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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
All rights reserved
INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.
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and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.
UMI 3404858
Copyright 2010 by ProQuest LLC. All rights reserved. This edition of the work is protected against
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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
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…………………………………….…………...199
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 B and Y ions are identified in blue…………………………… ……………200
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 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
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…………………………………………..……..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
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……………………………………….………...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
5.26 The proposed structure for A2E derived aldehyde (m/z 382)
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
5.28 The proposed structure for A2E derived aldehyde (m/z 366)
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
5.30 The proposed structure for A2E derived aldehyde (m/z 382)
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
731) are identified (blue).
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
731) are identified (blue).
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
Applied Potential (V)
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
Applied Potential (V)
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
Applied Potential (V)
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
Applied Potential (V)
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
B and Y ions are identified in blue.
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
B and Y ions are identified in blue.
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
Charge (z) MH+ Sequence
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
highlighted in red and the B and Y ions are identified in blue.
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
highlighted in red and the B and Y ions are identified in blue.
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
highlighted in red and the B and Y ions are identified in blue.
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
Charge (z) MH+ Sequence
182.416 2.000 364.831 [-]CSR[A] 592.696 1.000 592.696 [-]CSRAR[K] 240.217 3.000 720.868 [-]CSRARK[Q] 659.782 2.000 1319.564 [-]CSRARKQAASIK[V] 403.778 5.000 2018.335 [-]CSRARKQAASIKVAVSADR[-] 374.461 1.000 374.461 [R]ARK[Q] 973.385 1.000 973.156 [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[-]
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
365.416 1.000 365.431 [-]CSR[A] 592.696 1.000 592.696 [-]CSRAR[K] 720.217 1.000 720.868 [-]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[-]
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
Observed m/z 374 730
associated charge 1 1
Retention time 23 mins 32 mins
CSRARK
Observed m/z 720 711
associated charge 1 2
Retention time 43 mins 52 mins
QAASIK
Observed m/z 618 608
associated charge 1 2
Retention time 35 mins 41 mins
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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