laura murdaugh

ABSTRACT

THE BIOCHEMICAL STUDY OF AGE RELATED CHANGES IN HUMAN RETINAL PIGMENT EPITHELIUM AND BRUCH’S MEMBRANE

Laura S. Murdaugh, Ph.D. Department of Chemistry and Biochemistry

Northern Illinois University, 2010 Elizabeth R. Gaillard, Director

Age-related macular degeneration (AMD) is an ocular disease that causes

severe visual loss and legal blindness in the elderly population. The pathophysiology

of AMD is complex and may include genetic predispositions, accumulation of

lipofuscin and drusen, local inflammation and neovascularization. Therefore, specific

age-related changes in the retinal pigment epithelium (RPE) and Bruch’s membrane

have been investigated

The accumulation of lipofuscin has been shown to precede the death of

photoreceptor cells and the deterioration of the RPE. As a result, the determination of

the photosensitive components of lipofuscin have been of major interest. One of these

components, previously identified as a bis-retinoid pyridinium compound, is referred

to as A2E. A2E has been characterized by mass spectrometry and is known to have a

mass of 592 Da. The remaining chromaphores in RPE lipofuscin are structurally

related to A2E as determined by their fragmentation pattern with losses of M+/- 190,

174 and/or 150 Da. Analysis of lipofuscin from various donors indicates that the

extracts consist of as many as fifteen of these hydrophobic components which are

also observed to form spontaneously in vitro over extended periods of time.

Previous studies have shown that numerous structural changes are induced in

Bruch’s membrane with age. These changes may have a harmful effect on Bruch’s

membrane, resulting in damage to RPE cells and the onset of AMD. Recent research

has identified a commonly inherited variant of the complement factor H gene from

different groups of AMD patients linking the genetics of the disease to inflammation.

During inflammation there is activation of nitric oxide synthase and release of nitric

oxide, which could lead to non-enzymatic nitration within extracellular deposits

and/or intrinsic extracellular matrix (ECM) protein components of human Bruch’s

membrane. Two possible biomarkers for non-enzymatic nitration in aged human

Bruch’s membrane have been identified, which include 3-nitrotyrosine and nitrated

A2E. The presence of nitrated A2E could not be detected in RPE extracts, suggesting

that nitro-A2E may be a Bruch’s membrane specific biomarker. The nonenzymatic

glycation and nitration of the basement membrane protein laminin, as a model for

aging Bruch’s membrane, was also investigated. The results indicated that fragments

containing lysine and arginine residues were preferentially modified in the glycated

and irradiated samples. However, nitration of laminin fragments was not observed.

Instead several of the fragments ending in a lysine residue appeared to bind to other

fragments also ending in a lysine residue, indicating a polymerization-type reaction.

This study provides evidence that glycation, nitration, and the presence of A2E may

be involved in modifications to essential basement membrane proteins leading to

deleterious changes within the RPE ECM environment.

NOTHERN ILLINOIS UNIVERSITY DEKALB, ILLINOIS

MAY 2010

THE BIOCHEMICAL STUDY OF AGE-RELATED CHANGES IN HUMAN

RETINAL PIGMENT EPITHELIUM AND BRUCH’S MEMBRANE

BY

LAURA S. MURDAUGH ©2010 Laura S. Murdaugh

A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE

DOCTOR OF PHILOSOPHY

DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY

Doctoral Director: Elizabeth R. Gaillard

UMI Number: 3404858

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ACKNOWLEDGEMENTS

I would like to sincerely thank several individuals who made this work possible.

First, I would like to thank my advisor, Dr. Elizabeth Gaillard, for her guidance, support

and hard work. Her advising and encouragement have been invaluable and have made

me a better chemist. I would also like to thank the members of my dissertation

committee, Dr. James Dillon, Dr. Victor Ryzhov, Dr. James Horn, and Dr. Linda Yasui,

for their input and critically revising my dissertation. I want to give a special thank you

to Dr. James Dillon for always taking the time to answer my questions and assist with

technical problems and research.

I would also like to thank my family, Linda and Gaylord Murdaugh, Ross and

Judy Dill, and Christine and John Leal for their love, support, faith, and never-ending

reassurance throughout the years. Finally, I would like to thank my husband, Adam

Dill, for his love, patience, and understanding. His help and encouragement were

essential to finishing this work.

TABLE OF CONTENTS

Page LIST OF TABLES..................................................................................... ..... vii LIST OF FIGURES..................................................................................... ..... ix Chapter

1. INTRODUCTION…………………………………………... 1

The Visual System……………………………………..... 2

The Retinal Pigment Epithelium………………………. .. 7

Bruch’s Membrane…………………………………….. .. 14

Lipofuscin……………………………………………….. 19

Oxidative Stress and the antioxidant Glutathione…… .… 26 Inflammmation and AMD……………………………… . 32

Advanced Glycation Endproducts and AMD…………… 34

Dissertation Research…………… .................................... 38

2. MATERIALS AND METHODS………………………… … 40

Materials……………………………………………… … 40

Instrumentation……………………………………… ….. 41

Methods……………………………………………… …. 48

Synthesis of A2E………………………………… …. 48

iv Chapter Page

Isolation of Lipofuscin …………………………...... . 48 Auto-Oxidation of A2E…………............................. .. 55

Lipofuscin and A2E LC-MS Analysis……………… 55

Determination of the Water-Octanol Partition Coefficient of A2E: Log P…………………………... 56 Cyclic Voltammetry……………………………… ... 57

Reaction of A2E with Retinalaldehye…………… .... 58

Separation of a Compound with m/z 920 from A2E RAL Reaction Mixture……………………………... 58 Bruch’s Membrane Preparation…………………...… 59 Preparation of Organic Soluble Materials from Bruch’s Membrane………………………………………...… 60 Bruch’s Membrane LC-MS Analysis……………..… 60 Acid Hydrolysis……………………………………... 61 Bruch’s Membrane LC-MS Analysis After Acid Hydrolysis and Standard Addition of 3-Nitrotyrosine…………...........................................…. 62 Conditions of Tryptic Digests for Laminin Samples….63 Modifications with Glycolaldehyde to Laminin………63 Modifications to Laminin with A2E…………………..64 Modifications to Laminin with NaNO2………………. 64 LC-MS Analysis of Laminin Samples………………….65 Protein Prospector……………………………………. 66

v Chapter Page

Bioworks Browser………………………………...... 67

SEQUEST…………………………………………... 67

Data analysis………………………………………... 68

3. THE COMPOSITIONAL STUDIES AND MOLECULAR

MODIFICATIONS OF HUMAN RPE LIPOFSUCIN……. 69

Introduction…………………………………………... .. 69

Results………………………………………………... .. . 72

Discussion……………………………………………… 139

4. AGE-RELATED ACCUMULATION OF A2E AND NITRO-

A2E IN HUMAN BRUCH’S MEMBRANE………………..146

Introduction…………………………………………....... 146

Results………………………………………………..… .151

Indentification of tyrosine nitration in Bruch’s membrane………………………………………… ....151 Identification of nitro-A2E in Bruch’s membrane… ..154 Concentration of nitro-A2E in Bruch’s membrane samples from different decades of life…………… ....169

Discussion……………………………………………......176

5. MODIFICATIONS TO THE BASEMENT PROTEIN LAMININ

AND A2E: A MODEL FOR AGING IN BRUCH’S

MEMBRANE………………………………………………..181

vi Chapter Page

Introduction…………………………………………. 181

Results………………………………………………. 185

Laminin modified with glycolaldehyde……...185

Laminin modified with

carboxymethyllysine........................................203

Laminin modified with A2E……………....…209

Laminin modification with nitrite…… ...……228

Discussion……………………………………………243

6. CONCLUSIONS AND FUTURE WORK………………......248

Compositional studies of human retinal lipofuscin .... .249

Accumulation of 3-nitrotyrosine and nitro-A2E in Bruch’s membrane…………………………...........…250 Modifications to laminin……………………… ....... ..252

REFERENCES……………………………………………….......….255

LIST OF TABLES

Table Page 5.1 Laminin Control: Laminin fragments identified in the

control sample including the observed m/z, associated charge, parent ions (MH+), and corresponding amino acid sequences…………………………………………………… .....189

5.2 Glycated Laminin Sample: Laminin fragments (without modifications) identified in the glycated laminin sample including the observed m/z, associated charge, the MH+, and corresponding amino acid sequence………....196

5.3 Glycated Laminin: Most abundant laminin fragments

modified with glycolaldehyde identified by LC-MS/MS including the observed m/z of the unmodified sequence, the associated charge, the observed m/z of the sequence after modification with glycolaldehyde, the observed intensity associated with the modified m/z, and the corresponding amino acid sequence with site of modification highlighted………………………………………… .....197

5.4 Glycated Laminin: Most abundant laminin fragments

modified with CML identified by LC-MS/MS including the observed m/z of the unmodified sequence, the associated charge, the observed m/z of the sequence after modification with glycolaldehyde, the observed intensity associated with the modified m/z, and the corresponding amino acid sequence with site of modification highlighted……………………………………… ....….205

5.5 Laminin fragments identified in A2E incubated laminin

samples including the observed m/z of the laminin fragment, the associated charge, the MH+, and the corresponding amino acid sequence………………………………………………………...216

viiiTable Page 5.6 Laminin fragments modified with irradiated A2E

including the observed m/z of the laminin fragment, the corresponding amino acid sequence with the site of modification highlighted, the associated charge, and the observed masses of laminin with modification A2E aldehydes…………………………………………………….....217

5.7 Peptide fragment’s CSR, CSRAR, and CSRARK in the laminin control, glycated laminin, A2E laminin control, and irradiated A2E laminin samples including their corresponding observed m/z, associated charge, and retention time. The irradiated A2E sample also includes the mass of the corresponding A2E aldehyde modification……………………………………………………….....229

5.8 Control Laminin Sample: Laminin fragments identified

in the NaCl laminin sample including the observed m/z, associated charge, the MH+, and corresponding amino acid sequence……………………………………………………...…230

5.9 Nitrated Laminin Sample: Laminin fragments identified

in the nitrated sample including the observed m/z, associated charge, the MH+, and corresponding amino acid sequence……………………………………………………...…231

5.10 Peptide fragment’s ARK, CSRARK, and QAASIK in the

laminin control and nitrated laminin sample including their corresponding observed m/z, associated charge, and retention time……………………………………………… ...….233

LIST OF FIGURES

Figure Page 1.1 Anatomy of the human eye…………………………………………......3 1.2 The Retina……………………………………………………………... 5

1.3 The Retinal Pigment Epithelium cell structure showing the

relationship between the RPE cell and Bruch’s Membrane………….... 8

1.4 Formtion of phagolysosme and lipofuscin………………………….... 11

1.5 The visual cycle………………………………………………………. 12 1.6 The position and layers of Bruch’s membrane……………………….. 15 1.7 Transmission electron microscope image of drusen…………………. 18 1.8 Transmission electron microscope image of lipofuscin granule……... 21 1.9 Stucture of A2E and iso-A2E………………………………………....22

1.10 Synthesis of A2E in vivo....................................................................... 23 1.11 Structures of A2E and oxidized A2E with corresponding

aldehydes identified…………………………………………………... 27

1.12 Structure of glutathione (GSH) and its dimer (GSSG)……………….. 30 1.13 Maillard reaction………………………………………………………35 2.1 Electrospray Ionization……………………………………………….. 42 2.2 Taylor Cone…………………………………………………………... 43 2.3 Quadrupole Ion Trap…………………………………………………. 45 2.4 Electron multiplier……………………………………………………. 46

x

Figure Page 2.5 Chromatogram of the A2E reaction mixture using HPLC

with PDA detection. A2E and iso-A2E are identified……………….. .49

2.6 The UV-Vis spectra of A2E and iso-A2E……………………………. 50

2.7 The mass spectrum of purified A2E………………………………….. 51

2.8 The MS/MS spectrum of purified A2E………………………………. 52

2.9 Isolation of Lipofuscin……………………………………………….. 54

3.1 The TIC from the Folch extract of lipofuscin granules (top) And the corresponding PDA chromatogram (bottom) are shown. The chromatogram consists of A2E, oxidized A2E, and a complex mixture of components………………………………..73

3.2 The mass spectrum of the Folch extract of human lipofuscin at time 62.93 mins. Group I, II, and III identify the related clusters of higher molecular weight compounds with mass to charge ratios of approximately 800, 1000, and 1400 respectively. Highlighted in red are the additions of 14 amu starting with m/z 847.9………………………………………………. 75

3.3 The mass spectrum of the Folch extract of human lipofuscin at time 86.26 mins Group II, and III identify the related clusters of higher molecular weight compounds with mass to charge ratios of approximately 1000 and 1400 respectively………..... 76

3.4 The MS/MS scan for A2E identified in the Folch extract

of lipofuscin granules. Peaks corresponding to the mass of 592 (red) with the loss of, 106 (m/z 486.5), 150 (m/z 442), 174 (m/z 418), and 190 (m/z 402) are identified……………………... 77

3.5 The UV-visible spectrum of A2E…………………………………….. 78

3.6 Characteristic cleavages for the fragmentation of A2E………………. 79 3.7 The MS/MS scan of peak with m/z 814 from lipofuscin

sample. Peaks corresponding to the mass of 814 (red) with the loss of 106, 150, 174, and 190 are identified (blue)…............80

3.8 The UV-Vis absorption for the peak with m/z 814……………………81

xi Figure Page 3.9 Possible Structure of m/z 814 with cleavages identified……...............83

3.10 The MS/MS scan for m/z 1081 located in lipofuscin.

Peaks corresponding to the mass of 1081 (red) with the loss of 106 (m/z 975), 150 (m/z 931), 174 (m/z 907), and 190 (m/z 891) are identified (blue)…………………………….....84

3.11 The UV-Visible spectrum of m/z 1081 in lipofuscin………………. ...85

3.12 Possible structure of m/z 1081 with cleavages identified……………..86

3.13 The MS/MS results for the fragmentation of peak with m/z 1423 (red) in the lipofuscin sample. Peaks corresponding to the mass of 1423 with the loss of 174 (m/z 1249) and 190 (m/z 1233) are identified (blue)………………… 87

3.14 Possible structure for m/z 1424 with cleavages identified…………… 88

3.15 Calibration curve for Log P values of DDT, Triphenylamine,

Phenanthrene, Benzophenone, and Cinnamic Acid to determine the Log P of A2E and higher molecular weight products…………………………………………………………… .....90

3.16 Product from esterification reaction with A2E and R group. The R group being acetyl chloride, Hexanoyl chloride, or Cinnamoyl chloride………………………………………………… ...91

3.17 The MS/MS of A2E acetyl ester (m/z 634) with the corresponding structure………………………………………… ...…..92

3.18 The MS/MS of the A2E hexanoyl ester (m/z = 690.5) with the corresponding structure…………………………………… ...93

3.19 CID of main fragment m/z 548 (red) with losses of 150 (m/z 398), 174 (m/z 374), and 190 (m/z 358)(blue)………………… ..94

3.20 CID spectrum of species with m/z = 548 (red) with losses

of 150 (m/z 398), 174 (m/z 374), and 190 (m/z 358) (blue) in full mass spectrum of human lipofuscin sample………………… ...95

xii Figure Page

3.21 Rearrangement of esterification product yielding main

fragment with m/z 548…………………………………………….......96 3.22 Possible structure and fragmentations of peak with m/z = 548…....….97 3.23 MS/MS of Cinnamoyl chloride ester (m/z = 723)…………....……….98 3.24 Proposed product of Cinnamoyl chloride ester (m/z = 723)… ...…....100

3.25 MacLafferty rearrangement in species with m/z = 574……………. ..101

3.26 The mass spectrum of A2E mixture at 93.52 minutes of

chromatographic separation. Peaks found in lipofuscin mixture (Figures 3.2 and 3.3) are identified (blue)…………… ...…..102

3.27 The MS/MS of m/z 859 in A2E. Peaks corresponding to mass 859 (red) with the loss of 150 (m/z 709), 174 (m/z 685), and 190 (m/z 669) are identified (blue)…………… ...…..103

3.28 UV-visible spectrum of m/z 858 in aged A2E…………………… ....104

3.29 The MS/MS scan for m/z 1081 located in aged A2E. Peaks corresponding to the mass of 1081 (red) with the loss of 150 (m/z 931), 174 (m/z 907), and 190 (m/z 891) are identified (blue). The mass of A2E (m/z 592) and additional peaks corresponding to smaller molecular weight compounds (m/z 818 and 745) with similar losses identified in the same sample……………………....…105

3.30 The UV-Visible spectrum of m/z 1081 in aged A2E……………… ..106

3.31 The MS/MS of m/z 859 in reaction mixture for A2E synthesis. Peaks corresponding to mass 859 (red) with the loss of 150 (m/z 709), 174 (m/z 685), and 190 (m/z 669) are identified (blue)…………………………………...…..108

3.32 The MS/MS of m/z 920 in reaction mixture for A2E synthesis. Peaks corresponding to the mass of 920 (red) with the loss of 150 (m/z 771) and 190 (m/z 731) are identified (blue)………………………………………………… ...…109

xiii Figure Page

3.33 The MS/MS of 1189 in reaction mixture for A2E

sythesis. Peaks corresponding to the mass of 1189 with the loss of 150 (m/z 1039), 174 (m/z 1015), and 190 (m/z 999) are identified…………………………… ..……...110

3.34 The UV-Visible spectrum of m/z 859 in reaction mixture for A2E synthesis……………………………………… .......111 3.35 The UV-Visible spectrum of m/z 920 in reaction mixture

for A2E synthesis……………………………………………… ...….112 3.36 The UV-Vis of m/z 1189 in reaction mixture for A2E sythesis…… .113 3.37 The full mass spectrum of the reaction between A2E and

cinnamaldehyde……………………………………………………...114 3.38 The full mass spectrum of the reaction between A2E and

benzaldehyde…………………………………………………… ...…115

3.39 The MS/MS spectrum of the higher molecular weight compound (m/z = 790) in A2E and Cinnamaldehyde reaction mixture using 40 % collision energy. Peaks corresponding to the mass of 790 (red) with the loss of 150 (m/z 640), 174 (m/z 617), and 190 (m/z 556) are identified (blue)……………………………………………… .....116

3.40 The MS/MS spectrum of one of the higher molecular weight compounds in A2E benzaldehyde reaction mixture. Peaks corresponding to the mass of 794 (red) with the loss of 122 (m/z 672), 140 (m/z 654), 150 (m/z 644), and 190 (m/z 604) are identified (blue)………………………………………..117

3.41 Structure and fragmentation of one of the higher molecular weight compounds from reaction of oxidized A2E and cinnmaldehyde……………………………………………………….118

3.42 Possible structure and fragmentation of one of the higher molecular weight compounds from reaction of oxidized A2E and benzaldehyde……………………………………………....119

3.43 The mass spectrum of A2E reacted with all-trans-retinal……………121

xiv Figure Page

3.44 The MS/MS spectrum of m/z 920 from A2E RAL reaction.

Peaks corresponding to the mass of 920 (red) with the loss of 150 (m/z 771) and 190 (m/z 731) are identified (blue)………….. .122

3.45 The MS/MS spectrum of m/z 1189 from A2E RAL reaction.

Peaks corresponding to the mass of 1189 (red) with the loss of 150 (m/z 1039), 174 (m/z 1015), and 190 (m/z 999) are identified (blue)………………………………………………… ...…123

3.46 Possible structure of m/z 920 with cleavages identified………… .....124

3.47 Possible structure of m/z 1189……………………………………… 125

3.48 The MS/MS of m/z 859 in A2E and all-trans-retinal reaction.

Peaks corresponding to m/z 858 (red) with the loss of 150 (m/z 708), 174 (m/z 684), and 190 (m/z 668) are identified (blue)….126

3.49 Possible structure for m/z 859 with cleavages identified…………… 127 3.50 The chromatogram of the A2E RAL reaction mixture using

HPLC and PDA detection. Compound with m/z 920 eluted at 35 min…………………………………………………………….. 128

3.51 The full mass spectrum if peak that eluted at 35 min in

Figure 3.18…………………………………………………………...129 3.52 UV-Vis absorption for the peak with m/z 920……………………… 130 3.53 The MS/MS spectrum of m/z 920………………………………… ...131 3.54 The MS/MS spectrum of m/z 920 from Lipofuscin.

Peaks corresponding to the mass of 920 (red) with the loss of 150 (m/z 771) and 190 (m/z 731) are identified (blue)…….... 132

3.55 The voltammogram of TEAP background………………………….. 133 3.56 The voltammogram of ferrocene……………………………………. 135 3.57 The voltammogram of benzaldehyde……………………………….. 136 3.58 The voltammogram of cinnamaldehyde…………………………….. 137 3.59 The voltammogram of all-trans retinal……………………………… 138

xv Figure Page 4.1 Selected Reaction Monitoring (SRM) chromatogram of 3-NT

and acid hydrolysate of BM (SRM 227.1→181.1).3-NT and acid hydrolysate of BM was analyzed by LC/MS as described in method. The SRM scan of BM acid hydrolysate has a peak with molecular mass 227 and fragment 181 and similar retention time (51 minutes) to 3-NT which indicates the presence of 3-NT in BM acid hydrolysate……………………………………………... 152

4.2 The tandem mass spectra of standard 3-nitrotyrosine and

component with m/z 227.0 at RT 51mins in BM. The tandem mass spectrum of the component at RT 51mins from human BM extracted from 72-75 year old donors is very similar to the tandem mass spectrum of 3-NT. The inset gives the predicted fragmentation of 3-NT……………………………………………….153

4.3 The zoom scan of BM with the standard addition of

3-nitrotyrosine (m/z 227)…………………………………………….155 4.4 The SRM scan of m/z 227 181 from the standard addition

of 3-nitrotyrosine in BM samples from different age groups……….. 156 4.5 Integration of area under SRM scan from Figure 4.4……………….. 157 4.6 Calibration curve for 3-nitrotyrosine………………………………... 158 4.7 The concentration of 3-nitrotyrosine in BM samples from

ages of < 25, 40-60, and > 65 years………………………………….159 4.8 The selected ion chromatograms for synthetic A2E

and nitro-A2E……………………………………………………….. 160 4.9 The UV-Vis spectra for A2E (m/z 592.5) and for

nitro-A2E (m/z 637.5)………………………………………………. 162 4.10 Structures of A2E (m/z 592) and nitro-A2E (m/z 637)

showing characteristic cleavage points and the resulting fragment molecular weights………………………………………… 163

4.11 The tandem mass spectrum of synthetic nitro-A2E

induced dissociation to confirm the identification of nitro-A2E……………………………………………………………. 164

xvi Figure Page 4.12 The tandem mass spectrum of nitro-A2E isolated from 65 yrs

and older BM. Box = mass same in synthetic nitro-A2E and nitro-A2E isolated from 65 yrs and older BM……………………….166

4.13 Chromatogram of m/z 592.5 (A2E), m/z 637.5 (nitro A2E),

m/z 653.4 (nitro A2E plus oxygen), and m/z 682.5 (A2E with 2 nitro substitutions)…………………………………….. 167

4.14 The selected ion chromatograms for A2E (m/z 592) and

nitro-A2E (m/z 637) from RPE lipofuscin and BM extracts from human donor globes that were 65 yrs and older. Note that nitro A2E and A2E from the BM have similar concentrations, whereas no nitro-A2E could be detected from the RPE despite increasing the sensitivity of the detector…………...168

4.15 Integration of A2E in BM samples from different decades

of life (<20, 40, 50, 60, 70, and 80 yrs)……………………………...170 4.16 The concentration of A2E in BM samples from different

decades of life (<20, 40, 50, 60, 70, and 80 yrs)……………………..171 4.17 The SRM scans of Nitro-A2E from BM samples from different

decades of life (<20, 40, 50, 60, 70, and 80 yrs)……………………..172 4.18 Integration of Nitro-A2E A2E in BM samples from different

decades of life (<20, 40, 50, 60, 70, and 80 yrs)………………… .....173 4.19 The concentration of Nitro-A2E in BM samples from different

decades of life (<20, 40, 50, 60, 70, and 80 yrs)………………… .....174 4.20 The concentration of A2E and Nitro-A2E in BM samples

from <20, 40, 50, 60 70, and 80 decades of life and dry AMD……...175 5.1 The amino acid sequence of laminin with B and Y ions identified.....186 5.2 The reaction scheme for glycation of lysine and arginine within the

laminin fragment or with A2E and A2E derived aldehydes……..…..187 5.3 The TIC for a typical enzymatically digested laminin control sample

without modification is shown. The m/z ratios corresponding to fragments with amino acid sequences of CSRARK, AR, CSRARKQAASIKVAVSADR, and CSRAR are identified…..…….188

xvii

Figure Page 5.4 The MS/MS spectrum for digested laminin fragment,

CSR (m/z 365),with B and Y ions identified…………………… .….190 5.5 The MS/MS spectrum for digested laminin fragment,

ARK (m/z 374), with B and Y ions identified…………………..…...191 5.6 The MS/MS spectrum for digested laminin fragment,

QAASIK (m/z 618), with B and Y ions identified…………….….....192 5.7 The MS/MS spectrum for digested laminin fragment,

VAVSADR (m/z 718), with B and Y ions identified……….……….193 5.8 The MS/MS spectrum for digested laminin fragment,

CSRARKQAASIKVAVSADR (m/z 1009), with B and Y ions identified……………………………………………… .……….194

5.9 The MS/MS spectrum for digested laminin fragment,

CSR (m/z 204), modified by glycolaldehyde. The site of glycation is highlighted in red and the B and Y ions are identified in blue…………………………..…………198


CSRAR (m/z 634), modified by glycolaldehyde. The site of glycation is highlighted in red and the B and Y ions are identified in blue…………………………………….…………...199


CSRARK (m/z 762), modified by glycolaldehyde. The site of glycation is highlighted in red and the B and Y ions are identified in blue…………………………… ……………200


QAASIK (m/z 659), modified by glycolaldehyde. The site of glycation is highlighted in red and the B and Y ions are identified in blue………………………………….……………...201

5.13 The ms/ms spectrum for digested laminin fragment,

CSRARKQAASIKVAVSADR (m/z 1051), modified by glycolaldehyde. The site of glycation is highlighted in red and the B and Y ions are identified in blue… .………………..202

xviii Figure Page 5.14 The reaction scheme of glycolaldehyde with lysine producing

carboxymethyl lysine (CML) and then the modification of primary amines in laminin by CML…………………………… ..…..204

5.15 The MS/MS spectrum of CML located in the glycated

laminin sample. The inset is the structure of CML (m/z 205) with characteristic cleavages identified…………… .…….206


ARK (m/z 543), modified by CML. The site of modification is highlighted in red and the B and Y ions are identified in blue…………………………………………..……..207

5.17 The proposed structure for CML modification of ARK fragment ..…208 5.18 The MS/MS spectrum for digested laminin fragment,

CSRARK (m/z 906), modified by CML. The site of modification is highlighted in red and the B and Y ions are identified in blue………………………………………….……...210


QAASIK (m/z 803), modified by CML. The site of modification is highlighted in red and the B and Y ions are identified in blue……………………………………….………...211

5.20 The proposed structure for CML modification of CSRARK

Fragment………………………………………………….……….....212 5.21 The proposed structure for CML modification of QAASIK

Fragment……………………………………………….………….....213 5.22 The cleavage positions and the molecular masses of corresponding

aldehydes in A2E and oxidized A2E are shown…… ……………….214 5.23 The MS/MS spectrum for digested laminin fragment,

CSRAR (m/z 980), modified by A2E derived aldehyde with m/z 406. The site of modification is highlighted in red and the B and Y ions are identified in blue……… ………………….218

5.24 The proposed structure for A2E derived aldehyde (m/z 406)

modification of CSRAR fragment……………….…………………..220

xix Figure Page 5.25 The MS/MS spectrum for digested laminin fragment,

CSRAR (m/z 956), modified by A2E derived aldehyde with m/z 382. The site of modification is highlighted in red and the B and Y ions are identified in blue…………………………… …221


modification of CSRAR fragment……………….……………….….222 5.27 The MS/MS spectrum for digested laminin fragment,

KQAASIK (m/z 1058), modified by A2E derived aldehyde with m/z 366. The site of modification is highlighted in red and the B and Y ions are identified in blue…………………… …….223


modification of KQAASIK fragment……………………… ………..224 5.29 The MS/MS spectrum for digested laminin fragment,

KQAASIK (m/z 1058), modified by A2E derived aldehyde with m/z 382. The site of modification is highlighted in red and the B and Y ions are identified in blue……………… ………….225


modification of KQAASIK fragment………………… ……………..226 5.31 The HPLC total PDA trace of the laminin control, glycated

laminin, A2E laminin control, and irradiated A2E laminin samples are shown respectively. Selected fragments are identified in each chromatogram…………………… ……………….227

5.32 The MS/MS spectrum for digested laminin fragment

QAASIKKRA (m/z 973). The B and Y ions are identified in blue ....234 5.33 The MS/MS spectrum for digested laminin fragment

CSRARKKRARSC (m/z 711). The B and Y ions are identified in blue………………………………………………….….235

5.34 The ms/ms spectrum for digested laminin fragment

QAASIKKISAAQ (m/z 608). The B and Y ions are identified in blue…………………………………………….…...236

5.35 The MS/MS spectrum for digested laminin fragment

ARKKRA (m/z 729). The B and Y ions are identified in blue ……...237

xx Figure Page 5.36 The proposed structure for QAASIKKRA…………………… .…….238 5.37 The proposed structure for CSRARKKRARSC…………….……….239 5.38 The proposed structure for QAASIKKISAAQ…………… ………...240 5.39 The proposed structure for ARKKRA…………………….…………241 5.40 The HPLC total PDA trace of the laminin control and nitrated

laminin samples are shown respectively. Selected fragments, ARK, CSRARK, AND QAASIK, are identified in each Chromatogram………………………………………… …………….242

CHAPTER 1

INTRODUCTION

Age-related macular degeneration (AMD) is the predominant cause of

irreversible blindness in developed countries. Currently, 15 million Americans have

been diagnosed with the disease, with an estimated 2 million new cases each year

(Friedman, O'Colmain et al. 2004). Patients diagnosed with AMD lose their high

resolution central vision. Initially, patients may exhibit mild symptoms of blurring

and distortion but as the disease progresses a complete loss of their central vision

generally occurs.

AMD is characterized as two types: either atrophic (dry) or exudative (wet).

Atrophic AMD involves the degeneration of the retinal pigment epithelium (RPE)

and photoreceptor cells. This is the most common form, accounting for

approximately 85 % of all cases. The more rapidly progressing form, exudative

AMD, occurs in only a small percentage of patients. Choroidal neovascularization is

a predominant symptom associated with exudative AMD. These new blood vessels

created within the eye are generally weak and tend to break open, leading to

bleeding within the retina and a sudden loss in central vision. Previously, literature

has suggested that AMD is one disease with two forms and that a patient with the

atrophic form will often develop the more severe exudative form (Brown, Brown et

al. 2005). However, Hageman et al. recently proposed that AMD is actually two

2 separate and distinct diseases and, therefore, patient with atrophic AMD will not

necessarily develop exudative AMD (Hageman, Luthert et al. 2001). Because of

this ambiguity and the mutifactorial nature of AMD, the exact mechanism leading to

the death of photoreceptor cells and the onset of AMD is still unknown. Recent

research has suggested that age-related changes within the RPE and underlying

Bruch’s Membrane play a crucial role in the development of AMD (Dorey, Wu et al.

1989; Taylor, Munoz et al. 1990; Holz, Bellman et al. 2001). Therefore, age-related

changes to the RPE and Bruch’s Membrane and the mechanisms involved were

investigated. Increasing our understanding of the biochemical and cellular changes

occurring in the RPE and Bruch’s Membrane may aid in the development of new

therapies when diagnosing and treating patients with AMD.

The Visual System

The visual system is a series of complex interlinking processes that enable

an organism to see and is part of the central nervous system. The modular

arrangement of these processes includes lateralized, hierarchical, and parallel

processing. Together these processes enable an organism to discriminate between

colors, objects in motion, patterns, and dimensions. The basic anatomy of the eye,

illustrated in Figure 1.1, is fundamental in understanding how the visual system

works. As light enters the eye through the pupil, the cornea and lens focus the light

onto the retina. Once the light (photons) reaches the retina, photoreceptor cells

absorb the photons. The visual pigments convert these photons into an electrical

3

Figure 1.1 Anatomy of the human eye (McCarthy 2009)

4 signal that stimulates neurons in the retina. Retinal ganglion cell axons located in

the optic nerve then transmit the visual image to the brain.

The cornea is a transparent tissue of mainly collagen that protectively covers

the iris, pupil, and anterior chamber (Oyster 1999). There are five layers that make

up the cornea including the epithelium, Bowman’s membrane, the stroma,

Descemet’s membrane, and the endothelium. Together with the lens, the cornea

refracts light and provides two-thirds of the eye’s focusing power (Cassin and

Solomon 1990; Goldstein 2007). However, this focus is fixed. Therefore, small

adjustments to the eye’s focus are controlled by the lens, where the curvature can be

changed by the ciliary muscles. Located in the anterior segment, the lens also acts as

a UV-filter containing compounds that absorb light from 295 to 400 nm (Dillon,

Zheng et al. 1999; Gaillard, Zheng et al. 2000). Therefore, only light with

wavelengths longer than 400 nm can reach the retina, protecting the retina from

harmful UV-damage. The amount of light that enters the eye is controlled by the

iris and pupil. The iris is a pigmented fibrovascular tissue located in front of the lens.

At the center of the iris is a circular hole called the pupil. The iris regulates the size

of the pupil, effectively changing the intensity of an image, the field of view, and

the depth of focus.

The retina is a complex seven-layered structure located in the back of the

eye, as shown in Figure 1.2. Light initially enters through the ganglion cell layer

and must travel through all cell types before reaching and activating the

photoreceptor cells. The outer segments of both rods and cones contain the light-

5

Figure 1.2: The Retina (Molavi 1997)

6 sensing visual pigment and send signals to the cell bodies in the outer nuclear layer

to axons. These axons in the outer plexiform layer connect with dendrites from

bipolar and horizontal cells. The bipolar cells in the inner nuclear layer process the

signal from the photoreceptor and horizontal cells and then send it to their axons. In

the inner plexiform layer, the bipolar axons connect with ganglion cell dendrites and

amacrine cells. The ganglion cells then send the signal with their axons from the

ganglion cell layer through the optic fiber layer to the optic disc.

An adult retina is on average 22 mm in diameter and .5 mm thick and

contains approximately 7 million cone and 100 million rod photoreceptor cells. The

outer segment of each photoreceptor cell contains an opsin-retinal complex known

as the visual pigment. Each visual pigment contains the same chromophore of 11-

cis-retinal but the type of opsin differs between pigments. The visual pigment in

rods, which are responsible for low light vision, is called rhodopsin. The remaining

three pigments, responsible for color and bright light vision, are found in the

different types of cone cells (Wald 1961; Brown and Wald 1964). These

photoreceptor cells are unevenly distributed throughout the retina. Rods are located

in the peripheral retina whereas cone cells are located almost exclusively in the

fovea, which is responsible for high visual acuity. The area in and around the fovea

that has a yellow pigmentation is called the macula (Kolb, Nelson et al. 2001).

Damage to the macula can cause photoreceptor cells to die, which diminishes high

visual acuity and leads to severe loss of vision.

The 7th layer of the retina, the retinal pigment epithelium, is separated from

the choroid by a thin layer of tissue known as Bruch’s Membrane. The choroid

7 consists of four different layers, including: Haller’s layer, Sattler’s layer, the

choroidal capillaries, and Bruch’s Membrane. Each layer contains different size

blood vessels that supply the eye with oxygen and nutrients. The choroidal

capillaries contain the smallest blood vessels in the choroid. This structure controls

the transport of oxygen, nutrients, and waste by passive diffusion (Olver 1990;

Ramrattan, van der Schaft et al. 1994).

The Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a pigmented single layer of cells

that firmly attaches to the underlying choroid at the basal surface and weakly

attaches to overlying photoreceptor cells at the apical surface (Cassin and Solomon

1990; Boyer, Poulsen et al. 2000). These smooth, hexagonally shaped cells,

illustrated in Figure 1.3, are densely packed together with little extracellular space

between each cell. Rivet-like structures, known as hemidesmosomes, connect the

basal surface of the RPE to the basement membrane and may maintain the cohesive

properties between the RPE and Bruch’s Membrane (Miki, Bellhorn et al. 1975).

The apical surface of the RPE cells contains microvilli, which are cellular

membrane protrusions that increase surface area. These microvilli form a close-

fitting envelope around the ends of the photoreceptor cells (Schraermeyer and

Heimann 1999). RPE cells also develop differently from most monolayer epithelial

cells. Most epithelial cells develop adherin junctions several hours after cell-to-cell

8

Figure 1.3: The Retinal Pigment Epithelium cell structure showing the relationship between the RPE cell and Bruch’s Membrane (Schraermeyer and Heimann 1999).

9 contact is made; however, fully developed RPE cells require weeks after confluence

to develop mature junctions. In addition, RPE cells have been reported to express N-

cadherin instead of E-cadherin (Lagunowich and Grunwald 1989; Davis, Bernstein

et al. 1995; Huotari, Sormunen et al. 1995; Marrs, Andersson-Fisone et al. 1995;

McKay, Irving et al. 1997), which is common in nonepithelial cells. However,

Burke et al. reported that post confluent cultures of RPE cells contained both N- and

E-cadherin (Burke, Cao et al. 1999). The expression of E-cadherin occurs in late

confluency after N-cadherin is already present. Since E-cadherin is involved in

desmosome assembly and Na/K ATPase polarity (Nelson, Shore et al. 1990; Marrs,

Andersson-Fisone et al. 1995; Lewis, Wahl et al. 1997), the late expression of E-

cadherin may be responsible for the absence of desmosomes in the RPE and the

presence of RPE sodium pumps on the apical surface instead of the basolateral

membrane (Burke, Cao et al. 1999).

The RPE has numerous functions that are essential to maintaining the visual

system. Photoreceptor cells are continuously renewed, synthesizing approximately

10 % of the outer segment each day (Young 1971a; Young 1971b). Because of the

close proximity to photoreceptor cells, the RPE is responsible for the phagocytic

uptake and break down of these shed photoreceptor outer segments. New discs are

added to the base of the outer segment while the tips are engulfed and degraded by

the RPE cells sending the waste to the choroidal capillaries. Approximately 3 billon

discs can be engulfed by a single RPE cell in an average lifetime (Marshall 1987).

This continual process of renewal and degradation of the photoreceptor cell outer

segments is crucial in maintaining the viability of the photoreceptor cells because

10 they are continuously exposed to light and a relatively high oxygen concentration.

The photoreceptor cells are susceptible to oxidative damage from reactive oxygen

species (Winkler, Boulton et al. 1999).

The RPE cell phagolysosmal system is highly efficient at digesting large

quantities of the photoreceptor cell’s outer segment discs. These discs are visible in

the RPE cytoplasm as membrane-bound vesicles known as phagosomes. Once

inside the RPE, the phagosome can then fuse with a lysosome forming a

phagolysosome, as seen in Figure 1.4. Under normal circumstances, hydrolytic

digestion starts to break down the proteins, lipids, and polysaccharides within the

phagolysosome. In young eyes, these molecules are generally reduced to 50 % of

their original size in approximately 24 hrs. However, in older eyes, undigestible

material from the phagolysosme, known as lipofuscin, starts to accumulate (Feeney-

Burns and Eldred 1983; Feeney-Burns, Gao et al. 1988). As the eye ages, the

number of photoreceptor and RPE cells disproportionately decreases, which results

in a net increase in lipofuscin, melanosomes, and melanolipofuscin in the remaining

cells. This overloads the RPE cells and decreases the cytoplasmic free space. The

accumulation of lipofuscin, aging, and a variety of other factors changes the

chemical composition within the RPE cells and increases oxidative stress. This can

deleteriously affect their function and viability.

RPE cells are also significantly involved in the visual cycle. Illustrated in

Figure 1.5, the visual cycle involves the repeated movement of retinoids by the

interphotoreceptor retinoid-binding protein (IRBP) between photoreceptor cells and

11

Figure 1.4: Formtion of phagolysosme and lipofuscin (Feeney-Burns and Eldred 1983)

12

Figure 1.5: The visual cycle (Cornwall 2009)

13 the RPE. Since free retinoids damage cells, the IRBP acts as a two-way carrier

transporting retinoids through the interphotoreceptor matrix that separates the

photoreceptor cells and the RPE (Pepperberg, Okajima et al. 1993). The visual cycle

is initially activated when light is absorbed by rhodopsin, which causes the

chromophore, 11-cis-retinal, to undergo photoisomerization to all-trans-retinal. All-

trans-retinal is released from rhodopsin and then reduced to all-trans retinol by all-

trans-retinol dehydrogenase. The all-trans-retinol is then sent back to the RPE by

IRBP to recharge. Once in the RPE, the all-trans-retinol is esterified by lecithin

retinol acyltransferase and converted back to 11-cis-retinal by isomerohydrolase

RPE65. Then, 11-cis-retinal is transported back to the photoreceptor cells by IRBP

where it can bind with rhodopsin, restarting the visual cycle (Rando 2001).

In addition to the phagocytic breakdown of photoreceptor cell outer

segments and processing retinol in the visual cycle, the RPE has several other

specialized functions including creating ion gradients within the interphotoreceptor

matrix, uptake and storage of vitamin A for retinal synthesis, and building up the

blood retinal barrier (Heller and Bok 1976; Bridges, Alvarez et al. 1982;

Schraermeyer and Heimann 1999). The RPE also provides the only transport of

oxygen, nutrients, and waste between the photoreceptor cells and choroid

(Schraermeyer and Heimann 1999).

14 Bruch’s Membrane

Bruch’s Membrane is a thin extracellular membrane that is approximately 2-5 µm

thick depending on the age of the eye (Oyster 1999) and structurally separates the

choroid from the RPE. Illustrated in Figure 1.6, the membrane is composed of five

layers including the basal lamina of the RPE, the inner collagen layer, the elastin

layer, the outer collagen layer, and the basement membrane of the choroidal

capillaries. Although other small compounds are present, Bruch’s Membrane is

primarily composed of collagen, elastin, fibronectin, laminin, and heparin sulfate

(Robey and Newsome 1983; Das, Frank et al. 1990).

Bruch’s membrane has a fundamental association with RPE cells that is

mutually beneficial. Bruch’s Membrane acts as a support for RPE cell attachment

and survival. The RPE cells attach to the inner layer of Bruch’s membrane, the basal

lamina, which contains mostly laminin, Type IV collagen, and heparin sulfate.

Therefore, a significant portion of the extracellular matrix (ECM) environment of

RPE cells comes from Bruch’s membrane. Components in Bruch’s membrane

ECM send signals that trigger cell differentiation, migration, and proliferation.

Synergically with Bruch’s membrane, the RPE maintains Bruch’s membrane by

synthesizing, regulating, and degrading ECM proteins. Changes within this ECM

environment caused by oxidative stress, blue light damage, and lipofuscin or drusen

accumulation can detrimentally affect the cell signals and disrupt cellular function

15

Figure 1.6: The diagram shows the position and layers of Bruch’s Membrane (Anderson, Ozaki et al. 2001)

16 (Dorey, Wu et al. 1989; Winkler, Boulton et al. 1999; Sparrow, Nakanishi et al.

2000; Suter, Reme et al. 2000; Sparrow and Cai 2001; Liang and Godley 2003;

Wang, Paik et al. 2005).

In addition to providing a support and attachment site for RPE cells, Bruch’s

membrane also functions as a barrier that selectively filters nutrients between the

RPE and choroidal capillaries (Hewitt, Nakazawa et al. 1989). Together with the

closely packed RPE cells, Bruch’s membrane forms the retinal blood barrier. This

barrier is critical to maintaining the viability of the retina. Once the barrier is

damaged, fluid can leak into the surrounding structures, causing damage to cells.

Bruch’s membrane also undergoes a variety of structural and compositional

changes with aging. The thickness of Bruch’s membrane increases with age

primarily because of the accumulation of fibrous material within the inner collagen

layer (Mishima 1978; Newsome, Huh et al. 1987). Collagens and their components

within the lamina progressively start to cross-link, which decreases their solubility.

The central elastin layer becomes less organized, undergoing fragmentation and

calcification (Hogan 1972). In addition, previous studies have shown that the

phospholipid, triglyceride, fatty acid, and free cholesterol content in Bruch’s

membrane exponentially increases with age. Curcio et.al have reported that there is

a progressive accumulation of lipids and both esterified and unesterified cholesterol

in Bruch’s membrane (Curcio, Millican et al. 2001; Curcio, Presley et al. 2005).

These cholesterol esters frequently oxidize generating cytotoxic oxysterols, which

have proinflammatory properties (Fliesler 2002). The increase of these components

17 in Bruch’s membrane may be related to the development of drusen (Coffey and

Brownstein 1986).

Drusen are deposits of extracellular material located between the basal lamina of the

RPE and the inner collagen layer of Bruch’s membrane (Figure 1.7). These deposits

generally appear with age and are classified as either hard or soft with varying size,

abundance, and shape. Hard drusen are generally small, round, and have well

defined borders. Soft drusen, although larger than hard drusen, have varying size

and shape, lack well defined borders, and generally have a pale yellow color (Davis,

Gangnon et al. 2005; Ferris, Davis et al. 2005). The presence of soft drusen within

the macula is a common indicator of AMD (Crabb, Miyagi et al. 2002).

Although the exact composition of drusen is still unknown, several studies

have reported the presence of lipids, carbohydrates, and proteins (Hageman, Luthert

et al. 2001; Crabb, Miyagi et al. 2002). Newly formed drusen contain components

that are similar to those found in aging Bruch’s membrane, including membrane-

bound vesicles that rupture, releasing granular and vesicular material. Neutral lipids

found in drusen contain esterified and nonesterified cholesterol and account for

approximately 3 % of the dry weight (Rudolf, Clark et al. 2008). The remainder is a

mixture of proteins, carbohydrates, and cellular components of the RPE. The 129

proteins previously identified in drusen are a combination of blood, extracellular,

and intracellular proteins (Rudolf, Clark et al. 2008; D'Souza, Jones et al. 2009).

Recently, complement factors H and B gene sequence variants have been associated

with an increased risk of developing AMD. Therefore, research focused specifically

18

Figure 1.7: Transmission electron microscope image of drusen (2002)

19 on drusen proteins involved in inflammation (immunoglobulins, factor X, C5, and

C5b complex) and their relation to AMD has increased (Rudolf, Clark et al. 2008).

In addition to lipids and proteins, current data indicates that RPE and photoreceptor

cells are involved in the formation of drusen. Entire cells, cellular organelles and

fragments, basal lamina from the RPE, lipofuscin, and melanin have all been found

in drusen, suggesting a source of origin (Crabb, Miyagi et al. 2002). However, the

exact relationship between RPE dysfunction and drusen formation is still unknown.

Drusen may form as a result of RPE dysfunction initially caused by genetic and

environmental factors, or the accumulation of drusen may actually cause RPE

dysfunction by disrupting the exchange of nutrients and waste between the RPE and

choriodal capillaries, leading to choroidal neovascularization (Sarks 1982; Sarks,

Arnold et al. 1999; Chong, Keonin et al. 2005). The complex composition of drusen,

including proteins, lipids, carbohydrates, and cellular components indicates that its

pathogenesis may be a combined mechanism.

Lipofuscin

Lipofuscin is an autofluorescent heterogenous mixture present in the

cytoplasm of postmitotic cells and is an ordinary morphological sign of aging

(Feeney 1978; Kennedy, Rakoczy et al. 1995; Yin 1996). The composition of

lipofuscin varies between different cell types. In the RPE, it results from the

accumulation of undigestible material after phagocytosis of the photoreceptor cell

outer segments (Feeney-Burns, Hilderbrand et al. 1984; Katz, Drea et al. 1986;

20 Boulton, McKechnie et al. 1989) and forms clusters of granules as seen in Figure

1.8. In an average lifetime, these granules can occupy approximately 20 % of the

cytoplasmic free space (Haralampus-Grynaviski, Lamb et al. 2003).

Although extensive research has been performed involving the mechanism

of lipofuscin formation and its composition, the complex mixture is still relatively

unknown. The mixture has both organic and water-soluble fractions that exhibit

different fluorescence and UV-Visible absorption characteristics (Eldred and Katz

1988; Gaillard, Atherton et al. 1995). As the eye ages, the water-soluble portion

increases (Rozanowska, Pawlak et al. 2004). Initially, Eldred and Katz were able to

isolate 10 different fluorophores in human RPE extract (Eldred and Katz 1988).

Two of these fluorophores were later identified as retinyl palmitate (Lamb, Zareba

et al. 2001) and two isomers of a bis-retinoid pyridinium compound, A2E (2-[2,6-

dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E,5E,7E-octatetraenyl]-1-(2-

hydroxyethyl)-4-[4-methyl-6-- (2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-

hexatrienyl]—pyridinium) and iso-A2E, illustrated in Figure 1.9 (Eldred and Katz

1988; Sakai, Decatur et al. 1996). The chromophore was named A2E because the

synthesis requires two equivalents of all-trans-retinal and one equivalent

ethanolamine to produce the compound. Later, several other minor isomers of A2E

were identified (Parish, Hashimoto et al. 1998).

The mechanism for A2E synthesis in vivo is shown in Figure 1.10. Abundant

in photoreceptor cells outer segments, all-trans-retinal reacts with

phosphatidylethanolamine forming a Shiff’s base known as NRPE. The NRPE

reacts with a second molecule of all-trans-retinal, producing A2PE.

21

Figure 1.8: Transmission electron microscope image of lipofuscin granule (Haralampus-Grynaviski, Lamb et al. 2003)

22

Figure 1.9: Stucture of A2E and iso-A2E (Parish, Hashimoto et al. 1998)

23

Figure 1.10: Synthesis of A2E in vivo (Liu, Itagaki et al. 2000)

24 After the photoreceptor outer segment is phagocytized by the RPE, the phospholipid

group is then hydrolyzed by the enzymes in the RPE lysosomes, forming A2E (Liu,

Itagaki et al. 2000).

The accumulation of lipofuscin has previously been suggested to cause

retinal dysfunction by a variety of different mechanisms. Photochemically,

lipofuscin can generate reactive oxygen species. Boultan et al. first reported that

lipofuscin granules exposed to light generated significant amounts of superoxide

ions (Boulton, Dontsov et al. 1993). The photogeneration of hydrogen peroxide and

singlet oxygen was also observed in lipofuscin. Within the granules, free radicals

such as superoxide ions are primarily responsible for the oxidation of lipids.

Previous research reported that isolated granules exposed to visible light had a 30 %

increase in lipid peroxidation when compared to controls (Rozanowska, Jarvis-

Evans et al. 1995; Rozanowska, Wessels et al. 1998). In addition, lipofuscin

accumulation in RPE cells has previously been studied in vitro and found to be

photochemically toxic to RPE cells (Davies, Elliott et al. 2001). Lipofuscin is also

capable of enzyme inactivation and causes a decrease in the phagocytic capacity of

RPE cells (Sundelin, Wihlmark et al. 1998; Wassell, Davies et al. 1999).

The accumulation of A2E, a major chromophore in lipofuscin, has a harmful

effect on RPE cells. As seen in Figure 1.9, A2E structurally has two hydrophobic

side chains connected by a positively charged pyridinium ring, making the molecule

amphiphilic. This amphiphilic structure may disrupt the integrity of the RPE cell

membranes. Sparrow et al. reported that when high concentrations of A2E are added

to RPE cell membranes, the membranes become irregularly shaped and start to

25 bulge (Sparrow, Parish et al. 1999). Also, by acting as a detergent, A2E can disrupt

the ATP-driven proton pumps, which causes the lysosomal pH to increase,

preventing enzyme activity (Holz, Schutt et al. 1999; Bergmann, Schutt et al. 2004).

In addition, the viability and function of RPE cells change with the accumulation of

A2E. Under irradiation with blue light, A2E induces apoptosis in RPE cells and

damages DNA (Sparrow, Nakanishi et al. 2000; Suter, Reme et al. 2000; Sparrow,

Vollmer-Snarr et al. 2003). A2E was also reported to damage mitochondrial

activity by inhibiting cytocrome c oxygenase (Suter, Reme et al. 2000; Shaban,

Gazzotti et al. 2001), and cause membrane permeabilization inhibiting lysosomal

function (Finnemann, Leung et al. 2002). Recently, the accumulation of A2E was

suggested to cause specific phenotypic changes in the RPE, predisposing the retina

to choroidal neovascularization (Iriyama, Fujiki et al. 2008).

In addition to causing A2E-mediated damage, blue light causes oxidation of

A2E. The oxidation of A2E results in the formation of three different types of

compounds. The first group of compounds arises from the addition of oxygen to

A2E, resulting in masses of 608 amu, 624 amu, and 640 amu. A total of nine oxygen

additions have been observed (Ben-Shabat, Itagaki et al. 2002). These compounds

were initially believed to form epoxides but later were shown to undergo

rearrangement to the more stable furanoid oxides (Dillon, Wang et al. 2004). These

products were identified in RPE cells that were fed A2E and in the organic soluble

portion of human retinal lipofuscin. The second group of compounds results from

the loss or addition of two hydrogen atoms from the oxidized A2E series described

above. The third series of compounds were generated from cleavages along the

26 polyene chain in A2E and oxidized A2E, resulting in a series of lower molecular

weight aldehydes (Figure 1.11) (Ben-Shabat, Itagaki et al. 2002; Avalle, Wang et al.

2004). These aldehydes are highly reactive and are more toxic to the cell than the

oxidation products.

Oxidative Stress and the Antioxidant Glutathione

Oxidative stress results from an imbalance between the production of

reactive oxygen species (ROS) and a system’s ability to remove or repair the

damage caused by these reactive intermediates. Normally, ROS such as superoxide

anions, hydrogen peroxide, hydroxyl radicals, and singlet oxygen are produced at

low levels within a cell and therefore, the damage they create can easily be repaired.

Also, because of their involvement in redox signaling and their ability to kill

pathogens, ROS can be extremely beneficial. However, high levels of oxidative

stress can cause modifications to DNA, proteins, and lipids causing apoptosis or, in

extreme cases, necrosis.

Oxidative stress has been implicated in a variety of diseases including

Alzheimer’s, Parkinson’s and AMD. Because of the high oxygen consumption, the

constant exposure to light, and the presence of polyunsaturated fatty acids, the retina

is particularly susceptible to damage from oxidative stress (Beatty, Koh et al. 2000).

Oxidative stress has been shown to cause damage to irradiated or hyperoxic tissues,

suggesting that light irradiation and retinal damage are related. The high blood flow

27

NHO

+

472

432

406

O

N

HO

Correspondingaldehydes

472 432 406 366

Correspondingaldehydes

488 448 422 382

A2E

Oxidized A2E

+

Figure 1.11: Structures of A2E and oxidized A2E with corresponding aldehydes identified (Wang, Keller et al. 2006)

28 through the choroidal capillaries and the phagocytosis of photoreceptor cells by the

RPE generate a high concentration of hydrogen peroxide (Tate, Miceli et al. 1995).

The accumulation of lipofuscin within the RPE also produces reactive oxygen

species (Boulton, Dontsov et al. 1993; Gaillard, Atherton et al. 1995). Handa et al.

reported that advanced glycation endproducts accumulate in Bruch’s membrane

with age, providing direct evidence that oxidative stress occurs in the vicinity of the

RPE. In addition, RPE cells treated with hydrogen peroxide result in decreased

expression of RPE cell markers (Alizadeh, Wada et al. 2001) and a disruption of

RPE cell junctions and barrier integrity (Bailey, Kanuga et al. 2004).

Several studies have suggested that antioxidants reduce the risk of

developing or decrease the severity of AMD. Under normal conditions, enzymes

maintain a reducing environment within the cell. Changes to this normal redox state

can cause toxicity, resulting in damage to cellular components and disease. Previous

studies have shown that decreased levels of the macular pigments lutein and

zeaxanthin in the central retina result in an increased risk of developing AMD

(Mozaffarieh, Sacu et al. 2003; Ahmed, Lott et al. 2005). A decrease in vitamin E

was also shown to cause retinal degeneration (Hayes 1974). In addition, superoxide

dismutase, catalase, and glutathione peroxidase were reported to have significantly

lower levels in patients with AMD (Evereklioglu, Er et al. 2003).

Glutathione (GSH) is a ubiquitous tripeptide that decreases in concentration

with age, which has been associated with age-related chronic illnesses (Lang, Mills

et al. 2000). Although the exact mechanism for the age-related decrease is unknown,

a reduction in the enzyme activity of glutathione peroxidase, glutathione reductase,

29 and glutathione transferase are contributing factors (Sethna, Holleschau et al. 1982;

Katakura, Kishida et al. 2004; Sastre, Martin et al. 2005). Involved in several critical

cell processes, GSH is essential to maintain and regulate the redox status of a cell

(Hammond, Lee et al. 2001; Schafer and Buettner 2001; Ballatori, Hammond et al.

2005). The ratio of GSH to glutathione dimer (GSSG), Figure 1.12, is an important

indicator of the redox environment of a cell and changes to this ratio have been

associated with cellular proliferation (Suthanthiran, Anderson et al. 1990),

differentiation (Hansen, Carney et al. 2001), and apoptosis (Hammond, Lee et al.

2001). Within the cell, individual organelles have different redox requirements and

therefore, different GSH:GSSG ratios. The endoplasmic reticulum has an oxidizing

environment with a potential of -170 to -185 mV at pH 7.0, giving a ratio of

GSH/GSSG of 1:1 to 3:1 (Hwang, Sinskey et al. 1992). The cytosol has a reducing

environment with a potential of -290 mV at pH 7.0, giving a ratio of GSH/GSSG of

3,300:1 (Ostergaard, Tachibana et al. 2004). Isolated mitochondria have a redox

potential from -250 to -280 mV at a pH 7.8, giving a GSH/GSSG ratio of 20:1 to

40:1 (Outten and Culotta 2004; Rebrin, Zicker et al. 2005; Shen, Dalton et al. 2005;

Hu, Dong et al. 2008). Each cyclic voltammetry experiment used a standard

hydrogen electrode. However, effectively measuring the GSH/GSSG ratio in

mitochondria is difficult because separation of the matrix from the intermembrane

space is nearly impossible and oxidiation of GSH often occurs during cell lysis and

fractionation.

30

Figure 1.12: Structure of glutathione (GSH) and its dimer (GSSG) (King 2009)

31

Within the eye, GSH is found in high levels throughout the lens, cornea,

aqueous humor, and retina, protecting the eye from oxidative damage. The age-

related decrease of GSH levels has been associated with the development of

cataracts, glaucoma, and AMD. Nuclear cataracts progressively deteriorate as the

oxidation of methionine and cysteine residues and the loss of thiol groups in

structural proteins increase. High GSH levels are essential to protect the thiol groups

from ROS. Therefore, a decrease in GSH with an increase in GSSG causes an

imbalance in the GSH/GSSG ratio and extensive protein cross-linking and

insolubility occurs (David and Shearer 1984; Calvin, Medvedovsky et al. 1986).

Glaucoma results from an increase in intraocular pressure with progressive loss of

retinal ganglion cells by apoptosis. The production of ROS and a decrease in GSH

levels causes the apoptosis of the ganglion cells (Maher 2005). In patients with

exudative AMD, the total thiol and GSH concentration significantly decreases

(Coral, Raman et al. 2006), possibly because of insufficient GSH synthesis and

recycling in RPE cells’ (Sternberg, Davidson et al. 1993; Cohen, Olin et al. 1994).

Reduced antioxidant properties in the RPE cells may increase the RPE cells

susceptibility to oxidative stress. Compromised antioxidant defense systems are

associated with age-related eye diseases and therefore supplements replacing these

diminished antioxidants may be therapeutically beneficial.

32 Inflammation and AMD

Chronic inflammation has been implicated in age-related diseases including

Alzheimer’s disease, heart disease, atherosclerosis, and AMD. Inflammation

accelerates the production of free radicals, which can normally be controlled by

antioxidants. However, when inflammation becomes chronic, the accelerated

production of reactive oxygen species (ROS) and reactive nitrogen species (RNS)

leads to increased concentrations of these species and associated tissue damage.

Inflammatory mediators such as prostaglandins are a major source for the

production of superoxide, hydroxyl radicals, and hydrogen peroxide (Chung, Kim et

al. 2001). The activated phagocytes, neutrophils and macrophages, can also produce

significant quantities of superoxide and hydrogen peroxide while simultaneously

activating nitric oxide synthase to produce large increases in nitric oxide (NO)

(Carreras, Pargament et al. 1994; Miller and MacFarlane 1995). Although

considered relatively nonreactive, NO can form more toxic intermediates including

nitrite (NO2-), peroxynitrite (ONOO -), nitrogen dioxide (NO2), and dinitrogen

trioxide (N2O3). These highly reactive intermediates interact with macrophages,

hepatocytes, thiols, and a variety of enzymes causing DNA damage, neurotoxicity,

and apoptosis (Dawson 1995; Ignarro 1996). When superoxide is simultaneously

released with NO, peroxynitrite is produced, which can cause the oxidation of

proteins, lipid peroxidation, and tyrosine nitration. Previous research reported that

60 % of bovine RPE cells died approximately 6 hours after treatment with

peroxynitrite. Also, using an anti nitrotyrosine antibody, protein modification within

33 RPE cells was detected after treatment with peroxynitrite (Behar-Cohen, Goureau et

al. 1996). The nonenzymatic nitration of long-lived proteins has been notably

associated with inflammation (Bailey, Paul et al. 1998; Paik, Dillon et al. 2001).

Paik et al. reported the nonenzymatic nitration of extracellular matrix proteins by

nitrite at physiological pH (Paik, Ramey et al. 1997; Paik, Dillon et al. 2001). In

addition, the nonenzymatic nitration of extracellular matrix proteins was reported to

detrimentally affect RPE function and viability (Wang, Paik et al. 2005) and reduce

the RPE phagocytic capacity (Sun, Cai et al. 2007).

Hageman and colleagues were the first group to suggest that inflammation

and AMD were related based on the presence of immune response proteins in

drusen that were isolated from the retinas of AMD patients (Hageman, Luthert et al.

2001). Immunohistochemical studies later identified a variety of ultrastructural

components within drusen, including: immunoglobulins, components involved in

the complement system (C5a, C3, and C5b-9), molecules involved in the acute-

phase response to inflammation (vitronectin, Amyloid P, and clusterin), and proteins

that maintain and regulate the immune response (fibronectin, ubiquitin, and

apolipoprotein E) (Hageman and Mullins 1999; Hageman, Mullins et al. 1999;

Mullins and Hageman 1999; Rodrigues 2007). In addition, drusen contain

glycoprotein-rich domains from dendritic cells. Later, Johnson et al. proposed that

drusen formation starts after RPE degeneration initiates dendritic cells, which

causes the release of regulatory proteins and activates the complement cascade,

evoking an immune response (Hageman, Luthert et al. 2001; Johnson, Leitner et al.

2001).

34 Direct evidence supporting the relationship between inflammation and AMD

involved four independent genetic studies. The genomes of AMD patients examined

all had the same inherited variant, Y402H, on the same gene called complement

factor H (CFH), which significantly increases a patient’s risk for developing AMD.

CFH regulates inflammation and therefore the inherited variant may result in an

overactive inflammatory process. Since the identification of CFH, AMD

susceptibility variants have also been identified in complement component 2,

complement factor B, and complement component 3. In addition, chemokine

receptor 1, toll-like receptor 4, and the major histocompatibility complex class 1

genes have been suggested to play a role in the development of AMD.

Advanced Glycation Endproducts and AMD

Advanced glycation endproducts (AGEs) are a heterogeneous collection of

modifications, mainly oxidative, that result from the spontaneous reaction of

aldehydes and proteins through the Maillard reaction (Figure 1.13). The Maillard

reaction, or nonenzymatic glycation, refers to chemical reactions involving primary

or secondary amines and carbonyl compounds. In biological systems, the main

source of amines comes from N-terminal amino groups, primary amino groups on

free amino acids, and the ε-amino group on lysine residues within proteins. The

primary sources for carbonyl compounds are from reducing sugars such as glucose,

fructose, and lactose. Initially, there is a nucleophilic substitution between the

carbonyl on a reducing sugar and amino group within a protein producing a Schiff’s

35

Figure 1.13: Maillard reaction (Koldunov, Kononov et al.)

36 base. The Schiff’s base undergoes spontaneous rearrangement to produce a

relatively stable Amadori product. The Shiff’s base and Amadori product can then

further react through polymerization, cyclization, enolization, and oxidation to

produce numerous AGEs. The rate of AGE formation during aging is greater than

the rate predicted by first order kinetics. The rate of the reaction is dependent on the

pKa of the amino group, the location of the amino group within the protein, the

electrophilicity of the carbonyl carbon, and the ratio of the sugar cyclic to acyclic

form (Bunn and Higgins 1981; Baynes, Watkins et al. 1989; Labuza and Baisier

1992; Naranjo, Malec et al. 1998). Therefore over time, there is a significant

accumulation of AGEs on long-lived proteins. The accumulation of these

irreversible AGE adducts depends on the lifetime of the modified protein, oxidative

stress, redox status, and the availability of metal ions. Modification of proteins by

AGEs often leads to protein cross-linking, pigmentation, and fluorescence (Thorpe

and Baynes 2003).

The presence of oxygen can also influence the specificity and rate of AGE

formation. Previous studies have reported that the initial rate of glycation and

selectivity of amino groups within a protein are reduced in the presence of oxygen,

which was attributed to competitive parallel glycoxidation reactions with reducing

sugars (Yeboah, Alli et al. 1999; Yeboah, Alli et al. 2000). These competitive

reactions decrease the concentration of the reducing sugars available to react

through glycation, resulting in a decreased rate of the initial reaction. However,

when transition metal ions and oxygen are both present, oxidation of reducing

sugars easily occurs. For example, aldoses form glyoxal and glucosone, which are

37 more reactive oxidation products. Since these products are more effective at

glycating primary amines and the guanidine group on arginine residues, the rate of

glycation reactions will increase as their concentration increases (Hayase,

Yamamoto et al. 1996).

The formation and presence of AGEs has been reported to accelerate age-

related changes and contribute to age-related diseases including; arthritis, cataracts,

diabetic retinopathy, and AMD. Previous literature has reported that AGEs

accumulate in human BM and basal deposits. Specifically, carboxymethyllysine

(CML) was the first AGE identified in BM and drusen from AMD patients

(Ishibashi, Murata et al. 1998). CML and pentosidine, a fluorescent cross-linking

AGE, were reported to increase in BM with age (Handa, Verzijl et al. 1999; Glenn,

Beattie et al. 2007). AGEs have also been detected in the RPE as free adducts or as

AGE-modified proteins in lipofuscin granules (Schutt, Bergmann et al. 2003). RPE

cells that were grown on AGE-modified substrate accumulated an increased

quantity of lipofuscin, which is related to a decrease in lysosomal enzyme activity

(Glenn, Mahaffy et al. 2009). AGE receptors such as RAGE, AGE-R1, and AGE-

R3 have also been reported to increase in the RPE and photoreceptor cells or BM of

AMD patients (Howes, Liu et al. 2004; Gu, Yuan et al. 2009). The presence of

RAGE in vivo has been associated with chronic inflammation. The activation of

RAGE and AGEs changes CD59, a major regulatory protein, and increases the

inflammatory response (Cheng and Gao 2005). In addition, AGEs also occur at

relatively high concentrations in the membranes associated with choroidal

neovascularization (CNV) (Swamy-Mruthinti, Miriam et al. 2002). Elevated in BM

38 of AMD patients, the AGEs CML and carboxyethylpyrrole, promote

neovascularization in vivo by stimulating vascular endothelial growth factor

(VEGF) (Kobayashi, Nomura et al. 2007), which is related to wet AMD. Several

AGEs have also been reported to produce the expression of pro-angiogenic growth

factor in RPE in vitro (Zhou, Cai et al. 2005). Therefore, AGEs have been

implicated in the pathology of several retinal diseases, suggesting their potential

benefit as critical biomarkers in diagnosing a patient’s susceptibility to these

diseases.

Dissertation Research

Vision loss associated with AMD is currently the predominant cause of

irreversible blindness in developed countries. As a result of the increased number of

documented cases and the severity of the disease, research focused specifically on

the origin and progression of the disease is essential in order to treat patients

effectively. The characteristic central vision loss associated with AMD is caused by

photoreceptor cell death. However, the exact mechanism leading to the death of

these cells and the onset of AMD is still unknown. The retina consists of several

layers including the neural retina (neurons and photoreceptors), retinal pigment

epithelium (RPE), and Bruch’s membrane. Recent research has suggested that age-

related changes within the RPE and underlying Bruch’s membrane may play a

crucial role in the development of AMD. These changes include the accumulation

of debris called lipofuscin and its major chromophore, A2E, in the RPE and the

39 development of lipid-like deposits on and in Bruch’s membrane from the RPE.

Therefore, this dissertation will focus on investigation of these age-related changes

in the RPE and Bruch’s Membrane.

One of the major contributors to detrimentally affect RPE cell viability is the

accumulation of lipofuscin. However, the origin of lipofuscin granules is still

unknown. Therefore, the structures and reactivities of the higher molecular weight,

more hydrophobic relatives of A2E within lipofuscin granules, were investigated to

identify the compounds and suggest possible sources of formation. In addition to

damage caused by lipofuscin, RPE cells are also affected by alteration to Bruch’s

membrane including the accumulation of debris, chemical modifications, and

compounds involved in inflammation. This study also focuses on the formation of

A2E and A2E-related compounds within Bruch’s membrane and modifications to

extracellular matrix proteins by nitrite, glycolaldehyde, and A2E as possible sources

for age-related changes observed in patients with AMD. These results will increase

the understanding of biochemical and cellular changes occurring in RPE cells and

Bruch’s membrane in relation to AMD.

Chapter 2

MATERIALS AND METHODS

Materials

All chemicals used were of the highest possible purity commercially

available. All solvents used were HPLC grade and were purchased from Thermo

Fisher Scientific Inc. (Waltham, MA). All-trans-retinal, tryptophan, dithiothreitol,

ammonium bicarbonate, urea, iodoacetamide, glycolaldehyde, formic acid, acetic

acid, sodium nitrite, ammonium acetate, sodium chloride, sulfanilamide, N-naphyl-

ethylenediamine, hydrochloric acid, dichloro-diphenyl trichloroethane,

triphenylamine, phenanthrene, benzophenone, and cinnamic acid, ferrocene,

benzaldehyde, cinnamaldehyde, 3-nitrotyrosine, and the Cys-laminin α chain were

purchased from Sigma Aldrich Co. (St. Louis, MO). Ethanolamine was purchased

from ACROS Organics (Pittsburgh, PA). The sequencing grade modified trypsin

was purchased from Promega Corp. (Madison, WI). Water was purified by using a

Millipore Milli-Q Plus PUREpak 2 (18.2 MΩ) water purification system.

41Instrumentation

The UV-Visible absorption spectra were obtained from the Ocean Optics

spectrophotometer (Dunedin, FL). The HPLC system consists of Hewlett Packard

quaternary pump (Ti series, 1050, Hewlett Packard, France) with a diode array

detector. The column used for separations was a C-18 reverse phase (RP), 250 ×10

mm, and C-12 RP, 150 × 4.60, 4 μm size columns from Phenomenex (Torrance,

CA). For mass spectrometric analysis, a Thermo Finnigan LCQ Advantage with

Surveyor LC-pump (Thermo electron, San Jose, CA) was used. Steady-state

irradiation was performed using a Philips special blue light (Oriel, Stratford, CT,

model number 6292) in a quarter-inch acrylic glass irradiating chamber.

Electrospray Ionization Mass Spectrometry (ESI-MS) was used in all

proceeding studies to analyze human retinal lipofuscin, Bruch’s membrane, and the

modifications to laminin and A2E. Electrospray ionization is a powerful technique

usually coupled to mass spectrometry, which creates ions from a solution containing

the analytes of interest at atmospheric pressure (Figure 2.1). The sample is injected

through a sample loop or syringe into capillary tubing or emitter, where a high

voltage is applied (2-5 kV). The liquid sample then reaches the tip of the emitter

where a Taylor cone is formed. At the center of the cone a jet of liquid sample is

emitted, which ends in a fan-shaped plume (Figure 2.2). The initial sample generally

has acid such as acetic acid or TFA added to it to increase the conductivity of the

solution, which decreases the size of the droplets initially formed. The charged

droplets then undergo further nebulization by interacting with an inert gas such as

42

Figure 2.1: Electrospray Ionization (Gates 2004)

43

Figure 2.2: Taylor Cone (New Objective 2004)

44nitrogen. The charged particles then enter the first vacuum area of the mass

spectrometer through an ion transfer tube. This tube is heated, which heats the

counter flow nitrogen gas, increasing evaporation of the charged droplets. The

evaporation continues until the particles become unstable, reaching their Rayleigh

limit. At this critical limit, Coulombic explosion occurs, creating even smaller

droplets. This process continues until the analyte ions of interest are released from

the droplets. The charge on the droplets and subsequent ions formed depends on the

voltage initially applied. The charged ions are then carried to the mass analyzer,

which is a quadrupole ion trap (Figure 2.3) in the LCQ Advantage. The trap is made

up of two hyperbolic endcap electrodes with a ring electrode in between. Within the

quadrupole ion trap, constant direct current (DC) and radio frequencies (RF) and

oscillating alternating current (AC) electric fields are used to trap the ions. Ions are

then separated and sequentially ejected based on the stability of their trajectories in

the oscillating field. The ions are then carried to the detector, which is a continuous

dynode electron multiplier in the LCQ Advantage (Figure 2.4). After leaving the

mass analyzer, ions strike the starting electrode with enough energy to cause

secondary emission. The emitted electrons are then accelerated down the multiplier

where the electrons can strike again, producing even more electrons and amplifying

the signal. The secondary electrons are eventually collected at the end of the

electron multiplier at a second electrode known as the anode. The signal can then be

recorded and displayed.

The LCQ Advantage has several scan types that were used to analyze human

retinal lipofuscin, Bruch’s membrane, and laminin, which include single-stage full

45

Figure 2.3: Quadrupole Ion Trap (Gates 2004)

46

Figure 2.4: Electron Multiplier (Kvech 2000)

47scans, two-stage full scans, selected reaction monitoring, and zoom scans. The

single-stage full scan type has one stage of mass analysis. The ions formed from ESI

are stored in the mass analyzer. Then, these ions are sequentially scanned out of the

mass analyzer to produce a full mass spectrum. The two-stage full scan type has two

stages of mass analysis. In the first stage, the ions from ESI are stored in the mass

analyzer. Then, the ion of a certain mass-to-charge ratio, also called the parent ion,

is selected and all other ions are ejected from the mass analyzer. The precursor ion

is excited, causing collisions with the background gas (helium) that is present in the

mass analyzer. These collisions cause the parent ion to fragment, producing

fragment ions. These daughter ions are stored in the mass analyzer and then are

sequentially scanned out of the mass analyzer to produce a full product ion mass

spectrum (MS/MS or MS2). Selected reaction monitoring (SRM) is a two-stage

technique in which precursor ion and fragment ions are monitored. In the first stage

of mass analysis, the ions formed from ESI are stored in the mass analyzer. The

parent ion is selected and all other ions are ejected from the mass analyzer. Then,

the parent ion is excited and collides with helium. The collisions of the parent ion

cause fragmentation, generating the corresponding daughter ions. The parent and

corresponding daughter ions of interest are then stored in the mass analyzer and all

other ions are ejected. These selected ions are then sequentially scanned out of the

mass analyzer, producing the SRM product ion spectrum. Finally, to confirm the

molecular weight and charge state of certain compounds, higher resolution zoom

scans were also performed.

48Methods

Synthesis of A2E

A2E for all reactions was prepared from all-trans-retinal and ethanolamine in

acetic acid and ethanol as previously described by Parish et al. (Parish, Hashimoto

et al. 1998). The mixture was stirred in the dark for three days at room temperature.

After excess solvent was removed by drying under argon, the A2E was separated

from the initial reaction mixture using a HP 1050 Ti HPLC and a C18 RP column.

Using an isocratic gradient of MeOH:H2O (90:10) and a flow rate of 1.0 mL/min,

the retention time of A2E was approximately 28 min monitored with a photodiode

array detector at 430 nm, as shown in Figure 2.5. The concentration of the purified

A2E was determined by measuring the absorbance at 439 nm using an Ocean Optics

spectrometer, given an extinction coefficient of 36,900 L/mol•cm (Parish,

Hashimoto et al. 1998). The absorption spectra for A2E and iso-A2E are displayed

in Figure 2.6. After collection, the pure A2E fraction (Figure 2.7) was confirmed on

the LCQ Advantage mass spectrometer using collision induced dissociation (CID)

(Figure 2.8). The sample was then dried under argon and stored at -70 ºC for further

analysis.

Isolation of Lipofuscin

Human RPE lipofuscin granules were extracted and isolated from donor

49

Figure 2.5: Chromatogram of the A2E reaction mixture using HPLC with PDA detection. A2E and iso-A2E are identified.

50

Figure 2.6: The UV-Vis spectra of A2E and iso-A2E

200 250 300 350 400 450 500 550 600

nm

iso A2E A2E

0

200

400

1000

800

600

mAU

51

200 300 400 500 600 700 800 9000.00E+000

1.00E+008

2.00E+008

3.00E+008

4.00E+008

5.00E+008

6.00E+008

7.00E+008 592.5

In

tens

ity (

AU

)

m/z

Figure 2.7: The mass spectrum of purified A2E (m/z 592)

52

352.4364.4376.4

402.4

418.4

442.4

468.4

486.5

536.5576.5

592.6

300 400 500 600

0.0

2.0x105

4.0x105

6.0x105

8.0x105

1.0x106

1.2x106

1.4x106

1.6x106

1.8x106

2.0x106N

HO

124

150

174

190

402

442

418

468

Inte

nsity

(A

U)

m/z

MS/MS 592

Figure 2.8: The MS/MS spectrum of purified A2E (m/z 592)

53globes (Midwest Eye Banks and Transplantation Centers, Chicago, IL) as

previously described by Feeney-Burns (Feeney-Burns and Eldred 1983). Extraneous

fat and muscle were removed from the periphery of the eyeballs to reduce

contamination by other cells and to aid in dissection. The outside of the sclera was

cut mid-coronal, approximately one centimeter posterior to the cornea, with a razor

blade. An incision was made through the sclera and fine-tipped scissors were used

to cut the eyeball following the initial path from the razor blade. After the eyeball

was cut into two parts, the anterior portion of the eye was removed and discarded

along with the vitreous humor. A cold (4 oC) phosphate (0.1 M) buffered sucrose

solution (0.32 M) was pipetted into the eyecup so that the level was below the edge

of the incision (to prevent contamination of the eyecup) and the neural retina was

allowed to float up in the sucrose solution. The optic nerve was cut at the base

where it enters the interior of the eye and the neural retina was removed. The

eyecup was filled with approximately 1 mL of the sucrose solution and brushed

gently with a camel hair paint brush to remove the RPE cells, which were placed

into a 15 mL centrifuge tube. The eyecup was repeatedly brushed until all the cells

appeared to be removed and the sucrose solution appeared clear. The solution was

then pipetted out of the eyecup into the centrifuge tube, which was subsequently

centrifuged for 5 min at 100X G using a Beckman J2-HS centrifuge and a JA-20

rotor to remove unbroken cells and melanin. The supernatant was then layered on a

3 step sucrose density gradient of 0.63, 1.37, and 2.25 M, which was then

centrifuged for 15 min at 8,500X G. The interface between the top two layers was

then removed, lyophilized, and frozen at -70 oC (Figure 2.9). To obtain the organic-

54

Figure 2.9: Isolation of Lipofuscin (Feeney-Burns and Eldred 1983)

55soluble portion of lipofuscin, the samples were re-suspended and a Folch

extraction was performed using a 1:1:1 ratio of CHCl3:CH3OH:H2O. The organic

soluble portion was then dried under argon, resuspended in 1 mL MeOH, and

analyzed using ESI-MS/MS with with simultaneous PDA detection.

Auto-Oxidation of A2E

After synthesis and purification, 2 mL aliquots of 15 µM A2E was

transferred to a 4 ºC refrigerator in the dark. To determine the products formed

from the auto-oxidation of A2E over time, an aliquot (20 µL) was removed from the

sample at times 0, 30 and 60 days and analyzed on ESI-MS/MS with simultaneous

PDA detection.

Lipofuscin and A2E LC-MS Analysis

All samples were analyzed on a Thermo Finnigan LCQ Advantage mass

spectrometer. The mass spectrometer was set to positive ion mode with a capillary

temperature of 200 °C, source voltage of 4.0 kV, capillary voltage of 42 V, and a

tube lens offset of 50 V. The mass-to-charge ratios were collected from 200 to 2000

and a normalized collision energy between 30-40 % was used for the MS/MS data.

The lipofuscin and purified A2E samples were separated using the Surveyor LC

system with a Synergi Max-RP C12 column. The flow rate was set to 0.2 mL/min

56with a mobile phase of MeOH balanced with H2O (both containing 0.1 % formic

acid) using a gradient of 80 % MeOH for 30 min and 80-100% MeOH for 90 min.

Determination of the Water-Octanol Partition Coefficient of A2E: Log P

A stock solution of purified A2E was determined to have a concentration of

approximately 5 x 10-5 M by UV-Visible spectroscopy in methanol at 439 nm. A

0.5 mL aliquot of the purified A2E was added to 5.00 mL of octanol in a 30 mL

separatory funnel. The separatory funnel was inverted several times and allowed to

equilibrate for one hour. Next, the two layers were separated and collected for

HPLC analysis. Triplicate injections of each layer were performed on an Hewlett

Packard 1050 HPLC with a 100 µL sample loop and a Phenomenex Synergi (4 µm,

15 mm x 5 mm) column while monitoring the absorbance at 439 nm. The peak area

of each injection was recorded and used for calculation of the partition coefficient.

The theoretical Log P value was also calculated using the Sparc software program

and was then compared to the experimental Log P value for A2E.

To determine the approximate partition coefficients for the higher molecular

weight compounds located in the lipofuscin samples, A2E, dichloro-diphenyl

trichloroethane, triphenylamine, phenanthrene, benzophenone, and cinnamic acid

were separated by HPLC using a flow rate of 1.0 mL/min on a C12 RP column and

an isocratic gradient of 90:10 MeOH:H2O while monitoring the UV-Vis. The

elution times and known partition coefficients were plotted to form a linear least-

squares calibration curve (Figure 3.15), which was then used to determine the

57approximate partition coefficients for the higher molecular weight compounds in

the lipofuscin sample based on extrapolated linear correlation to the compound

elution time.

Cyclic Voltammetry

All voltammograms were obtained using a three electrode system

comprising a platinum button working electrode, a platinum wire counter electrode,

a silver wire as a quasi-reference electrode, a BioAnalytical Systems, Inc. CV27

Potentiostat, and an analog-to-digital converter equipped with computer data

acquisition. The scan rate was set at 50 mV/s. Also, each scan was started at 0.0 V

in forward (positive potential) direction and the gain was constant throughout the

experiments. All half-cell potentials were reported with respect to the

ferrocene/ferrocenium half-cell potential (vs. Ef(Fc+/Fc)) for reversible reactions.

For non-reversible reactions, the simple peak potential was reported vs. Ef(Fc+/Fc).

A background solution containing 0.1 M tetraethylammonium perchlorate (TEAP)

in acetonitrile was prepared. All acetonitrile was dried using 5°A molecular sieves

prior to electrochemical analysis. This solution was then analyzed with the CV27

potentiostat to determine the working potential window. The potential window used

for analysis was from -1.75 to 1.6 V. An internal standard containing solution was

prepared containing 0.05 M ferrocene and 0.1M TEAP in acetonitrile. This internal

standard containing solution was then analyzed using the above system with

identical analysis parameters to identify the position of the ferrocene/ferrocenium

58redox couple. Also, this solution was used as the solvent to prepare the analyte-

containing solutions. To prepare these solutions, the appropriate masses of the

analytes--benzaldehyde (0.05 M), cinnamaldehyde (0.05 M), and all-trans retinal

(0.015 M)--respectively--were added to a 100 mL volumetric flask and diluted to

volume using the internal standard containing solution.

Reaction of A2E with Retinaldehyde (RAL)

After synthesis and purification, a 5 mL aliquot of 50 µM A2E was mixed

with 100 µM RAL and a catalytic amount of acetic acid. The mixture was then

bubbled with argon and irradiated for 1 h. An aliquot was then removed and diluted

with methanol and analyzed on ESI-MS/MS with simultaneous PDA detection.

Separation of a Compound with m/z 920 from A2E RAL Reaction Mixture

Once the A2E and RAL reaction was complete, a 200 μL aliquot was

injected onto a HP 1050 Ti HPLC using a C18 RP column. Using a flow rate of 1

mL/min, the compound with m/z 920 eluted at approximately 35 min with a

gradient of 80:20 MeOH/H2O for 15 min followed by a linear increase to 100%

MeOH over 10 min, which was then maintained for an additional 35 min. The peak

that eluted at 35 min was collected and confirmed using direct injection on the LCQ

Advantage mass spectrometer. This procedure was then repeated multiple times

59until approximately 5 mg was collected. The sample was then dried and

resuspended in deuterated chloroform for future analysis.

Bruch’s Membrane Preparation

Donor globes were purchased from Chicago Eye Bank (Midwest Eye Banks

and Transplantation Centers). BM tissues from different decades, including donors

of 18, 40, 50, 60, 70, and 80 years of age, and patients diagnosed with dry AMD

were used. The preparation of BM followed the method described by Karwatowski

et al.(Karwatowski, Jeffries et al. 1995). The eye globe was opened by

circumferential incision along the iris. The lens and vitreous humor were separated.

The neuronal retina was removed and the BM and choroid complex was incubated

in 0.01 vol% trypsin in 10 mM phosphate buffer solution (PBS, pH 7.4) for 10 min

at 37 oC and subsequently rinsed in PBS. The tissue was then gently brushed to

remove the RPE cells and choroidal tissue. According to Karwatowski et al.

(Karwatowski, Jeffries et al. 1995), this treatment removed most of the debris from

the basement membrane and left only Bruch’s membrane and some choriodal

capillaries, and only a small amount of collagen (4%) was released during this

treatment. Once the RPE was removed, BM was gently cut out.

60Preparation of Organic Soluble Materials from Bruch's Membrane

Isolated Bruch’s membrane was cut into small pieces and placed in a

homogenizer. An equal amount of CHCl3:CH3OH:H2O was added and gently

homogenized to extract the organic soluble components. Glass wool was inserted

into a Pasteur pipette and the homogenized Bruch’s membrane sample was filtered

through the pipette, separating the solid from the supernatant. The organic layer of

the extract was separated from the water-soluble layer by decanting. The organic

supernatant was centrifuged for 15 min at a speed of 5000 rpm. The supernatant

from the centrifuged solution was collected and the excess solvent was evaporated

under argon. Approximately 50 μL of methanol was added to the dried extract and

20 μL of the extract solution was injected and analyzed by liquid chromatography –

tandem mass spectrometry (LC-MS/MS).

Bruch’s Membrane LC-MS Analysis

To investigate the synthesized nitro-A2E and organic solvent extracts of

Bruch's membranes, samples were dissolved in methanol as described above and

analyzed by LC-ESI-MS/MS. The conditions for mass spectrometry for the organic

soluble extract of BM were: positive polarity, capillary temperature of 200 oC,

source voltage of 4.5 kV, capillary voltage of 43 V, and tube lens offset of 50 V,

m/z range: 200-1,000, and a normalized collision energy of 25%. The separation

was carried out on a 1504.6 mm Synergi Max-RP C12 column using a linear

61gradient of 85% to 96% methanol for 60 min and 96%-100% methanol for 10 min

with a balance of water containing 0.1% trifluoro acetic acid (TFA) and a flow rate

of 0.3 mL/min. For synthesized nitro A2E analysis, the separation was carried out

using an isocratic mobile phase of 5% methanol for 10 min and linear gradient of 5-

100% methanol for 30 min balanced with water with 0.1% formic acid and a flow

rate of 0.3 mL/min (monitored at 430 nm, 350 nm, and 250 nm). The compounds

with m/z values of 592, 637, 653 and 682 were selected for subsequent MS/MS

scans using normalized collision energy of 52%. These are the molecular weights of

A2E, nitrated A2E, nitrated A2E plus one oxygen, and A2E with two sites of

nitration. The mass spectrometer was set as source voltage 4 kV, capillary voltage

3.3 V, capillary temperature 200 oC, and tube lens voltage of 25 V.

Acid Hydrolysis

Bruch’s membranes from different decades of life were pooled into three

samples including < 25 yrs, 40-60 yrs, and >65 yrs. These samples were dissected

and prepared as previously described and then hydrolyzed in 6 M HCl at 110 oC for

24 hours using homemade glass tubes with Teflon-lined screw caps. Before

hydrolysis, deoxygenation of the samples was achieved by six freeze-pump-thaw

cycles. After samples were placed in the tubes, air was removed by applying a

vacuum for approximately 5 min. After hydrolysis, excess acid was evaporated

using argon gas. The samples were then resuspended in 50L H2O and spiked with

6250 µL of 100 µM 3-nitrotyrosine. The samples were then analyzed by LC-MS

and the concentration was calculated using standard addition.

Bruch’s Membrane LC-MS Analysis After Acid Hydrolysis and Standard Addition

of 3-Nitrotyrosine

HPLC separation was performed using a Synergi Max-RP C12 column (150

×4.6 mm). To analyze the acid hydrolysates of Bruch's membrane, the LC mobile

phase was acetonitrile (ACN) balanced with H2O (both containing 0.1% TFA) with

the following gradients: 1-10% ACN for 50 min, 10-60% ACN for 30 min, 60-

100% ACN for 20 min and a flow rate 0.2 mL/min. The conditions for mass

spectrometry (Thermo Finnigan LCQ Advantage and Surveyor LC system, San Jose,

CA) were: positive polarity, capillary temperature of 200 oC, source voltage of 4.5

kV, capillary voltage of 43 V, and tube lens offset of 50 V, m/z range: 200-1,000,

normalized collision energy of 30% to investigate whether 3-nitrotyrosine (m/z of

[MH]+ is 227.1) was present within the sample. The MS method contains one zoom

scan (m/z 222.1-232.1), one MS2 scan with a parent mass of 227.1 and a selective

reaction monitoring (SRM) scan with a parent mass of 227.1 and a fragment mass of

181.1 (corresponding to loss of a nitro group).

63Conditions of Tryptic Digests for Laminin Samples

Enzymatic digests were performed on all laminin samples. Each protein

was prepared to have a concentration of 1mg/mL in water. A solution of 8 M urea

and 0.4 M ammonium bicarbonate (pH 7.5- 8.5) was prepared as the digestion

buffer. An aliquot of 150 µL of the protein was added to 200 µL of the urea and

ammonium bicarbonate solution. A 50 µL sample of 50 mM dithiothreitol was

added to the sample and then allowed to incubate at 50 ºC for 15 mins. After

cooling to room temperature, a 50 µL sample of 100 mM iodoacetamide was added

to the protein sample and left to react in the dark for 15 mins. The sample was then

diluted by adding 350 µL of Milli-Q water. The Promega grade trypsin was

suspended in 200 µL of 50 mM acetic acid and a 25 µL aliquot was removed and

added to the diluted protein sample. The sample was then allowed to incubate at 37

ºC overnight or up to a total of 24 hrs.

Modifications with Glycolaldehyde to Laminin

The Cys-laminin α chain, CSRARKQAASIKVAVSADR, was dissolved in

1 mL of Milli-Q water and divided into two equivalent samples. The first sample

was digested with trypsin for 18 hrs and then dried under argon. Glycolaldehyde

modified laminin was prepared by adding 150 µL of 50 mM glycolaldehyde

solution to 150 µL of 0.1 mg/mL Cys-laminin α chain. The concentration of

glycolaldehyde was selected based on previous literature reports regarding the

64glycation of proteins with glycolaldehyde (Nagai, Matsumoto et al. 2000;

Nakajou, Horiuchi et al. 2005). The mixture was then incubated for 12 hrs at 37 ºC.

All aliquouts were dialyzed using PBS (1mM KH2PO4, 10 mM Na2HPO4, 137 mM

NaCl, 2.7 mM KCl, pH 7.4) to removed unreacted glycolaldehyde.

Modifications to Laminin with A2E

An additional sample of 1.0 mg/mL Cys-laminin α chain was dissolved in 1

mL of Milli-Q water. The purified A2E (1 mL, 18 µM) was then added to the

laminin peptide in the dark. The mixture was divided into two equivalent aliquots.

The first aliquot was kept in the dark at room temperature for 60 min. The second

aliquot was irradiated through a quarter-inch piece of acrylic glass with a Phillips

“Special Blue” bilirubin bulb for 60 min. The bilirubin bulb produces a narrow

bandwidth of blue light approximately 420-480 nm that is used to treat

hyperbilirubinemia (Sarici, Alpay et al. 1999).

Modifications to Laminin with NaNO2

A suspension of 1 mg of laminin in 200 mM NaNO2 or 200 mM NaCl

dissolved in 10 mM phosphate buffer (pH 7.4) was prepared. Both samples were

incubated in the dark for approximately 7 days at 37 °C. The excess salt was then

removed by dialysis against 10 mM phosphate buffer. The dialysis was stopped

once the solution surrounding the dialysis tubing was Griess assay negative for

65nitrite modification (Tsikas, Gutzki et al. 1997; Romitelli, Santini et al. 2007).

Initially, sulfanilamide (2mg/ml) and 4 N HCl were added to 1 ml aliquot of the

dialysis solution in a 1:1:1 ratio. Next, 1 ml of N-naphyl-ethylenediamine (NED)

(1mg/ml) was added to the previous mixture. The nitrite in the solution reacts with

the sulfanilamide in acid to form a diazonium salt. The salt then reacts with the

NED, producing a stable azo compound, which has an intense purple color. The

dialysis solution was determined to be Griess assay negative once the solution

remained clear after the addition of these compounds. The proteins within the

dialysis tubing were then removed and a tryptic digest was preformed followed by

analysis with LC-MS.

LC-MS Analysis of Laminin Samples

All samples were prepared in triplicate and then separated and analyzed on a

ThermoFinnigan LCQ Advantage and Surveyor LC system using a Synergi Max-RP

C12 column. The mass spectrometer was set to positive polarity, a capillary

temperature of 200 °C, source voltage of 4.0 kV, capillary voltage of 42 V, and a

tube lens offset of 50 V. The mass-to-charge ratios were collected from 200 to 2000

and a normalized collision energy of 35 % was used for the tandem mass

spectrometry data. The laminin control and glycolaldehyde modified laminin

samples were separated using a flow rate 0.2 mL/min and a mobile phase of MeOH

balanced with H2O (both containing 0.1 vol% formic acid) with the following

gradient: 1-10% MeOH for 60 min, 10-70% MeOH for 60 min, and 70-100% for 60

66min. The A2E and laminin samples were separated using the same flow rate and

mobile phase but the gradient started with 40-70% MeOH for 60 min, 70-90%

MeOH for 60 min, and 90-100% MeOH for 60 min. For each sample, a data-

dependent method was designed to acquire one full MS scan and three MS/MS

scans for the three most abundant peaks in the full MS scan. The data generated

from the mass spectrometer was then analyzed using information regarding enzyme

digests, Protein Prospector, and SEQUEST software. The data from all control

samples is provided as supplemental material.

Protein Prospector

(http://prospector.ucsf.edu/prospector/mshome.htm)

Protein Prospector is a proteomics tool for searching sequence databases

to compare data from mass spectrometry experiments. The MS-Digest option was

used to obtain and compare the results from the enzymatic digest for laminin control.

The minimum and maximum fragment masses were set to 100 and 4000 Da with a

minimum fragment length of one amino acid. The enzyme was set to trypsin with a

maximum number of missed cleavages set to 5 and multiple charges reported. The

peptide fragment entered was CSRARKQAASIKVAVSADR with the instrument

set to ESI-ION-TRAP.

67Bioworks Browser

The Bioworks browser program 3.1 enables the analysis of raw data files

generated in X-calibur. All chromatograms were analyzed using the pepmap and

pepmatch software within the Bioworks browser software package. Pepmap was

used to identify separated digest fragments of peptides resulting from enzyme

digestion. Pepmap matches the acquired spectrum against the predicted digest

fragment masses. The parameters were set to 5 % threshold, a scan width of 1, a

mass tolerance of 1.5, and a maximum of 5 incomplete digest with no disulfide

bonds. Pepmatch was used to predict the product ions of a peptide analyzed by CID.

Pepmatch calculates the mass-to-charge ratio of each predicted fragment and

matches those masses to peaks in the displayed mass spectrum. The parameters

were set to display and compare generated B and Y ions, with a threshold of 2 %,

and multiple charges. The identification of these fragments was then compared and

confirmed using SEQUEST.

SEQUEST

SEQUEST is a proteomics tool which correlates tandem mass spectra data

with amino acid sequences from protein databases. A protein database was

generated for the laminin fragment and used as the reference. The parameters for a

positive match on SEQUEST were set to a delta correlation (DelCn) of 0.1, a

preliminary score (Sp) of 200, and the ion probability of 70 % coverage. The cross

68correlation value (Xcorr) was set to the standard 1.9 for +1, 2.2 for +2, and 3.75

for +3 charged peptides. Modifcations from nitrite were identified based on the

mass addition of 45 and corresponding absorption spectra from the PDA output. The

modifications from glycolaldehyde were based on the mass additions of 42 and 102,

which correspond to the addition of one or two molecule of glycolaldehyde with the

loss of water. The modifications from A2E had to be confirmed by identifying the

peaks that were not present in the control and analyzing the peaks in the MS/MS

data set.

Data Analysis

A standard t-test was used for all statistical analysis with a p<0.05 indicating

that the difference between groups was statistically significant. In addition, ANOVA

one-way statistical analysis with a 95 % confidence level was performed on the

Bruch’s membrane samples from different decades.

CHAPTER 3

THE COMPOSITIONAL STUDIES AND MOLECULAR MODIFICATIONS OF

HUMAN RPE LIPOFUSCIN

Introduction

As organism’s age, many metabolically active post mitotic cells accumulate

autofluorescent lysosomal storage bodies known as lipofuscin. Lipofuscin is a

brown-yellow, electron-dense, aging pigment that is composed of a complex

heterogeneous mixture of lipid-protein aggregates that form clusters of granules in

the RPE. Within the human eye, these granules are believed to be formed from the

indigestible material of phagocytized photoreceptor outer segments (Feeney-Burns

and Eldred 1983; Boulton, McKechnie et al. 1989) and may account for up to 19 %

of the cytoplasmic volume by the age of 80 (Feeney-Burns, Hilderbrand et al. 1984;

Weiter, Delori et al. 1986; Davies, Elliott et al. 2001).

Lipofuscin has also been shown to generate a series of reactive oxygen

species (ROS), which include singlet oxygen, hydrogen peroxide, and superoxide

anions (Boulton, Dontsov et al. 1993; Gaillard, Atherton et al. 1995; Rozanowska,

Wessels et al. 1998; Wassell, Davies et al. 1999; Davies, Elliott et al. 2001).

Considered photochemically toxic, lipofuscin was found to decrease phagocytic

70capacity (Sundelin, Wihlmark et al. 1998). Previous studies have shown that both

the isolated granules and the organic soluble extract of lipofuscin are extremely

photoreactive (Gaillard, Atherton et al. 1995; Rozanowska, Wessels et al. 1998;

Winkler, Boulton et al. 1999).

One of the major fluorophores of lipofuscin, A2E, has been extensively

studied since it was first isolated by Eldred et al. Structurally, A2E is a pyridinium

bis-retinoid, which is synthesized using two moles of all-trans-retinal (RAL) and

one mole of ethanolamine (Eldred and Katz 1988; Eldred and Lasky 1993; Parish,

Hashimoto et al. 1998). After A2E could be chemically synthesized, research

focused on the effect A2E had on cellular function. Previous literature reported that

visible and UV radiation can cause lesions on the neural retina and RPE cells. The

RPE cells are unusually susceptible to damage by wavelengths corresponding to the

blue region of the visible spectrum, which is where A2E has the strongest

absorbance. Previous research has reported that RPE cells fed A2E were severely

damaged or killed after they were irradiated with blue light.

Another possible source of light-induced A2E mediated damage is related to

the photo-oxidation of A2E, which generates ROS, such as peroxide and superoxide

radicals (Reszka, Eldred et al. 1995; Ragauskaite, Heckathorn et al. 2001). Ben-

Shabat et al. were the first to propose that the photo-oxidation of A2E results in

higher molecular weight compounds that differ by 16 amu, resulting in multiple

epoxide formation along the polyene chain (Ben-Shabat, Itagaki et al. 2002).

However, because of the acidic environment within a lysosome, the allylic epoxides

would be unstable and undergo rearrangement. Dillon et al. proposed that these

71allylic epoxides of A2E would rearrange forming a furanoid oxide structure,

which is relatively stable (Dillon, Wang et al. 2004). Furthermore, oxidative

cleavage of side chains results in the formation of highly reactive aldehydes and

ketones, which could readily react with cell constituents and cause irreversible

damage (Wang, Keller et al. 2006). This explanation is based on the structural

similarities between carotenoids and A2E. In addition, the amphiphilic structure of

A2E may be responsible for detergent-like action in membrane disruption. The

quaternary amine structure of A2E may also aid in the inhibition of lysosomal

function by complexing to specific lysosomal enzymes (Eldred and Katz 1988).

Even though the formation and composition of lipofuscin and the major

fluorophore A2E have received notable attention, the origin of the granules and the

identity of most of the compounds and the consequence of A2E accumulation within

the granules are still unknown. One hypothesis suggested that A2E could exist in a

free or esterified form. In the RPE, all-trans retinol, produced from the visual cycle,

is converted to all-trans retinyl ester, which then self-aggregates into a retinosome

(Imanishi, Gerke et al. 2004). This prevents hydrophobic interactions with cellular

components that would disrupt normal cell function. Since A2E is extremely

hydrophobic and accumulates within RPE lysosomes, A2E was suggested to

undergo a similar esterification reaction (Mandal 2008). In addition to the

esterification reactions, a second hypothesis involving the modification of A2E by

A2E derived aldehydes was also suggested. Within the acidic lysosomal

environment, A2E undergoes rearrangements and oxidation, generating aldehydes

and ketones that are structurally similar to ß-carotene oxidation products

72(Sommerburg, Langhans et al. 2003). These aldehydes are extremely reactive and

in the presence of A2E may interact, forming higher molecular weight products.

Therefore, in this study, lipofuscin was analyzed using a reversed-phase

HPLC with an electrospray ionization mass spectrometer (ESI-MS) to investigate

the hydrophobic compounds that elute later than A2E and that absorb radiation with

wavelengths greater than 400 nm (Figure 3.1). The results indicate that a large

quantity of the components of lipofuscin have mass spectra analogous to that of

A2E, but with higher molecular weights as determined by their fragmentation

pattern with losses of 190, 174 and/or 150 amu and the formation of fragments of ca

592 amu. The vast majority of the relatively hydrophobic components correspond

to derivatized A2E with discrete molecular weights of 800-900 m/z, 970-1080 m/z

and above 1200 m/z regions. These modified components increase the

hydrophobicity of A2E and may explain the formation of lipofuscin granules in the

RPE. The present study is part of a continuing effort to identify the molecular

modifications to the structure of A2E (Dillon, Wang et al. 2004; Wang, Keller et al.

2006) and their mechanisms of formation.

Results

To study the composition of lipofuscin, samples were isolated from donor

globes and analyzed on LC-MS as previously described in Chapter 2. The total ion

chromatogram (TIC) and total absorbance from the Folch extract of lipofuscin

granules are displayed in Figure 3.1. The chromatogram consists of A2E, oxidized

73

Figure 3.1: The TIC from the Folch extract of lipofuscin granules (top) and the corresponding PDA chromatogram (bottom) are shown. The chromatogram

consists of A2E, oxidized A2E, and a complex mixture of components

74A2E, and a complex mixture of components. Integration of the peak areas

indicated that A2E comprised only approximately 5-10 % by volume relative to the

complex mixture. We assumed that all molecules in the mixture have similar

ionization efficiency and all of the instrumental parameters, flow rate and solvent

composition remained constant. After further analysis of the mass spectral data of

compounds that eluted from 50-100 mins, the mass spectrum revealed a series of

closely related compounds that differed by a mass of 14 amu. Representative mass

spectra of compounds that eluted at approximately 60 mins and 80 mins are

displayed in Figures 3.2 and 3.3, respectively. There are three clusters of eluting

masses, labeled I, II, and III, in the range of 800, 1000, and 1400 amu that exhibit

the characteristic addition of 14 amu.

To determine if these components were structurally related to A2E, the

MS/MS data was analyzed. The MS/MS spectrum and the total absorbance of A2E

are displayed in Figures 3.4 and 3.5, respectively. The fragmentation pattern

displayed characteristic losses of 150, 174, and 190 amu from the parent ion mass of

592 amu. These distinctive cleavages are illustrated in Figure 3.6. Once identified,

these losses were compared to the MS/MS data of the components located within

the complex mixture of the lipofuscin sample and all of the material that eluted

between 50-80 mins and approximately 50 % of the material between 80-110 min

had analogous spectra.

Figures 3.7 and 3.8 display the MS/MS and absorbance spectrum of peak

with m/z 814, which is representative of components that eluted at approximately 62

min in the TIC displayed in Figure 3.1. Once analyzed, Figure 3.7 displayed ions

75

700 750 800 850 900 950 1000 1050 1100 1150 1200

0.0

2.0x105

4.0x105

6.0x105

8.0x105

1.0x106

1.2x106

1.4x106

1.6x106

1.8x106

2.0x106

Inte

nsity

(A

U)

m/z

862.8

874.9

876.9

878.8

860.8

831.0

1020.8

1022.9998.9

971.0

847.9

814.2

Figure 3.2: The mass spectrum of the Folch extract of human lipofuscin eluted at time 62.93 mins. Groups I, II, and III identify the related clusters of higher

molecular weight compounds with mass to charge ratios of approximately 800, 1000, and 1400, respectively. Highlighted in red are the additions of 14 amu starting with

m/z 847.9.

76

400 600 800 1000 1200 1400 1600 1800 2000

0.0

2.0x105

4.0x105

6.0x105

8.0x105

1.0x106

1.2x106

1.4x106

In

ten

sity

(A

U)

m/z

904.9

927.0

948.9

1083.2

1081.1

1277.0

1455.0

1863.61472.0

Figure 3.3: The mass spectrum of the Folch extract of human lipofuscin eluted at time 86.26 min. Groups II and III identify the related clusters of higher molecular weight compounds with mass to charge ratios of approximately 1000 and 1400,

respectively.

77

200 250 300 350 400 450 500 550 600

0

1x106

2x106

3x106

4x106

Inte

nsi

ty (

AU

)

m/z

592.6

402.4

418.4

442.4

392.4

352.3

486.5

536.4

Figure 3.4: The MS/MS scan for A2E identified in the Folch extract of lipofuscin granules. Peaks corresponding to the m/z of 592 (red) with the loss of 106 (m/z

486.5), 150 (m/z 442), 174 (m/z 418), and 190 (m/z 402) are identified.

78

200 250 300 350 400 450 500 550 600

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

1.2x105

Inte

nsi

ty (

AU

)

Wavelength (nm)

Figure 3.5: The UV-visible absorbance spectrum of A2E

79

Figure 3.6: Characteristic cleavages for the fragmentation of A2E

80

358.3384.4434.5

450.4488.5

508.5

532.5

558.5

598.5

624.6

640.6

663.6

708.6722.6

758.7798.7

813.79 6 6 3 1 8

400 600 800

0.0

5.0x105

1.0x106

1.5x106

2.0x106

Inte

nsi

ty (

AU

)

m/z

Figure 3.7: The MS/MS scan of peak with m/z 814 from lipofuscin sample. Peaks corresponding to the mass of 814 (red) with the loss of 106, 150, 174, and 190 are

identified (blue).

81

300 400 500 600

0

1x104

2x104

3x104

4x104

5x104

6x104

7x104

Inte

nsity

(A

U)

Wavelength (nm)

Figure 3.8: The UV-Vis absorption for the peak with m/z 814

82with masses of 663, 640, and 634, which correspond to losses of 150, 174, and

190 from the parent ion of 814. The proposed structure is displayed in Figure 3.9,

which could form from the addition of one molecules of all-trans retinal to A2E

with the loss of water and the ethanol group on A2E. Next the components that

eluted with masses in the range of 1000 and 1400 amu were analyzed, and the

MS/MS data was again compared to the fragmentation pattern of A2E. Figures 3.10

and 3.11 present the MS/MS and absorbance spectra for m/z 1081. The

fragmentation pattern for m/z 1081 displayed ions with masses of 931, 907 and 891,

which correspond to the characteristic losses of 150, 174, and 190 from the parent

ion. The proposed structure is displayed in Figure 3.12, which represents the

addition of two molecules of all-trans retinal to A2E with the loss of water and

ethanol group. Figure 3.13 presents the MS/MS spectrum of m/z 1424. The

fragmentation pattern displayed losses of 174 and 190 displaying fragments with

masses 1249 and 1233. The proposed structure is displayed in Figure 3.14, which

represents the addition of one molecule of A2E aldehyde with m/z 472 and one

molecule of A2E aldehyde with m/z 422 to A2E with the loss of the ethanol group

on A2E and water. The spectra for m/z 1081 and 1423 also displayed fragmentation

ions that were the same as the compounds, m/z 757, 803, and 814, located within

group I of the lipofuscin samples. These data suggest that components in group II

and III result from the polymerization of derivatives from group I.

In addition to MS/MS data, the Log P of A2E was measured to determine

the aggregative characteristics of A2E in aqueous environments. Using HPLC, the

absorbance was measured and peak area was used to calculate the Log P of 7.3 +/-

83

Figure 3.9: Possible Structure of m/z 814 with cleavages identified

84

400 500 600 700 800 900 1000

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

1.2x105

1.4x105

1.6x105

Inte

nsity

(A

U)

m/z

1081

592

891813

907

931

825

865799

767487 663

729975

Figure 3.10: The MS/MS scan for m/z 1081 located in lipofuscin. Peaks corresponding to the mass of 1081 (red) with the loss of 106 (m/z 975), 150 (m/z

931), 174 (m/z 907), and 190 (m/z 891) are identified (blue).

85

300 400 500 600

0

1x105

2x105

3x105

4x105

5x105

6x105

7x105

Inte

nsi

ty (

AU

)

Wavelength (nm)

Figure 3.11: The UV-Visible spectrum of m/z 1081 in lipofuscin

86

Figure 3.12: Possible structure of m/z 1081 with cleavages identified

87

400 600 800 1000 1200 1400

0.0

5.0x104

1.0x105

1.5x105

2.0x105

Re

lativ

e A

bu

nd

an

ce (

AU

)

m/z

1423

1233757

863

795

1057

1393

931 1019 1135 1249

1219

1203592

566620

Figure 3.13: The MS/MS results for the fragmentation of peak with m/z 1423 (red) in the lipofuscin sample. Peaks corresponding to the mass of 1423 with the loss of

174 (m/z 1249) and 190 (m/z 1233) are identified (blue).

88

Figure 3.14: Possible structure for m/z 1424 with cleavages identified

89 1, which was in agreement with computational values using the Sparc software

program (Log P = 8.2 +/- 1). Using this value for A2E, a calibration curve of

compounds with similar Log P values (Figure 3.15), and elution times from the TIC

of lipofuscin, the approximate Log P values for the higher molecular weight

components were calculated. Group I was determined to have an approximate Log P

value of 8.3 +/- 0.5 followed by group II and III, which were approximately 9.2 +/-

0.7 and 10.2 +/- 1, respectively.

To investigate the possibility that these modifications of A2E resulted from

esterification, A2E was first treated with either acetyl chloride or hexanoyl chloride

to synthesize the esters as indicated in Figure 3.16. The MS/MS obtained from

esterification reaction of acetyl A2E is displayed in Figure 3.17 with the structure

displayed as an inset. The MS/MS for the A2E hexanoyl ester is displayed in Figure

3.18 with the proposed structure displayed as an inset. The resulting spectra for

A2E acetyl and hexanoyl esters gave a major fragment with m/z = 548. Further

fragmentation of this major peak (m/z = 548) yielded fragments, with m/z = 358,

410, 374 (Figure 3.19), which was also located in the human lipofuscin sample

(Figure 3.20). This fragment can readily be explained by the rearrangement depicted

in Figure 3.21, giving the structure displayed in Figure 3.22. However, the parent

ions from the A2E esterified products are not seen in the lipofuscin sample and,

therefore, these products are not structurally related to those found in the lipofuscin

mixture.

A2E was further esterified with cinnamoyl chloride, which more closely

structurally resembles A2E. Figure 3.23 displays the MS/MS data for the A2E

90

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

Log K

Lo

g P

Figure 3.15: Calibration curve for Log P values of DDT, Triphenylamine, Phenanthrene, Benzophenone, and Cinnamic Acid to determine the Log P of A2E

and higher molecular weight products.

91

Figure 3.16: Product from esterification reaction with A2E and R group. The R group is acetyl chloride, Hexanoyl chloride, or Cinnamoyl chloride (Mandal 2008).

92

100 200 300 400 500 600 700

0

1x105

2x105

3x105

4x105

5x105

358.3

548.4634.4

NO

CO

CH3

Inte

nsi

ty (

AU

)

m/z

Figure 3.17: The MS/MS of A2E acetyl ester (m/z 634) with the corresponding structure (Mandal 2008)

93

200 300 400 500 600 700

0.0

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

3.0x105

358.3

548.4

574.5

N O O

Inte

nsity

(A

U)

m/z

Figure 3.18: The MS/MS of the A2E hexanoyl ester (m/z = 690.5) with the corresponding structure (Mandal 2008)

94

200 300 400 5000.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

198.2

241.2

358.3

374.3410.2

506.4

548

Inte

nsity

(A

U)

m/z

398.2

Figure 3.19: CID of main fragment m/z 548 (red) with losses of 150 (m/z 398), 174 (m/z 374), and 190 (m/z 358)(blue) (Mandal 2008)

95

200 300 400 5000.0

5.0x104

1.0x105

1.5x105

2.0x105

241.2

348.4

358.3

398.4

410.4

442.5 492.4

548.6

Inte

nsi

ty (

AU

)

m/z

374.2

Figure 3.20: CID spectrum of species with m/z = 548 (red) with losses of 150 (m/z 398), 174 (m/z 374), and 190 (m/z 358) (blue) in full mass spectrum of human

lipofuscin sample

96

N

O

H O

NH

O

O

m/z 548

Figure 3.21: Rearrangement of esterification product yielding main fragment with m/z 548 (Mandal 2008)

97

412372

346

242

358

Figure 3.22: Possible structure and fragmentations of peak with m/z = 548

98

400 500 600 700

0.0

2.0x103

4.0x103

6.0x103

8.0x103

1.0x104

1.2x104

359.3 483.2

533.3

575.5

617.4723.4

Inte

nsity

(A

U)

m/z

549.2

3.23: MS/MS of Cinnamoyl chloride ester (m/z = 723) (Mandal 2008)

99cinnamoyl ester with the proposed structure displayed in Figure 3.24. The

spectrum is relatively simple, giving one major fragment (m/z 575), which was

interpreted as resulting from a McLafferty rearrangement (Figure 3.25) (Mandal

2008). Again, these results were also not consistent with the compounds found in

lipofuscin.

A second hypothesis that may account for the hydrophobic mixture in

lipofuscin involved reactions with A2E-derived aldehydes (Figure 2.4). It was

proposed that, once formed, these aldehydes could then react with other A2E

molecules, forming higher molecular weight species. To investigate this hypothesis,

A2E reaction mixture was allowed to incubate at 4 °C for 60 days. This sample led

to a complex mixture, which included many of the compounds found in vivo. The

aged A2E samples kept at 4 ºC for 0, 30 and 60 days were then analyzed to

determine if similar products were formed from pure A2E over time. The TIC for

the aged A2E sample after 60 days displayed peaks similar to the lipofuscin sample,

including ions with m/z of 859 and 1081 (Figure 3.26). The MS/MS for 859 and

corresponding absorbance spectrum are displayed in Figures 3.27 and 3.28,

respectively. The MS/MS for 1081 and corresponding absorbance spectrum are

displayed in Figures 3.29 and 3.30, respectively. Both figures displayed peaks

corresponding to losses of 150, 174, and 190 from the parent ion. In addition, the

absorption spectra show two peaks with maxima at 330 and 500 nm. However, the

intensity of the ion generated for the 1081 peak was smaller than the intensity of the

ions generated for the 859 peak. Also, after 60 days, the more hydrophobic higher

100

Figure 3.24: Proposed product of Cinnamoyl chloride ester (m/z = 723)(Mandal 2008)

101

N

O

N

O

H

Figure 3.25: MacLafferty rearrangement in species with m/z = 574 (Mandal 2008)

102

813.8831.8

839.9

859.4

887.9

907.1 1032.2

1081.7

1113.1

1188.1

1203.1

1236.1

131

800 1000 1200

0.0

5.0x105

1.0x106

1.5x106

2.0x106

2.5x106

3.0x106

3.5x106

4.0x106

4.5x106

Inte

nsi

ty (

AU

)

m/z

1070.1981.4

784.3

Figure 3.26: The mass spectrum of A2E fraction that eluted at 93.52 minutes of chromatographic separation. Peaks found in lipofuscin mixture (Figures 3.2 and 3.3)

are identified (blue).

103

400 600 800

0

1x105

2x105

3x105

4x105

685Inte

nsi

ty (

AU

)

m/z

415

669

859643

709

753721603531493 630

Figure 3.27: The MS/MS of m/z 859 in A2E. Peaks corresponding to mass 859 (red) with the loss of 150 (m/z 709), 174 (m/z 685), and 190 (m/z 669) are identified

(blue).

104

300 400 500 600

0.0

2.0x103

4.0x103

6.0x103

8.0x103

1.0x104

1.2x104

Inte

nsity

(A

U)

wavelength (nm)

m/z 859

Figure 3:28: UV-visible absorption spectrum of m/z 858 in aged A2E

105

400 500 600 700 800 900 1000 1100

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

Inte

nsi

ty (

AU

)

m/z

1008.7

1051.7

907.6

891.6

818.6

754.5

663.3

689.6

642.2

591.8 931.6

1081

Figure 3.29 The MS/MS scan for m/z 1081 located in aged A2E. Peaks corresponding to the mass of 1081 (red) with the loss of 150 (m/z 931), 174 (m/z 907), and 190 (m/z 891) are identified (blue). The mass of A2E (m/z 592) and

additional peaks corresponding to smaller molecular weight compounds (m/z 818 and 745) with similar losses identified in the same sample.

106

300 400 500 600

0

1x104

2x104

3x104

4x104

5x104

6x104

7x104

Inte

nsi

ty (

AU

)

Wavelength (nm)

Figure 3.30: The UV-Visible absorption spectrum of m/z 1081 in aged A2E

107molecular weight compounds located within the lipofuscin sample were either

absent from the aged A2E sample or were not abundant enough for adequate

identification. The MS/MS of three of the major peaks with m/z = 859, m/z = 920,

and m/z = 1188 are displayed in Figures 3.31, 3.32, and 3.33 with characteristic

losses of 150, 174 and 190 identified. The corresponding absorbance spectra for

each compound are displayed in Figures 3.34, 3.35, and 3.36, respectively. Based on

these spectra, the compounds with m/z 920 and 859 are clearly related.

To investigate the specific mechanism of formation of the higher molecular

weight compounds formed from the reaction of A2E with aldehydes, A2E was

reacted with specific aldehydes, either cinnamaldehyde or benzaldehyde, for

approximately 12 h in the dark. The resulting full mass spectra from

cinnamaldehyde and benzaldehyde showed completely oxidized A2E and peaks

with the oxidized A2E and attached aldehydes (Figures 3.37 and 3.38). As in

human lipofuscin, the reactions with A2E appear as a series of discrete groups. For

both the cinnamaldehyde and benzaldehyde reactions, group I is A2E and its

oxidation products, group II is group I plus the addition of one aldehyde, and group

III is the addition of a second aldehyde.

The fragmentation patterns of one of the higher molecular weight

compounds in A2E cinnamaldehyde and benzaldehyde reactions are displayed in

Figure 3.39 and Figure 3.40 with corresponding proposed structures in Figures 3.41

and 3.42. The fragmentation of major peaks showed similar patterns with losses of

190, 174, and 150, which were also observed in oxidized A2E. The loss of 148 in

108

414.4454.4

478.4530.3 602.5

642.5

658.6

668.5

684.6708.6

828.6

5 4 5

400 600 800

0.0

2.0x104

4.0x104

6.0x104

Inte

nsity

(A

U)

m/z

859.6

Figure 3.31: The MS/MS of m/z 859 in reaction mixture for A2E synthesis. Peaks corresponding to mass 859 (red) with the loss of 150 (m/z 709), 174 (m/z 685), and

190 (m/z 669) are identified (blue).

109

565.5605.5 657.5

669.6

684.5708.2

730.6770.5

793.6

859.6

890.6

920.6

600 800

0

1x105

2x105

3x105

4x105

Inte

nsi

ty (

AU

)

m/z

Figure 3.32: The MS/MS of m/z 920 in reaction mixture for A2E synthesis. Peaks corresponding to the mass of 920 (red) with the loss of 150 (m/z 771) and 190 (m/z

731) are identified (blue).

110

709.8

749.8

770.7810.7849.7

873.8

925.7

937.8998.8

1014.8

1039.7

1082.8

1127.8

800 1000

0.0

2.0x105

4.0x105

6.0x105

8.0x105

1.0x106

1.2x106

Inte

nsi

ty (

AU

)

m/z

Figure 3.33: The MS/MS of 1189 in reaction mixture for A2E sythesis. Peaks corresponding to the mass of 1189 with the loss of 150 (m/z 1039), 174 (m/z 1015),

and 190 (m/z 999) are identified.

111

250 300 350 400 450 500 550 600

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

1.2x105

1.4x105

Inte

nsi

ty (

AU

)

wavelength (nm)

Figure 3.34: The UV-Visible absorption spectrum of m/z 859 in reaction mixture for A2E synthesis

112

200 300 400 500

1x105

2x105

3x105

4x105

5x105

6x105M+ 920

Inte

nsi

ty (

AU

)

Wavelength (nm)

Figure 3.35: The UV-Visible absorption spectrum of m/z 920 in reaction mixture for A2E synthesis

113

300 400 500

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

3.0x105

3.5x105

Inte

nsity

(A

U)

Wavelength (nm)

M+ 1189

Figure 3.36: The UV-Visible absorption spectrum of m/z 1188 in reaction mixture for A2E sythesis

114

400 600 800 1000

0.0

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

3.0x105

Ox A2E + 2CAL

Ox A2E + CAL

Inte

nsi

ty (

AU

)

m/z

551.2

641

642.1

788.9

952.5836.7

772.9

686.9

708

657805

625

Ox A2E

Figure 3.37: The full mass spectrum of the reaction between A2E and cinnamaldehyde (Mandal 2008)

115

500 600 700 800 900 1000

0.0

5.0x105

1.0x106

1.5x106

2.0x106

2.5x106

3.0x106

3.5x106

Ox A2E + 2BALOx A2E + BAL

Inte

nsity

(A

U)

m/z

557.4

617.3

672.9

750.7

794.8

810.8

826.8

842.7

916.7

932.7

964.6

Ox A2E

Figure 3.38: The full mass spectrum of the reaction between A2E and benzaldehyde (Mandal 2008)

116

400 500 600 700 8000

2000

4000

6000

8000

Inte

nsi

ty (

AU

)

m/z

346422.2 512.3

556.2

640.4

762.4

684.3

617540454.5374 791

Figure 3.39: The MS/MS spectrum of the higher molecular weight compound (m/z = 790) in A2E and Cinnamaldehyde reaction mixture using 40 % collision energy. Peaks corresponding to the mass of 790 (red) with the loss of 150 (m/z 640), 174

(m/z 617), and 190 (m/z 556) are identified (blue) (Mandal 2008).

117

450 500 550 600 650 700 750 8000

10000

20000

30000

40000

50000

60000

70000

Inte

nsity

(A

U)

m/z

672.4

766.4

722.5

654.5574.3

490468 561508

794

644604

Figure 3.40 The MS/MS spectrum of one of the higher molecular weight compounds in A2E benzaldehyde reaction mixture. Peaks corresponding to the mass of 794 (red) with the loss of 122 (m/z 672), 140 (m/z 654), 150 (m/z 644), and 190

(m/z 604) are identified (blue) (Mandal 2008).

118

Figure 3.41 Possible structure and fragmentation of one of the higher molecular weight compounds from reaction of oxidized A2E and cinnmaldehyde (Mandal

2008)

119

Figure 3.42: Possible structure and fragmentation of one of the higher molecular weight compounds from reaction of oxidized A2E and benzaldehyde (Mandal 2008).

120the A2E cinnamaldehyde spectrum could be attributed to cinnamic acid and the

loss of 122 in the A2E benzaldehyde spectrum could be due to the loss of the

benzoic acid moiety. These fragments signify that the side chains of A2E are intact

and the modifications are occurring at the ends of the polyene chain (Mandal 2008).

The photolysis of all-trans-retinal in the presence of A2E was also

performed. The full mass spectrum displayed in Figure 3.43 indicates the formation

of two main products with m/z = 920 and 1188 after one hour of irradiation. The

fragmentation pattern of m/z = 920 and 1188 are displayed in Figures 3.44 and 3.45

with the proposed structures displayed in Figures 3.46 and 3.47, respectively. The

same characteristic losses of 150, 174, and 190 and a major fragment with m/z =

858 (Figures 3.48 and 3.49) are identified. This reaction was also performed

without irradiation; however, the formation of products with m/z = 920 and 1188

was much slower, appearing after 18 hrs. Once the A2E and RAL reaction was

complete, the mixture was injected onto an HPLC to separate and collect the

compound with m/z = 920 (Figure 3.50). The compound that eluted at 35 mins was

directly injected into the mass spectrometer (Figure 3.51) and confirmed by UV-Vis

(Figure 3.52) and MS/MS (Figure 3.53) to be the same compound identified in the

A2E reaction mixture (Figures 3.33 and 3.34) and lipofuscin samples (Figure 3.54).

To further investigate the chemical reactions involved in producing the

higher molecular weight products found in lipofuscin, cyclic voltammetry was

performed on benzaldehyde, cinnamaldehyde and all-trans retinal. Initially, a

background was taken of the working solution (Figure 3.55). This solution

contained an electrolyte (0.1 M TEAP) added to 100 ml of anhydrous acetonitrile to

121

592.9

727.5

920.1

1188.2

400 600 800 1000 1200 1400

0.0

2.0x106

4.0x106

6.0x106

8.0x106

Inte

nsity

(A

U)

m/z

A2E + RAL rxn

858.9

Figure 3.43: The mass spectrum of A2E reacted with all-trans-retinal

122

400 600 800 1000

0.0

2.0x103

4.0x103

6.0x103

8.0x103

1.0x104

1.2x104

1.4x104

1.6x104

1.8x104

771.8

Inte

nsi

ty (

AU

)

m/z

565.4

669.6

730.6

793.6

833.7

920.8

859.7

Figure 3.44: The MS/MS spectrum of m/z 920 from A2E RAL reaction. Peaks corresponding to the mass of 920 (red) with the loss of 150 (m/z 771) and 190 (m/z


123

800 1000 1200

0

1x103

2x103

3x103

4x103

Inte

nsi

ty (

AU

)

m/z

1127.9

1159

749.6 861.6

873.7

937.8

998.8

1014.8

1038.8

1082.9

1115.8

1188

1129.0

Figure 3.45: The MS/MS spectrum of m/z 1188 from A2E RAL reaction. Peaks corresponding to the mass of 1188 (red) with the loss of 150 (m/z 1038), 174 (m/z

1014), and 190 (m/z 998) are identified (blue).

124

Figure 3.46: Possible structure of m/z 920 with cleavages identified

125

Figure 3.47: Possible structure and fragmentation pattern of m/z 1188

126

440.3466.3

654.5

400 600 800

0.0

2.0x103

4.0x103

6.0x103

8.0x103

1.0x104

1.2x104

1.4x104

1.6x104

1.8x104

2.0x104

Inte

nsi

ty (

AU

)

m/z

858.6668.6

684.4

708.5

602.4532.2

Figure 3.48: The MS/MS of m/z 858 in A2E and all-trans-retinal reaction. Peaks corresponding to m/z 858 (red) with the loss of 150 (m/z 708), 174 (m/z 684), and

190 (m/z 668) are identified (blue).

127

Figure 3.49: Possible structure for m/z 858 with cleavages identified

128

Figure 3.50: The chromatogram of the A2E RAL reaction mixture using HPLC and PDA detection. Compound with m/z 920 eluted at 35 min.

min 10 20 30 40 50

mAU

0

500

1000

1500

2000

920

129

920.97

400 600 800 1000 1200 1400 1600 1800 2000

0

1x107

2x107

3x107

4x107

5x107

6x107

Inte

nsi

ty (

AU

)

m/z

Figure 3.51: The full mass spectrum of peak that eluted at 35 min. in Figure 3.18

130

200 300 400 500

0

1x105

2x105

3x105

4x105

5x105

6x105R

ela

tive

Ab

sorb

anc

e (A

U)

Wavelength (nm)

Figure 3.52: UV-Vis absorption for the peak with m/z 920

131

440.9502.8

565.8606

669.9

685.8708.9

793.8

859.8

600 800

0.0

5.0x105

1.0x106

1.5x106

2.0x106

2.5x106

3.0x106

3.5x106

Inte

nsi

ty (

AU

)

m/z

920.5

Figure 3.53: The MS/MS spectrum of m/z 920

132

459.3473.4

572.9

668.9

730.2

770.6

858.5

920.4

400 600 800 1000

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

1.2x105

Inte

nsity

(A

U)

m/z

Figure 3.54: The MS/MS spectrum of m/z 920 from Lipofuscin. Peaks corresponding to the mass of 920 (red) with the loss of 150 (m/z 771) and 190 (m/z


133

ACN with 0.1 M TEAP Background 50 mV/s Scan

-50

-40

-30

-20

-10

0

10

-2 -1.5 -1 -0.5 0 0.5 1 1.5 2

Applied Potential (V)

Cu

rren

t (u

A)

Figure 3.55: The voltammogram of TEAP background

134ensure sufficient conductivity. The redox couple ferrocenium/ ferrocene (Fc+/Fc)

(0.05 M) was added to each of the sample solutions to serve as an internal standard,

including the initial working solution (Figure 3.56), the benzaldehyde (Figure 3.57),

the cinnamaldehyde (Figure 3.58), and the all-trans retinal (Figure 3.59).

Benzaldehyde appears to undergo two irreversible reductions at approximately -

0.958 and -1.817 V vs. Ef(Fc+/Fc). These reductions could be irreversible as a result

of kinetic considerations, the reduction being much more highly favored than the

oxidation, or because of subsequent chemical processes, such as decomposition of

the reduction product prior to the oxidation. Cinnamaldehyde appears to undergo a

reversible redox reaction at -1.920 V vs. Ef(Fc+/Fc). However, from the

disproportionate intensity of the redox peaks (the reduction peak being much more

intense than the oxidation peak) it appears that the reduction is a more highly

favored reaction. Alternatively, the reduction product could also be more stable and

simply take more time to decompose, leaving a smaller amount of cinnamaldehyde

reduction product to be subsequently oxidized. All-trans retinal appears to undergo

an irreversible oxidation at 0.721 V and two irreversible reduction at -1.830 V and

-1.641 V vs. Ef(Fc+/Fc). However, further analysis of these compounds is still

needed to confirm the potentials. In addition, to determine the redox environment

within the lipofsucin granules, cyclic voltammetry should be performed on A2E and

the higher molecular weight products.

135

ACN with 0.1 M TEAP + 0.05 M Ferrocene 50 mV/s Scan Run 01

-60

-40

-20

0

20

40

60

-2 -1.5 -1 -0.5 0 0.5 1 1.5 2


Cu

rren

t (u

A)

Figure 3.56: The voltammogram of ferrocene

136

ACN with 0.1 M TEAP + 0.05 M Benzaldehyde 50 mV/s Scan

-1.2883, -26.327

-1.2542, -50.208

-0.3948, -17.296 0.5024, -21.448

0.6231, 40.879

-80

-60

-40

-20

0

20

40

60

-2 -1.5 -1 -0.5 0 0.5 1 1.5 2


Cu

rren

t (u

A)

Figure 3.57: The voltammogram of benzaldehyde

137

ACN with 0.1 M TEAP + 0.05 M Cinnamaldehyde 50 mV/s Scan

-1.4484, -69.931

-1.1891, 3.912

0.5252, -20.372

0.6763, 36.122

-80

-60

-40

-20

0

20

40

60

-2 -1.5 -1 -0.5 0 0.5 1 1.5 2


Cu

rren

t (u

A)

Figure 3.58: The voltammogram of cinnamaldehyde

138

ACN with 0.1 M TEAP + 0.015 M All-Trans 50 mV/s Scan

-1.2307, -62.862

-1.076, -2.622

-1.0556, -25.102

1.3153, 86.879

0.5333, -17.211

0.665, 36.012

-80

-60

-40

-20

0

20

40

60

80

100

120

-2 -1.5 -1 -0.5 0 0.5 1 1.5 2


Cu

rren

t (u

A)

0

Figure 3.59: The voltammogram of all-trans retinal (blue) and control (red)

139Discussion

In this chapter, the composition of lipofuscin and the individual components

of lipofuscin have been investigated. The results reported support the hypothesis

that the reason A2E is being sequestered within lipofuscin granules is to minimize

damage to the RPE, and that the higher molecular weight products originate from

the reaction of A2E derived aldehydes with other molecules of A2E present in the

lipofuscin mixture, not esterification reactions.

Previously, research has suggested that retinal lipofuscin is extremely

phototoxic. However, when RPE cells were fed lipofuscin granules, the RPE cells

did not show an appreciable amount of damage (Ligget 2007), suggesting that the

individual compounds within the lipofuscin granules are responsible for observed

phototoxicity and not the granules themselves. A2E has been reported to cause

damage to RPE cells by photochemically initiating free radical reactions and acting

as a detergent by disrupting cell membranes. Sparrow et al. reported that A2E

mediates blue light-induced apoptosis and that blue light damages DNA in A2E-

laden RPE cell solutions (Sparrow, Parish et al. 1999; Sparrow and Cai 2001).

However, these cells were fed free A2E and not lipofuscin granules. Boulton et al.

later reported that the physiological concentration of A2E in lipofuscin granules was

too small to account for the blue light-induced phototoxicity observed when RPE

cells are fed lipofuscin granules (Davies, Elliott et al. 2001). Nevertheless, the cells

were still damaged after irradiation with blue light, indicating that the major

phototoxic component was not A2E and still is unidentified. This is supported by

140research that discovered that A2E was excited by energy transfer within the

lipofuscin granules and consequently could not be the dominant blue-absorbing

chromophore (Haralampus-Grynaviski, Lamb et al. 2003). The comparison of

fluorescence spectra of lipofuscin and A2E were similar, but variations in lipofuscin

spectra suggest that multiple components contribute to the absorbance and

fluorescence of lipofuscin. Therefore, the biological activity of the lipofuscin

granules and the damaging effects associated with the granules, A2E, and other

components of lipofuscin are still controversial subjects.

Numerous compounds including A2E, oxidized A2E, and a complex mixture

of hydrophobic components have been identified in Lipofuscin (Figure 3.1). This

complex mixture of higher molecular weight compounds accounts for a large

portion of the lipofuscin sample. Analysis of the corresponding spectra revealed a

series of closely related compounds that differed by 14 amu, which most likely

result from the addition of methylene groups. Eluting between 50 and 110 mins

with 100 % methanol, these components were also determined to be relatively

hydrophobic. The collision-induced dissociation (CID) and corresponding

absorbance spectra for several of these higher molecular weight compounds were

analyzed and then compared to the fragmentation pattern and absorbance spectra of

A2E within the lipofuscin sample. Upon CID, the parent ion of A2E exhibits

characteristic losses of 150, 174, and 190. These characteristic losses and the parent

ion mass were observed in components of the complex mixture. All of the

components that eluted between 50-80 min and approximately 50 % of the material

that eluted from 80-110 min had analogous spectra to A2E, suggesting that these

141higher molecular weight products are derivatized A2E. In addition, numerous

spectra located within group II and III in the lipofuscin samples displayed ions

consistent with compounds present in group I. For example, Figure 3.2 displays ions

with m/z 814 and 863, which are also present in mass spectra of fragments found in

group II (m/z 1081) and III (m/z 1424) of the lipofuscin sample (Figure 3.10 and

3.13), suggesting that these higher molecular weight products are the result of a

polymerization reaction.

Since hydrophobic substances like all-trans retinol are stored in the RPE as

esters, esterification of A2E was previously investigated to identify the higher

molecular weight products in lipofuscin. Initially, esterification of A2E was

performed with acetyl chloride and hexanoyl chloride. Both reactions were used as

model systems to represent the possible short and long chain fatty acids that exist in

the retina (RPE) that could derivatize A2E to form esters. The fragmentation pattern

for both esters could not be found in the human lipofuscin extract. However, the

major fragment, m/z = 548, was identified in the full mass spectrum of lipofuscin

and had a similar fragmentation pattern to the synthesized A2E ester, suggesting a

similar structure. The predicted structure for species with m/z = 548 was A2E with

the loss of the ethanol group. The loss of the ethanol group was also later seen in

compounds with m/z 814 (Figure 3.9) and 1081 (Figure 3.12). Since both

esterification products displayed the same fragmentation pattern, A2E was treated

with structurally similar cinnamoyl chloride. The fragmentation pattern of the A2E

cinnamoyl chloride ester differed from the acetyl and hexanoyl esters, the major

peak had m/z = 575, which was attributed to a McLafferty rearrangement. This

142mechanism could not be traced in human RPE samples, indicating that A2E is

not stored as esters (Mandal 2008).

To investigate the relationship between these higher molecular weight

compounds and A2E, samples of pure A2E were aged for 60 days. The TIC of the

aged A2E samples displayed similar clusters of peaks that were located in the

lipofuscin sample (Figure 3.26). The CID of these peaks displayed in Figures 3.27

and 3.29 were also similar to the CID of the peaks located within lipofuscin

displayed in Figures 3.7, 3.10, and 3.13. These figures show ions corresponding to,

in most cases, A2E and losses of 150, 174, and 190 from the parent ion, which is

also observed in the CID of A2E. However in the aged A2E, the peak with m/z =

859 had a greater abundance than peak with m/z = 1081. These data suggest that as

A2E ages, the compound forms higher molecular weight derivatives and that these

derivatives increase in abundance with age. The cluster of peaks with m/z of

approximately 1400 in the lipofuscin sample was not observed in the synthetically

aged A2E, which was attributed to the sample not being aged long enough. Ions

corresponding to compounds located within group I were also observed in the

MS/MS spectra from compounds in group II within the aged A2E sample, which

supports a polymerization reaction. In addition, the reaction mixture of

ethanolamine and all-trans retinal that produces A2E generated higher molecular

weight products found in the lipofuscin sample. Since the esters previously

synthesized were not consistent with the compounds found in lipofuscin, a second

hypothesis involving the reaction of A2E with aldehydes was tested. The auto-

oxidation of A2E in the presence of cinnamaldehyde and benzaldehyde yielded a

143series of compounds including oxidation and addition products. The

fragmentation patterns and characteristic losses of these products were similar to

those found in oxidized A2E (Mandal 2008). The photolysis of retinal in the

presence of A2E also generated compounds with the same characteristic

fragmentation patterns with losses of 150, 174, and 190 found in aged A2E, the A2E

reaction mixture, and human retinal lipofuscin. The absorption spectra show two

peaks with maxima at 330 and 500 nm, which is in agreement with previously

reported compounds located in photoreceptor cell out segments (Bui, Han et al.

2006). The compound with m/z 920 was suggested to be A2E plus the addition of

one molecule of all-trans-retinal and one molecule of CH2COOH with the loss of

oxygen (Figure 3.46). The compound with m/z 1188 was suggested to be 2

molecules of all-trans-retinal and one molecule of CH2COOH with the loss of two

oxygens (Figure 3.47). The MS/MS of 858 was consistent with the addition of one

molecule all-trans-retinal to A2E with the loss of water (Figure 3.49). The

spectroscopic characteristics and fragmentation patterns associated with these

compounds supports the hypothesis that A2E is reacting with aldehydes such as all-

trans-retinal (Figures 3.46, 3.47, and 3.49), A2E-derived aldehydes (Figure 3.14),

cinnamaldehyde and benzaldehyde (Figures 3.41 and 3.42) to form the higher

molecular weight compounds found in lipofuscin.

Also, the previously described cyclic voltammetric experiments support

these observations. All three aldehydes--benzaldehyde, cinnamaldehyde, and all-

trans-retinal--undergo irreversible reductions, as previously described. The fact that

these aldehydes undergo irreversible reductions suggests that the reduction products

144are highly reactive and that the rate of their disappearance—potentially through

subsequent reactions—is quite fast. Previous studies have shown that

electrochemical electron transfer to benzaldehyde produces a radical anion that can

dimerize with an identical radical anion or the parent molecule, forming a higher

molecular weight product (Armstrong, Quinn et al. 1974; Yeh 1977; Fawcett and

Lasia 1981). This same type of polymerization reaction has been shown with many

aromatic aldehydes and, in cases with aldehydes similar to benzaldehyde, have been

shown to undergo further polymerization initiated by radical anion formation (Yeh,

Liu et al. 2004).

In addition, Simon et al. reported that the surface of lipofuscin granules

contained small distinctive areas that were separated by thin layers, indicating that

lipofuscin is an aggregated material (Haralampus-Grynaviski, Lamb et al. 2003).

One of the major fluorescent components in the hydrophobic fraction of lipofuscin,

A2E, was determined to have a log P of approximately 7.3, indicating that A2E is

lipophilic and in aqueous solution will aggregate, minimizing contact with water or

other polar substances. However, contrary to previous literature, A2E is not the

dominant blue-absorbing chromophore or yellow-emitting fluorophore in lipofuscin.

A2E becomes electronically excited mainly by energy transfer (Haralampus-

Grynaviski, Lamb et al. 2003). The lack of fluorescence suggests that A2E may self

quench as it aggregates, forming higher molecular weight products (Ragauskaite,

Heckathorn et al. 2001). This supports our results indicating that the majority of

components in the hydrophobic portion of RPE lipofuscin granules consist of

derivatized A2E generating a series of relatively hydrophobic compounds from

145auto-oxidation. The higher molecular weight compounds identified result from

an Aldol type condensation. These A2E modifying reactions assist in self-

aggregation to form hard granules, which sequester A2E, diminishing its destructive

ability.

CHAPTER 4

AGE-RELATED ACCUMULATION OF 3-NITROTYROSINE AND NITRO-A2E

IN HUMAN BRUCH’S MEMBRANE

Introduction

Recently four independent research groups used different methods to screen

the genomes from different groups of AMD patients. All four studies discovered a

commonly inherited variant (Y402H) of the complement factor H (CFH) gene that

significantly increases the risk of AMD (Edwards, Ritter et al. 2005; Hageman,

Anderson et al. 2005; Haines, Hauser et al. 2005; Klein, Zeiss et al. 2005). This

finding links genetics and inflammation. Before this finding, the study of the

components of drusen had provided compelling evidence that inflammatory and

immune-mediated events participate in the development of drusen and progression

of AMD. Protein components of drusen include immunoglobulins, components of

the complement pathway (e.g., C5 and C5b-9), molecules involved in the acute-

phase response to inflammation (e.g., Amyloid P component), and proteins that

modulate the immune response (e.g., vitronectin, clusterin, and apolipoprotein E)

(Hageman and Mullins 1999; Hageman, Mullins et al. 1999; Johnson, Ozaki et al.

2000; Mullins, Russell et al. 2000). The finding that macrophages are important in

choroidal neovascularization (CNV) also supports the involvement of inflammation

147 in AMD (Grossniklaus, Ling et al. 2002). Recent research provided further evidence

that inflammation is involved in the development of AMD (Chen, Forrester et al.

2007; Laine, Jarva et al. 2007; Schaumberg, Christen et al. 2007; Skerka, Lauer et al.

2007) and the link between inflammation, drusen and oxidative stress (Wu, Lauer et

al. 2007; Hollyfield, Bonilha et al. 2008; Wang, Ohno-Matsui et al. 2008).

During inflammation, large fluxes of nitric oxide (NO) are released through

the activation of inducible nitric oxide synthase (Marletta, Yoon et al. 1988;

Carreras, Pargament et al. 1994). Nitrite concentration is reported to be nearly

doubled in the diabetic retina (El-Remessy, Behzadian et al. 2003). Cigarette

smoking, which has been strongly associated with the development of AMD

(Solberg, Rosner et al. 1998), is also an important chronic contributor to human NO

exposure (Council 1986; Borland and Higenbottam 1987). Patients with AMD have

significantly higher plasma NO levels than control subjects (Evereklioglu, Er et al.

2003). NO itself is a relatively unreactive radical; however, it is able to form other

reactive intermediates including nitrite (NO2-), peroxynitrite (ONOO-), NO2, and

N2O3, etc that can modify proteins, lipids and other compounds. Nitrite is one of the

major NO metabolic products and has been used as a marker of NO production

(Farrell, Blake et al. 1992; Gaston, Reilly et al. 1993). In addition, nonenzymatic

nitration of long-lived proteins such as extracellular matrix proteins is a well known

pathway that has been associated with inflammation (Bailey, Paul et al. 1998; Paik,

Dillon et al. 2001). The extracellular matrix proteins such as collagen and elastin

have been reported to be nonenzymatically modified by nitrite at physiological pH

(Paik, Ramey et al. 1997; Paik, Dillon et al. 2001). It has been reported that nitrite-

148 modification of basement membrane-like extracellular matrix proteins can impart

deleterious effects on adjacent epithelial cell function and viability (Wang, Paik et al.

2005) and impair phagocytic capacity (Sun, Cai et al. 2007).

Bruch’s membrane lies between the choroidal capillary bed and retinal

pigment epithelial (RPE) cells. The exchange of various materials between the

underlying choriocapillaris and overlying RPE occurs through Bruch’s membrane

(Lyda, Eriksen et al. 1957; Sellner 1986). Bruch’s membrane is permeable to

macromolecules up to 300kD in size in healthy eyes, but there are numerous

examples of pathological processes in which larger macromolecules or even cells,

including macrophages and leukocytes, can traverse Bruch’s membrane in the

diseased eye (Crane and Liversidge 2008). In addition to Bruch’s membrane,

trafficking of material from the RPE to the choriocapillaris is limited in the healthy

eye by tight junctions between adjacent cells of the RPE monolayer. This outer

blood-retinal barrier is part of the specialized ocular microenvironment that confers

protection or immune privilege to mitigate the effect of deleterious immune

responses (Streilein 2003). Nevertheless, this barrier is altered in pathological

circumstances, and breakdown of the outer blood retinal barrier, including

macrophage and leukocyte infiltration of the retina, are implicated in many diseases

including AMD (Jha, Bora et al. 2007). Several investigators have suggested that

age-related damage to Bruch’s membrane allows for the accumulation of abnormal

extracellular deposits, called drusen, between the basal lamina of the RPE and the

inner collagen layer of Bruch’s membrane (Newsome, Huh et al. 1987; Pauleikhoff,

Barondes et al. 1990; Mullins, Russell et al. 2000; Crabb, Miyagi et al. 2002). The

149 accumulation of drusen is thought to elicit a local inflammatory response (Anderson,

Mullins et al. 2002; Yasukawa, Wiedemann et al. 2007; Hollyfield, Bonilha et al.

2008).

Recently, research has shown that age-related changes in human Bruch’s

membrane can exert significant deleterious effects on RPE function that are

independent of cell aging, including impairing the ability of cultured RPE to

phagocytize photoreceptor outer segments (Sun, Cai et al. 2007). A similar effect on

RPE function is observed after nonenzymatic nitration of RPE basement membrane

in tissue culture (Wang, Paik et al. 2005). We hypothesize that inflammation will

produce reactive nitrogen species that will modify intrinsic extracellular matrix

proteins and/or extrinsic deposits accumulated in Bruch's membrane. Surprisingly,

there have been no studies that have reported nitrite modification occurring in

intrinsic Bruch’s membrane proteins or extrinsic deposits, although tyrosine

nitration has been shown to occur in photoreceptor cells (Miyagi, Sakaguchi et al.

2002). However, previous studies have demonstrated that numerous structural and

molecular alterations occur within human Bruch’s membrane as a function of age.

These changes, which disrupt the normal molecular architecture of Bruch’s

membrane, include: (1) structural changes in the main collagen framework,

including cross-linking and deposition of long-spaced collagen (Yamamoto and

Yamashita 1989), qualitative and quantitative changes in the native extracellular

matrix molecules (Pauleikhoff, Wojteki et al. 2000), deposition of abnormal

extrinsic molecules including fluorescent products that accumulate in drusen

(Ruberti, Curcio et al. 2003), macromolecular changes in the structure of Bruch’s

150 membrane, such as calcification, cracks or loss of inner layers due to inadequate

basal membrane regeneration as in geographic atrophy (Feeney-Burns and

Ellersieck 1985; Grossniklaus, Hutchinson et al. 1994), and changes in the physical

characteristics of Bruch’s membrane, such as an age-dependent increase in trans-

membrane hydraulic conductivity (Moore, Hussain et al. 1995) and age-related

linear decline in collagen solubility, an index of deleterious cross-linking

(Karwatowski, Jeffries et al. 1995),

3-nitrotyrosine is a specific marker for inflammation-induced oxidative

damage to proteins. In addition to proteins, Bruch's membrane also contains lipids,

lipofuscin and carbohydrates (Hageman, Luthert et al. 2001; Yasukawa, Wiedemann

et al. 2007). Lipofuscin is a mixture of autofluorescent material that accumulates in

the RPE cells and is reported to photochemically generate a series of reactive

oxygen species, including singlet oxygen, hydrogen peroxide, and superoxide

anions (Gaillard, Atherton et al. 1995; Rozanowska, Wessels et al. 1998) that may

enhance oxidative stress in the RPE. One of the major organic soluble

chromophores in lipofuscin is A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-

cyclohexen-1-yl)-1E, 3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6--

(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]—pyridinium). In this

chapter, liquid chromatography-mass spectrometry (LC-MS) was used to investigate

the modifications to intrinsic and extrinsic proteins and A2E in human Bruch's

membrane by reactive nitrogen species released during inflammation. We have

identified an increasing accumulation of 3-nitrotyrosine and nitro-A2E in human

Bruch's membrane with advancing patient age. Detection of nitro-A2E within

151 human Bruch’s membrane may serve as a specific biomarker for inflammation and

non-enzymatic nitration.

Results

Identification of tyrosine nitration in Bruch's membrane

To determine if tyrosine nitration occurs in Bruch's membrane, Bruch's

membrane was acid hydrolyzed and analyzed by LC-MS. 3-nitrotyrosine (3-NT),

which is an important biomarker of nonenzymatic nitration, is stable under acid

hydrolysis (Crowley, Yarasheski et al. 1998). The m/z of the quasimolecular ion

([MH]+) of 3-nitrotyrosine is 227.0. This molecule easily loses a nitro group under

collision-induced dissociation (CID), forming a fragment with m/z 181.0. Therefore,

we used selective reaction monitoring (SRM) (parent ion m/z = 227.0 with daughter

ion m/z = 181.0) to specifically monitor the presence of 3-nitrotyrosine. Figure 4.1

gives the results of selective reaction monitoring scans of the acid hydrolysate of

Bruch's membrane and standard 3-nitrotyrosine. The SRM scan of the acid

hydrolysate of Bruch’s membrane has a peak with similar retention time to the peak

of 3-nitrotyrosine. The tandem mass spectrum of the compound in this peak is also

similar to the tandem mass spectrum of 3-nitrotyrosine (Figure 4.2) (Wang 2005).

Identical experiments were performed on three samples of human Bruch’s

membranes from different donors to determine the relative concentration of 3-

nitrotyrosine within the human Bruch’s membrane samples as a function of patient

152

0 20 40 60 80 100 120

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105Inte

nsi

ty

Retention time

Bruch's membrane

0 20 40 60 80 100 120

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

1.2x105

Standard 3-NT

Figure 4.1: Selected Reaction Monitoring (SRM) chromatogram of 3-NT and acid hydrolysate of BM (SRM 227.1→181.1).3-NT and acid hydrolysate of BM was

analyzed by LC/MS as described in method. The SRM scan of BM acid hydrolysate has a peak with m/z 227 and fragment 181 and similar retention time (51 minutes)

to 3-NT which indicates the presence of 3-NT in BM acid hydrolysate (Wang 2005).

153

100 120 140 160 180 200 220

0

1x104

2x104

3x104

4x104

5x104Inte

nsi

ty

M/Z

22

6.9

20

9.8

181

.0

Bruch's membrane

100 120 140 160 180 200 220

0.0

2.0x106

4.0x106

6.0x106

8.0x106

C

HO

NH2

HO

O

O2N

m/z 227

210181

22

6.9

209

.8

18

1.0Standard 3-NT

Figure 4.2: The tandem mass spectra of standard 3-nitrotyrosine and component with m/z 227.0 at RT 51min in BM. The tandem mass spectrum of the component at RT 51mins from human BM extracted from 72-75 year old donors is very similar to the tandem mass spectrum of 3-NT. The inset gives the predicted fragmentation of

3-NT (Wang 2005).

154 age. Approximately six pieces of Bruch’s membrane from four different donors

from the decades <25 yrs, 40-60 yrs, and >65 yrs were obtained. These samples

were then extracted as previously described in Chapter 2. To quantify the actual

concentration of 3-nitrotyrosine, the standard addition of 50 µM solution of 3-

nitrotyrosine was added to each of the samples before analysis with LC-MS. The

Zoom and SRM scans for each sample are displayed in Figures 4.3 and 4.4,

respectively. The peaks were then integrated (Figure 4.5) and and the concentration

was calculated using standard addition with a calibration curve (Figure 4.6). Figure

4.7 displays the concentrations of 3-nitrotyrosine in the different decades. The

presence of 3-nitrotyrosine is negligible in the < 25 yrs sample of BM. There was a

small increase in the BM sample between the ages of 40 to 60 yrs followed by a

substantial increase in the BM sample > 65 yrs. The exponential increase of 3-

nitrotyrosine in BM, observed in Figure 4.7, suggests that tyrosine nitration occurs

in human Bruch’s membrane as a function of age, which may be related to the

inflammatory response.

Identification of nitro-A2E in Bruch’s membrane

To investigate our hypothesis that one of the major components in lipofuscin,

A2E, may be modified by reactive nitrogen species resulting in the formation of

nitro-A2E, nitro-A2E was synthesized as described in Materials and Methods and

then analyzed by mass spectrometry. To confirm the presence of A2E and nitro-

155

20 40 60 800.0

2.0x106

4.0x106

6.0x106 Standard

Time (min)

0.02.0x106

4.0x106

6.0x106 BM < 25 yrs

Inte

nsity

(A

U)

0.0

2.0x106

4.0x106

BM 40-60 yrs

0.02.0x106

4.0x106

6.0x106

BM > 65 yrs

Figure 4.3: The zoom scan of BM with the standard addition of 3-nitrotyrosine (m/z 227).

156

20 40 60 80

01x1062x1063x1064x1065x106

Inte

nsity

(A

U)

Time (min)

Standard

01x105

2x105

3x105

BM < 25 yrs

0

1x106

2x106

BM 40-60 yrs

01x1062x1063x1064x106

BM > 65 yrs

Figure 4.4: The SRM scan of m/z 227 181 from the standard addition of 3-nitrotyrosine in BM samples from different age groups.

157

-20 0 20 40 60 80 100 120 140-5.0x105

0.0

5.0x105

1.0x106

1.5x106

2.0x106

2.5x106

3.0x106

3.5x106

4.0x106

4.5x106

Inte

nsi

ty (

AU

)

Time (min)

Standard 1 BM < 25 yrs BM 40-60 yrs Standard 2 BM > 65 yrs Standard 3

Figure 4.5: Integration of area under SRM scan from Figure 4.4.

158

Calibration Curve of 3-nitrotyrosine Standards

-2.00E+06

-1.00E+06

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

7.00E+06

0 5 10 15 20 25 30

Volume (ml)

Are

a

Figure 4.6: Calibration curve for 3-nitrotyrosine

159

Concentration 3-nitrotyrosine in BM

0.00E+00

2.00E-05

4.00E-05

6.00E-05

8.00E-05

1.00E-04

1.20E-04

1.40E-04

1.60E-04

1.80E-04

2.00E-04

BM <25 yrs BM 40-60 yrs BM > 65yrs

Co

nce

ntr

atio

n (

M)

Figure 4.7: The concentration of 3-nitrotyrosine in BM samples from ages of < 25, 40-60, and > 65 years.

160

0 20 40 60 80 100 120

0.0

5.0x106

1.0x107

1.5x107

2.0x107

2.5x107

3.0x107

3.5x107

m/z 637.5

Time (min)

51.660 20 40 60 80 100 120

0.05.0x107

1.0x108

1.5x108

2.0x108

2.5x108

3.0x108

3.5x108

4.0x108

Inte

nsity

m/z 592.552.86

Figure 4.8: The selected ion chromatograms for synthetic A2E (top) and nitro-A2E

161 A2E, the total ion chromatogram for synthetic nitro-A2E filtered for m/z 592.5 and

637.5 are displayed in Figure 4.8 with corresponding retention times. The

ultraviolet-visible absorption spectra of m/z 592.5 (A2E) and 637.5 (nitro-A2E)

were also compared (Figure 4.9). A2E had absorption peaks at 335 and 430 nm

(Figure 4.9a), which corresponds to previously reported results (Parish, Hashimoto

et al. 1998). The absorption spectrum of nitro-A2E (Figure 4.9b) is very similar to

the absorption spectrum observed in Figure 5a for A2E. However, the two most

intense absorption peaks were located at 330 and 415 nm (Figure 4.9b), indicating

that nonenzymatic nitration induces an expected slight blue shift in the absorption

spectrum.

The structure of A2E is compared to the predicted structure of nitro-A2E in

Figure 4.10 with characteristic cleavages identified (Dillon, Wang et al. 2004). The

m/z of synthetic nitro-A2E measured by mass spectrometry is 637.5, which is in

agreement with this predicted structure. Figure 4.11 displays the tandem mass

spectrum of synthetic nitro-A2E. The major fragments from the CID spectrum

match the predicted structure and characteristic fragmentations shown in Figure

4.10.

To investigate the possible presence of nitro-A2E in vivo, the organic soluble

components in Bruch’s membrane from 70-yr-old donor globes were extracted and

analyzed by LC/MS. The total ion chromatogram revealed a peak with m/z 592,

which was identified as A2E based on its absorption spectrum and characteristic

fragmentation pattern. A peak with m/z 637.5 within the total ion chromatogram

was also seen at approximately the same retention time as the peak with m/z 637.5

162

#

200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e A

bsor

banc

e

335.0 430.0

395.0

235.0

#

200 250 300 350 400 450 500 550 600wavelength (nm)

0

20

40

60

80

100

Rel

ativ

e A

bsor

banc

e

235.0

330.0285.0415.0425.0

#

200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e A

bsor

banc

e

335.0 430.0

395.0

235.0

#

200 250 300 350 400 450 500 550 600wavelength (nm)

0

20

40

60

80

100

Rel

ativ

e A

bsor

banc

e

235.0

330.0285.0415.0425.0

#

200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e A

bsor

banc

e

335.0 430.0

395.0

235.0

#

200 250 300 350 400 450 500 550 600wavelength (nm)

0

20

40

60

80

100

Rel

ativ

e A

bsor

banc

e

235.0

330.0285.0415.0425.0

a

b

m/z 592.5

m/z 637.5

Figure 4.9: The UV-Vis spectra for A2E (m/z 592.5) and for nitro A2E (m/z 637.5).

163

A2E (m/z 592) Nitro-A2E (m/z 637)

Figure 4.10: Structures of A2E (m/z 592) and nitro-A2E (m/z 637) showing characteristic cleavage points and the resulting fragment molecular weights

.

164

300 400 500 6000

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

danc

e (A

U)

m/z

MS/MS 637.5

637.4

605.3

591.4

576.4

619.4401.5 469.2

376.2 487.3

418.2 442.3

Figure 4.11: The tandem mass spectrum of synthetic nitro-A2E induced dissociation to confirm the identification of nitro-A2E.

165 located in the synthetic nitro-A2E sample, suggesting the presence of nitro-A2E

within the Bruch’s membrane sample. This peak was then fragmented by collision-

induced dissociation to confirm the identification of nitro-A2E. Figure 4.12 displays

the tandem mass spectrum of the component with m/z 637.5 located within the

Bruch’s membrane extract. The major fragments correspond to characteristic

cleavages illustrated in Figure 4.10 and also observed in the authentic nitro-A2E

sample (Figure 4.11). The total ion chromatogram also contained samples with

molecular weights of m/z 653 and 682 (Figure 4.13), which suggests the presence of

oxidized-nitrated A2E and doubly nitrated A2E, but the amounts were insufficient

to acquire a full CID spectrum.

We then sought to determine whether A2E was nitrated within RPE

lipofuscin and then transported to Bruch’s membrane, or whether nitration of A2E

occurred after A2E accumulation within Bruch’s membrane. To address this issue,

approximately ten samples of the organic soluble extract of lipofuscin and the

organic soluble extract of Bruch’s membrane from three donors were analyzed by

LC-MS and compared. Figure 4.14 displays filtered total ion chromatograms for

A2E (m/z 592) and nitro-A2E (m/z 637) in RPE lipofuscin and Bruch’s membrane.

The presence of several peaks in the chromatograms result from several isomers co-

existing (Parish, Hashimoto et al. 1998). The highest concentration of A2E was

observed in RPE lipofuscin followed by a significantly lower concentration (30-40

fold) within Bruch’s membrane extract. Nitro-A2E was absent from the lipofuscin

samples tested but nitro-A2E was detected within human Bruch’s membrane, thus

166

300 400 500 6000

20

40

60

80

100

Rel

ativ

e A

bund

ance

(A

U)

m/z

MS/MS 637.8 in BM

637.8

619.4

591.5

535.4

517.2

487.2

252.2

273.2

317.6

358.1

469.3

418.4 442.4

Figure 4.12: The tandem mass spectrum of nitro-A2E isolated from 65 yrs and older BM. Box = mass same in synthetic nitro-A2E and nitro-A2E isolated from 65 yrs

and older BM.

167

0 20 40 60 80 100 120

0.02.0x1034.0x1036.0x1038.0x1031.0x104

Inte

nsity

(A

U)

Time (min)

m/z 682.5

0 20 40 60 80 100 120

0.05.0x1031.0x1041.5x1042.0x104

m/z 653.4

0 20 40 60 80 100 120

01x105

2x105

3x105

m/z 637.2

0 20 40 60 80 100 120

01x1062x1063x1064x106

m/z 592.5

Figure 4.13: The selected ion chromatogram of m/z 592.5 (A2E), m/z 637.5 (nitro A2E), m/z 653.4 (nitro A2E plus oxygen), and m/z 682.5 (A2E with 2 nitro

substitutions).

168

0 20 40 60 80 100 120

0.0

2.0x106

Time (min)

m/z 592 in BM

0 20 40 60 80 100 120

0.05.0x1051.0x1061.5x1062.0x106

m/z 637 in BM

0 20 40 60 80 100 120

0.0

2.0x106

4.0x106

Inte

nsity

m/z 592 in lipofuscin

0 20 40 60 80 100 120

01x1072x1073x1074x1075x107

m/z 637 in lipofuscin

Figure 4.14: The selected ion chromatograms for A2E (m/z 592) and nitro-A2E (m/z 637) from RPE lipofuscin and BM extracts from human donor globes that were

65 yrs and older. Note that nitro A2E and A2E from the BM have similar concentrations, whereas no nitro-A2E could be detected from the RPE despite

increasing the sensitivity of the detector.

169 providing strong evidence that the nitration of A2E is specific to Bruch’s membrane

and does not occur within RPE lipofuscin.

Concentration of Nitro-A2E in Bruch’s membrane samples from different decades

of life

We then determined the relative concentration of A2E within the human

Bruch’s membrane samples as a function of patient age. Approximately 8 pieces of

Bruch’s membrane from 4 different donors from each decade (including <20s, 40s,

50s, 60s, 70s, 80s), and clinically diagnosed nonexudative AMD were obtained.

These samples were then extracted as previously described in Chapter 2. To

quantify the actual concentration of A2E and nitro-A2E, an internal standard of 50

µM tryptophan was added to each of the samples before analysis with LC-MS. The

peaks were then integrated (Figure 4.15) and concentration calculated for A2E

(Figure 4.16). The SRM scans of Nitro-A2E in each decade are displayed in Figure

4.17. Once analyzed by LC-MS, the corresponding peaks were integrated (Figure

4.18) and the concentration in each sample was calculated (Figure 4.19). The

concentrations of A2E and nitro-A2E throughout the different decades were then

compared to concentrations found in AMD samples (Figure 4.20). The

accumulation of both A2E and nitro-A2E is negligible up to the 4th decade of life.

However, between the 4th and 5th decades there is a substantial increase in the

concentrations of both A2E and nitro-A2E, which continues to rise throughout the

6th, 7th, and 8th decades. To determine if these results were relevant to AMD, the

170

0 20 40 60 80 100 120 140 160 180 200

0.0

5.0x105

1.0x106

1.5x106

2.0x106

2.5x106

3.0x106

3.5x106

4.0x106

4.5x106

5.0x106

5.5x106

6.0x106

Inte

nsity

(A

U)

TIme (min)

< 20 yrs 40 yrs 50 yrs 60yrs 70 yrs 80 yrs AMD

Figure 4.15: Integration of A2E in BM samples from different decades of life (<20, 40, 50, 60, 70, and 80 yrs)

171

10 20 30 40 50 60 70 80-0.0001

0.0000

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

0.0007

0.0008

Co

nce

ntr

atio

n (

M)

Time (years)

Figure 4.16: The concentration of A2E in BM samples from different decades of life (<20, 40, 50, 60, 70, and 80 yrs)

172

0 20 40 60 80 100

0

1x104

Inte

nsity

(A

U)

<20 yrs

0.04.0x1038.0x1031.2x104

40 yrs

0.04.0x1038.0x1031.2x104

50 yrs

01x1042x104 60 yrs

01x1052x1053x105 70 yrs

0.02.0x1054.0x1056.0x105 80 yrs

01x1072x1073x1074x107

AMD

Figure 4.17: The SRM scans of Nitro-A2E from BM samples from different decades of life (<20, 40, 50, 60, 70, and 80 yrs)

173

0 20 40 60 80 100 120 140 160 180 200

0.0

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

3.0x105

3.5x105

4.0x105

4.5x105

5.0x105

5.5x105

Inte

nsi

ty (

AU

)

Time (min)

< 18 yrs 40 yrs 50 yrs 60 yrs 70 yrs 80 yrs AMD

Figure 4.18: Integration of Nitro-A2E A2E in BM samples from different decades of life (<20, 40, 50, 60, 70, and 80 yrs)

174

10 20 30 40 50 60 70 80-0.00002

0.00000

0.00002

0.00004

0.00006

0.00008

0.00010

0.00012

0.00014

0.00016

0.00018

Con

cent

ratio

n of

nitr

o-A

2E (

M)

Time (years)

Figure 4.19: The concentration of Nitro-A2E in BM samples from different decades of life (<20, 40, 50, 60, 70, and 80 yrs)

175

0.00E+00

2.00E-04

4.00E-04

6.00E-04

8.00E-04

1.00E-03

1.20E-03

18 40 50 60 70 80 AMD

Time (years)

Co

nce

ntr

atio

n (

M)

A2E

Nitro-A2E

Figure 4.20: The concentration of A2E and Nitro-A2E in BM samples from <20, 40, 50, 60 70, and 80 decades of life and dry AMD.

176 concentrations of A2E and nitro-A2E throughout the different decades were also

compared to the concentrations found in nonexudative AMD, as shown in Figure

4.19. The nonexudative AMD samples had the highest concentration of A2E and

nitro-A2E. Patients in the 8th decade of life displayed similar concentration of both

the A2E and nitro-A2E, as seen in the nonexudative AMD samples.

Discussion

Bruch's membrane is located between the endothelium layer of the

choriocapillaris and a monolayer of retinal pigment epithelium. In the normal eye

Bruch’s membrane serves as an attachment surface for the RPE. The outer blood-

retinal barrier is formed by tight junctions between adjacent RPE; Bruch’s

membrane is partially responsible for limiting the movement of large molecules and

cells from the choriocapillaris to the outer retina. This barrier is broken down during

inflammation and inflammatory cells such as monocytes, macrophages,

lymphocytes (Dua, McKinnon et al. 1991) and inflammatory mediators including

complement components (Hollyfield, Bonilha et al. 2008) can traverse Bruch’s

membrane and accumulate within this structure. Nitric oxide released by these

inflammatory cells together with the high oxygen concentration in the retina is

expected to cause oxidative stress to many components in Bruch’s membrane and

could lead to nonenzymatic nitration of intrinsic proteins and extrinsic products that

accumulate within Bruch’s membrane as a function of age. To our knowledge, the

finding of 3-nitrotyrosine and A2E nitration in Bruch’s membrane in this study

177 provides the first clear demonstration of non-enzymatic nitration of proteins and

age-related deposits (A2E) within human Bruch’s membrane.

Numerous changes develop within human Bruch’s membrane as a function

of increasing patient age, including collagen cross-linking (Yamamoto and

Yamashita 1989) and the accumulation of abnormal deposits such as drusen

(Ruberti, Curcio et al. 2003). Physiological collagen cross linking provides

structural stability to this important structural protein, whereas nonphysiological

collagen cross linking is an imprecisely controlled process that impairs collagen

structure and function (Bailey, Paul et al. 1998). Nonenzymatic collagen cross

linking can be induced by nitrite, and nitration of protein tyrosine residues to form

3-nitrotyrosine is a hallmark of tissue injury caused by inflammation. 3-

nitrotyrosine has been identified in many diverse pathological conditions such as

atherosclerosis, pulmonary and heart disease, viral infections, and neurological

disorders (Ischiropoulos 1998). Recent studies have established that 3-nitrotyrosine

serves as a “marker” of reactive nitrogen species formation and can alter protein

function. For example, modification of tyrosine residues can affect the

phosphorylation and dephosphorylation of tyrosine, an important mechanism of cell

regulation (Di Stasi, Mallozzi et al. 1999). Tyrosine nitration in Bruch's membrane

may affect the degree of phosphorylation of some important proteins and further

affect the migration of inflammatory cells through the blood retinal barrier

(Erickson, Sundstrom et al. 2007). Nitrite-induced modification of extracellular

proteins can be induced in vitro (Paik, Ramey et al. 1997; Paik, Dillon et al. 2001),

and RPE cell viability and phagocytic ability decrease on nitrite-treated extracellular

178 matrix (Wang, Paik et al. 2005; Sun, Cai et al. 2007). Nitrite-induced changes in

normal basement membrane mimic the deleterious effects of aging Bruch’s

membrane on RPE function (Wang, Paik et al. 2005; Sun, Cai et al. 2007).

Lipofuscin and other RPE cellular components have been found in drusen,

the extracellular deposits located between the basal lamina of the RPE and the inner

collagenous layer of the Bruch’s membrane (Hageman, Luthert et al. 2001; Crabb,

Miyagi et al. 2002). One of the major components of lipofuscin is A2E, and this

study demonstrates the presence of A2E in human Bruch’s membrane. A2E is not

normally a component of Bruch’s membrane in young eyes, and we did not identify

significant levels of A2E or nitro A2E in samples obtained from patients in the

second decade of life (Figure 4.19). In addition, the concentration of A2E clearly

increases with patient age (Figure 4.19), thus demonstrating that A2E deposition is a

nonphysiological process that does not occur, or occurs to a very limited extent, in

young individuals. The mechanism for A2E accumulation is not known. It is

believed that RPE ordinarily does not extrude or exocytose active lysosomes or

lysosomal enzymes although aged RPE extrude cytoplasm with active lysosomes

into Bruch's membrane (Feeney-Burns, Gao et al. 1987). We could not determine if

the A2E identified in Bruch’s membrane is part of this normal extrusion process.

Lipofuscin and other cellular debris accumulated in Bruch’s membrane may

contribute to the decreasing hydraulic conductivity observed with age (Moore,

Hussain et al. 1995) and also may stimulate chronic inflammation.

Our results clearly demonstrate that 3-nitrotyrosine is present within proteins

that are present within human Bruch’s membrane that is isolated using previously

179 described techniques. Previous studies using scanning and transmission electron

microscopy of Bruch’s membrane preparations demonstrate that the Bruch’s

membrane isolated in these preparations contains all 5 layers of Bruch’s membrane

(i.e., basal lamina of the RPE, inner collagen layer, elastin, outer collagen layer, and

basal lamina of the choriocapillaris). Scanning electron microscopy demonstrates

the preparation contains extracellular deposits on both the inner and the outer

aspects of the RPE basal lamina (Del Priore and Tezel 1998; Tezel, Del Priore et al.

2004). Since our preparation contains intrinsic Bruch’s membrane proteins as well

as extracellular deposits, additional studies are required to determine if the 3-

nitrotyrosine that we have detected represents modifications of intrinsic Bruch’s

membrane proteins, proteins located in extracellular deposits such as drusen, or both.

However, it should be noted that nitro-A2E is present within the Bruch’s membrane

preparation but we did not detect nitro-A2E in lipofuscin extracted from human

RPE (Figure 4.13). This suggests that nitration of A2E occurs after A2E has

accumulated within Bruch’s membrane. Thus, non-enzymatic nitration of A2E must

occur within Bruch’s membrane, possibly due to nitric oxide and/or related nitrating

agents such as peroxynitrite. It is likely that non-enzymatic nitration of both

intrinsic Bruch’s membrane proteins and extracellular deposits would occur by a

similar mechanism.

To our knowledge, the current study represents the first clear demonstration

of inflammation-related chemical modifications detected in human Bruch’s

membrane. The presence of 3-nitrotyrosine and nitro-A2E may be important

biomarkers for immune-mediated processes during aging, and the role of this

180 process in the development of age-related macular degeneration. Further

experiments are needed to evaluate other aspects of this process, such as: (1) the

relationship between the degree of nitration and the age/medical history of the

donor; (2) the effect of nitration on the turnover of intrinsic extracellular matrix

proteins; and (3) determination of the three-dimensional structural changes resulting

from nitration and the effects of these changes on cellular function.

CHAPTER 5

MODIFICATIONS TO THE BASEMENT MEMBRANE PROTEIN LAMININ

AND A2E: A MODEL FOR AGING IN BRUCH’S MEMBRANE

Introduction

The dry form of age-related macular degeneration (AMD) is a multifactoral

disease characterized by central vision loss attributed to photoreceptor cell death as

a result of the degeneration of the retinal pigment epithelium (RPE). Oxidative

stress and blue light-mediated damage related to lipofuscin, and particularly its

major chromophore A2E, are considered some of the possible mechanisms

underlying the degeneration of the RPE (Dorey, Wu et al. 1989; Winkler, Boulton

et al. 1999; Sparrow, Nakanishi et al. 2000; Suter, Reme et al. 2000; Sparrow and

Cai 2001; Liang and Godley Bernard 2003). The accumulation of drusen and basal

deposits between the RPE and Bruch’s membrane have also been shown to have

deleterious effects on RPE cell viability (Nakaizumi 1964; Sarks 1976; Newsome,

Huh et al. 1987; Pauleikhoff, Barondes et al. 1990; Ho and Del Priore 1997; Mullins,

Russell et al. 2000). Furthermore, previous research has suggested that modification

of basement membrane proteins may also affect the physiology and attachment of

RPE cells (Ho and Del Priore 1997; Ivert, Keldbye et al. 2005; Wang, Paik et al.

2005). The accumulation of fluorescent lysosomal storage bodies, lipofuscin, in the

182 RPE is considered one of the major changes associated with age and may be related

to the onset of AMD (Dorey, Wu et al. 1989). A2E (2-[2,6-dimethyl-8-(2,6,6-

trimethyl-1-cyclohexen-1-yl)-1E, 3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-

methyl-6-- (2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]—

pyridinium), a major fluorescent component of lipofuscin, is a bis-retinoid

pyridinium salt that induces blue light damage in RPE cells (Sparrow, Nakanishi et

al. 2000; Sparrow and Cai 2001) and nonphotochemically induces apoptosis in RPE

cells at physiological concentrations (Suter, Reme et al. 2000). A2E, shown in

Figure 1.9, has also been reported to photochemically initiate free radical reactions

in organized media (Ragauskaite, Heckathorn et al. 2001) and in solution produce

superoxide and peroxyl radicals in the presence of oxygen, which are toxic to cells

(Reszka, Eldred et al. 1995). We have recently reported that A2E has also been

detected in human BM extracts (Dillon, Wang et al. 2006).

As a result of these age-related changes and the relationship between the

RPE and Bruch’s membrane, studies involving modifications to basement

membrane proteins such as laminin and type IV collagen have been of interest.

Wang et al. have shown that glycation and nitration of Type IV collagen leads to

damaging effects on RPE cell function and viability (Wang, Paik et al. 2005). Also,

nonenzymatic glycation of laminin has been shown to decrease self assembly,

binding to type IV collagen, and binding of heparin sulfate proteoglycan due to

structural deformations following glycation (Federoff, Lawrence et al. 1993).

Handa et al. reported the increase of pentosidine and carboxymethyllysine,

advanced glycation endproducts, in Bruch’s membrane from older human samples

183 (Handa, Verzijl et al. 1999). The presence of AGEs has also been reported in drusen

and basal laminar deposits. The accumulation of drusen is considered the

predominant morphological change that occurs in aged Bruch’s membrane, which

may be related to inflammation (Anderson, Mullins et al. 2002). Hagemen et al.

reported that several proteins located within drusen were related to the inflammatory

or immune response (Hageman, Luthert et al. 2001). During inflammation, large

fluxes of nitric oxide (NO) are released through the activation of inducible nitric

oxide synthase (Marletta, Yoon et al. 1988; Carreras, Pargament et al. 1994). In

addition, nitrite concentration is nearly doubled in the diabetic retina (El-Remessy,

Behzadian et al. 2003); serum nitrite levels are elevated in people who smoke and

cigarette smoking has been strongly associated with the development of AMD

(Solberg, Rosner et al. 1998). Patients with AMD were reported to have

significantly higher plasma NO levels over control subjects (Evereklioglu, Er et al.

2003). NO itself is a relatively unreactive radical; however, it is able to form other

reactive intermediates including nitrite (NO2-), peroxynitrite (ONOO-), NO2, and

N2O3 that can modify proteins, lipids and other compounds. Nitrite is one of the

major NO metabolic products and has been used as a marker of NO production

(Farrell, Blake et al. 1992; Gaston, Reilly et al. 1993). In addition, nonenzymatic

nitration of long-lived proteins such as extracellular matrix (ECM) proteins is a well

known pathway that has been associated with inflammation (Bailey, Paul et al.

1998; Paik, Dillon et al. 2001). The ECM proteins, such as collagen and elastin,

have been reported to be nonenzymatically modified by nitrite at physiological pH

(Paik, Ramey et al. 1997; Paik, Dillon et al. 2001).

184 Studies of basement membrane proteins are experimentally problematic,

often hindered by small samples sizes and complex sample compositions. Previous

studies have concentrated on creating antibodies that are generally expensive and

frequently unreliable. To mitigate these complications, it is advantageous to create

model systems that are biologically relevant and then compare these results to data

obtained from living tissues.

Therefore in this chapter, nonenzymatic glycation and nitration of the

basement membrane protein laminin was performed using the Cys-laminin α-chain

synthetic peptide as a model compound for the modification of basement membrane

proteins, which is comparable to the alpha chain of Laminin type 1 within Bruch’s

membrane (Kanemoto, Reich et al. 1990; Aisenbrey, Zhang et al. 2006). In addition,

modifications by A2E are also studied. Following the enzymatic digests, all

samples, including control groups, were analyzed using liquid chromatography-

electrospray ionization mass spectrometry (LC/ESI-MS). The results explicitly

indicated that fragments containing lysine and arginine residues were preferentially

modified in the glycated and irradiated samples. However, nitration of laminin

fragments was not observed. Instead several of the fragments ending in a lysine

residue appeared to bind to other fragments also ending in a lysine residue,

indicating a polymerization-type reaction.

185 Results

Laminin modification with glycolaldehyde

The non-enzymatic glycation of basement membrane proteins via the

Maillard reaction is of particular interest since this type of modification has been

implicated in retinal dysfunction. To simulate the generation of advanced glycation

endproducts, one sample of the cys-laminin α chain, CSRARKQAASIKVAVSADR

(Figure 5.1), was incubated with glycolaldehyde. The reaction scheme for

glycolaldeyhe modification of a primary amine is displayed in Figure 5.2. Following

tryptic digests, both samples were analyzed via LC-MS and the resulting total ion

chromatograms (TIC), were compared to elucidate the glycated fragments. Figure

5.3 displays a typical TIC of the unmodified tryptically digested laminin segment

with corresponding mass-to-charge (m/z) ratios of identified fragments. Laminin

fragments identified via SEQUEST software in the MS/MS experiments for the

control and glycolaldehyde incubated samples were identified using the B and Y

ions generated for each fragment (Figure 5.1). The identified fragments from each

sample were then compared. The peptides generated were a result of fully and

partially enzymatically digested protein. The most abundant peptides identified in

the laminin control are displayed in Table 5.1. The most abundant fragments in the

control were identified as CSR (Figure 5.4), ARK (Figure 5.5), QAASIK (Figure

5.6), VAVSADR (Figure 5.7), and CSRARKQAASIKVAVSADR (Figure 5.8).

The unmodified laminin fragments from the glycolaldehyde incubated laminin

186

Figure 5.1: The amino acid sequence of laminin fragment with B and Y ions identified. (B2 and Y17 are examples of fragments generated after cleavage)

187

Figure 5.2: The reaction scheme for glycation of lysine and arginine within the laminin fragment or with A2E and A2E derived aldehydes

188

0 20 40 60 80 100 120

0.0

5.0x107

1.0x108

1.5x108

2.0x108

2.5x108

Re

lativ

e A

bso

rba

nce

(A

U)

Time (min)

404

180

592

246

Figure 5.3: The TIC for a typical enzymatically digested laminin control sample without modification is shown. The m/z ratios corresponding to fragments with amino acid sequences of CSRARK, AR, CSRARKQAASIKVAVSADR, and

CSRAR are identified.

189

Table 5.1 – Laminin Control: Laminin fragments identified in the control sample including the observed m/z, associated charge, parent ions (MH+), and

corresponding amino acid sequences.

m/z

Charge (z) (MH+) Sequence

365.431 1.000 365.431 [-]CSR[A] 592.696 1.000 592.696 [-]CSRAR[K] 180.217 4.000 720.868 [-]CSRARK[Q] 329.89 4.000 1319.564 [-]CSRARKQAASIK[V] 403.667 5.000 2018.335 [-]CSRARKQAASIKVAVSADR[-] 246.288 1.000 246.288 [R]AR[K] 324.385 3.000 973.156 [R]ARKQAASIK[V] 417.982 4.000 1671.927 [R]ARKQAASIKVAVSADR[-] 248.63 3.000 745.891 [R]KQAASIK[V] 722.332 2.000 1444.663 [R]KQAASIKVAVSADR[-] 617.718 1.000 617.718 [K]QAASIK[V] 1316.490 1.000 1316.490 [K]QAASIKVAVSADR[-] 717.795 1.000 717.795 [K]VAVSADR[-]

190

200 250 300 350

0

1x104

2x104

3x104

4x104

Inte

nsity

(A

U)

m/z

CSR

175.2

190.2

245.5365.3

347.4

Y1

B2

B3

Y3

Y2

Figure 5.4: The MS/MS spectrum for digested laminin fragment, CSR (m/z 365), with B and Y ions identified.

191

220 240 260 280 300 320 340 360 380

0

1x105

2x105

3x105

4x105

5x105

Inte

nsi

ty (

AU

)

m/z

ARK

228

286.3

339.1 356.4 374.4

B2

Y2*

B3* B

3 Y3

Figure 5.5: The MS/MS spectrum for digested laminin fragment, ARK (m/z 374), with B and Y ions identified.

192

254.3260.5313.5

323.4

341.3

347.4

408.2

418.4

426.3

436.1

454.1

582.3

601.2

250 300 350 400 450 500 550 600 650

0

1x105

2x105

3x105

4x105

5x105

6x105

7x105

Inte

nsi

ty (

AU

)

m/z

QAASIK

618.2B

6

B6*

B5*

Y4

Y2

B3*

Y4*Y

3*

Figure 5.6: The MS/MS spectrum for digested laminin fragment, QAASIK (m/z 618), with B and Y ions identified.

193

373.4428.4

431.3

448.4

530.5

699.5718.4

300 400 500 600 700

0.0

2.0x105

4.0x105

6.0x105

8.0x105

1.0x106

1.2x106

1.4x106

Inte

nsi

ty (

AU

)

m/z

VAVSADR

Y3*

Y4*

618.2

Y5

B7*

Y2 544.5

B6

B5 Y

3

271.2B

3

Figure 5.7: The MS/MS spectrum for digested laminin fragment, VAVSADR (m/z 718), with B and Y ions identified.

194

400 500 600 700 800 900 1000

0.0

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

3.0x105

3.5x105

4.0x105

Inte

nsi

ty (

AU

)

m/z

CSRARKQAASIKVAVSADR

1009

865

958

1000922

914.56402

457509

566

637

701

728785

801835

B6

Y7

B9*

B10

B11

Y12

B13

B14

*

B15

Y14

Y15

B17

Y16

B18

Y17

B19

267

308

Figure 5.8: The MS/MS spectrum for digested laminin fragment, CSRARKQAASIKVAVSADR (m/z 1009), with B and Y ions identified.

195 sample that were identified vary slightly from the control (Table 5.2). This slight

variation is a result of different charges associated with each peptide and the

abundance of fragments with incomplete proteolytic digestion of a protein sample,

resulting in fragments containing internal cleavage sites.

The most abundant modified peptides from the glycolaldehyde incubated

sample identified using MS/MS data are reported in Table 5.3, including the

unmodified m/z ratios, charge state, and relative intensity of each fragment. In the

third column of Table 5.3, the modified masses are reported as the m/z of the

laminin fragment with the addition of glycolaldehyde and the loss of water.

Fragments identified primarily ended with a lysine or arginine residue, which was

expcted. The MS/MS spectra of the most abundant fragments, CSR (Figure 5.9),

CSRAR (Figure 5.10), CSRARK (Figure 5.11), QAASIK (Figure 5.12) and

CSRARKQAASIKVAVSADR (Figure 5.13) are presumably the result of an

incomplete tryptic digest. Because trypsin cleaves peptides at arginine and lysine

residues, the incomplete digest can be attributed to trypsin not being able to

recognize the arginine and lysine residues of each fragment after modification. The

additional fragments reported were consistent with a single site of glycation and a

loss of water. The identified sites of modification are highlighted in column four

with the identified sequence (Table 5.3). These sites of modification were identified

using the B and Y ions generated for the MS/MS scan of each sequence (Hunt,

Yates et al. 1986; Mann and Wilm 1994). Modified sites displayed B and Y ions

196

Table 5.2 – Glycated Laminin Sample: Laminin fragments (without modifications) identified in the glycated laminin sample including the observed m/z, associated

charge, the MH+, and corresponding amino acid sequence.

m/z

Charge (z) MH+ Sequence

365.431 1.000 365.431 [-]CSR[A] 197.56 3.000 592.696 [-]CSRAR[K] 180.217 4.000 720.868 [-]CSRARK[Q] 263.913 5.000 1319.564 [-]CSRARKQAASIK[V] 403.667 5.000 2018.335 [-]CSRARKQAASIKVAVSADR[-] 246.288 1.000 246.288 [R]AR[K] 187.231 2.000 374.461 [R]ARK[Q] 324.385 3.000 973.156 [R]ARKQAASIK[V] 417.982 4.000 1671.927 [R]ARKQAASIKVAVSADR[-] 248.63 3.000 745.891 [R]KQAASIK[V] 722.332 2.000 1444.663 [R]KQAASIKVAVSADR[-] 717.795 1.000 717.795 [K]VAVSADR[-]

197

Table 5.3 – Glycated Laminin: Most abundant laminin fragments modified with glycolaldehyde identified by LC-MS/MS including the observed m/z of the

unmodified sequence, the associated charge, the observed m/z of the sequence after modification with glycolaldehyde, and the corresponding amino acid sequence with

site of modification highlighted.

m/z Charge

(z)

m/z After Modification

[((M+unmod) + 60 -18)/ z) = (m/zmod)]

Sequence of Laminin Fragment (Site of Modification

Highlighted)

365.43 2 203.7 CSR 592.70 3 211.57 CSRAR 720.87 2 381.43 CSRARK 617.72 2 329.86 QAASIK 2018.34 2 1051.17 CSRARKQAASIKVAVSADR

198

100 120 140 160 180 200

0

1x103

2x103

3x103

4x103

5x103

6x103

Inte

nsity

(A

U)

m/z

CSR

87.7

B2*

142.9

Y2*

152.1Y

2

186.9B

3*

195.9

204.2Y

3*

Figure 5.9: The MS/MS spectrum for digested laminin fragment, CSR (m/z 204), modified by glycolaldehyde. The site of glycation is highlighted in red and the B

and Y ions are identified in blue.

199

229.7294.7

335

389.7

402.7

460.6

634.6

616.2

200 250 300 350 400 450 500 550 600 650

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

1.2x105

1.4x105

1.6x105

Inte

nsi

ty (

AU

)

m/z

CSRAR

Y2*

B3

Y3

B4

B5

Y4

531

Figure 5.10: The MS/MS spectrum for digested laminin fragment, CSRAR (m/z 634), modified by glycolaldehyde. The site of glycation is highlighted in red and the

B and Y ions are identified in blue.

200

300 400 500 600 700

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

1.2x105

1.4x105

1.6x105

Inte

nsity

(A

U)

m/z

CSRARK

762.8

745.9

727.6

659.8642.7

599.7555.2399.4330.1

B3* Y

3*

418.2

B4

Y4*

572.6

Y4

B5

Y5

Y5*

B6*

Y6*

Figure 5.11: The MS/MS spectrum for digested laminin fragment, CSRARK (m/z 762), modified by glycolaldehyde. The site of glycation is highlighted in red and the


201

200 300 400 500 600

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

1.2x105

1.4x105

1.6x105

1.8x105

Inte

nsi

ty (

AU

)

m/z

QAASIK

254.2341.1

372.0

659.2

642.9

514.4

471.2

442.1

B3*

B4*

Y3*

Y4*

B5

Y5*

B6

Y4

Y3

B4

Figure 5.12: The MS/MS spectrum for digested laminin fragment, QAASIK (m/z 659), modified by glycolaldehyde. The site of glycation is highlighted in red and the


202

400 500 600 700 800 900 1000

0.0

5.0x102

1.0x103

1.5x103

2.0x103

2.5x103

3.0x103

3.5x103

4.0x103

Inte

nsi

ty (

AU

)

m/z

CSRARKQAASIKVAVSADR

1051

350

907

1000

1043964

956

373309

444499

551608

679

743770828

843

878

Y5

Y6*

B6

Y7

B9*

B10

B11

Y8

B13

B14

*

B15

Y15

Y16

B17

Y17

B18

Y18

B19

Figure 5.13: The MS/MS spectrum for digested laminin fragment, CSRARKQAASIKVAVSADR (m/z 1051), modified by glycolaldehyde. The site of

glycation is highlighted in red and the B and Y ions are identified in blue.

203 that contained an additional mass of 42, which corresponded to the addition of

glycolaldehyde with the loss of water. By reconstructing the peptide from the

individual B and Y ions, the site of modification was determined. The

corresponding B and Y ions identified for the most abundant fragments are

displayed in blue in their corresponding spectra.

Laminin modification with Carboxymethyllysine (CML)

In addition to modifications of laminin by glycolaldehyde, the data

generated for the glycated laminin sample was also analyzed to determine if

common advanced glycation end products were present. Specifically, CML was

identified in the sample, which forms from nonenzymatic glycation followed by

oxidation of proteins (Figure 5.14). The most abundant CML modified peptides

from the glycolaldehyde incubated sample identified using MS/MS data are reported

in Table 5.4 including the unmodified m/z ratios, charge state, and relative intensity

of each fragment. In the third column of Table 5.4, the modified masses are

reported as the m/z of the laminin fragment with the addition of CML and the loss

of water. Fragments identified primarily ended with a lysine residue. The MS/MS

spectrum of CML (m/z 205) with characteristic cleavages is displayed in Figure

5.15. The most abundant laminin fragments modified by CML were ARK,

CSRARK, and QAASIK. The MS/MS spectrum and proposed structure of ARK are

displayed in Figures 5.16 and 5.17, respectively. The B and Y ions associated with

204

Figure 5.14: The reaction scheme of glycolaldehyde with lysine producing carboxymethyl lysine (CML) and then the modification of primary amines in

laminin by CML

205

Table 5.4 - Glycated Laminin: Most abundant laminin fragments modified with CML identified by LC-MS/MS including the observed m/z of the unmodified

sequence, the associated charge, the observed m/z of the sequence after modification with glycolaldehyde, and the corresponding amino acid sequence with

site of modification highlighted.

MH+unmod Charge

(z)

m/z After Modification [((M+unmod) + 204-18)/ z) =

(m/zmod)]

Sequence of Laminin Fragment (Site of Modification

Highlighted)

374.43 1 560.43 ARK 720.80 1 906.8 CSRARK 617.87 1 803.87 QAASIK 974.72 2 580.36 ARKQAASIK 2018.34 2 1195.17 CSRARKQAASIKVAVSADR

206

140 160 180 200

0

1x104

2x104

3x104

4x104

5x104

6x104

7x104

NH2

HN

O

OH

OH

O

187

159 130145

Inte

nsity

(A

U)

m/z

CML

130.2

145.8 159.3

188.0

205.8

189.8

Figure 5.15: The MS/MS spectrum of CML located in the glycated laminin sample. The inset is the structure of CML (m/z 205) with characteristic cleavages identified.

207

250 300 350 400 450 500 550

0.0

2.0x104

4.0x104

6.0x104

8.0x104

ARK

Inte

nsity

(A

U)

m/z

228.3 316.3333.4

525.6

543.6

B1 Y

1*

Y1

B2*

358.3

Figure 5.16: The MS/MS spectrum for digested laminin fragment, ARK (m/z 543), modified by CML. The site of modification is highlighted in red and the B and Y

ions are identified in blue.

208

Figure 5.17: The proposed structure for CML modification of ARK fragment

209 the cleavages of each amino acid are identified in blue and the site of modification

is highlighted in red. The carbonyl carbon forms a double bond with the primary

amine on lysine’s side chain with the subsequent loss of water. In addition to ARK,

the MS/MS spectra for laminin fragments, CSRARK (Figure 5.18) and QAASIK

(5.19), displayed similar additions in their identified B and Y ions, resulting in

structural similarities (Figure 5.20 and 5.21). Both the CSRARK and QAASIK

fragments showed that the carbonyl compound of CML formed a double bond with

the primary amine on a lysine residue with the loss of water.

Laminin modification with A2E

The bis-retinoid pyridium compound A2E is also of interest as a potential

modifier of basement membrane proteins. A2E has previously been shown to

mediate blue light-induced apoptosis in RPE cells and to non-photochemically

induce apoptosis in RPE cells at physiological concentrations (Sparrow, Nakanishi

et al. 2000; Sparrow and Cai 2001). In addition, the photo-oxidation and auto-

oxidation of A2E has been reported to yield a complex mixture of lower molecular

weight products, which are generated from a series of cleavages along the acyclic

chain forming aldehydes displayed in Figure 5.22 (Wang, Keller et al. 2006). The

aldehydes formed could potentially modify basement membrane proteins and,

therefore, cause damage to RPE cells. As a result, laminin was incubated with A2E

210

200 300 400 500 600 700 800 900

0.0

2.0x103

4.0x103

6.0x103

8.0x103

1.0x104

1.2x104

1.4x104

1.6x104

Inte

nsity

(A

U)

m/z

CSRARK

906.6

889.9

803.6

786.8704.7

699.7560.2333.4191.1

B*

Y1

401.2

B4

Y4*

574.6

Y3

B5

Y5*

Y5

B6

347.3

B2

489.4Y

2

Figure 5.18: The MS/MS spectrum for digested laminin fragment, CSRARK (m/z 906), modified by CML. The site of modification is highlighted in red and the B and

Y ions are identified in blue.

211

200 300 400 500 600 700 800

0.0

2.0x103

4.0x103

6.0x103

8.0x103

1.0x104

1.2x104

1.4x104

1.6x104

1.8x104

Inte

nsity

(A

U)

m/z

QAASIK

254.2341.3

516.6

803.92

785.9

658.76

471.53

587.68

B3*

B4*

Y3*

Y4*

B5

Y5*

B6

B4

615.4

Figure 5.19: The MS/MS spectrum for digested laminin fragment, QAASIK (m/z 803), modified by CML. The site of modification is highlighted in red and the B and

Y ions are identified in blue.

212

Figure 5.20: The proposed structure for CML modification of CSRARK fragment

213

Figure 5.21: The proposed structure for CML modification of QAASIK fragment

214

Figure 5.22: The cleavage positions and the molecular masses of corresponding aldehydes in A2E and oxidized A2E are shown.

215 and subjected to blue light irradiation to compare these effects with modifications

from glycolaldehyde. To differentiate between modifications from irriadiated A2E,

a non-irradiated control sample was analyzed in conjunction with the irradiated

sample. The unmodified fragments identified in the sample irradiated in the

presence of A2E differ from the control sample only by the charge associated with

each fragment and abundance of fragments with incomplete proteolytic digestion

(Table 5.5). Once identified, these peaks were eliminated from both chromatograms,

leaving only peaks that were possible sites of modification. The chromatogram of

the A2E laminin sample without irradiation, the dark control, contained an abundant

A2E peak and multiple laminin peaks; however, the presence of modifications from

A2E on the protein fragments was absent, indicating that there are no dark reactions

detectable with our experimental conditions. The data for the most abundant

modifications resulting from the A2E-laminin sample after irradiation are

highlighted in column 2 of Table 5.6. The A2E-laminin sample primarily displayed

modifications to arginine and lysine residues, which agreed with the glycated

laminin results. However, the number of modifications identified with A2E was

significantly more extensive and is evidence of the high reactivity and structural

diversity of A2E-derived oxidation products. The modified laminin fragments are

consistent with additions of A2E derived aldehydes that predominantly result from

cleavages closest to the pyridinium ring in A2E and oxidized A2E. These A2E

derived aldehydes correspond to masses of 366, 382, 406, and 422 Da and are

216

Table 5.5 – Laminin fragments identified in A2E incubated laminin samples including the observed m/z of the laminin fragment, the associated charge, the MH+,

and the corresponding amino acid sequence.

m/z


182.716 2.000 365.431 [-]CSR[A] 592.696 1.000 592.696 [-]CSRAR[K] 180.217 4.000 720.868 [-]CSRARK[Q] 659.782 2.000 1319.564 [-]CSRARKQAASIK[V] 672.778 3.000 2018.335 [-]CSRARKQAASIKVAVSADR[-] 374.461 1.000 374.461 [R]ARK[Q] 324.385 3.000 973.156 [R]ARKQAASIK[V] 745.891 1.000 745.891 [R]KQAASIK[V] 722.332 2.000 1444.663 [R]KQAASIKVAVSADR[-] 617.718 1.000 617.718 [K]QAASIK[V] 438.83 3.000 1316.490 [K]QAASIKVAVSADR[-] 717.795 1.000 717.795 [K]VAVSADR[-]

217

Table 5.6 – Laminin fragments modified with irradiated A2E including the observed m/z of the laminin fragment, the corresponding amino acid sequence with the site of

modification highlighted, the associated charge, and the observed masses of laminin with modification A2E aldehydes.

Laminin Sequence ChargeMasses of Known A2E

Aldehydes (Da)

MH+ Fragments (Site of

Modification Highlighted)

(Z) 366 382 406 422

374.46 ARK 1 722.46 738.46 ----- 778.46

592.73 CSRAR 1 ----- 956.73 ----- -----

720.87 CSRARK 2 ----- 542.4 554.4 562.4

973.16 ARKQAASIK 2 660.58 668.58 ----- 688.58

1319.56 CSRARKQAASIK 2 ----- 841.78 ----- 861.78

592.73 CSRAR 2 470.4 478.4 490.4 ---

218

200 300 400 500 600 700 800 900 1000

0

1x103

2x103

3x103

4x103

5x103

6x103

7x103

8x103

9x103 CSRAR (mod. 406)

Inte

nsity

(A

U)

m/z

174.2

191.3

229.3

325.5

390455

544.1

589

654.7

735.4

789.5830.7

877.6

945.6

963.7980.4

529.2

Y1

B2

Y2*

Y3*

B3

B4

Y4

B4*

Y5*

Y5

Figure 5.23: The MS/MS spectrum for digested laminin fragment, CSRAR (m/z 980), modified by A2E-derived aldehyde with m/z 406. The site of modification is

highlighted in red and the B and Y ions are identified in blue.

219 displayed in Figure 5.22. Each laminin fragment appeared in the TIC multiple

times; however, the A2E aldehyde modification associated with each fragment

differed. For example, the MS/MS spectrum for CSRAR (Figure 5.23) displayed a

fragmentation pattern with B and Y ions indicating that the arginine residue was

modified by A2E aldehyde with m/z 406 (Figure 5.24). An additional MS/MS

spectrum for this same fragment, CSRAR (Figure 5.25), displayed a fragmentation

pattern with B and Y ions indicating that the same arginine residue was modified by

a different A2E derived aldehyde with m/z 382 (Figure 5.26). The MS/MS for

laminin fragment KQAASIK (Figure 5.27) displayed a fragmentation pattern with

ions indicating a modification to the lysine residue by A2E-derived aldehyde with

m/z 360 (Figure 5.28). An additional MS/MS spectrum for the same fragment,

KQAASIK (Figure 5.29), displayed a fragmentation pattern with B and Y ions

indicating the modification to the same lysine residue by A2E-derived aldehyde

with m/z 366 (Figure 5.30). Therefore, each laminin fragment displayed in Table 5.6

yielded multiple peaks in the TIC corresponding to modifications of the same amino

acid in the same laminin fragment but with a different aldehyde attached.

The PDA chromatograms for the laminin control, glycated laminin, and the

irradiated A2E laminin samples are displayed in Figure 5.31. The UV-vis maxima

for the laminin control and glycated sample were approximately 265, 270, 280, and

295 nm. However, the UV-maxima for the irradiated A2E laminin samples shifted

to 330, 340, 360, and 380 nm. The CSR, CSRAR, CSRARK, and

CSRARKQAASIKVAVSADR fragments for each sample are identified in the

individual chromatograms as representative peaks for sites of modification. These

220

Figure 5.24: The proposed structure for A2E derived aldehyde (m/z 406) modification of CSRAR fragment

221

200 300 400 500 600 700 800 900

0

1x103

2x103

3x103

4x103

5x103

6x103

7x103

8x103

9x103 CSRAR (mod. 382)

Inte

nsi

ty (

AU

)

m/z

175.2

191.3

229.3

296

325.5

364

402.5529.1

544.3

589

694.7

711.4

789.5765.7

836.5

853.6

921.6

939.7

956.6

Y1

B2

Y2*

Y3

B3*

B3

B4*

Y4

Y4*

B5*

Y5*

Figure 5.25: The MS/MS spectrum for digested laminin fragment, CSRAR (m/z 956), modified by A2E derived aldehyde with m/z 382. The site of modification is


222

Figure 5.26: The proposed structure for A2E derived aldehyde (m/z 382) modification of CSRAR fragment

223

400 500 600 700 800 900 1000

0.0

2.0x103

4.0x103

6.0x103

8.0x103

1.0x104

1.2x104

1.4x104

Inte

nsity

(A

U)

m/z

KQAASIK (mod. 366)

352.1401.4

472.1

489.6 600.3617.3

676.9

728.2

745.2

817.2

834.7Y3*

Y5*

Y5

Y6*

Y6

B3

1058.1947.3

B4*

Y7

B5*

B5 B

6

Figure 5.27: The MS/MS spectrum for digested laminin fragment, KQAASIK (m/z 1058), modified by A2E derived aldehyde with m/z 366. The site of modification is


224

Figure 5.28: The proposed structure for A2E derived aldehyde (m/z 366) modification of KQAASIK fragment

225

400 500 600 700 800 900 1000 1100

0.0

2.0x103

4.0x103

6.0x103

8.0x103

1.0x104

1.2x104

1.4x104

Inte

nsity

(A

U)

m/z

KQAASIK (mod. 382)

366.9401.3

472.0

489.6 600.2617.2

693.9

728.2

745.2

834.2

851.7 964.6

10751092.3Y

4*

Y5*

Y5

Y6*

Y6

Y7*

Y7

B5*

B5

B3

B6

B7*

Figure 5.29: The MS/MS spectrum for digested laminin fragment, KQAASIK (m/z 1092), modified by A2E derived aldehyde with m/z 382. The site of modification is


226

Figure 5.30: The proposed structure for A2E derived aldehyde (m/z 382) modification of KQAASIK fragment

227

0 20 40 60

01x1042x1043x1044x1045x104

0.05.0x1041.0x1051.5x1052.0x1052.5x1053.0x105

494326

Time (min)

A2E Laminin Irradiated554

0.05.0x1041.0x1051.5x1052.0x105

A2E Laminin Control

1051

Re

lativ

e A

bun

dn

ce (

AU

)

Glycated Laminin381

317

183

180A2E

0.02.0x1054.0x1056.0x1058.0x1051.0x106

1009

180Laminin Control

296365

296

A2E

Figure 5.31: The HPLC total PDA trace of the laminin control, glycated laminin, A2E laminin control, and irradiated A2E laminin samples are shown respectively.

Selected fragments are identified in each chromatogram.

228 CSRARKQAASIKVAVSADR fragments for each sample are identified in the

individual chromatograms as representative peaks for sites of modification. These

three fragments only varied by specific mass modifications, elution times, and

charge states, which are displayed in Table 5.7.

Laminin modification with nitrite

Because nonenzymatic nitration of long lived-proteins has also been

associated with retinal dysfunction, laminin was also treated with nitrite and the

control was treated with NaCl. After dialysis and tryptic digests, both samples were

analyzed via LC-MS and the resulting total ion chromatograms (TIC) were

compared to elucidate the nitrated fragments. Tables 5.8 and 5.9 display the

unmodified laminin fragments that were found in the control sample and nitrated

samples. The unmodified laminin fragments from the nitrated laminin sample that

were identified vary slightly from the control. This slight variation is a result of

different charges associated with each peptide and the abundance of fragments with

incomplete proteolytic digestion of a protein sample, resulting in fragments

containing internal cleavage sites. Once identified, peaks common to both samples

were removed and the remaining peaks in the nitrated laminin sample were further

analyzed. Initially the remaining spectra were analyzed to determine if an addition

of 45 or multiple of 45, corresponding to the addition of NO2, to the parent mass

was observed. However, this addition could not be found in any of the spectra,

suggesting that

229

Table 5.7 – Peptide fragment’s CSR, CSRAR, and CSRARK in the laminin control, glycated laminin, A2E laminin control, and irradiated A2E laminin samples

including their corresponding observed m/z, associated charge, and retention time. The irradiated A2E sample also includes the mass of the corresponding A2E

aldehyde modification.

Peptide Fragment Laminin Control

Glycated Laminin

A2E Laminin Control

Irradiated A2E

Laminin CSR

Observed m/z 365 203.7 183 Not Found

associated charge 1 2 2 ----

Retention time 8 mins 10 mins 10 mins ---- Mass of A2E aldehyde

CSRAR

Observed m/z 278 317.3

Not Found

326

associated charge 2 2 ---- 3

Retention time 10 mins 12 mins ---- 14 mins Mass of A2E aldehyde 406 Da CSRARK Observed m/z 180 381.4 180 554.0

associated charge 4 2 4 2

Retention time 10 mins 14 mins 10 mins 17 mins Mass of A2E aldehyde 406 Da CSRARKQAASIKVAVSADR

Observed m/z 1009 1051

Not Found

494

associated charge 2 2 ---- 5

Retention time 35 38 ---- 36 Mass of A2E aldehyde 472

230

Table 5.8 – Control Laminin Sample: Laminin fragments identified in the NaCl laminin sample including the observed m/z, associated charge, the MH+, and

corresponding amino acid sequence.

m/z



231

Table 5.9 – Nitrated Laminin Sample: Laminin fragments identified in the nitrated sample including the observed m/z, associated charge, the MH+, and corresponding

amino acid sequence.

m/z

Charge (z)

MH+ Sequence


232 nitration of the amino acids did not occur. This may be attributed to the lack of

aromatic amino acids within the laminin sequence or possibly the fragments were

undergoing another type of reaction. Next, the fragmentation patterns for several of

the larger parent ions were analyzed. This data suggested that the laminin fragments

ending in a lysine residue were further reacting with other laminin fragments also

ending in a lysine residue. The primary amine on a lysine side chain binds to the

carbonyl carbon on the second lysine residue with the loss of water. Table 5.10

displays the most abundant fragments that formed in this type of reaction, which

include the sequences QAASIKKRA, CSRARKKRARSC, QAASIKKISAAQ, and

ARKKRA. The MS/MS spectra for these fragments are displayed Figures 5.32, 5.33,

5.34, and 5.35 followed by the proposed structures in Figures 5.36, 5.37, 5.38, and

5.39. The corresponding B and Y ions for each fragment are identified in blue for

each spectra and were used to determine each structure.

The PDA chromatograms for the laminin control and the nitrated laminin

samples are displayed in Figure 5.40. The UV-vis maxima for the laminin control

and nitrated laminin sample were approximately 265, 270, and 280 nm. The ARK,

CSRARK, and QAASIK fragments for each sample are identified in the individual

chromatograms as representative peaks for sites of modification. These three

fragments only varied by specific mass modifications, elution times, and charge

states, which are displayed in Table 5.10.

233

Table 5.10 – Peptide fragment’s ARK, CSRARK, and QAASIK in the laminin control and nitrated laminin sample including their corresponding observed m/z, associated charge, and retention time.

Peptide Fragment

Laminin Control

Laminin Dimer (nitration sample)

ARK


associated charge 1 1

Retention time 23 mins 32 mins

CSRARK




QAASIK




234

200 300 400 500 600 700 800 900

0.0

2.0x103

4.0x103

6.0x103

8.0x103

1.0x104

1.2x104

1.4x104

1.6x104

Inte

nsity

(A

U)

m/z

QAASIKKRA

341.1

271.3384.5

478.4

523.4

557.6599.5

625.5

651.3

673.5

692.4

725.4

742.4782.5

898.9

937.7

955.8

973.1

B3

B4*

B9

Y5*

B8

Y4

Y6

Figure 5.32: The MS/MS spectrum for digested laminin fragment QAASIKKRA (m/z 973). The B and Y ions are identified in blue.

235

200 300 400 500 600 700

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

1.2x105

1.4x105

1.6x105

1.8x105

Inte

nsity

(A

U)

m/z

CSRARKKRARSC

201.2 278.9 343.4

352.4

416.9

407.5

485.6

494.1521 599.2608

642.8

651.7

703.4

711.8

B4* B

5* B

6*

Y5*

Y6*

B7*

B12

B11

B11

*

B10

B8*

B9*

Y10

*

Figure 5.33: The MS/MS spectrum for digested laminin fragment CSRARKKRARSC (m/z 711). The B and Y ions are identified in blue.

236

250 300 350 400 450 500 550 600 650

0.0

5.0x103

1.0x104

1.5x104

2.0x104

Inte

nsity

(A

U)

m/z

QAASIKKISAAQ

236.8

291.8

364.9

464.6

491.6

535.7

600.2

608.7

300.8

355.9

412.5421.3

456.0

500.1

527.1

591.2

Y4*

B6*

B6

Y7*

B7*

B12

*

B11

Y11

*

B8*

Y8

B9

B11

*

B10

Figure 5.34: The MS/MS spectrum for digested laminin fragment QAASIKKISAAQ (m/z 608). The B and Y ions are identified in blue.

237

200 300 400 500 600 700

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

Inte

nsity

(A

U)

m/z

ARKKRA712.8

729.9211.2228.3 339.4 357.4

467.6 485.6

502.2531.3583.9

622.7641.8

B6

B5

B5*B

2*

Y1*

B3*

Y2*

B4*

Y4

Y4*

Figure 5.35: The MS/MS spectrum for digested laminin fragment ARKKRA (m/z 729). The B and Y ions are identified in blue.

238

Figure 5.36: The proposed structure for QAASIKKRA

239

Figure 5.37: The proposed structure for CSRARKKRARSC

240

Figure 5.38: The proposed structure for QAASIKKISAAQ

241

Figure 5.39: The proposed structure for ARKKRA

242

0 20 40 60 80 100 120 140

0

1x105

2x105

3x105Inte

nsi

ty (

AU

)

Time (min)

Nitrated Laminin

608

711

730

0 20 40 60 80 100 120 140

0

1x105

2x105

3x105

4x105

5x105

Laminin control

720

618374

Figure 5.40: The HPLC total PDA trace of the laminin control and nitrated laminin

samples are shown respectively. Selected fragments, ARK, CSRARK, AND QAASIK, are identified in each chromatogram

243 Discussion

Laminins are ubiquitous multifunctional basement membrane proteins that

are the most abundant noncollagenous structural glycoproteins within extracellular

matrices. Laminins are involved in copious intricate interactions with themselves,

components of the basal lamina, and a variety of other cells facilitating the

formation, design, and integrity of the basement membranes (Aumailley and Smyth

1998). The non-enzymatic modifications of proteins via the Maillard reaction have

been shown to form advanced glycation endproducts and disrupt function (Ahmed,

Dunn et al. 1988; Vlassara, Bucala et al. 1994; Stitt and Vlassara 1999). In this

reaction scheme, the carbonyl on a reducing sugar reacts with the nucleophilic

amino group on an amino acid, preferentially lysine or arginine, producing a variety

of AGEs (Ulrich and Cerami 2001; Yan, Ramasamy et al. 2003). Initially, a

condensation reaction produces a Schiff base followed by Amadori rearrangement

to produce Amadori products. The Schiff-base and Amadori products can further

react through cyclization, enolization, and oxidation to produce numerous AGEs.

The type, quantity, and physiological relevance of AGEs produced will depend on

the source of the primary amine and the type of sugar used as the reducing agent

(Honda, Farboud et al. 2001; Nagaraj, Biswas et al. 2008).

Modifications to the sequence or changes to the structure of laminin caused

by AGEs can disrupt control of cellular functions (Charonis, Reger et al. 1990;

Charonis and Tsilbary 1992; Federoff, Lawrence et al. 1993). Crosslinking

mediated via AGEs decreases interactions between the components within the

244 basement membranes, leading to irreversible changes to the integrity and structure

of the membrane, tissue rigidity, and reduction in enzymatic susceptibility (Tarsio,

Reger et al. 1988; Tsilibary, Charonis et al. 1988; Charonis, Reger et al. 1990).

Recent studies have shown that abnormal adhesion, spreading, and proliferation of

vascular endothelial cells occurs when cultured on AGE-modified substrates

(Haitoglou, Tsilibary et al. 1992; Kalfa, Gerritsen et al. 1995; Paul and Bailey 1999).

Since adequate interactions between the vascular cells and the basement membrane

are required for normal cell function, AGE accumulation contributes to cell

dysfunction by altering receptor recognition (Grant, Kleinman et al. 1990). In

addition, Glenn et al. have recently reported an increase in accumulation of

autofluorescent material on AGE-modified BM attributed to a decrease in RPE

lysosomal enzyme activity (Glenn, Mahaffy et al. 2008), providing evidence that

components of the RPE and BM interact. Interestingly, Maeda et al. reported that

undigested photoreceptor cell outer segments and basal laminar deposits within the

RPE invaded BM (Maeda, Maeda et al. 2008). We have also observed the presence

of A2E and its derivatives in organic soluble extracts of human BM, suggesting that

A2E does come into contact with material within BM (Dillon, Wang et al. 2006). In

addition, previous work has shown that the photo-oxidation of A2E, a bis-retinoid

pyridinium compound, forms highly reactive aldehydes (Wang, Keller et al. 2006)

that are small enough to diffuse to and in Bruch’s membrane. These highly reactive

species were hypothesized to cause comparable modifications to laminin as the

glycated sample. As a result, studies were performed which showed that A2E

preferentially modifies laminin fragments ending in a lysine or arginine residue with

245 aldehydes generated from the two closest cleavages to the pyridinium ring of A2E

(m/z = 592) and oxidized A2E (m/z = 608) displayed in Figure 5.22. The presence

of modifications to A2E and oxidized A2E indicated that the irradiated sample

underwent photo-oxidation and/or auto-oxidation. The extent of modification to

laminin by A2E was greater than that of the glycated laminin sample because of the

high reactivity of A2E after photo-oxidation.

The major modifications were determined by comparing the laminin control,

glycolaldehyde-incubated laminin, nitrated laminin and A2E irradiated laminin

samples. Unlike typical post translational modifications, these changes result from

oxidative stress and are therefore not present in current databases. As a result,

sample specific modifications were determined by eliminating control spectra and

analysis of remaining peaks instead of typical databases searches. The results for the

glycated and A2E irradiated laminin indicated that fragments containing lysine and

arginine residues were preferentially modified within this protein. However, in the

nitrated laminin sample there was no evidence that nitration occurred. Instead, the

lysine residues in the fragments appeared to react by forming longer chains of

amino acids. This may suggest that in the presence of NO2, the lysine is reacting to

form an adduct similar to the AGEs formed in the glycation reactions. Although

several studies have reported that proteins can undergo deaminitation and nitrite-

induced crossing linking (Paik and Dillon 2000; Deng 2006; Paik, Saito et al. 2006),

studies involving polymierization reactions in the presence of nitrite could not be

found, making this a novel reaction. In addition to the comparison of these

modifications to laminin, the presence of CML was also identified in the glycated

246 sample. The CML, a common AGE, also caused modification to the laminin

fragments, suggesting that oxidative stress accelerates the production of AGEs and

modification to proteins, which can cause dysfunction.

In a variety of retinal diseases, including AMD, basement membranes are

susceptible to alterations in structure and function. These modifications can lead to

a decrease in the ability of basement membrane proteins to function normally,

leading to a decrease in the associations of components that make up the membrane

network, a decrease in the protein present in the membrane, and possibly a more

permeable membrane (Charonis, Reger et al. 1990; Charonis and Tsilbary 1992). It

is also possible that the protein will exhibit abnormal crosslinking, a decrease in

enzymatic susceptibility, and a decrease in solubility. These factors may have a

harmful effect on Bruch’s membrane, resulting in damage to RPE cells. When the

cellular attachments between the RPE cells and basement membrane are disrupted,

the RPE and photoreceptor cells die, leading to the onset of AMD. Previous studies

have shown that numerous structural changes are induced in Bruch’s membrane

with age including thickening of the membrane (Pauleikhoff, Harper et al. 1990;

Zarbin 2004) and the accumulation of hydrophobic patches of debris related to

drusen (Moore, Hussain et al. 1995). The study of modifications from A2E,

glycolaldehyde, or other components located within the extra cellular matrix is still

incomplete; however, this study provides suggestive evidence that A2E may be

involved in modifications to essential basement membrane proteins leading to

deleterious changes within the RPE-ECM environment. These preliminary

experiments are essential in the identification of such changes in vivo because they

247 give predictable chromatographic and spectroscopic characteristics of those

modifications.

CHAPTER 6

CONCLUSIONS AND FUTURE WORK

As a result of the increased number of documented cases and severity of the

disease, research focused specifically on the origin and progression of AMD is

essential in order to treat patients effectively. Currently, medical treatment to stop

the progression of the disease is limited. The characteristic central vision loss

associated with AMD is caused by photoreceptor cell death. In wet AMD, the death

of these cells is associatd with neovascularization, whereas in dry AMD the death of

photoreceptor cells is attributed to damage of the RPE by extracellular deposits such

as drusen. However, the exact mechanism leading to the death of these cells and the

onset of AMD is still unknown. Recent research has suggested that age-related

changes within the RPE and underlying BM may play a crucial role in the

development of AMD. Therefore, in this work, the biochemical and cellular changes

occurring in the RPE and Bruch’s membrane were studied. Our results suggest that

multiple factors, including age-related changes, contribute to the pathogenesis of

AMD.

249 Compositional Studies of Human Retinal Lipofuscin

The formation and composition of lipofuscin and the major fluorophore A2E

have received notable attention. However, the origin of the granules and the identity

of most of the compounds and the consequence of A2E accumulation within the

granules are still unknown. One hypothesis suggested that A2E could exist in a free

or esterified form. In the RPE, all-trans retinol, produced from the visual cycle, is

converted to all-trans retinyl ester, which then self-aggregates into a retinosome

(Imanishi, Gerke et al. 2004). This prevents hydrophobic interactions with cellular

components disrupting normal cell function. Since A2E is extremely hydrophobic

and accumulates within RPE lysosomes, A2E was suggested to undergo a similar

esterification reaction (Mandal 2008). In addition to the esterification reactions, a

second hypothesis involving the modification of A2E by A2E-derived aldehydes

was also suggested. Within the acidic lysosomal environment, A2E undergoes

rearrangements and oxidation, generating aldehydes and ketones that are structurally

similar to ß-carotene oxidation products. These aldehydes are extremely reactive

and in the presence of A2E may interact, forming higher molecular weight products.

Therefore, in this study, lipofuscin was analyzed to investigate the

hydrophobic compounds that chromatographically elute later than A2E and that

absorb radiation with wavelengths greater than 400 nm. The results indicate that a

large quantity of the components of lipofuscin have mass spectra analogous to that

of A2E, as determined by their fragmentation pattern with losses of 190, 174 and/or

150 amu and the formation of fragments of ca 592 amu but with higher molecular

250 weights. The vast majority of the relatively hydrophobic components correspond to

derivatized A2E with discrete molecular weights of m/z 800-900, m/z 970-1080 and

above m/z 1200 regions. The majority of modifications are much more hydrophobic

than A2E, increasing its log P, and probably explain the formation of lipofuscin

granules in the RPE. The spectroscopic characteristics and fragmentation patterns

associated with these compounds supports the hypothesis that A2E is reacting with

aldehydes such as all-trans-retinal, A2E-derived aldehydes, by studying model

reactions with cinnamaldehyde and benzaldehyde to form the higher molecular

weight compounds found in lipofuscin. This study is part of a continuing effort to

identify the molecular modifications to the structure of A2E. Further experiments

are being performed to confirm the structure of some of these higher molecular

weight products such as the compounds with m/z 920 and 1188. However, this

process is complicated by small sample sizes. One of the main challenges is to

collect enough pure A2E to react with RAL, producing mM quantities of these

higher molecular weight products so that 1H and 13C NMR can be performed. In

addition, the fragmentation patterns of additional higher molecular weight

compounds in the lipofuscin sample are still being analyzed to determine the

structures.

Accumulation of 3-nitrotyrosine and nitro-A2E in Bruch’s membrane

The inflammatory response has also been associated with the development

of AMD. Recently four independent research groups have identified a commonly

251 inherited variant (Y402H) of the complement factor H gene in the genome from

different groups of AMD patients (Edwards, Ritter et al. 2005; Hageman, Anderson

et al. 2005; Haines, Hauser et al. 2005; Klein, Zeiss et al. 2005). The Y402H variant

of CFH significantly increases the risk of AMD and links the genetics of the disease

with inflammation. During inflammation there is activation of inducible nitric oxide

synthase and release of nitric oxide (Marletta, Yoon et al. 1988; Carreras,

Pargament et al. 1994), which in principle could lead to non-enzymatic nitration

within extracellular deposits and/or intrinsic extracellular matrix protein

components of human Bruch’s membrane. Modifying ECM proteins in Bruch’s

membrane can result in changes to the ECM proteins similar to age-related changes,

such as protein yellowing and cross-linking. Therefore, the second part of this work

investigated the modifications to intrinsic proteins and extrinsic deposits and A2E in

human Bruch's membrane by reactive nitrogen species released during inflammation.

We have identified an increasing accumulation of 3-nitrotyrosine and nitro-A2E in

human Bruch's membrane with advancing patient age. To our knowledge, the

current study represents the first clear demonstration of inflammation-related

chemical modifications detected in human Bruch’s membrane. The presence of 3-

nitrotyrosine and nitro-A2E may be important biomarkers for immune-mediated

processes during aging, and the role of this process in the development of age-

related macular degeneration. However, further experiments are needed to evaluate

other aspects of this process, including the relationship between the degree of

nitration and the age/medical history of the donor, the effect of nitration on the

turnover of intrinsic extracellular matrix proteins, and determination of the three-

252 dimensional structural changes resulting from nitration and the effects these changes

have on cellular function.

Modifications to Laminin

Chemical modifications to basement membrane proteins may deleteriously

affect Bruch’s membrane, leading to the development of AMD. These modifications

can lead to a decrease in the basement membrane proteins’ ability to function

normally, leading to a decrease in the association of components that make up the

membrane network, a decrease in the protein present in the membrane, and possibly

a more permeable membrane (Charonis, Reger et al. 1990; Charonis and Tsilbary

1992). It is also possible that the protein will exhibit abnormal crosslinking, a

decrease in enzymatic susceptibility, and a decrease in solubility. These factors may

have a harmful effect on Bruch’s membrane, resulting in damage to RPE cells and

the onset of AMD. Previous studies have shown that numerous structural changes

are induced in Bruch’s membrane with age including the accumulation of

hydrophobic patches of debris, which may be related to drusen (Moore, Hussain et

al. 1995). In addition, the formation of advanced glycation endproducts (AGEs) and

associated damage has been associated with a variety of diseases including diabetic

retinopathy.

Therefore, the purpose of this study was to investigate modifications from

AGEs (glycolaldehyde-derived AGEs and CML), inflammation, (nitrite), and AMD

(A2E) on the membrane-like protein fragment laminin as a model for aging of

253 Bruch’s membrane in age-related eye diseases. Modifications to laminin by

glycolaldehyde, CML, and A2E occurred preferentially on lysine or arginine

residues. The A2E-modified laminin fragments are consistent with additions of

A2E-derived aldehydes resulting from cleavages closest to the pyridinium ring in

A2E and oxidized A2E. Although direct nitration of the laminin fragment was not

observed, the laminin fragments ending in a lysine residue appeared to undergo

dimerization. This suggests that in the presence of NO2, the laminin fragments may

react to create higher molecular weight products similar to AGEs, which may result

in damage to Bruch’s membrane and the overlying RPE. This is a novel reaction

induced by nitration. These results provide evidence that A2E and AGEs may be

involved in modifications to essential basement membrane proteins leading to

deleterious changes in the retinal pigment epithelium extracellular matrix (RPE-

ECM) environment. The study of modifications from A2E, glycolaldehyde, or other

components located within the extracellular matrix is still incomplete. Further

analysis of other AGEs, A2E, and A2E-derived aldehydes present in the laminin

samples is being performed. However, this study provides suggestive evidence that

A2E may be involved in modifications to essential basement membrane proteins

leading to deleterious changes.

The pathogenesis of AMD is multifactorial. RPE cells and Bruch’s

membrane are functionally and structurally interdependent. This work focused on

the study of biochemical and cellular changes to the RPE and Bruch’s membrane in

order to understand the modifications and mechanism of formation in vivo, which

254 may provide important information related to the development of AMD and the

development of an effective treatment.

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