Generation of Fluorescent Adducts of Malondialdehyde and Amino Acids: Toward an Understanding of Lipofuscin

Article abstract of DOI:10.1562/0031-8655(2004)79<21:GOFAOM>2.0.CO;2

Lipofuscin is a yellow-brown, highly fluorescent pigment that undergoes an age-related progressive accumulation in animal cells, mainly in postmitotic cells. It is a heterogeneous, high-molecular weight material associated with proteins, lipids and nucleic acids. Lipofuscin is implicated in many aspects of human health, including aging, oxidative stress, macular degeneration, lipid peroxidation, atherosclerosis, dementia (Alzheimer's Disease) and diseases associated with prions. Although the fluorescent properties of lipofuscin have long been recognized, neither histologists nor chemists have yet isolated the pigments themselves or characterized their optical properties. We have prepared lipofuscinlike species by reacting malondialdehyde (MDA) with cysteine (Cys). MDA: Cys adducts 3:2 and 2:2 are two of those that have been identified among the many that were present in the reaction. Whereas previous attempts to synthesize lipofuscinlike species resulted in compounds that were either nonfluorescent or emitted principally in the blue, the MDA:Cys adducts reported in this study are not only fluorescent but also emit over a broader range.

Full text of DOI:10.1562/0031-8655(2004)79<21:GOFAOM>2.0.CO;2

Generation of Fluorescent Adducts of Malondialdehyde and Amino Acids: Toward
an Understanding of Lipofuscin
Author(s): Pramit K. Chowdhury, Mintu Halder, Prabir K. Choudhury, George A. Kraus, Meera J. Desai,
Daniel W. Armstrong, Thomas A. Casey, Mark A. Rasmussen, and Jacob W. Petrich
Source: Photochemistry and Photobiology, 79(1):21-25.
Published By: American Society for Photobiology
DOI: http://dx.doi.org/10.1562/0031-8655(2004)79<21:GOFAOM>2.0.CO;2
URL: http://www.bioone.org/doi/full/10.1562/0031-8655%282004%2979%3C21%3AGOFAOM
%3E2.0.CO%3B2
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Photochemistry and Photobiology, 2004, 79(1): 21–25
Generation of Fluorescent Adducts of Malondialdehyde
and Amino Acids: Toward an Understanding of Lipofuscin{
Pramit K. Chowdhury1, Mintu Halder1, Prabir K. Choudhury1, George A. Kraus1, Meera J. Desai1,
Daniel W. Armstrong1, Thomas A. Casey2, Mark A. Rasmussen2 and Jacob W. Petrich*1
1Department of Chemistry, Iowa State University, Ames, IA and
2National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA
Received 4 July 2003; accepted 20 October 2003
Many biomolecules, particularly polyunsaturated fatty acids, are
ABSTRACT
highly susceptible to oxygen radicals. Free radical–induced lipid
Lipofuscin is a yellow-brown, highly fluorescent pigment that
undergoes an age-related progressive accumulation in animal
cells, mainly in postmitotic cells. It is a heterogeneous, high–
molecular weight material associated with proteins, lipids
and nucleic acids. Lipofuscin is implicated in many aspects
of human health, including aging, oxidative stress, macular
degeneration, lipid peroxidation, atherosclerosis, dementia
(Alzheimer’s Disease) and diseases associated with prions.
Although the fluorescent properties of lipofuscin have long
been recognized, neither histologists nor chemists have yet
isolated the pigments themselves or characterized their optical
properties. We have prepared lipofuscinlike species by
reacting malondialdehyde (MDA) with cysteine (Cys). MDA:
Cys adducts 3:2 and 2:2 are two of those that have been
identified among the many that were present in the reaction.
Whereas previous attempts to synthesize lipofuscinlike species
resulted in compounds that were either nonfluorescent or
emitted principally in the blue, the MDA:Cys adducts
reported in this study are not only fluorescent but also emit
over a broader range.
peroxidation processes have been suggested as an origin for the
formation of lipofuscin; i.e. after the initiation of lipid peroxidation
by reactive oxygen species, lipid hydroxyperoxides and cyclic
peroxides are formed, which degrade into various carbonyl
compounds, mainly aldehydes. Unsaturated aldehydes are often
cytotoxic, and many of them are precursors of fluorescent products.
The most extensively studied are malondialdehyde (MDA) and 4-
hydroxynonenal (HNE) (2,3) (Table 1). Terman and Brunk
proposed that the accumulation of nondegradable oxidized material
(mainly from the autophagocytosis of mitochondria) in lysosomes
may be essential for the generation of lipofuscin granules. Their
proposal suggests that lipofuscin granules are residual bodies
containing the products of previous autodigestive processes. The
accumulation of these granules is most likely to be favored in long-
lived postmitotic cells, such as neurons (1,4–10).
It is known that in humans with Creutzfeldt–Jakob’s disease
(CJD) and in experimentally induced instances of CJD, the amount
of neuronal lipofuscin is increased. Boellaard et al. (4) have
demonstrated a relationship among lipofuscin production, the
presence of autophagocytosis and the experimental induction of
CJD in mice (4). It has been argued that the accumulation of
incompletely digested material, e.g. lipofuscin, interferes with
cellular functions and increases the probability of cell death.
Because cell division results in a dilution of nondegraded material,
age-related cell damage and death is most likely to occur when
cellular development is complete. Consequently, postmitotic cells,
not proliferating cells, are most susceptible. This phenomenon has
recently been referred to as the ‘‘garbage catastrophe theory of
aging’’ (5,8).
INTRODUCTION
As early as 1842 it had been recognized that aging process in
humans and animals was accompanied by the progressive accumu-
lation of yellow-brown intracellular pigments (1). These age-related
fluorescent pigments are classically known as ‘‘lipofuscin.’’
Lipofuscin is a granular substance accumulating mainly in post-
mitotic cells, such as neurons, cardiac muscle and retinal epithelium.
Lipofuscin is believed to be the product of ‘‘oxidative stress.’’
In the past few years, attempts have been made to produce and to
identify lipofuscinlike molecules in vitro. As discussed in great
detail in the review by Yin (1), synthetic attempts to produce
central nervous system (CNS) lipofuscin have resulted in blue-
green emitters, and fluorometry of extracts has failed to detect
yellow emission. Table 1 presents a summary of the emission
maxima of lipofuscin pigments prepared in the laboratory—
excluding those of the retina. It is very well established that a major
component of lipofuscin in the eye is the ‘‘orange’’-emitting fluoro-
phore, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,
3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-
trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium
(A2E) (11–14). Considerable effort has been devoted to
{Posted on the website on 26 October 2003.
*To whom correspondence should be addressed at: Department of
Chemistry, Iowa State University, Ames, IA 50011, USA. Fax: 515-
294-0105; e-mail: jwp@iastate.edu
Abbreviations: A2E, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-
yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-
trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium; arbs,
arbitrary units; CJD, Creutzfeldt–Jakob’s disease; CNS, central nervous
system; Cys, cysteine; HNE, 4-hydroxynonenal; HPLC, high-perfor-
mance liquid chromatography; LC-MS, liquid chromatographic–mass
spectrometric analysis; MDA, malondialdehyde; NMR, nuclear magnetic
resonance.
Ó 2004 American Society for Photobiology 0031-8655/04
$5.00þ0.00
21

22 Pramit K. Chowdhury et al.
Table 1. Some previous attempts to prepare lipofuscinlike species
em
max
Oxidant
Reactant
Ribonuclease
Human low-density lipoprotein
Human low-density lipoprotein
Na-Hyppuryllysine
Lysine
Boc-Lys
Gly, Val, Leu
NAc-lysine
NAc-Gly-Lys-CO2Me
Hexanal–hexylamine
k
(nm)
Ref.
HNE
HNE
HNE
HNE
HNE
MDA
MDA
MDA
MDA
H2O2
435
440
445
430*
430
(28)
(29)
(30)
(31)
(32)
(27)
(26)
(33)
(3)
Nonfluorescent
450
455
450*à
430
(34)
*Cited detection wavelength for HPLC analysis.
The product is (Boc-Lys)2MDA.
àThe product is 1-[5-carboxymethyl-5-(N-acetylglycylamino)pentyl]-3-[1-
(5-carboxymethyl-5-(N-acetylglycylamino)pentyl)-1,4-dihydropyridin-
4-yl]pyridinium.
Figure 2. Front-faced fluorescence spectra from brain and spinal cord.
Twelve samples of each type were analyzed. All samples were solid. The
shape of the spectra in each case is highly conserved. kex 5 480 nm. A
cutoff filter at 505 nm was placed before the detector to eliminate scattered
light.
understanding the origin of lipofuscin generation in the eye, given
its association with age-related macular degeneration (13,15–18).
On the other hand, the identification of orange-emitting species
from other tissues is incomplete. Retinoyl complexes have been
found in diseased cerebral cortex (19), and retinoid-binding
proteins have been isolated in adult CNS (20).
electron microscopy of fixed cells or tissues, qualitative fluorom-
etry based on observing emission as a function of cutoff filters and
size-exclusion chromatography (with traditional Sephadex-type
media) of tissue extracts (1). Although these approaches have been
beneficial, lipofuscin is still not well understood in terms of its
composition, properties, physicochemical behavior and origin.
Clearly, more exacting and definitive experimental approaches and
instrumental methodologies would be useful for our understanding
of this enigmatic but important material. (We note that more
quantitative studies have been conducted on the lipofuscin
pigments found in the eye [13,15–18,21–23].) Ironically, analytical
chemists seem to have regarded the CNS pigments as a nuisance
rather than a researchable resource (24). This article presents an
effort to isolate and characterize lipofuscin as well as to synthesize
lipofuscinlike molecules as an approach to understand the
formation of lipofuscin.
The methods most often used to characterize and to quantify
lipofuscin substances in CNS tissue have been transmission
MATERIALS AND METHODS
Synthesis of MDA adducts. Preparation of cysteine (Cys) adducts of MDA
was according to the detailed experimental conditions of Buttkus (25) at
258C. The Cys solution was prepared by dissolving 5 g (0.028 moles) of
Cys-HCl in 4 mL of water and adding 13.5 mL of 2 N KOH under an argon
atmosphere. The pH was then adjusted to 6.5. MDA was prepared by
Figure 1. (A) Excitation spectra of spinal cord tissue extract in
chloroform–methanol (2:1) mixture with emission monochromator at (a)
420 nm, (b) 480 nm and (c) 580 nm. Spectrum (a) has been scaled up by
a factor of 20. (B) Emission spectra of the spinal cord tissue sample in
chloroform–methanol (2:1) mixture: (a) kex 5 330 nm, (b) kex 5 420 nm
and (c) kex 5 500 nm.
Figure 3. (i) Absorption spectrum of Cys-MDA adduct in water. (ii)
Fluorescence spectra of Cys-MDA adduct in water: kex 5 320 nm (solid
line) and kex 5 420 nm (dashed line).

Photochemistry and Photobiology, 2004, 79(1) 23
Figure 4. LC-MS analysis of MDA:Cys adducts (panel A). The HPLC mobile phase was 67:33 (water–ethanol), flow rate 5 0.15 mL minꢀ1, column 5 150
34.6 mm ID C-18 from Astec, Inc. (Whippany, NJ). The HPLC peak at 26.9 min may contain a protonated 2:2 (MDA:Cys) adduct at m/z 333 (upper portion
of panel B). The HPLC peaks at 33.1, 43.5, 58.5 and 73.3 min are thought to be higher molecular weight adducts. For example, the second spectrum shown
corresponds to the peak at 58.5 min and has a very strong MS response at 694.5 m/z (lower portion of panel B), corresponding to an unidentified higher
molecular weight adduct from the Cys:MDA reaction. Such adducts most likely result from reaction products evolving over time, as indicated in the text.
hydrolyzing 8 mL (0.033 moles) of 1,1,3,3-tetraethoxypropane with 3 mL
of 1 N HCl at 458C until miscible and clear (20 min). After the addition of 2
mL of water, the solution was held for an additional 20 min at 458C. The
solution was cooled to room temperature and pH was adjusted to 6.5 with
10 mL of 2 N KOH. When the MDA solution was added to the Cys
solution, the pH of the resulting reaction mixture dropped to 5.0. After 15
min, the pH of the solution was raised to 7.0 with 2 N KOH, and the
solution was kept at pH 7.0 for another 15 min. The reaction mixture was
then acidified slowly with 1 N HCl to pH 3.0. The product precipitated as
a yellow colored material. The solid was washed with cold distilled water
and then with acetone. It is important to note that the 3:2 and 2:2 adducts
were not separable and appear to be mixtures of diastereomers. For the
combined adducts—IR: 2360, 1734, 1594, 1419 and 1236 cmꢀ1; nuclear
magnetic resonance (NMR): 2.15–2.80 (multiplet), 3.25–3.7 (multiplet),
4.4–5.3 (multiplet) and 7.8–8.1 (multiplet). The structures of certain
adducts were determined by liquid chromatographic–mass spectrometric
analysis (LC-MS) (below).
Preparation of extract. Bovine brain and spinal cord tissue (provided
by the National Animal Disease Center, United Stated Department of
Agriculture laboratory) was suspended in a 2:1 chloroform–methanol
mixture and was sonicated for 20 min with the sample being immersed in an
ice bath throughout to prevent excessive local heating. The resulting
suspension was centrifuged at 20 000 g for 10 min. This was followed by
careful decanting of the clear supernatant liquid, which was then used for
the steady-state measurements.
Auxiliary and sheath gases were 0 and 25 arbs, respectively. All other
parameters remained the same as above.
RESULTS AND DISCUSSION
Fluorescence properties of naturally occurring lipofuscin
Figure 1 presents fluorescence excitation and emission spectra of
lipofuscin extracted from bovine spinal cord. As expected, variation
in the excitation spectra with emission wavelength and the variation
in emission spectra with excitation wavelength indicate that the
extract in Fig. 1 is very heterogeneous. A comparison with the
spectra of specific MDA adducts (26) (Table 1) indicates that
lipofuscin isolated from bovine tissues emits at very long wave-
lengths, much longer than had been suggested in the literature (1).
There are significant differences among the solution phase
emission spectrum shown in Fig. 1B and the spectra of the tissue
samples presented in Fig. 2. This is not surprising. The broad
distribution of pigments may be inhomogeneous in the tissue. It is
also likely that in the tissue there are significant self-quenching
effects from closely packed pigments.
Although a rather clear picture of at least some of the lipofuscin
pigments in the eye is emerging and quantitative studies are
currently in progress (13,15–18,21–23), we believe that Figs. 1 and
2 (below) present the first optical spectra (emission intensity vs
wavelength) recorded for CNS tissue. This is remarkable con-
sidering the importance of these pigments as an indicator of aging
and disease.
Steady-state measurements. Steady-state emission and excitation spectra
were obtained on a SPEX Fluoromax (Jobin-Yvon/Spex, Edison, NJ) with
a 4 nm band-pass filter and corrected for detector response. A 1 cm path-
length quartz cuvette was used for the measurements.
LC-MS analysis. LC-MS data were acquired using a Thermo Finnigan
(San Jose, CA) Surveyor liquid chromatography system coupled to
a Thermo Finnigan LCQ Advantage ion trap mass spectrometer with
electrospray as the ion source. Spectra were collected in positive-ion mode
with nitrogen used as both the auxiliary and sheath gas. The MS parameters
were as follows: auxiliary gas, 10 arbitrary units (arbs); sheath gas, 35 arbs;
source voltage, 4.50 kV; capillary voltage, 34.0 V; capillary temp, 2008C;
tube lens, 30.0 V; microscans, 3 and injection time, 50.0 ms. Full-scan mass
spectra were acquired over the mass range 150–800 m/z. MS infusion of
samples was performed in positive-ion mode (full scan 150–2000).
Synthesis of lipofuscinlike pigments
A number of structures have been advanced for the chromophore
responsible for lipofuscin fluorescence. Three of the most
commonly proposed and studied structures are listed below. The
first two might arise from the combination of MDA, a reactive
dialdehyde produced from the oxidation of lipids, with amines
from commonly available amino acids such as lysine. The third

24 Pramit K. Chowdhury et al.
fraction of the total material. The adducts shown above could be
transformed into a pyridinium salt by cleavage of the C–S bond
and double-bond migration.
Figures 4 and 5 show the LC-MS analysis of the mixture of the
MDA:Cys adducts. The high-performance liquid chromatography
(HPLC) peak at 26.9 min may contain a protonated 2:2
(MDA:Cys) adduct at m/z 333. We believe that the HPLC peaks
at 33.1, 43.5, 58.5 and 73.3 min are higher molecular weight
adducts. For example, the peak at 58.5 min has a very strong MS
response at 694.5 m/z, corresponding to an unidentified higher
molecular weight adduct from the Cys:MDA reaction. The most
important feature of the data presented in these figures, at least in
the context of this article, is to stress that the reaction of MDA and
Cys produces a wide variety of products of higher and higher
molecular weight. It is this feature of the reaction that we believe
may be the most relevant from a physiological perspective.
It will be interesting to study the behavior of the MDA adducts
over time at physiologically relevant temperature ranges. This may
be significant for the production of lipofuscin because the
molecular weight of the adduct of MDA with Cys increased over
time at room temperature (data not shown). This suggests some
dynamic evolution of structure that might mirror the production of
the lipofuscins in cells. Importantly, mass spectral comparison of
our freshly prepared MDA:Cys adduct with an adduct allowed to
stand at room temperature for 1 week showed the presence of
higher molecular weight fractions in the older material. Figure 3
presents the absorption and fluorescence spectra of the MDA:Cys
adducts. It is significant, in that the adducts are yellowish, as
opposed to those reported in Table 1, emitting farther to the red
than those described by Chio and Tappel (26). In other words, they
tend to exhibit characteristics more like those of natural lipofuscin.
Figure 5. Direct infusion of MDA:Cys adducts into the mass spectrometer.
The sample was dissolved in water and continuously introduced into the
electrospray at 10 lL minꢀ1. The MS was operated in positive-ion mode
full-scan from 150 to 2000 m/z. The m/z of 333.1 appears to be the
protonated 2:2 (MDA:Cys) adduct. The peak at 369.3 corresponds to the
first proposed structure of the 3:2 (MDA:Cys) adduct (I). The most
abundant peak at 387.2 m/z corresponds to the second proposed structure of
the 3:2 (MDA:Cys) adduct (II).
structure is proposed to come from 4-hydroxy-2-nonenal, an
aldehyde that is produced by lipid peroxidation, plus an amine such
as the e-amino group in lysine. The amino acrolein structure suffers
from the observation that simple 1:1 adducts such as 1 are not
fluorescent (27).
CONCLUSIONS
In this study synthetic adducts of MDA with Cys have been
subjected to various analyses. They emit over a broader range than
other synthetically prepared lipofuscin. A better understanding of
the structure of natural lipofuscin is important for the rational
design of experimental systems that will mimic the synthesis of
naturally produced lipofuscin. This will enable researchers to
develop and test strategies that may prevent the accumulation of
these pigments as cells age.
In 1969 Buttkus reported that Cys formed an unusual adduct
with MDA (25). He noted that the molecular weight corresponded
to a structure comprising two molecules of Cys and three
molecules of MDA. The adduct was yellow and did not contain
free SH groups. He observed that the molecular weight increased
on storage at room temperature. Moreover, he found that
methionine (which contains an SMe group) formed only a 1:1
adduct with MDA. (Buttkus did not report the fluorescence spectra
of the adducts.) We reproduced these experiments with Cys and
obtained a fluorescent yellow solid (Fig. 3). By NMR we
determined that it was a mixture of isomers. On the basis of
observations of Buttkus (no free SH groups) and our NMR, UV
and mass spectra (Figs. 4 and 5), we assign the structures shown
below to three adducts in the reaction mixture.
Acknowledgements—Part of this work was supported by DOD contract
DAMD17-03-1-0460 to J.W.P. We thank Professor Mark Hargrove and
Dr. Suman Kundu for assistance with sample preparation and for the use
of their sonicator and centrifuge.
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