Plasma Protein Pentosidine and Carboxymethyllysine, Biomarkers for Age-related Macular Degeneration
2009; Elsevier BV; Volume: 8; Issue: 8 Linguagem: Inglês
10.1074/mcp.m900127-mcp200
ISSN1535-9484
AutoresJiaqian Ni, Xianglin Yuan, Jiayin Gu, Xiuzhen Yue, Xiaorong Gu, Ram H. Nagaraj, John W. Crabb,
Tópico(s)Retinal Imaging and Analysis
ResumoAge-related macular degeneration (AMD) causes severe vision loss in the elderly; early identification of AMD risk could help slow or prevent disease progression. Toward the discovery of AMD biomarkers, we quantified plasma protein Nε-carboxymethyllysine (CML) and pentosidine from 58 AMD and 32 control donors. CML and pentosidine are advanced glycation end products that are abundant in Bruch membrane, the extracellular matrix separating the retinal pigment epithelium from the blood-bearing choriocapillaris. We measured CML and pentosidine by LC-MS/MS and LC-fluorometry, respectively, and found higher mean levels of CML (∼54%) and pentosidine (∼64%) in AMD (p < 0.0001) relative to normal controls. Plasma protein fructosyl-lysine, a marker of early glycation, was found by amino acid analysis to be in equal amounts in control and non-diabetic AMD donors, supporting an association between AMD and increased levels of CML and pentosidine independent of other diseases like diabetes. Carboxyethylpyrrole (CEP), an oxidative modification from docosahexaenoate-containing lipids and also abundant in AMD Bruch membrane, was elevated ∼86% in the AMD cohort, but autoantibody titers to CEP, CML, and pentosidine were not significantly increased. Compellingly higher mean levels of CML and pentosidine were present in AMD plasma protein over a broad age range. Receiver operating curves indicate that CML, CEP adducts, and pentosidine alone discriminated between AMD and control subjects with 78, 79, and 88% accuracy, respectively, whereas CML in combination with pentosidine provided ∼89% accuracy, and CEP plus pentosidine provided ∼92% accuracy. Pentosidine levels appeared slightly altered in AMD patients with hypertension and cardiovascular disease, indicating further studies are warranted. Overall this study supports the potential utility of plasma protein CML and pentosidine as biomarkers for assessing AMD risk and susceptibility, particularly in combination with CEP adducts and with concurrent analyses of fructosyl-lysine to detect confounding factors. Age-related macular degeneration (AMD) causes severe vision loss in the elderly; early identification of AMD risk could help slow or prevent disease progression. Toward the discovery of AMD biomarkers, we quantified plasma protein Nε-carboxymethyllysine (CML) and pentosidine from 58 AMD and 32 control donors. CML and pentosidine are advanced glycation end products that are abundant in Bruch membrane, the extracellular matrix separating the retinal pigment epithelium from the blood-bearing choriocapillaris. We measured CML and pentosidine by LC-MS/MS and LC-fluorometry, respectively, and found higher mean levels of CML (∼54%) and pentosidine (∼64%) in AMD (p < 0.0001) relative to normal controls. Plasma protein fructosyl-lysine, a marker of early glycation, was found by amino acid analysis to be in equal amounts in control and non-diabetic AMD donors, supporting an association between AMD and increased levels of CML and pentosidine independent of other diseases like diabetes. Carboxyethylpyrrole (CEP), an oxidative modification from docosahexaenoate-containing lipids and also abundant in AMD Bruch membrane, was elevated ∼86% in the AMD cohort, but autoantibody titers to CEP, CML, and pentosidine were not significantly increased. Compellingly higher mean levels of CML and pentosidine were present in AMD plasma protein over a broad age range. Receiver operating curves indicate that CML, CEP adducts, and pentosidine alone discriminated between AMD and control subjects with 78, 79, and 88% accuracy, respectively, whereas CML in combination with pentosidine provided ∼89% accuracy, and CEP plus pentosidine provided ∼92% accuracy. Pentosidine levels appeared slightly altered in AMD patients with hypertension and cardiovascular disease, indicating further studies are warranted. Overall this study supports the potential utility of plasma protein CML and pentosidine as biomarkers for assessing AMD risk and susceptibility, particularly in combination with CEP adducts and with concurrent analyses of fructosyl-lysine to detect confounding factors. Age-related macular degeneration (AMD) 1The abbreviations used are:AMDage-related macular degenerationAGEadvanced glycation end productAccQ6-aminoquinolyl-N-hydroxysuccinimidyl carbamateCEPcarboxyethylpyrroleCIconfidence intervalCMLNε-carboxymethyllysineCNVchoroidal neovascularizationORodds ratioPECS-β(4-pyridylethyl)-l-cysteinePTCphenylthiocarbamylROCreceiver operating characteristicRPEretinal pigment epitheliumR.S.D.relative S.D.RAGE and AGE-Rreceptors for AGEsMRMmultiple reaction monitoring. 1The abbreviations used are:AMDage-related macular degenerationAGEadvanced glycation end productAccQ6-aminoquinolyl-N-hydroxysuccinimidyl carbamateCEPcarboxyethylpyrroleCIconfidence intervalCMLNε-carboxymethyllysineCNVchoroidal neovascularizationORodds ratioPECS-β(4-pyridylethyl)-l-cysteinePTCphenylthiocarbamylROCreceiver operating characteristicRPEretinal pigment epitheliumR.S.D.relative S.D.RAGE and AGE-Rreceptors for AGEsMRMmultiple reaction monitoring. is a progressive, multifactorial disease and a major cause of severe vision loss in the elderly (1Jager R.D. Mieler W.F. Miller J.W. Age-related macular degeneration.N. Engl. J. Med. 2008; 358: 2606-2617Crossref PubMed Scopus (1176) Google Scholar). Deposition of debris (drusen) in the macular region of Bruch membrane, the extracellular matrix separating the choriocapillaris from the retinal pigment epithelium (RPE), is an early, hallmark risk factor of AMD. The disease can progress to advanced dry AMD (geographic atrophy), which is characterized by regional degeneration of photoreceptor and RPE cells, or to advanced wet AMD (choroidal neovascularization (CNV)), which is characterized by abnormal blood vessels growing from the choriocapillaris through Bruch membrane beneath the retina. CNV accounts for over 80% of debilitating vision loss in AMD; however, only 10–15% of AMD cases progress to CNV. age-related macular degeneration advanced glycation end product 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate carboxyethylpyrrole confidence interval Nε-carboxymethyllysine choroidal neovascularization odds ratio S-β(4-pyridylethyl)-l-cysteine phenylthiocarbamyl receiver operating characteristic retinal pigment epithelium relative S.D. receptors for AGEs multiple reaction monitoring. age-related macular degeneration advanced glycation end product 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate carboxyethylpyrrole confidence interval Nε-carboxymethyllysine choroidal neovascularization odds ratio S-β(4-pyridylethyl)-l-cysteine phenylthiocarbamyl receiver operating characteristic retinal pigment epithelium relative S.D. receptors for AGEs multiple reaction monitoring. There is growing consensus that AMD is an age-related inflammatory disease involving dysregulation of the complement system; however, triggers of the inflammatory response have yet to be well defined. Oxidative stress appears to be involved as smoking significantly increases the risk of AMD (2Seddon J.M. Willett W.C. Speizer F.E. Hankinson S.E. A prospective study of cigarette smoking and age-related macular degeneration in women.JAMA. 1996; 276: 1141-1146Crossref PubMed Google Scholar), antioxidant vitamins can selectively slow AMD progression (3Age-Related Eye Disease Study Group A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss.Arch. 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Sci. 2007; 48 (E-abstract 34)Google Scholar, 11Gu J. Paeur G.J. Yue X. Narendra U. Sturgill G.M. Bena J. Gu X. Peachey N.S. Salomon R.G. Hagstrom S.A. Crabb J.W. Clinical Genomic And Proteomic Study Group Assessing susceptibility to age-related macular degeneration with proteomic and genomic biomarkers.Mol. Cell. Proteomics. 2009; 10.1074/mcp.M800453-MCP200Abstract Full Text Full Text PDF Scopus (89) Google Scholar). Oxidative protein modifications like carboxyethylpyrrole (CEP) and Nε-carboxymethyllysine (CML), both elevated in AMD Bruch membrane, stimulate neovascularization in vivo (12Okamoto T. Tanaka S. Stan A.C. Koike T. Kase M. Makita Z. Sawa H. Nagashima K. Advanced glycation end products induce angiogenesis in vivo.Microvasc. Res. 2002; 63: 186-195Crossref PubMed Scopus (42) Google Scholar, 13Ebrahem Q. Renganathan K. Sears J. Vasanji A. Gu X. Lu L. Salomon R.G. Crabb J.W. Anand-Apte B. 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Systemic complement activation in age-related macular degeneration.PLoS ONE. 2008; 3: e2593Crossref PubMed Scopus (298) Google Scholar). Support for this hypothesis also comes from mounting evidence that advanced glycation end products (AGEs) may play a role in AMD (4Ishibashi T. Murata T. Hangai M. Nagai R. Horiuchi S. Lopez P.F. Hinton D.R. Ryan S.J. Advanced glycation end products in age-related macular degeneration.Arch. Ophthalmol. 1998; 116: 1629-1632Crossref PubMed Scopus (149) Google Scholar, 5Hammes H.P. Hoerauf H. Alt A. Schleicher E. Clausen J.T. Bretzel R.G. Laqua H. N(epsilon)(carboxymethyl)lysin and the AGE receptor RAGE colocalize in age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 1999; 40: 1855-1859PubMed Google Scholar, 7Howes K.A. Liu Y. Dunaief J.L. Milam A. Frederick J.M. Marks A. Baehr W. Receptor for advanced glycation end products and age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 2004; 45: 3713-3720Crossref PubMed Scopus (111) Google Scholar, 18Handa J.T. Verzijl N. Matsunaga H. Aotaki-Keen A. Lutty G.A. te Koppele J.M. Miyata T. Hjelmeland L.M. Increase in the advanced glycation end product pentosidine in Bruch's membrane with age.Invest. Ophthalmol. Vis. Sci. 1999; 40: 775-779PubMed Google Scholar, 19Yamada Y. Ishibashi K. Ishibashi K. Bhutto I.A. Tian J. Lutty G.A. Handa J.T. The expression of advanced glycation endproduct receptors in rpe cells associated with basal deposits in human maculas.Exp. Eye Res. 2006; 82: 840-848Crossref PubMed Scopus (89) Google Scholar). AGEs are a heterogeneous group of mostly oxidative modifications resulting from the Maillard nonenzymatic glycation reaction that have been associated with age-related diseases and diabetic complications (20Baynes J.W. The role of AGEs in aging: causation or correlation.Exp. Gerontol. 2001; 36: 1527-1537Crossref PubMed Scopus (284) Google Scholar, 21Goh S.Y. Cooper M.E. Clinical review: the role of advanced glycation end products in progression and complications of diabetes.J. Clin. Endocrinol. Metab. 2008; 93: 1143-1152Crossref PubMed Scopus (766) Google Scholar). In 1998, CML was the first AGE to be found in AMD Bruch membrane and drusen (4Ishibashi T. Murata T. Hangai M. Nagai R. Horiuchi S. Lopez P.F. Hinton D.R. Ryan S.J. Advanced glycation end products in age-related macular degeneration.Arch. Ophthalmol. 1998; 116: 1629-1632Crossref PubMed Scopus (149) Google Scholar). Other AGEs have since been detected in AMD ocular tissues (5Hammes H.P. Hoerauf H. Alt A. Schleicher E. Clausen J.T. Bretzel R.G. Laqua H. N(epsilon)(carboxymethyl)lysin and the AGE receptor RAGE colocalize in age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 1999; 40: 1855-1859PubMed Google Scholar, 7Howes K.A. Liu Y. Dunaief J.L. Milam A. Frederick J.M. Marks A. Baehr W. Receptor for advanced glycation end products and age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 2004; 45: 3713-3720Crossref PubMed Scopus (111) Google Scholar, 18Handa J.T. Verzijl N. Matsunaga H. Aotaki-Keen A. Lutty G.A. te Koppele J.M. Miyata T. Hjelmeland L.M. Increase in the advanced glycation end product pentosidine in Bruch's membrane with age.Invest. Ophthalmol. Vis. Sci. 1999; 40: 775-779PubMed Google Scholar) and in Bruch membrane, drusen, RPE, and choroidal extracellular matrix from healthy eyes (6Crabb J.W. Miyagi M. Gu X. Shadrach K. West K.A. Sakaguchi H. Kamei M. Hasan A. Yan L. Rayborn M.E. Salomon R.G. Hollyfield J.G. Drusen proteome analysis: an approach to the etiology of age-related macular degeneration.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 14682-14687Crossref PubMed Scopus (959) Google Scholar, 22Farboud B. Aotaki-Keen A. Miyata T. Hjelmeland L.M. Handa J.T. Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in Bruch's membrane of the aging eye.Mol. Vis. 1999; 5: 11PubMed Google Scholar). CML, a nonfluorescent AGE, and pentosidine, a fluorescent cross-linking AGE, increase with age in Bruch membrane (18Handa J.T. Verzijl N. Matsunaga H. Aotaki-Keen A. Lutty G.A. te Koppele J.M. Miyata T. Hjelmeland L.M. Increase in the advanced glycation end product pentosidine in Bruch's membrane with age.Invest. Ophthalmol. Vis. Sci. 1999; 40: 775-779PubMed Google Scholar, 23Glenn J.V. Beattie J.R. Barrett L. Frizzell N. Thorpe S.R. Boulton M.E. McGarvey J.J. Stitt A.W. Confocal Raman microscopy can quantify advanced glycation end product (AGE) modifications in Bruch's membrane leading to accurate, nondestructive prediction of ocular aging.FASEB J. 2007; 21: 3542-3552Crossref PubMed Scopus (101) Google Scholar). Receptors for AGEs (RAGE and AGE-R1) appear elevated on RPE and photoreceptor cells in early and advanced dry AMD (7Howes K.A. Liu Y. Dunaief J.L. Milam A. Frederick J.M. Marks A. Baehr W. Receptor for advanced glycation end products and age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 2004; 45: 3713-3720Crossref PubMed Scopus (111) Google Scholar) especially in RPE overlying drusen-like deposits on Bruch membrane (19Yamada Y. Ishibashi K. Ishibashi K. Bhutto I.A. Tian J. Lutty G.A. Handa J.T. The expression of advanced glycation endproduct receptors in rpe cells associated with basal deposits in human maculas.Exp. Eye Res. 2006; 82: 840-848Crossref PubMed Scopus (89) Google Scholar). AGE-R3, also known as galectin-3, is elevated in AMD Bruch membrane (24Gu X. Yuan X. Crabb J.S. Shadrach K. Hollyfield J.G. Crabb J.W. Comparison of proteins in dry and wet AMD Bruch's membrane.Invest. Ophthalmol. Vis. Sci. 2009; 50 (E-abstract 2343)Google Scholar). Although AMD susceptibility genes now account for over 50% of AMD cases (25Fritsche L.G. Loenhardt T. Janssen A. Fisher S.A. Rivera A. Keilhauer C.N. Weber B.H. Age-related macular degeneration is associated with an unstable ARMS2 (LOC387715) mRNA.Nat. Genet. 2008; 40: 892-896Crossref PubMed Scopus (317) Google Scholar), many individuals with AMD risk genotypes may never develop advanced disease with severe vision loss. Nevertheless the prevalence of advanced AMD is increasing (26Friedman D.S. O'Colmain B.J. Muñoz B. Tomany S.C. McCarty C. de Jong P.T. Nemesure B. Mitchell P. Kempen J. Prevalence of age-related macular degeneration in the United States.Arch. Ophthalmol. 2004; 122: 564-572Crossref PubMed Scopus (76) Google Scholar). Toward the discovery of better methods to detect those at risk for advanced AMD, we quantified CML and pentosidine in plasma proteins from AMD and control patients and compared their discriminatory accuracy with plasma CEP biomarkers. CEP biomarkers have been shown to enhance the AMD predictive accuracy of genomic AMD biomarkers (11Gu J. Paeur G.J. Yue X. Narendra U. Sturgill G.M. Bena J. Gu X. Peachey N.S. Salomon R.G. Hagstrom S.A. Crabb J.W. Clinical Genomic And Proteomic Study Group Assessing susceptibility to age-related macular degeneration with proteomic and genomic biomarkers.Mol. Cell. Proteomics. 2009; 10.1074/mcp.M800453-MCP200Abstract Full Text Full Text PDF Scopus (89) Google Scholar). This report shows CML and pentosidine to be elevated in AMD plasma proteins and demonstrates their potential biomarker utility in assessing AMD risk and susceptibility especially in combination with CEP biomarkers. Clinically documented AMD and control blood donors were recruited prospectively between 2005 and 2008 from the Cole Eye Institute, Cleveland Clinic Foundation with Institutional Review Board approval and according to Declaration of Helsinki principles. All patients received a comprehensive eye examination by a clinician in the Clinical Study Group and provided written informed consent. Blood samples were collected after clinical examination and diagnosis and without the possibility of systematic bias. Human identifiers were removed, and the specimens were encoded by the Clinical Study Group to protect donor confidentiality. The study design a priori was to compare CML and pentosidine plasma protein levels in a statistically significant number of control, early/mid-stage AMD, and advanced AMD plasma from age- and gender-matched donors. AMD disease progression was categorized based on fundus examination, and patients were included in the study from Age-Related Eye Disease Study AMD categories 2, 3, and 4 (3Age-Related Eye Disease Study Group A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss.Arch. Ophthalmol. 2001; 119: 1417-1436Crossref PubMed Scopus (2519) Google Scholar). Briefly early/mid-stage AMD patients (Age-Related Eye Disease Study categories 2 and 3) exhibited multiple small to intermediate drusen in the macula or one or more large drusen, RPE pigmentary abnormalities, or any combination of these in one or both eyes or geographic atrophy that did not involve the macula and at least one eye having visual acuity 20/30 or better. AMD category 4 patients exhibited advanced AMD with substantial CNV or geographic atrophy involving the macula in one or both eyes; this cohort included 28 CNV and three geographic atrophy patients. Control donors lacked macular drusen and exhibited no clinical evidence of any retinal disorder. Study population characteristics are summarized in Table I, including age, gender, race, smoking status, and health history.Table ICharacteristics of the study populationProperty and categoryControl (n = 32)Early/mid-stage AMD (n = 27)Advanced AMD (n = 31)Age (year)Mean ± S.D.72 ± 673 ± 974 ± 6Range56–8457–9164–89GenderMale15 (46.9%)14 (51.9%)16 (51.6%)Female17 (53.1%)13 (48.1%)15 (48.4%)RaceCaucasian32 (100%)27 (100%)31 (100%)Smoking statusNon-smoker30 (93.8%)21 (77.8%)30 (96.8%)Smoker2 (6.2%)6 (22.2%)1 (3.2%)Health historyHypertension21 (65.6%)14 (51.9%)20 (64.5%)Hyperlipidemia15 (46.9%)14 (51.9%)10 (32.3%)Diabetes0 (0.0%)3 (11.1%)2 (6.5%)Cardiovascular disease6 (18.8%)7 (25.9%)5 (16.1%) Open table in a new tab Nonfasting blood specimens were collected in BD Vacutainer® K2EDTA tubes, and plasma was prepared within 6 h and aliquoted to vials containing the antioxidant butylated hydroxytoluene (22 µg/ml plasma) and a protease inhibitor mixture (Sigma product number P 8340; 10 µl/ml plasma) (11Gu J. Paeur G.J. Yue X. Narendra U. Sturgill G.M. Bena J. Gu X. Peachey N.S. Salomon R.G. Hagstrom S.A. Crabb J.W. Clinical Genomic And Proteomic Study Group Assessing susceptibility to age-related macular degeneration with proteomic and genomic biomarkers.Mol. Cell. Proteomics. 2009; 10.1074/mcp.M800453-MCP200Abstract Full Text Full Text PDF Scopus (89) Google Scholar). The plasma was flushed with argon, quench frozen in liquid nitrogen immediately, and stored at −80 °C until analysis. For the plasma used in this study storage time at −80 °C ranged from 4 to 34 months and averaged 13 months. All samples were frozen and thawed only once. Plasma (∼200 µl; ∼10-mg protein) was transferred to 6 × 50-mm glass hydrolysis tubes, and protein was precipitated with 2 volumes of cold acetone. After incubation at 4 °C for 10–20 min, the preparation was centrifuged briefly in a Microfuge, the supernatant was discarded, and the pellet was washed once with 67% acetone (400 µl) and vacuum-dried (27Crabb J.W. West K.A. Dodson W.S. Hulmes J.D. Amino acid analysis.in: Coligan J.E. Ploegh H.L. Smith J.A. Speicher D.W. Current Protocols in Protein Science. John Wiley & Sons, Inc., New York1997: 11.09.01-11.09.42Google Scholar). Plasma protein was prepared for hydrolysis by adding 60 µl of 6 n HCl to each dried pellet, and then the hydrolysis tubes were placed in a 40-ml screw cap vial containing ∼300 µl of 6 n HCl with a few small crystals of phenol. The 40-ml vial was capped with a Mininert slide valve, the valve was connected to a vacuum pump and argon source via a three-way stopcock, and the vial was alternately evacuated and flushed with argon three times and then sealed under vacuum (27Crabb J.W. West K.A. Dodson W.S. Hulmes J.D. Amino acid analysis.in: Coligan J.E. Ploegh H.L. Smith J.A. Speicher D.W. Current Protocols in Protein Science. John Wiley & Sons, Inc., New York1997: 11.09.01-11.09.42Google Scholar). Protein was hydrolyzed at 110 °C for 16 h, then vacuum-dried, flushed with argon, and stored at −20 °C until analysis. Protein was quantified by PTC amino acid analysis (∼80 µg was derivatized, and ∼3 µg was analyzed) using an Agilent 1100 HPLC system, a Haisil PTC C18 column (220 × 2.1 mm; Applied Biosystems), and a Gilson model 116 UV detector (27Crabb J.W. West K.A. Dodson W.S. Hulmes J.D. Amino acid analysis.in: Coligan J.E. Ploegh H.L. Smith J.A. Speicher D.W. Current Protocols in Protein Science. John Wiley & Sons, Inc., New York1997: 11.09.01-11.09.42Google Scholar). Protein was also quantified by AccQ·TagTM amino acid analysis (∼3.5 µg was derivatized, and ∼35 ng was analyzed) using an Acquity Ultra Performance LC system (Waters) and AccQ·Tag Ultra column (100 × 2.1 mm) according to the vendor (28Cohen S.A. Michaud D.P. Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography.Anal. Biochem. 1993; 211: 279-287Crossref PubMed Scopus (843) Google Scholar). Bovine serum albumin from the National Bureau of Standards was used as a protein standard and hydrolysis control. Amino acid calibration standards were obtained from Pierce and Thermo Scientific. Fructosyl-lysine was quantified as furosine derivative (ε-N-(2-furoylmethyl)lysine) by duplicate AccQ·Tag amino acid analyses (∼15 µg was derivatized, and ∼2.7 µg was analyzed) using the Acquity LC system (Waters) described above. Furosine contains a primary and a secondary amino group, and both are derivatized by the AccQ·Tag reagent, yielding two different monoderivatized forms and a diderivatized species. The apparent diderivatized species was well separated from other amino acids, exhibited a constant response factor up to ∼55 pmol derivatized, and was used for quantification of furosine in protein hydrolysates. Furosine standard was purchased from NeoMPS, Inc. Plasma protein HCl hydrolysates (∼8 mg in 40 µl of H2O) were spiked with a S-β(4-pyridylethyl)-l-cysteine (PEC) internal standard (50 pmol) and fractionated on LC system 1 composed of an Agilent 1100 HPLC system, a HypercarbTM porous graphite carbon column (5-µm particles, 50 × 10 mm; Thermo Scientific) maintained at 30 °C with an Applied Biosystems 112A column oven, and aqueous trifluoroacetic acid/acetonitrile solvents using gradient elution (0–100% acetonitrile over 13 min) and a flow rate of 1 ml/min. The eluant was monitored for fluorescence (335-nm excitation, 385-nm emission) with a WatersTM 474 scanning fluorometer and initially split with 20% directed to an API 3000 triple quadrupole electrospray mass spectrometer (Applied Biosystems) and 80% directed to a fraction collector. After determining reproducible elution times using control plasma spiked with standard AGEs and PEC, 100% of the eluant was directed to the fraction collector and three fractions were collected, one each for CML, PEC, and the co-eluting pentosidine plus argpyrimidine. The fractions were vacuum dried and re-fractionated on LC system 2 composed of the same HPLC equipment but with an aqueous normal phase Cogent Diamond Hydride™ Column (4.2 µm particles, 150 × 2.1 mm) used at room temperature. Fractions containing CML were re-chromatographed using aqueous acetic acid/acetonitrile solvents and gradient elution (95–0% acetonitrile in 13.6 min) at a flow rate of 400 µl/min. Fractions containing PEC, argpyrimidine and pentosidine were re-chromatographed using 10 mm ammonium acetate, pH 6.0/acetonitrile solvents, gradient elution (100–0% acetonitrile in 15 min) at a flow rate of 400 µl/min. Aqueous normal phase chromatography was monitored by fluorescence detection followed by 100% of the eluant directed to the mass spectrometer. CML and PEC were quantified by multiple reaction monitoring (MRM), and pentosidine and argpyrimidine were measured by fluorescence; final CML and pentosidine amounts were adjusted based on the recovery of the PEC internal standard. Plasma protein argpyrimidine concentrations were below reliable detection limits in this analytical system and are not reported. Calibration curves were developed in triplicate each day of analysis using LC system 2 and external standards. CML standard was purchased from NeoMPS, Inc., pentosidine was obtained from the International Maillard Reaction Society (Case Western Reserve University, Cleveland, OH), argpyrimidine was prepared in our laboratories by R. H. N., and PEC was purchased from Sigma. The mass spectrometer was operated with Analyst 1.4.1 software (Applied Biosystems); MS/MS spectra were generated on singly charged precursor ions for CML, pentosidine, argpyrimidine, and PEC; and specific transition ions for each modified amino acid were analyzed by MRM. The declustering potential, focusing potential, collision energy, and exit potential were optimized for each ion to ±0.1 Da and ±1 V. Ion spray voltage was set at 5300 V, and source temperature was set at 425 °C in LC system 1 and at 490 °C in LC system 2. The m/z of the precursor ions and each of the transitions ions and their optimized voltages were transcribed, respectively, into an LCsync method in the Analyst 1.4.1 software. CML was quantified by MRM using precursor ion 205.1 and product ion
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