Compositional studies of human RPE lipofuscin: Mechanisms of molecular modifications

Article abstract of DOI:10.1002/jms.1865

The accumulation of lipofuscin has previously been implicated in several retinal diseases including Best's macular dystrophy, Stargardt's disease and age-related macular degeneration (AMD). Previously one of the major fluorophores of lipofuscin was identi

Full text of DOI:10.1002/jms.1865

Research Article
Received: 6 July 2010
Accepted: 1 November 2010
Published online in Wiley Online Library: 23 December 2010
(wileyonlinelibrary.com) DOI 10.1002/jms.1865
Compositional studies of human RPE
lipofuscin: mechanisms of molecular
modifications
L. S. Murdaugh,a S. Mandal,a A. E. Dill,a† J. Dillon,a,b J. D. Simonc
and E. R. Gaillarda,b∗
The accumulation of lipofuscin has previously been implicated in several retinal diseases including Best’s macular dystrophy,
Stargardt’s disease and age-related macular degeneration (AMD). Previously one of the major fluorophores of lipofuscin was
identified as a bis-retinoid pyridinium salt called A2E, which is known to photochemically cause damage. In addition to A2E,
there are numerous components in RPE lipofuscin that are unidentified. These compounds were determined to be structurally
related to A2E by their fragmentation pattern with losses of 106, 190, 174 and/or 150 amu from the parent ion and the formation
of fragments of ca 592 amu. The vast majority consists of relatively hydrophobic components corresponding to derivatized A2E
with molecular weights in discrete groups of 800–900, 970–1080 and >1200 m/z regions. In order to determine the mechanism
of these modifications, A2E was chemically modified by; (1) the formation of specific esters, (2) reaction with specific aldehydes
and (3) spontaneous auto-oxidation. The contribution of ester formation to the naturally occurring components of lipofuscin
was discounted since their fragmentation patterns were different to those found in vivo. Alternatively, reactions with specific
aldehydes result in nearly identical products as those found in vivo. Artificial aging of RPE lipofuscin gives a complex mixture of
structurally related components. This results from the auto- and/or photooxidation of A2E to form aldehydes, which then back
react with A2E giving a series of higher molecular weight products. The majority of these modifications result in compounds
that are much more hydrophobic than A2E. These higher molecular weight materials have increased values of log Pcompared to
A2E. This increase in hydrophobicity most likely aids in the sequestering of A2E into granules with the concomitant diminution
c
of its reactivity. Therefore, these processes may serve as protective mechanisms for the RPE. Copyright ꢀ 2010 John Wiley &
Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: lipofuscin; A2E; blue light damage; oxidative stress; age-related macular degeneration
Introduction
One of the major fluorophores of lipofuscin, A2E, was first
isolated from lipofuscin granules by Eldred.[20] Structurally, A2E
is a pyridinium bis-retinoid, which is synthesized using 2 mol
of all-trans-retinal (RAL) and 1 mol of ethanolamine.[21–23] The
photooxidation of A2E results in the generation of ROS, such as
peroxide and superoxide radicals.[24,25] Ben-Shabat et al.[26] were
the first to propose the photooxidation of A2E results in higher
molecular weight compounds that differ by 16 amu resulting in
epoxide formation along the polyene chain. However, the survival
of these epoxides in the acidic environment of lysosomes could
notbeexplained. Dillonet al.[27] proposedthattheepoxidesofA2E
would undergo rearrangement to form furanoid oxide structures,
As organisms age, many metabolically active post-mitotic cells
accumulate autofluorescent lysosomal storage bodies known
as lipofuscin. Lipofuscin is a brown-yellow, electron-dense,
age-related pigment that is composed of a complex heteroge-
neous mixture of lipid–protein aggregates that form clusters of
granules in the RPE. The accumulation of lipofuscin has previously
been implicated in several retinal diseases including Best’s
macular dystrophy, Stargardt’s disease and age-related macular
degeneration (AMD).[1–8] Within the human eye, the granules are
believed to form from the indigestible material of phagocytized
photoreceptor outer segments[9,10] and may account for up to
19% of the cytoplasmic volume by the age of 80.[3,11,12] Lipofuscin
has also been shown to generate a series of reactive oxygen
species (ROS), which include singlet oxygen, hydrogen peroxide
and superoxide anions.[4,11,13–15] Considered photochemically
toxic, lipofuscin was found to decrease phagocytic capacity.[16]
Previous studies have shown that both the isolated granules and
the organic soluble extract of lipofuscin are photoreactive.[4,14,17]
Additional studies indicate that light exposure, the accumulation
of these photoreactive granules, photoreceptor cell death and
the onset of AMD are all related.[2,18,19]
∗
Correspondence to: E. R. Gaillard, Department of Chemistry and Biochemistry,
Northern Illinois University, DeKalb, IL 60115, USA. E-mail: gaillard@niu.edu
†
a
Current address: A. E. Dill, GreatPoint Energy, 2225 Harrison St., Chicago, IL
60612, USA.
DepartmentofChemistryandBiochemistry,NorthernIllinoisUniversity,DeKalb,
IL, USA
b
c
Department of Ophthalmology, Columbia University, New York, NY, USA
Department of Chemistry, Duke University, Durham, NC, USA
c
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Compositional studies of human RPE lipofuscin
which are relatively stable in the acidic lysosomal environment.
Furthermore, oxidativecleavageofsidechainsresultsintheforma-
tion of highly reactive aldehydes and ketones, which could readily
react with cell constituents and cause irreversible damage.[28]
This explanation is based on the structural similarities between
carotenoidsandA2E.Inaddition,theamphiphilicstructureofA2Eis
responsible for detergent-like action in membrane disruption. The
quaternary amine structure also aids in the inhibition of lysosomal
function by complexing to specific lysosomal enzymes.[21]
Besides A2E, there are numerous components in RPE lipofuscin
that are structurally related to A2E as determined by their
fragmentation pattern with losses of 106, 190, 174 and/or 150 amu
from the parent ion and the formation of fragments of ca 592 amu.
The vast majority consists of relatively hydrophobic components
corresponding to derivatized A2E with molecular weights in
discrete groups of 800–900, 970–1080 and >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[27,28] and their
mechanism of formation.
Reaction of A2E with cinnamaldehyde and benzaldehyde
A2E collected from the HPLC was dried under argon. A2E
(30 µmol/l) was then resuspended in 4 ml of acetonitrile and then
treated with cinnamaldehyde (15 µM) or benzaldehyde (15 µM).
The reaction was run for approximately 12 h, an aliquot was then
diluted with acetonitrile and analyzed with ESI-MS with the same
instrument settings as below.
Lipofuscin
Human RPE lipofuscin granules were extracted and isolated from
donor globes (Midwest Eye Banks and Transplantation Centers) as
previously described by Feeney-Burns and Eldred.[10] The organic
solubleportionwasobtainedfromthesamplesbyFolchextraction
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 a PDA detector.
Artificially aged (auto-oxidized) A2E
After synthesis and purification, a 2-ml aliquot of 15 µM A2E was
transferred to a 4 ◦C refrigerator in the dark. An aliquot (20 µl) was
removed from the sample at times: 0, 30 and 60 days and analyzed
on ESI-MS/MS with PDA detector.
Materials and Methods
Chemicals
Reaction of A2E with all-trans-retinal (RAL)
The chemicals used for the experiments were of the highest purity
that are commercially available. All solvents used were HPLC grade
and were purchased from Thermo Fisher Scientific (Waltham, MA,
USA). RAL was obtained from A.G. Scientific (San Diego, CA,
USA). Ethanolamine was obtained from ACROS Organics (NJ, USA).
Water was purified using a Millipore Milli-Q Plus PurePak 2 water
purification system.
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 using
a Philips special blue light (Oriel, Stratford, CT, USA, model number
6292) in a glass irradiating chamber fitted with a quarter inch sheet
of plexiglass cover to block the small amount of UV output from
the lamp. An aliquot was then removed and diluted with methanol
and analyzed on ESI-MS/MS with PDA detector.
Synthesis of A2E
LC–MS analysis
A2E was prepared from RAL and ethanolamine in acetic acid and
ethanol as previously described by Parish et al.[23] The mixture was
stirred in the dark for 3 days at room temperature. After excess
solvent was removed, the A2E was separated from the initial
reaction mixture using 1050 Ti HPLC (Hewlett Packard, France)
and a phenomenex 4-µm 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. 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.[23] After collection, the pure A2E
fraction was dried under argon and stored at −70 ◦C until further
analysis.
All samples were analyzed on a Thermo Finnigan LCQ Advantage.
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 normalized collision
energy of 35% was used for the MS/MS data. The lipofuscin and
purified A2E samples were separated using Surveyor LC system
with a Synergi Max-RP C12 column. The flow rate was set to
0.2 ml/min with 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.
Results
Synthesis of A2E esters
Besides A2E, there are numerous components in RPE lipofuscin
that are structurally related to A2E. This is determined by their
fragmentation pattern with losses of 106, 190, 174 and/or 150 amu
from the parent ion and the formation of fragments of ca 592 amu.
The vast majority consists of relatively hydrophobic components
corresponding to derivatized A2E with molecular weights in
discrete groups of 800–900, 970–1080 and >1200 m/z regions.
Since the RPE stores retinal as a fatty acid ester, our first hypothesis
was that the hydrophobic components of A2E were a series of
fatty acid derivatives.
HPLC purified A2E was used for this reaction. A2E was dissolved
in chloroform and transferred to a three-neck flask. The flask was
continuously purged with argon gas on one side and fitted to
a drying tube on the other side to absorb the hydrochloric acid
from the reaction. A2E was reacted with acetyl chloride, hexanoyl
chloride and cinnamoyl chloride separately. The reactions were
quenched with sodium hydroxide and water and dried under
argon. Analysis of the three reaction mixtures was performed
using ESI/MS by re-dissolving in acetonitrile.
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L. S. Murdaugh et al.
which are similar to the lipofuscin mixture. The MS/MS of one of
the major peaks with m/z 858 is displayed as the inset in Fig. 4
with characteristic losses of 106, 150, 174 and 190 identified.[30] To
further investigate the formation of the higher molecular weight
compounds formed from the reaction of A2E with aldehydes, A2E
was reacted with specific aldehydes such as cinnamaldehyde, ben-
zaldehyde and RAL for approximately 12 h in the dark or A2E was
photolyzed for 3 h with a subsequent addition of the aldehyde.
The resulting full mass spectra from reaction with cinnamaldehyde
showed completely oxidized A2E and peaks with the oxidized A2E
and attached aldehydes (Fig. 5(a) and (b)). The benzaldehyde reac-
tion led to similar products (Supporting Information Fig. 2). Similar
to the human lipofuscin data, the reactions with A2E appear as
a series of discrete groups.[30] 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.
Figure 1. Product from esterification reaction with A2E and alkyl chloride.
R equals acetyl, hexanoyl or cinnamoyl (C9H7ClO).
The fragmentation pattern of one of the higher molecular
weight compounds in the A2E–cinnamaldehyde (m/z 790)
condensation is displayed in Fig. 5(b). The fragmentation of
major peaks showed similar patterns as seen in the lipofuscin
components with the loss of 150, but did not have the peaks
corresponding to losses of 190 and 174 fragments. The loss of
148 in the A2E–cinnamaldehyde spectrum could be attributed to
cinnamic acid. The loss of 106 also signifies that the side chains
of A2E are intact and the modifications are occurring at the ends
of the polyene chain. The loss of xylene is commonly observed
in polyene compounds that have more than four conjugated
bonds.[31,32]
The photolysis of A2E and RAL was also performed. The full
mass spectrum displayed in Fig. 6 indicates the formation of three
main products with m/z 858, 920 and 1188 after 1 h of irradiation.
The fragmentation pattern of m/z 858 displayed in Fig. 7 shows
the same characteristic losses of 106, 150, 174 and 190 and the
parent ion with m/z 858 as seen in human retinal lipofuscin (Fig. 8).
Fragmentationofcompoundswith m/z 1188and920bothcontain
a major daughter ion with m/z 858 (Supporting Information
Figs 4 and 5). This reaction was also performed without irradiation;
however, the formation of products with m/z 920 and 1188 was
much slower, appearing after 18 h. The peak with m/z 920 was also
found in lipofuscin but with a lower abundance compared with
the artificially aged A2E.
Figure 2. The MS/MS of cinnamoyl ester (m/z 722).
A2E wasfirsttreatedwithacetyl, hexanoylorcinnamoylchloride
to synthesize the esters as indicated in Fig. 1, whereas Fig. 2
displays the MS/MS obtained from the cinnamoyl ester. All esters
showed the characteristic losses of 106 and 190[29] but also gave
spectra with major fragments of m/z 548 and/or 574. Further
fragmentation of this major peak (m/z 548) yielded fragments,
with m/z 358, 410 and 374 (Supporting Information Fig. 1). The
two major daughter fragments of the esters can readily be
explained by the rearrangement depicted in Fig. 3 resulting from
de-esterification with the loss of ethanol to form m/z 548 (a) or a
McLafferty rearrangement resulting in m/z 574 (b). In addition, it
is possible to form m/z 548 by the loss of 174 from the parent m/z
722 for the cinnamoyl ester. These fragments are not found in the
lipofuscin components.
A second hypothesis that may account for the hydropho-
bic mixture in lipofuscin involved reactions with A2E-derived
aldehydes.[28] It was proposed that once formed these aldehydes
could then react with other A2E molecules forming higher molec-
ular weight species. To investigate this hypothesis, synthesized
A2E was allowed to incubate at 4 ◦C for 60 days. This artificially
aged A2E sample led to a complex mixture, which included many
of the compounds found in vivo and appeared as clusters in three
m/z regions (centered at 850, 1100 and 1400). Figure 4 displays
some of the compounds found in the artificially aged A2E sample,
Discussion
Hydrophobic substances, like lipids, have the ability to bind to
cellular membranes and hydrophobic proteins, which disrupt the
normal cellular function. Hence, these hydrophobic substances
are stored in small pockets known as lipid droplets or adiposomes.
In the eye, the retinyl ester storage particles in the RPE are called
retinosomes. These storage particles are inert and prevent any
interaction of hydrophobic lipids with cellular components.[33]
In the RPE, RAL, an intermediate of the visual cycle is converted
to all-trans retinyl ester by lecithin retinal acyl transferase
(L-RAT).[34–37] The retinyl ester then self-aggregates into a
retinosome,[33] which later reacts with visual enzymes to produce
11-cis-retinal. Similarly, A2E that accumulates in the RPE lysosomes
may also be subjected to esterification or modification with
the reactive aldehydes that result from oxidation of A2E.[27]
Furthermore, the accumulation of A2E is indirectly associated
with decreased efflux of cholesterol from the RPE cells.[38] Since
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Compositional studies of human RPE lipofuscin
McLafferty Rearrangement:
.
+
R1
.
H
R1
+
OH
O
+
R
R
Figure 3. Scheme for the formation of the two major fragments (m/z 574 and 548) observed in the MS/MS spectrum of esterified A2E. The m/z 548 species
is proposed to be formed through de-esterification and the loss of ethanol. The m/z 574 species is proposed to form through McLafferty rearrangement
(see inset).
accelerated by age-dependent thickening of Bruch’s membrane
(BM). BM is located between the choriocapillaris and the RPE and
is found to accumulate extracellular matrix materials that can
interact with lipoproteins.[40]
Esterification of A2E was performed with acetyl chloride and
hexanoyl chloride. Both the reactions were used as model systems
to represent the possible short and long chain fatty acids that
exist in the lipofuscin extract that could derivatize A2E to form
esters. The fragmentation pattern for both the esters had patterns
that could not be found in the human lipofuscin extract. However,
the major fragment with m/z 548 was identified in the full mass
spectrum of human lipofuscin extract and had similar fragments
to that of the fragment with similar m/z from A2E ester. The
structural make up of both the compounds could be similar.
Figure 3 illustrates a possible structure for the species with m/z
548, which is A2E after the loss of the ethanol group. Supporting
information (supplemental figure 1) displays the MS/MS data for
the compound with m/z 548, which includes a major fragment
Figure 4. The mass spectrum of artificially aged A2E at retention time
89.18 min (inset MS/MS 858 from the companion paper[30]).
with m/z 358. Since both the esterification products displayed the
same fragmentation pattern, A2E was treated with the aromatic
cinnamoyl chloride, which has structural similarity to A2E. The
fragmentationpatternoftheA2Ecinnamoylchlorideesterdiffered
from the acetyl and hexanoyl esters, the major peak had m/z 574,
which was attributed to McLafferty rearrangement illustrated in
Fig. 3. This fragmentation was not observed in human RPE samples
indicating that A2E is probably not stored as esters.
the phagocytic activity of RPE is reduced considerably with the
accumulation of lipofuscin, the major transcription factors such as
A2-P and PPARγ ,[39] which maintains the acid lipase activity and
transcription of major cholesterol transporters, are less expressed
leading to decreased cholesterol efflux and increased cholesteryl
ester formation.[38] Based on the activity of the acid lipase and
RPE lysosomal enzymes, A2E could exist as both an A2E ester
and free A2E. In addition, cholesterol accumulation can be further
Oxidation of A2E results in the formation of highly reactive
aldehydes that result from A2E side chain cleavage. The higher
molecular weight compounds are not stored as esters but may
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L. S. Murdaugh et al.
(a)
Figure 6. ThemassspectrumofA2Ephotolyzedinthepresenceofall-trans-
retinal.
(b)
Figure 5. (a) The full mass spectrum of the reaction between A2E
and cinnamaldehyde (CAL) and (b) the MS/MS of m/z 790 from the
A2E–cinnamaldehyde reaction mixture.
Figure 7. The MS/MS of m/z 858 from the A2E–all-trans-retinal reaction.
Peaks corresponding to m/z 858 with the loss of 106 (m/z 754), 150 (m/z
708), 174 (m/z 684) and 190 (m/z 668) are identified.
be a result of the attachment of aldehydes to oxidized A2E
structures. When A2E was allowed to incubate for 60 days, several
compoundsareformedthatarealsoobservedinhumanlipofuscin.
The auto-oxidation of A2E in the presence of cinnamaldehyde
and benzaldehyde yielded a series of compounds including
oxidation and addition products. The fragmentation patterns
and characteristic losses of these products were similar to those
found in oxidized A2E. The photolysis of A2E in the presence of
retinalalsogeneratedcompoundsthathadthesamecharacteristic
fragmentation patterns with losses of 106, 150, 174 and 190 found
in human retinal lipofuscin.[29]
In addition, Simon et al. recently reported that lipofuscin is an
aggregatedmaterialconsistingofavarietyofdifferentcompounds
including blue-absorbing chromophores and yellow-emitting
fluorophores. However, in contrast to previous literature reports,
A2E is not the dominant blue-absorbing chromophore or yellow-
emitting fluorophore in lipofuscin. Excited state A2E becomes
deactivated by energy transfer.[41] This absence of fluorescence
suggests that A2E may self-quench as it aggregates and forms
higher molecular weight products. This supports our result
indicating that the majority of components in the hydrophobic
portion of RPE lipofuscin granules consist of derivatized A2E.
We hypothesized that auto- and/or photooxidation of A2E forms
aldehydes which then further react with A2E to yield a series
of higher molecular weight compounds that are much more
hydrophobic than A2E. These compounds have increasing values
of log P (hydrophobicity factor) which induces the sequestering of
these derivatives into granules with a concomitant diminution in
reactivity. These reactions also trap resultant aldehydes which
would oxidize cellular components with concomitant cellular
damage.
Supporting information
Supporting information may be found in the online version of this
article.
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Compositional studies of human RPE lipofuscin
[17] B. S. Winkler, M. E. Boulton, J. D. Gottsch, P. Sternberg. Oxidative
damage and age-related macular degeneration. Mol. Vis. 1999,
5, 32.
[18] F. G. Holz, C. Bellman, S. Staudt, F. Schutt, H. E. Volcker. Fundus
autofluorescence and development of geographic atrophy in age-
related macular degeneration. Invest. Ophthalmol. Vis. Sci. 2001, 42,
1051.
[19] H. R. Taylor, B. Munoz, S. West, N. M. Bressler, S. B. Bressler,
F. S. Rosenthal. Visible light and risk of age-related macular
degeneration. Trans. Am. Ophthalmol. Soc. 1990, 88, 163; discussion
173.
[20] G. E. Eldred. Age pigment structure. Nature 1993, 364, 396.
[21] G. E. Eldred, M. L. Katz. Fluorophores of the human retinal pigment
epithelium: separation and spectral characterization. Exp. Eye Res
1988, 47, 71.
[22] G. E. Eldred, M. R. Lasky. Retinal age pigments generated by self-
assembling lysosomotropic detergents. Nature 1993, 361, 724.
[23] C. A. Parish, M. Hashimoto, K. Nakanishi, J. Dillon, J. Sparrow.
Isolationandone-steppreparationofA2Eandiso-A2E,fluorophores
from human retinal pigment epithelium. Proc. Natl. Acad. Sci. USA
1998, 95, 14609.
[24] L. Ragauskaite, R. C. Heckathorn, E. R. Gaillard. Environmental
effects on the photochemistry of A2-E, a component of human
retinal lipofuscin. Photochem. Photobiol. 2001, 74, 483.
[25] K. Reszka, G. E. Eldred, R. H. Wang, C. Chignell, J. Dillon. The
photochemistry of human retinal lipofuscin as studied by EPR.
Photochem. Photobiol. 1995, 62, 1005.
Figure 8. The MS/MS of m/z 858 in lipofuscin. Peaks corresponding to m/z
858 with the loss of 106 (m/z 752), 150 (m/z 708), 174 (m/z 684) and 190
(m/z 668) are identified.
[26] S. Ben-Shabat, Y. Itagaki, S. Jockusch, J. R. Sparrow, N. J. Turro,
K. Nakanishi. Formation of a nonaoxirane from A2E, a lipofuscin
fluorophore related to macular degeneration, and evidence of
singlet oxygen involvement. Angew. Chem. (Int. Ed.) 2002, 41, 814.
[27] J. Dillon, Z. Wang, L. B. Avalle, E. R. Gaillard. The photochemical
oxidation of A2E results in the formation of a 5,8,5ꢁ,8ꢁ-bis-furanoid
oxide. Exp. Eye Res. 2004, 79, 537.
References
[1] F. C. Delori, D. G. Goger, C. K. Dorey. Age-related accumulation and
spatial distribution of lipofuscin in RPE of normal subjects. Invest.
Ophthalmol. Vis. Sci. 2001, 42, 1855.
[28] Z. Wang, L. M. Keller, J. Dillon, E. R. Gaillard. Oxidation of A2E results
in the formation of highly reactive aldehydes and ketones.
Photochem. Photobiol. 2006, 82, 1251.
[29] E. R. Gaillard, L. B. Avalle, L. M. Keller, Z. Wang, K. J. Reszka,
J. P. Dillon. A mechanistic study of the photooxidation of A2E, a
component of human retinal lipofuscin. Exp. Eye Res. 2004, 79, 313.
[30] L. S. Murdaugh,L. B. Avalle,S. Mandal,A. E. Dill,J. Dillon,J. D. Simon,
E. R. Gaillard. J. Mass Spectrom. 2010, 45, 1139.
[31] C. R. Enzell, R. A. Appleton, I. Wahlberg. Terpenes and terpenoids.
In Biochemical Applications of Mass Spectrometry, G. R. Waller (Ed.).
Wiley: New York, 1972, 375.
[32] G. Waller. Biochemical Applications of Mass Spectrometry. Wiley-
Interscience: New York, 1972, 377.
[33] Y. Imanishi, V. Gerke, K. Palczewski. Retinosomes: new insights into
intracellular managing of hydrophobic substances in lipid bodies.
J. Cell Biol. 2004, 166, 447.
[34] R. J. Barry, F. J. Canada, R. R. Rando. Solubilization and partial
purification of retinyl ester synthetase and retinoid isomerase from
bovine ocular pigment epithelium. J. Biol. Chem. 1989, 264, 9231.
[2] C. K. Dorey, G. Wu, D. Ebenstein, A. Garsd, J. J. Weiter. Cell loss in the
aging retina. Relationship to lipofuscin accumulation and macular
degeneration. Invest. Ophthalmol. Vis. Sci. 1989, 30, 1691.
[3] L. Feeney-Burns, E. S. Hilderbrand, S. Eldridge. Aging human RPE:
morphometric analysis of macular, equatorial, and peripheral cells.
Invest. Ophthalmol. Vis. Sci. 1984, 25, 195.
[4] E. R. Gaillard, S. J. Atherton, G. Eldred, J. Dillon. Photophysical
studies on human retinal lipofuscin. Photochem. Photobiol. 1995,
61, 448.
[5] F. G. Holz, F. Schutt, J. Kopitz, G. E. Eldred, F. E. Kruse, H. E. Volcker,
M. Cantz. Investigative ophthalmology &visualscience 1999, 40, 737.
[6] P. F. Lopez, I. H. Maumenee, Z. de la Cruz, W. R. Green. Autosomal-
dominant fundus flavimaculatus. Clinicopathologic correlation.
Ophthalmology 1990, 97, 798.
[7] M. F. Rabb, M. O. Tso, G. A. Fishman. Cone-rod dystrophy. A clinical
and histopathologic report. Ophthalmology 1986, 93, 1443.
[8] T. A. Weingeist, J. L. Kobrin, R. C. Watzke. Histopathology of Best’s
macular dystrophy. Arch. Ophthalmol. 1982, 100, 1108.
[9] M. Boulton, N. M. McKechnie, J. Breda, M. Bayly, J. Marshall. The
formation of autofluorescent granules in cultured human RPE.
Invest. Ophthalmol. Vis. Sci. 1989, 30, 82.
[10] L. Feeney-Burns, G. E. Eldred. The fate of the phagosome:
conversion to ‘age pigment’ and impact in human retinal pigment
epithelium. Trans. Ophthalmol. Soc. UK 1983, 103(Pt 4), 416.
[11] S. Davies, M. H. Elliott, E. Floor, T. G. Truscott, M. Zareba, T. Sarna,
F. A. Shamsi, M. E. Boulton. Free radical biology & medicine 2001, 31,
256.
[12] J. J. Weiter, F. C. Delori, G. L. Wing, K. A. Fitch. Retinal pigment
epithelial lipofuscin and melanin and choroidal melanin in human
eyes. Invest. Ophthalmol. Vis. Sci. 1986, 27, 145.
[13] L. B. Avalle, J. Dillon, E. R. Gaillard. Action spectrum for singlet
oxygen production by human retinal lipofuscin. Photochem.
Photobiol. 2005, 81, 1347.
[14] M. Rozanowska, J. Wessels, M. Boulton, J. M. Burke, M. A. Rodgers,
T. G. Truscott, T. Sarna. Free radical biology & medicine 1998, 24,
1107.
[35] P. N. MacDonald, D. E. Ong. Evidence for
a
lecithin-retinol
acyltransferase activity in the rat small intestine. J. Biol. Chem. 1988,
263, 12478.
[36] R. R. Rando. The biochemistry of the visual cycle. Chem. Rev. 2001,
101, 1881.
[37] J. C. Saari, D. L. Bredberg. Lecithin : retinol acyltransferase in retinal
pigment epithelial microsomes. J. Biol. Chem. 1989, 264, 8636.
[38] A. Lakkaraju, S. C. Finnemann, E. Rodriguez-Boulan. The lipofuscin
fluorophoreA2Eperturbscholesterolmetabolisminretinalpigment
epithelial cells. Proc. Natl. Acad. Sci. USA 2007, 104, 11026.
[39] A. V. Ershov, N. Parkins, W. J. Lukiw, N. G. Bazan. Modulation of early
response gene expression by prostaglandins in cultured rat retinal
pigment epithelium cells. Curr. Eye Res. 2000, 21, 968.
[40] C. A. Curcio, C. L. Millican, T. Bailey, H. S. Kruth. Accumulation of
cholesterol with age in human Bruch’s membrane. Invest.
Ophthalmol. Vis. Sci. 2001, 42, 265.
[41] N. M. Haralampus-Grynaviski, L. E. Lamb, C. M. Clancy, C. Skumatz,
J. M. Burke, T. Sarna, J. D. Simon. Proceedings of the National
Academy of Sciences of the United States of America 2003, 100,
3179.
[15] J. Wassell, S. Davies, W. Bardsley, M. Boulton. The photoreactivity of
the retinal age pigment lipofuscin. J. Biol. Chem. 1999, 274, 23828.
[16] S. Sundelin, U. Wihlmark, S. E. Nilsson, U. T. Brunk. Lipofuscin
accumulation in cultured retinal pigment epithelial cells reduces
their phagocytic capacity. Curr. Eye Res. 1998, 17, 851.
c
J. Mass. Spectrom. 2011, 46, 90–95
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