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 - 9 160 M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 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 M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 161 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. 162 M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 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 M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 163 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 164 M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 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 M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 165 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. 166 M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 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. M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 167 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). 168 M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 Figs. 4 – 7. M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 169 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. 170 M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 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 M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 171 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 172 M.L. Katz et al. / Mechanisms of Ageing and De6elopment 92 (1996) 159–174 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. References [1] E.A. Porta and W.S. 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