Lipid peroxidation generates body odor component trans-2-nonenal covalently bound to protein in vivo

Article abstract of DOI:10.1074/jbc.M109.068023

trans-2-Nonenal is an unsaturated aldehyde with an unpleasant greasy and grassy odor endogenously generated during the peroxidation of polyunsaturated fatty acids. 2-Nonenal covalently modified human serum albumin through a reaction in which the aldehyde preferentially reacted with the lysine residues. Modified proteins were immunogenic, and a specific monoclonal antibody (mAb) 27Q4 that cross-reacted with the protein covalently modified with 2-nonenal was raised from mouse. To verify the presence of the protein-bound 2-nonenal in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole limpet hemocyanin was raised. It was found that a novel 2-nonenal-lysine adduct, cis- and trans-N∈-3-[(hept-1-enyl)-4-hexylpyridinium] lysine (HHP-lysine), constitutes an epitope of the antibody. The immunoreactive materials with mAb 27Q4 were detected in the kidney of rats exposed to ferric nitrilotriacetate, an iron chelate that induces free radical-mediated oxidative tissue damage. Using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for detection of the cis- and trans-HHP-lysine and confirmed that the 2-nonenallysine adducts were indeed formed during the lipid peroxidation- mediated modification of protein in vitro and in vivo. Furthermore, we examined the involvement of the scavenger receptor lectin-like oxidized low density lipoprotein receptor-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand.

Full text of DOI:10.1074/jbc.M109.068023

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 20, pp. 15302–15313, May 14, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Lipid Peroxidation Generates Body Odor Component
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S
trans-2-Nonenal Covalently Bound to Protein in Vivo*
Received for publication, September 22, 2009, and in revised form, March 7, 2010 Published, JBC Papers in Press, March 8, 2010, DOI 10.1074/jbc.M109.068023
Kousuke Ishino‡, Chika Wakita‡, Takahiro Shibata‡, Shinya Toyokuni§, Sachiko Machida¶, Shun Matsudaʈ,
Tomonari Matsudaʈ, and Koji Uchida‡1
From the ‡Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, the §Department of Pathology and
Biological Responses, Graduate School of Medicine, Nagoya University, Nagoya 466-8550, the ¶National Food Research Institute,
ʈ
2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, and the Research Center for Environmental Quality Management, Kyoto
University, 1-2 Otsu, 520-0811, Japan
trans-2-Nonenal is an unsaturated aldehyde with an un-
pleasant greasy and grassy odor endogenously generated during
the peroxidation of polyunsaturated fatty acids. 2-Nonenal
covalently modified human serum albumin through a reaction
in which the aldehyde preferentially reacted with the lysine res-
idues. Modified proteins were immunogenic, and a specific
monoclonal antibody (mAb) 27Q4 that cross-reacted with the
protein covalently modified with 2-nonenal was raised from
mouse. To verify the presence of the protein-bound 2-nonenal
in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole
limpet hemocyanin was raised. It was found that a novel 2-non-
saturated fatty acids has also been implicated in the pathogen-
esis of many types of liver injuries and especially in the hepatic
damage induced by several toxic substances. The lipid peroxi-
dation leads to the formation of a broad array of different prod-
ucts with diverse and powerful biological activities. Among
them are a variety of different aldehydes (2). The primary prod-
ucts of lipid peroxidation, lipid hydroperoxides, can undergo
carbon-carbon bond cleavage via alkoxyl radicals in the pres-
ence of transition metals giving rise to the formation of short-
chain, unesterified aldehydes or a second class of aldehydes still
esterified to the parent lipid. These reactive aldehydic interme-
diates readily form covalent adducts with cellular macromole-
cules, including protein, leading to the disruption of important
cellular functions. The important agents that give rise to the
modification of protein may be represented by ␣,␤-unsaturated
aldehydic intermediates, such as 2-alkenals, 4-hydroxy-2-
alkenals, and 4-oxo-2-alkenals (3, 4).
⑀
enal-lysine adduct, cis- and trans-N -3-[(hept-1-enyl)-4-hexyl-
pyridinium]lysine (HHP-lysine), constitutes an epitope of the
antibody. The immunoreactive materials with mAb 27Q4 were
detected in the kidney of rats exposed to ferric nitrilotriacetate,
an iron chelate that induces free radical-mediated oxidative tis-
sue damage. Using high performance liquid chromatography
with on-line electrospray ionization tandem mass spectrometry,
we also established a highly sensitive method for detection of the
cis- and trans-HHP-lysine and confirmed that the 2-nonenal-
lysine adducts were indeed formed during the lipid peroxida-
tion-mediated modification of protein in vitro and in vivo. Fur-
thermore, we examined the involvement of the scavenger
receptor lectin-like oxidized low density lipoprotein receptor-1
in the recognition of 2-nonenal-modified proteins and estab-
lished that the receptor recognized the HHP-lysine adducts as a
ligand.
2-Alkenals represent a group of highly reactive aldehydes
containing two electrophilic reaction centers. A partially posi-
tive carbon 1 or 3 in such molecules can attack nucleophiles,
such as protein. It has been suggested that these aldehydes pri-
marily react with the sulfhydryl group of cysteine, the ⑀-amino
group of lysine, and the imidazole group of histidine in the
proteins (2, 3). Among the 2-alkenals, 2-nonenal (Fig. 1A) has a
characteristically unpleasant greasy and grassy odor. It is also a
major contributor to the unpleasant cardboard flavor in aged
beer. It was previously shown that 2-nonenal could be formed
through lipid peroxidation as a minor product in peroxide-
mediated oxidation of high concentrations of linoleic acid
hydroperoxide or from liver microsomes treated with ADP/
iron in vitro (2). Toyokuni et al. (5) also reported the production
of C2–C12 saturated and unsaturated aldehydes, including
2-nonenal, in the kidney of rats exposed to Fe3ϩ-NTA.2 More
Lipid peroxidation in tissue and in tissue fractions represents
a degradative process, which is the consequence of the produc-
tion and propagation of free radical reactions primarily involv-
ing membrane polyunsaturated fatty acids, and has been impli-
cated in the pathogenesis of numerous diseases, including
atherosclerosis, diabetes, cancer, and rheumatoid arthritis, as
well as in drug-associated toxicity, postischemic reoxygenation
injury, and aging (1). The peroxidative breakdown of polyun-
2 The abbreviations used are: Fe3ϩ-NTA, ferric nitrilotriacetate; AcLDL, acetylated
LDL; BSA, bovine serum albumin; DiD, 1,1Ј-dioctadecyl-3, 3,3Ј,3Ј-tetramethyl-
indodicarbocyanine perchlorate; ELISA, enzyme-linked immunosorbent
assay; HHP-lysine, N⑀-3-[(hept-1-enyl)-4-hexylpyridinium]lysine; HMBC,
1H-detected multiple-bond connectivity; KLH, keyhole limpet hemocya-
nin; LDL, low density lipoprotein; LOX-1, lectin-like oxidized LDL receptor;
HSA, human serum albumin; LC/ESI/MS/MS, high performance liquid chro-
matography with on-line electrospray ionization tandem mass spectrome-
try; mAb, monoclonal antibody; MRM, multiple reaction monitoring; PBS,
phosphate-buffered saline; HPLC, high pressure liquid chromatography; CFP,
cyan fluorescent protein; CHO, Chinese hamster ovary; Fmoc, N-(9-fluorenyl)-
methoxycarbonyl; HSA, human serum albumin.
* This work was supported by a grant-in-aid for scientific research on innova-
tive areas (research in a proposed research area), from the Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan (to K. U.).
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The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S22.
1 To whom correspondence should be addressed. Fax: 81-52-789-5741;
E-mail: uchidak@agr.nagoya-u.ac.jp.
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Lipid Peroxidation Generates Protein-bound 2-Nonenal
EXPERIMENTAL PROCEDURES
recently, Haze et al. (6) analyzed the body odor components
that adhered to the shirts of the subjects by gas chromatogra-
phy/mass spectrometry and demonstrated that 2-nonenal is
present in increasing amounts in the body odors of persons 40
years or older. They have also suggested that cis-2-nonenal and
trans-2-nonenal are formed from the oxidative degradation of
polyunsaturated fatty acids, such as palmitoleic acid. However,
it is still not clear how 2-nonenal could be formed in vivo.
Because of its insolubility in water, 2-nonenal is less reactive
with proteins than other 2-alkenals, such as acrolein and cro-
tonaldehyde, and therefore has received relatively little atten-
tion as a causative agent for modification of proteins. Only
inhibition of enzymes, such as platelet membrane-bound phos-
photyrosine phosphatase (7) and liver microsomal glucose-6-
phosphatase (8), has been reported.
A structurally diverse protein supergroup called scavenger
receptors mediates the cellular uptake of modified lipoproteins.
Scavenger receptors are expressed by endothelial cells, macro-
phages, and smooth muscle cells and mediate recognition,
internalization, and physiological responses to a wide range
of ligands, including phospholipids, lipoprotein particles,
apoptotic cells, and pathogens. Several oxidized low density
lipoprotein (LDL) receptors have been identified so far,
including SR-A I/II, CD36, SR-BI, Fc␥RII, lectin-like oxi-
dized LDL receptor (LOX-1), macrosialin, SR expressed by
endothelial cells, etc. Among them, LOX-1 is characterized
as the major receptor for oxidized LDL in the endothelial
cells of large arteries. Its inducible expression (9) might be
involved in the oxidized LDL-mediated endothelial dysfunc-
tion. LOX-1 exhibits a binding activity for multiple ligands.
LOX-1 is capable of interacting with a variety of structurally
and functionally distinct ligands, including oxidized LDL,
platelets, aged red blood cells, apoptotic cells, advanced gly-
cation end products, heat shock protein 70, bacteria, and
phosphatidylserine (10). LOX-1 binds and internalizes a
diverse array of macromolecules, although their structures
are not always related to each other.
In this study, to understand the mechanism underlying the
formation of a covalently modified protein with 2-nonenal in
vivo, we raised a monoclonal antibody (mAb) against protein-
bound 2-nonenal and identified a novel 2-nonenal-lysine
adduct as the epitope. Our immunohistochemical studies dem-
onstrated the localization of the immunoreactive materials in
the kidney of rats exposed to Fe3ϩ-NTA, an iron chelate that
induces acute renal proximal tubular necrosis, a consequence
of free radical-mediated oxidative tissue damage, eventually
leading to a high incidence of renal adenocarcinoma in rodents.
Importantly, using high performance liquid chromatography
with on-line electrospray ionization tandem mass spectrometry
(LC/ESI/MS/MS), we also showed evidence that the 2-nonenal-
lysine adduct is indeed accumulated during the lipid peroxida-
tion-mediated modification of protein in vitro and in vivo. Fur-
thermore, to evaluate the biological implication of the
␣
Materials—trans-2-Nonenal (2-nonenal), N -acetyl-L-ly-
sine, and human serum albumin (HSA) were obtained from the
Sigma. Acrolein was obtained from Tokyokasei (Tokyo, Japan).
␣
⑀
N -[U-13C6,15N2]Fmoc-N -boc-lysine (where boc is t-butoxy-
carbonyl) was obtained from the Cambridge Isotope Laborato-
ries (Andover, MA). The keyhole limpet hemocyanin (KLH)
was obtained from Pierce. Ferric nitrate nonahydrate, sodium
carbonate, and bovine serum albumin (BSA) were from Wako
(Osaka, Japan), and the nitrilotriacetic acid disodium salt was
from Nacalai Tesque, Inc. (Kyoto, Japan). The Fe3ϩ-NTA solu-
tion was prepared immediately before use by the method
described in a previous study (11). The stock solutions of 4-hy-
droxy-2-nonenal were prepared by the acid treatment (1 mM
HCl) of 4-hydroxy-2-nonenal dimethyl acetal, which was syn-
thesized according to the procedure of De Montarby et al. (12).
4-Oxo-2-nonenal was synthesized by the oxidation of 4-hy-
droxy-2-nonenal dimethyl acetal with pyridinium dichloro-
chromate, followed by HCl hydrolysis (13). The sodium salt of
malondialdehyde was prepared by the Dowex hydrolysis of
malondialdehyde bis(diethyl acetal) (14). Other aldehydes were
purchased from Wako.
In Vitro Modification of HSA—Modification of the protein by
2-nonenal and its related short-chain aldehydes was performed
by incubating HSA (1.0 mg/ml) with 0–10 mM 2-nonenal in 1
ml of 50 mM sodium phosphate buffer, pH 7.2, at 37 °C for 24 h.
The Fe2ϩ-catalyzed oxidation of unsaturated fatty acids in the
presence of HSA was performed by incubating HSA (1 mg/ml)
with 2 mM unsaturated fatty acids in the presence of 50 ␮M Fe2ϩ
and 1 mM ascorbic acid in 1 ml of 50 mM sodium phosphate
buffer, pH 7.2, in atmospheric oxygen at 37 °C. The reaction
was terminated by the addition of 1 mM butylated hydroxytolu-
ene and 100 ␮M diethylenetriaminepentaacetic acid.
In Vitro Peroxidation of LDL—LDL (1.019–1.063 g/ml) was
prepared from the plasma of healthy humans by sequential
ultracentrifugation and then extensively dialyzed three times
against phosphate-buffered saline (PBS, 10 mM sodium phos-
phate buffer, pH 7.2, containing 150 mM NaCl) containing
0.01% EDTA at 4 °C. LDL used for the oxidative modification by
Cu2ϩ was dialyzed five times against a 1000-fold volume of PBS
at 4 °C. The oxidation of LDL was performed by incubating 0.5
mg of LDL with CuSO4 (5 ␮M) in 1 ml of PBS for 24 h at 37 °C.
The reaction was terminated by the addition of 1 mM EDTA and
then stored at 4 °C.
Amino Acid Analysis—An aliquot (0.2 ml) of the protein
samples (1 mg/ml) incubated for 24 h at 37 °C in the absence or
presence of 2-nonenal was treated with 10 mM EDTA (20 ␮l), 1
N NaOH (20 ␮l), and 100 mM sodium borohydride (20 ␮l). After
incubation for 1 h at room temperature, 20 ␮l of 2 N HCl was
added to the mixture to stop the reaction, and the mixture was
then incubated for 60 min at room temperature after adding
280 ␮l of 20% trichloroacetic acid. After centrifugation at
5,000 ϫ g for 10 min at 4 °C, the proteins were hydrolyzed in
2-nonenal modification of protein, we examined the involve- vacuo with 2 ml of 6 N HCl for 24 h at 110 °C. The hydrolysates
ment of LOX-1, a member of the scavenger receptor family, in were then dried and dissolved in sodium citrate buffer, pH 3.15.
the recognition of the 2-nonenal-lysine adducts.
The amino acid analysis was performed using a JEOL JLC-500
MAY 14, 2010•VOLUME 285•NUMBER 20
JOURNAL OF BIOLOGICAL CHEMISTRY 15303

Lipid Peroxidation Generates Protein-bound 2-Nonenal
amino acid analyzer equipped with a JEOL LC30-DK20 data 0.8 ml/min. The elution profiles were monitored by absorbance
analyzing system.
at 239 nm. The NMR analyses were performed using a Bruker
Preparation of Monoclonal Antibody against 2-Nonenal- AMX400 (400 MHz) instrument. For P-1, 1H NMR (400 MHz,
modified Protein—The immunogen was prepared by incubat- MeOD): ␦ 0.85–0.92 (6H, m), 1.25–1.50 (22H, m), 1.66 (2H, m),
ing the KLH (1.0 mg/ml) with 10 mM 2-nonenal in 3 ml of PBS 1.70–2.08 (7H, m), 2.13 (2H, q), 2.87 (2H, t), 4.36 (1H, q), 4.55
at 37 °C for 24 h. We immunized the female BALB/c mice (2H, t), 6.16 (1H, m), 6.51 (1H, d, J ϭ 11.6 Hz), 7.93 (1H, d, J ϭ
(Chubu Kagaku Shizai Co., Ltd., Nagoya, Japan) on day 1 with 6.4 Hz), 8.66 (1H, s), and 8.70 (1H, d, J ϭ 5.4 Hz). For P-2, 1H
complete Freund adjuvant and 0.06 mg of immunogen (2-non- NMR (MeOD, 600 MHz): ␦ 0.89–0.94 (6H, m), 1.30–1.52
enal-modified KLH) and boosted on days 11, 21, and 31 with (11H, m), 1.40–1.52 (2H, m), 1.56 (2H, m), 1.66 (2H, m), 1.70–
incomplete Freund adjuvant and 0.02 mg of immunogen by 1.92 (2H, m), 1.96 (3H, s), 2.01–2.06 (2H, m), 2.36 (2H, q), 2.93
emulsifying and intraperitoneal injection. Titers to 2-nonenal- (2H, t), 4.39 (1H, q), 4.53 (2H, q), 6.54 (1H, m), 6.66 (1H, d, J ϭ
modified BSA in the immunized mice sera were measured by an 16.2 Hz), 7.84 (1H, d, J ϭ 6.0 Hz), 8.61 (1H, d, J ϭ 6.0 Hz), 8.91
enzyme-linked immunosorbent assay (ELISA) (15). Two (1H, s); 13C NMR (150 MHz, MeOD): ␦ 14.3 (2C), 22.4, 23.4,
months after the initial immunization, the immunized mice 23.5 (2C), 29.5, 30.0, 30.2, 31.6, 32.1, 32.5, 32.6, 34.0, 34.2, 53.0,
were given an intraperitoneal boost of 0.06 mg/ml 2-nonenal- 62.0, 122.4, 129.2, 139.4, 142.2 (2C), 142.5, 160.7, 173.3, and
modified KLH. Three days later, the spleen cells from the 175.1.
immunized mice were fused with P3/U1 murine myeloma cells
Immunohistochemical Detection of 2-Nonenal Adducts in
in the presence of polyethylene glycol and cultured in hypox- Vivo—Male Wistar rats (Shizuoka Laboratory Animal Center,
anthine/amethopterin/thymidine selection medium. The cul- Shizuoka, Japan), weighing 130–150 g (6 weeks old), were used.
ture supernatants of the hybridoma were screened using an The animals received a single intraperitoneal injection of Fe3ϩ
-
ELISA, employing pairs of wells of microtiter plates on which NTA (15 mg of iron/kg of body weight) or repeated administra-
were absorbed 2-nonenal-treated BSA as the antigen (0.5 ␮g of tion as described previously (5). They were sacrificed by decap-
protein/well). After incubation with 100 ␮l of the hybridoma itation at 0, 1, 6, 24, and 48 h after a single injection or 3 weeks
supernatants, and with intervening washes with PBS/Tween, after the repeated administration. Both kidneys of each animal
the wells were incubated with alkaline phosphatase-conjugated were immediately removed. One of them was fixed in Bouin’s
goat anti-mouse IgG, followed by a substrate solution contain- solution, embedded in paraffin, cut in 3-mm thick slices, and
ing 0.5 mg/ml 1,2-phenylenediamine. Hybridoma cells corre- used for the immunohistochemical analyses by an avidin-biotin
sponding to the supernatants that were positive on the 2-non- complex method with alkaline phosphatase. Briefly, after
enal-modified BSA and negative on the native BSA were then deparaffinization with xylene and ethanol, normal rabbit serum
cloned by limited dilution. After repeated screening, four (Dako; diluted to 1:75) for the inhibition of the nonspecific
clones were obtained. Among them, clone 27Q4 showed the binding of the secondary antibody, a primary antibody (20
most significant recognition of the 2-nonenal-modified BSA. ␮g/ml), biotin-labeled rabbit anti-mouse IgG serum (Vector
Competitors were prepared by incubating 50 mM amino acid Laboratories; diluted to 1:300), and avidin-biotin complex
␣
␣
␣
derivatives, N -acetyl-L-lysine, N -acetylhistidine, or N -ace- (Vector; diluted to 1:100) were sequentially used. Procedures,
tylcysteine, in the presence or absence of 2-nonenal (50 mM), in using PBS or the IgG fraction (0.5 mg/ml) of the normal mouse
50 mM sodium phosphate buffer, pH 7.2, for 24 h at 37 °C.
serum instead of mAb 27Q4, showed no response or negligible
Isolation and Structural Analysis of Antigenic 2-Nonenal-Ly- positive responses.
sine Adducts—The reaction mixture (6 ml) contained 200 mM
Preparation of Isotope-labeled HPH-lysines—To prepare the
␣
␣ ⑀
trans-2-nonenal and 200 mM N -acetyl-L-lysine in 50 mM [U-13C6,15N2]HHP-lysine, 1 mM N -[U-13C6,15N2]Fmoc-N -
sodium phosphate buffer, pH 7.2. After incubation for 72 h at Boc-lysine was treated with 1 ml of trifluoroacetic acid for 2 h at
37 °C, a portion of the reaction mixture was analyzed by a room temperature, applied to a C-18 Sep-Pak column, and
reverse-phase HPLC using a Develosil ODS-HG-5 column eluted with 5 ml of methanol. The eluate was evaporated and
(4.6 ϫ 250 mm, Nomura Chemicals) equilibrated in a solution redissolved in 1 ml of 50 mM sodium phosphate buffer, pH 7.4.
of 5% acetonitrile in 0.01% trifluoroacetic acid. The chromato- The obtained N -[U-13C6,15N2]Fmoc-lysine was modified with
␣
graphic separation was performed by a gradient elution as fol- 1 mM 2-nonenal for 24 h at 37 °C and subsequently treated with
lows (solvent A was water and solvent B was acetonitrile, both 20% piperidine for 2 h at room temperature to remove the Fmoc
containing 0.01% trifluoroacetic acid): 0–10 min, linear gradi- moiety. The resulting mixture was purified by HPLC, carried
ent to 40% B; 10–40 min, 40% B; 40–41 min, linear gradient to out under the same HPLC conditions as already described.
100% B; 41–45 min, hold; flow rate ϭ 0.8 ml/min. The isolation LC/ESI/MS/MS analysis with MRM mode of the isotope-la-
and purification of the antigenic adducts (P-1 and P-2) were beled HPH-lysines showed that that the standards contained no
carried out under the same HPLC conditions and finally endogenous (nonlabeled) adducts (supplemental Fig. S1).
obtained 2.3 and 2.9 mg of P-1 and P-2, respectively. Their
LC/ESI//MS/MS—Mass spectrometric analyzes were per-
structures were characterized by LC/MS and 1H and 13C NMR. formed using a Quattro Ultima triple stage quadrupole mass
The LC/MS was measured by a Jasco PlatformII-LC instru- spectrometer (Waters-Micromass, Manchester, UK) equipped
ment. The LC/MS analysis was performed using a Develosil with an ESI probe and interfaced with a Shimadzu HPLC sys-
ODS-HG-5 column (4.6 ϫ 250 mm) eluted with a linear gradi- tem (Shimadzu, Kyoto, Japan). The sample injection volumes of
ent from 100% water containing 0.1% acetic acid to 100% ace- 20 ␮l each were separated on a Shim-pack VP-ODS 2.0 ϫ 150
tonitrile containing 0.1% acetic acid for 60 min at a flow rate of mm, 5-␮m column (Shimadzu) at a flow rate of 0.3 ml/min. A
15304 JOURNAL OF BIOLOGICAL CHEMISTRY
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Lipid Peroxidation Generates Protein-bound 2-Nonenal
discontinuous gradient was used by solvent A (H2O containing
0.1% formic acid) with solvent B (acetonitrile) as follows: iso-
cratic elution with 20% solvent B from 0 to 5 min; increasing to
50% solvent B from 5 to 20 min; increasing to 70% solvent B
from 20 to 25 min; and then isocratic elution with 70% sol-
vent B from 25 to 30 min. Mass spectrometric analyzes were
performed on line using ESI/MS/MS in the positive ion
mode with MRM mode (cone potential 35 eV/collision
energy 24 eV). The MRM transitions monitored were as fol-
lows: [U-13C6,15N2]HHP-lysine, m/z 397 3 261; cis- and trans-
HHP-lysine, m/z 389 3 260. L-Glutamate upon reaction with
2-nonenal gave similar MRM transitions but MRM transitions
(360 3 260) were detected in the reaction mixture of 2-non-
enal/glutamate; however, their elution times were completely
different from those of standard HHP-lysines (supplemen-
tal Fig. S2). The amount of each HHP-lysine adduct was quan-
tified by the ratio of the peak area of the target adducts and of
the HHP-lysine-stable isotope. The HHP-lysine standard con-
centration was determined by assay of the amino groups using a
2,4,6-trinitrobenzenesulfonic acid assay (16). Briefly, to 30 ␮l of
hydrolyzed HHP-lysine solution was added 30 ␮l of 4%
NaHCO3 and 30 ␮l of 0.1% 2,4,6-trinitrobenzenesulfonic acid.
The solution was allowed to react at 37 °C for 2 h, and 30 ␮l of
1% SDS and 15 ␮l of 1 N HCl were then added. The absorbance
of the solution was read at 340 nm. The standard curve (using
valine as a standard) was linear in the range 0–3.3 nmol of NH2.
QuanLynx (version 4.0) software (Waters-Micromass) was
used to create standard curves (supplemental Fig. S3) and to
calculate the adduct concentrations.
DiD-labeled AcLDL Uptake Assays—DiD (1,1Ј-dioctadecyl-
3,3,3Ј,3Ј-tetramethylindodicarbocyanine perchlorate)-labeled
AcLDL was prepared from human LDL according to the meth-
ods previously described for 1,1Ј-dioctadecyl-3, 3,3Ј,3Ј-tetra-
methylindocarbocyanine perchlorate labeling (17). CHO cells
that stably expressed CFP-tagged LOX-1 (18) were grown on
coverslips. For the cells transiently expressing CFP, CHO cells
suspended at a density of 1 ϫ 104 cells/ml were plated on a
coverslip 24 h prior to transfection. The cells (40% confluent)
were transfected with pECFP-N1 (Clontech) using Lipo-
fectamine reagent according to the manufacturer’s protocol
and then incubated for 48 h. Cells were treated with each con-
centration of adducts prepared in F-12 medium without fetal
calf serum for 10 min at 37 °C in a 5% CO2 humidified atmo-
sphere, followed by incubation with DiD-AcLDL (0.4 ␮g/cov-
erslip) for another 15 min. The cells were then fixed with 2%
formaldehyde. Each coverslip was inverted onto a glass slide
with a spacer, and the cells were examined using a Leica DM
IRE2 microscope (Leica Microsystems, Wetzler, Germany)
equipped with a ϫ100, NA 1.4 objective and a Cool SNAP-HG
digitalized cooled CCD camera (Roper Scientific, Trenton, NJ)
driven by MetaMorph software (Universal Imaging, Downing-
town, PA). The expression level of LOX-1 was examined by the
CFP fluorescence intensity using an E4 filter with excitation at
436 nm (7-nm bandpass) and a 470-nm long pass emission fil-
ter. Images were acquired at 0.2 s exposure time. The DiD-
AcLDL uptake was analyzed by the amount of the DiD-derived
fluorescence intensity using a Y5 filter, with excitation using a
620 nm (50-nm bandpass) and 700 nm (75-nm bandpass) emis-
sion filters. The images were acquired at a 2-s exposure.
For the LC/ESI/MS/MS analysis of the HHP-lysines in vitro,
the protein samples were treated with an equal volume of 20%
trichloroacetic acid. After centrifugation at 5,000 ϫ g for 10
min at 4 °C, the proteins were hydrolyzed in vacuo with 2 ml
of 6 N HCl for 24 h at 110 °C. The internal standard,
[U-13C6,15N2]HHP-lysine, was added to the samples prior to
the acid hydrolysis. After the acid hydrolysis, the samples were
partially separated with Oasis hydrophilic-lipophilic balance
(HLB) cartridges (Waters, Milford, MA). After the sample load-
ing, the HLB cartridges were washed with 2 ml of 50% metha-
nol, and the HHP-lysines were eluted with 2 ml of 80% metha-
nol. The samples were then dried, dissolved in methanol, and
subjected to LC/ESI/MS/MS analysis.
Images of over 100 cells from three independent experiments
were acquired. After subtracting the background emission level
with respect to 436 and 620 nm from the fluorescence image,
the fluorescence intensity of each cell was determined from the
average pixel value of the whole cell. The expression level of
LOX-1 was calculated from the CFP intensity, and the level of
uptake of AcLDL was calculated from the DiD intensity (18).
For each experimental condition, the level of AcLDL uptake
was averaged per expression level of LOX-1.
RESULTS
Monoclonal Antibody against Protein-bound 2-Nonenal—To
assess the reactivity of 2-nonenal to proteins, HSA (1 mg/ml)
was exposed to 2-nonenal (0–10 mM) under physiological con-
ditions in vitro, and changes in the amino acid composition of
the protein were examined by an amino acid analysis. As shown
in Fig. 1B, 2-nonenal preferentially reacted with the lysine res-
idues, indicating that 2-nonenal can covalently modify protein
spontaneously, and the likely mechanism is a nucleophilic
attack by the protein.
For the LC/ESI/MS/MS analyses of the HHP-lysines in vivo,
male ddY mice (Shizuoka Laboratory Animal Center, Shi-
zuoka), weighing 25–35 g (6 weeks of age), were used. Animals
received a single intraperitoneal injection of Fe3ϩ-NTA (5 mg
of iron/kg of body weight). They were sacrificed at 0, 1, 3, 6, and
24 h after the administration. Both kidneys of each animal were
immediately removed. The kidneys were homogenized in a Tef-
lon homogenizer in 10 volumes of PBS containing butylated
hydroxytoluene (1 mM). The homogenate was centrifuged at
18,000 ϫ g for 15 min, and the precipitates were hydrolyzed in
vacuo with 6 N HCl (2 ml) and [U-13C6,15N2]HHP-lysine for
24 h at 110 °C. The hydrolysates were then dried and dissolved
in methanol. After the acid hydrolysis, the samples were par-
tially separated with HLB cartridges and subjected to LC/ESI/
MS/MS analysis.
To determine whether modification of proteins by 2-nonenal
occurs in vivo, we attempted to develop a mAb against 2-non-
enal-modified protein. To this end, the keyhole limpet hemo-
cyanin that had been exposed to 2-nonenal was administered to
mice at 10-day intervals. During the preparation of the mAb,
hybridomas were selected by comparing the reactivities of the
culture supernatant to the 2-nonenal-modified BSA. Among
the four obtained clones, the clone 27Q4 showed the most dis-
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Lipid Peroxidation Generates Protein-bound 2-Nonenal
the antibody. We then determined
whether the 2-nonenal-modified
proteins contained the 27Q4
epitope. The mAb 27Q4 immuno-
reacted with not only 2-nonenal/
BSA, but also with 2-nonenal/HSA
and 2-nonenal/lactoglobulin, where-
as the unmodified proteins were not
recognized by the antibody (Fig.
1D), suggesting that the 2-nonenal-
modified proteins contained a com-
mon adduct structure recognized by
mAb 27Q4. The time course study
of formation of the 2-nonenal
epitope during the incubation of
BSA with 2-nonenal in vitro was
determined by direct ELISA using
mAb 27Q4. When BSA was incu-
bated with 1 mM 2-nonenal at 37 °C,
the immunoreactive materials rap-
idly increased within 4 h (Fig. 1E).
To identify the possible attacking
nucleophiles that form antigenic
structures, we investigated the reac-
tion of 2-nonenal with individual
amino acids likely to form stable
antigenic products. We studied
lysine, histidine, and cysteine, each
protected at the ␣-amino group as
an acetamide, leaving only the side-
chain nucleophile as a potential
reactant. As shown in Fig. 1F, bind-
ing of the 2-nonenal-modified pro-
tein to the antibody was hardly
inhibited by the reaction mixtures
of 2-nonenal/histidine and 2-non-
enal/cysteine but was significantly
inhibited by the reaction mixture of
2-nonenal/lysine, suggesting that
the mAb might recognize a 2-non-
enal-lysine adduct as the epitope.
None of the original amino acid
derivatives, histidine, cysteine, and
lysine, cross-reacted with mAb
27Q4.
FIGURE 1. Monoclonal antibody against protein-bound 2-nonenal. A, chemical structure of 2-nonenal.
B, loss of lysine residues in protein treated with 2-nonenal. HSA (1 mg/ml) was incubated with 0–10 mM
2-nonenal in 50 mM sodium phosphate buffer, pH 7.2, at 37 °C. C, immunoreactivity of mAb 27Q4 to aldehyde-
modified proteins. Affinity of mAb 27Q4 was determined by a direct ELISA using aldehyde-modified BSA as
the absorbed antigen. A coating antigen (0.5 ␮g/well) was prepared by incubating 1 mg of BSA with 1 mM
aldehydes in 1 ml of 50 mM sodium phosphate buffer, pH 7.2, for 24 h at 37 °C. ACR, acrolein; CRA, crotonalde-
hyde; MDA, malondialdehyde; HNE, 4-hydroxy-2-nonenal; ONE, 4-oxo-2-nonenal. D, immunoreactivity of mAb
27Q4to2-nonenal-modifiedproteins. Acoatingantigen(0.005–0.5 ␮g/well)waspreparedbyincubating1mg
of protein with 1 mM 2-nonenal in 1 ml of 50 mM sodium phosphate buffer, pH 7.2, for 24 h at 37 °C. Proteins are
as follows: open square, native lactoglobulin; closed triangle, 2-nonenal-modified HSA; closed circle, 2-nonenal-
modified BSA; closed square, 2-nonenal-modified lactoglobulin. E, time-dependent increase in the immunore-
activity of protein treated with 2-nonenal. HSA (1 mg/ml) was incubated with 1 mM 2-nonenal in 50 mM sodium
phosphate buffer, pH 7.2, at 37 °C. F, competitive ELISA analysis with the reaction mixtures of amino acid
derivatives and 2-nonenal. Competitors were prepared by incubating 50 mM amino acid derivatives, N␣-acetyl-
lysine, N␣-acetylhistidine, or N␣-acetylcysteine, in the presence or absence of 2-nonenal (50 mM), in 50 mM
sodium phosphate buffer, pH 7.2, for 24 h at 37 °C. Competitors are as follows: open circle, N␣-acetyllysine; open
triangle, N␣-acetylhistidine; open square, N␣-acetylcysteine; closed circle, 2-nonenal/N␣-acetyllysine; closed tri-
angle, 2-nonenal/N␣-acetylhistidine; closed square, 2-nonenal/N␣-acetyllysine.
Isolation and Structural Char-
acterization of the Antigenic 2-
Nonenal-Lysine Adducts—Because
characterization of the ability of
antibodies to recognize specific
molecular targets in their native
three-dimensional conformation is
critical to the use of these reagents,
tinctive recognition of the 2-nonenal-modified BSA. The spec- we sought to identify the intrinsic epitope recognized by mAb
ificity of the obtained antibody was characterized. As shown in 27Q4. To identify the 2-nonenal-lysine adduct recognized by
Fig. 1C, the monoclonal antibody showed a strong immunore- mAb 27Q4, the immunoreactivity with the reaction products of
␣
activity not only with 2-nonenal but also with 2-octenal and 2-nonenal with N -acetyllysine was characterized. As shown in
2-heptenal, whereas the unmodified BSA was not recognized by Fig. 2A, the ELISA analysis of the HPLC fractions for immuno-
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Lipid Peroxidation Generates Protein-bound 2-Nonenal
1.52 ppm; ␦C 23.4 or 23.5 ppm), the
Hf proton with the Hg proton (␦H
2.01–2.06 ppm; ␦C 31.6 ppm), and
the Hg proton with the Hh proton
(␦H 4.53 ppm; ␦C 62.0 ppm). The
13C NMR spectrum of the product
showed five signals (␦C 129.2, 139.4,
142.2 (two carbon signals), and
160.7 ppm) in the aromatic carbon
region. In the HMBC spectrum,
cross-peaks between the ␦-proton
of the lysine residue, Hh, and car-
bons (Hh-Ci (142.2 ppm) and
Hh-Cm (142.2 ppm)), cross-peaks
between Hi (8.61 ppm, J ϭ 6.0 Hz)
and carbons (Hi-Ch, Hi-Cj (129.2
ppm) and Hi-Ck (160.7 ppm)), and
cross-peaks between Hm (8.91
ppm) with carbons (Hm-Ch,
Hm-Ci, Hm-Ck, and Hm-Cl (139.4
ppm)). The coupling constant and
HMBC spectrum indicated that Hi
and Hj (7.84 ppm, J ϭ 6.0 Hz) are
adjacent. Moreover, in the HMBC
spectrum, the cross-peaks Hj and
carbons (Hj-Ci, Hj-Ck, and Hj-Cl)
are observed. The Ck and Cl were
missing from the distortionless
enhancement by polarization trans-
fer spectrum. This indicates that Ck
FIGURE 2. Isolation and structural characterization of the antigenic 2-nonenal-lysine adducts. A, compet-
and Cl are quaternary carbons.
These results suggest that the aro-
matic part of this product seemed to
be a pyridinium ring, in which Ck
and Cl are substituted. The coupling
constant for the vinyl proton (Ht,
itive ELISA analysis of HPLC fractions for immunoreactivity with mAb 27Q4. The reaction was performed by
incubating 200 mM N␣-acetyllysine with 200 mM 2-nonenal in 1 ml of 50 mM sodium phosphate buffer, pH 7.2,
at 37 °C for 24h. Solid line, profileofUVabsorbanceat200–650nm. Bar, competitiveELISA analysis. B, chemical
structures of cis-HHP-lysine and (P-1) and trans-HHP-lysine (P-2). C, competitive ELISA analysis with cis- and
trans-HHP-lysine. Competitors are as follows: closed circle, N␣-acetyl-trans-HHP-lysine (P-2); closed triangle,
N␣-acetyl-cis-HHP-lysine (P-1); open circle, N␣-acetyllysine.
reactivity with mAb 27Q4 showed that the antibody primarily 6.66 ppm) was J ϭ 16.2 Hz for the trans-proton. In the HMBC
had an immunoreactivity with two fractions, P-1 and P-2. The spectrum, the cross-peaks between the Ht and carbons (Ht-Ck,
LC/MS total ion current analysis of P-2 detected one product, Ht-Cl, and Ht-Cm) and cross-peaks between the Hn and car-
which showed a 242-Da increase in the mass value of the unmod- bons (Hn-Cj, Hn-Ck, and Hn-Cl) were observed. These results
ϩ
ified lysine derivative and gave the [M ϩ H] peak at m/z 431.0 indicated that the substituted quaternary carbons, Ck and Cl,
(supplemental Fig. S4), suggesting that it was composed of one were adjacent to Cn and Ct, respectively. The residual parts of
␣
molecule of N -acetyllysine and two molecules of 2-nonenal.
P-2 were the (E)-1-heptenyl and hexyl groups. The signals at
Because P-2 was a major immunoreactive fraction, we first 0.89–0.94 ppm were assigned to Hs and Hz. The correlation
attempted to identify this product. To characterize the chemi- spectroscopy experiments were performed to determine the
cal structure, isolation by HPLC on the reverse-phase column alkyl chain connectivity. With the correlation spectroscopy
was carried out. After purification, the structure of the products spectrum, starting from the methyl groups, the spin systems for
was characterized by an NMR analysis. The assignments of pro- an (E)-1-heptenyl chain and a hexyl chain were determined.
tons and carbons were based on the result of the HMBC and After establishing the connectivities for the alkyl chains, the
distortionless enhancement by polarization transfer experi- HMBC experiments were performed. Based on these charac-
ments. Compared with the 13C NMR and H NMR spectra teristics, it was determined that P-2 was the novel 2-nonenal-
1
␣
␣
⑀
between N -acetyllysine and the product (supplemental Figs. lysine adduct, N -acetyl-N -3-[(hept-1-enyl)-4-hexylpyridini-
␣
S5–S10),the Cb (173.3 ppm), the Cd (175.1 ppm), the Ha proton um]lysine (N -acetyl-HHP-lysine) (Fig. 2B). The LC/MS total
(1.96 ppm), and the Hc proton (␦H 4.39 ppm; ␦C 53.0 ppm) ion current analysis of P-1 detected one product, which showed
remained in the 13C NMR and 1H NMR spectra of the product. a 242-Da increase in the mass value of the unmodified lysine
ϩ
The correlation spectroscopy spectrum showed correlations derivative and gave the [M ϩ H] peak at m/z 430.9
between the Hc proton with the He (␦H 1.70–1.92 ppm; ␦C (supplemental Fig. S11), suggesting that P-1 is a cis isomer of
␣
32.1 ppm) proton, the He proton with the Hf proton (␦H 1.40– N -acetyl-HHP-lysine. To confirm the structure of P-1, the
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Lipid Peroxidation Generates Protein-bound 2-Nonenal
in some of the renal proximal tubular cells 1, 6, 24, and 48 h (Fig.
3, B–E) after the administration of 15 mg of iron/kg of body
weight of Fe3ϩ-NTA. No immunoreactivity was observed after
the repeated (3 weeks) administrations of Fe3ϩ-NTA (Fig. 3F).
These patterns of distribution in the rat kidney are consistent
with those of the distribution of membrane lipid peroxidation
products and their conjugates with cytosolic proteins, suggest-
ing a correlation between the production of the 2-nonenal/2-
alkenals and oxidative stress. Pre-absorption of the antibody
with HHP-lysine completely abolished the immunostaining
(data not shown).
LC/ESI/MS/MS Analysis of HHP-lysine—To obtain direct
evidence for the formation of the antigenic HHP-lysine adducts
in vivo, we first attempted to establish a method for the detec-
tion of adducts using LC/ESI/MS/MS. The acid hydrolysis of
N -acetyl-HHP-lysine gave a single product, which was identi-
fied to be the expected de-acetylated product (HHP-lysine)
(supplemental Figs. S18 and S19). Collision-induced dissocia-
tion of the HHP-lysine produced relevant daughter ions at m/z
344, 260, 130, and 84 (Fig. 4A). These ions were applied to the
structure shown in Fig. 4B. The product ions at m/z 84 and 130
were confirmed to be the product ions of a lysine moiety, and
the ions at m/z 344 and 389 originated from the HHP-lysine.
Fig. 4C demonstrates the LC/ESI/MS/MS analysis of the HHP-
lysine (0–10 nM) in the positive ion mode using MRM between
␣
ϩ
the transition from the protonated parent ion [M ϩ H] to the
FIGURE 3. Immunohistochemistry of renal cortex with mAb 27Q4 (؋400).
A, untreated control; B, 1 h; C, 6 h; D, 24 h; E, 48 h after single intraperitoneal
injection of Fe3ϩ-NTA; F, 3 weeks after repeated intraperitoneal injection of
Fe3ϩ-NTA. Refer to “Experimental Procedures” for details.
characteristic daughter ion (m/z 389.6 3 260.2), allowing
detection of both the cis- and trans-HHP-lysines. Chromato-
grams for the internal standard are shown in supple-
mental Fig. S20. Using the LC/ESI/MS/MS technique, we
attempted to detect HHP-lysine in the 2-nonenal-modified
HSA. No adducts were detected in the native HSA, whereas the
treatment of HSA with 0–10 mM 2-nonenal in 50 mM sodium
phosphate buffer, pH 7.2, for 24 h at 37 °C gave 0.61 and 0.86
mol of the cis- and trans-HHP-lysines, respectively, per mol of
protein (Fig. 4, D and E).
NMR experiments were performed (supplemental Figs.
S12–S17) and showed that the coupling constant for the vinyl
proton (6.51 ppm) was J ϭ 11.6 Hz for the cis proton. Selected
ion-current chromatograms obtained from the LC/MS analysis
␣
showed that, when 200 mM N -acetyllysine was incubated with
200 mM 2-nonenal for 72 h, P-1 and P-2 were formed in the
␣
In Vivo Formation of HHP-lysine via Lipid Peroxidation—It
is known that 2-nonenal is generated by the lipid peroxidation
of unsaturated fatty acids, such as palmitoleic acid (6). To
examine the involvement of the lipid peroxidation during the
formation of HHP-lysine, we sought to detect HHP-lysine in
the Cu2ϩ-oxidized LDL using LC/ESI/MS/MS. Incubation of
LDL with Cu2ϩ led to the oxidation of the LDL as assessed by
the formation of thiobarbituric acid-reactive substances (data
not shown). As shown in Fig. 5A, the Cu2ϩ-induced peroxida-
tion of LDL dramatically enhanced the formation of HHP-ly-
sine with the levels of the trans-HHP-lysine exceeding those of
the cis-HHP-lysine by 2-fold.
ratio of 1:1.4, indicating that the cis isomer of N -acetyl-HHP-
lysine can be formed from the trans-2-nonenal-mediated
reaction.
As shown in Fig. 2C, the antibody showed a specificity toward
both P-1 and P-2 but was rather specific to P-2. Approximately
50 pmol of P-2/well (100 ␮l) caused a 50% inhibition of the
antibody binding to the 2-nonenal-modified protein, whereas
at least 100-fold higher concentrations of P-1 were necessary
for the same inhibition.
Formation of Immunoreactive Materials with mAb 27Q4 in
Vivo—We next sought to explore whether immunoreactive
materials with mAb 27Q4 are formed in vivo. For this purpose,
we used Fe3ϩ-NTA-treated rats, an established animal model
of oxidant injury to the kidney. It has been established that
Fe3ϩ-NTA induces acute renal proximal tubular necrosis, a
consequence of oxidative tissue damage, that eventually leads
to a high incidence of renal adenocarcinoma in rodents (11).
Previously, we have shown that the levels of 2-nonenal mark-
edly increase after the administration of Fe3ϩ-NTA (5). In the
control rat kidney (Fig. 3A), an almost negligible level of immu-
To further examine which fatty acids are involved in the for-
mation of the HHP-lysine, several unsaturated fatty acids were
incubated with an iron/ascorbate-mediated free radical gener-
ating system in the presence of HSA, and HHP-lysine generated
on the protein molecule was analyzed by LC/ESI/MS/MS fol-
lowing acid hydrolysis. As shown in Fig. 5B, the iron/ascorbate-
mediated oxidation of palmitoleic acid and ␻6-polyunsaturated
fatty acids, such as linoleic acid, ␥-linolenic acid, and arachi-
noreactivity was observed. The immunoreactivities were found donic acid, in the presence of HSA resulted in a dramatic
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Lipid Peroxidation Generates Protein-bound 2-Nonenal
that each of the appropriate transi-
tions showed co-chromatography
(supplemental Figs. S21 and S22). In
addition, the ratio of the different
parent 3 daughter transitions was
also checked (Fig. 5C). As shown in
Fig. 5D, the levels of both the cis-
and trans-HHP-lysines markedly
increased 3 h after the administra-
tion of Fe3ϩ-NTA. The yields of the
cis- and trans-HHP-lysines at 3 h
were 366.5 and 357.8 fmol/g wet tis-
sue weight, respectively.
Recognition of the 2-Nonenal-Ly-
sine Adduct by LOX-1—Finally, we
evaluated the biological implication
of the 2-nonenal modification of
protein. To this end, based on the
previous findings that a scavenger
receptor LOX-1 might be involved
in the processing of aldehyde-mod-
ified proteins (19) and that, among
the aldehyde-treated proteins, the
2-nonenal-modified protein can be
very efficiently incorporated into
the LOX-1-overexpressing cells,3
we sought to examine if LOX-1
recognizes the 2-nonenal-lysine
adduct. AcLDL was used as an alter-
native ligand to the oxidized LDL,
which shows a comparable affinity
to LOX-1 (20) to avoid ambiguous
effects from variations in the extent
of the oxidized LDL oxidation (21,
22). In this assay, the amount of
incorporated DiD-labeled AcLDL
directly reflected the binding activ-
ity of LOX-1, which was confirmed
by an independent analysis as
described previously (23). The rate
FIGURE 4. LC/ESI/MS/MS analysis of HHP-lysine. A, collision-induced dissociation of the [M ϩ H]ϩ of trans-
of DiD-AcLDL taken up by CHO
stably expressing CFP/LOX-1 was
assessed by a fluorescence micro-
scope equipped with a cooled CCD
camera. In CHO cells transfected
with pECFP-N1, the CFP signal was
detected in the cytoplasm, and no
DiD-AcLDL uptake was detected
HHP-lysine at m/z 389.5 at a collision energy of 25 V. B, proposed structures of individual ions. C, LC/ESI/MS/MS
analysis of authentic cis- and trans-HHP-lysines. The ion current tracing of HHP-lysine using LC/ESI/MS/MS with
MRM is shown. D, LC/ESI/MS/MS analysis of cis- and trans-HHP-lysines generated in the 2-nonenal-modified
HSA. The ion current tracing of cis- and trans-HHP-lysines using LC/ESI/MS/MS with MRM is shown. E, dose-de-
pendent formation of cis- and trans-HHP-lysines in the 2-nonenal-modified HSA. Symbols used are as follows:
open circle, cis-HHP-lysine; closed circle, trans-HHP-lysine. D and E, HSA (1.0 mg/ml) was incubated with 2-non-
enal (0–10 mM) in 50 mM sodium phosphate buffer, pH 7.2, for 24 h at 37 °C. The native and modified HSA were
analyzed by LC/ESI/MS/MS with MRM mode followed by acid hydrolysis.
increase in the levels of HHP-lysine with levels of trans-HHP- (Fig. 6A, top). The stable cell line expressed a moderate amount
lysine exceeding those of cis-HHP-lysine by 3–9-fold. of LOX-1, and a fluorescence derived from DiD-AcLDL was
We then sought to explore whether HHP-lysine is formed in observed (Fig. 6A, middle). The effect of the 2-nonenal-modi-
the kidney of animals exposed to Fe3ϩ-NTA in vivo. Kidneys fied BSA upon DiD-AcLDL uptake through LOX-1 was exam-
from the untreated and treated animals collected 1 and 3 h after ined in the competition assay to evaluate the LOX-1 recogni-
the administration of Fe3ϩ-NTA were analyzed for HHP-lysine tion of modified proteins. The native BSA failed to show any
using LC/ESI/MS/MS. The MRM transitions monitored were inhibitory effects (date not shown), whereas the protein modi-
as follows: [U-13C6,15N2]HHP-lysine, m/z 397 3 261; cis- and
trans-HHP-lysine, m/z 389 3 260. Multiple distinct MRM
channels (parent 389.1 3 344.1, 260.2, 130.0, and 83.9) showed 3 S. Machida and K. Uchida, manuscript in preparation.
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Lipid Peroxidation Generates Protein-bound 2-Nonenal
reactive aldehydes derived from
lipid peroxidation have been impli-
cated as causative agents in cyto-
toxic processes initiated by the
exposure of biological systems to
oxidizing agents (2). On the basis of
a large number of reports concern-
ing the detection of lipid peroxida-
tion-specific adducts as biomarkers
in human diseases, there is no doubt
that the steady-state levels of lipid
peroxidation products increase in
pathophysiological states associated
with oxidative stress. Considerable
progress has also recently been
made toward understanding the
mechanisms of action of lipid per-
oxidation products. The quantita-
tive and analytical importance of
lipid peroxidation-specific adducts
has prompted the development of
methods to specifically analyze
these adducts to understand their
chemical nature, formation path-
way, and distribution level in vivo.
The results presented herein
show that 2-nonenal, a body odor-
ant originating from lipid peroxida-
tion, covalently modifies proteins. A
spontaneous chemical reaction was
observed in vitro. In addition, we
obtained a murine monoclonal anti-
body, mAb 27Q4, that clearly distin-
guished the 2-nonenal-modified
protein from the native protein.
This antibody appeared to be highly
specific for the protein-bound
2-alkenals, including 2-heptenal,
2-octenal, and 2-nonenal. Using the
antibody, the formation of the
2-nonenal-modified proteins in vivo
was tested in the kidney of rats
exposed to Fe3ϩ-NTA. The iron
FIGURE 5. In vivo formation of HHP-lysine via lipid peroxidation. A, LC/ESI/MS/MS analysis of cis- and
trans-HHP-lysines in oxidized LDL (oxLDL). LDL (0.5 mg) was incubated with 5 ␮M Cu2ϩ in 1 ml of PBS at 37 °C.
The native LDL and oxidized LDL were analyzed by LC/ESI/MS/MS with MRM mode followed by acid hydrolysis.
B, LC/ESI/MS/MS analysis of cis- and trans-HHP-lysines in protein exposed to lipid peroxidation. The metal-
catalyzed oxidation of unsaturated fatty acids in the presence of HSA was performed by incubating HSA (1
mg/ml)with2mM unsaturatedfattyacidsinthepresenceof50␮M Fe2ϩ and1mM ascorbicacidin1mlof50mM
sodium phosphate buffer, pH 7.2, in atmospheric oxygen at 37 °C. The native and modified HSA were analyzed
by LC/ESI/MS/MS with MRM mode followed by acid hydrolysis. The abbreviations used are as follows: PA,
palmitoleic acid; LA, linoleic acid; LNA, ␥-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA,
docosahexaenoic acid. C, LC/ESI/MS/MS analysis of cis-[U-13C6,15N2]- and trans-[U-13C6,15N2]HHP-lysines in the
kidneys of mice exposed to Fe3ϩ-NTA. The ion current tracing of cis-[U-13C6,15N2]- and trans-[U-13C6,15N2]HHP-
lysines LC/ESI/MS/MS with MRM is shown. D, LC/ESI/MS/MS analysis of cis- and trans-HHP-lysines in the kidneys
of mice exposed to Fe3ϩ-NTA. The ion current tracing of HHP-lysine using LC/ESI/MS/MS with MRM is shown.
Panel a, 0 h after administration; panel b, 1 h after administration; panel c, 3 h after administration; panel d,
internal standards.
fied with 2-nonenal significantly inhibited the uptake of AcLDL chelate was originally used for an experimental model of iron
(Fig. 6A, bottom). Finally, we evaluated the purified cis- and overload (24). Repeated intraperitoneal injections of Fe3ϩ
-
trans-HHP-lysines for their recognition by LOX-1 in the com- NTA were reported to induce acute and subacute renal proxi-
petition assay, and we observed that they served as a ligand for mal tubular necrosis and a subsequent high incidence (60–
LOX-1, as evidenced by their ability to effectively compete with 92%) of renal adenocarcinoma in male rats and mice (25, 26). A
AcLDL (Fig. 6, B–D). There was no effect by the 1% DMSO single injection of Fe3ϩ-NTA causes a number of time-depen-
carried from the stock solution of the HHP-lysine (data not dent morphological alterations in the structure and the func-
shown).
tion of the renal proximal tubular cells and their mitochondria.
During the early stage of injury, typical cellular changes are the
loss of the brush border, cytoplasmic vesicles, mitochondrial
DISCUSSION
Lipid peroxidation proceeds by a free radical chain reaction disorganization, and dense cytoplasmic deposits in the proxi-
mechanism and yields lipid hydroperoxides as major initial mal tubular cells. Most of the damaged epithelia show the typ-
reaction products. Subsequently, the decomposition of the lipid ical appearance of necrotic cells, and more than half of the
hydroperoxides generates a number of breakdown products proximal tubular cells are gone. It has been suggested that oxi-
that display a wide variety of damaging actions. A number of dative stress is one of the basic mechanisms of Fe3ϩ-NTA-in-
15310 JOURNAL OF BIOLOGICAL CHEMISTRY
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Lipid Peroxidation Generates Protein-bound 2-Nonenal
duced acute renal injury and is closely associated with renal
carcinogenesis (27, 28). The present in vivo study has shown
that lipid peroxidation generates 2-nonenal covalently bound
to proteins in the renal proximal tubules of rats treated with
Fe3ϩ-NTA (Fig. 3). To the best of our knowledge, this is the first
report of the in vivo formation of protein-bound 2-nonenal/2-
alkenals in the target organ of the carcinogenic protocol. It was
also striking that the immunoreactivities with mAb 27Q4 were
detected even 48 h after the administration of Fe3ϩ-NTA (Fig.
3E). Long retention of this aldehyde may play a role in the Fe3ϩ
-
NTA-induced renal carcinogenesis.
Upon investigation of an antigenic adduct recognized by the
antibody (mAb 27Q4), we unexpectedly identified novel lysine-
pyridinium adducts, cis- and trans-HHP-lysines. Of interest,
the monoclonal antibodies raised against protein-bound 2-
alkenals, such as acrolein and crotonaldehyde, recognize pyri-
dinium-containing adducts as the major epitopes (29, 30). It is
likely that, due to the placement of a fixed, positive charge on
the ⑀-amino group, the pyridinium-containing adducts could
be an important immunological epitope generated in 2-alkenal-
modified proteins. The formation of the 3,4-substituted pyri-
dinium adducts has also been reported to be a dominant path-
way for modification of the primary amine with 2-alkenals, such
as 2-hexenal and 2-octenal (31, 32). Based on these studies,
HHP-lysine is likely to be formed through the formation of the
2-nonenal-lysine Schiff base (Fig. 7). After the formation of the
Schiff base adduct, the C3 position of the initial Schiff base may
be attacked by the C2 of the enolate anion from the second
aldehyde, followed by dehydration and cyclization to form the
isomeric pyridinium adduct. On the other hand, trans-cis
isomerization of ␣,␤-unsaturated compounds is known to be
catalyzed by amines (addition-elimination). Thus, the isomer-
ization required for the formation of cis-HHP-lysine may occur
at the stage of the free 2-nonenal or following Schiff base
formation.
Due to the fact that the core structures of the pyridinium-
containing lysine adducts are resistant to the conventional acid
hydrolysis of proteins, we established a highly sensitive method
for the detection of cis- and trans-HHP-lysine using LC/ESI/
MS/MS. In vitro studies of the detection of HHP-lysine demon-
strated that the 2-nonenal-lysine adducts were generated in the
oxidized LDL (Fig. 5A). In addition, substantial amounts of the
2-nonenal-lysine adducts, mainly the trans-HHP-lysine, were
detected in the metal-catalyzed peroxidation of unsaturated
FIGURE 6. Effect of the 2-nonenal-lysine adducts on ligand uptake by
LOX-1. A, CHO cells transfected with vector carrying only CFP (top). LOX-1
expression level and AcLDL uptake ability of stable cell line (middle) is shown.
Effect of 2-nonenal-modified BSA (4 ␮g/ml) on AcLDL uptake (bottom) is
shown. B, effect of cis-HHP-lysine (10–100 ␮M) on AcLDL uptake. C, effect of
trans-HHP-lysine (10–100 ␮M) on AcLDL uptake. Images were acquired using
ϫ100 objective. Panels are as follows: left, phase contrast; middle, CFP/LOX-1
(excitation at 436 nm; exposure time, 0.2 s); right, DiD-AcLDL (excitation at
620 nm; exposure time, 2 s). D, quantitative measurement of fluorescence
intensity of each cell. CHO cells stably expressing CFP/LOX-1 were treated
with each concentration of adducts and followed by incubation with DiD-
AcLDL. The cell-associated fluorescence intensity derived from CFP and DiD
(pixel value) was determined as described under “Experimental Procedures.”
The specific uptake was expressed as the fluorescence intensity derived from
DiD (pixel value) per the fluorescence intensity derived from CFP (pixel value)
of each cell. The values represent the means of over 100 cells from three
independent experiments.
MAY 14, 2010•VOLUME 285•NUMBER 20
JOURNAL OF BIOLOGICAL CHEMISTRY 15311

Lipid Peroxidation Generates Protein-bound 2-Nonenal
cis- and trans-HHP-lysines competed for the uptake of AcLDL.
These data suggested that LOX-1 might serve as a receptor for
the 2-nonenal-lysine adducts generated on oxidized LDL mol-
ecules. Data from the mesenteric vein injection of an aldehyde-
modified protein also suggest that LOX-1 may be involved to a
certain degree in these processes (38). Thus, it is likely that
LOX-1 is involved in the recognition of 2-nonenal-modified
proteins. However, the possibility still exists for other receptors
to be involved in the processing of 2-nonenal-modified
proteins.
In summary, to assess the formation of 2-nonenal generation
under oxidative stress in vivo, we raised a new murine mono-
clonal antibody, mAb 27Q4, against the 2-nonenal-modified
KLH. The model system provides a detailed structural charac-
terization of the epitopes, the cis- and trans-HHP-lysines,
formed during the reaction of the lysine side-chain amino
groups with 2-nonenal. We proved that the immunoreactive
materials with mAb 27Q4 were indeed generated in an animal
model of oxidative stress in vivo. In addition, using LC/ESI/MS/
FIGURE 7. Proposed mechanism for the formation of HHP-lysine.
fatty acids in the presence of protein (Fig. 5B). These data sug- MS, the biological levels of the 2-nonenal-lysine adduct in vitro
gest that covalent modification of proteins by 2-nonenal, gen- and in vivo were accurately estimated. Finally, we examined the
erating the pyridinium-containing lysine adducts, could have involvement of the scavenger receptor LOX-1 in the recogni-
diagnostic applications. Because oxidative degradation of tion of 2-nonenal-modified proteins and established that the
unsaturated fatty acids, accelerated by lipid peroxides, may be receptor recognized the HHP-lysine adducts as a ligand. The
involved in the formation of 2-nonenal, resulting in deteriora- present results not only offer structural insights into protein
tion of the body odors for the middle-aged and the elderly (6), modification by lipid peroxidation products but also provide a
plasma or red blood cell levels of a long-lived 2-nonenal adduct platform for the chemical analysis of protein-bound aldehydes
could provide a measure of past exposure and aid in the man- in vitro and in vivo.
agement of one’s health condition. Such a reporter function
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MAY 14, 2010•VOLUME 285•NUMBER 20
JOURNAL OF BIOLOGICAL CHEMISTRY 15313

Lipids:
Lipid Peroxidation Generates Body Odor
Component trans-2-Nonenal Covalently
Bound to Protein in Vivo
Kousuke Ishino, Chika Wakita, Takahiro
Shibata, Shinya Toyokuni, Sachiko Machida,
Shun Matsuda, Tomonari Matsuda and Koji
Uchida
J. Biol. Chem. 2010, 285:15302-15313.
doi: 10.1074/jbc.M109.068023 originally published online March 8, 2010
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