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Journal of Lipid Research, Vol. 42, 1187-1196, August 2001
Copyright © 2001 by Lipid Research, Inc.

Original Article

Immunochemical detection of a lipofuscin-like fluorophore derived from malondialdehyde and lysine

Satoshi Yamadaa, Shigenori Kumazawab, Takeshi Ishiib, Tsutomu Nakayamab, Koichi Itakurac, Noriyuki Shibatad, Makio Kobayashid, Kensuke Sakaie, Toshihiko Osawae, and Koji Uchidae
a Tsukuba Research Laboratory, NOF Company, Tsukuba 300-2635, Japan
b Department of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8529, Japan
c Faculty of Education, Aichi University of Education, Kariya 448, Japan
d Department of Pathology, Tokyo Women's Medical University, Tokyo 162-8666, Japan
e Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

Correspondence to: Koji Uchida, To whom correspondence should be addressed., uchidak{at}agr.nagoya-u.ac.jp (E-mail)


  ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

The accumulation of fluorescent age pigment or lipofuscin is a frequently observed age-associated cellular alteration in a variety of postmitotic cells of many species. These pigments are observed within granules composed, in part, of damaged protein and lipid. Modification of various biomolecules by aldehyde products of lipid peroxidation is believed to contribute to lipofuscin and ceroid formation. In the present study, we raised a monoclonal antibody (MAb 1F83) directed to the malondialdehyde-modified protein and identified a lipofuscin-like dihydropyridine fluorophore as the major epitope. This antibody was used to conclusively demonstrate that the fluorophore forms on oxidatively modified low density lipoproteins. In addition, we demonstrated that the materials immunoreactive to MAb 1F83 indeed constituted the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells.

The results of this study suggest that the reaction between the lipid peroxidation-derived aldehyde and primary amino groups of protein might represent a process common to the formation of the lipofuscin-like fluorophore during aging and its related diseases. — Yamada, S., S. Kumazawa, T. Ishii, T. Nakayama, K. Itakura, N. Shibata, M. Kobayashi, K. Sakai, T. Osawa, and K. Uchida. Immunochemical detection of a lipofuscin-like fluorophore derived from malondialdehyde and lysine. J. Lipid Res. 2001. 42: 1187;–1196.

Supplementary key words: lipid peroxidation, protein modification, dihydropyridine, monoclonal antibody


  INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

Progressive accumulation of fluorescent pigments, generally referred to as lipofuscin, within the cytoplasm of long-lived cells is the most conspicuous cytological change associated with aging and its related diseases. Fluorescence ascribed to lipofuscin has been observed in postmitotic cells from a variety of organisms within intracellular granules composed, in part, of protein and lipid (1) (2) (3). Because of its age-related increase and seemingly universal occurrence, lipofuscin is considered a hallmark of aging. However, relatively little direct chemical evidence as to the identity of this substance exists, and it is therefore difficult to assess the functional significance of lipofuscin in aging and its related diseases.

There is a hypothesis that lipid peroxidation, which has been implicated in the pathogenesis of numerous diseases, including atherosclerosis, diabetes, and aging, is involved in the formation of lipofuscin (4). Lipid peroxidation proceeds by a free radical chain reaction mechanism and yields lipid hydroperoxides as major initial reaction products. Subsequently, decomposition of lipid hydroperoxides generates a number of breakdown products that display a wide variety of damaging actions (5). There is increasing evidence that reactive aldehydes among them are causally involved in most of the pathophysiological effects associated with oxidative stress in cells and tissues (5) (6). Chio and Tappel (7) (8) have reported that malondialdehyde (MDA), which is the most abundant individual aldehyde resulting from lipid peroxidation, can target a wide range of primary amine-containing cellular components to give fluorescent pigments with spectral properties nearly identical to those found for age pigment. Kikugawa and associates (9) (10) (11) have proposed that lipofuscin fluorophore consists mainly of dihydropyridine derivatives, which arise by a series of condensation reactions of MDA with amines. The phenomenon of fluorescent pigment accumulation is the experimental observation most often quoted to support the free radical theory of aging; that is, the concept that aging is a result of free radical-initiated reactions, such as lipid peroxidation. Thus, it is important to critically examine the experimental evidence in support of the relationship between in vivo lipid peroxidation and accumulation of lipofuscin.

To understand the mechanism of the oxidative modification of proteins in vivo, we initiated studies of the identification of covalently modified amino acids generated during in vitro incubation of the proteins with lipid peroxidation products. In the present study, we have raised a monoclonal antibody (MAb) directed to protein-bound MDA and identified an MDA-derived lipofuscin-like fluorophore to be a major epitope of the antibody. Using this antibody, formation of the fluorophore in oxidized human LDL is characterized. In addition, we show evidence that this fluorophore is indeed formed in atherosclerotic lesions of human aorta.


  MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES

Materials
The sodium salt of MDA was prepared by Dowex hydrolysis of malondialdehyde bis(diethyl acetal) (12). Keyhole limpet hemocyanin (KLH) was obtained from Pierce (Rockville, IL). Horseradish peroxidase-linked anti-rabbit IgG immunoglobulin and enhanced chemiluminescence (ECL) Western blotting detection reagents were obtained from Amersham (Arlington Heights, IL). N {alpha}-Acetyl-L-lysine, bovine insulin, and BSA were obtained from Sigma (St. Louis, MO).

General procedure
NMR spectra were recorded with a Bruker (Billerica, MA) AMX600 (600 MHz) instrument. Ultraviolet absorption spectra were measured with a Hitachi (Tokyo, Japan) U-Best-50 spectrophotometer, and fluorescence spectra were recorded with a Hitachi F-2000 spectrometer. Fast atom bombardment-mass spectrometry (FAB-MS) was measured with a JEOL (Akishima, Japan) JMS-700 (MStation) instrument. Liquid chromatography-mass spectrometry (LC-MS) was measured with a Jasco (Tokyo, Japan) PlatformII-LC instrument.

Reaction of N {alpha}-acetyllysine with MDA
N {alpha}-acetyllysine (100 mM) was incubated with 10 mM MDA in 10 ml of 0.1 M sodium phosphate buffer (pH 7.2) at 37°C for 24 h. Separation of products was carried out on a Jasco Gulliver HPLC with a Jasco MD-910 UV-visible photodiode array detector, using a Develosil ODS-UG-5 column (4.6 x 250 mm) (Nomura Chemical, Seto, Japan). The adducts were eluted with a linear gradient of 1% acetic acid in water (solvent A)-methanol (solvent B) (time = 0, 100% A; 40 min, 80% solvent A; 45;–50 min, 0% solvent A), at a flow rate of 1 ml/min. The elution profiles were monitored by absorbance at 215 nm and by fluorescence intensity (excitation, 387 nm; emission, 455 nm). The antigenic adduct (product c; see Fig 2) was isolated and its chemical structure was characterized by 1H-NMR, FAB-MS, and liquid chromatography-mass spectrometry (LC-MS).



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  		Figure 1. Immunoreactivity of MAb 1F83. A: Immunoreactivity of MAb 1F83 to the aldehyde-treated proteins. Affinity of MAb 1F83 was determined by a direct ELISA using aldehyde-treated BSA as the absorbed antigen. A coating antigen was prepared by incubating 1 mg of BSA with 1 mM aldehyde in 1 ml of 50 mM sodium phosphate buffer, pH 7.4, for 2 h at 37°C. B: Immunoreactivity of MAb 1F83 to MDA-treated proteins. Affinity of MAb 1F83 was determined by a direct ELISA using MDA-treated proteins as the absorbed antigen. A coating antigen was prepared by incubating 1 mg of protein with 1 mM MDA in 1 ml of 50 mM sodium phosphate buffer, pH 7.4, for 2 h at 37°C. Open circles, BSA; solid circles, human serum albumin; open triangles, lysozyme; solid triangles, ribonuclease; open squares, transferrin; solid squares, hemoglobin.



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  		Figure 2. Analysis of MDA-N {alpha}-acetyllysine preparations by HPLC. The reaction was performed by incubating 100 mM N {alpha}-acetyllysine with 10 mM MDA in 10 ml of 0.1 M sodium phosphate buffer (pH 7.2) at 37°C for 24 h. A: Profile of UV absorbance at 215 nm. Peaks a;–c, primary products of the reaction between MDA and N {alpha}-acetyllysine. B: Profile of fluorescence intensity (excitation, 387 nm; emission, 455 nm). C: Competitive ELISA analysis of HPLC fractions for immunoreactivity with MAb 1F83.

Reaction of insulin with MDA
Insulin (0.1 mg/ml) was incubated with 1 mM MDA in 10 ml of 0.1 M sodium phosphate buffer (pH 7.2) at 37°C for 24 h.

In vitro modification of LDL
LDL (1.019;–1.063 g/ml) was prepared from the plasma of healthy humans by sequential ultracentrifugation and then extensively dialyzed against PBS (10 mM sodium phosphate buffer, pH 7.2, containing 150 mM NaCl) containing 0.01% EDTA at 4°C. MDA modification of LDL was performed by incubating 0.5 mg of LDL with 1 mM MDA in 1 ml of 50 mM sodium phosphate buffer (pH 7.2) at 37°C. LDL used for oxidative modification by Cu2+ was dialyzed against a 1,000-fold volume of PBS at 4°C. Oxidation of LDL was performed by incubating 0.5 mg of LDL with CuSO4 (5 µM) in 1 ml of 50 mM sodium phosphate buffer (pH 7.2) at 37°C.

Preparation of the MAb
The antigen (MDA-modified KLH) was prepared by the reaction of 1 mg of KLH with 1 mM MDA in 1 ml of 0.1 M sodium phosphate buffer (pH 7.2) for 24 h at 37°C. Female BALB/c mice were immunized three times with the MDA-treated KLH. Spleen cells from the immunized mice were fused with P3 murine myeloma cells and cultured in hypoxanthine-aminopterin-thymidine (HAT) selection medium. Culture supernatants of the hybridoma were screened by ELISA, employing pairs of wells of microtiter plates on which were absorbed MDA-treated BSA as antigen (1 µg of protein per well). After incubation with 100 µl of hybridoma supernatants, and with intervening washes with Tris-buffered saline, pH 7.8, containing 0.05% Tween 20 (TBS-Tween), the wells were incubated with alkaline phosphatase-conjugated goat anti-mouse IgG, followed by a substrate solution containing p-nitrophenyl phosphate (1 mg/ml). Hybridoma cells corresponding to supernatants that were positive on MDA-modified BSA were then cloned by limiting dilution. After repeated screening, eight clones were obtained. Among them, clone 1F83 showed the most distinctive recognition of MDA-modified BSA.2

ELISA
Direct ELISA. A coating antigen was prepared by incubating 1 mg of BSA with 10 mM aldehydic compounds in 1 ml of 50 mM sodium phosphate buffer, pH 7.2, for 24 h at 37°C. A 100 µl aliquot of the antigen solution containing 0.4 mg of protein was added to each well of a 96-well microtiter plate and incubated overnight at 4°C. The antigen solution was then removed, and the plate was washed with PBS-Tween. Each well was filled with 200 µl of 0.5% gelatin solution for 1 h at 37°C. A 1 µg/ml solution of the primary antibody (MAb 1F83) was then added to the wells at 100 µl/well for 3 h at 37°C. The plate was then washed once with PBS-Tween. After discarding the supernatants and washing three times with PBS-Tween, 100 µl of a 5 x 103 dilution of goat anti-mouse IgG conjugated to horseradish peroxidase in PBS-Tween was added. After incubation for 1 h at 37°C, the supernatant was discarded, and the plates were washed three times with PBS-Tween. Enzyme-linked antibody bound to the well was revealed by adding, at 100 µl/well, 1,2-phenylenediamine (0.5 mg/ml) in 0.1 M citrate-phosphate buffer (pH 5.0) containing 0.003% H2O2. The reaction was terminated by the addition of 50 µl of 2 M sulfuric acid, and the absorbance at 492 nm was read on a micro-ELISA plate reader.

Competitive ELISA. A competitor was incubated with the antibody (MAb 1F83) for 20 h at 4°C to yield competitor-antibody mixtures containing antibody at 1 µg/ml and variable concentrations of the competitor. A 100 µl aliquot of the competitor-antibody mixture was added to each well and incubated for 1 h at 37°C. After discarding the supernatants and washing three times with PBS-Tween, the second antibody was added, and the enzyme-linked antibody bound to the well was revealed as previously described. Results were expressed as the ratio B/B0, where B = [absorbance in the presence of the competitor - background absorbance (no antibody)] and B0 = (absorbance in the absence of the competitor - background absorbance).

SDS-PAGE/immunoblot analysis
SDS-PAGE was performed according to Laemmli (13). Native and modified LDL with MDA were treated with Laemmli sample buffer for 3;–5 min at 100°C and then run on two 6% SDS-polyacrylamide slab gels. One gel was used for staining with Coomassie Brilliant Blue; the other was transblotted to nitrocellulose membranes, incubated with 2% BSA in TBS-Tween for blocking, washed, and treated with MAb 1F83 (2 µg/ml). This procedure was followed by addition of horseradish peroxidase conjugated to goat anti-rabbit IgG immunoglobulin and ECL reagents. The bands were visualized by exposure of membranes to autoradiography film.

Agarose gel electrophoresis/immunoblot analysis
Agarose gel electrophoresis of LDL was performed with the Helena TITAN GEL high resolution protein system (Helena Laboratories, Saitama, Japan). The samples were run on two separate gels. One gel was used for staining with Fat Red 7B; the other was transblotted to nitrocellulose membranes, incubated with Block Ace (40 mg/ml) for blocking, washed, and treated with the primary antibody (MAb 1F83). This procedure was followed by the addition of horseradish peroxidase conjugated to a goat anti-mouse IgG F(ab')2 fragment and ECL reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK). The bands were visualized by exposure of the membranes to autoradiography film.

Immunohistochemistry
Aortic wall samples were obtained at autopsy from five cases of arterial atherosclerosis without diabetes mellitus, and used for histopathological and immunohistochemical examinations. The postmortem time before starting autopsy varied from 1 to 3 h. We have not seen any changes in the positivities at least within this time period. Each autopsy was performed at Tokyo Women's Medical College (Tokyo, Japan).

Consecutive 6 µm-thick sections, for staining with hematoxylin-eosin and immunostaining with the antibodies to MDA and CD68, were cut from formalin (10%)-fixed, paraffin-embedded tissues. The sections were deparaffinized in xylene and ethanol, rehydrated in distilled water, quenched for 10 min with 3% hydrogen peroxide, rinsed in PBS (pH 7.6), and pretreated for 30 min with 3% nonimmune animal serum in PBS. Before immunostaining for CD68, the paraffin sections were treated for 30 min at 37°C with 0.1% trypsin in PBS for antigen retrieval, according to the manufacturer instructions. These sections were incubated overnight at 4°C with the primary antibodies. Sections processed with omission of the antibodies were included as negative reaction controls. Immunoreaction was detected by the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) using 3,3'-diaminobenzidine tetrahydrochloride as the chromogen. Immunostained sections were counterstained with hematoxylin.

All authors approve and warrant that informed consent was obtained under the study approved by our institutional review board. The study was performed in accordance with the Helsinki Declaration of 1975, as revised in 1983.


  RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES

MAb directed to the protein-bound MDA
During the lipid peroxidation process, decomposition of lipid hydroperoxides leads to the generation of many compounds, including MDA, as reactive intermediates. In turn, these can bind to lysine residues of proteins or to aminophospholipids, generating relatively stable end products. To evaluate the contribution of protein modification by MDA in the pathogenesis of various diseases associated with oxidative stress, we attempted to raise an MAb specific to the protein-bound MDA. Mice were immunized with MDA-modified KLH and, during the preparation of the monoclonal antibodies, hybridomas were selected by the reactivities of the culture supernatant to MDA-modified BSA. We finally obtained one clone (1F83) that showed the most distinctive recognition of MDA-modified BSA.

MDA-lysine adducts recognized by MAb 1F83
In spite of an extensive screening of hybridomas that produce monoclonal antibodies specific to the MDA-modified BSA, it is still conceivable that the antibody recognizes epitopes originating from other lipid peroxidation products. Hence, we examined the immunoreactivity of the antibody to aldehyde-treated BSA by a direct ELISA and found that, among the aldehydes tested, MDA was the only source of immunoreactive materials generated in the protein ( Fig 1A). In addition, the antibody recognized epitopes that were generated on any of a variety of different proteins treated with MDA (Fig 1B). To identify the epitope structure recognized by MAb 1F83, its immunoreactivity with the products produced by reaction of MDA with N {alpha}-acetyllysine was characterized. As shown in Fig 2A and Fig B, the reaction of MDA with N {alpha}-acetyllysine mainly provided three products, including a predominant nonfluorescent product (Fig 2, peak a) eluted at 22 min and two fluorescent products (Fig 2, peaks b and c) eluted at 24 and 39 min. The ELISA analysis of HPLC fractions for immunoreactivity with MAb 1F83 showed that the antibody had immunoreactivity with the main product a and the fluorescent product c (Fig 2C). However, the ratio of immunoreactivities to the yields of each products suggested that the antibody predominantly reacted with product c. The competitive ELISA analysis indeed showed that the binding of the MDA-modified protein to the antibody (MAb 1F83) was scarcely inhibited by products a and b but was significantly inhibited by product c ( Fig 3).



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  		Figure 3. Competitive ELISA analysis of the purified MDA-N {alpha}- acetyllysine adducts for immunoreactivity with MAb 1F83. Competitors: 119open circles, N {alpha}-acetyllysine; open squares, N {alpha}-acetyl-N {varepsilon}-(2-propenal)lysine (peak a in Fig 2); solid triangles, N {alpha}-acetyl-N {varepsilon}-lysyl-3,5-diformyl-2,6-dihydropyridin-4-yl-pyridinium derivative (peak b in Fig 2); solid circles, N {alpha}-acetyl-N {varepsilon}-lysyl-4-methyl-2,6-dihydropyridine-3,5-dicarbaldehyde derivative (peak c in Fig 2).

Identification of epitope structure
After purification, the structure of the major antigenic product c was characterized by FAB-MS, LC-MS, and 1H-NMR. The product was fluorescent at 387 nm (excitation) and 455 nm (emission) and exhibited a UV absorption maximum at 396 nm. FAB-MS gave a molecular ion peak at m/z 322.9 [MH+]. The 1H-NMR spectrum exhibited the following signals; {delta}H (D2O): 1.05 (3H, d), 1.20;–1.98 (4H, m), 1.91 (3H, s), 3.37 (2H, m), 3.49 (3H, t), 3.82 (1H, m), 4.33-4.45 (1H, m), 7.10 (2H, s), 9.25 (2H, s). These data suggested that product c was identical to a 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde (dihydropyridine) derivative ( Fig 4), which has been putatively identified as the lipofuscin-like age pigment (9) (10) (11). Thus, it was determined that the structure of the epitope recognized by MAb 1F83 was the lipofuscin-like dihydropyridine fluorophore.



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  		Figure 4. Structures of N {alpha}-acetyllysine and MDA-N {alpha}-acetyllysine adducts [(a), (b), and (c) refer to peaks a;–c in Fig 2].

The UV spectra and LC-MS analysis indicated that products a and b corresponded to a 2-propenal-type lysine derivative (UV {lambda}max, 282 nm; MH+ m/z 242.8) (14) and a 3,5-diformyl-1,4-dihydropyridin-4-yl-pyridinium derivative (UV {lambda}max, 235, 260, and 398 nm; MH+ m/z 557.2) (15), respectively.

Formation of dihydropyridine fluorophores in MDA-modified insulin
To examine the formation of the dihydropyridine fluorophore in polypeptide, the reaction of human insulin, which contains one lysine and two N-terminal amino acid residues per polypeptide chain, with MDA was characterized. The covalent binding of MDA to insulin was assessed by loss of primary amino groups. As shown in Fig 5A, incubation of insulin (1 mg/ml) with 1 mM MDA resulted in a time-dependent loss of primary amino groups of the protein. Approximately 80% of the amino groups were lost after 24 h of incubation, suggesting that MDA reacted not only with the lysine residue but also with the N-terminal amino acid residues in insulin. The loss of amino groups of insulin was accompanied by an increase in protein fluorescence (Fig 5B) and immunoreactivity with MAb 1F83 (Fig 5C), suggesting that the MAb may recognize a dihydropyridine fluorophore generated in the MDA-modified insulin. This was also supported by the observation that the ELISA analysis of HPLC fractions of MDA-insulin for immunoreactivity with MAb 1F83 yielded a profile similar to the fluorescence profile (data not shown).



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  		Figure 5. MDA modification of insulin. Insulin (1 mg/ml) was incubated with 1 mM MDA in 50 mM sodium phosphate buffer (pH 7.4) at 37°C. A: Loss of primary amino group. B: Increase in fluorescence intensity (excitation, 387 nm; emission, 455 nm). C: Immunoreactivity with MAb 1F83.

Fluorescent properties of MDA-modified and Cu2+-oxidized LDL
Various lines of evidence have indicated that an important part of the pathogenesis of atherosclerosis is the oxidative modification of plasma LDL (16) (17). Oxidative modification of LDL is associated with the formation of a large number of lipid peroxidation products that could covalently bind to LDL apolipoprotein B (apoB) (5) (6). The resulting oxidized LDL may be taken up by cells and may be the ultimate source of the lipids that accumulate in atherosclerotic lesions. The fact that the formation of fluorescent properties is a well-documented change in oxidized LDL (18) (19) (20) led us to assess the involvement of dihydropyridine fluorophores in the fluorescence of oxidized LDL. As shown in Fig 6A, exposure of LDL to 1 mM MDA at 37°C indeed resulted in a time-dependent increase in the fluorescence intensity of LDL. The MDA-modified LDL exhibited the same characteristics as those of proteins treated with MDA (7) (8). Like the MDA modification of LDL, LDL peroxidation resulted in the formation of fluorescent characteristics in the oxidized LDL. As shown in Fig 6B, when LDL was incubated with 5 µM Cu2+ at 37°C, a significant increase in the fluorescence intensity of LDL was observed. A linear relationship between enhanced fluorescence and the thiobarbituric acid-reactive substance (TBARS) content of the oxidized LDL was also observed (data not shown). As compared with the MDA-modified LDL, the oxidized LDL showed a relatively broad fluorescent spectra (data not shown), suggesting that more than one fluorescent product is formed during LDL peroxidation. In view of the facts that MDA is one of the most abundant aldehydes generated during oxidative modification of LDL (21) and that oxidative modification of LDL in vitro yields an LDL particle with many of the properties of MDA-modified LDL (22), it was anticipated that a common fluorophore was formed in both MDA-modified and Cu2+-oxidized LDL.



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  		Figure 6. Fluorescence production in MDA-modified LDL (A) and Cu2+-oxidized LDL (B). LDL (0.5 mg) was incubated with 1 mM MDA or 5 µM Cu2+ in 1 ml of 50 mM sodium phosphate buffer (pH 7.2) at 37°C.

Formation of dihydropyridine fluorophores in MDA-modified and Cu2+-oxidized LDL
To determine whether the dihydropyridine fluorophores are formed in MDA-modified LDL, LDL treated with 1 mM MDA was subjected to an SDS-PAGE/immunoblot analysis with MAb 1F83. As shown in Fig 7A, little or no immunoreactivity was detected with native LDL apoB, whereas the MDA-modified LDL apoB showed intense immunoreactivity. The immunoreactivity of LDL apoB clearly depends on incubation time, although treatment of LDL with MDA barely affected the pattern of electrophoretic mobilities of apolipoproteins detected by Coomassie blue staining. Incubation of LDL with Cu2+ led to oxidation of the LDL as assessed by the formation of TBARS (data not shown). After separation by agarose gel electrophoresis (Fig 7B, left), the native form of the LDL appeared as a single protein band that was readily visualized by Fat Red 7B staining; however, the LDL incubated with 5 µM Cu2+ exhibited enhanced anodic mobility compared with the native LDL, indicating an increased negative charge of the molecule, probably caused by the modification of the {varepsilon}-amino group of the lysine residues. An agarose gel electrophoresis/immunoblot analysis of the Cu2+-oxidized LDL, using MAb 1F83, revealed the formation of immunoreactive materials that were not detected in the native LDL (Fig 7B, right).



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  		Figure 7. Immunoblot analysis of MDA-modified and Cu2+-oxidized LDL. LDL (0.5 mg) was incubated with 1 mM MDA or 5 µM Cu2+ in 1 ml of 50 mM sodium phosphate buffer (pH 7.2) at 37°C. A: SDS-PAGE/immunoblot analysis of MDA-modified LDL: left, SDS-PAGE; right, immunoblot analysis with MAb 1F83. B: Agarose gel electrophoresis/immunoblot analysis of oxidized LDL: left, agarose gel electrophoresis; right, agarose gel electrophoresis/immunoblot analysis with MAb 1F83.

Localization of dihydropyridine fluorophores in human atherosclerotic lesions
The findings that the level of MDA-modified LDL increases in the plasma of patients with atherosclerosis (23) and that the monoclonal antibodies raised against MDA-modified LDL bind to epitopes in atherosclerotic lesions (24) (25) suggest that the protein-bound MDA is most likely to be accumulated in an atherosclerotic lesion. Hence, atherosclerotic aorta were immunohistochemically examined for the dehydropyridine fluorophores, using MAb 1F83. No immunoreaction product deposits were detected in sections with omission of the primary antibody or with incubation with the preabsorbed antibody (data not shown). The macrophages appearing in the atherosclerotic plaques were identified in hematoxylin-eosin-stained ( Fig 8A) or CD68-immunostained sections (Fig 8B). The cytoplasm of most of the foamy or spindle macrophages was immunoreactive with MAb 1F83 (Fig 8C). To avoid the oxidative modification during procedural handling, EDTA was included during preparation of the atherosclerotic aorta for immunohistochemical analyses and the frozen sections without acetone postfixation were immunostained. Nevertheless, cell-associated stainings were clearly observed (data not shown). These data suggest that generation of epitopes recognized by the antibody during the dehydration, embedding, rehydration, and staining processes is unlikely and that the observed immunoreactivities reflect endogenous products generated in vivo. Thus, the detection of the dihydropyridine fluorophore in atherosclerotic plaques supports the notion that the reaction between MDA and primary amines might represent a process common to the formation of the lipofuscin-like fluorophore during aging and its related diseases (23) (24) (25).



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  		Figure 8. Immunohistochemical detection of dehydropyridine fluorophores in atherosclerotic aorta. Arterial tissue specimen with atherosclerosis was immunostained with hematoxylin-eosin (A), anti-CD68 antibody (B), and MAb 1F83 (C). Original magnification: A;–C: x100.


  DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES

This study demonstrates for the first time that MDA-derived dihydropyridines contribute to the presence of elevated levels of fluorescence not only in protein incubated with MDA but also in oxidatively modified LDL and human atherosclerotic aorta. Lipofuscin is a general term used to describe fluorescent material that accumulates in cells as a function of age (2) (3). Lipid peroxidation has been regarded generally as being involved in the formation of lipofuscin with characteristic fluorescence (26). Because their fluorescence spectra (excitation maxima at 370;–400 nm, emission maxima at 450;–470 nm) are similar to those of lipofuscin (excitation maxima at 340;–390 nm, emission maxima at 430;–490 nm), the structure of the fluorescent components found in the lipofuscin complex had been assumed to be the conjugated Schiff base (1-amino-3-iminopropene) between MDA and primary amino groups of proteins and phospholipids (7) (8). Significantly, however, this putative lipofuscin fluorophore has not been detected directly by NMR at neutral pH in water, despite extensive chemical research (7) (27) (28) (29). The cross-link has always been detected as a borohydride-reduced product (7) (27), whereas other fluorescent compounds, the dihydropyridine derivatives, have been isolated in crystalline form from the reaction of MDA with primary amines (9) (10) (11). Slatter et al. (28) (30) have suggested that the dihydropyridine, rather than the 1-amino-3-iminopropene derivatives, may represent the major fluorescent products of the reaction of MDA with amines. This was also supported by the present data that the major fluorescent products detected in the reaction of N {alpha}-acetyllysine with MDA were dihydropyridine (Fig 2, product c) and its cross-linked derivatives (Fig 2, product b).

Formation of dihydropyridine fluorophores in the MDA-modified protein was suggested by the observation that the ELISA analysis of HPLC fractions of MDA-treated insulin for immunoreactivity with MAb 1F83 yielded a profile similar to the fluorescence profile (data not shown). To obtain further evidence that the fluorescent MDA-insulin adduct contains the dihydropyridine fluorophore, the major fluorescent and immunoreactive product was analyzed by LC-MS. Unmodified insulin gave a molecular mass of 5,807.0 Da, which was in agreement with the theoretical molecular mass derived from the sequence for insulin, whereas the LC-MS analysis of the immunoreactive peak gave a molecular mass of 6,049.0 Da (S. Kumazawa, T. Nakayama, and K. Uchida, unpublished data). The peaks in the mass spectrum differed in molecular mass by 243 Da. The corresponding mass shift suggested that at least three molecules of MDA might be involved in the formation of the major fluorescent and immunoreactive MDA-polypeptide adduct and seemed to be reasonably explained by the formation of one dihydropyridine and two propenal derivatives; however, further efforts to detect the fragments containing the dihydropyridine derivative of an amino acid by tandem MS analysis remain unsuccessful, probably because of the lability of the fluorophore in analysis.

It should be noted that, although treatment of LDL with MDA barely affected the pattern of electrophoretic mobilities of apolipoproteins detected by Coomassie blue staining, MDA caused significant modification of LDL apoB, leading to the formation of dihydropyridine (Fig 7A, right). Notably, MDA immediately forms the high molecular weight protein band, which probably corresponds to the dimer of apoB. In addition, a progressively broad distribution of immunoreactivity was also observed. The formation of these immunoreactive bands of lower and higher mobilities may be explained, at least in part, by intramolecular and intermolecular cross-linkage reactions, resulting in the formation of more globular and polymerized forms, respectively. Furthermore, MDA is likely to mediate the formation of cross-linkages between apoB and aminophospholipids, which may significantly affect the mobility of apoB. It is also interesting that the migrated proteins seemed to be preferentially stained with the antibody (MAb 1F83). This suggests the possibilities that i) the migrated proteins contain more epitope (dihydropyridine) than the nonmigrated proteins and ii) the formation of dihydropyridine is closely associated with the formation of cross-links. These may not be unlikely because the aldehyde groups of the dihydropyridine derivative could react further with the primary amines, generating cross-links (28).

During incubation of LDL with cells, the LDL molecule undergoes a large number of structural changes that alter its metabolism (16) (17). Although the detailed mechanism for oxidative modification of LDL has not been established, it is generally accepted that the primary generation of lipid hydroperoxide derivatives initiates a reaction cascade leading to rapid propagation and amplification of the number of reactive oxygen species formed; this leads ultimately to extensive fragmentation of the fatty acid chains (20) and conversion of the LDL to a more atherogenic form (31). Oxidation of LDL leads to significant changes in structural and functional properties, including an increased electronegative charge, loss of lysine residues, and enhanced recognition by macrophages (16) (17). It is also known that the remarkable features of oxidized LDL include fluorescence (18) (19) (20). Because of its high sensitivity, this fluorescence is widely used for monitoring the oxidative modification of LDL. It has been suggested that fluorophores formed in oxidized LDL are generated by covalent binding of lipid peroxidation products to the lysine {varepsilon}-amino groups of the LDL apolipoprotein. The fluorescence has been ascribed to the reaction of reactive aldehydes, such as MDA, 4-hydroxy-2-alkenals, and 2,4-alkadienals, generated during LDL peroxidation; however, there have been few detailed insights into the fluorescence characteristics of oxidized LDL. The present study demonstrated that dihydropyridines could be generated in the Cu2+-oxidized LDL in vitro (Fig 7). However, the fluorescence of oxidized LDL cannot be entirely explained by this fluorophore, because fluorescent characteristics between oxidized LDL (excitation maximum at 365;–375 nm, emission maximum at 430;–450 nm) and dihydropyridines (excitation maximum at 375;–405, emission maximum at 435;–465 nm) are slightly different. These data suggest that the dihydropyridine fluorophores may be partially responsible for the fluorescence of oxidized LDL.

It has previously shown that ultraviolet laser-induced autofluorescence is detected in the ceroid deposits in atherosclerotic plaques (32). Notably, the emission spectra of ceroid in atherosclerotic plaques appear to be similar in wavelength specificity to those of dihydropyridine fluorophores. This may support the notion that the fluorescence of age pigments, such as ceroid, results at least in part from the formation of MDA adducts consequent to lipid peroxidation (33). Furthermore, the present observations are consistent with, and extend the previous findings by, Mitchinson and coworkers (34) (35) that the ceroid accumulation in macrophages is significantly induced by oxidized LDL in vitro.

Antibodies recognizing MDA-linked protein have been published in multiple contexts. Using the MAb (MDAlys) against MDA-lysine residues, Haberland, Fong, and Cheng (24) demonstrated immunocytochemically the presence of MDA-lysine residues in atherosclerotic lesions that colocalized with apoB. In addition, by immunocytochemical application of different antibodies, Palinski et al. (25) (36) and Rosenfeld et al. (37) confirmed the presence of MDA-lysine in atherosclerotic lesions and demonstrated that several different oxidation-specific epitopes are found in lesioned but not in normal areas of rabbit aortas. In the present study, the materials immunoreactive to the MAb (MAb 1F83) raised against MDA-modified protein indeed constituted the atherosclerotic lesions of human aorta. Thus, the monoclonal antibodies developed in these studies commonly recognize the MDA linked to lysine residues in arterial lesions. However, because of the different experimental procedures for antigen generation (e.g., concentration of MDA, pH, time of incubation, and protein carrier), it is likely that they may recognize different MDA-lysine epitopes. The possibility that MDA may play a role in the pathogenesis of atherosclerosis has also been suggested by the facts that i) high concentrations of MDA can be generated during the oxidation of LDL phospholipids (5) (19) (20); ii) the structural and functional changes associated with the oxidation of LDL can also be produced by direct interaction of LDL with MDA (38); iii) the reaction of MDA with a critical number of lysine residues of LDL apoB produces internalization by the scavenger receptor of human monocyte-macrophages and subsequent intracellular accumulation of lipoprotein-derived cholesteryl ester (39) (40) (41); iv) the level of MDA-modified LDL increases in the plasma of patients with atherosclerosis (23); and v) antioxidant therapy slows the progress of early atherosclerotic lesions (42) (43). None of these items rigorously indicate that MDA-induced modifications or dihydropyridine adducts cause disease to any greater extent than dozens of other structural changes accompanying lipoprotein oxidation; however, the possible significance of fluorescent pigment as a causal factor in cell death and functional decrements has been suggested (44).

In summary, we raised MAbs directed to protein-bound MDA and obtained a new murine MAb, 1F83, that clearly distinguished the MDA-modified protein from the native protein. In addition, it appeared that MAb 1F83 is specific to the lipofuscin-like fluorophore, dihydropyridine. The observation that this MAb did not cross-react with proteins that had been treated with the reactive aldehydes but only with MDA suggested that this aldehyde is the only source of fluorophores. Using MAb 1F83, we showed that the fluorophores were indeed formed not only in the oxidized LDL but also in the atherosclerotic lesions of human aorta. These data suggested that the reaction between lipid peroxidation products and primary amines of proteins and phospholipids might represent a process common to the formation of lipofuscin-like fluorophores during aging and its related diseases. The results of this study and the methods developed are therefore critical to future investigations aimed at elucidating i) the relative contribution of MDA-derived fluorophores to lipofuscin and ceroid accumulation in vivo, ii) the subcellular origin(s) of damaged cellular components present in these heterogeneous, fluorescent pigments, iii) the effects of fluorophore formation on specific physiological functions, and iv) mechanisms by which cells respond to free radical and lipid peroxidation damage.


  FOOTNOTES

Abbreviations: apoB, apolipoprotein B-100; FAB-MS, fast atom bombardment-mass spectrometry; KLH, keyhole limpet hemocyanin; LC-MS, liquid chromatography-mass spectrometry; MDA, malondialdehyde; WHHL rabbit, Watanabe heritable hyperlipidemic rabbit. Back
2 The monoclonal antibody (MAb 1F83) can be obtained from the corresponding author for this article: Koji Uchida, Ph.D., Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. Fax: 81-52-789-5741. e-mail: uchidak{at}agr.nagoya-u.ac.jp. Back


  ACKNOWLEDGMENTS

This work was supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). We thank Shunpei Sakamoto of Nagoya University for technical support.

Manuscript received December 4, 2000; and in revised form April 6, 2001


  REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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