Portal de Periódicos da CAPES

Formation of N ε-(Hexanonyl)lysine in Protein Exposed to Lipid Hydroperoxide

1999; Elsevier BV; Volume: 274; Issue: 29 Linguagem: Inglês

10.1074/jbc.274.29.20406

1083-351X

Yoji Kato, Yoko Mori, Yuko Makino, Yasujiro Morimitsu, Sadayuki Hiroi, Toshitsugu Ishikawa, Toshihiko Osawa,

Electron Spin Resonance Studies

The objectives of this study were to estimate the structure of the lipid hydroperoxide-modified lysine residue and to prove the presence of the adducts in vivo. The reaction of lipid hydroperoxide toward the lysine moiety was investigated employingN-benzoyl-glycyl-l-lysine (Bz-Gly-Lys) as a model compound of Lys residues in protein and 13-hydroperoxyoctadecadienoic acid (13-HPODE) as a model of the lipid hydroperoxides. One of the products, compound X, was isolated from the reaction mixture of 13-HPODE and Bz-Gly-Lys and was then identified as N-benzoyl-glycyl-N ε-(hexanonyl)lysine. To prove the formation of N ε-(hexanonyl)lysine, named HEL, in protein exposed to the lipid hydroperoxide, the antibody to the synthetic hexanonyl protein was prepared and then characterized in detail. Using the anti-HEL antibody, the presence of HEL in the lipid hydroperoxide-modified proteins and oxidized LDL was confirmed. Furthermore, the positive staining by anti-HEL antibody was observed in human atherosclerotic lesions using an immunohistochemical technique. The amide-type adduct may be a useful marker for the lipid hydroperoxide-derived modification of biomolecules. The objectives of this study were to estimate the structure of the lipid hydroperoxide-modified lysine residue and to prove the presence of the adducts in vivo. The reaction of lipid hydroperoxide toward the lysine moiety was investigated employingN-benzoyl-glycyl-l-lysine (Bz-Gly-Lys) as a model compound of Lys residues in protein and 13-hydroperoxyoctadecadienoic acid (13-HPODE) as a model of the lipid hydroperoxides. One of the products, compound X, was isolated from the reaction mixture of 13-HPODE and Bz-Gly-Lys and was then identified as N-benzoyl-glycyl-N ε-(hexanonyl)lysine. To prove the formation of N ε-(hexanonyl)lysine, named HEL, in protein exposed to the lipid hydroperoxide, the antibody to the synthetic hexanonyl protein was prepared and then characterized in detail. Using the anti-HEL antibody, the presence of HEL in the lipid hydroperoxide-modified proteins and oxidized LDL was confirmed. Furthermore, the positive staining by anti-HEL antibody was observed in human atherosclerotic lesions using an immunohistochemical technique. The amide-type adduct may be a useful marker for the lipid hydroperoxide-derived modification of biomolecules. During lipid peroxidation, biomolecules such as proteins or aminolipids can be covalently modified by lipid decomposition products. For the case of aliphatic aldehydes (alkanals) such as 1-hexanal or 1-nonanal, the N ε-amino groups of the lysine residues in protein can be modified through the formation of a Schiff base. α,β-Unsaturated aldehydes (alkenals) such as acrolein or 4-hydroxy-2-nonenal react with lysine, cysteine, and histidine through a Michael-type addition (1Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5903) Google Scholar, 2Uchida K. Kanematsu M. Sakai K. Matsuda T. Hattori N. Mizuno Y. Suzuki D. Miyata T. Noguchi N. Niki E. Osawa T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4882-4887Crossref PubMed Scopus (551) Google Scholar). On the other hand, lipid hydroperoxide might covalently react with protein without serious decomposition of its structure (3Fruebis J. Parthasarathy S. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10588-10592Crossref PubMed Scopus (108) Google Scholar). Keto fatty acid (4Kühn H. Wiesner R. Rathmann J. Schewe T. Eicosanoids. 1991; 4: 9-14PubMed Google Scholar), which is one of the products by lipoxygenase reaction, can also react with protein and amino acids as previously suggested (5Fukuzawa K. Kishikawa K. Tokumura A. Tsukatani H. Shibuya M. Lipids. 1985; 20: 854-861Crossref PubMed Scopus (32) Google Scholar, 6Bull A.W. Bronstein J.C. Earles S.M. Blackburn M.L. Life Sci. 1996; 25: 2355-2365Crossref Scopus (12) Google Scholar, 7Blackburn M.L. Ketterer B. Meyer D.J. Juett A.M. Bull A.W. Chem. Res. Toxicol. 1997; 10: 1364-1371Crossref PubMed Scopus (21) Google Scholar). In addition, the pyrrole compounds from long chain epoxides and lysine were identified (8Hidalgo F.J. Zamora R. J. Lipid Res. 1995; 36: 725-735Abstract Full Text PDF PubMed Google Scholar). However, the mechanism of lipid hydroperoxide-derived protein modification is not so clear. To estimate the structure after lipid hydroperoxide-derived lysine modification, the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) 1The abbreviations 13-HPODE13-hydroperoxyoctadecadienoic acidBz-Gly-LysN-benzoyl-glycyl-l-lysineAGLMEN-acetyl-glycyl-l-lysine methyl esterNHSN-hydroxysuccinimideHELN ε-(hexanonyl)lysineLDLlow density lipoproteinsulfo-NHSN-hydroxysulfosuccinimideEDC1-ethyl-3-(3-dimethylaminopropyl)carbodiimideKLHkeyhole limpet hemocyanin15-HPETE15-hydroperoxyeicosatetraenoic acidBSAbovine serum albuminLC-MSliquid chromatography-mass spectrometryPBSphosphate-buffered salineAAPH2,2′-azobis(2-amidinopropane)dihydrochlorideELISAenzyme-linked immunosorbent assay13-HODE13-hydroxyoctadecadienoic acidFAB-MSfast-atom bombardment MSESPelectrospray ionizationMLmethyl linoleateMLOOHML hydroperoxideHPLChigh performance liquid chromatography withN-benzoyl-glycyl-l-lysine (Bz-Gly-Lys) was investigated. In this study, a novel compound,N-benzoyl-glycyl-N ε-(hexanonyl)lysine (named HEL), was identified as one of the lipid hydroperoxide-modified lysine residues. The formation of HEL in lipid hydroperoxide-modified proteins including oxidatively modified LDL was confirmed using the specific antibody to the HEL residue. In addition, the HEL moiety was detected in human atherosclerotic plaques by immunohistochemical approach. As far as we know, the formation of an amide-type adduct has not been previously reported. This novel adduct derived from lipid hydroperoxide may become an initial marker for the oxidative damage of biological molecules in vivo. 13-hydroperoxyoctadecadienoic acid N-benzoyl-glycyl-l-lysine N-acetyl-glycyl-l-lysine methyl ester N-hydroxysuccinimide N ε-(hexanonyl)lysine low density lipoprotein N-hydroxysulfosuccinimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide keyhole limpet hemocyanin 15-hydroperoxyeicosatetraenoic acid bovine serum albumin liquid chromatography-mass spectrometry phosphate-buffered saline 2,2′-azobis(2-amidinopropane)dihydrochloride enzyme-linked immunosorbent assay 13-hydroxyoctadecadienoic acid fast-atom bombardment MS electrospray ionization methyl linoleate ML hydroperoxide high performance liquid chromatography The chemicals used were from the following sources. Bz-Gly-Lys and N-acetyl-glycyl-l-lysine methyl ester (AGLME) were purchased from Peptide, Inc. Soybean lipoxygenase, lipid-free BSA (product number A7511, initial fractionation by cold alcohol precipitation, ≥97% albumin, essentially fatty acid free), arachidonic acid, methylglyoxal, 2-hexenal, cardiolipin, andN ε-carboxybenzoyl-l-lysine methyl ester were obtained from Sigma. Linoleic acid, glyoxal, 1-nonanal, 2-nonenal, hexanoic acid, acetic acid, N-hydroxysuccinimide (NHS), and benzoyl-glycine were purchased from Wako Pure Chemicals Industries. Methyl linoleate and 1-hexanal were obtained from Nacarai Tesque, Inc. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC),N-hydroxysulfosuccinimide (sulfo-NHS), and keyhole limpet hemocyanin (KLH) were obtained from Pierce. 4-Hydroxy-2-nonenal was synthesized and provided by Dr. Koji Uchida (Nagoya University). Propionic acid, butyric acid, valeric acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, and undecanoic acid were purchased from GL Science, Inc. Monomethyl azelate was supplied by Larodan Fine Chemicals. Gluatric anhydride and malonaldehyde bis(dimethylacetal) were purchased from Aldrich. 13-HPODE was prepared by the enzymatic reaction of lipoxygenase with linoleic acid (9Kato Y. Makino Y. Osawa T. J. Lipid Res. 1997; 38: 1334-1346Abstract Full Text PDF PubMed Google Scholar, 10Kato Y. Osawa T. Arch. Biochem. Biophys. 1998; 351: 106-114Crossref PubMed Scopus (27) Google Scholar). 15-Hydroperoxyeicosatetraenoic acid (15-HPETE) was prepared as described previously (9Kato Y. Makino Y. Osawa T. J. Lipid Res. 1997; 38: 1334-1346Abstract Full Text PDF PubMed Google Scholar, 10Kato Y. Osawa T. Arch. Biochem. Biophys. 1998; 351: 106-114Crossref PubMed Scopus (27) Google Scholar). Methyl linoleate hydroperoxide (MLOOH) was prepared by the reaction of soybean lipoxygenase with methyl linoleate (ML). A 200-mg sample of ML and sodium deoxycholate (1.62 g) was dissolved in 240 ml of 200 mm borate buffer (pH 9.0). Lipoxygenase (100 mg, Sigma type I-B) was added to the solution and reacted for 3 h at room temperature. The formed peroxide was extracted twice with an equal amount of chloroform/methanol (1:1). The collected chloroform layer was evaporated. The obtained peroxide was purified by thin layer chromatography (TLC) and developed with n-hexane/ether (6:4). The peroxide was extracted with CHCl3 and then evaporated. The amount of MLOOH was calculated from the molar coefficient, ε234 nm=25000 m−1cm−1 using the value of linoleic acid hydroperoxide (11Verhagen J. Bouman A.A. Vliegenthart J.F.C. Boldingh J. Biochim. Biophys. Acta. 1977; 486: 114-120Crossref Scopus (54) Google Scholar). Bz-Gly-Lys (5 mm) and lipid-free BSA (5 mg/ml) were typically incubated with 13-HPODE, 15-HPETE, or MLOOH (5 mm) at 37 °C in 0.1 mphosphate buffer (pH 7.4) for 3 days. The lipid hydroperoxide-modified proteins were isolated by ethanol precipitation as already described (9Kato Y. Makino Y. Osawa T. J. Lipid Res. 1997; 38: 1334-1346Abstract Full Text PDF PubMed Google Scholar, 10Kato Y. Osawa T. Arch. Biochem. Biophys. 1998; 351: 106-114Crossref PubMed Scopus (27) Google Scholar). To obtain 13-HPODE-modified Bz-Gly-Lys, 5 mm Bz-Gly-Lys was incubated with 5 mm 13-HPODE for 3 days at 37 °C in phosphate buffer and then freeze-dried. The sample was extracted with methanol to remove the large amounts of inorganic salts. The extract was evaporated, dissolved in H2O, and then applied to gel filtration chromatography (TOYOPEAL HW-40F, 1.5 × 50 cm) with H2O as an eluent at a flow rate of 0.8 ml/min. The fractions (5 ml each) were monitored by absorbance at 234 nm and lipofuscin-like fluorescence (excitation, 350 nm; emission, 420 nm) using a JASCO Ubest-50 UV-visible spectrophotometer and Hitachi F2000 fluorescence spectrophotometer, respectively. The fluorescent fractions 29–34 were used for further identification because the fluorescence might be considered as a marker of lipid amine adducts. The fluorescent fractions were concentrated and then applied to a Sep-Pak cartridge (Waters) with 0–100% methanol (20% stepwise) elution. The 20% methanol fraction was used for the isolation of the modified lysine derivative, because it had the strongest fluorescence. The fraction was next applied to reversed-phase HPLC (Develosil ODS-HG-5 (8 × 250 mm), Nomura Chemical Co.) and then fractionated using gradient elution (solvent A, 0.1% acetic acid/CH3CN (7/3); solvent B, 0.1% acetic acid/CH3CN (1/1)) at a flow rate of 2.0 ml/min. The gradient program was as follows: 0 min (B 0%), 10 min (B 0%), 50 min (B 100%), 60 min (B 100%), and 61 min (B 0%). The elution was monitored by UV absorbance at 234 nm. The peak (retention time 30 min) was further purified by repeated reversed-phase HPLC. The obtained compound X weighed 1.3 mg. Spectral data of the isolated compound X are as follows: 1H NMR (CD3OD) (ppm) 0.80 (t, J = 6.9Hz, 3H), 1.19 (m, 2H), 1.23 (m, 2H), 1.32 (m, 2H), 1,42 (m, 2H), 1.49 (m, 2H), 1.62 (m, 1H), 1.83 (m, 1H), 2.06 (t, J = 7.7Hz, 2H), 3.05 (t, J = 6.7Hz, 2H), 3.99 (m, 2H), 4.30 (m, 1H), 7.37 (t, J = 5.1Hz, 2H), 7.45 (t, J = 5.1Hz, 1H), 7.78 (d, J = 7.1Hz, 2H); FAB+-MSm/z 406 (M+H)+, 428 (M+Na)+. For the first step, benzoyl-glycine (1 eq.) and the Nε-(carboxybenzoyl)lysine methyl ester (1 eq.) were conjugated in dimethylformamide (DMF) with EDC (1.1 eq.) in the presence of an enhancer, NHS (1.1 eq.), as described previously (12Grabarek Z. Gergely J. Anal. Biochem. 1990; 185: 131-135Crossref PubMed Scopus (726) Google Scholar) with some modifications. After an overnight reaction at room temperature, the reaction mixture was dissolved with ethyl acetate and then washed with equal amounts of 1 N HCl, water, 5% NaHCO3, and then water. The residual ethyl acetate layer was passed through Na2SO4 for dehydration. The eluent was concentrated, and the crude N-benzoyl-glycyl-Nε-(carboxybenzoyl)lysine methyl ester was crystallized with water/ethanol at 4 °C for 3 h. The removal of carboxybenzoyl from the purified peptide was performed using Pd-C under H2 for 3 h at room temperature in water/methanol. The obtained N-benzoyl-glycyl-l-lysine methyl ester was purified by preparative reversed-phase HPLC (Develosil ODS-5 (20 × 250 mm), Nomura Chemical Co.) using 0.1% trifluoroacetic acid, CH3CN (5/3) as the eluent. Hexanoic acid and the N-benzoyl-glycyl-l-lysine methyl ester were conjugated with EDC and NHS as described previously. The reaction mixture was washed as already described, and the residual product was purified by reversed-phase HPLC on a Develosil ODS-5 (20 × 250 mm) using 0.1% trifluoroacetic acid, CH3CN (5/3) as the eluent. TheN-benzoyl-glycyl-N ε-(hexanonyl)lysine methyl ester was treated with 0.25 n NaOH at 37 °C for 1 h to remove the methyl ester. The obtained compound was purified by reversed-phase HPLC on the column using 0.1% trifluoroacetic acid, CH3CN (5/3) as the eluent. The identification was performed by 1H NMR and mass spectroscopy. The spectral data of the syntheticN-benzoyl-glycyl-N ε-(hexanonyl)lysine are as follows: 1H NMR (CD3OD) (ppm) 0.80 (t,J = 6.9 Hz, 3H), 1.20 (m, 2H), 1.24 (m, 2H), 1.34 (m, 2H), 1.42 (m, 2H), 1.49 (m, 2H), 1.66 (m, 1H), 1.84 (m, 1H), 2.06 (t,J = 7.4 Hz, 2H), 3.07 (t, J = 6.9 Hz, 2H), 3.99 (m, 2H), 4.34 (m, 1H), 7.37 (t, J = 7.2 Hz, 2H), 7.45 (t, J = 5.2 Hz, 1H), 7.77 (d,J = 7.2 Hz, 2H); FAB+-MSm/z 406 (M + H)+, 428 (M + Na)+. N-Benzoyl-glycyl-N ε-(hexanonyl(D-11))lysine derivative was prepared using D-11-hexanoic acid as follows. Briefly, benzoyl-glycyl-l-lysine was conjugated with D-11-hexanoic acid using EDC and NHS as coupling reagents (12Grabarek Z. Gergely J. Anal. Biochem. 1990; 185: 131-135Crossref PubMed Scopus (726) Google Scholar). The obtained benzoyl-glycyl-N ε-(hexanonyl)lysine was isolated and purified by reversed-phase HPLC on a Develosil ODS-HG-5 (8 × 250 mm) equilibrated with 0.1% trifluoroacetic acid, CH3CN (5/3) at a flow rate of 2.0 ml/min. The elution was estimated by UV absorbance at 234 nm, and identification of the synthetic deuterified hexanonyl compound was performed by liquid chromatography-mass spectrometry (LC-MS). TheN-acetyl-glycyl-N ε-(hexanonyl)-l-lysine methyl ester (N ε-hexanonyl AGLME) was prepared by conjugation between AGLME and hexanoic acid using EDC as the coupling reagent and NHS as the enhancer, as described previously. The synthetic compound could not be separated with ethyl acetate/water fractionation because of its high water solubility. Therefore, the reaction mixture was diluted with 0.1% trifluoroacetic acid and passed through a Sep-Pak cartridge. The cartridge was washed with 0.1% trifluoroacetic acid, and the products were then eluted with 0.1% trifluoroacetic acid, CH3CN (1/1). The eluent was concentrated and applied to preparative reversed-phase HPLC (Develosil ODS-5 (20 × 250 mm)) using 0.1% trifluoroacetic acid, CH3CN (7:3) as the eluent. The elution was monitored by absorbance at 215 nm. The peak was collected and concentrated. The obtainedN-acetyl-glycyl-N ε-(hexanonyl)-l-lysine methyl ester (N ε-hexanonyl AGLME) was identified by 1H NMR and mass spectroscopy (LC-MS). The spectral data of N ε-hexanonyl AGLME are as follows: 1H NMR (CD3OD) (ppm) 0.71 (t,J = 7.1 Hz, 3H), 1.09 (m, 2H), 1.14 (m, 2H), 1.20 (m, 2H), 1.31 (m, 2H), 1.40 (m, 2H), 1.49 (m, 1H), 1.63 (m, 1H), 1.80 (s, 3H), 1.97 (t, J = 7.7 Hz, 2H), 2.96 (t,J = 6.9 Hz, 2H), 3.51 (s, 3H), 3.68 (m, 2H), 4.22 (m, 1H); LC-MS (ESP+) m/z 358 (M + H)+. Samples were hydrolyzed with 6n HCl in vacuo at 105 °C. The hydrolysates were dried, dissolved in citrate buffer (pH 2.2), and then applied to an amino acid analyzer, JLC-500 (JEOL). The conjugation of hexanoic acid with proteins was performed as follows. Hexanoic acid (2.3 mg), EDC (4.5 mg), and sulfo-NHS (5 mg) were dissolved in 400 μl of dimethylformamide, and the reaction mixture was incubated for 24 h at room temperature. To the solution, 0.95 ml of KLH or BSA (10 mg in 0.1 mphosphate buffer (pH 7.4)) was added and further incubated for 4 h at room temperature. The obtained hexanonyl proteins were dialyzed against phosphate-buffered saline (PBS) for 3 days at 4 °C. The hexanonyl KLH was emulsified with an equal volume of complete Freund's adjuvant to a final concentration of 0.5 mg/ml, and 1 ml of the solution was then intramuscularly injected into a New Zealand White rabbit. After 4 weeks, 1 ml of the hexanonyl KLH emulsified with an equal volume of incomplete adjuvant (0.5 mg/ml) was injected as a booster every 2 weeks until an adequate antibody generation occurred. Hexanonyl BSA was used for the evaluation of the antibody generation specific to hexanonyl protein. Conjugates of acetic acid (C2), propionic acid (C3), butyric acid (C4), valeric acid (C5), heptanoic acid (C7), octanoic acid (C8), nonanoic acid (C9), decanoic acid (C10), and undecanoic acid (C11) with BSA were prepared using EDC and NHS as coupling agents as described previously. Glutaric acid-BSA was prepared as follows (13Murakami M. Horiuchi S. Tanaka K. Morino Y. J. Biochem. 1987; 101: 729-741Crossref PubMed Scopus (59) Google Scholar). Briefly, lipid-free BSA (4 mg/ml) in PBS was mixed with an equal volume of saturated sodium acetate. Under ice-cool conditions, glutaric anhydride (3 mm) was added and reacted for 1 h. The modified BSA was dialyzed against water at 4 °C for 24 h. Azelaic acid-BSA conjugate was prepared as follows. First, the monomethylazelaic acid (50 mg), EDC (52.2 mg), and NHS (31.3 mg) in dimethylformamide (1 ml) were incubated at room temperature for 24 h. Five milliliters of BSA solution (30 mg/ml in 0.1 m phosphate buffer (pH 7.4)) was then added to the solution and then incubated at room temperature for 16 h. The reaction mixture was dialyzed against PBS at 4 °C for 3 days. Azelaic acid-BSA conjugate was prepared from obtained monomethylazelaic acid-BSA by saponification. Alkaine solution (0.25 m NaOH) was added to the monomethylazelaic acid-BSA and further incubated for 1 h. After neutralization with HCl, the reaction mixture was dialyzed against PBS at 4 °C for 24 h. These conjugations were evaluated by the trinitrobenzenesulfonic acid method (14Steinbrecher U.P. J. Biol. Chem. 1987; 262: 3603-3608Abstract Full Text PDF PubMed Google Scholar). The losses (%) of lysine residue were as follows: C2, 72%; C3, 40%; C4, 17%; C5, 90%; C6 (hexanonyl BSA), 91%; C7, 91%; C8, 89%; C9, 89%; C10, 94%; C11, 90%; glutaric acid-BSA, 38%; azelaic acid-BSA, 62%. Oxidized lipid-modified proteins were prepared as follows. Lipid (linoleic acid and arachidonic acid, 5 mm; cardiolipin, 1 mg/ml) was oxidized by 5 mm ascorbic acid and 0.05 mm FeCl3 for 24 h at 37 °C in PBS containing 20% methanol. To the reaction mixture, lipid-free BSA (final concentration, 5 mg/ml) was added and further incubated at 37 °C for 3 days. To isolate the modified proteins, an equal amount of CHCl3:CH3OH (2:1) was added, vigorously mixed, and then centrifuged for 10 min at 4 °C. The lower layer was discarded, and an equal amount of CHCl3 was added and mixed. After centrifugation, the lower layer was discarded again. To the residual upper layer, 9 volumes of ice-cool ethanol was added and kept for 45 min at 4 °C. After centrifugation, the pellet was dissolved in water with sonication. The protein solution was dialyzed against water for 2 days at 4 °C. Aldehyde-modified proteins were prepared as already described (9Kato Y. Makino Y. Osawa T. J. Lipid Res. 1997; 38: 1334-1346Abstract Full Text PDF PubMed Google Scholar, 10Kato Y. Osawa T. Arch. Biochem. Biophys. 1998; 351: 106-114Crossref PubMed Scopus (27) Google Scholar). The preparation of lipid hydroperoxide-modified proteins was as follows. The hydroperoxides (13-HPODE or 15-HPETE) were incubated with lipid-free BSA at 37 °C in 0.1 m phosphate buffer (pH 7.4) for 3 days. Oxidized BSA was prepared by the incubation of lipid free-BSA with hydrogen peroxide/metal ion (9Kato Y. Makino Y. Osawa T. J. Lipid Res. 1997; 38: 1334-1346Abstract Full Text PDF PubMed Google Scholar, 10Kato Y. Osawa T. Arch. Biochem. Biophys. 1998; 351: 106-114Crossref PubMed Scopus (27) Google Scholar). Modified proteins were dialyzed against PBS at 4 °C for 2 days. The concentration of all modified proteins was measured by a BCA assay kit (Pierce). Human LDL was isolated from healthy volunteers using density centrifugation (15Kato Y. Tokunaga K. Osawa T. Biochem. Biophys. Res. Commun. 1996; 226: 923-927Crossref PubMed Scopus (17) Google Scholar). The modification of LDL was performed by copper ion and 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) as described previously (9Kato Y. Makino Y. Osawa T. J. Lipid Res. 1997; 38: 1334-1346Abstract Full Text PDF PubMed Google Scholar). The modification by copper ion was performed by incubation of LDL (0.2 mg/ml) with 50 μmCuSO4 in PBS at 37 °C. AAPH-induced oxidation of LDL was carried out by incubation of AAPH (0–5 mm) with LDL (0.2 mg/ml) in PBS at 37 °C for 24 h. The reaction was terminated by the addition of 10 μm butylated hydroxytoluene and 1 mm EDTA. The measurements of lipid peroxidation were performed by the following two methods. The generation of thiobarbituric acid reactive substance was measured as described previously (9Kato Y. Makino Y. Osawa T. J. Lipid Res. 1997; 38: 1334-1346Abstract Full Text PDF PubMed Google Scholar, 10Kato Y. Osawa T. Arch. Biochem. Biophys. 1998; 351: 106-114Crossref PubMed Scopus (27) Google Scholar). The formation of lipid peroxide was measured by a Determiner LPO kit (Kyowa medix), a colorimetric method based on the reaction of lipid peroxides with a methylene blue derivative in the presence of hemoglobin (16Yagi K. Kiuchi K. Saito Y. Miike A. Kayahara N. Tatano T. Ohishi N. Biochem. Int. 1986; 12: 367-371PubMed Google Scholar). The sample was applied to a liquid chromatograph on a Develosil ODS-HG-5 (4.6 × 250 mm), which was connected with a mass spectrometer (PLATFORM II, VG Biotech). The separation was performed by a two-pump gradient. The solvent A for AGLME was 0.1% acetic acid; solvent B for AGLME was CH3CN. For the Bz-Gly-Lys system, solvent A was 0.1% acetic acid, CH3CN(7/3), and solvent B was 0.1% acetic acid, CH3CN (3:7). The gradient programs were as follows: AGLME, 0 min, A 100%; 70 min, A 30%; 75 min, A 30%; 80 min, A 100%. Bz-Gly-Lys, 0 min, A 100%; 30 min, A 0%; 35 min, A 0%; 40 min, A 100%. The electrospray ionization (positive) mode was used for the detection. For the measurements of theN ε-hexanonyl derivative of Bz-Gly-Lys, deuterified hexanonyl Bz-Gly-Lys was added to samples at a concentration of 19 μm before the analysis as an internal standard. Indirect noncompetitive ELISA was performed as already described (9Kato Y. Makino Y. Osawa T. J. Lipid Res. 1997; 38: 1334-1346Abstract Full Text PDF PubMed Google Scholar, 10Kato Y. Osawa T. Arch. Biochem. Biophys. 1998; 351: 106-114Crossref PubMed Scopus (27) Google Scholar). Briefly, 50 μl of antigen in PBS was dispensed into a well and kept at 4 °C overnight. After blocking with Block Ace (Dainihon Seiyaku, Osaka, Japan), 100 μl of antiserum (1/5000 in PBS containing 0.5% BSA) was added to the well. The binding of the antibody on the coated antigen was evaluated as already described (9Kato Y. Makino Y. Osawa T. J. Lipid Res. 1997; 38: 1334-1346Abstract Full Text PDF PubMed Google Scholar, 10Kato Y. Osawa T. Arch. Biochem. Biophys. 1998; 351: 106-114Crossref PubMed Scopus (27) Google Scholar). The cross-reactivity of the low molecular weight compound with antibody was investigated by indirect competitive ELISA (9Kato Y. Makino Y. Osawa T. J. Lipid Res. 1997; 38: 1334-1346Abstract Full Text PDF PubMed Google Scholar, 10Kato Y. Osawa T. Arch. Biochem. Biophys. 1998; 351: 106-114Crossref PubMed Scopus (27) Google Scholar). As a coating agent, 50 μl of hexanonyl BSA (0.5 μg/ml) was pipetted onto wells and kept at 4 °C overnight. At the same time, 50 μl of antiserum (1/2500 in PBS containing 1% BSA) and 50 μl of sample were mixed in an Eppendolf tube and reacted at 4 °C overnight. The plate was washed, and 90 μl of the reacted solution was pipetted onto a well. The binding of the residual antibody on coated hexanonyl BSA was estimated as described previously (9Kato Y. Makino Y. Osawa T. J. Lipid Res. 1997; 38: 1334-1346Abstract Full Text PDF PubMed Google Scholar, 10Kato Y. Osawa T. Arch. Biochem. Biophys. 1998; 351: 106-114Crossref PubMed Scopus (27) Google Scholar). The effects of the preincubation of 13-HPODE on the formation of N ε-(hexanonyl)lysine were investigated as follows: 13-HPODE (50 mm) was incubated in 0.1 m phosphate buffer (pH 7.4) containing 20% methanol at 37 °C. Fifty μl of the incubated solution was withdrawn, and a 10-times diluted sample was reacted with 5 mm substrate (Bz-Gly-Lys/BSA) in 0.1 m phosphate buffer at 37 °C for 3 days. The reaction mixture of Bz-Gly-Lys and preincubated 13-HPODE was stored at −70 °C until LC-MS analysis (see above). The "preincubated 13-HPODE"-modified BSA was isolated from the reaction mixture by ice-cool ethanol precipitation and used for ELISA as described in the previous section. At the same time, an aliquot of the incubated solution of 13-HPODE was used for the measurement of the loss of 13-HPODE. Fifty μl of the preincubated solution was reduced with 100 μl of 100 mm NaBH4 in 1 mNaOH and further incubated for 1 h at room temperature. The reduction was terminated by the addition of 200 μl of 1nHCl, and the amount of 13-HODE obtained was measured by reversed-phase HPLC on a Develosil ODS-HG-5 (4.6 × 250 mm) equilibrated with 0.1% trifluoroacetic acid, methanol (1/3) at a flow rate of 0.8 ml/min. The detection was monitored by the absorbance at 234 nm. Tissue sections were prepared from frozen arteries (8 μm thick). Before immunostaining, frozen sections were fixed by incubation in ice-cold acetone for 20 min. Sections were incubated with 10% normal goat serum in PBS (20 min) to block nonspecific binding before staining and then with primary antibody (1:350 dilution) for 1 h at room temperature. Sections were incubated with 5% normal rabbit serum or the anti-HEL antibody preabsorbed with hexanonyl BSA instead of the primary antibody as negative controls. Immunostaining was performed with anti-rabbit antibody peroxidase-label (1:50 dilution, DAKO) with hydrogen peroxide and 3,3-diaminobenzidine tetrahydrochloride as chromogen. Sections were counterstained with aqueous hematoxylin. To search for the specific hydroperoxide-derived lysine modification, we used Bz-Gly-Lys as the substrate, and the isolation of the 13-HPODE-modified lysine derivative was performed. To remove large amounts of unreacted Bz-Gly-Lys, the reaction mixture was concentrated and then applied to gel filtration chromatography using TOYOPEAL HW-40 (TOSOH) as a gel. The fractions were monitored by absorbance at 234 nm and lipofuscin-like fluorescence (Fig.1 A). As a result of the HPLC analysis of each fraction, fractions 35–40 contained the unreacted substrate, Bz-Gly-Lys. Fractions 29–34 had a lipofuscin-like fluorescence, which could be considered as a marker of lipid decomposition product-modified molecules. Therefore, the fluorescent fractions were used for further isolation. The mixed fractions 29–34 were applied to a Sep-Pak cartridge with stepwise methanol elutions. The HPLC profile of the 20% methanol fraction is shown in Fig.1 B. A compound, labeled X, was then isolated by repeated reversed-phase HPLC. The molecular weight of X, 405, was confirmed by FAB-MS. Compound X was hydrolyzed with 6 n HCl at 105 °Cin vacuo and submitted for amino acid analysis. Interestingly, both Gly and Lys were completely recovered from the acid hydrolysates of compound X (Gly/Lys = 0.96). This suggested that the bond between the lipid-derived structure and Bz-Gly-Lys was acid liable such as an amide bond or a Schiff base. The structure of X was elucidated using 1H NMR. The proposed structure of X with the parent molecules is shown in Fig.2 A. To confirm the structure of compound X, the synthesis of theN ε-(hexanonyl)lysine adduct was performed by carbodiimide conjugation of the lysine derivative with hexanoic acid. The instrumental analysis of the syntheticN-benzoyl-glycyl-N ε-(hexanonyl)lysine almost agreed with that of isolated compound X. Neither the isolated compound X nor the synthetic N ε-hexanonyl adduct had any fluorescence. The time-dependent changes in Bz-Gly-Lys during incubation with 13-HPODE were examined. As shown in Fig. 2 B, a loss of Bz-Gly-Lys was observed in a time-dependent fashion, and the formation ofN-benzoyl-glycyl-N ε-(hexanonyl)lysine, compound X, was confirmed. The conversion yield of compound X from the loss of Bz-Gly-Lys after a 3-day incubation with 13-HPODE was 5.6%. To further confirm the formation of theN ε-(hexanonyl)lysine named "HEL" in the 13-HPODE-modified Lys, the AGLME was incubated with 13-HPODE, and the formation of the HEL derivative was investigated. After a 3-day incubation, an aliquot of the reaction mixture was applied to LC-MS. The product, which shows m/z 358 as an (M + H)+ ion, corresponding toN ε-hexanonyl AGLME (M r357), was eluted at a retention time of 40.79 min, and this completely agreed with the elution time of the syntheticN ε-hexanonyl AGLME (Fig.3). The mass charts of both the product and the synthetic N ε-hexanonyl AGLME showed the same fragmentation pattern. H