Structural characterization of bisretinoid A2E photocleavage products and implications for age-related macular degeneration

Article abstract of DOI:10.1073/pnas.0913112107

Fluorescent bisretinoids, such as A2E and all-trans-retinal dimer, form as a by-product of vitamin A cycling in retina and accumulate in retinal pigment epithelial (RPE) cells as lipofuscin pigments. These pigments are implicated in pathological mechanisms involved in several vision-threatening diseases including age-related macular degeneration. Efforts to understand damaging events initiated by these bisretinoids have revealed that photoexcitation of A2E by wavelengths in the visible spectrum leads to singlet oxygen production and photooxidation of A2E. Here we have employed liquid chromatography coupled to electrospray ionization mass spectrometry together with tandem mass spectrometry (MS/MS), to demonstrate that A2E also undergoes photooxidation-induced degradation and we have elucidated the structures of some of the aldehyde-bearing cleavage products. Studies in which A2E was incubated with a singlet oxygen generator yielded results consistent with a mechanism involving bisretinoid photocleavage at sites of singlet molecular oxygen addition. We provide evidence that one of the products released by A2E photodegradation is methylglyoxal, a low molecular weight reactive dicarbonyl with the capacity to form advanced glycation end products. Methylglyoxal is already known to be generated by carbohydrate and lipid oxidation; this is the first report of its production via bisretinoid photocleavage. It is significant that AGE-modified proteins are detected in deposits (drusen) that accumulate below RPE cells in vivo; drusen have been linked to age-related macular degeneration pathogenesis. Whereas various processes play a role in drusen formation, these findings are indicative of a contribution from lipofuscin photo-oxidation in RPE.

Full text of DOI:10.1073/pnas.0913112107

Structural characterization of bisretinoid A2E
photocleavage products and implications
for age-related macular degeneration
a
a
b
b
a,c,1
Yalin Wu , Emiko Yanase , Xidong Feng , Marshall M. Siegel , and Janet R. Sparrow
a
c
b
Department of Ophthalmology, Columbia University, 630 West 168th Street, New York, NY 10032; Wyeth Research, Pearl River, NY 10965; and
Department of Pathology and Cell Biology, Columbia University, 630 West 168th Street, New York, NY 10032
Edited* by Nicholas J. Turro, Columbia University, New York, NY, and approved March 11, 2010 (received for review November 16, 2009)
Fluorescent bisretinoids, such as A2E and all-trans-retinal dimer,
form as a by-product of vitamin A cycling in retina and accumulate
in retinal pigment epithelial (RPE) cells as lipofuscin pigments.
These pigments are implicated in pathological mechanisms in-
volved in several vision-threatening diseases including age-related
macular degeneration. Efforts to understand damaging events in-
itiated by these bisretinoids have revealed that photoexcitation of
A2E by wavelengths in the visible spectrum leads to singlet oxygen
production and photooxidation of A2E. Here we have employed
liquid chromatography coupled to electrospray ionization mass
spectrometry together with tandem mass spectrometry (MS/MS),
to demonstrate that A2E also undergoes photooxidation-induced
degradation and we have elucidated the structures of some of the
aldehyde-bearing cleavage products. Studies in which A2E was in-
cubated with a singlet oxygen generator yielded results consistent
with a mechanism involving bisretinoid photocleavage at sites of
singlet molecular oxygen addition. We provide evidence that one
of the products released by A2E photodegradation is methylglyox-
al, a low molecular weight reactive dicarbonyl with the capacity to
form advanced glycation end products. Methylglyoxal is already
known to be generated by carbohydrate and lipid oxidation; this
is the first report of its production via bisretinoid photocleavage. It
is significant that AGE-modified proteins are detected in deposits
low for absorption and excitation by light in the short wavelength
region of the visible spectrum (A2E: λmax, 439 nm; A2-DHP-PE:
λmax 490 nm; all-trans-retinal dimer: λmax, 430 nm). In the case of
all-trans-retinal dimer-PE and all-trans-retinal dimer-E, an addi-
tional red-shift to 510 nm is associated with protonation of the
Schiff base nitrogen (11).
Photochemical reactions initiated by these bisretinoid pig-
ments likely contribute to the adverse effects of RPE lipofuscin
accumulation. Specifically, A2E and all-trans-retinal dimer are
photosensitizers for the production of singlet oxygen; of these
two bisretinoids all-trans-retinal dimer is the more efficient pro-
ducer (11, 12). Both of these bisretinoids also chemically quench
singlet oxygen with the result that polyoxidized forms of these
compounds are generated. The participation of singlet oxygen
in the photooxidation of A2E and all-trans-retinal dimer is indi-
cated by the finding that exposure to a singlet oxygen generator
produced the same forms of oxidized A2E as was observed with
irradiation by short wavelength visible light. Moreover, photoox-
idation is potentiated when the lifetime of singlet oxygen is ex-
tended in the presence of deuterium oxide (12, 13). Further
evidence comes from the inhibitory effects of known quenchers/
scavengers of singlet oxygen (14). At least in the case of A2E,
electron spin resonance spectroscopy indicates that superoxide
anion is also generated following 430 nm irradiation of the com-
pound (15). The oxygen-containing moieties ensuing from the
photoexcitation of A2E and all-trans-retinal dimer include epox-
ides, furanoid oxides, and cyclic peroxides (11–13, 16, 17). Oxi-
dized forms of A2E and all-trans-retinal dimer have been
detected in human and mouse RPE cells (11, 13).
(
drusen) that accumulate below RPE cells in vivo; drusen have
been linked to age-related macular degeneration pathogenesis.
Whereas various processes play a role in drusen formation, these
findings are indicative of a contribution from lipofuscin photo-
oxidation in RPE.
advanced glycation end products ∣ lipofuscin ∣ photofragmentation ∣
photooxidation ∣ retinal pigment epithelial cells
By utilizing liquid chromatography (LC) coupled to
electrospray ionization mass spectrometry (ESI-MS) together
with tandem mass analysis (MS/MS), we have characterized
photooxidation-induced cleavage products of A2E and we have
shown that photolysis occurs at sites of singlet oxygen addition.
The detection of these photo-products was facilitated by utilizing
A2E that was tagged either by introducing a bromine label to the
pyridinium head-group of A2E (A2E-bromine; A2E-Br) or by
labeling A2E with a dipeptide linked to the pyridinium ring
luorescent bisretinoid pigments are amassed as lipofuscin in
F
retinal pigment epithelial (RPE) cells in association with aging
although individuals vary with respect to the extent of accumula-
tion (1). The excessive deposition of these compounds in RPE
cells is also considered to lead to retinal degeneration in early
onset blinding disorders associated with mutations in the genes
encoding ABCA4 (ATP-binding cassette subfamily A member 4)
(
2, 3), ELOVL4 (4), and BEST-1 (5). Moreover, the deposition of
(
A2E-lysine/leucine; A2E-Lys/Leu) (Fig. 1). These nonphysiolo-
these pigments likely also contributes to the etiology of age-
related macular degeneration (AMD) (6, 7). Whereas these bis-
retinoids constitute a complex mixture, all appear to originate
from reactions of all-trans-retinal and some have been identified,
including A2E (Fig. 1) and its isomers (8); A2-dihydropyridine-
phosphatidylethanolamine (A2-DHP-PE) and A2-dihydropyri-
dine-ethanolamine (A2-DHP-E) (9); and all-trans-retinal dimer,
all-trans-retinal dimer-phosphatidylethanolamine (all-trans-
retinal dimer-PE), and all-trans-retinal dimer-ethanolamine (all-
trans-retinal dimer-E) (10, 11). Structural features common to all
of these pigments are the alternating single and double carbon–
carbon bonds that originate from an aromatic head group, and
extend along the two sidearms of the molecules and into the
terminal β-ionone rings. These extended conjugation systems al-
gical tags signaled cleavage products that carried the pyridinium
head group of A2E. By observing similar cleavage products
using multiple strategies, we demonstrated the reproducibility
of our findings.
Author contributions: J.R.S. designed research; Y.W., E.Y., X.F., M.M.S., and J.R.S.
performed research; Y.W., E.Y., M.M.S., and J.R.S. analyzed data; and Y.W. and J.R.S. wrote
the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1
To whom correspondence should be addressed. Email: jrs88@columbia.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0913112107/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.0913112107
PNAS ∣ April 20, 2010 ∣ vol. 107 ∣ no. 16 ∣ 7275–7280

Fig. 1. Structures of A2E, A2E-Lysine/Leucine (A2E-Lys/Leu) and A2E-bro-
mine (A2E-Br).
Results
Photooxidation of A2E, A2E-Br, and A2E-Lys/Leu. Fast atom bombard-
ment ionization mass spectrometry (FAB-MS) analysis revealed
that 430 nm irradiation of A2E, A2E-Br and A2E-Lys/Leu
(
Fig. 1), lead to spectra characterized by m/z peaks at 592,
7
30/732, and 952 attributable to A2E, A2E-Br, and A2E-Lys/
Leu, respectively, together with a series of m∕z þ n ꢀ 16 peaks
indicating starting compounds modified by addition or insertion
of oxygen atoms (Fig. S1). These results confirmed that tagged-
A2E underwent photooxidation in the same fashion as A2E. The
mass spectra generated after A2E, A2E-Br, or A2E-Lys/Leu were
incubated with the singlet oxygen generator, endoperoxide of 1,4-
dimethylnaphthalene, also reflected profiles indicative of oxida-
tion (Fig. S1).
LC/ESI-MS and ESI-MS Analysis of Photooxidation-Associated
Cleavage Products of A2E. The mixture of lower molecular weight
photo-induced cleavage products generated by irradiation of
tagged A2E (A2E-Br) (Fig. 1) was subsequently studied by
LC/MS (Fig. 2) using an electrospray ionization (ESI) source op-
erating in positive ion mode. Because bromine imparts a charac-
teristic isotope pattern in the mass-spectrum—two peaks of
7
9
almost equal height separated by 2 m/z units due to isotopes Br
8
1
Fig. 2. LC/ESI-MS analysis of photooxidation-induced cleavage of bromine-
tagged A2E. (A2E-Br; m/z 730) (A) Overlay of total ion chromatogram and
mass chromatogram for m/z 730 recorded from unirradiated sample of
A2E. (B) Total ion chromatogram of irradiated (430 nm; 10 min) sample of
A2E. (C–J) Mass chromatograms at m/z 560/562, 570/572, 576/578, 586/588,
and Br—the bromine tag was indicative of cleavage products
that included the pyridinium head group of A2E-Br. Fig. 2 shows
an overlay of total ion chromatogram and mass chromatogram of
samples of A2E-Br that were unirradiated and irradiated for
1
0 min and monitored at m/z 730 (Fig. 2 A and B). The peak
6
02/604, 608/610, 626/628, and 642/644 for unirradiated (Left) and irradiated
at 20.9 min is attributable to A2E-Br; this signal was greatly di-
minished by 430 nm irradiation. Importantly, selected molecular
ion monitoring (Fig. 2 C–J) revealed 8 peaks (m/z 562/560, 572/
(
Right) samples of A2E-Br. Molecular ion monitoring was performed for bro-
mide isotopes Br-79 (Upper Traces) and Br-81 (Lower Traces). Mass chroma-
tograms were acquired in positive ESI mode and recorded as a function of
retention time.
5
70, 578/576, 588/586, 604/602, 610/608, 628/626, and 644/642)
that carried the bromine tag and were lower in mass than m/z
30; these peaks were also not present in unirradiated samples
7
470, 488, and 504) corresponding to the photocleavage products
generated with A2E-Br (Table 1). Several other lower molecule
weight species were also detected.
of A2E-Br and eluted earlier than A2E-Br, consistent with oxi-
dized products that are more polar than the parent compound. It
should be noted that in some cases multiple peaks of equal abun-
dance were detected at a given mass but different retention times.
Some of these peaks are likely cis/trans isomers, isobaric with the
major components and others could reflect isomers generated by
photocleavage at one or other of the side-arms of A2E-Br. LC/
ESI-MS analysis of irradiated samples of A2E-Lys/Leu (m/z 952)
revealed five m/z signals (m/z 782, 792, 808, 824, 892) (Fig. S2)
that matched photocleavage products generated with A2E-Br,
after the mass of the Br and Lys/Leu tags were accounted for
Characterization of A2E Photocleavage Products by Tandem
Mass Spectrometry. For structural characterization of
8
photooxidation-induced A2E cleavage products identified by
LC/ESI-MS (m/z 422, 432, 438, 448, 464, 470, 488, and 504), tan-
dem mass spectrometric analysis (MS/MS) was performed in the
ESI positive-ion mode (Fig. 4). The MS/MS fragmentation pro-
cesses leading to structural assignments for the photocleavage
products corresponding to the precursor molecular ions (Fig. 4
A–H, Insets) involved rearrangements, eliminations, cyclizations,
and bond cleavages; these MS/MS mechanisms are presented in
Fig. S3, Fig. S4, Fig. S5, and Fig. S6 and are described in SI Text.
In assigning structures to the precursor molecular ions, we were
informed both by our previous studies of A2E (13), and by mass
spectrometry principles. Specifically, our earlier work indicated
(
Table 1).
Guided by our findings from LC/ESI-MS, we turned to direct-
injection ESI-MS to test the reproducibility of the photo-induced
cleavage pattern we had observed. Accordingly, ESI-MS analysis
of photo-induced fragments generated from untagged A2E
(Fig. 3), revealed 8 photoproducts (m/z 422, 432, 438, 448, 464,
7
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Wu et al.

Table 1. Mass values (m/z) for A2E, photooxidized A2E (þ16 m∕z)
and photooxidation-induced cleavage products. Corresponding
m/z values for A2E and two forms of tagged A2E: A2E-Br and
A2E-Lys/Leu
despite O–O bond photocleavage (13). In addition, the structures
proposed for the precursor molecular ions were guided by mass
spectrometry principles (18): (i) direct MS/MS cleavage at double
bonds does not occur because of the high energy requirements
(
19); (ii) MS/MS fragmentation processes generally involve a loss
A2E½Mꢂþ
A2E-Br½Mꢂþ
A2E-Lys∕Leu½Mꢂþ
of water from a substituted ethanol group and a loss of CO from
an aldehyde; in the case of an aldehyde conjugated to the pyri-
dinium ring, no loss of CO is observed; (iii) even-mass fragment
ions containing the positive charge of the pyridinium group would
form via homolytic cleavages at charge remote sites; (iv) odd-
mass product ion peaks would be radicals due to charge remote
radical homolytic cleavages wherein the charge resides on the
pyridinium group and a free radical resides at the fragmentation
site; (v) homolytic charge remote site reactions would involve hy-
drogen and/or methyl rearrangements; (vi) oxygens could be
added to aldehyde structures to satisfy elemental compositions
and peroxides could undergo losses of atomic oxygen; and (vii)
m/z
592
608
730/732
746/748
762/764
778/780
794/796
810/812
826/828
842/844
858/860
874/876
560/562
570/572
576/578
586/588
602/604
608/610
626/628
642/644
952
968
984
oxidation (+16)
6
6
6
6
6
7
7
7
24
40
56
72
88
04
20
36
1000
1016
1032
1048
1064
1080
1096
782
792
798
808
824
photocleavage
422
4
4
4
4
4
4
5
32
38
48
64
70
88
04
0
0
the C12–C13 and C14 –C15 single bonds of A2E would be
0
0
strengthened by the C11–C12 and C13 –C14 double bonds, re-
spectively, that are conjugated with double bonds situated in the
aromatic ring (20, 21). Importantly, the proposed structures of
the precursor and product ions of the 8 photocleavage products,
were also supported by exact-mass measurements performed uti-
lizing ESI-Fourier transform-ion cyclotron resonance (FT-ICR)
MS and MS/MS. The most probable elemental compositions
for these ions were obtained with a high degree of confidence:
Based on the difference between the experimental mass and
the predicted mass, the errors were within ꢁ2.7 ppm (Table S1).
Included within the complex mixture of photocleavage pro-
ducts was a species at m/z 470 (RT 10.98 min) that was present
in low abundance (Fig. 4F). The proposed structure of this
830
848
864
that the first oxidation would occur at the C5–C6 double bond on
the β-ionone ring of the short arm and that the next oxidation
0
0
would be at the C5 –C6 double bond on the long arm. In addi-
0
0
tion, the presence of a stable 5; 8∕5 ; 8 -monofuran would be con-
sistent with previously described mechanisms (13, 17) whereas
the C–C single bond associated with the peroxide at the 5, 6
0
0
(
or 5 , 6 ) position in the cyclohexenyl ring would remain intact
Fig. 3. ESI mass spectral analysis of A2E (untagged). Samples of A2E (m/z 592) were unirradiated (A) and irradiated at 430 nm (hv) for 30 min (B); expanded
view of panel B in the range of m/z 300–540 (C). The series of peaks at m/z 608, 624, 640, 656, 672, 688, 704, 720, and 736 reflect photooxidation (m∕z þ n ꢀ 16;
n ¼ 1; 2; …; 9) at carbon–carbon double bonds (B). Lower mass peaks (<m∕z592) correspond to A2E fragments forming due to photooxidation-induced clea-
vage (panels B and C). Peaks from m/z 422 to 504 that are labeled in bold, correspond to photocleavage products also identified by LC-MS. (Upper Left)
Structure of A2E. Absorbance peaks at 439 nm and 338 nm can be assigned to the long and short arms of the molecule, respectively.
Wu et al.
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þ
Fig. 4. ESI-MS/MS spectra and proposed structures of photooxidation-induced cleavage products of A2E. Product ion spectra of ½Mꢂ ions at m/z 422 (A), 432
(
B), 438 (C), 448 (D), 464 (E), 470 (F), 488 (G), and 504 (H). Proposed MS/MS mechanisms operating in the formation of the structurally informative product ions
are presented in Fig. S3, Fig. S4, Fig. S5, and Fig. S6. The retention times indicated were obtained from LC∕ESI-MS: × n, abundance multiplication factor versus
the parent ion.
product revealed an unstable photo-fragment that originated
from an A2E molecule that had undergone peroxide formation
m/z 366, 382, and 398 (Fig. 6C) that were observed with irradiated
A2E in the absence of 4-NPH (Fig. 6B and Fig. 3C). The eradica-
tion of these peaks by reaction with 4-NPH confirmed that these
photo-fragments were aldehyde-bearing. Subsequent analysis by
nanoelectrospray FT-ICR MS established that the experimental
mass of the nitrophenyl hydrazone product was 343.11484, con-
0
0
at at least 3 sites on the long arm (Fig. 5): (i) at the C5 –C6 dou-
ble bonds on the β-ionone ring where the first oxidation is known
0
0
to occur (13); (ii) at the C11 –C12 double bond where cleavage
occurred producing the aldehyde group; and (iii) at the C13′–
C14′ double bond. We expect that the m/z 470 photocleavage pro-
duct was of low abundance because further cleavage would occur
1
þ
sistent with the predicted elemental formula (C H N O ; the-
1
5
15
6
4
oretical mass 343.11493; error, 0.26 ppm) and confirmatory of the
reaction of 4-NPH with the photo-product methylglyoxal.
0
0
following opening of the C13 –C14 peroxide ring. Photocleavage
here would result in a photo-fragment of mass 398 Da together
with a dicarbonyl molecule (methylglyoxal; calculated molecular
weight 72 Da) (Fig. 5). Indeed, this photocleavage mechanism
likely occurs because a species at m/z 398 is detectable by ESI-
MS (Fig. 3). Further corroboration was obtained by nanoelectros-
pray FT-ICR MS, as the experimental mass of this product ion
was shown to be 398.2329, consistent with the predicted ele-
LC/ESI-MS Following Incubation of A2E with a Singlet Oxygen Genera-
tor. Photocleavage along the side-arms of A2E could occur at sites
where endoperoxide (O–O) moieties form subsequent to addi-
tion of singlet molecular oxygen at carbon–carbon double bonds.
Evidence in support of this mechanism was obtained from sam-
ples of A2E-bromine incubated with the singlet oxygen generator
endoperoxide of 1,4-dimethylnaphthalene. As presented in
SI Text and Fig. S8, LC/ESI-MS analysis of the resulting photo-
cleavage products revealed 5 peaks (m/z 572/570, 578/576, 604/
602, 610/608, and 644/642) that were lower in mass than the par-
ent ion A2E-Br (m/z 730/732). These bromine isotope doublet
peaks also corresponded to molecular ions (m/z 432, 438, 464,
470, and 504) detected in samples of A2E (untagged) that were
irradiated at 430 nm (Fig. 3 and Table 1). Cleavage at the per-
oxide sites would result in the formation of aldehyde (-CHO)
functional groups (Fig. S7B). Consistent with this mechanism,
þ
mental formula (C H NO ) (theoretical mass 398.2326; error,
2
4
32
4
0
.1 ppm). It should be mentioned that methylglyoxal is highly
toxic and is an agent involved in formation of advanced glycation
end (AGE) products (22, 23). To obtain additional evidence for
methylglyoxal release following A2E photo-oxidation, we trapped
this volatile fragment with 4-nitrophenylhydrazine (4-NPH), a
compound well known for reacting with carbonyl groups. As
shown in Fig. 6, reaction of 4-NPH with commercially available
methylglyoxal, yielded the expected 2-(4-nitrophenyl)hydrazone
þ
product (Fig. S7A) at m/z 343.1 (½M þ Hꢂ ). Pooled samples
1
of irradiated A2E were then incubated/not incubated with
the H-NMR spectrum of irradiated samples of A2E exhibited
4
-NPH and evaluated by ESI-MS. Accordingly, we observed a
a broad resonance at 9.3 ppm indicative of a proton signal specific
to an aldehyde moiety; this signal was not present in a sample of
unirradiated A2E. These data are consistent with a previous re-
port of aldehyde formation following A2E oxidation (17).
production peak at m/z 343.1 (Fig. 6C) that in the absence of
-NPH was not produced (Fig. 6B); this peak could be explained
4
by the facile reaction of 4-NPH with the released photo-product
methylglyoxal. Incubation with 4-NPH also eliminated peaks at
Discussion
The extensive conjugation systems situated along the side-arms of
A2E are linked through a pyridinium ring that houses a nitro-
gen carrying an inherent positive charge. This system renders
the compound amenable to ESI in the positive ionization mode.
Accordingly, we have utilized ESI-MS to identify products of
photocleavage occurring within the polyenic structures that
comprise the side-arms of A2E. Taken together, the results
reported here are consistent with a mechanism by which A2E
photocleavage occurs at sites of singlet molecular oxygen addition
to carbon–carbon double bonds (Fig. S7B). At each of these sites,
opening of both O–O and C–C bonds gives rise to two fragments
each bearing an aldehyde moiety. Aldehydes are reactive species
Fig. 5. Photochemical mechanism proposed for conversion of m/z 470
photocleavage product to m/z 398 photo-product. Cleavages at the O–O
bond and C–C single bond at the C13 –C14 peroxide ring yields an alde-
hyde-bearing photo-product with m/z 398 and a smaller dicarbonyl molecule
0
0
(
methylglyoxal) with calculated molecular weight 72 (Inset).
7
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Wu et al.

result in the addition or loss of these compounds (24). On the
other hand, it is clear that considerable degradation of the bisre-
tinoids can occur as a consequence of photo-oxidation; over time
this process could result in considerable loss of bisretinoid
pigment even as newly formed pigment is being deposited. Con-
sequently, measurements of A2E, all-trans-retinal dimer com-
pounds and A2-DHP-PE in the eye at any given time, do not
likely reflect the true burden placed on the RPE cells by the de-
position of these bisretinoids throughout the life of an individual.
Products of A2E photooxidative cleavage are implicated as
agents that perturb the RPE cell and its surroundings. For in-
stance, we have shown that cellular injury can be induced by
photooxidized A2E under conditions that eliminate singlet oxy-
gen or other reactive forms of oxygen as the immediate cause of
damage (25). It is significant that the current work has revealed
that photocleavage of A2E can produce a low molecular weight
product (m/z 72), methylglyoxal; this reactive dicarbonyl can alter
molecular structure and function by forming AGE-adducts with
proteins, phospholipids, and nucleotides. Methylglyoxal is known
to form as a by-product of metabolic pathways such as glycolysis
and lipid peroxidation (26, 27). Here we present evidence for an
additional unique source—the bisretinoids of RPE lipofuscin.
Because there are 3 methyl groups distributed along the side-
arms of A2E and the double bonds both proximal and distal
to these methyl groups have the potential to undergo photoclea-
vage (Fig. 1), three moles of methylglyoxal per mole of A2E
could be released in this way. AGE-modified proteins con-
tributes to age-related inflammatory disease (28) and along
with complement-related proteins, cholesterol, and other mole-
cules, have been detected in aged Bruch’s membrane and
drusen (29–31), the subRPE deposits that have been linked to
AMD (32).
Diffusion of A2E photocleavage products such as methylglyox-
al is suggested by the finding that photooxidation of intracellular
A2E is associated with DNA damage even though the parent bis-
retinoid is housed in lysosomes (33). We have also previously ob-
served that when cultured RPE cells that have accumulated A2E
are irradiated (430 nm), the extracellular fibronectrin substrate
on which the cells are grown becomes AGE-modified (34). The
observation that the complement protein C3 can be activated in
serum overlying irradiated-A2E-laden RPE cells is also consis-
tent with the view that diffusible reactive cleavage products of
A2E are generated by photooxidation (35, 36). Moreover, direct
exposure of complement-containing serum to photooxidized
forms of A2E or all-trans-retinal dimer also leads to C3 activa-
tion. These findings are significant because genetic association
studies have demonstrated links between DNA sequence variants
in complement factor H, factor B (BF)/complement component
C2 and complement component C3, and protection or risk for
AMD (37–41). Together these genetic studies implicate comple-
ment dysregulation in the presence of an activating agent such as
the products of A2E photocleavage, as an underlying cause of
AMD pathogenesis in a significant number of cases.
RPE bisretinoid lipofuscin is a target for therapies that aim
to combat vision loss in atrophic AMD. Several approaches under
investigation involve limiting bisretinoid formation (42–44). A
number of reports, including from the Age-Related Eye Dis-
ease Study, have also demonstrated a role for antioxidant
intake in protecting against AMD progression (45–47). Evidence
from our work (14, 25, 48, 49) indicates that antioxidants may
operate at least in part by suppressing RPE bisretinoid photoox-
idation. The current report also lends support to epidemiological
studies that suggest a link between AMD and sunlight expo-
sure (50, 51).
Fig. 6. Detection of methylglyoxal by derivatization with 4-nitrophenylhy-
drazine (4-NPH). ESI-MS spectra in the range m/z 200–400. (A) Incubation of 4-
NPH with methylglycoxal (obtained commercially) in ethanol yielded the m/z
3
43.1 product 2-(4-nitrophenyl)hydrazone (inset). (B) Cleavage products of
A2E in photooxidized sample (irradiation at 430 nm). 4-NPH. The presence
of 2-(4-nitrophenyl)hydrazone (m/z 343.1) is indicative of the reaction of
4
-NPH with methylglyoxal in the mixture of A2E photodegradation products.
Inset, MS profile expanded between m/z 300 and 380.
that form Schiff bases with amino groups and can be expected to
play havoc in biological systems. Photocleavage products bearing
the pyridinium ring moiety carried a positive charge and were
readily monitored. Equally interesting, however, are the comple-
mentary noncharged residual photo-products (Fig. S7B) that
would be released upon photofragmentation; some of these would
bear a β-ionone ring. However, because the fragments originating
distal to the cleavage site are uncharged, they were undetectable by
ESI-MS. Nevertheless, it is clear that numerous photo-products of
a variety of molecular weights were generated (Fig. 3), with many
neutral photocleavage products having molecular weights comple-
menting the detectable products (Fig. 5). In addition, because our
research indicates that atomic oxygen can also insert at the car-
bon–carbon double bonds (12, 15) (Fig. 3), many of these
photo-fragments could carry furanoid or epoxide moieties (Fig. 4).
Because we have identified the oxidized forms of A2E and all-
trans-retinal dimer in human and mice eyes, it is clear that these
photooxidation processes are ongoing in vivo (11, 13).
Methods
In mice that have accumulated RPE lipofuscin bisretinoids
while raised under cyclic light, subsequent dark-rearing does not
Synthesis. A2E-Br (M.W. 730/732) and A2E-Lys/Leu (M.W. 952) were synthe-
sized by incubating mixtures of all-trans-retinal and 4-bromophenethylamine
Wu et al.
PNAS
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April 20, 2010
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vol. 107
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no. 16
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7279

in ethanol or all-trans-retinal and Cbz-Lys-Leu-OEt in ethanol espectively, as
described in SI Text. 1H-NMR assignments of synthesized A2E-Lys/Leu and
A2E-Br are provided in SI Text. For synthesis of 2-(4-nitrophenyl)hydrazone,
LC/ESI-MS and LC/ESI-MS/MS. Analysis was performed on a Waters Alliance
2690 HPLC interfaced with a Waters, MicroMass ZQ mass spectrometer as de-
scribed in SI Text. In addition, an Agilent 1100 HPLC system was used with a
reversed phase column, linear gradients of H2O and acetonitrile with formic
acid and ESI-MS data aquisition via a Waters-Micromass LCT Premier XE Time-
of-Flight mass spectrometer in the positive ionization mode as described in
SI Text. Tandem MS (ESI-MS/MS) employing collision-induced dissociation was
performed using a Waters-Micromass Q-TOF Micro mass spectrometer in the
positive ionization mode; details in SI Text. Fourier transform mass spectro-
metry was performed to obtain high-resolution exact mass, as described in
SI Text.
4
-NPH was incubated with methylglyoxal in the presence of sulfuric acid
(
98%, 0.5 ml) (SI Text).
Photooxidation and Singlet Oxygen Oxidation. A2E, A2E-Br, and A2E-Lys/Leu
(
1
100 μM in water with 1% DMSO) were irradiated (430 ꢁ 20 nm,
2
.3 mW∕cm , 10–30 min) or incubated with the singlet oxygen generator
1
,4-endoperoxide of 1,4- dimethylnaphthalene (40 mM) (52) in deuterated
methanol overnight at room temperature. To identify carbonyl groups within
irradiated A2E, samples of the latter were incubated with 0.2 M 4-NPH as
detailed in SI Text.
ACKNOWLEDGMENTS. We thank Dr. Jiangao He for LC-MS assistance. This
work was supported by National Institutes of Health Grant EY 12951 and
a grant to the Department of Ophthalmology from Research to Prevent
Blindness.
FAB-MS. FAB-MS was performed as described in (SI) Text.
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