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• W2068986375 abstract "Diverse oxidative pathways, such as direct oxidation of amino acids, glycoxidation, and lipoxidation could contribute to Alzheimer disease pathogenesis. A global survey for the amount of structurally characterized probes for these reactions is lacking and could overcome the lack of specificity derived from measurement of 2,4-dinitrophenylhydrazine reactive carbonyls. Consequently we analyzed (i) the presence and concentrations of glutamic and aminoadipic semialdehydes, Nϵ-(carboxymethyl)-lysine, Nϵ-(carboxyethyl)-lysine, and Nϵ-(malondialdehyde)-lysine by means of gas chromatography/mass spectrometry, (ii) the biological response through expression of the receptor for advanced glycation end products, (iii) the fatty acid composition in brain samples from Alzheimer disease patients and agematched controls, and (iv) the targets of Nϵ-(malondialdehyde)-lysine formation in brain cortex by proteomic techniques. Alzheimer disease was associated with significant, although heterogeneous, increases in the concentrations of all evaluated markers. Alzheimer disease samples presented increases in expression of the receptor for advanced glycation end products with high molecular heterogeneity. Samples from Alzheimer disease patients also showed content of docosahexaenoic acid, which increased lipid peroxidizability. In accordance, Nϵ-(malondialdehyde)-lysine formation targeted important proteins for both glial and neuronal homeostasis such as neurofilament L, α-tubulin, glial fibrillary acidic protein, ubiquinol-cytochrome c reductase complex protein I, and the β chain of ATP synthase. These data support an important role for lipid peroxidation-derived protein modifications in Alzheimer disease pathogenesis. Diverse oxidative pathways, such as direct oxidation of amino acids, glycoxidation, and lipoxidation could contribute to Alzheimer disease pathogenesis. A global survey for the amount of structurally characterized probes for these reactions is lacking and could overcome the lack of specificity derived from measurement of 2,4-dinitrophenylhydrazine reactive carbonyls. Consequently we analyzed (i) the presence and concentrations of glutamic and aminoadipic semialdehydes, Nϵ-(carboxymethyl)-lysine, Nϵ-(carboxyethyl)-lysine, and Nϵ-(malondialdehyde)-lysine by means of gas chromatography/mass spectrometry, (ii) the biological response through expression of the receptor for advanced glycation end products, (iii) the fatty acid composition in brain samples from Alzheimer disease patients and agematched controls, and (iv) the targets of Nϵ-(malondialdehyde)-lysine formation in brain cortex by proteomic techniques. Alzheimer disease was associated with significant, although heterogeneous, increases in the concentrations of all evaluated markers. Alzheimer disease samples presented increases in expression of the receptor for advanced glycation end products with high molecular heterogeneity. Samples from Alzheimer disease patients also showed content of docosahexaenoic acid, which increased lipid peroxidizability. In accordance, Nϵ-(malondialdehyde)-lysine formation targeted important proteins for both glial and neuronal homeostasis such as neurofilament L, α-tubulin, glial fibrillary acidic protein, ubiquinol-cytochrome c reductase complex protein I, and the β chain of ATP synthase. These data support an important role for lipid peroxidation-derived protein modifications in Alzheimer disease pathogenesis. Oxidative stress-induced molecular alterations affect all sorts of biological molecules, including especially sensitive amino acid residues in proteins, such as tyrosine, methionine, arginine, proline, and lysine, among others (1Amici A. Levine R.L. Tsai L. Stadtman E.R. J. Biol. Chem. 1988; 365: 3341-3346Google Scholar). Brain aging is associated with changes increasing the risk of Alzheimer disease (AD). 1The abbreviations used are: AD, Alzheimer disease; DNP, 2,4-dinitrophenylhydrazine; GSA, glutamic semialdehyde; AASA, aminoadipic semialdehyde; CEL, Nϵ-(carboxyethyl)-lysine; CML, Nϵ-(carboxymethyl)-lysine; AGE, advanced glycation end products; PUFA, polyunsaturated fatty acids; MDAL, Nϵ-malondialdehyde-lysine; Aβ, amyloid β-peptide; RAGE, receptor for advanced glycation end products; CHAPS, 3-[(cholamidopropyl)dimethylamino]-1-propanesulfonate; DTT, dithiothreitol; GC/MS, gas chromatography/mass spectrometry; UFA, unsaturated fatty acids; MUFA, monounsaturated fatty acids; ACL, average chain length; DHA, docosahexaenoic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; MALDI-reTOF, matrix-assisted laser desorption/ionization reflectron time-of-flight. A corollary of this fact is that AD shows an acceleration of those phenomena underlying aging. Accordingly, an increased amount of protein bound 2,4-dinitrophenylhydrazine (DNP)-reactive carbonyls, generated by protein oxidation are a common finding in brain samples from AD patients (2Aksenov M.Y. Aksenova M.V. Butterfield D.A. Geddes J.W. Markesbery W.R. Neuroscience. 2001; 103: 373-383Crossref PubMed Scopus (442) Google Scholar, 3Smith C.D. Carney J.M. Starke-Reed P.E. Oliver C.N. Stadtman E.R. Floyd R.A. Markesbery W.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10540-10543Crossref PubMed Scopus (1171) Google Scholar). All these works rely on the reaction of DNP with carbonyl groups. However, this assay has been criticized because of the possibility of artifacts (4Cao G. Cutler R.G. Arch. Biochem. Biophys. 1995; 320: 106-114Crossref PubMed Scopus (128) Google Scholar). The direct measure of the concentration of structurally characterized products could overcome this fact and it may be used as a complement for assessing the effects of oxidative stress in vivo. Glutamic semialdehyde (GSA) derives from the metal-catalyzed oxidation of proline and arginine, whereas aminoadipic semialdehyde (AASA) results from lysine oxidation (5Requena J.R. Chao C.C. Levine R.L. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 69-74Crossref PubMed Scopus (378) Google Scholar). These products are among the main carbonyl products of metal-catalyzed oxidation of proteins (5Requena J.R. Chao C.C. Levine R.L. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 69-74Crossref PubMed Scopus (378) Google Scholar), thus specific probes of oxidation of amino acids in protein. However, their presence and the factors affecting their concentrations in human brains are unknown to date. The chemical pathways linking increased free radical efflux and protein structural modification also involve third-party molecules, which may give rise to increased DNP-reactive carbonyls in proteins (6Berlett B.S. Stadtman E.R. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2809) Google Scholar). Particularly, carbohydrates, when reacting with free radicals generate highly reactive dicarbonyl compounds, such as glyoxal and methylglyoxal (7Thornalley P.J. Langborg A. Minhas H.S. Biochem. J. 1999; 344: 109-116Crossref PubMed Scopus (1026) Google Scholar). In the cellular context, these may also be derived from glycolysis, triose phosphate metabolism, acetone metabolism (7Thornalley P.J. Langborg A. Minhas H.S. Biochem. J. 1999; 344: 109-116Crossref PubMed Scopus (1026) Google Scholar), lipid peroxidation (8Fu M.X. Requena J.R. Jenkins A.J. Lyons T.J. Baynes J.W. Thorpe S.R. J. Biol. Chem. 1996; 271: 9982-9986Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar), and hypochlorite-mediated reactions (9Anderson M.M. Requena J.R. Crowley J.R. Thorpe S.R. Heinecke J.W. J. Clin. Investig. 1999; 104: 103-113Crossref PubMed Scopus (325) Google Scholar). These compounds generate stable adducts reacting with lysine, arginine, and cysteine in proteins. Nϵ-(Carboxyethyl)-lysine (CEL) and Nϵ-(carboxymethyl)-lysine (CML) are two of these adducts, first described as advanced glycation end products (AGE), later named glycoxidation products and now recognized as mixed AGEs-advanced lipoxidation products. Despite that CML has been detected in AD lesions by immunohistochemical analyses (reviewed in Ref. 10Kikuchi S. Shinpo K. Takeuchi M. Yamagishi S. Makita Z. Sasaki N. Tashiro K. Brain Res. Rev. 2003; 41: 306-323Crossref PubMed Scopus (147) Google Scholar), no chemical evidence have been reported for this product in AD samples or models. Polyunsaturated fatty acids (PUFA) are other third-party molecules. PUFA are molecules very susceptible to the oxidative action of free radicals (11Cosgrove J.P. Church D.F. Pryor W.A. Lipids. 1987; 22: 299-304Crossref PubMed Scopus (465) Google Scholar), generating specific reactive aldehydes, such as malondialdehyde or 4-hydroxynonenal, among others (12Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5936) Google Scholar). These aldehydes could react with proteins, generating also DNP-reactive moieties (6Berlett B.S. Stadtman E.R. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2809) Google Scholar). The important PUFA content in brain and its high oxygen consumption support the possible significance of lipid peroxidation-derived processes in brain aging and AD pathogenesis (2Aksenov M.Y. Aksenova M.V. Butterfield D.A. Geddes J.W. Markesbery W.R. Neuroscience. 2001; 103: 373-383Crossref PubMed Scopus (442) Google Scholar). Analogously to the other modifications pointed out above, evidence for lipid peroxidation-derived protein damage in AD comprise immunohistochemistry (13Butterfield D.A. Castegna A. Lauderback C.M. Drake J. Neurobiol. Aging. 2002; 203: 655-664Crossref Scopus (609) Google Scholar). However, there is no chemical evidence for lipid peroxidative damage of proteins in AD, based on structurally identified compounds and supported by mass spectrometry. Concerning the pathogenic role of these products, many recent studies of mechanisms underlying cellular dysfunction in Alzheimer disease have focused on amyloid β-peptide (Aβ). Persistent chronic inflammation appears to have a significant role in AD pathogenesis and evidence suggests that the sustained microglial response to Aβ could play a role in this process (14Akiyama H.S. Barger S. Barnum S. Bradt B. Bauer J. Cole G.M. Cooper N.R. Eikelenboom P. Emmerling M. Fiebich B.L. Finch C.E. Frautschy S. Griffin W.S. Hampel H. Hull M. Landreth G. Lue L. Mrak R. Mackenzie I.R. McGreer P.L. O'Banion M.K. Pachter J. Pasinetti G. Plata-Salaman C. Rogers J. Rydel R. Shen Y. Streit W. Strohmeyer R. Tooyoma I. Van Muiswinke F.L. Veerhuis R. Walker D. Webster S. Wegryniak B. Wenk G. Wysscoray T. Neurobiol. Aging. 2000; 21: 383-421Crossref PubMed Scopus (3733) Google Scholar). In AD brains, the most highly activated microglia is observed in close association with Aβ plaques (15Itagaki S. McGreer P.L. Akiyama H. Zhu S. Selkoe D. J. Neuroimmunol. 1989; 24: 173-182Abstract Full Text PDF PubMed Scopus (770) Google Scholar). In addition, many inflammatory mediators detected in AD brains are of microglial origin (14Akiyama H.S. Barger S. Barnum S. Bradt B. Bauer J. Cole G.M. Cooper N.R. Eikelenboom P. Emmerling M. Fiebich B.L. Finch C.E. Frautschy S. Griffin W.S. Hampel H. Hull M. Landreth G. Lue L. Mrak R. Mackenzie I.R. McGreer P.L. O'Banion M.K. Pachter J. Pasinetti G. Plata-Salaman C. Rogers J. Rydel R. Shen Y. Streit W. Strohmeyer R. Tooyoma I. Van Muiswinke F.L. Veerhuis R. Walker D. Webster S. Wegryniak B. Wenk G. Wysscoray T. Neurobiol. Aging. 2000; 21: 383-421Crossref PubMed Scopus (3733) Google Scholar). In vitro, interaction of Aβ with microglia has been shown to cause the induction of a range of inflammatory products, including proinflammatory cytokines, neurotoxic factors, reactive oxygen species, and complement pathway proteins (15Itagaki S. McGreer P.L. Akiyama H. Zhu S. Selkoe D. J. Neuroimmunol. 1989; 24: 173-182Abstract Full Text PDF PubMed Scopus (770) Google Scholar, 16Haga S. Ikeda K. Sato M. Ishii T. Brain Res. 1993; 601: 88-94Crossref PubMed Scopus (130) Google Scholar). Multiple Aβ-binding protein or receptors have been identified on a number of different cell types, including microglia. They cover cell surface heparan proteoglycans and signal transduction receptors (17Yaar M. Zhai S. Pilch P.F. Doyle S.M. Eisenhauer P.B. Fine R.E. Gilchrest B.A. J. Clin. Investig. 1999; 100: 2333-2340Crossref Scopus (304) Google Scholar). The receptor for advanced glycation end products (RAGE) is one of these cell surface Aβ-binding proteins (18Brett J. Schmidt A.M. Yan S.D. Zou Y.S. Weidman E. Pinski D. Nowygrod R. Neeper M. Przysiecki C. Shaw A. Am. J. Pathol. 1993; 143: 1699-1712PubMed Google Scholar). RAGE is a member of the immunoglobulin superfamily of cell surface molecules whose expression is up-regulated at sites of diverse pathologies from atherosclerosis to AD (19Schmidt A.M. Yan S.D. Wautier J.L. Stern D.M. Circ. Res. 1999; 84: 489-497Crossref PubMed Scopus (710) Google Scholar). The generation of reactive oxygen species, an early event after ligation of the receptor, may be fundamental for many RAGE-induced changes in cellular properties. In the case of neurons, RAGE-induced cellular activation ultimately results in induction of programmed cell death (20Schmidt A.M. Yan S.D. Yan S.F. Stern D.M. Biochim. Biophys. Acta. 2000; 1498: 99-111Crossref PubMed Scopus (609) Google Scholar). In this work, we attempted to identify and quantify the amount of oxidation-derived modified amino acids in proteins from the frontal cerebral cortex of human brains with AD. Prior, we evaluated the distribution of these protein modifications by Western blot, the possible influence of sample preparation in DNP-based outcomes, and studied part of the biological response to these products by examining RAGE expression. Finally, given the importance of PUFA in lipid peroxidation, we also studied the fatty acid profile from these samples and the identification of major targets of lipoxidative damage in brain cortex. Human Brain Specimens and Reagents—Brain samples were obtained from the Institute of Neuropathology Brain bank following the guidelines of the local ethics committee. The brains of 8 patients with AD (four men and four women) and 5 age-matched controls (three women and two men) were obtained from 2 to 13 h after death, and were immediately prepared for morphological and biochemical studies. The agonal state was short with no evidence of acidosis or prolonged hypoxia. AD changes were categorized following the nomenclature of Braak and Braak (21Braak H. Braak E. Cerebral Cortex: Neurodegenerative and Age-related Changes in Structure and Function of the Cerebral Cortex. 14. Kluwer Academic/Plenum Press, NY1999Google Scholar). Stage C of amyloid A4 deposition implicates involvement of the whole neocortex, whereas stages V and VI of neurofibrillary degeneration indicate moderate and severe involvement of the neocortex. No additional vascular or degenerative anomalies were present in these cases. Age-matched controls did not show neuropathological anomalies, particularly considering the absence of amyloid deposits often considered as normal old-age changes. Frozen samples of the frontal cortex (area 8) were used for biochemical studies. Samples of control and diseased brains were processed in parallel. Summary of the main clinical and neuropathological aspects is shown in Table I. Unless otherwise specified, all reagents were from Aldrich or Sigma, of the highest purity available.Table ISummary of the main clinical and neuropathological findings in the present series Alzheimer disease, stages C of amyloid-βA4 deposition, and stages V and VI of neurofibrillary degeneration (NFT) according to Braak and Braak (21Braak H. Braak E. Cerebral Cortex: Neurodegenerative and Age-related Changes in Structure and Function of the Cerebral Cortex. 14. Kluwer Academic/Plenum Press, NY1999Google Scholar) M: male, F: female.PatientDiseaseGenderAgeDuration of the diseasePost-mortemBraak stagesAmyloid-βA4NFTyearsyearsh1ControlF735.32ControlM7563ControlF7974ControlF803.35ControlM7066ADM6986CV7ADF821310CV8ADF8482CV9ADF86810CV10ADM93107.2CV11ADM71613CVI12ADM7276CVI13ADF7285CVI Open table in a new tab Distribution of Protein Modifications by Western Blotting and Derivatization of Proteins for Carbonyl Detection—Samples were homogenized in a buffer containing 180 mm KCl, 5 mm MOPS, 2 mm EDTA, 1 mm diethylenetriaminepentaacetic acid, and 1 μm butylated hydroxyl toluene, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, pH 7.3 (Potter-Elvehjem device, at 4 °C). After a brief centrifugation (500 × g, 5 min) to pellet cellular debris, protein concentrations were measured in the supernatants using the Lowry assay (Bio-Rad). Prior to electrophoresis, samples were derivatized with DNP as previously described (22Portero-Otin M. Pamplona R. Ruiz M.C. Cabiscol E. Prat J. Bellmunt M.J. Diabetes. 1999; 48: 2215-2220Crossref PubMed Scopus (57) Google Scholar). Briefly, to 15-μl homogenates adjusted to 3.75 μg/μl of protein, SDS was added to a final concentration of 6%, and after boiling for 3 min, 20 μl of 10 mm DNP in 10% trifluoroacetic acid were added. After 7 min at room temperature, 20 μl of a solution containing 2 m Tris base, 30% glycerol, and 15% β-mercaptoethanol were added for neutralization and sample preparation for loading onto SDS-PAGE gels. For sample clarification, prior to derivatization, chloroform (2:1, v/v) was added to an aliquot of the homogenates. Samples were then vortexed for 1 min and centrifuged at 13,000 × g for 10 min. The proteins in the resulting supernatant were measured using the Bio-Rad method and equalized. Immunodetection of Protein-bound 2,4-Dinitrophenylhydrazones, AGEs and CML—For immunodetection, after SDS-PAGE, proteins were transferred using a Mini Trans-Blot Transfer Cell (Bio-Rad) to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA). Immunodetection was performed using as primary antibodies: a rabbit anti-DNP antiserum (1:4000, Dako, Carpenteria, CA); 6D12, a monoclonal anti-CML antibody (1:2000, Transgenic Inc., Kumamoto, Japan); and a polyclonal anti-AGE antiserum, raised against glyoxilic acid-treated keyhole limpet hemocyanin (1:2000). Peroxidase-coupled secondary antibodies were used from the Tropix chemiluminescence kit (Tropix Inc., Bedford, MA). Luminescence was recorded and quantified in Lumi-Imager equipment (Boehringer), using the Lumianalyst software. Control experiments showed that EDTA presence does not lead to generation of artifactual oxidation and that omission of the derivatization step, primary or secondary antibody addition produced blots with no detectable signal (data not shown). Immunodetection of RAGE—A slightly modified protocol was used. Briefly, samples (0.2g) from diseased and control cases were homogenized in a glass homogenizer in 10 volumes of ice-cold lysis buffer (Hepes, pH 7.5, 250 mm sucrose, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol (DTT), 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride), and centrifuged at 5,000 × g for 10 min at 4 °C. Pellet fractions were discarded and protein concentrations of the supernatants were determined by the bicinchoninic acid method with bovine serum albumin as a standard. Samples containing 50 μg of protein were loaded onto 10% SDS-PAGE gels. Proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose filters. After transfer, the filters were blocked by incubation with 5% nonfat dry milk in 100 mm Tris-buffered saline-Tween, containing 140 mm NaCl, 0.1% Tween 20, pH 7.4 (TBS-T), 1 h at room temperature. Then the filters were incubated overnight with TBS-T containing 3% bovine serum albumin and anti-RAGE antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 1:100 dilution. Next, the filters were washed three times in TBS-T and incubated with TBS-T containing 5% skimmed milk and horseradish peroxidase-linked goat immunoglobulins (Dako) diluted 1:1000 for 45 min at room temperature. Filters were washed several times in TBS-T, and immunoreactivity was detected using an enhanced chemiluminescence Western blot detection system (Amersham Biosciences), followed by exposure to ECL HYPER film (Amersham Biosciences). Mouse monoclonal anti-α-tubulin at a dilution of 1:5000 was used to ensure equal loading of samples. Two-dimensional Electrophoresis—Cortex samples were homogenized in a denaturation buffer (9 m urea, 4% CHAPS, 0.8% 3–11 IPG NL Buffer (Amersham Biosciences), 1% DTT and protein was quantified. An aliquot containing 50 μg of protein was adjusted to a final volume of 200 μl using rehydration buffer (8 m urea, 0,5% CHAPS, 0,5% 3–11 IPG NL Buffer, 18, 15 mm DTT, and 2% bromphenol blue) and applied overnight to 3–11 NL 11-cm IPG Strips (Amersham Biosciences). Isoelectric focusing was performed as follows: 500 V for 3 h, a linear gradient to 1000 V for 1 h, and finally 6000 V for 3 h in a Bio-Rad system. Strips were then incubated for 10 min in 37.5 mm Tris-HCl, pH 8.8, containing 6 m urea, 2% (w/v) SDS 20% (v/v) glycerol, and 0.5% DTT, and then re-equilibrated for 10 min in the same buffer except that DTT was replaced with 4.5% iodoacetamide. The equilibrated strips were loaded in a 11% 20-cm long SDS-PAGE gel and run as described in one-dimensional electrophoresis. Immunoblotting was performed as described above, using a Semidry transfer system and an anti-MDAL polyclonal antibody (Academy Biomedical Co., Houston, TX) as primary antibody (1:2000). For gel staining, a MS-modified silver staining method (Amersham Biosciences) was used as described by the manufacturer. For membrane staining, a silver staining method based on the Gallyas intensifier was used according to previously described procedures (23Sorensen B.K. Hojrup P. Ostergard E. Jorgensen C.S. Enghild J. Ryder L.R. Houen G. Anal. Biochem. 2002; 304: 33-41Crossref PubMed Scopus (50) Google Scholar). Image Analysis—The gels (4 for each group) and polyvinylidene difluoride blots (n = 5) were scanned using a GS800 Calibrated Densitometer (Bio-Rad). PDQuest two-dimensional analysis software (Bio-Rad) was used for matching and analysis of silver-stained gels and membranes. The average mode of background subtraction was chosen to compare protein and MDAL immunoreactivity content between cortex samples from AD patients and control individuals. Normalized intensities of each protein spot from individual gels (or membranes) were compared between groups by the Student's t test. Mass Spectrometry for Protein Identification—After excision from gel, proteins were reduced with 10 mm DTT and alkylated with 55 mm iodoacetamide. Enzymatic digestion was performed with trypsin (Promega, Madison, WI) following conventional procedures as described (24Forne I. Carrascal M. Martinez-Lostao L. Abian J. Rodriguez-Sanchez J.L. Juarez C. J. Biol. Chem. 2003; 278: 50641-50644Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). After evaporation and redissolution in methanol/water (1:2 v/v), 1% acetic acid, digests were analyzed by matrix-assisted laser desorption/ionization reflectron time-of-flight (MALDI-reTOF MS). The MALDI-reTOF MS analysis of the samples was performed using a Voyager DE-PRO MALDI-reTOF mass spectrometer (Applied Biosystems, Foster City, CA). The instrument was run in reflectron mode with an average resolution of 12,000 full-width half-maximum at m/z 1500. A 5 mg/ml α-cyano-4-hydroxycinnamic solution was used as MALDI matrix. Spectra were externally calibrated using a standard peptide mixture consisting of angiotensin II, substance P, bombesin, and adrenocorticotropic hormone. When the ions corresponding to known trypsin autolytic peptides (m/z 842.5100 and 2111.1046) were detected, a second automatic internal calibration of the spectra was performed using these ions as reference. Data Base Search—The Protein Prospector software version 4.0.1 (University of California San Francisco, Mass Spectrometry Facility) was used to identify proteins from the peptide mass fingerprinting obtained from MALDI-reTOF MS. Swiss-Prot (European Bioinformatics Institute, Heidelberg, Germany) and GenBank™ (National Center for Biotechnology Information) data bases were used for the search. Measurement of GSA, AASA, CML, CEL, and MDAL—GSA, AASA, CML, CEL, and MDAL concentrations in total proteins from cerebral cortex homogenates were measured by gas chromatography/mass spectrometry (GC/MS) as previously described (25Pamplona R. Portero-Otin M. Requena J. Gredilla R. Barja G. Mech. Ageing Dev. 2002; 123: 1437-1446Crossref PubMed Scopus (110) Google Scholar). Samples containing 0.75–1 mg of protein were delipidated using chloroform/methanol (2:1, v/v), and proteins were precipitated by adding 10% trichloroacetic acid (final concentration) and subsequent centrifugation. Protein samples were reduced overnight with 500 mm NaBH4 (final concentration) in 0.2 m borate buffer, pH 9.2, containing 1 drop of hexanol as an anti-foam reagent. Proteins were then reprecipitated by adding 1 ml of 20% trichloroacetic acid and subsequent centrifugation. The following isotopically labeled internal standards were then added: [2H8]lysine (d8-Lys; CDN Isotopes); [2H4]CML (d4-CML), [2H4]CEL (d4-CEL), and [2H8]MDAL (d8-MDAL), prepared as described (8Fu M.X. Requena J.R. Jenkins A.J. Lyons T.J. Baynes J.W. Thorpe S.R. J. Biol. Chem. 1996; 271: 9982-9986Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar, 26Requena J.R. Fu M.X. Ahmed M.U. Jenkins A.J. Lyons T.J. Baynes J.W. Thorpe S.R. Biochem. J. 1997; 322: 317-325Crossref PubMed Scopus (270) Google Scholar); and [2H5]5-hydroxy-2-aminovaleric acid (for GSA quantization) and [2H4]6-hydroxy-2-aminocaproic acid (for AASA quantization) prepared as described in Ref. 5Requena J.R. Chao C.C. Levine R.L. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 69-74Crossref PubMed Scopus (378) Google Scholar. The samples were hydrolyzed at 155 °C for 30 min in 1 ml of 6 n HCl, and then dried in vacuo. The N,O-trifluoroacetyl methyl ester derivatives of the protein hydrolysate were prepared as previously described (5Requena J.R. Chao C.C. Levine R.L. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 69-74Crossref PubMed Scopus (378) Google Scholar). GC/MS analyses were carried out on a Hewlett-Packard model 6890 gas chromatograph equipped with a 30-m HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm) coupled to a Hewlett-Packard model 5973A mass selective detector (Agilent, Barcelona, Spain). The injection port was maintained at 275 °C; the temperature program was 5 min at 110 °C, then 2 °C/min to 150 °C, then 5 °C/min to 240 °C, then 25 °C/min to 300 °C, and finally hold at 300 °C for 5 min. Quantification was performed by external standardization using standard curves constructed from mixtures of deuterated and non-deuterated standards. Analytes were detected by selected ion-monitoring GC/MS. The ions used were: lysine and d8-lysine, m/z 180 and 187, respectively; 5-hydroxy-2-aminovaleric acid and d5–5-hydroxy-2-aminovaleric acid (stable derivatives of GSA), m/z 280 and 285, respectively; 6-hydroxy-2-aminocaproic acid and d4–6-hydroxy-2-aminocaproic acid (stable derivatives of AASA), m/z 294 and 298, respectively; CML and d4-CML, m/z 392 and 396, respectively; CEL and d4-CEL, m/z 379 and 383, respectively; and MDAL and d8-MDAL, m/z 474 and 482, respectively. The amounts of products were expressed as the ratio of micromole of glutamic semialdehyde, aminoadipic semialdehyde, CML, CEL, or MDAL/mol of lysine. Fatty Acid Analysis—Fatty acid analysis was performed as previously described (25Pamplona R. Portero-Otin M. Requena J. Gredilla R. Barja G. Mech. Ageing Dev. 2002; 123: 1437-1446Crossref PubMed Scopus (110) Google Scholar). Total lipids from human brain homogenates were extracted with chloroform/methanol (2:1, v/v) in the presence of 0.01% butylated hydroxytoluene. The chloroform phase was evaporated under nitrogen, and the fatty acids were transesterified by incubation in 2.5 ml of 5% methanolic HCl for 90 min at 75 °C. The resulting fatty acid methyl esters were extracted by adding 2.5 ml of n-pentane and 1 ml of saturated NaCl solution. The n-pentane phase was separated, evaporated under nitrogen, redissolved in 75 μl of carbon disulfide, and 1 μl was used for GC/MS analysis. Separation was performed in a SP2330 capillary column (30 m × 0.25 mm × 0.20 μm) in a Hewlett-Packard 6890 Series II gas chromatograph (Agilent). A Hewlett-Packard 5973A mass spectrometer was used as detector in the electron-impact mode. The injection port was maintained at 220 °C, and the detector at 250 °C; the temperature program was 2 min at 100 °C, then 10 °C/min to 200 °C, then 5 °C/min to 240 °C, and finally hold at 240 °C for 10 min. Identification of fatty acid methyl esters was made by comparison with authentic standards and based on mass spectra. Results are expressed as mol %. From fatty acid composition, the following indexes were calculated: saturated fatty acids = Σ % of saturated fatty acids; unsaturated fatty acids (UFA) = Σ % unsaturated fatty acids; monounsaturated fatty acids (MUFA) = Σ % of monoenoic fatty acids; polyunsaturated n-3 fatty acids (PUFAn-3) = Σ % of polyunsaturated fatty acids n-3 series; polyunsaturated n-6 fatty acids (PUFAn-6) = Σ % of polyunsaturated fatty acids n-6 series; average chain length = [(Σ % Total14 × 14) +... + (Σ % totaln × n)]/100 (n = carbon atom number); peroxidizability index = [(Σ mol % monoenoic × 0.025) + (Σ mol % dienoic × 1) + (Σ mol % trienoic × 2) + (Σ mol % tetraenoic × 4) + (Σ mol % pentaenoic × 6) + (Σ mol % hexaenoic × 8)] (11Cosgrove J.P. Church D.F. Pryor W.A. Lipids. 1987; 22: 299-304Crossref PubMed Scopus (465) Google Scholar). Statistical analyses—All statistics were performed using the SPSS software (SPSS Inc., Chicago, IL). Differences between groups were analyzed by" @default.
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