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Proc. Natl. Acad. Sci. USA
Vol. 95, pp. 14609–14613, December 1998
Biochemistry
Isolation and one-step preparation of A2E and iso-A2E,
fluorophores from human retinal pigment epithelium
CRAIG A. PARISH*, MASARU HASHIMOTO*, KOJI NAKANISHI*
†
,JAMES DILLON
‡
, AND JANET SPARROW
‡
*Department of Chemistry, Columbia University, New York, NY 10027; and
‡
Department of Ophthalmology, Columbia University, New York, NY 10032
Communicated by Ronald Breslow, Columbia University, New York, NY, October 12, 1998 (received for review August 31, 1998)
ABSTRACT Age-related macular degeneration, a major
cause of blindness for which no satisfactory treatments exist,
leads to a gradual decrease in central high acuity vision. The
accumulation of fluorescent materials, called lipofuscin, in
retinal pigment epithelial cells of the aging retina is most
pronounced in the macula. One of the fluorophores of retinal
pigment epithelial lipofuscin has been characterized as A2E,
a pyridinium bis-retinoid, which is derived from two mole-
cules of vitamin A aldehyde and one molecule of ethanolamine.
An investigation aimed at optimizing the in vitro synthesis of
A2E has resulted in the one-step biomimetic preparation of
this pigment in 49% yield, readily producing more than 50 mg
in one step. These results have allowed for the optimization of
HPLC conditions so that nanogram quantities of A2E can be
detected from extracts of tissue samples. By using 5% of the
extract from individual aged human eyes, this protocol has led
to the quantification of A2E and the characterization of
iso-A2E, a new A2E double bond isomer; all-trans-retinol and
13-cis-retinol also have been identified in these HPLC chro-
matograms. Exposure of either A2E or iso-A2E to light gives
rise to 4:1 A2E:iso-A2E equilibrium mixtures, similar to the
composition of these two pigments in eye extracts. A2E and
iso-A2E may exhibit surfactant properties arising from their
unique wedge-shaped structures.
Aging changes in retinal pigment epithelial (RPE) cells are
generally assumed to contribute to the pathogenesis of age-
related macular degeneration (AMD) (1–3), the leading cause
of acquired visual loss in persons older than 65 years of age
(4–7) for which no cure exists. The most characteristic feature
of aging in the RPE is the progressive cellular accumulation of
lipofuscin (2, 3, 8). The deposition of this autofluorescent
material is known to occur as a consequence of the role of RPE
cells in phagocytosing packets of membrane that are shed by
photoreceptor cells as part of the process by which photore-
ceptor outer segments undergo complete turnover every 2
weeks (9–11). In 1993, Eldred and coworkers (12, 13) pro-
posed a bis-Schiff base structure (N-retinyl-N-retinylidene-
ethanolamine) as one of the fluorescent pigments isolated
from the pooled lipofuscin of a large number of aged human
eyes. We later revised the proposed structure as the pyridinium
bis-retinoid A2E (Fig. 1) (14) and succeeded in confirming this
structure by a total chemical synthesis, which involved the
coupling of the pyridine framework with the retinoid side
chains (15).
The genesis of A2E (Fig. 2), although speculative, could
involve initial Schiff base formation between all-trans-retinal
and ethanolamine or phosphatidylethanolamine to give (I), a
[1,6]-proton tautomerization to enamine (II) followed by
Schiff base generation with a second molecule of retinal to
(III). This intermediate would undergo a [3,3]-sigmatropic
rearrangement to (IV) and subsequent autooxidation to yield
fluorescent pigment A2E or its phosphatidylethanolamine-
linked adduct (which would then be hydrolyzed to A2E). The
formation of A2E has been simulated in vitro by mixing two
equivalents of all-trans-retinal with ethanolamine in organic
solvent (14), although the yield was quite low (,1%) (13).
Retinal and ethanolamine are both components of photore-
ceptor outer segment membranes with 11-cis-retinal serving as
the chromophore of the visual pigment rhodopsin and phos-
phatidylethanolamine being an abundant membrane phospho-
lipid. In vivo, absorption of light leads to the isomerization of
11-cis-retinal to all-trans-retinal followed by release of the
latter from rhodopsin and its rapid reduction to all-trans-
retinol. Thus, only all-trans-retinal, which evades reduction,
would be available for A2E formation.
Because the biological properties of A2E and its relationship
to AMD have yet to be clarified, the availability of an efficient
and simple method for the preparation of large quantities of
A2E becomes critical for further chemical and biological
studies. Although A2E has been prepared by both biomometic
(14) and total chemical syntheses (15), the yield according to
the former route was not only ,1% but also required extensive
chromatography, whereas the latter was labor-intensive, re-
quired several chemical transformations, and provided only
modest overall yields. In the following, we report a biomimetic
preparation of A2E that directly yields substantial quantities of
this fluorophore and can be used for extensive studies aimed
at clarifying the role of this pigment in AMD.
The optimization of HPLC conditions, which can detect as
little as 5 ng has been accomplished. This HPLC protocol has
led to the isolation and quantification of A2E in human eyes
by using one-twentieth of the extract from a single eye. In
addition, these experiments have allowed for the isolation of an
additional fluorophore from RPE lipofuscin, a double bond
isomer of A2E, iso-A2E. Preliminary studies on the photo-
chemistry of A2E and iso-A2E indicate that they exist in a
photoequilibrium of 4:1 A2E:iso-A2E and that this equilib-
rium most probably exists under physiological conditions.
MATERIALS AND METHODS
A2E and iso-A2E from all-trans-Retinal and Ethanolamine.
A mixture of all-trans-retinal (100 mg, 352
m
mol) and etha-
nolamine (9.5 mg, 155
m
mol) in ethanol (EtOH) (3.0 ml) was
stirred in the presence of acetic acid (9.3
m
l, 155
m
mol) at room
temperature with a sealed cap in the dark for 2 days. After the
mixture was concentrated in vacuo, the residue was purified by
silica gel column chromatography. After elution with methanol
(MeOH):CH
2
Cl
2
(5:95), further elution with MeOH:CH
2
Cl
2
:
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accordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1998 by The National Academy of Sciences 0027-8424y98y9514609-5$2.00y0
PNAS is available online at www.pnas.org.
Abbreviations: AMD, age-related macular degeneration; A2E, pyri-
dinium bis-retinoid formed from two molecules of vitamin A aldehyde
and one molecule of ethanolamine; iso-A2E, double bond isomer of
A2E; RPE, retinal pigment epithelium; TFA, trifluoroacetic acid; HR
FAB-MS, high resolution fast atom bombardment mass spectrometry;
t
R
, retention time.
†
To whom correspondence should be addressed. e-mail: kn5@
columbia.edu.
14609
trifluoroacetic acid (TFA) (8:92:0.001) gave A2E (53.8 mg,
76.3
m
mol, 49%), which contained '5% iso-A2E as estimated
by
1
H-NMR. Pure samples were obtained by HPLC purifica-
tion [Cosmosil C18, 4.6 3 150 mm, 85–96% H
2
OyMeOH
(0.1% TFA) for 20 min, 1.0 mlymin flow detected at UV 430
nm]. A2E and iso-A2E were detected at retention time (t
R
) 5
16.7 min and t
R
518.8 min, respectively. Collection of each
fraction provided pure A2E and iso-A2E for further analysis.
A2E was identical in all respects to material previously de-
scribed (14). Because A2E appeared to be sensitive to trace
hydrochloric acid, which may be present in CDCl
3
, NMR
analysis in CD
3
OD was preferable. A2E:
1
H-NMR (CD
3
OD,
500 MHz)
d
1.09, 1.10 (each 6H, s, C5-(CH
3
)
2
and C59-
[(CH
3
)
2
], 1.53 (4H, m, C2-H
2
and C29-H
2
), 1.68 (4H, m, C3-H
2
C39-H
2
), 1.75, 1.77 (each 3H, s, C1-CH
3
and C19-CH
3
), 2.07
(3H, s, C9-CH
3
), 2.10 (4H, m, C4-H
2
and C49-H
2
), 2.19 (3H,
s, C13-CH
3
), 2.20 (3H, s, C99-CH
3
), 3.94 (2H, t, J 5 5.0 Hz,
CH
2
-O), 4.56 (2H, t, J 5 5.0 Hz, N-CH2), 6.20 (1H, d, J 5 16.0
Hz, C8-H), 6.27 (1H, J 5 11.5 Hz, C10-H), 6.30 (1H, d, J 5 16.5
Hz, C89-H), 6.37 (1H, br d, J 5 16.0 Hz, C7-H), 6.43 (1H, d,
J 5 11.5 Hz, C109-H), 6.57 (1H, br d, J 5 16.5 Hz, C79-H), 6.63
(1H, d, J 5 15.5 Hz, C12-H), 6.71 (1H, s, C14-H), 6.78 (1H, d,
J 5 15.0 Hz, C129-H), 7.15 (1H, dd, J 5 11.0, 15.0 Hz, C11-H),
7.89 (1H, d, J 5 0.8 Hz, C139-CH), 7.95 (1H, dd, J 5 0.8, 7.0
Hz, C149-H), 8.00 (1H, dd, J 5 11.5, 15.5 Hz, C119-H), and 8.55
(1H, d, J 5 7.0 Hz, C159-H). High resolution fast atom
bombardment (HR FAB)-MS (3-nitrobenzylalcohol) myz 5
592.4505 (calculated for C
42
H
58
ON, 592.4521, [M]
1
) iso-A2E:
1
H-NMR (500 MHz, CD
3
OD) is shown in Fig. 5. HR FAB-MS
(myz 5 592.4491; calculated for C
42
H
58
ON, 592.4521, [M]
1
).
UV (methanol):
l
max 430 nm («
M
31,000), 335 («
M
27,000).
Extraction of Human RPE Pigments. Individual human
donor eyes were obtained from the Eye Bank for Sight
Restoration (New York). From eyes ranging in age from 44 to
80 years (male and female, all Caucasian, died of cancer or
cardiovascular disease, and no significant ophthalmologic his-
tory), the RPE and choroidal layers were isolated and homog-
enized in a tissue homogenizer with a solution of chloroformy
methanolyPBS (2.0 mly1.0 ml). Each extract was filtered
through cotton and passed through a reversed phase (C18
Sep-Pak, Millipore) cartridge with 0.1% TFA in methanol.
After removing all solvent in vacuo, each sample was redis-
solved in a small amount of methanol (200
m
l) before HPLC
analysis.
HPLC Analysis of Eye Extracts. Samples extracted from
human RPEychoroid tissue were injected onto a reversed
phase (C18) column (Cosmosil 5C18, Nacalai Tesque, 150
mm 3 4.6 mm), and eluted with a gradient of methanol in
water (85–96% methanol 1 0.1% TFA, 1 mlymin, Waters
600E HPLC system). A photodiode array detector (Waters
996) was used to obtain an UV spectrum of each eluted
fraction. Specific wavelength detection was used for monitor-
ing A2E (430 nm) and retinols (320 nm).
Photoisomerization of A2E and iso-A2E. A solution of
HPLC-purified A2E or iso-A2E in methanol ('40
m
M) was
subjected to either room light or monochromatic (430 nm)
light from a monochromator (5–10 nm slit width). The extent
of isomerization at each time point was analyzed by RP-HPLC
by using the conditions described above.
RESULTS AND DISCUSSION
An investigation aimed at optimizing the yield of A2E (Fig. 1)
from all-trans-retinal and ethanolamine was performed by
using a wide range of reaction conditions. Typical results are
summarized in Table 1. When this reaction was carried out in
the absence of acetic acid, only a trace amount of A2E was
observed after 1 week (run 1), whereas conditions using
ethanolamine hydrochloride in place of the free amine gave no
A2E (run 2). The previously used biomimetic conditions
included acetic acid (AcOH) as an additive, and, in fact, A2E
was obtained under conditions similar to those initially re-
ported (AcOH, CH
2
Cl
2
, 20 min) (13), although this protocol
gave the desired product in only '0.5% yield. These results
indicated that the preparation of A2E is very sensitive to the
nature of the acid additive as well as to the overall pH of the
reaction mixture. A more detailed analysis of the preparation
of A2E and the role of acetic acid in the biomimetic synthesis
indicated that improved yields were obtained when only one
equivalent of acid was used in the reaction mixture. As shown
in run 3 (Table 1), prolonging the reaction time from 20 min
to 4 days, under otherwise identical reaction conditions,
increased the yield up to 8%.
The use of ethanol as the solvent in the biomimetic proce-
dure led to a dramatic improvement, affording A2E in 49%
yield (.50 mg, run 4); moreover, only a single silica gel column
chromatography was required to give essentially pure A2E,
which contained only a small amount of an A2E isomer. It
appears that the A2E formation cascade (Fig. 2) is not favored
under strongly acidic or basic conditions, an observation which
is consistent with the fact that A2E is formed under physiologic
conditions. The use of these reaction conditions has led to the
preparation of
14
C-labeled A2E from [1,2-
14
C]-ethanolamine
hydrochloride (data not shown), which will be used in further
biochemical studies.
Because the biological properties of A2E and its relationship
with AMD are unclear, the development of efficient methods
for the microscale analysis of A2E was essential for further
biological and chemical studies. The optimization of HPLC
conditions for the analysis of A2E was carried out by using the
sample previously obtained. It was found that RP- (C18)
Table 1. Optimization of A2E synthesis
Run* HOC
2
H
4
NH
2
Additives Solvents Time Results
†
1 Free amine None EtOH 7 days trace
2 HCl salt None EtOH 7 days 0%
3 Free amine AcOH (1 eq) CH
2
Cl
2
4 days 8%
4 Free amine AcOH (1 eq) EtOH 2 days 49%
*All reactions were performed open to the air and under dim red light.
The concentration of ethanolamine was 50–100
m
molyml.
†
The yields are calculated as TFA salts because they were isolated by
silica gel chromatography eluted with TFA-MeOH-CH
2
Cl
2
.
FIG. 1. Structures of A2E and iso-A2E. Molecular modeling on
each compound was performed by optimizing (MOPAC 93, AM1)
initial conformers, which were constructed according to the NOE
data.
14610 Biochemistry: Parish et al. Proc. Natl. Acad. Sci. USA 95 (1998)
HPLC by using a linear gradient of methanol and water (85 to
96% methanol) containing 0.1% of TFA provided a sharp peak
corresponding to A2E at 16.7 min., the detection limit of A2E
under these conditions being '5 ng.
Because the reported isolation from a pool of 250 aged
human eyes provided '100
m
g of A2E (13), it was reasonable
to expect that the quantity of A2E from a single aged eye
would be more than sufficient to be detected by HPLC. The
RPE and choroidal layers were isolated from individual human
eyes, the donors of which were greater than forty years of age.
Each sample was extracted with chloroformymethanol mix-
tures to obtain the lipophilic materials present in the tissue.
The entire extraction was performed under dim light to
prevent photochemical degradation of fluorophores. This sim-
ple procedure minimizes the extent of degradation, as well as
the amount of sample loss, which might occur during extended
protocols. As expected, the HPLC conditions thus developed
were able to detect the A2E isolated from 5% of the sample
from one aged eye. An example of the HPLC profile is shown
in Fig. 3. The major component of all samples was A2E (peak
1) based on UV absorbance. The UV spectrum of synthetic
A2E in methanol (Fig. 4) contained two peaks with
l
max 439
nm («
M
36, 900) and 334 nm («
M
25, 600). The analysis at
various other wavelengths between 300 and 450 nm provided
similar chromatograms with slight alterations in peak intensity.
While A2E can be observed with UV detection over 400 nm,
retinols (
l
max 5 325 nm) and retinals (
l
max 5 365 nm) are
only observed at shorter wavelengths. In addition, because
retention times may vary slightly between injections, coinjec-
tion of authentic material with the eye extract was used to
confirm the identity of the A2E peak.
HR FAB-MS of peak 1 (Fig. 3) collected from an injection
of the extracted material (10% of the total solution) provided
a molecular ion peak of myz 5 592.4505 (calculated for
C
42
H
58
ON, 592.4521). The UV of peak 1 as obtained from the
photodiode array detector (Fig. 4B) was identical to that
obtained from synthetic A2E (Fig. 4A). All eyes from seven
adult donors contained substantial amounts of A2E. The
quantity of A2E isolated from each eye, as determined from
integrated peak intensities was found to range from 200 to 800
ng. No direct comparison between the amount of A2E and age
has been made due to the small sample size and the lack of
appropriate control eyes. However, this protocol would allow
for a thorough analysis of the relationship between A2E and
age in a more extensive study. The isolated amounts of A2E
corresponded well with the estimate made from the previous
protocol (13). In contrast to the adult eyes, the extracts of
choroidyRPE isolated from two fetal eyes (18 and 20 weeks of
gestation; Anatomic Gift Foundation, White Oaks, GA) did
not contain A2E. Even when the entire fetal eye sample was
injected, none of this pigment was observed (Fig. 3), indicating
that A2E accumulates with increasing age. Recently, rat eye
extracts have been analyzed for A2E by HPLC at the short
wavelength of 200 nm, a nonselective wavelength where pig-
mented as well as other compounds would be detected (16).
However, those conditions do not provide a clear indication of
the quantity of A2E present in the rat samples due to the short
wavelength (200 nm) used for detection.
Because A2E can be generated efficiently from all-trans-
retinal and ethanolamine under biomimetic conditions, it was
necessary to exclude the possibility that the pigment was being
generated during isolation. Thus, we sought to determine
whether retinal was present in the eye extracts. If A2E was
being generated after extraction of the eye tissues, significant
quantities of retinal would be observed in these samples. First,
it was established that all-trans-retinal could pass through a
reversed phase (C18) cartridge under conditions used in the
extraction procedure. While the retention time of authentic
all-trans-retinal (
l
max 5 365 nm) was 14.5 min under these
HPLC conditions, none was observed in any eye sample. The
absence of all-trans-retinal from RPEychoroid was not sur-
prising since all-trans-retinal is reduced to all-trans-retinol
within the photoreceptor outer segment before being trans-
ported to the RPE (17). All-trans-retinol (Fig. 3, peak 3, t
R
5
13.1 min) and 13-cis-retinol (Fig. 3, peak 4, t
R
5 13.0) were
identified in the HPLC profile by their UV (
l
max 5 325) and
by coinjection with authentic samples. The single maximum of
these UV (Fig. 4C) clearly indicates that these fractions
correspond to retinols and not structures that are analogous to
A2E. The retinol peaks could be detected in five of the seven
aged eyes that have been investigated to this point. It was
expected that retinyl palmitate would be present in the RPEy
choroid extract (12). However, since authentic retinyl palmi-
tate was retained on the C18 column under these conditions,
FIG. 2. Biogenesis of A2E from retinal and (phosphatidyl)ethanol-
amine.
FIG. 3. HPLC of aged and fetal eye extracts. Samples extracted
from human RPEychoroid tissue were injected onto a reversed phase
(C18) column (Cosmosil 5C18, Nacalai Tesque, 150 mm 3 4.6 mm),
eluting with a gradient of methanol in water (85–96% methanol 1
0.1% TFA, 1 mlymin). The displayed chromatograms correspond to
absorbances at 320 nm. Upper trace: The HPLC of extracted pigments
(10
m
lofa200
m
l extract) from an 80-year-old eye contained a mixture
of pigments, which have been partially identified: peak 1, A2E; peak
2, iso-A2E; peak 3, all-trans-retinol; peak 4, 13-cis-retinol; and p,
unidentified pigment. All-trans-retinal has a retention time of 14.5 min
under these conditions. Lower trace: The HPLC of fetal eye extracts
(entire extract) did not contain any detectable amount of these
materials.
Biochemistry: Parish et al. Proc. Natl. Acad. Sci. USA 95 (1998) 14611
the presence of this material in the eye extracts can only be
confirmed by using a modified HPLC solvent system.
In addition to A2E, a second peak with considerable peak
height, corresponding to a slightly less polar pigment, was
visible in all aged eye extracts (Fig. 3, peak 2, t
R
5 18.8 min).
The UV spectrum of this fraction (Fig. 4B) contained two
peaks with
l
max 5 430 and 330 nm, similar to that of A2E, but
different in relative intensity. HR FAB-MS (myz 5 592.4491;
calculated for C
42
H
58
ON, 592.4521) indicated that this com-
pound was an isomer of A2E. However, it was not possible to
obtain an interpretable
1
H-NMR of this compound when
isolated from donor eyes since ,100 ng could be obtained
from each extract. Interestingly, it was found that the HPLC
chromatogram of the biomimetic synthesis of A2E described
earlier (see above) contained material, which was identical to
peak 2 of the eye extract as judged by HPLC coinjection, UV
(Fig. 4A) and FAB-MS. Therefore, the synthetic sample was
studied by
1
H-NMR and identified as iso-A2E, the Z-isomer of
A2E at the C13–C14 double bond (Fig. 5). The UV spectrum
of iso-A2E in methanol (Fig. 4A) contained two peaks with
l
max 426 nm («
M
31,000) and 335 nm («
M
27,000). The
1
H-NMR spectrum was very similar to, but not identical to,
A2E (Fig. 5).
1
H-
1
H COSY (homonuclear shift correlation
spectroscopy) allowed for the complete identification of
methyl and olefinic proton resonances. A significant correla-
tion peak was observed between H
a
(7.88 ppm, d, J 5 1.5 Hz)
and H
b
(6.56 ppm, d, J 5 15.0 Hz) by rotating frame nuclear
Overhauser and exchange spectroscopy (ROESY) suggesting
a C13-C14 Z-geometry. This correlation peak was not ob-
served in A2E when the structure was fully described by ROE
techniques (14). All other double bonds were identified as
being in E configurations based on large coupling constants
(J
7, 8
5 16.0 Hz, J
11, 12
5 15.0, J
79,89
5 16.5, J
119,129
5 15.5) and
ROESY correlations (8-H910-H, 13-CH
3
914-H, 89-H9109-H)
(see numbering in Fig. 1).
While iso-A2E in methanol was stable when stored at 270°C
for more than 1 month in the dark, exposure of this solution
to room light for 30 min at room temperature led to isomer-
ization of most of the compound to A2E. Under these
conditions, HPLC-purified A2E and iso-A2E both undergo
photoequilibration to provide '4:1 mixtures of A2E:iso-A2E
without significant decomposition to other products, as deter-
mined by HPLC (Fig. 6). This isomerization also was observed
when A2E and iso-A2E were exposed to monochromatic (430
nm) light. For example, a gradual conversion of iso-A2E to
FIG.5.
1
H-NMR spectra of A2E and iso-A2E. Region of
1
H-NMR
(CD
3
OD, 500 MHz) spectra of A2E (A) and iso-A2E (B) correspond-
ing to aromatic and olefinic protons. The critical ROESY correlation
peak between H
a
and H
b
of iso-A2E is identified. This correlation peak
is absent in A2E (14). The assignment of all
1
H-NMR chemical shifts
as well as the COSY and ROESY correlation peaks, which were
important for assigning the configuration of each double bond, are
indicated.
FIG. 6. Photoisomerization of A2E and iso-A2E. Methanolic
solutions of purified (A) A2E and (C) iso-A2E were exposed to room
light for 30 min. The samples were reanalyzed by HPLC and resulted
in similar product mixtures containing approximately 4:1 A2E:iso-
A2E: (B) A2E 1 light; (D) iso-A2E 1 light. A small impurity (p)is
present in the synthetic iso-A2E sample.
FIG. 4. UV spectra of synthetic and extracted pigments. (A) UV spectra of A2E (solid line) and iso-A2E (dashed line) in methanol. A2E:
l
max
439 nm («
M
36, 900), 336 («
M
25, 600); iso-A2E:
l
max 426 nm («
M
31,000), 335 («
M
27,000); (B) UV spectra of peak 1 (A2E) and peak 2 (iso-A2E)
from eye extracts. The weak iso-A2E spectrum is due to the limited quantity of iso-A2E present in the extracts. (C) UV spectra of peak 3
(all-trans-retinol) and peak 4 (13-cis-retinol) from eye extracts. The
l
max of retinol is '325 nm. The spectra provided in B and C were obtained
by PDA detection in the HPLC eluent, '10% methanol in water containing 0.1% TFA.
14612 Biochemistry: Parish et al. Proc. Natl. Acad. Sci. USA 95 (1998)
A2E resulted from increasing light exposure: 15 sec exposure,
A2Eyiso-A2E 3y97; 2 min, 22y78; 7 min, 58y42; and 15 min,
78y22. Thus, although the human lens absorbs light below 400
nm (18), longer wavelength light reaching the retina would
have the capacity to photoisomerize A2E generated in vivo.
Furthermore, because iso-A2E was present in an eye extracted
in the dark, it appears that this fluorophore is also present in
RPE lipofuscin, as opposed to being generated by photoi-
somerization after isolation. The photochemistry of A2E is
such that the generation of radical species could be involved in
this isomerization in vivo (19). It also is possible that radicals
generated by photoactivated A2E may lead to cellular damage
and a decrease in RPE function.
This work describes the optimization of conditions for the
single-step preparation of A2E in high yield providing large
(.50 mg) quantities of material. An efficient HPLC protocol
allows for the identification of the fluorophores present in
RPE lipofuscin by using extracts from a single human eye. This
procedure has resulted in the identification of one novel
pigment (iso-A2E) present in aged eye RPE lipofuscin. The
presence of all-trans- and 13-cis-retinol in most extracts also
has been confirmed. While the role of A2E in AMD remains
unclear, the amount of this natural product in each eye can be
easily quantified by this procedure. The ability to determine
the amount of A2E in the RPE of an individual is of special
interest for epidemiological studies. A substantial source of
A2E and iso-A2E will enable the further clarification of the
role of these fluorophores in AMD. Preliminary experiments
incubating cultured human RPE cells with synthetic A2E have
confirmed that A2E is incorporated into the cells (data not
shown). The cellular effects of A2E exposure are currently
being characterized. In addition to the possibility that these
pigments can lead to oxidativeyradical damage in cells, the
amphiphilic character of the uniquely wedge-shaped A2E and
the narrower, more streamlined iso-A2E (Fig. 1, molecular
models) would suggest that these detergent-like molecules
could readily interact with membranes and perturb membrane
integrity. The identification of additional lipofuscin fluoro-
phores, the photochemical properties of A2E, and the effect of
this pigment on membrane and cell function are under inves-
tigation.
This work was supported by National Institutes of Health Grant
34509 (to K.N.), a National Institutes of Health National Research
Service Award postdoctoral fellowship (to C.A.P.), and an unrestricted
grant from Research to Prevent Blindness to the Department of
Ophthalmology, Columbia University.
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