C57BL6 mice were used), while

comparable sections of age-matched C57BL6 mice showed much less staining.

Additionally, serial sections of the slides mentioned above were also stained with pre-

immune antibodies, that served as controls. The green channel was used to detect the

interfering autofluorescence of the sections. Nuclear staining was performed using 4',6-

diamidino-2-phenylindole (DAPI).

171

Iso[4]LGE2 Pre-Immune

DBA/2J C57BL6

Iso[4]LGE2

A

C D

E

F

B

Iso[4]LGE2 Pre-Immune

DBA/2J C57BL6

Iso[4]LGE2

A

C D

E

F

B

Figure 5.5. Histochemical localization of isoLGE2 modified proteins in TM.

Representative immunohistochemical analyses with a rabbit polyclonal anti-isoLGE2 are

shown. Rhodamine conjugated secondary antibody with red fluorescence (Alexa 594)

was used for detection of iso[4]LGE2-modified proteins. A. DBA/2J mice TM, 8 months

(green channel) B. DBA/2J mice TM, 8 months(red channel) C. C57BL6 mice TM,

8months (green channel). D. C57BL6 mice TM, 8 months (red channel). Red channel –

immunofluorescence of iso[4]LGE2 modified proteins; green channel – autofluorescence.

5.2.4. Immunoprecipitation of iso[4]LGE2-modified proteins. To identify the proteins

that were modified by iso[4]LGE2, protein extracts from the TM were immunoprecipitat-

ed using rabbit anti-iso[4]LGE2-pAb linked to protein A sepharose beads. The beads

were prepared using protocols described in Chapter 4. Each of the IP experiments utilized

172

protein extracts from TM of both eyes from one DBA/2J mouse. The Coomassie blue

stained gels showed the protein bands that were immunoprecipitated as well as the long

chain of the IgGs. As mentioned in Chapter 4, The bands detected at ~55 kDa that is

common to both the lane 2 and 4 (shown with dotted lines in Figure 5.6) corresponds to

the long chain of IgG originating from the cleavage of IgG chains from the antibody-

coupled beads as reported previously.48

11898

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IgG

Lane 1 2 3 4 5

11

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IgG

Lane 1 2 3 4 5

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Figure 5.6: Anti-iso[4]LGE2 immunoprecipitation of TM proteins from an 8 month old

DBA/2J mouse. Immunoprecipitations (IPs) utilized antibody coupled beads (10 µg) and

TM protein extracts (10 µg). They were analyzed by SDS-PAGE and mass spectrometry.

A Coomassie blue stained gel is shown: lane 1, molecular weight markers; lane 2,

antibody coupled beads without TM; lane 3, TM protein extract (5 µg); lane 4, anti-

iso[4]LGE2 IP products; lane 5, supernatant containing unbound proteins that did not

immunoprecipiate. Bands corresponding to proteins excised and identified in Table 5.1

are numbered 1-6.

173

5.2.5. Identification of modified proteins using LC-MS/MS. Identification of the

immunoprecipitation products was performed as described earlier.

Table 5.1: Proteins immunoprecipitated from TM extract of DBA/2J mice using

iso[4]LGE2-pAb. Gel slices from top to the bottom of lane 4 of the gel in Figure 5.6 were

digested using trypsin and the proteins identified using LC-MS/MS and MassLynx™

software with the Swissprot database.

Proteins Lysine

Content(%)

BandNo.

Peptides Matched

Calculated1 MW(kDa)

Observed MW(kDa) AccessionNo.

Actin 5.0 3 1 42 120 P68134

Calcium/calmodulin-dependent

phosphodiesterase 6 5 1 64 67 Q61481

Collagen alpha 1(I) 3.8 1 1 137 ≥2204 P11087

F-box/LRR-repeat protein 20 4.4 1 1 48 ≥2204 Q9CZV8

GFAP2 4.7 1 1 50 ≥2204 P03995

Hemoglobin 7.8 1 4 15 ≥2204 P01942

Histone H1 27 1,5 1 21 67, ≥2204 P15864

Histone H2 10 1,5 1 14 67, ≥2204 P22752

Keratin, cytoskeletal 3.7 6 5 58 65 P02535

Liprin-beta 2 6.8 4 1 98 98 O35711 PI4K3 5.2 2 1 90 170 Q8BKC8

Serum albumin 8.4 5 1 68 68 P07724

Alpha-tubulin 1 4.2 2,5 2 50 170 P68369

Enamelin precursor 4.0 3 1 128 120 O97939

1 For monomeric protein 2GFAP – Glial acidic fibrillary protein; 3PI4K - Phosphatidylinositol 4-kinase; 4

High molecular weight bands at the top of the gel

174

The proteins identified included extracellular matrix proteins as well as some proteins

from blood. There were fewer proteins detected from IP of DBA/2J TM extracts than

from IP of human TM proteins probably due to the lesser amount of IP proteins in mouse

TM than from the human TM. However, a lack in overlap between the proteins identified

from DBA/2J and human was not completely understood. The observed molecular weight

is an estimate based on SDS PAGE and the molecular weight maker positions using the

Digitizer® function of the program Origin® version 7.5.

175

5.3. Discussion

The importance of oxidative stress as a contributing factor in the progression of

glaucoma has been previously hypothesized. This study was directed towards

understanding the pathological consequences of oxidative stress in glaucoma.49 Animal

models to study the basic aspects of the disease have benefits, both in terms of time and

effort. DBA/2J has been well established as mouse model of glaucoma with a well-

defined period of increase in the intraocular pressure followed by irreversible damage to

the optic nerve. Herein we sought to determine whether there is an increase in the levels

of oxidative damage in the DBA/2J mouse TM concurrently with damage of the optic

nerve.

Western analyses of the TM extracts from DBA/2J mice at different ages

compared to age matched control C57BL6 mice using iso[4]LGE2 antibodies shows a

distinct increase in the amount of immunoreactive bands in older DBA/2J mice. Also, of

note, a number of immunoreactive bands appear in the higher molecular weight region of

the SDS PAGE. This indicates the presence of cross-linked proteins. Since the amount of

protein in the higher molecular weight region of the Coomassie stained gel appeared to be

very low (Figure 5.4), we concluded that the identification of proteins from this 1D

PAGE would not be easy. So, to concentrate and characterize the iso[4]LGE2-modified

protein, we performed immunoprecipitation with isoLGE2 antibody coupled protein A

sepharose beads. Iso[4]LGE2 immunoprecipitation of the DBA/2J mouse TM extract

enriched some proteins compared to that of the total protein extract (Figure 5.6). A lack

of iso[4]LGE2-modified proteins was demonstrated in C57BL6 and the putative

iso[4]LGE2-modified proteins were not immunoprecipitated with preimmune rabbit

176

serum (see Appendix 5.1S). The immunoprecipitated proteins from DBA/2J mice (n=5)

examined were identified by using LC-MS/MS and are listed in the Table 5.1.

Because they appear as oligomers on a denaturing PAGE gel (Figure 5.4 and

Table 5.1), many of the proteins from the IP may be crosslinked. Some of the

immunoprecipitated proteins that were identified are actin, collagen I, liprin, keratin

(cytoskeletal) and tubulin, which are involved in the structural organization of the TM.

The structural protein actin (band 3, Figure 5.4) appears at ~120 kDa, while it’s

molecular weight is 42 kDa. This implies that it may be crosslinked with itself or other

proteins. Collagen I, is also a structural protein present in TM. There are reports that the

levels of collagen I increase with hydrostatic pressure in cell cultures,50 a model of

increased intraocular pressure (IOP). It is tempting to speculate that the increased IOP

associated with glaucomas promote lipid oxidation and consequent protein

oligomerization and accumulation. Both Liprin-β2, which functions as a cytoskeletal

anchoring protein, and GFAP, a class-III intermediate filament in astrocytes,51 appear to

be modified. GFAP appears to be extensively oligomerized.

Modification of nuclear proteins may have profound effects on the molecular

processes that are involved in cell division and cell differentiation. Tubulin is a major

constituent of microtubules that is involved in mitosis. Dr. Salomon’s lab has previously

shown that this protein can be crosslinked, by exogenous LGE2, and LGE2 also inhibits

microtubule assembly in vitro and cell division in vivo.52 In the present mouse model of

glaucoma, we found tubulin to be crosslinked, possibly as a trimer. Histones are nuclear

proteins that are associated with DNA and play an important role in structural

organization of nucleosomes and regulation of transcription. Histones are highly enriched

177

with lysine residues (10-27%), which are targets for LGs. In the IP, histones (H1 and H2)

appear in the very high molecular region (band 1 and 2, Figure 5.6); possibly, due to high

lysine content that makes them prone to extensive crosslinking. Previously, LGs were

shown to crosslink DNA in vivo with protein that is presumably histones.43,53

Another protein that appears to be modified extensively (perhaps oligomerized) is

the F-box protein Fbx2, which binds specifically to proteins attached to N-linked high-

mannose oligosaccharides and subsequently contributes to ubiquitination of N-

glycosylated proteins.54 This implies that this protein is involved in the substrate

recognition of ubiquitin-proteosomal degradation machinery and it’s modification can

possibly lead to cellular instability.55

Phosphoinsitol 4-kinase (PI4K) plays a vital role in cell growth and is expressed

in higher amounts during cell stress.56 The IP also shows that PI4K (band 2, Figure 5.6) is

significantly modified and is perhaps crosslinked as a dimer.

Preliminary results using Western blot analysis using anti-proteasomal antibody

that recognizes the 23 kDa representing proteasome 20S also suggested that the

proteasome is also modified in the TM of 8-month-old DBA/2J mice (Figure 5.7). It

appears as a rather narrow band with an apparent molecular weight of 120 kDa, which

suggests a pentamer. Earlier reports of chronic inhibition of proteasome in TM cultures in

response to oxidative stress has been noted.57

178

2.5

mon

ths

8 m

onth

s

11898

56

37

29

20

2.5

mon

ths

8 m

onth

s

Iso[4

]LGE 2

Prot

easo

me

Western Blots

2.5

mon

ths

8 m

onth

s

11898

56

37

29

20

11898

56

37

29

20

2.5

mon

ths

8 m

onth

s

Iso[4

]LGE 2

Prot

easo

me

Western Blots

Figure 5.7. Proteasome crosslinked in 8-month-old DBA/2J mice. A. Western blot using

iso[4]LGE2 polyclonal antibody (portion of Figure 5.4). B. The immunoblot in Figure

5.4A was probed with anti-rabbit proteasome polyclonal antibody that detects the 23 kDa

representing the proteasome 20S (arrow shows the position of high molecular weight

band immunoreactive for the proteasome antibody).

These studies suggest that: (1) there is an increase in the oxidative stress levels of

the glaucomatous DBA/2J mice and (2) the structural proteins are modified extensively.

The oxidative modifications that we detected are those produced through a free radical

pathway. Further studies are needed to establish whether free radical and enzyme

catalyzed lipid oxidation are important in glaucoma pathogenesis.

179

5.4. Experimental procedures

5.4.1. Tissue procurement. DBA/2J and C57BL/6 control mice were obtained (Jackson

ImmunoResearch Laboratories Inc., West Grove, PA) and bred in the Cole Eye Institute

animal facility. Trabecular meshwork tissues were excised from these mice at different

stages of their lifetime in accordance with the animal procedures approved by CCF

IACUC and stored at -80º C. Whole eyes from these mice were removed and fixed in 4%

formaldehyde solution and embedded using standard protocols for immunohistochemical

analysis.

5.4.2. Protein extraction. The excised TM tissue was incubated on ice in 50 µL of 100

mM Tris-Cl (pH 7.8) containing 8 mM SnCl2, 50 mM NaHPO4, 5 mM DTT

(dithiothreitol), 1mM DTPA (diethlenetriaminepentacetic acid), 100 mM BHT (butylated

hydroxytoluene) and 0.5% SDS (sodium dodecylsulphate) for 30 minutes. The tissue was

homogenized using a hand held homogenizer. The solution was subsequently centrifuged

at 8K rpm for 2 min. The concentration of the soluble protein fraction was determined

using the Bradford method 58 and amino acid quantified BSA (National Bureau of

Standards) as standard. The solution were stored at -80 ºC until analyzed.

5.4.3. Western analysis. Electrophoretic separation of protein extracts (~5 µg) from TM

tissue, in Laemmli sample buffer59, was performed on a 10% SDS PAGE with a 4%

stacking gel using the Bio-Rad Mini-Protein II system following standard protocols.60

The gel was electroblotted onto a polyvinyl difluoride (PVDF) membrane (Millipore,

Bedford, MA), and probed with anti-iso[4]LGE2 polyclonal antibodies (pAb)

180

antibodies.45 To evaluate whether equal amounts of protein were used for Western

analyses, PVDF membranes were stained with Coomassie blue post-transfer. Western

analyses were performed with antibodies to modifications of proteins by iso[4]LGE245

and anti-rabbit proteasome polyclonal antibody. The anti-proteasome polyclonal antibody

that detects the 23 kDa proteasome subunit was obtained from Abcam Ltd (Cambridge,

MA). Iso[4]LGE2 modified BSA was used as positive control. The immunoreactive bands

were quantified using GS-710 Densitometer scanner and QuantityOne ® software from

Biorad™ (Hercules, CA).

5.4.4. Immunohistochemistry. Eyes fixed in 4% paraformaldehyde were cryoprotected

by incubating them sequentially in 10%, 20% and 30% sucrose for 1 h each until the eyes

completely sank. The eyes were then incubated in 30% sucrose in O.C.T. embedding

medium for 15 min. The eyes were quickly embedded into O.C.T. medium on liquid

nitrogen. After the O.C.T. solidified, the eyes were stored in a -20 ºC freezer only to be

removed when ready to section onto slides. The sections (7-10 µm) were then placed onto

microscopic slides. The sections were incubated in phosphate buffered saline (PBS)

solution followed by a solution of 100 mM glycine in PBS (pH 7.4) for 1 h each. The

tissues were then covered with the antibody to be probed at 1:100 dilution in PBS for 1 h.

The tissue was then washed thrice with PBS solution for 10 min each, and then incubated

in 10 ng secondary antibody conjugated with Alexa 594 or Alexa 488 (Molecular Probes

Inc, Eugene, OR) for 1 h. The sections were sealed with Vectashield® Mounting Medium

(H-1000) and analyzed with a Nikon EFD-3 fluorescence microscope attached to a CCD

181

camera. Hematoxylin stained slides were used to locate the TM in the sections. The

microscopic images were assembled in Adobe® Photoshop CS.

5.4.5. Immunoprecipitation. Antibody-coupled protein A beads , prepared using

suitable modifications of standard protocols,61 were used for all immunoprecipitations.

About 100 µg of swelled protein A sepharose CL-4B beads (Amersham Pharmacia

Biotech, CA) was coupled with 100 µg of antibody using 4 x 25 mg

dimethylpimelimidate, each time with a 2 h incubation at room temperature for each

aliquot, in 50 mM sodium borate buffer pH 8.3. Following incubation, the beads were

washed, and then incubated for 2 h with 200 mM ethanolamine pH 8.0. Beads (20 µg

antibody-coupled beads) were finally washed in phosphate buffered saline pH 7.4 and

incubated with protein extracts (~20 µg) prepared in 100 mM Tris-Cl buffer pH 7.5, 50

mM NaCl and 0.01% genapol. After incubation, the beads were recovered by

centrifugation at 2500 x g for 5 minutes and washed 3 x 500 µl of 100 mM Tris-Cl buffer

pH 7.5, 100 mM NaCl and 0.02% genapol. The beads were boiled with 30 µL Laemmli

buffer59 for 2 min, and then separated on a 10% SDS-PAGE. The gels were subjected to

staining with Coomassie stain and subsequent LC MS/MS of in-gel tryptic digested

proteins bands or Western analyses.

5.4.6. LC MS/MS analysis. Following electrophoresis, the gel bands were excised in

1mm slices and destained and processed using standard protocol for in-gel digestion 60 in

a 96-well plate. The in-gel tryptic digested proteins were subjected to LC-MS/MS

analysis using a quadrupole-time of flight (QToF2) mass spectrometer equipped with a

182

CapLC system (Micromass, Milford, MA), ProteinLynx™ Global Server acquisition and

processing software. Peptide digests were trapped on a precolumn (0.3 x 1mm, 5µC18,

LC Packing) with 0.1% formic acid in 2% acetonitrile as loading solvent, and then eluted

onto a capillary column (PicoFrit 0.050 x 50mm, 5µ tip ID, New Objective Inc).

Chromatography was performed with a flow of 250 nl/min with aqueous

acetonitrile/formic acid solvents and 100% of the eluant was directed towards the mass

spectrometry. The mass spectrometer was operated in standard MS/MS switching mode

with the three most intense ions in each survey scan subjected to MS/MS analysis.

183

5.5. References

(1) Quigley, H. A. Br J Ophthalmol 1996, 80, 389-93. (2) http://www.glaucoma.org/learn/; Vol. 2005. (3) Oyster, C. W. The Human Eye Structure and Function; Sinauer Associates, Inc.:

Sunderland, Massachusetts., 1999. (4) Chew, S. In The Glaucomas: Basic Sciences, Anatomy and Pathology; 2 ed.;

Krupin, T., Ed.; Mosby: New York, 1996; Vol. 1. (5) Green, K. Ophthalmic Res 1995, 27 Suppl 1, 143-9. (6) Li, N.; Karin, M. Faseb J 1999, 13, 1137-43. (7) Schreck, R.; Rieber, P.; Baeuerle, P. A. Embo J 1991, 10, 2247-58. (8) Bowie, A. G.; Moynagh, P. N.; O'Neill, L. A. J Biol Chem 1997, 272, 25941-50. (9) Verhasselt, V.; Goldman, M.; Willems, F. Eur J Immun 1998, 28, 3886-3890. (10) Liao, F.; Andalibi, A.; deBeer, F. C.; Fogelman, A. M.; Lusis, A. J. J Clin Invest

1993, 91, 2572-9. (11) Liao, F.; Andalibi, A.; Qiao, J. H.; Allayee, H.; Fogelman, A. M.; Lusis, A. J. J

Clin Invest 1994, 94, 877-84. (12) Finkel, T.; Holbrook, N. J. Nature 2000, 408, 239-47. (13) Zhou, X.; Li, F.; Kong, L.; Tomita, H.; Li, C.; Cao, W. J Biol Chem 2005, 280,

31240-8. (14) Chandrasekar, B.; Colston, J. T.; de la Rosa, S. D.; Rao, P. P.; Freeman, G. L.

Biochem Biophys Res Commun 2003, 303, 1152-8. (15) Mandelkorn, R. M.; Hoffman, M. E.; Olander, K. W.; Zimmerman, T. J.; Harsha,

D. Ophthalmic Paediatr Genet 1985, 6, 325-31. (16) Stone, E. M.; Fingert, J. H.; Alward, W. L. M.; Nguyen, T. D.; Polansky, J. R.;

Sunden, S. L. F.; Nishimura, D.; Clark, A. F.; Nystuen, A.; Nichols, B. E.; Mackey, D. A.; Ritch, R.; Kalenak, J. W.; Craven, E. R.; Sheffield, V. C. Science 1997, 275, 668-670.

(17) Fingert, J.; Heon, E.; Liebmann, J.; Yamamoto, T.; Craig, J.; Rait, J.; Kawase, K.; Hoh, S.; Buys, Y.; Dickinson, J.; Hockey, R.; Williams-Lyn, D.; Trope, G.; Kitazawa, Y.; Ritch, R.; Mackey, D.; Alward, W.; Sheffield, V.; Stone, E. Hum Mol Genet 1999, 8, 899-905.

(18) Stoilov, I. R.; Costa, V. P.; Vasconcellos, J. P.; Melo, M. B.; Betinjane, A. J.; Carani, J. C.; Oltrogge, E. V.; Sarfarazi, M. Invest Ophthalmol Vis Sci 2002, 43, 1820-7.

(19) Vincent, A. L.; Billingsley, G.; Buys, Y.; Levin, A. V.; Priston, M.; Trope, G.; Williams-Lyn, D.; Heon, E. Am J Hum Genet 2002, 70, 448-60.

(20) Liu, B.; Neufeld, A. H. Arch Ophthalmol 2001, 119, 240-5. (21) Lam, T. T.; Abler, A. S.; Kwong, J. M.; Tso, M. O. Invest Ophthalmol Vis Sci

1999, 40, 2391-7. (22) Surgucheva, I.; McMahan, B.; Ahmed, F.; Tomarev, S.; Wax, M. B.; Surguchov,

A. J Neurosci Res 2002, 68, 97-106. (23) Tezel, G.; Hernandez, R.; Wax, M. B. Arch Ophthalmol 2000, 118, 511-8. (24) Ikeda, Y.; Ohguro, H.; Maruyama, I. Jpn J Ophthalmol 2000, 44, 648-52. (25) Schori, H.; Kipnis, J.; Yoles, E.; WoldeMussie, E.; Ruiz, G.; Wheeler, L. A.;

Schwartz, M. Proc Natl Acad Sci U S A 2001, 98, 3398-403.

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http://www.glaucoma.org/learn/;

(26) Dyka, F. M.; May, C. A.; Enz, R. J Neurochem 2004, 90, 190-202. (27) Jacobi, P. C.; Dietlein, T. S.; Krieglstein, G. n. K. Ophthalmology 2000, 107, 417-

421. (28) John, S. W.; Smith, R. S.; Savinova, O. V.; Hawes, N. L.; Chang, B.; Turnbull,

D.; Davisson, M.; Roderick, T. H.; Heckenlively, J. R. Invest Ophthalmol Vis Sci 1998, 39, 951-62.

(29) Schuettauf, F.; Quinto, K.; Naskar, R.; Zurakowski, D. Vision Res 2002, 42, 2333-7.

(30) Anderson, M. G.; Smith, R. S.; Hawes, N. L.; Zabaleta, A.; Chang, B.; Wiggs, J. L.; John, S. W. Nat Genet 2002, 30, 81-5.

(31) Chang, B.; Smith, R. S.; Hawes, N. L.; Anderson, M. G.; Zabaleta, A.; Savinova, O.; Roderick, T. H.; Heckenlively, J. R.; Davisson, M. T.; John, S. W. Nat Genet 1999, 21, 405-9.

(32) Ishida, Y.; Migita, K.; Izumi, Y.; Nakao, K.; Ida, H.; Kawakami, A.; Abiru, S.; Ishibashi, H.; Eguchi, K.; Ishii, N. FEBS Lett 2004, 569, 156-60.

(33) Zhang, B.; Wu, K. F.; Cao, Z. Y.; Rao, Q.; Ma, X. T.; Zheng, G. G.; Li, G. Leuk Res 2004, 28, 91-5.

(34) Salomon, R. Acc Chem Res 1985, 18, 294 (35) Salomon, R. G.; Miller, D. B.; Zagorski, M. G.; Coughlin, D. J. J Am Chem Soc

1984, 106, 6049. (36) Salomon, R. G.; Miller, D. B. Adv Prostaglandin Thromboxane Leukot Res 1985,

15, 323-6. (37) Morrow, J. D.; Harris, T. M.; Roberts, L. J., 2nd Anal Biochem 1990, 184, 1-10. (38) Hamberg, M.; Svensson, J.; Samuelsson, B. Proc Natl Acad Sci USA 1974, 71,

3824-3828. (39) Nugteren, D.; Hazelhof, E. Biochim Biophys Acta 1973, 326, 448-61. (40) Nugteren, D.; Christ-Hazelhof, E. Adv Prostaglandin Thromboxane Res 1980, 6,

129-37. (41) Brame, C. J.; Salomon, R. G.; Morrow, J. D.; Roberts, L. J., 2nd J Biol Chem

1999, 274, 13139-46. (42) Foreman, D.; Zuk, L.; Miller, D. B.; Salomon, R. G. Prostaglandins Other Lipid

Mediators 1987, 34, 91-8. (43) Murthi, K. K.; Friedman, L. R.; Oleinick, N. L.; Salomon, R. G. Biochemistry

1993, 32, 4090-7. (44) Salomon, R. G.; Subbanagounder, G.; O'Neil, J.; Kaur, K.; Smith, M. A.; Hoff, H.

F.; Perry, G.; Monnier, V. M. Chem Res Toxicol 1997, 10, 536-45. (45) Salomon, R. G.; Batyreva, E.; Kaur, K.; Sprecher, D. L.; Schreiber, M. J.; Crabb,


(46) Iyer, R. S.; Ghosh, S.; Salomon, R. G. Prostaglandins 1989, 37, 471-80. (47) Davies, S. S.; Amarnath, V.; Montine, K. S.; Bernoud-Hubac, N.; Boutaud, O.;

Montine, T. J.; Roberts, L. J., 2nd Faseb J 2002, 16, 715-7. (48) Doolittle, M. H.; Ben-Zeev, O.; Briquet-Laugier, V. J Lipid Res 1998, 39, 934-

942. (49) Chen, J. Z.; Kadlubar, F. F. Am J Med 2003, 114, 697-8. (50) Yang, J.; Neufeld, A.; Zorn, M.; Hernandez, M. Exp Eye Res 1993, 56, 567-74.

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(51) Nielsen, A. L.; Jorgensen, A. L. J Biol Chem. 2004, 279, 41537-41545. (52) Murthi, K. K.; Salomon, R. G.; Sternlicht, H. Prostaglandins 1990, 39, 611-22. (53) Boutaud, O.; Andreasson, K.; Zagol-Ikapitte, I.; Oates, J. Brain pathol (Zurich,

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Matsuoka, K.; Yoshida, M.; Tanaka, K.; Tai, T. Nature (London) 2002, 418, 438-42.

(55) Ciechanover, A.; Brundin, P. Neuron 2003, 40,427-46. (56) Audhya, A.; Emr, S. Dev Cell 2002, 2, 593-605. (57) Caballero, M.; Liton, P. B.; Epstein, D. L.; Gonzalez, P. Biochem Biophys Res

Commun 2003, 308, 346-52. (58) Bradford, M. M. Anal Biochem 1976, 72, 248-54. (59) Laemmli, U. K. Nature 1970, 227, 680-5. (60) Miyagi, M.; Sakaguchi, H.; Darrow, R. M.; Yan, L.; West, K. A.; Aulak, K. S.;


(61) Harlow, E.; Lane, D. Antibodies: a laboratory manual; Cold Spring Harbor Laboratory, 1988.

186

Chapter 6

Part A: Pilot Studies Towards Identification of Levuglandin Modified Proteins in

Macrophages

Part B: Initial Studies Towards Developing a Model System to Differentiate Enzyme

Mediated and Free- Radical Mediated Formation of Levuglandins

187

6.1. Background

6.1.1. Macrophages, inflammation and atherosclerosis. Monocytes present in the

blood are progenitors of macrophages. Insults to the endothelial layer of the blood

vessels, such as hypercholesterolemia or cytokines, can stimulate the production of

leukocyte chemo-attractant molecules and expression of adhesion molecules that bind to

the monocytes and make them adhere to the endothelial layer through integrins (Figure

6.1).1 A family of low molecular weight chemotactic cytokine proteins, called

chemokines, e.g., monocyte chemoattractant protein-1 (MCP-1), recruit the monocytes to

the site of inflammation.2 Once the monocytes are inside the arterial intima, they are

differentiated into macrophages through the influence of the cytokine, macrophage-

colony stimulating factor (MCS-F).3 Macrophages are involved in both specific

immunity, e.g., engulf and degrade foreign material, as well as non-specific immunity,

e.g., process and present antigens to immune cells.

A crucial role of macrophages in atherosclerosis was established through a mouse

model that is deficient for both apoE and MCS-F, which showed dramatic protection

from atherosclerosis.4 Macrophages can be activated by cytokines, including interferon

(INF) γ and tumor necrosis factor (TNF) α. Furthermore, activated macrophages can

promote the production of cytokines.5 It has been shown that apoE deficient mice lacking

the INFγ receptor, which down regulates expression of the ABCA-1 receptor and

consequently inhibits cholesterol efflux from the macrophages. This is proatherogenic,

because it accelerates the formation of foam cells.6 Altered cholesterol homeostasis is

critical factor in foam cell formation as the macrophage internalizes large amounts of

cholesterol derived from lipoproteins. Uptake of oxidized or modified lipoproteins occurs

188

through scavenger receptors expressed on macrophages. Components of oxLDLs are

degraded and processed by macrophages to present the antigens present in them and

additionally, these oxLDL can act as activators of the macrophages itself. For example,

the oxLDL may contain strongly proinflammatory platelet activating factor (PAF)-like

molecules which can activate the macrophages as well as endothelial cells.7 A role for

oxLDL in the atherosclerosis is discussed in the following sections of this chapter.

igure 6.1: Schematic description of one of the factors contributing to foam cell

LDL

monocytes

macrophage oxLDL

Foam cell

Lum

enVe

ssel

wal

l

Endothelial layerradicals

peroxides

LDL

monocytes

macrophage oxLDL

Foam cell

Lum

enVe

ssel

wal

l

Endothelial layerradicals

peroxides

F

formation through endocytosis of oxLDL by macrophages.

189

6.1.2. Macrophages and COX-2. Cyclooxygenase enzymes are involved in the

rmal

X-1,

n

itial studies of atherogenic processes using non specific inhibitors of COX

enzyme be

s,

.1.3. Lipopolysaccharides (LPS) and cyclooxygenase (COX) expression. LPS is a

s

ation

formation, from fatty acids (vide supra), of eicosanoids, that are involved in the no

physiological functions. They are known to modulate physiological processes in

atherosclerosis and thrombosis. Two-isoforms of the enzymes are known viz, CO

which is constitutively expressed in most human tissues,8 and COX-2, whose expressio

is readily induced by inflammatory stimuli such as LPS and cytokines in a variety of

cells.9,10

In

s showed its anti-atherogenic effects in animal models.11 COX-2 is known to

upregulated in activated macrophages through proinflammatory agents like IL-1, TNFα,

LPS and TGFβ. COX-2 mediated production of PGs by activated macrophages in arterial

wall has been linked to atherosclerosis through a number of mechanisms including

activation of chemotaxis, propagation of the inflammatory cascade through cytokine

and induction of vascular permeability.11 More direct evidence for the role of COX-2 in

atherosclerosis was provided by a study using an LDL receptor negative (LDLR-/-) mouse

that is deficient in the COX-2 gene had significantly fewer atherosclerotic lesions

compared an LDLR-/- mouse with the COX-2 gene.12

6

structural element of Gram-negative bacterial cell wall consisting of lipids and polymer

of carbohydrates. LPS is an endotoxin, which acts as a ligand for receptors that can

trigger proinflammatory cytokines in many cell types.13 Among the other systemic

effects that are caused by LPS are the generation of secondary mediators of inflamm

190

viz, prostaglandins (PGs) and platelet activating factors (PAFs).14 In the case of

macrophages, LPS stimulation causes the secretion of PGs (mainly PGE2) and

proinflammatory cytokines, which, in turn, act in an autocrine or paracrine man

regulate the host response.

ner to

in the synthesis of PGs by LPS stimulation is the enzyme

cycloox

to

.1.4. Distinguishing levuglandins and isolevuglandins. Cyclooxygenation of

des

2,

rm

α, β-

.

15

The rate-limiting step

ygenase (COX).16 COX-2 expression in response to LPS in human and mouse

macrophages has been reported to be induced via NF-κB, indicating a transcriptional

mechanism for this potent stimulus.17 LPS-treated macrophages have also been shown

release the proinflammatory cytokines IL-1ß and TNF-α.15

6

polyunsaturated fatty acids (PUFAs) is known to generate prostanoid endoperoxi

through enzymatic or non-enzymatic processes. The prostaglandin endoperoxide, PGH

is converted enzymatically into a wide range of products that are receptor ligands in

normal physiological processes. PGH2 is also known to spontaneously rearrange to fo

PG isomers, PGD2 and PGE2. Additionally, Dr. Salomon discovered that PGH2

rearranges nonenzymatically to give γ-ketoaldehyde functional compounds with

substituted prostanoid chains. These γ-ketoaldehydes were named levuglandins (LGs)

Free radical mediated cyclooxygenation of the PUFAs can also generate PGs and the

corresponding LGs. The isomers of LGs generated by free radical processes include a

variety of structural isomers that are not produced through the enzyme-mediated proces

which only generates

s,

stereoisomers of PGs (Scheme 1.7, page 9). Since, wide ranges of

PUFAs are present in the LDL particle; oxidation of LDL can generate an array of PGs as

191

well as isoLGs. Differences in the amounts of LGs that are generated via enzyme-

mediated or free radical mediated processes can potentially be an effective tool to

compare their contributions towards the total oxidative stress and pathobiology.

The LGs and isoLGs can modify proteins with greater avidity, forming “stable”

adducts

ls

y mass

estigate the pharmacological and clinical significance of various LG

derived

m the

ove, LPS can trigger the expression of COX-2 and subsequently

generat

, than most other lipid oxidation products e.g., HNE and MDA.18 This feature

makes them an attractive “bio-marker” tool to assess the oxidative stress in the tissues.

Our lab has generated antibodies against several of the LG adducts to measure their leve

by immunochemical techniques. Using these antibodies, levels of adducts formed by LGs

or isoLGs, have been shown to be elevated in atherosclerosis19 and glaucoma.20

Elevated levels of LG adducts in Alzheimer’s disease have been recently shown b

spectrometric methods.21 Adduction by LGs have shown to cause loss of protein function,

e.g., loss of tublin function,22 and formation of protein crosslinks23 and DNA-protein

crosslinks.24

To inv

adducts requires an in vivo model system. The model system could also be

utilized in distinguishing the levels of enzyme mediated lipid oxidation products fro

free radical mediated processes under a variety of conditions. This can be achieved

immunochemically by using antibodies that are specific to LG adducts derived from

either of the processes.

As mentioned ab

e LGs or PGs. Part A of this chapter describes the use of this method in cultured

mouse peritoneal macrophages (MPM) to identify the proteins that are susceptible to

oxidative modifications. In part B of this chapter, cornea is used as an in vivo model

192

system to study the LPS-induced formation of LGs and isoLGs through corneal

inflammation or keratitis.

193

6.2. Results.

PART A

6.2.1. LPS stimulates formation of LGE2 and iso[4]LGE2 modified proteins in mouse

peritoneal macrophage cell cultures. Macrophages were cultured in the presence or

absence of LPS (10 µg/mL) for 24 h and probed for LGE and iso[4]LGE modified

proteins by Western blot analysis. The proteins from the LPS treated cells showed

increased immunoreactivity with anti-LGE2-KLH polyclonal antibodies (pAb) compared

that of the cells that were not treated (Figure 6.2).

20

2837

50

88114250

LMW

1 2 1 2LMW

LMW low molecular weight marker1-control2-LPS

Coomassie Western blotA B C

32

68

0

100

Control LPS

20

2837

50

88114250

LMW

1 2 1 2LMW


Coomassie Western blotA B C

32

68

0

100

Control LPS

Figure 6.2: LGE2-modified proteins in LPS stimulated macrophages. A. Coomassie

stained SDS PAGE proteins from macrophages. B. Western blot analysis for LGE2

immunoreactivity in the electroblotted PVDF membrane. C. Densitometric quantification

of the total immunoreactive bands in each of the lanes (as relative intensity). 1- control

macrophages, 2 - LPS (10 µg/mL) treated macrophages (n=2).

194

When the cell culture was treated with indomethacin, a COX inhibitor, before and

after LPS treatment, staining by the anti-LGE2 pAb was absent (see Appendix). The

electroblots were also probed with anti-iso[4]LGE2 pAb and a small elevation in the

levels of immunoreactivity was observed in LPS treated compared to untreated cells

(Figure 6.3, B & C).

LMW

2029

36

50

89

114250

LMW

Coomassie Western blotA B

1 2 1 2


C

4456

0

100

Control LPS

LMW

2029

36

50

89

114250

LMW

Coomassie Western blotA B

1 2 1 2


C

4456

0

100

Control LPS

Figure 6.3: Iso[4]LGE2-modified proteins in LPS stimulated macrophages. A.

Coomassie stained SDS PAGE of proteins from macrophages. B. Iso[4]LGE2

immunoreactivity of the electro-blotted PVDF membrane. C. Densitometric

quantification of the total immunoreactive bands in each of the lanes (as relative

intensity).

This suggests that the LPS may induce COX-2 expression, which results in the

generation of LGE2. However, some contribution of the free radical-induced oxidation is

also indicated by the small elevation in iso[4]LGE2 immunoreactivity that ensues upon

195

treatment with LPS. Thus, the immunoreactivity shown in the lane 2 of Figure 6.2

represents the proteins that are modified by LGs through the COX-2 pathway.

6.2.2. Separation and identification of modified protein by 2D PAGE and mass

spectrometry. Proteins from the macrophages treated with LPS were electrophoretically

separated by isoelectric focusing and subsequently on a SDS PAGE. The separated

proteins were then partially electroblotted on to a PVDF membrane and probed with anti-

LGE2-KLH pAb (Figure 6.4).

pI

A

B

20

28

37

51

88114

20

28

37

51

88

114

34

578

911

1213

14 18

34

578

911 12

13

14 18

kDa

3 10987654pI

A

B

20

28

37

51

88114

20

28

37

51

88

114

34

578

911

1213

14 18

34

578

911 12

13

14 18

kDa

3 10987654

Figure 6.4: Separation of proteins extracted from macrophages treated with LPS. A.

Western blot using anti-LGE2-KLH polyclonal antibody. B. Coomassie blue stained 2D

SDS PAGE.

196

The Western blots were aligned with the Coomassie stained gel and the protein

spots corresponding to immunoreactive spots were excised. The gel pieces were analyzed

for proteins using the protocol reported earlier.25 The proteins identified by MS/MS

using NCBI database are listed in Table 6.2.

Table 6.1: List of LGE2 – modified proteins electrophoretically separated by 2D gel

electrophoresis and identified by mass spectrometry.

Molecular Mass (kDa/pI) Spot No

Proteins (immunoreactive to

LGE2)

Peptides Matched Measured Calculated

Accession No.

3 β-actin 3 1 42.5/5.3 41.7/5.4 P53486

3 IgG 1 42.5/5.4 11.5/5.8 P01834

3 Tubulin β-1 1 42.5/5.5 49.8/4.8 P09203

4 Rab11 1 43.0/5.2 69.5/9.4 Q8R361

4 Serpin 12 1 43.0/5.3 45.7/6.1 Q9JK88

4 Eucaryotic initiation factor 1 43.0/5.4 44.5/5.3 P87206

5,7,18 Rab11 1 49.5/5.1 92/8.4, 175/7,

55/6.5 Q8R362

8-13 Vimentin 5 55.0/5.1 52-97/7.3-8.7 P48670

14 Keratin, cytoskeletal 4 77.0/5.2 65.0/9.1 P04104

The cytoskeletal proteins viz, actin (actin 3, spot 3), microtubules (tubulin β-1,

spot 3), and two major intermediate filaments vimentin (spots 8-13) and cytokeratin I

(spot 14) were identified. Vimentin and Rab11 have altered masses that correspond to

dimers or trimers (Table 6.2). A detailed discussion is presented in section 6.4.

197

6.2.3. Immunohistochemical analysis of macrophages. Difference in the amount of

LGE2 modified proteins in naive macrophages compared to the cells treated with LPS or

with LPS and indomethacin were found using immunohistochemical analysis. The

macrophages were labeled with 4’,6-diamidino-2-phenylindole (DAPI), a nucleus

specific stain with blue fluorescence and the LGE2-modified proteins were labeled using

a green fluorescent tagged secondary antibody, Alexa 488.

B

A C

DB

A C

D

Figure 6.5: Immunohistochemical analysis of macrophages for LGE2 modified proteins.

macrophages grown to confluency were plated on to a glass slide and treated with or

without LPS. A. Cells treated with LPS and stained with pre-immune serum. B. Controls

cells stained with anti-LGE2-KLH pAb. C. Cells treated with LPS stained with anti-

LGE2-KLH pAb D. Cells treated with LPS along with indomethacin for 24 h and stained

with anti-LGE2-KLH pAb (green fluorescence). Macrophage nuclei are stained with

DAPI (blue fluorescence).

198

As shown in Figure 6.5, there is an increase in the staining (green fluorescence)

of cells treated with LPS (panel C, Figure 6.5) compared to the untreated control cells

(panel B, Figure 6.5). Also, macrophages that were treated with indomethacin before and

after LPS treatment appeared to show less fluorescence (panel D, Figure 6.5) compared

to LPS treated slides, but more than the control cells. This suggests that only some, if

any, LGE2 immunoreactivity is produced through COX pathway, and some is produced

through free radical-induced cyclooxygenation.

199

PART B

6.2.4. Protein modifications in LPS treated cornea – 1D SDS PAGE. Cornea of

C57BL/6 mouse was treated in vivo with LPS 2 µg (2 µL volume) by intrastromal

injection. The cornea was then excised after 24 h. The proteins were extracted from

cornea using the extraction buffer described in Part A of this chapter (page 234) and

separated electrophoretically on a 10% polyacrylamide gel with subsequent

electroblotting to a PVDF membrane. Densitometric calculation of immunoreactive

bands in the Western blots (Figure 6.6) showed relatively increased amounts of LGE2

modified proteins in LPS treated cornea compared to the naïve or PBS treated cornea

(Figure 6.7).

2029

36

50

89

114250

Untre

ated

PBS

LPS

Untre

ated

PBS

LPS

Untre

ated

PBS

LPS

A B C

2029

36

50

89

114250

2029

36

50

89

114250

Untre

ated

PBS

LPS

Untre

ated

PBS

LPS

Untre

ated

PBS

LPS

A B C

Figure 6.6: Western blot using anti-LGE2 pAb of mouse corneal protein extract (2 µg)

from C57BL/6 treated with PBS or LPS (A, B, C are replicates of same experiment).

Lane 1. Untreated cornea. Lane 2. PBS treated cornea. Lane 3. LPS (10 µg/mL) treated

cornea.

200

0

20

40

60

80

Untreated PBS LPS

Rel

ativ

e in

tens

ity (%

)

Figure 6.7: Relative amount of LGE2 immunoreactivity. Densitometric quantification of

immunoreactive bands of the Western blots probed with LGE2-pAb. untreated – corneal

proteins of untreated C57BL/6 mouse, PBS – corneal proteins of mice treated with PBS,

LPS - corneal proteins of mice treated with LPS (10 µg/mL). Error bars indicate standard

deviation for three experiments.

6.2.5. Immunohistochemical analysis. To localize the LGE2 and iso[4]LGE2 modified

proteins in the cornea and to determine the effect of LPS on the generation of LGs,

sections of cornea were stained by treatment with the corresponding antibodies. The

stained section shows increased staining in corneal stroma in the LPS treated cornea

compared to that of untreated or PBS treated cornea. The PBS treated cornea shows some

immunoreactivity that could be contributed by intra-stromal injection-induced

inflammation. The amount of green fluorescence in the stromal region of LPS treated

corneal sections stained with LGE2 –pAb (panel L, Figure 6.8), was visibly greater than

201

that of the corresponding stromal region of the iso[4]LGE2 stained sections (panel K,

Figure 6.8).

LGE2-pAbPreimmune

Unt

reat

ed24

h PB

S6h

LP

S24

h LP

S

Iso[4]LGE2-pAb

A

D

G

J K

H

E

B C

F

I

L

Epithelial layer

Stroma

LGE2-pAbPreimmune

Unt

reat

ed24

h PB

S6h

LP

S24

h LP

S

Iso[4]LGE2-pAb

A

D

G

J K

H

E

B C

F

I

L

Epithelial layer

Stroma

Figure 6.8: Cornea of C57BL/6 mouse was treated with LPS or PBS in vivo and

preserved after 6 h and 24 h. Sections (5 µm) of cornea was stained with LGE2 and

iso[4]LGE2 pAb. Untreated corneal sections were used as additional controls.

A,B,C – Untreated cornea; D,E,F – PBS treated (24 h); G,H,I – LPS treated (6 h); J,K,L

– LPS treated (24 h). Panels treated with preimmune, LGE2 and iso[4]LGE2 pAbs are

indicated in the figure.

202

6.3. Discussion.

PART A

Oxidative damage of LDL particles and their subsequent internalization by macrophages

through scavenger receptors leads to foam cell formation, an early step in the

pathobiology of atherosclerosis. Dr. Salomon’s lab previously reported that LDL

modified with LGE2 can bind to the macrophage scavenger receptor26 and additionally,

has identified a set of oxidized phospholipids that can act as ligands for the scavenger

receptor CD36.27 Inefficient processing of oxidatively modified LDL leads to

accumulation of oxLDL in macrophages, and formation of “foam cells”. To understand

the pathological alterations that interfere with metabolism of endocytosed oxLDL by

macrophages, identification of the modified proteins that are present in macrophages can

be helpful. Part A of this chapter is a pilot project in identifying the proteins that are

modified by levuglandins in macrophages as a consequence of activation by

lipopolysaccharides (LPS). This study was conducted in collaboration with Dr. Eugene

Podrez (Cleveland Clinic Foundation).

LPS induced increase in the COX-2 expression along with increase in the

expression of inflammatory cytokines has been reported for various cell types including

macrophages. Mouse peritoneal macrophage cells were cultured and exposed to LPS.

Increases in the amount of COX-2 can subsequently catalyze the formation of LGs. A

definite difference in the amount of LGE2-modified proteins in the LPS stimulated

macrophages compared to the untreated cells was detected by Western blot analysis of

the cellular proteins. Proteins from macrophages pre-treated with a COX inhibitor,

203

indomethacin, showed no immunoreactivity for LGE2 pAb upon exposure to LPS. This

establishes that the protein modification was promoted by COX.

Identification of the LGE2-modified proteins was performed by 2D PAGE

separation followed by identification of the immunoreactive proteins by mass

spectrometric analysis. The proteins identified included several cytoskeletal proteins

crucial to cell stability, cell migration, cell division, intracellular transport, vesicular

trafficking, and organelle morphogenesis. All three types of protein filaments were

affected (Table 6.2): actin, microtubules, and two major intermediate filaments vimentin

and cytokeratin I. Of these, vimentin is critical for post-lysosomal trafficking of lipo-

protein cholesterol in SR-BI-expressing cells.28,29 and tubulin is also associated with lipid

efflux.30 We suggest that, if the function of these proteins is compromised by covalent

modification, deficient delivery of cholesterol for efflux could result. We also detected

modification of Rab11 interacting protein (Rip 11) and eukaryotic initiation factor 4A

(elF4A). Rip11 appears to regulate the trafficking of Rab11,31 which in turn is an

essential regulator of the dynamics of recycling endosomes that is critical in cellular

cholesterol transport.32 ElF4A is a subunit of the initiation factor complex elF4F that

mediates binding of mRNA to the ribosome.33 Consequently, modification of these two

proteins suggests that LGs can affect, respectively, cholesterol trafficking and/or the gene

expression profile in macrophages.

204

PART B

Cornea is a clear transparent tissue that is ideal for in vivo visualization and

quantitation of biological experiments through fluorescently-tagged probes. This feature

of cornea is exploited in studying models of angiogenesis,34 tumor growth,35 tissue

preservation,36 and inflammation.37 Here, part B of this chapter deals with developing

cornea as an in vivo model system for generation of LGs and isoLGs upon activation by

LPS (in collaboration with Dr. Eric Carlson and Dr. Victor Perez at Cleveland Clinic

Foundation).

Intrastromal injection of LPS in the cornea is known to induce COX-2

expression.38 We extracted the proteins from cornea 6h and 24 h after LPS injection and

analyzed for LGE2 immunoreactivity using Western blot analysis. There were increased

amounts of LGE2 modified proteins (62%) compared to the untreated (15%) or PBS

(22%) treated cornea (Figure 6.7). Also, immunohistochemical data suggests that there

was an increased amount of modified proteins in LPS treated cornea compared to the

controls (Figure 6.8).

Further experiments in continuation of this pilot project will be focused on identification

of methods to inhibit COX-2 and study the influence of light on generation of various

LGs and isoLGs in cornea.

205

6.4. Experimental Procedures.

6.4.1. General methods. The following commercially available materials were used as

received: bovine serum albumin (BSA, fraction V, 96-99%) and human serum albumin

(HSA, fraction V) were from Sigma (St. Louis, MO); goat anti-rabbit IgG-alkaline

phosphatase (Boehringer-Mannheim); anti-rabbit IgG coupled with Alexa 488 (a green

fluorescent tag) (Molecular probes, OR); Phosphate buffered saline (PBS) was prepared

from a pH 7.4 stock solution containing 0.2 M NaH2PO4/Na2HPO4, 3.0 M NaCl, and

0.02% NaN3 (w/w). This solution was diluted to 10 mM or as needed. Anti-LGE2-KLH

antibodies19 and anti-iso[4]LGE2-KLH39 antibodies were prepared as described

previously. Iso[4]LGE2 was prepared by Mr. Jim Laird in our lab. The iso[4]LGE2 –

HSA and iso[4]LGE2-BSA40 were prepared as described previously.

6.4.2. Protein extraction. Thioglycollate-elicited mouse peritoneal macrophage (MPM)

cells from wild-type (C57BL/6) mice were isolated and cultured by Dr. Podrez.41 The

cells were scraped, pelleted and stored at -20 ºC until used. The pelleted cells were

extracted by homogenization in 100 mM Tris-Cl buffer pH 7.8 containing 5 mM

dithiothreitol, 1mM SnCl2, 50 mM NaHPO4, 1mM diethylenetriaminepentaacetic acid,

100 mM butylated hydroxytoluene and 0.5% SDS. Insoluble material was removed by

centrifugation (8000g for 5 min). Soluble protein was quantified by the Bradford assay42

using amino acid quantified BSA as standard, yielding ~1 µg/µL of soluble protein.

Protein extracts were subjected to SDS-PAGE on 10% gels (Bio-Rad Laboratories,

Hercules, CA) and the gels were used either for Western analyses or for mass

spectrometric proteomic analyses.43,44

206

6.4.3. Western analysis. Electrophoresis and Western blotting methods were carried out

according to published protocols.43 Protein extract (10 µg) was suspended in Laemmli

sample buffer 45 and fractionated on a 10% SDS PAGE and partially electro blotted onto

polyvinyl difluoride (PVDF) membrane (Millipore, Bedford, MA). The PVDF

membranes were blocked with 5% NFDM milk and probed using various antibodies: 10

µg rabbit polyclonal anti-LGE2 and 4.5 µg of rabbit polyclonal anti-iso[4]LGE246 as

described in Chapter 4 and 10 -100 ng anti-rabbit secondary antibody.

6.2.4. Histochemical analysis. Cultured macrophages were fixed on slides and processed

for immunohistochemical analysis. The cells were blocked with 1% BSA solution in PBS

for 1 h and washed with PBS (10 mM) thrice. Following which the cells were incubated

with 1:100 dilution antibody solutions (0.8 µg/µL rabbit polyclonal anti-Iso[4]LGE2 or

1.5 µg/µL anti-LGE2) for 1 h. The sections were then washed with a solution of 1% BSA

in PBS (3 x 5 min) and subsequently incubated with 10 ng anti-rabbit secondary antibody

conjugated with Alexa 488 (Molecular Probes Inc, Eugene, OR) for one hour at room

temperature. Sections were washed with 1% BSA in PBS as mentioned above, sealed

with Vectashield®, and analyzed with a Nikon EFD-3 fluorescence microscope attached

to a CCD camera. The microscopic images were assembled in Adobe® Photoshop® CS.

(Experiment performed by Dr. Eugene Podrez)

Immunohistochemical analyses to localize modified proteins in corneal tissue

were performed with eyes enucleated from C57BL/6 mice. The corneal processing was

done by enucleating the eye at the specific time period and snap freezing it in OCT®.

5 µm corneal sections were then cut, fixed with 4% paraformaldehyde. The sections were

207

serially incubated in 1% BSA in phosphate buffered saline pH 7.5, then with 10-100 ng

primary antibody (rabbit polyclonal anti-Iso[4]LGE2 or anti-LGE2) overnight at 4°C. The

sections were then washed with a solution of 1% BSA in PBS (3 x 5 min) and

subsequently incubated with 10 ng anti-rabbit secondary antibody conjugated with Alexa

488 (Molecular Probes Inc, Eugene, OR) for one hour at room temperature. Sections

were washed with 1% BSA in PBS as mentioned above, and sealed with Vectashield®,

and analyzed with a Nikon EFD-3 fluorescence microscope attached to a CCD camera.

The microscopic images were assembled in Adobe® Photoshop® CS. (Experiment

performed by Dr. Eric Carlson)

6.4.5. 2D gel electrophoresis. Protein extract from LPS treated macrophages (80 µg)

were used for the two dimensional electrophoresis as discussed in Chapter 4 (page 151).

6.4.6. LC MS/MS analysis and protein identification. Following the SDS PAGE, the

Gelcode Blue® stained gel bands were excised in 1 mm3 slices and destained according

to standard protocol for in-gel digestion.43 The tryptic digested proteins were analyzed by

LC-MS/MS using a quadrupole-time of flight (QTof2) mass spectrometer equipped with

a CapLC system (Waters, Millford, MA), ProteinLynx Global Server acquisition and

processing software. Peptide digests were trapped on a precolumn (0.3 x 1 mm, 5 µC18,

LC Packing) with 0.1% formic acid in 2% acetonitrile as loading solvent then eluted onto

a capillary column (PicoFrit 0.050 x 50 mm, 5 µ tip ID; New Objective Inc., Woburn,

MA). Chromatography was performed at 250 nL/min with aqueous acetonitrile/formic

acid solvents and 100% of the eluant was directed towards the mass spectrometry. The

208

MS/MS data for representative peptides were collected for the mass range of 50-2000.

Protein identifications from MS/MS data utilized ProteinLynxTM Global Server (Waters

Corporation) and Mascot (Matrix Science) search engines and the Swiss-Protein and

NCBI protein sequence databases.43

209

6.5. Reference.

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211

Appendix

213

Chapter 2

O OTs

2.14

a)

b)

Figure 2.1S: a) 13C NMR and b) 1H NMR of 2.14

214

O

OTs

OHO

2.36

Figure 2.2S: 1H NMR of 2.38 (crude)

OC5H11

OHO

2.37


215

HO

O

O

OTs

2.17 a)

b)


216

O

OTBDMS

OHO

2.38 a)

b)


217

O OHO

OTBDMS

2.21

Figure 2.6S: 1H NMR of 2.21

218

a)

HO

O

O

OTBDMS

2.22

b)


219

O

OTHP

OHO

2.39


220

O

O

O

OTHP

2.40 a)

b)

Figure 2.9S: a) 13C (APT) NMR and b) 1H NMR of sec-butyl ester of 2.40

221

2.27


222

2.26

Figure 2.11S : 1H NMR of 2.26

223

Chapter 3

HD2C

D2C O

SO

O

3.3


224

HD2C

D2C I

3.4


225

a)

OOHD2C

CD2 OH

3.6

b)

Figure 3.3S: a) 1H NMR b) 13C NMR of 3.6

226

OHD2C

CD2 OH

3.7


227

OHD2C

CD2 OH

3.7a)

b)

Figure 3.5S: a) 1H NMR and b) 13C NMR of 3.7

228

TOF – time of flight analyzerMSMS – tandem mass 260.00 – mass of parent ion analyzedES+ - Electrospray positive mode

Intensity ions formed

TOF – time of flight analyzerMSMS – tandem mass 260.00 – mass of parent ion analyzedES+ - Electrospray positive mode

Intensity ions formed

Figure 3.6S: How to read a mass spectra?

H2NOH

O

N

NH

O

198 m/z

CH2CO

H3NOH

O

N

NH

156 m/z

HCOOH

HN

N

NH2

110 m/z

OHO

N

NH

139 m/z

HN

NH

82 m/z

HCN

H2N

N

NH

O

154 m/z

NH

H2N

44 m/z

44 m/z

NH3

229

Scheme 3.1S: Fragmentation of N-acetyl histidine

HNOH

O

N

N

O

O

H2NOH

O

N

N

O

C15H25N3O4Exact Mass: 311.1845


N

N

N

O

O


-carboxyl

H3N

N

N

O

HCOOH

C12H19N3O•+

Exact Mass: 221.1523

HO

HOHO

HO

-acetyl

N

NH

OHO

C14H23N2O2+


C14H24N3O2+


NH3

1,2-hydride shift

HN

N

N

OH

HN

N

NH

OHO

-acetyl

-acetyl -carboxyl

Scheme 3.2S: Fragmentation of (N-acetyl-His)-HNE Michael adduct

HN

O

H2N

ONH

OO

O

HO

HNO

NH2

O

NHO

O

OHN

O

HN

ONH

OO

HO

HO

O

-H20

Retro-Michael

H2NO

NH

HN

O

O

HO

O

McL -acetyl

-H20

m/z 416

m/z 139

m/z 372 m/z 372

m/z 398

230

Scheme 3.3S: Suggested fragmentation of (N-acetyl-Gly-Lys-OMe)-HNE Michael (1:1)

adduct.

HNO

N

O

NH

O

OOH

H2NO

N

NH

O

OOH

-acetyl

N

NH3

O

O

m/z 281

NOH

O

m/z 250

Nm/z 204

NOH

222 m/z

m/z 354

H2O

Amide cleavage

CO2

-NH3, -CH3

H2O

m/z 398

Scheme 3.5S: Suggested fragmentation of (N-acetyl-Gly-Lys-OMe)-HNE Schiff base

adduct

231

NHO

N

OHN

O

O

NH3

O

N

HN

O

O

N

N

NH3O

O

m/z 380

m/z 338

m/z 204 m/z 281

Scheme 3.6S: Fragmentation of (N-acetyl-Gly-Lys-OMe)-HNE pyrrole adduct

His-HNE 10ug0.00000000

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00Time0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

HIS_HNE-07 SIR of 13 Channels ES+ 696

8.17e411.16

10.291.68

17.4415.8414.5712.10 13.90 18.38


8.37e417.44

15.7013.7011.901.28

19.58


7.66e418.5815.84

14.6313.7011.83

1.61

17.57


3.70e519.51

15.8414.70 18.44


1.70e515.70

14.63

13.6317.51 19.64


4.60e515.70

14.6319.78

232

Figure 3.7S: ESI-TOF-SIR of N-acetyl-His/HNE reaction mixture. Channels shown here

m/z 696, m/z 692, m/z 688, m/z 684, m/z 678 and m/z 674.

Figure 3.8S: ESI-TOF-SIR of (N-acetyl-Gly-Lys-OMe)/HNE reaction mixture. Channels

shown here are from m/z 384, 402, 420, 580, 640, 644, 652, 656, 736 and 740.

233

HN

OHO

S

O

NH

HO O

S

O

HN

OHO

S

O

S HN

OHO

S

O

NH2

HO O

S

m/z 281m/z 208

HN

OHO

S

O

NH

O

S

O

m/z 307

m/z 324-H2O

-acetylα−cleavage

Scheme 3.7S: Fragmentation of (N-acetyl-Cys)-(N-acetyl-Cys) disulphide adduct

HN

HO

O

SO

OHO

NHHO

O

SH

O

O

NH3HO

O

S

O

NH2

O

S

O

m/z 302

m/z 260m/z 242

m/z 319

-acetyl

-H2O

-H2O

Scheme 3.8S: Fragmentation of (N-acetyl-Cys)-HNE Michael adduct

234

Cys-HNE10ug0.00000000

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00Time0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

CystHNE-10 SIR of 13 Channels ES+ 360

4.12e611.03

10.43 15.5711.5013.63


4.35e610.16 10.89

11.5013.70


4.42e511.09

10.89

15.7715.1711.50 14.03


3.23e511.76

10.161.34 10.49

17.3114.17

13.63 15.77


2.38e59.76

8.822.280.2117.3114.0311.5610.96

13.03 16.3715.7718.17

CystHNE-10 SIR of 13 Channels ES+ TIC

1.05e711.09

9.8311.50

15.7715.2413.70 17.31

Figure 3.9S: ESI-TOF-SIR of (N-acetyl-Cys)/HNE reaction mixture. Channels shown

here are from channels monitored at m/z 320, m/z 326, m/z 352, m/z 356 and m/z 360.

235

O OH

HNOH

O

N

N

O

OH

O

OHOH

H3NOH

O

N

N

O

O

m/z 468

OHOH

H2N

N

N

O

O

m/z 422

OHOH

H2N

N

N

O

O

O

m/z 466

OHOH

OHO

N

N

O

O

m/z 451

OHOH

N

N

O

O

m/z 407

-acetyl

-carboxyl

-NH3

m/z 510

-acetyl -carboxyl

-acetyl

Scheme 3.9S: Suggested fragmentation of (N-acetyl-His)-HNE 1:2 adduct

OHOH

HNOH

O

N

N

O

O

HO

O

O

OHOH

HNOH

O

N

N

O

O

m/z 394

OHOH

HNOH

O

N

N

O

O

m/z 466

m/z 665

O OHOH

HO

O

O

m/z 467

Scheme 3.10S: Suggested fragmentation of (N-acetyl-His)-HNE 1:2 adduct

236

Table 3.1 Summary of tandem MS/MS fragments incorporating the C5 alkyl chain of

HNE derived from the precursor ions 354, 358, 510, 514, and 510 thus showing the 4 Da

difference.

Fragments with mass difference of 4 Da

Precursor ion (m/z)

Mass Range (Da)

354

358

510

514

518

550-500 - - 510 514 518

500-450 - - 464, 466, 468 468, 470, 472 472, 474, 476

450-425 - - 422 426 430

425-400 - - 407 411 415

375-345 354 358 354

348

354, 358

348, 352

358

352

345-300 308, 312

336

312, 316

340

308, 312

336

312, 316

336, 340

312, 316

340

300-275 277

290, 294

281

294, 298

277

290,294

277, 281

290, 294, 298

281

294, 298

275-245 248, 251

259, 266

252,255

263, 270

248, 251

259, 266

248,251,252,255

259,263,266,270

252,255

263, 270

245-200

-

221, 233

-

-

225, 237

-

209

221, 233

239

209,213

221,225, 233,237

239,243

213

225, 237

243

200-100 139 143 139 139,143 143

237

Chapter 4

43FW

77M

W

85FW

52M

W

49M

W

85FW

62FW

67FW

62FW

77M

W

46FW

77M

W

85FW

81FW

82FW

63M

W

86FW

57FW

61FW

59FW

74M

W


11488

50

35

28

20

25043

FW

77M

W

85FW

52M

W

49M

W

85FW

62FW

67FW

62FW

77M

W

46FW

77M

W

85FW

81FW

82FW

63M

W

86FW

57FW

61FW

59FW

74M

W


11488

50

35

28

20

250

11488

50

35

28

20

250

Figure 4.1S: Western blot analyses of normal and POAG trabecular meshwork using

anti-iso[4]LGE2 antibodies as described in Section 4.4.4.

43FW

77M

W

85FW

52M

W

49M

W

85FW

62FW

67FW

62FW

77M

W

46FW

77M

W

85FW

81FW

82FW

63M

W

86FW

57FW

61FW

59FW

74M

W


11488

50

35

28

20

250

43FW

77M

W

85FW

52M

W

49M

W

85FW

62FW

67FW

62FW

77M

W

46FW

77M

W

85FW

81FW

82FW

63M

W

86FW

57FW

61FW

59FW

74M

W


11488

50

35

28

20

250

11488

50

35

28

20

250


anti-HNE antibodies as described in Section 4.4.4.

238

88M

W

74 M

W

87 M

W

84 M

W

80 F

W

72 M

W

87 M

W

87 M

W

LMW

c

11488

50

35

28

20


11488

50

35

28

20

88M

W

74 M

W

87 M

W

84 M

W

80 F

W

72 M

W

87 M

W

87 M

W

LMW

c

11488

50

35

28

20

11488

50

35

28

20


11488

50

35

28

20

11488

50

35

28

20


anti-argpyrimidine antibodies as described in Section 4.4.4.

LMW

Prot

ein e

xtrac

t

IP u

sing

iso[4

]LGE

2 pA

bIP

usin

g Pr

eimm

une

se

114

88

50

35

28

20

250

LMW

Prot

ein e

xtrac

t

IP u

sing

iso[4

]LGE

2 pA

bIP

usin

g Pr

eimm

une

se

LMW

Prot

ein e

xtrac

t

IP u

sing

iso[4

]LGE

2 pA

bIP

usin

g Pr

eimm

une

se

114

88

50

35

28

20

250

114

88

50

35

28

20

250

Figure 4.4S: Immunoprecipitation of POAG trabecular meshwork using anti-iso[4]LGE2

pAb and preimmune serum antibodies as described in Section 4.4.6

239

Chapter 5

A

B

7 da

ys

15 d

ays

3 w

eeks

1 m

onth

s

2.5

mon

ths

8 m

onth

s

9 m

onth

s

BS

A-Is

o[4]

LGE

2

LMWA

B

7 da

ys

15 d

ays

3 w

eeks

1 m

onth

s

2.5

mon

ths

8 m

onth

s

9 m

onth

s

BS

A-Is

o[4]

LGE

2

LMW

Figure 5.1S: Western blot of TM proteins from DBA/2J mouse of different age groups..

5 µg of protein was electrophoretically separated on a SDS PAGE and electroblotted on

a PVDF membrane and probed with iso[4]LGE2 pAb. Time course of DBA/2J mice. A.

Western blot. B. Coomassie stained SDS PAGE.

240

Prot

ein e

xtrac

t

IP u

sing

iso[4

]LGE

2 pA

b

1148

50

35

28

20

250

LMW

Prot

ein e

xtrac

t

IP u

sing

iso[4

]LGE

2 pA

bIP

usin

g Pr

eimm

une

seru

m

Prot

ein e

xtrac

t

IP u

sing

iso[4

]LGE

2 pA

b

1148

50

35

28

20

250

1148

50

35

28

20

250

LMW

Prot

ein e

xtrac

t

IP u

sing

iso[4

]LGE

2 pA

bIP

usin

g Pr

eimm

une

seru

m

Figure 5.2S: Immunoprecipitation of trabecular meshwork proteins from a 8 month old

DBA/2J mouse using anti-iso[4]LGE2 pAb and preimmune serum antibodies as described

in Section 5.4.6.

241

Chapter 6

1 2LMW

LMW low molecular weight marker1-control2-LPS3-treated with indomethacin

Western blot

31 2LMW

LMW low molecular weight marker1-control2-LPS3-treated with indomethacin

Western blot

3

Figure 6.1S: LGE2-modified proteins in LPS stimulated MPM cells. Western blot

analysis for LGE2 immunoreactivity in the electroblotted PVDF membrane.

242

Thesis Conclusion

This thesis primarily focuses on the levels of lipid derived protein modifications

in glaucoma. Previously, other studies, used spectrophotometric methods to quantify the

lipid oxidation products in glaucoma, or investigated the biological consequences of

oxidative stress in cell culture models of glaucoma. The present study, using antibodies

against protein modifications (iso[4] levuglandin E2 and 4-hydroxynonenal) derived from

lipid oxidation, provides the first direct evidence for elevated levels of lipid-derived

oxidative protein modifications in the trabecular meshwork of primary open angle

glaucoma compared to the controls. The oxidatively modified proteins were identified

using immunoprecipitation, 2D gel electrophoresis and mass spectrometry. Proteins

identified to be putatively modified, included several structural proteins, which may be

important in maintaining the structure as well as the function of the trabecular meshwork.

Future work may be focused on the role of these modified proteins in the increase of

intraocular pressure that may augment our understanding of glaucoma pathogenesis.

Additionally, an increase in the levels of proteins modified by iso[4] levuglandin

E2 was observed in the trabecular meshwork of glaucomatous DBA/2J mice, a model for

glaucoma. This model can be valuable for studying the role of the modified proteins in

glaucoma pathogenesis. Using models for these analyses is desirable, inter alia, because

tissue samples from human glaucoma patients are difficult to obtain.

Multiple additions of the lipid oxidation product 4-hydroxynonenal (HNE) to side

chain residues of lysine and histidine were detected and characterized using deuterated

HNE and mass spectrometry. Future work may focus on the detection of such multiple

243

HNE adducts in vivo and determine their role in detoxification and/or pathology of

oxidative insult.

244

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LIPID-BASED OXIDATIVE PROTEIN MODIFICATIONS IN ...

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