Mechanisms of Ageing and Development
92 (1996) 159 – 174

Formation of lipofuscin-like ﬂuorophores by
reaction of retinal with photoreceptor outer
segments and liposomes
Martin L. Katz*, Chun-Lan Gao, Laura M. Rice
Uni6ersity of Missouri School of Medicine, Mason Eye Institute, Columbia, MO 65212, USA
Received 14 September 1996; revised 14 September 1996; accepted 8 November 1996

Abstract
During the aging process the retinal pigment epithelium (RPE) accumulates autoﬂuorescent lysosomal storage bodies (lipofuscin). Data from previous studies led to the hypothesis
that at least one of the ﬂuorescent components of RPE lipofuscin is formed by reaction of
vitamin A aldehyde with phosphatidylethanolamine (PE) in the photoreceptor outer segments. Experiments were performed to test this hypothesis. All-trans retinaldehyde was
incubated with isolated bovine photoreceptor outer segments and with synthetic liposomes.
Liposomes were made with two different lipid compositions. One type of liposome consisted
of a mixture of lipids, including phosphatidylcholine (PC), none of which contained a
primary amine. The other liposome type was identical in composition except that some of the
PC was replaced with an equimolar amount of phosphatidylethanolamine (PE). After
incubation of the samples, aliquots were examined with ﬂuorescence microscopy to assess
whether any lipofuscin-like ﬂuorescence had developed. Lipids were extracted from additional aliquots of the samples and analyzed with thin layer chromatography. Photoreceptor
outer segments incubated with retinaldehyde developed an intense golden yellow ﬂuorescent
emission when illuminated with 395 – 440 nm light. Similar ﬂuorescence developed in the
liposomes containing PE, whereas the liposomes lacking PE or any other primary amine did
not develop any detectable ﬂuorescence. The development of ﬂuorescence in the samples in
situ correlated with the appearance of an orange colored component in the lipid extracts that
displayed a weak red emission upon ultraviolet light illumination. Incorporation of this
component into liposomes resulted in the appearance of a golden yellow ﬂuorescent
* Corresponding author. Fax: + 1 573 8844100.
0047-6374/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved.
PII S 0 0 4 7 - 6 3 7 4 ( 9 6 ) 0 1 8 1 7 - 9

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emission. The results of these experiments suggest that retinal, generated during visual
pigment bleaching, can react with PE in the photoreceptor outer segments to form a
ﬂuorophore, a derivative of which subsequently accumulates in RPE lipofuscin. An RPE
lipofuscin ﬂuorophore was previously shown to be identical to a reaction product of retinal
and ethanolamine. This ﬂuorophore is probably derived from the reaction product of outer
segment PE and retinal. © 1996 Elsevier Science Ireland Ltd.
Keywords: Aging; Retina; Vitamin A; Age pigment; Retinal pigment epithelium; Lipofuscin;
Phospholipids

1. Introduction
During the aging process almost all multicellular animals, including humans,
exhibit a build-up of lysosomal storage bodies in many of their postmitotic cell
types [1]. A distinctive feature of these storage bodies, also known as age-pigment
or lipofuscin, is that they emit a golden-yellow ﬂuorescence when examined under
near ultraviolet or blue light illumination [2]. Neither the molecular composition of
lipofuscin nor its mechanism of formation has been completely elucidated for any
tissue.
The most thorough studies on lipofuscin formation have been performed on the
pigment that accumulates in the retinal pigment epithelium (RPE) of mammalian
eyes. It appears that the constituents of RPE lipofuscin are derived primarily from
molecular components of the adjacent photoreceptor cells that are phagocytosed by
the RPE as part of the normal turnover process [3–8]. These photoreceptor cell
components do not exhibit detectable lipofuscin-like ﬂuorescence properties prior to
being engulfed by the RPE. Thus, the ﬂuorophores must either be concentrated in
the RPE after their uptake, or they must form within the phagolysosomal system.
Among the major molecular constituents of the photoreceptor outer segments are
the forms of vitamin A involved in the visual cycle. Dietary studies performed in
rats have indicated that vitamin A plays a critical role in RPE lipofuscin formation.
Animals fed diets deﬁcient in the forms of vitamin A utilized by the photoreceptor
cells in visual transduction show a greatly reduced accumulation of lipofuscin in the
RPE [9 – 12]. The accumulation of the ﬂuorescent components of RPE lipofuscin in
particular appear to be dependent on the availability of the visual cycle retinoids
[13]. Incorporation of vitamin A into lipofuscin-like inclusions in the RPE has been
demonstrated by autoradiography [14]. In addition, structural analysis of one of the
ﬂuorescent compounds present in human RPE lipofuscin indicates that at least
some of the RPE lipofuscin ﬂuorophores are derived from vitamin A aldehyde
[15,16].
On the basis of these data, it appeared likely that vitamin A aldehyde, produced
in the outer segments during visual pigment bleaching, can react with some of the
other molecular species in the outer segments to form the ﬂuorophores that
subsequently appear in RPE lipofuscin. To evaluate this possibility, all-trans retinal
was reacted with rod outer segments isolated from bovine eyes. The outer segments

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161

were then evaluated for the formation of lipofuscin-like ﬂuorophores. Similar
reactions were carried out with synthetic liposomes to determine whether outer
segment phosphatidylethanolamine (PE) was one of the molecular species with
which retinal could react to generate at least one of the RPE lipofuscin
ﬂuorophores.

2. Materials and methods

2.1. Isolation of bo6ine rod outer segments
Fresh bovine eyes were obtained from the University of Missouri Food Science Department abattoir and from Jennings Meat (New Franklin, MO). The
eyes were placed on ice immediately after enucleation, and were kept cold until
they were dissected. The neural retinas were dissected from the eyes, and the rod
outer segments were isolated from the retinas using a modiﬁcation of the technique described by Farnsworth and Dratz [17]. Each retina was placed in a vial
containing 3 ml of 34% sucrose in a base buffer consisting of 10 mM Hepes, 1
mM MgCl2, 0.15 mM CaC12, 0.1 mM Na2 EDTA, and 130 mM NaCl at a pH
of 7.40. The samples were then vortex mixed at high speed for 80 s each to
detach the photoreceptor outer segments from the retinas. The remainders of the
retinas and any large debris were removed by centrifugation at 5000 rpm on a
Sorvall SS34 rotor for 4 min. All centrifugation steps were carried out at 4°C.
The supernatants were diluted 1:1 with base buffer, and the photoreceptor outer
segments were pelleted by centrifugation for 20 min at 10 000 rpm on the SS34
rotor. Affer discarding the supernatants, the pellets were suspended in 24% sucrose in the base buffer. Using a syringe with a Teﬂon needle, 5 ml of 34%
sucrose in the base buffer was injected below 5 ml of sample suspension in a 15
ml polycarbonate tube. The samples were then centrifuged on a Sorvall HB-4
swinging bucket rotor at 12 000 rpm for 90 min. After centrifugation, the band
at the interface between the 24 and 34% sucrose layers was collected, diluted
with two volumes of base buffer, and centrifuged on the SS34 rotor at 10 000
rpm for 20 min. The resulting pellet containing puriﬁed photoreceptor outer
segments was used in the retinal reaction experiments.
To evaluate the purity of the outer segment preparations, an aliquot was ﬁxed
and examined by electron microscopy. A fraction of the outer segment pellet was
suspended in the base buffer and transferred into a centrifuge tube containing a
mixed aldehyde ﬁxative [18]. The suspension was centrifuged at 7200× g for 5
min to pellet the sample. After gently dislodging the pellet from the bottom of
the tube, the sample was ﬁxed with gentle mixing for 2 h at room temperature. After primary ﬁxation, the sample was post-ﬁxed in OsO4, embedded in
epoxy resin, sectioned, and stained with uranyl acetate and lead citrate. The
sections were then examined and photographed in a JEOL 1200EX electron
microscope.

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2.2. Isolation of human RPE lipofuscin
Human RPE lipofuscin was isolated so that any ﬂuorophores normally present in
RPE age pigment could be compared to those generated by reacting retinal with the
outer segments. Human donor eyes were obtained from the Missouri Lions Eye
Research Foundation eye bank. All eyes were enucleated within several h of death
of the donor and were dissected within 24 h of death. The anterior segments and
neural retinas of the eyes were removed. The remainders of the eyecups were lined
with RPE. Into each eyecup was placed 1 ml of 10 mM Hepes, 0.1 mM Na2 EDTA,
pH 7.40. Using a camel-hair brush, the RPE was brushed off the choroid into the
buffer. The resulting suspension, containing both intact and ruptured RPE cells,
was collected in a glass vial. An additional 0.5 ml Hepes buffer was added to the
eyecup, and the brushing and collection steps were repeated. The RPE preparations
from both eyes from the same donor were pooled into one vial. The samples were
layered with argon and stored at − 70°C until used.
RPE preparations from four donors were thawed and pooled into a 15 ml glass
homogenization sleeve. The samples were homogenized using 10 strokes of a
motor-driven Teﬂon pestle. The homogenates were then centrifuged for 3 min at
1000 rpm in a Sorvall HB-4 rotor. The supernatants were layered on top of 3-step
sucrose gradients consisting of 2.25, 1.37, and 0.63 molal sucrose in 10 mM Hepes,
1 mM Na2 EDTA, 150 mM NaCl, pH 7.40 (isolation buffer). After centrifugation
for 40 min at 10 000 rpm in the HB-4 rotor, the material that accumulated at the
interface between the 0.63 and 1.37 molal sucrose layers was collected. This
material was diluted with at least 4 parts isolation buffer and then centrifuged at
10 000 rpm on the HB-4 rotor for 15 min. The resulting RPE lipofuscin pellets were
stored under argon until used for ﬂuorophore extraction. Aliquots of pellets from
several such preparations were examined with ﬂuorescence and electron microscopy
to assess their purity. Sample preparation and examination with electron microscopy were as described for the outer segment preparations. For ﬂuorescence
microscopy, aliquots of the preparations were suspended in isolation buffer, placed
on glass slides and covered with coverslips. The samples were examined and
photographed as described below for the outer segment and liposome samples.

2.3. Preparation of liposomes
Experiments were performed to determine whether the major lipid ﬂuorophore
formed by reaction of retinal with the outer segments was due to reaction between
retinal and PE. This assessment was made by incubating retinal with liposomes
either containing or lacking PE. Large liposomes were prepared by a modiﬁcation
of the technique of Kim and Martin [19]. For liposomes lacking PE (designated PC
liposomes), chloroform and diethyl ether solutions were prepared each containing
phosphatidylcholine (PC), cholesterol, and phosphatidylglycerol, triolein, and h-tocopherol in a 16:16:8:3:1 molar ratio at a total lipid concentration of 3.6 vmol/ml.
A one ml aliquot of the chloroform solution was transferred to a one-dram vial
with a Teﬂon-lined cap. To this was added 1.0 ml of 300 mM aqueous sucrose in

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three equal aliquots with stirring between each addition to prevent formation of a
chloroform-in-water emulsion. The vial was then vortex mixed for 45 s. A 0.5 ml
aliquot of the ether solution was added to 2.5 ml of 300 mM sucrose and vortex
mixed for 15 s. To the latter solution was added 1.0 ml of the chloroform-in-water
dispersion, and the mixture was vortex mixed immediately for 10 s. The mixture
was then transferred to a round-bottomed ﬂask. The ﬂask was placed in a
mechanical rotator and rotated at : 30 rpm in a water bath at 37°C under a gentle
stream of argon. The residue remaining after 30 min was mixed with an equal
volume of 5% glucose. The liposomes were pelleted by centrifugation at 6000 rpm
on a Sorvall SS-34 rotor for 30 min. They were stored under argon at − 70°C until
used for incubation with retinal. The liposomes containing PE (designated PE
liposomes) were prepared in an identical manner, except that molar lipid composition in the chloroform and ether solutions was PC:PE:cholesterol phosphatidylglycerol:triolein:h-tocopherol 11:5:16:8:3:1.

2.4. Reaction of outer segments and liposomes with retinal
For evaluating their reactivity with retinal, the outer segments from four cow
eyes were suspended in 1.0 ml of citrate buffer (20 mM citric acid, 1 mM MgCl2,
0.15 mM CaC12, 0.1 mM Na2 EDTA, 120 mM NaCl, pH 4.80). The suspension
was divided into two equal aliquots which were placed in amber vials. To one vial
was added 15 vl of a 2 mg/ml solution of all-trans retinal in ethanol. On the basis
of the expected yield of opsin per eye [20], this amount of retinal (106 nmol)
represents a 6- to 10-fold molar excess over the amount of opsin present in the
sample. To the other vial, which served as the control, was added 15 vl of ethanol
alone. The samples were vortex mixed brieﬂy and then incubated at room temperature for 90 min. After this period of incubation the samples were either examined
or analyzed immediately or were stored under argon at −70°C until further
analyses were carried out.
Reactions of liposomes with retinal were carried out under similar conditions to
those used for the outer segments. Both PC and PE liposomes were suspended in
citrate buffer at a concentration of approximately 4.5 vmol total lipid/ml. To a 0.7
ml aliquot of each suspension was added either 35 vl ethanol or retinal dissolved in
ethanol at a concentration of 2 mg/ml. The samples were vortex mixed brieﬂy and
then incubated at room temperature for 3.5 h. As with the outer segment preparations, the samples were then either analyzed immediately or stored under argon at
−70°C until further analyses were carried out.

2.5. Fluorescence microscopy
After incubation with either retinal or the ethanol vehicle alone, the outer
segments or liposomes were examined immediately for the presence of lipofuscin-like ﬂuorescence. A small aliquot of each sample was placed on a glass slide
and covered with a glass coverslip. The samples were then examined in a Zeiss
Axiophot microscope equipped for epiﬂuorescence. ﬂuorescent emissions were

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stimulated with light from a 50 W high-pressure mercury vapor source.
Examination and photography of the specimens were performed using a
Plan-Neoﬂuor objective with a 1.30 numerical aperture, a 395–440 nm bandpass
exciter ﬁlter, an FT 460 chromatic beam splitter, and an LP 515 barrier ﬁlter.
Photography was performed using Kodak Ektachrome 200 ﬁlm and a ﬁxed
exposure time of 6 s.

2.6. Lipid extraction and thin-layer chromatography
After incubation of the outer segments or liposomes with retinal or ethanol
alone, lipids were extracted from the preparations and from RPE lipofuscin samples
for thin layer chromatographic (TLC) analysis of any ﬂuorophores present. The
samples were pelleted by centrifugation at 10 000 rpm on the SS34 rotor for 25 min.
After removal of the supernatants, the outer segment, liposome, and lipofuscin
preparations were each suspended in 400 vl water. To each sample was added 500
vl CHCl3 and 1.0 ml methanol. The samples were sonicated for 10 s on ice followed
by vortex mixing for 30 s. An additional 500 vl CHCl3 was added to each sample
followed by vortex mixing for 30 s. This was followed by the addition of 500 vl
water and vortex mixing for another 30 s. The samples were then centrifuged at
1000 × g for 5 min. The bottom organic phase was collected from each sample and
transferred to a new vial. The upper phase was washed twice with 1.0 ml aliquots
of CHCl3, and the resulting lower phases were pooled with that obtained as
described above. Solvent was removed from the samples by vacuum evaporation,
and the residues were each reconstituted in 60 vl 2:1 CHCl3:CH3OH.
Thin layer chromatography was carried out using channeled silica gel G plates
with a 250 vm coating and a preabsorabant zone (Analtech, Newark, DE). A 20 vl
aliquot of each sample was applied to the plate and the chromatograms were
developed for 20 min in a solvent mixture consisting of dichloromethane, methanol,
water, and acetic acid in a ratio of 300:60:6:1.8 (v/v/v/v). After development, the
plates were air dried and photographed under white and ultraviolet light as
described previously [5]. The ultraviolet source used to illuminate the TLC plates
for photography had a maximum energy output at 366 nm.

2.7. Incorporation of retinal– PE reaction product into liposomes
An orange-colored product was observed in the thin layer chromatograms of
organic solvent extracts of the outer segments and PE liposomes after reaction with
retinal. Experiments were performed to determine whether this product could be
responsible for the golden-yellow ﬂuorescent emissions observed in the outer
segments and liposomes reacted with retinal. The colored compound formed after
reaction of retinal with PE liposomes was separated from the other lipids with TLC
as described in the previous section. The compound was eluted from the silica
stationary phase with 2:1 chloroform:methanol. The solvent was removed by
evaporation under vacuum, and the residue was dissolved in 200 vl chloroform.
The resulting solution was combined with 900 vl of the chloroform solution of

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either the PC or PE lipid mixes described earlier. These mixtures were then used to
make liposomes with the methods outlined in a previous section.

3. Results

3.1. Quality of outer segment and RPE lipofuscin preparations
Electron microscopic examination of the bovine outer segments indicated that
they were intact and devoid of contamination by other cellular constituents (Fig. 1).
Human RPE lipofuscin prepared as described separates into two fractions. The
more dense fraction is located on the sucrose gradient at the interface between the
1.37 and 2.25 molal sucrose layers. The granules in this fraction appear to be
complexes between lipofuscin and melanin. The less dense fraction at the interface
between the 0.63 and 1.37 molal sucrose layers is almost pure lipofuscin without
melanin inclusions (Fig. 2A). Only the latter fraction was used in the present study.
When examined with ﬂuorescence microscopy, the isolated lipofuscin granules
displayed the golden-yellow emission typical of these RPE inclusions (Fig. 2B).

3.2. Reaction products of retinal and rod outer segments
Isolated rod outer segments incubated with ethanol alone displayed a weak
yellow ﬂuorescent emission when illuminated with 395–440 nm light (Fig. 3A). The

Fig. 1. Electron micrograph of bovine photoreceptor outer segments isolated for incubation with retinal.

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Fig. 2. (A) Electron micrograph of human RPE lipofuscin isolated from a sucrose density gradient. (B)
Fluorescence micrograph of the same lipofuscin preparation.
Fig. 3. Fluorescence micrographs of (A) bovine photoreceptor outer segments incubated without
exogenously added retinal, and (B) outer segments after incubation in the presence of added retinal for
90 min.

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intensity of this ﬂuorescence was greatly enhanced when exogenous retinal was
present in the incubation mixture (Fig. 3B). TLC analysis of the lipids from
the outer segments incubated in the absence of added retinal revealed the
presence of two adjacent orange bands with Rf of 0.59 to 0.67 (Fig. 4A,
lane 1). The intensity of these bands were greatly enhanced in the lipid extract of the outer segments incubated in the presence of added retinal (Fig. 4A,
lane 2). Two additional faint colored bands were present in the latter extract,
as was a band corresponding to unreacted retinal at Rf 0.90–0.96. When
the TLC plates were examined under ultraviolet illumination, the prominent colored bands at Rf of 0.59–0.67 displayed a relatively weak red emission
(Fig. 4B, lanes 1 and 2) that did not correspond exactly to any of the
ﬂuorophores present in a lipid extract of isolated RPE lipofuscin granules (Fig.
4B, lane 3).

3.3. Reaction products of retinal and liposomes
Based on a report that one of the ﬂuorophores present in RPE lipofuscin
appears to be identical to a reaction product of retinal and ethanolamine [15],
experiments were conducted to determine whether the in situ ﬂuorescence and
prominent colored compound observed after reaction of retinal with the photoreceptor outer segments could be due to formation of an adduct of retinal to PE.
Retinal was reacted with synthetic liposomes either containing or lacking PE (PE
and PC liposomes respectively). After incubation with retinal for 3 h, the PE
liposomes developed a bright golden-yellow ﬂuorescent emission (Fig. 5). No
corresponding ﬂuorescence was observed when the PE liposomes were incubated
with the ethanol vehicle alone. Likewise, PC liposomes incubated either in the
presence or absence of retinal failed to develop any detectable in situ ﬂuorescence.
After reaction with retinal, the liposomes were extracted with chloroform–
methanol, and the extracts were analyzed with TLC. In the extracts of the PC
liposomes, the only colored compound observed was unreacted retinal (Fig. 6A,
lane 1). The extracts from the PE liposomes, on the other hand, also contained
two adjacent orange-colored constituents with an Rf of 0.44–0.53 vg (6A, lane
2). Like the orange-colored products formed by reaction of retinal with the outer
segments, these components displayed a weak red ﬂuorescence when examined
under ultraviolet illumination.
An experiment was conducted to determine whether the latter components
were responsible for the golden-yellow ﬂuorescence observed in the PE liposomes
after reaction with retinal. The orange-colored products were eluted from the
TLC plate and reconstituted into both PC and PE liposomes. The liposomes
were then examined with ﬂuorescence microscopy. In both cases, the liposomes
into which these products were incorporated displayed a golden-yellow ﬂuorescence emission (Fig. 7).

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Figs. 4 – 7.

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169

4. Discussion
Although the age-related accumulation of autoﬂuorescent storage bodies in
post-mitotic cells has been well known for decades, the precise mechanisms responsible for the formation of the constituent ﬂuorophores of lipofuscin have not been
elucidated for any cell type. The most detailed studies on the mechanisms of
lipofuscin formation have been carried out on the RPE pigment. A variety of
evidence clearly indicates that the primary precursors of RPE lipofuscin are
molecular constituents of the photoreceptor outer segments that are phagocytosed
by the RPE. For example, it has been shown that in retinas lacking photoreceptor
outer segments, either due to a genetic defect or as a result of light damage, the rate
of lipofuscin accumulation in the RPE is reduced dramatically [5,7]. In addition,
cultured RPE cells allowed to phagocytose photoreceptor outer segments accumulate lipofuscin-like autoﬂuorescent inclusions, whereas very few such inclusions are
observed in control cultures not provided with outer segments [21].
Of the molecular constituents of the outer segments, the retinoids have been most
strongly implicated as potential precursors of RPE lipofuscin ﬂuorophores. Experiments conducted by Robison et al. demonstrated that vitamin E deﬁciency greatly
accelerates the rate of autoﬂuorescent pigment accumulation in the RPE [11]. The
pigment accumulation was prevented to a large extent if the animals had also been
depleted of the retinoids involved in the visual cycle. The normal age-related
accumulation of RPE lipofuscin was also greatly slowed by depleting animals of
visual cycle retinoids [9]. Additional evidence that vitamin A may be directly
involved in lipofuscin ﬂuorophore formation was provided by experiments in which
animals were given intraocular injections of the protease inhibitor leupeptin. These
animals showed a rapid and dramatic accumulation of outer segment-derived
inclusions that had lipofuscin-like ﬂuorescence properties [8]. The development of
ﬂuorescence in these inclusions was almost completely prevented if the eyes had
been depleted of visual cycle retinoids prior to the leupeptin treatment [13]. It was
recently demonstrated by electron microscopic autoradiography that vitamin A is
directly incorporated into the autoﬂuorescent inclusions that accumulate in the
RPE after leupeptin treatment [14].
Chromatographic and absorbance, ﬂuorescence and mass spectral analyses indicated that one of the major ﬂuorophores present in human RPE lipofuscin is a
Fig. 4. Thin layer chromatogram of chloroform – methanol extracts of bovine photoreceptor outer
segments incubated in the presence or absence of exogenous all-trans retinal. (A) Chromatogram
photographed under white light illumination. (B) Chromatogram photographed under UV light illumination. Lanes labeled (1) are samples incubated without the addition of retinal. Lanes labeled (2) are
samples incubated in the presence of added retinal. Lanes labeled (3) are extracts of isolated human RPE
lipofuscin granules.
Fig. 5. Fluorescence micrograph of PE liposomes incubated in the presence of all-trans retinal.
Fig. 6. Thin layer chromatograms of chloroform – methanol extracts of PC liposomes (lanes one) and PE
liposomes (lanes two) that had been reacted with all-trans retinal. (A) Chromatogram photographed
under white light illumination. (B) Chromatogram photographed under UV light illumination.
Fig. 7. Fluorescence micrograph of PC liposomes formulated with the TLC-puriﬁed PE – retinal reaction
product.

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covalent complex between retinaldehyde and ethanolamine [15]. This was conﬁrmed
by in vitro synthesis of this ﬂuorophore by reacting all-trans retinal with
ethanolamine [15]. The structure of this retinal–ethanolamine reaction product was
reported recently [16]. It was proposed that this compound formed by reaction of
retinal with ethanolamine in the phagolysosomes of the RPE [16]. However, it is
also possible that retinal in the outer segments reacts with the head group of
phosphatidylethanolamine and that the ethanolamine–retinal moiety is subsequently cleaved from the remainder of the molecule after uptake by the RPE. The
data from the present study are consistent with the latter hypothesis. Reaction of
retinal with isolated photoreceptor outer segments produced a substantial increase
in the intensity of a lipofuscin-like ﬂuorescence in the photoreceptor outer segments. This increase in ﬂuorescence correlated with an increase in the amount of
two colored weakly ﬂuorescent components present in the chloroform–methanol
extracts of the outer segments. The two components observed on TLC are likely to
be isomers. The in situ ﬂuorescence could have arisen from reactions between
retinal and any of several of the molecular species present in the outer segments. To
evaluate the possibility that this ﬂuorescence arose, at least in part, from reaction
between retinal and PE, retinal was reacted with synthetic liposomes either containing or lacking PE. Since retinal is expected to react predominantly with primary
amines, the liposomes were made such that PE was the only amine present. No
amines were present in the control liposomes. An intense lipofuscin-like ﬂuorescence appeared only in those liposomes containing PE. Chloroform–methanol
extraction of these liposomes yielded a component with similar spectral and
chromatographic properties to the colored weakly ﬂuorescent component extracted
from outer segments after reaction with retinal. These data strongly suggest that
retinal can react with PE in the outer segments to form a ﬂuorescent precursor of
the ethanolamine – retinal complex associated with RPE lipofuscin. The orange
colored product obtained after reaction of retinal with the PE liposomes differed
somewhat in its chromatographic mobility from that of the colored product formed
upon reaction of retinal with the outer segments. This difference was likely due to
the difference in fatty acid compositions between the outer segment and liposome
PE. The synthetic liposome PE contained exclusively oleic acid, whereas each
molecule of outer segment PE contains on average one saturated fatty acid and one
unsaturated fatty acid, with most of the unsaturated fatty acid being docosahexaenoic [22].
The control outer segments that were incubated in the absence of exogenous
retinal were not completely devoid of lipofuscin-like ﬂuorescence. Normally such
ﬂuorescence is not detectable in the outer segments of whole freshly isolated eyes.
It is likely that this weak ﬂuorescence arose as a result of reaction between
endogenous retinal and outer segment PE. No precautions were taken to control
bleaching of the visual pigment during the processing of the bovine eyes for
isolation of the outer segments. In vivo, retinal released during bleaching is rapidly
reduced to retinol through the action of an outer segment retinol dehydrogenase
enzyme using NADPH as a co-factor [23,24]. Perhaps as soon as the blood supply
to the retina was interrupted, but certainly once the outer segments were detached

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from the retinas, the metabolic regeneration of NADPH in the outer segments
would be expected to cease. Any subsequent bleaching of the visual pigment would
produce retinal that was not reduced to retinol, and thus was available to react with
PE.
The in situ ﬂuorescence generated by reacting retinal with either outer segments
or PE containing liposomes was similar in color to that of RPE lipofuscin.
However, upon chloroform –methanol extraction, the most prominent colored
product that was generated by reaction with retinal displayed only a weak red
ﬂuorescence (Figs. 4 and 6). This suggests that this ﬂuorophore is sensitive to
solvent effects in its spectral properties and quantum yield. The dependence of
ﬂuorescence properties on local environment or solvent is typical of many ﬂuorescent compounds. To determine whether the major colored product detected by TLC
was responsible for the golden-yellow ﬂuorescence generated in the PE liposomes
by reaction with retinal, this compound was incorporated into liposomes in the
absence of retinal. The resulting liposomes displayed the same ﬂuorescence properties as photoreceptor outer segments or PE liposomes reacted with retinal. Thus,
the compound isolated with TLC appears to be responsible for the retinal-induced
ﬂuorescence in both types of preparation.
It is important to note that no ﬂuorescent products of lipid autoxidation were
observed in either the photoreceptor outer segment or liposome preparations. When
retinal tissues or puriﬁed lipids and lipid–protein mixtures are subjected to oxidizing conditions in vitro, a number of autoﬂuorescent products are generated [25–28].
However, these ﬂuorescent products all display a blue emission when illuminated
with ultraviolet light [25 – 28]. No blue-emitting ﬂuorophores were observed in any
of the extracts analyzed in the present study. Thus lipid autoxidation is unlikely to
have played a role in the formation of the observed products of reaction between
retinal and the liposome or photoreceptor outer segment preparations.
Based on the ﬁndings from these and previous studies, the model illustrated in
Fig. 8 is proposed to explain the mechanism by which vitamin A and other factors
inﬂuence the rate of lipofuscin ﬂuorophore accumulation in the RPE [29]. According to this model, a retinal adduct to the amine of photoreceptor outer segment PE
is a precursor for the retinal–ethanolamine reaction product identiﬁed in RPE
lipofuscin [15,16]. This model is strongly supported by the ﬁnding that retinal can
react with outer segments or PE-containing liposomes to form a product that
displays lipofuscin-like ﬂuorescence in situ. The ﬂuorescent product formed by
reaction between retinal and PE in the outer segments in vitro probably also forms
in vivo at a low rate. Apparently the ethanolamine–retinal portion is cleaved from
the remainder of the molecule in the RPE where it accumulates in lipofuscin. It is
likely that under normal conditions in vivo, very little free retinal is available for
reaction with PE. However, even if only a small fraction of the retinal in the outer
segments undergoes this reaction, it may be sufﬁcient to account for the slow
accumulation of lipofuscin ﬂuorophores in the RPE over a life time. The rate of
formation of this retinal – PE reaction product would be enhanced by factors that
could increase the steady state concentration of retinal in the outer segments, such

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as oxidative stress, which would be expected to deplete NADPH and thus the
ability to reduce retinal to retinol.
No quantitative analysis of the chemical composition of RPE lipofuscin has yet
been undertaken. Thus it is not possible to estimate the fraction of this pigment
that consists of retinal derived ﬂuorophores. However, previous studies have
indicated that the total amount of lipofuscin in the RPE, as determined by
quantitative microscopic analyses, is directly related to the availability of visual
cycle retinoids [9 – 12]. Thus, it is likely that retinal-derived compounds make up a
substantial fraction of the RPE lipofuscin mass.
The involvement of retinal in the formation of at least one of the RPE lipofuscin
ﬂuorophores suggested by previous studies and supported by the present ﬁndings
does not necessarily imply that similar mechanisms are involved in the formation of
age pigments in other tissues. The involvement of retinal in lipofuscin formation
may be speciﬁc to the retina. Although elucidation of the molecular mechanisms of
RPE lipofuscin formation may not be applicable to other tissues, it is none the less
important, particularly in light of the potential role of RPE lipofuscin accumulation
in age-related retinal degeneration [30].

Fig. 8. Model describing the hypothesized mechanism by which all-trans retinal that is generated during
visual pigment bleaching participates in lipofuscin ﬂuorophore formation.

M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174

173

Acknowledgements
The authors thank the Missouri Lions Eye Research Foundation for supplying
the human donor eyes that were used in this research. Supported by USA Public
Health Service research grant EY08813, by a grant from the University of Missouri
Research Board, and by an unrestricted departmental grant from Research to
Prevent Blindness.

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