Mechanisms of Ageing and Development
99 (1997) 61 – 78

Preparation of artiﬁcial ceroid/lipofuscin by
UV-oxidation of subcellular organelles
Evalill Nilsson, Dazhong Yin *
Department of Pathology II, Faculty of Health Sciences, Linkoping Uni6ersity,
¨
¨
S-581 85 Linkoping, Sweden
Received 8 February 1997; received in revised form 22 May 1997; accepted 26 May 1997

Abstract
Recent studies have consistently shown that, during oxidative damage, glycation, and
other oxygen stress-related reactions, various biomolecules are converted into ceroid- and
lipofuscin-like ﬂuorescent pigments. In this study, artiﬁcial ceroid/lipofuscin was produced
by exposing rat liver fractions to UV-light overnight. Thiobarbituric acid reactive substances
(TBARS) were formed in increasing amounts during the early stages of the process, but
decreased as the material was later converted into a polymeric structure with few remaining
peroxides. In the transmission electron microscope the artiﬁcial pigment showed lamellar
structures and was osmiophilic. By energy-dispersive X-ray analysis the material was found
to contain Ca and Fe in the same way as natural ceroid/lipofuscin. Moreover, it exhibited
ceroid/lipofuscin-like, greenish-yellowish autoﬂuorescence when assayed by microﬂuorometry, with a ﬂuorescence maximum consistently found at 430 nm when excited at 350 nm.
Identical ﬂuorescence maxima were found for each fraction of rat liver that was used as the
origin of the pigments, i.e. nuclei, mitochondria, lysosomes and microsomes. Extracts with
either chloroform–methanol, or sodium dodecylsulphate, showed identical complex ﬂuorescence. When the pigments were extracted by chloroform – methanol, ﬁve ﬂuorescent bands
were obtained after thin-layer chromatographic separation. Fibroblasts were found to
endocytose the material, a process that converted them into lipofuscin-loaded cells of an
Abbre6iations: BHT, butylated hydroxytoluene; MDA, malondialdehyde; PBS, phosphate buffered
saline; SDS, sodium dodecyl sulphate; SEM, scanning electron microscopy; TBARS, thiobarbituric acid
reactive substances; TCA, trichloroacetic acid; TEM, transmission electron microscopy; TLC, thin-layer
chromatography; UAc, uranyl acetate; UV, ultraviolet.
* Corresponding author. Tel.: + 46 13 221517; fax: + 46 13 221529; e-mail: eva-lill.nilsson@pat.liu.se
0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 0 0 9 1 - 2

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aged phenotype as observed by light and electron microscopy. Similar ﬂuorescence emission
spectra were obtained from cells grown at 40% O2, in order to stimulate endogenous
lipofuscin-formation, and from cells exposed to artiﬁcial ceroid/lipofuscin. The described
technique for creating artiﬁcial ceroid/lipofuscin is relatively easy to perform and should
provide a useful new tool to study the possible inﬂuences of ceroid/lipofuscin on lysosomal
and cellular functions. © 1997 Elsevier Science Ireland Ltd.
Keywords: Aging; Lipofuscin; Lysosomes; Oxygen radicals

1. Introduction
Lipofuscin, or age pigment, is a yellowish-brown autoﬂuorescent material that
accumulates progressively with age in secondary lysosomes of post-mitotic cells,
such as neurones and cardiomyocytes. Although this age pigment has been known
since the 19th century, its origin and composition is still disputed (Baynes and
Monnier, 1989; Eldred, 1987; Eldred and Katz, 1988, 1989; Elleder, 1981; Kikugawa, 1990; Sohal, 1987; Strehler, 1964; Tappel, 1975; Yin and Brunk, 1991). The
chemical composition of lipofuscin may vary among different types of cells and
among the same cell during different conditions, such as aging, pathological
changes, and, probably, variations in the status of nutrition (Baynes and Monnier,
1989; Eldred and Katz, 1988; Labuza et al., 1994; Yin and Brunk, 1991). Of great
interest is the fact that short-lived animals accumulate lipofuscin within their
post-mitotic cells much faster than do long-lived ones (Brizzee et al., 1969; Porta
and Hartroft, 1969; Reichel, 1968; Reichel et al., 1968; Strehler et al., 1959). This
suggests that genetic factors are involved in lipofuscinogenesis, which may be
reﬂected in variations among different species with respect to their metabolic rate,
antioxidative defence systems, detoxiﬁcation enzymes, and repair processes (Cutler,
1995; Davies, 1988; Jazwinski, 1996; Sohal, 1996; Yin and Brunk, 1995).
Recently, signiﬁcant advances have been made in the understanding of lipofuscinogenesis (Brunk et al., 1992; Eldred and Lasky, 1993; Yin, 1996). Lipofuscin
may now be considered to be mainly an end-product of non-enzymatic glycation
and oxygen radical-induced lipid- and protein-oxidation. As shown in Fig. 1, when
lipids, proteins, carbohydrates, and other biomolecules are oxidatively modiﬁed,
various carbonylic compounds, such as malondialdehyde, hydroxyalkenals, and
deoxyosones, are created (Baynes and Monnier, 1989; Esterbauer et al., 1991;
Janero, 1990; Kikugawa, 1990). These carbonyls, particularly unsaturated aldehydes, may conjugate with amino groups of proteins and nucleic acids, to introduce
cross-links between these biomolecules and, thus, form complex polymerised
macromolecules. Although such cross-linking reactions are slow, and even partially
reversible, they are both cytotoxic and genotoxic (Baynes and Monnier, 1989;
Brunk et al., 1992; Esterbauer et al., 1991; Janero, 1990; Labuza et al., 1994).
The inverse relationship between the rate of lipofuscin accumulation and
longevity points to the possibility that lipofuscin might have deleterious effects on

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63

important lysosomal functions (Brunk et al., 1992; Glees and Hasan, 1976; Siakotos
and Armstrong, 1975). Some studies have suggested that the basic amines of Schiff
bases, that are present in lipofuscin, may interfere with lysosomal stability (deDuve
et al., 1974; Eldred, 1995; Matsumoto et al., 1989; Seglen, 1983). However, most
workers consider lipofuscin to be a harmless by-product (Brunk and Ericsson, 1972;
Glees and Hasan, 1976; Rubin and Farber, 1994; Siakotos and Armstrong, 1975).
This contemporary opinion is reﬂected in the name often given to lipofuscin-loaded
secondary lysosomes, i.e. residual bodies. Nevertheless, the idea that lipofuscin
accumulation may be a dangerous burden to cells would be worth testing. To do so
would require an experimental model system consisting of cells of the same type
and age, differing only with respect to their degree of lipofuscin accumulation.
Feeding artiﬁcial lipofuscin to cells in culture, which would endocytose the material
and transport it to their secondary lysosomes, should constitute such a model
system.
In this study we present an easy way to produce artiﬁcial ceroid/lipofuscin.
Moreover, we show that the artiﬁcial ceroid/lipofuscin is taken up by ﬁbroblasts,

Fig. 1. Formation mechanisms of ceroid/lipofuscin (age pigment).

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and transforms them into an aged phenotype. Such artiﬁcially aged cells are
presently being used to test functions related to their acidic vacuolar compartment.

2. Materials and methods

2.1. UV-irradiation of rat li6er fractions
Male Sprague Dawley rats (about 200 g) were starved for 24 h before they were
killed with carbon dioxide. Liver slices were homogenised 1:10 in 0.3 M sucrose on
ice and centrifuged in several steps at 300, 1000, 3000, 17 000 and 100 000× g for
10, 10, 10, 20 and 45 min, respectively, to obtain fractions containing mainly nuclei
(fraction 1), heavy mitochondria (fraction 2), light mitochondria and lysosomes
(fraction 3), and microsomes (fraction 4). The ﬁrst pellet, at 300×g, containing
debris and unbroken cells, was discarded. The other pellets were resuspended in
phosphate buffered saline (PBS), without Ca2 + and Mg2 + , and again centrifuged,
to wash away the sucrose before a ﬁnal resuspension in PBS. The whole process
was performed at 0 – 4°C. The protein content of the suspensions was measured
using the Bicinchoninic acid protein assay kit (Sigma, St Louis, MO) and adjusted
to 3 mg/ml. The different suspensions were then put into Petri dishes (10 ml/100
mm dish) without lids, and placed under UV-light in a Laminar Air Flow Bench for
up to 15 h to allow peroxidation to take place. Finally, the resulting dried,
ceroid/lipofuscin-like material was resuspended in sterile water to the original
volume. Further homogenisation was carried out by sonication.

2.2. TBARS measurement
The content of hydroperoxides within the material under peroxidation was
assayed by the formation of thiobarbituric acid reactive substances (TBARS)
during heating according to Buege and Aust (1977). In brief, 15% w/v
trichloroacetic acid (TCA) and 0.375% w/v thiobarbituric acid (TBA) were dissolved in 0.25 M HCl. Butylated hydroxytoluene (BHT) in ethanol was added to
the TBA reagent at a ﬁnal BHT concentration of 0.01%. The TBA reagent (2 ml)
was mixed with the irradiation products (1 ml) and heated to 96°C for 15 min in a
water bath. After cooling on ice, the solution was centrifuged at 1000× g for 10
min. The absorbance of the UV-irradiated supernatant material was measured with
a Varian DMS-100 spectrophotometer at 532 nm after 0, 3, 6, 9, 12 and 15 h of
UV-irradiation. The concentration of TBARS was calculated using an extinction
coefﬁcient of 156/mM/cm.

2.3. Transmission and scanning electron microscopy
The rat liver fractions, both non-irradiated and UV-irradiated for 15 h, were
ﬁxed in 2% glutaraldehyde (0.1 M Na–cacodylate, 0.1 M sucrose, pH 7.2),
postﬁxed in 1% OsO4 (0.15 M Na–cacodylate) and embedded in a 2% agar gel (to

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65

facilitate further processing). Small pieces of the gel were stained en bloc with
uranyl acetate (UAc), dehydrated, and embedded in Epon-812. Finally, thin
sections were cut with a diamond knife, stained with lead citrate, and examined in
a Jeol 2000-EX transmission electron microscope (Jeol, Tokyo, Japan), operated at
100 kV.
Fibroblasts were prepared for TEM as described before (Abok et al., 1988) while,
for SEM, they were subcultivated onto 12 mm cover slips and ﬁxed as above, when
they had settled and spread. After dehydration, in an ethanol series, critical point
drying from liquid CO2 was carried out using a Polaron E 3000 critical point
apparatus. The specimens were, ﬁnally, mounted on SEM-stubs and metal coated
with 10 nm platinum in an Edwards vacuum evaporator equipped with a magnetron sputter coater (Microvac, NSW, Australia) and examined in a Jeol JSM-840
scanning electron microscope, operated at 20 kV.

2.4. Energy-dispersi6e X-ray microanalysis
The artiﬁcial ceroid/lipofuscin (from liver-fraction 3, suspended in PBS) was
washed in distilled water to remove the buffer salts, air-dried, and then collected on
carbon-conductive tabs, attached to aluminium stubs for SEM. Energy dispersive
X-ray microanalysis was performed (20 kV, 300 s) in a JSM-840 scanning electron
microscope, connected to a Link AN 10 000 analysis system.

2.5. Spectroﬂuorometry
The ﬂuorescence spectra of the artiﬁcial ceroid/lipofuscin samples were assayed
using a Shimadzu RF-540 spectroﬂuorometer. The slit used for both excitation and
emission was 10 nm, and the ﬂuorescence emission was scanned from 250–600 nm.
Because ﬂuorophores at high concentrations often cause a quenching effect,
which disturbs the spectral observation, the ﬂuorescent material was diluted (1:100)
before the measurements. For some experiments, extraction in chloroform–
methanol (2:1) was performed.
Because the ﬂuorescence obtained in this model is in the blue-light region,
spectral correction does not enhance the spectral data and, therefore, was not
applied in any of the experiments.

2.6. Microﬂuorometry
Artiﬁcial ceroid/lipofuscin was smeared on slides for microﬂuorometric examination. Relatively condensed aggregates were collected in order to obtain enough
ﬂuorescent materials. The samples were covered with cover slips and inspected,
either immediately, or when the material had dried. Blue light excitation in
combination with a 550 nm barrier ﬁlter was used to view the artiﬁcial lipofuscin,
which shows greenish-yellow ﬂuorescence.

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2.7. Thin-layer chromatography (TLC)
TLC plates (Merck pre-coated silica gel 60) were used for separation of the
different ﬂuorophores produced. The artiﬁcial ceroid/lipofuscin, dissolved either in
chloroform – methanol (2:1) or in 1% sodium dodecyl sulphate (SDS) was separated
on TLC plates using a mobile phase consisting of 25% toluene, 30% chloroform,
15% methanol, 25% propanol-2 and 5% acetic acid. All chemicals were from Merck
(Darmstadt, Germany). Known Schiff-bases (reaction products of MDA–glycine)
(Yin, 1994) and an MDA-polymer (Gutteridge et al., 1977) were used as references.

2.8. Cell culture
Human foreskin ﬁbroblasts (AG-1518, passage 20–22) from Coriell Institute
(Camden, NJ), were grown in 35 mm plastic Petri dishes in Eagles Minimal
Essential Medium (Flow, Rickmansworth, UK) supplemented with 10% foetal calf
serum (GIBCO), 2 mM glutamine, 50 UI/ml penicillin, and 50 vg/ml streptomycin.
Some cultures were daily exposed to 50 vl artiﬁcial ceroid/lipofuscin (from fraction
3, see above) in fresh medium under standard culture conditions for two weeks.
Liver-fraction 3 was chosen because it predominately contains mitochondria and
lysosomes, which are believed to be dominating in normal, autophagocytosis-related, formation of lipofuscin. Other cultures were kept at 40% O2, in order to
stimulate endogenous lipofuscin-formation (Brunk et al., 1992), for up to four
months. Medium was changed three times a week.

2.9. Confocal microscopy
Fibroblasts fed artiﬁcial ceroid/lipofuscin were subcultivated onto 22 mm cover
slips (pre-washed in Decon) and, when the cells had settled and spread, ﬁxed for 15
min in 4% formaldehyde in PBS. The cover slips were then inverted onto micro-culture slides before assessment by a Zeiss LSM 410 Inverted Confocal Laser Scanning
Microscope (Carl Zeiss AG, Jena, Germany) equipped with a 488 nm Argon-laser.

2.10. Recording of ﬂuorescence emission spectra
Uncorrected ﬂuorescence emission spectra (520–700 nm) from individual cells
were recorded with a Leitz MPV 2 microscope equipped with a spectral Monochromator analysing unit (Leitz, Weizlar, Germany) (Gao et al., 1994). Cells were
prepared as described above for confocal microscopy.

3. Results
All the different fractions of rat liver were readily peroxidised during UV-irradiation. A substantial increase in TBARS was observed after 3 h of exposure to
UV-light, followed by a decrease before levelling off at a somewhat higher level

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67

Fig. 2. Yield of TBARS during UV-irradiation of the different rat liver fractions (for explanation of
‘fractions’ see Section 2). The values are from one typical experiment out of three.

than the initial one (Fig. 2). In some experiments irradiation was continued for as
long as two days, but no further change in TBARS was observed after the initial 15
h of irradiation.
All irradiated fractions showed emission maxima at about 430 nm when excited
at 350 nm (results not shown), which is typical of ceroid/lipofuscin (Yin, 1996). Fig.
3 shows the development of this ﬂuorescence peak during irradiation of fraction 3.
Native protein ﬂuorescence, at 280/335 nm, was detected both before and after
UV-irradiation. Excitation too close to 280 nm could, therefore, result in disturbances of the typical ceroid/lipofuscin ﬂuorescence peak and should be avoided.
Turbidity, introduced by small aggregates of the sample, often results in diffraction
causing both Raman and Rayleigh scattering. Such scattering-induced disturbances
have been discussed in other recent publications from our group (Yin and Brunk,
1997) and were consequently distinguished in this study. The shoulder seen in the
emission spectrum, close to 400 nm, is due to Raman scattering.

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Another shoulder, at about 515 nm, is mainly due to ﬂavonoids. This is most
prominent in the non-irradiated material.
When examined by TEM (Fig. 4) the fractions, after oxidation by UV-irradiation, were transformed into a homogeneous, osmiophilic material, resembling
age-pigment as it is usually seen in lysosomes of post-mitotic cells such as neurones
and cardiac myocytes. Following uptake by ﬁbroblasts the material within the
lysosomes became further condensed and even more lipofuscin-like (see below).
Energy dispersive X-ray analysis of the artiﬁcial ceroid/lipofuscin gave several
obvious peaks including those for calcium, potassium, chlorine and iron (Fig. 5).

Fig. 3. Increase in ﬂuorescence of fraction 3 (light mitochondria and lysosomes) during UV-irradiation.
Irradiation was carried out at about 23°C for up to 15 h. The material was then resuspended and diluted
for measurements. Fluorescence intensity is given in arbitrary units (a.u.). Arrow-head: typical lipofuscin
emission maximum (at 430 nm when excited at 350 nm). Arrow: Raman scattering (at approximately 400
nm). Double arrow: native protein excitation peak (at 280 nm).

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69

Fig. 4. Transmission electron micrograph of rat liver fraction 3 (light mitochondria and lysosomes)
before (A) and after (B) UV-irradiation for 15 h. The material was ﬁxed in glutaraldehyde, post-ﬁxed in
OsO4, embedded in agar gel, stained en bloc with UAc, dehydrated, embedded in Epon-812, cut and
counterstained with lead citrate. Bar = 1 vm.

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Fig. 5. X-ray analysis spectrum of artiﬁcial ceroid/lipofuscin, obtained from liver-fraction 3 (light
mitochondria and lysosomes), showing elemental peaks. Note the peaks for iron, calcium, potassium and
chlorine.

Identical ﬂuorescence spectra characteristics (350/430 nm) were also observed
when the irradiated material from the different fractions was extracted with either
chloroform – methanol (Fig. 6) or 1% SDS (results not shown). A compact residue
of yellow ﬂuorescent material still remained after either chloroform–methanol or
SDS extraction, which indicates that some ﬂuorophores are not extractable, because
they occur as highly complex polymers. Fluorescence was also seen in the aqueous
phase following chloroform – methanol extraction (results not shown).
The extracted ﬂuorophores were separated using TLC on pre-coated silica gel
plates. A comparison of that material with known Schiff-bases, and a MDA-polymer, is shown in Fig. 7.
When human ﬁbroblasts in culture were fed artiﬁcial ceroid/lipofuscin, the cells
were converted into an ‘old age’ phenotype. A heavy lysosomal-type of accumulation became obvious after some days, showing yellow auto-ﬂuorescence when
excited with blue light (Fig. 8). Fluorescence emission spectra of identical type,
having peaks around 550 nm, were found in cells fed artiﬁcial ceroid/lipofuscin as
well as in cells that had accumulated endogenous lipofuscin at 40% O2 (Fig. 9). The
ultrastructural ﬁndings in both TEM and SEM modes of lipofuscin-loaded cells are
given in Figs. 10 and 11.

4. Discussion
In the present study, UV-irradiation was selected as a simple and quick photooxidative technique to create highly polymerised, artiﬁcial, ceroid/lipofuscin, ready
to use in a cell culture model system. UV-irradiation has been widely used in studies
involving oxidative stress-related damage, such as eye- and skin damages, LDL-ox-

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Fig. 6. Fluorescence excitation/emission spectra of chloroform – methanol-extracted artiﬁcial ceroid/lipofuscin. All fractions were UV-irradiated for 15 h at about 23°C and then resuspended before extraction.
The extracts were diluted before measurements. Fluorescence intensity is given in arbitrary units (a.u.).

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idation, and carcinogenesis. Oxygen-derived radicals, singlet oxygen and hydrogen
peroxide are the main damaging species formed during such irradiation (Costanzo
et al., 1995; Girotti, 1990). Free radical-induced lipid peroxidation, as well as
glycoxidation, are also considered to be important mechanisms behind, e.g. atherogenesis, age-related macular degeneration, and aging in general (Wihlmark et al.,
1996).
In an early study, Chio et al. (1969), obtained lipofuscin-like ﬂuorescent substances by shaking rat liver fractions in oxygen. Their technique, however, is
difﬁcult to repeat because of problems in keeping the oxygen tension stable during
sampling, the ﬂuctuating pH of the suspension, and difﬁculties in obtaining sterile
materials for further use in cell culture experiments.

Fig. 7. TLC separation of chloroform–methanol (chl – met) extracted artiﬁcial ceroid/lipofuscin compared with known Schiff-bases. From left to right: (A), irradiated (15 h) fraction 2 (heavy mitochondria),
extracted in chl–met; (B), non-irradiated fraction 3 (light mitochondria and lysosomes); (C), irradiated
(15h) fraction 3 (light mitochondria and lysosomes), extracted in chl – met; (D), irradiated (15 h) fraction
3 (light mitochondria and lysosomes), extracted in SDS; (E), MDA – glycine, 200 mM (initial concentrations), in phosphate buffer, pH 7.0, 37°C; (F), MDA polymer, 2 M (initial concentration). Several
ﬂuorescent bands are seen, especially in column C, E, and F. In column C, excitation/emission maxima
of the bands (indicated in the ﬁgure) are at 350/445 nm (band 1), 450/510 nm (band 2), 345/425 nm
(band 3), 355/435 nm (band 4), and at 350/425 nm (band 5, remaining at the starting point). In column
E and F the components of the ﬂuorophores are not characterised.

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Fig. 8. Confocal laser scanning micrograph showing ﬁbroblasts fed sonicated, artiﬁcial ceroid/lipofuscin
(obtained from rat liver fraction 3) daily for 2 weeks. Note the heavy, lysosomal-type accumulation of
the strongly autoﬂuorescent material. Bar =25 vm.

UV-irradiation induced rapid alterations in all of the rat liver fractions. The yield
of oxidation-induced reactive carbonyls, mainly unsaturated aldehydes, such as
MDA and 4-hydroxynonenal, was measured as TBARS. During the early stages of
the UV-light exposure, TBARS formed in increasing amounts. Later the amount of
TBARS decreased, leaving a polymeric structure, with few remaining peroxides
(Fig. 2). The rise and fall in the amount of TBARS reﬂects the peroxidation,
fragmentation and eventual polymerisation of the material.
As shown by spectral ﬂuorometry (Figs. 3 and 6), the same type of ﬂuorescent
artiﬁcial ceroid/lipofuscin can be produced from different fractions of rat liver, and
give almost identical ﬂuorescent maxima. This similarity may indicate that speciﬁc
protein- or lipid-related ﬂuorescent pigments are predominant in the material, or it
could be due to a combination of various ﬂuorophores. However, the exact
interpretation of the similarity is still unclear to us. Since the process was already
completed after overnight treatment at about 23°C, the artiﬁcial ceroid/lipofuscin
created by UV-irradiation would mainly originate from an oxidative stress-type of
damage, rather than from Maillard-type reactions, which usually evolve very
slowly.
Lipofuscin has been shown to contain small amounts of metals, mostly calcium,
iron, zinc, and copper (Jolly et al., 1995). The presence of iron shown by X-ray
analysis (Fig. 5), within the artiﬁcial ceroid/lipofuscin, thus, adds to the growing
body of evidence that links this substance to natural lipofuscin.

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Protein autoﬂuorescence, at 280/335 nm in the tested cellular fractions, was taken
into consideration when evaluating the ﬂuorescence spectra of artiﬁcial lipofuscin.
When Figs. 3 and 6 are compared, the 280 nm peak in the excitation spectra of Fig.
3 is hardly observable in Fig. 6. This clearly demonstrates the difference between
extracted (Fig. 6) and non-extracted (Fig. 3) pigment.
Chloroform – methanol extracts are commonly used to assay lipofuscin-like
ﬂuorescence. We ﬁnd that the direct measurement of ceroid/lipofuscin in suspension
provides signiﬁcant improvement in the ﬂuorescence characterisation of such
ﬂuorophores. This approach is especially advantageous when comparing spectra
obtained by spectroﬂuorometry or microspectroﬂuorometry.
Although early studies have shown substantial effects of pH on various age
pigment-like ﬂuorophores, pH-adjustment does not signiﬁcantly affect the ﬂuorescence intensity of the chloroform–methanol extracted ﬂuorophores (results not
shown). This is consistent with the involvement of very complex ﬂuorescent
pigments, as was conﬁrmed by TLC (Fig. 7). Various ﬂuorescent bands were
formed, although ﬂuorophore separation was not perfect. Several blue bands, but
only one yellow band (band 2), were obtained, the latter maybe representing
derivatives of retinoids or ﬂavonoids. Signiﬁcant differences were seen when the
TLC-bands of artiﬁcial ceroid/lipofuscin were compared with those of long-stored
poly-MDA, and the ﬂuorescent product of the MDA–glycine reaction, emphasising

Fig. 9. Uncorrected ﬂuorescence emission spectra of ceroid/lipofuscin from individual ﬁbroblasts. Note
similar spectra from cells fed artiﬁcial ceroid/lipofuscin (obtained from rat liver fraction 3) daily for 2
weeks, and cells grown at 40% O2 (in order to stimulate endogenous lipofuscin-formation) for 4 months.

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Fig. 10. TEM micrographs of (A) control ﬁbroblasts, (B) cells fed sonicated artiﬁcial ceroid/lipofuscin
(obtained from rat liver fraction 3) daily for 2 weeks and (C) cells grown at 40% O2 (to stimulate
endogenous lipofuscin-formation) for four months. Cells were ﬁxed in glutaraldehyde, post-ﬁxed in
OsO4, stained en bloc with UAc, dehydrated, embedded in Epon-812, cut, and counterstained with lead
citrate. Note heavy accumulation of ceroid/lipofuscin in B and C. Part of the artiﬁcial ceroid/lipofuscin
is still not very condensed (B) (arrows) and is believed to represent recently endocytosed artiﬁcial
pigment (compare with Fig. 4B). Bar = 1 vm

the complexity of lipofuscin. The complicated products that result from even such
simple reactions as those between MDA and glycine need to be studied further.
In the ﬂuorescence microscope, the artiﬁcial ceroid/lipofuscin-suspension showed
intensively ﬂuorescent aggregates of many different sizes. Intensive sonication
fragmented the aggregates into smaller pieces which were endocytosed by ﬁbroblasts and transferred into their lysosomes. Consequently, lysosomes were rapidly
transformed into ‘residual bodies’ containing condensed lipofuscin while the cells
took on the morphology of lipofuscin-loaded, aged post-mitotic cells (Figs. 8, 10

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and 11). The similar pigment ﬂuorescence emission spectra of these cells (containing
artiﬁcial ceroid/lipofuscin) and that of cells grown for several months at 40% O2 (to
accumulate large amounts of endogenous lipofuscin), further conﬁrms the similarity
between artiﬁcial and natural ceroid/lipofuscin (Fig. 9). Such cells are now being
used to study the possible inﬂuence of ceroid/lipofuscin on the lysosomal functions
of aged cells.

Fig. 11. SEM micrographs (A–C) of the same type of cells as shown in Fig. 10. Cells were ﬁxed in
glutaraldehyde, post-ﬁxed in OsO4, dehydrated, critical point dried, and ﬁnally coated with platinum.
Note perinuclear aggregates (arrows) in B and C, believed to represent lipofuscin-ﬁlled lysosomes within
cells which have partly collapsed during the critical-point drying before metal-coating. Bar =10 vm.

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Acknowledgements
We wish to thank Drs Ulf Brunk and Sven Hammarstrom for their assistance
¨
and advice during the study and Sailesh Surapureddi, Karin Roberg, Bengt-Arne
Fredriksson and Uno Johansson for their excellent technical assistance. This
research was supported by the Swedish Medical Research Council, grant no. 4481.

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