Structure of a Synthetic Glucose Derived Advanced Glycation End

Yousef Al-Abed* and Richard Bucala. The Picower Institute for Medical Research, 350 Community Drive, Manhasset, New York 11030. Received May 20, 1999;...
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Bioconjugate Chem. 2000, 11, 39−45

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Structure of a Synthetic Glucose Derived Advanced Glycation End Product That Is Immunologically Cross-Reactive with Its Naturally Occurring Counterparts† Yousef Al-Abed* and Richard Bucala The Picower Institute for Medical Research, 350 Community Drive, Manhasset, New York 11030. Received May 20, 1999; Revised Manuscript Received September 6, 1999

Glucose reacts nonenzymatically with the amino groups of proteins to form stable, cross-linking adducts called advanced glycation end products or AGEs. While several lines of evidence have established that AGEs accumulate in tissues and contribute to the pathological sequelae of diabetes and aging, the identity of the major cross-link(s) that forms in vivo has remained enigmatic. This has been considered to be due to the labile nature and to the low fluorescence properties of this cross-link, despite the fact that fluorescence has been generally associated with AGE formation in vivo. Accordingly, the few AGE adducts that have been isolated thus far from proteins in vivo or from model systems in vitro have been found to account for only a fraction of the glucose-derived crosslinks that occur in tissues. This situation has been further underscored by the development of a wellcharacterized class of antibodies that recognize in vivo AGEs but which fail to react with the structurally defined AGEs that have been identified to date. This particular class of anti-AGE antibodies has proven valuable in the quantification of AGE-modified forms of hemoglobin, low-density lipoprotein (LDL), and β-amyloid peptide, and can provide prognostic information on the course of certain diabetic complications. To obtain insight into the structure of this immunoreactive, AGE adduct, we used an anti-AGE antibody (RU) as a probe to isolate novel AGE(s) that formed within a reaction mixture of glucose and the model glycation substrate, NR-CBZ-Arg-Lys. HPLC purification of an immunoreactive fraction that accumulated in this preparation showed the presence of a compound that was determined by 1H NMR and electrospray ionization mass spectrometry (ESI-MS) to be a stable, imidazole-based cross-link (termed arginine-lysine imidazole or ALI). The properties of ALI, immunoreactivity, acid-lability, nonfluorescence, and inhibition of formation by aminoguanidine, suggest that ALI is likely to typify an important class of the AGE cross-links that form in vivo.

INTRODUCTION

The posttranslational modification process known as advanced glycation begins with the nonenzymatic addition of reducing sugars to the free amino groups of macromolecules. Over time, the initial Schiff base and Amadori products that form undergo rearrangement, dehydration, and fragmentation reactions to produce more complex and irreversibly bound structures that can exhibit varied spectral and covalent cross-linking properties. These later products, termed advanced glycation end products or AGEs,1 have been shown to adversely affect the functional properties of proteins, lipids, and DNA (1-3). The significance of AGE formation in biological systems has become increasingly apparent over the last † Portions of this work were presented at the 6th International Symposium on the Maillard Reaction, London, U.K. (July 2730, 1997), and published in the proceeding volume from that symposium. * To whom correspondence should be addressed. Phone: (516) 562-9461. Fax: (516) 365-5090. E-mail: [email protected]. 1 Abbreviations: AFGP, 1-alkyl-2-formyl-3,4-diglycosylpyrrole; AGE, advanced glycation endproduct; AL, NR-CBZ-Arg-Lys; ALAP, NR-CBZ-Arg-Lys-Amadori product; ALI, Arg-Lys-imidazole; AP, Amadori product; BSA, bovine serum albumin; CML, carboxy-methyllysine; CP, cyclic pentosidine; ESI-MS, electrospray ionization mass spectrometry; FFI, 4-furanyl-2-furoyl-1Himidazole; IA, imidazolium adduct; NR-CBZ-Arg-Lys, N-carboxybenzoyloxy-Arg-Lys; P, pentosidine; PA, pyrimidinium adduct; PY, pyrraline; RNase, ribonuclease.

15 years, and it is now evident that AGEs accumulate in situ in living tissues and play an important role in protein turnover, tissue remodeling, and certain of the pathological sequelae of diabetes, renal insufficiency, and aging (1, 3). AGEs have been identified to form in vivo on basement membrane collagen (4), red cell hemoglobin (5), the serum protein 2-microglobulin (6), and the Alzheimer’s disease-amyloid peptide (7, 8). AGEs also have been shown to modify the apolipoprotein and the lipid components of low-density lipoprotein (LDL), and to inhibit the normal, receptor-mediated clearance of LDL from the circulation (9, 10). While several lines of evidence relying on glucosedependent increases in protein cross-linking, fluorescence, and amino group modification have established that AGEs exist in living tissue, the identity of the major AGE cross-link(s) that forms in vivo has remained unknown. This situation has been attributed in large part to the chemical complexity of the cross-linking process, which follows the principles of the Maillard reaction 2, and the inherent instability of the cross-links to the hydrolysis procedures that are necessary to liberate the adduct structure from the macromolecular backbone (1-4). One AGE-cross-link, pentosidine, that has been purified from human dura collagen appears to form as the condensation product of lysine, arginine, and a reducing sugar precursor (4, 11). Nevertheless, measurements of pentosidine content indicate that this product accounts for only a small percentage (1%) of the glucose-

10.1021/bc990061q CCC: $19.00 © 2000 American Chemical Society Published on Web 12/10/1999

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derived cross-links that exist in biological samples (11). There is also data to suggest that the pathologically relevant cross-links may not themselves be fluorescent, a property that has been associated historically with AGE formation and used almost universally as an indicator of the Maillard reaction in vivo (1, 11-14). Several years ago, we utilized hyperimmunization techniques against an AGE-cross-linked antigen to produce polyclonal and monoclonal antibodies that recognize in vivo formed AGEs (15). These antibodies made possible the development of immunohistochemical and ELISAbased technologies that were free of many of the specificity and sensitivity problems associated with fluorescencebased assays and provided for the first assessment of advanced glycation in living systems (5). Remarkably, these anti-AGE antibodies were found to recognize a class of AGEs that formed in vivo, but which were immunochemically distinct from previously characterized adducts such as pentosidine, pyrraline, carboxymethyllysine (CML), or (1-alkyl-2-formyl-3,4-diglycosylpyrrole) AFGP (15). Of importance, the specific AGE epitope recognized by these antibodies increased as a consequence of hyperglycemia on various proteins such as collagen, hemoglobin, and LDL (5, 10, 15-17). One particular polyclonal antibody species, designated “RU”, has been employed in human clinical studies to examine the efficacy of the pharmacological inhibitor of AGE formation, aminoguanidine (5, 10), and has been shown to provide prognostic information on the course of diabetic renal disease (17). To obtain insight into the chemical structure of this potentially important in vivo cross-link(s), we have used the RU anti-AGE antibody as a molecular probe to isolate the immunoreactive and potentially novel AGEs(s) contained within reaction mixtures prepared by the in vitro reaction of glucose with the model glycation substrate, N-CBZ-Arg-Lys. We report herein the isolation and characterization of a novel, imidazole-based argininelysine cross-link. This AGE is nonfluorescent, acid-labile, and appears to represent an important class of the AGE cross-links that form in vivo. EXPERIMENTAL PROCEDURES

Preparation of AGE Reaction Mixtures. To a solution of NR-CBZ-Arg-Lys (1 g, 2.25 mmol, Princeton Biochemical, Cleveland OH) in 500 mL of aqueous 0.2 M phosphate buffer (pH 7.4) was added D-glucose (22.5 mmol). The reaction mixture was stirred at 37 °C for 5 weeks. At intervals, 10 µL of the reaction mixture was analyzed by HPLC using an analytical primesphere column (5C18 MC, 5 micron, 250 × 4.6 mm, Phenomenex, Torrance, CA) and a binary solvent gradient consisting of 0.05% TFA in H2O (solvent A) and methanol (solvent B). Solvent was delivered at a flow rate of 1.0 mL/min as follows: 0-30 min, a linear gradient from A:B (95:5) to A:B (25:75); 30-45 min, a linear gradient from A:B (25:75) to A:B (0:100). Detection was by monitoring UV absorption at 214, 254, 280, 320, and 350 nm. For preparative HPLC, a 250 × 21.2 mm primesphere column 5C18 MC was employed. Solvent was delivered at a flow rate of 10 mL/min using the same gradient as described above. One milliliter fractions were collected and concentrated 10-fold, and 50 L aliquots subjected to competitive ELISA as described below. A prominent immunoreactive peak was identified and further purified by repetitive HPLC. Spectroscopic analysis of this subfraction revealed the structure of compound ALI, and in a high purity (>95%): ESI-MS m/z 545 [M]+; 1H NMR (270

Al-Abed and Bucala

MHz, D2O) 1.27-1.63 (10 H, m, -CH2-), 2.46 (1H, dd, J ) 18.3, 1.2 Hz, Hb), 2.89 (2H, m, -CH2-N), 2.91 (1H, d, J ) 17.6 Hz, Hb), 3.12 (2H, m, -CH2-N), 4.06 (1H, m, -NCHCO-), 4.23 (1H, m, -NCHCO-), 4.30 (1H, dd, J ) 10.4, 1.0 Hz, Hd), 4.48 (1H, dd, J ) 10.4, 4.2 Hz, Hd), 4.66 (1H, m, Hc), 5.07 (2H, s, -OCH2Ph), 7.36 (6H, m, -Ph and Ha). The ability of aminoguanidine to inhibit ALI formation was tested by including 50 mM aminoguanidine-HCl (Alteon Inc, Ramsey, NJ) in the incubation described above together with NR-CBZ-Arg-Lys and D-glucose. The acid stability of ALI was tested by subjecting a preparation of purified ALI to standard acid hydrolysis conditions (6 N HCl, 110 °C, overnight), removing the acid by lyophilization, and subjecting a neutralized solution of the preparation to 1H NMR and ELISA analysis. Immunochemical Detection of AGEs. HPLC fractions and purified compounds were analyzed by an AGEspecific ELISA following methods described previously (15). This ELISA employed a polyclonal anti-AGE antibody raised by hyperimmunization against an AGE-crosslinked preparation of ribonuclease (AGE-RNase). Total IgG was prepared by protein-G affinity chromatography and the RNase backbone specificities removed by immunoabsorption against an RNase-linked affinity column. For assaying AGE immunoreactivity, 96-well roundbottom microtiter plates (Costar, Cambridge, MA) first were coated with AGE-modified BSA (AGE-BSA, 3 mg/ mL, dissolved in 0.1 M sodium bicarbonate, pH 9.6). After washing, the unbound sites were blocked with SuperBlock following the manufacturer’s recommendations (Pierce, Rockford, IL). Dilutions of test antigen together with anti-AGE IgG then were added and the plates incubated at room temperature for 1 h. The plates were washed again and incubated with a secondary antibody (alkaline phosphatase-conjugated anti-rabbit IgG) at 37 °C for 1 h. The unbound antibodies were removed by extensive washing, and the bound antibodies were detected by incubation with p-nitrophenyl phosphate (pNPP) substrate for 30-60 min and recording the optical density at 405 nm by an ELISA reader (EL309, Bio-Tek Instruments Inc., Burlington, VT). Results were expressed as B/B0, calculated as [experimental OD - background OD (i.e., no antibody)]/[total OD (i.e., no competitor) background OD] (15). The various test antigens included 4-furanyl-2-furoyl1H-imidazole (FFI) (18), carboxy-methyllysine (CML) (19), 1-alkyl-2-formyl-3,4-diglycosylpyrrole (AFGP) (20), pyrraline (PY) (21), pentosidine (P) (4), cyclic pentosidine (CP) (22), NR-CBZ-Arg-Lys (AL), NR-CBZ-Arg-Lys-AP (ALAP), a pyrimidinium AGE adduct (PA) (23), and an imidazolium AGE adduct (IA) (13). Histidine and LysHis dipeptide were purchased from Bachem (Torrance, CA). Analytical Methods. NMR spectra were recorded in D2O on a JEOL (270 MHz) spectrometer. HPLC was performed using a Hewlett-Packard model 1090 and Waters Instruments model 626 pump and 490 E multiwavelength detector. Electrospray ionization (ESI) samples were run on a Quattro triple quadrupole mass spectrometer. Loop injection samples were performed using an ABI model 140B syringe pump employing H2O/CH3CN at a flow of 15 µL/ min, a Rheodyne model 7125 valve with a 10 µL loop, and a Micromass Megaflow ESI probe using nitrogen for the nebulizer/drying gas. LC/MS samples were run employing the ABI pump and Rheodyne valve with a 20 µL loop at a flow of 50 µL/min.

AGE-Cross-Link

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Figure 1. Reversed-phase HPLC profile of the reaction products formed by the incubation of NR-carboxybenzoyloxy-Arg-Lys (NRCBZ-Arg-Lys) with 10 equivalents of glucose at 37 °C. The elution position of NR-CBZ-Arg-Lys (AL) and the N-CBZ-Arg-Lys-Amadori product (ALAP) is indicated. The lower panel shows an expansion of the chromatogram from 33 to 46 min (-) and the reactivity of collected fractions when analyzed by competitive ELISA with the anti-AGE antibody species “RU”. The ELISA immunoreactivity is expressed as B/Bo (9). No immunoreactivity was demonstrable elsewhere in the chromatogram. RESULTS

We selected as a target substrate for AGE formation the model dipeptide NR-carboxybenzoyloxy-Arg-Lys (NCBZ-Arg-Lys). The close, intramolecular association of the arginine and lysine side chains provides for an important proximity effect that promotes cross-link formation, and a synthetic strategy employing an ArgLys dipeptide has been used recently to isolate a cyclic pentosidine in high yield (22). We incubated NR-CBZ-ArgLys together with 10 equiv of glucose in 0.2 M phosphate buffer (pH 7.4) for up to 5 weeks at 37 °C. Analysis of this mixture by reversed-phase HPLC revealed a number of chromatographically distinct reaction products (Figure 1). Each fraction was isolated, concentrated, and analyzed by 1H NMR and for its reactivity with anti-AGE antibody by competitive ELISA. The product(s) contained in fractions corresponding to the 34.5-34.7 min peak (and present in 1.5% yield) were found to completely block antibody binding and showed both dose dependence and a steep inhibition curve (Figure 1, and data not shown). Further purification of this fraction by repetitive HPLC using a semipreparative column showed the presence of a single UV-active compound (0.6% yield). The complete UV spectrum of the purified compound was unremarkable, and similar to that of the starting material. No fluorescence emission properties were noted. A 1H NMR spectrum in D2O showed, in addition to the NR-CBZ-ArgLys protons, five aliphatic protons that resonate between 2.46 and 4.65 ppm (5H), and an olefinic proton that resonates at 7.3 ppm within the CBZ-group (Figure 2).

NMR analysis revealed a geminal coupling of ∼18 Hz between the two protons at 2.46 and 2.91 ppm. A geminal coupling of the magnitude 10.4 Hz also was found across the two protons which resonate at 4.30 and 4.48 ppm. Of note, each proton of the methylene sets displayed an additional vicinal coupling to a single methine proton [-CH2-CH(OH)-CH2-]. The methine proton resonates at 4.65 ppm as multiplet however, with coupling constants of 1.24, 5.4, and 4.2 Hz. Overall, these data confirmed the presence of a glucose-derived adduct involving the side chain amines of NR-CBZ-Arg-Lys. The electrospray ionization mass spectrometry of the N-CBZ-Arg-Lys adduct displayed a molecular ion of m/z 545.3 [MH]+, an increase of 108 Da compared to the starting material, NR-CBZ-Arg-Lys (mol. mass: 435 Da) (Figure 3). Taken together, these data are consistent with the structure of a imidazole-derived cross-link in which the guanidino group of the arginine side chain is linked covalently to the Amadori product-derived moiety. We propose the following mechanism of formation for this imidazole cross-link, which we have assigned the trivial name arginine-lysine imidazole (ALI) (Figure 4). Glucose reacts with NR-CBZ-Arg-Lys to give an Amadori product (AP) at the amino group. The NR-CBZ-Arg-Lys Amadori product (ALAP) forms in 18% yield under the reaction conditions used in these studies, based on HPLC isolation and 1H NMR characterization. Dehydration of the hydroxyl group at the C-4 position then occurs to produce the obligate, 1,4-dideoxy-1-alkylamino-2,3-hexodiulose reactive intermediate (ALAP-dione). The revers1H

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Figure 2.

1H

Al-Abed and Bucala

NMR spectrum recorded in D2O of the purified, immunoreactive adduct ALI.

Figure 3. ESI mass spectrum of purifed ALI obtained as described in the Experimental Procedures.

ible addition of the guanidino moiety to the dicarbonyl then yields the 2-amino-4,5-dihydroxyimidazole, which undergoes dehydration to deliver the stable ALI. ALI is nonfluorescent and destroyed by acidification (6 N HCl). ALI also was not identified by HPLC and 1H NMR, or by immunoreactivity, in reaction mixtures supplemented with the glycation inhibitor aminoguanidine (data not shown). Figure 5 shows the dose-dependent reactivity of pure ALI in a competitive, ELISA utilizing the anti-AGE species “RU”. This antibody has been shown previously to recognize a major class of in vivo-formed AGEs which are sensitive to pharmacological inhibition by aminoguanidine (10, 13). The binding curve is steep, shows 50% inhibition at 500 nmol, and is almost coincident with that of AGE-modified BSA, suggesting an immunochemical relationship between ALI and AGE-BSA. For comparison purposes, we also studied the immunochemical reactivity of several structurally defined AGEs [4-furanyl2-furoyl-1H-imidazole (FFI) (18), carboxy-methyllysine (CML) (19), 1-alkyl-2-formyl-3,4-diglycosylpyrrole (AFGP) (20), pyrraline (PY) (21), pentosidine (P) (4), cyclic pen-

tosidine (CP) (22), a pyrimidinium (non-cross-linking) AGE adduct (PA) (13), an imidazolium (non-cross-linking) adduct (IA) (13, 23)], two precursor products [NR-CBZArg-Lys (AL), NR-CBZ-Arg-Lys-AP (ALAP)], and related epitopes [histidine (H), Lys-His (LH)]. With the exception of ALI and consistent with prior studies (15), none of these compounds showed detectable crossreactivity with the RU anti-AGE antibody species. DISCUSSION

AGEs form by chemical principles first described for the Maillard reaction 2. In the case of protein glycation, the initial event is the reaction of a reducing sugar such as glucose with the N-terminus of a protein or the amino group of a lysine to form an aldimine or Schiff base. The Schiff base can hydrolyze back to its reactants or undergo an Amadori rearrangement to form the more stable N-(1deoxy-1-fructosyl)lysine AP. The reaction pathway leading to reactive, cross-linking moieties (i.e., AGE formation) commences by further rearrangement of the AP. Possible routes include loss of the 4-hydroxyl group of the AP by dehydration to give a 1,4-dideoxy-1-alkyl-

AGE-Cross-Link

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Figure 4. Proposed scheme for the formation of ALI, showing the important role of dehydration in producing the reactive, ALAPdione intermediate. ALAP, N-CBZ-Arg-Lys-Amadori product.

Figure 5. ELISA competition curves for ALI, selected AGEs, precursors, and related compounds utilizing anti-AGE antibody species “RU”, shown previously to recognize in vivo formed AGEs (13). Assays employed glucose-derived, AGE-albumin (AGE-BSA) as the absorbed antigen and were performed as described in the Experimental Procedures. All points represent the mean of triplicate determinations and each curve is representative of at least two independent experiments. (O) AGEBSA, (b) ALI, (2) CML, (9) common line defined by the reactivity of AL, ALAP, AFGP, CP, FFI, IA, H, LH, P, PA, PY. No detectable crossreactivity was observed with acid-treated ALI, or Na-CBZ-Arg-Lys incubated with glucose in the presence of the advanced glycation inhibitor aminoguanidine.

amino-2,3-hexodiulose (AP-dione) (2), and an AP-dione with the structure of an amino-1,4-dideoxyosone has been isolated by trapping model Amadori products with the AGE inhibitor aminoguanidine (2, 24). Among the biological activities of early- and late-forming (i.e., AGE) glycation products, the formation of stable cross-links is considered to be their most important pathological manifestation (1, 3). In an important study (4), Sell and Monnier isolated from human dura collagen the fluorescent cross-link pentosidine, which is a condensation product of lysine, arginine, and a reducing sugar precursor. While the exact mechanism of pentosidine formation in vivo remains uncertain, cross-link formation requires that the sugarderived intermediate contain a dicarbonyl functionality that can react irreversibly with the guanidinium group of arginine. Short-chain, dicarbonyl-containing compounds such as methylglyoxal and glyoxal are known to participate in condensation reactions with the side chains of arginine and lysine, and the addition of methylglyoxal to the guanidine moiety of arginine in vitro has been

shown to produce pyrimidinium (13) and imidazol-4-one (13, 22) adducts. Similarly, the dicarbonyls 1- and 3-deoxyglucosone have been implicated in the formation of certain AGE cross-links in vitro (25, 26). Despite these mechanistic insights, the identification of the major cross-links that form in vivo has been problematic. While pentosidine has been identified to form in collagen (4, 11), circulating serum proteins (27), and the central nervous system plaques of Alzheimer’s disease plaque (8, 28), it does not appear to account for more than a small percentage of the glucose-derived cross-links that exist in aged or diabetic tissues (4, 11, 27). Indirect assessments of protein cross-linking based on collagen digestion and fluorescence measurements argue that the major AGE cross-link that forms in vivo is nonfluoresecent and acid labile (1, 11, 12). This conclusion has been reinforced by the development of a class of anti-AGE antibodies, such as RU, which can identify AGE-modified species of collagen, serum proteins, hemoglobin, and LDL that form in vivo in a glucose- and age-dependent fashion but which does not recognize pentosidine or any of the other model AGE structures that have been elucidated to date (15). Moreover, the AGE epitope(s) defined by the RU antibody can be inhibited from forming in human subjects by the pharmacological inhibitor of AGE formation, aminoguanidine (5, 10). By utilizing the RU anti-AGE antibody as a selection criteria for the identification of immunoreactive AGE structures, we have identified a novel, AGE cross-link, termed ALI, that forms in a synthetic mixture consisting of glucose and the N-blocked dipeptide, NR-CBZ-Arg-Lys. The structure of ALI is that of an imidazole-based crosslink and fulfills the criteria proposed for that of the major, AGE cross-link that forms in vivo: nonfluorescence, acidlability, and inhibition of formation by aminoguanidine. While we cannot exclude the possibility that additional AGEs, perhaps also bearing an imidazole cross-link structure, may yet be uncovered, the identification of ALI should assist in the further molecular analysis of the glucose-derived, macromolecular damage that occurs in vivo. When analyzed for immunoreactivity, ALI displayed an antibody inhibition curve that was virtually coincident, on a molar basis, with that of AGE-modified

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albumin (AGE-BSA). This suggests a potentially close immunochemical relationship between ALI and AGEBSA. Interestingly, a major site of AGE-modification within albumin has been proposed to be at an Arg-Lys motif (22). This could produce an intramolecular, AGEcross-link within albumin (22), and explain the relatively poor intermolecular cross-linking activity of albumin when compared to other AGE-substrates such as ribonuclease or collagen (11, 29). Of note, ribonuclease can form intramolecular cross-linking via the arginine and lysine side chain residues due to their proximity in the following sequence arginine-serine-lysine (31-33) and arginine-cysteine-lysine (39-41). The structure of ALI further affirms the important role of arginine in effecting glucose-mediated protein crosslinking (2). By this pathway, the amino group of lysine or the free amino terminus of a protein acts to initiate AGE formation by generating the Amadori and the subsequent dehydration products that constitute the reactive, cross-linking intermediates. The guanidinium group of arginine then traps the dicarbonyl moiety to produce a stable, covalent cross-link. The AP-dione is thus a critical intermediate in this pathway, supporting prior, pharmacologically based studies that have implicated both the AP-dione and its dehydration product, the AP-ene-dione in the formation of stable protein-protein cross-links (30). These dehydration products also have been shown to be involved in the formation of the cyclization product cypentodine (31) and to contribute to an additional pathway of protein cross-linking that may occur by the Michael addition of lysine, histidine, or cysteine (2). In conclusion, the identification of the nonfluorescent cross-link ALI fills an important gap in our understanding of the advanced glycation process. Further studies of ALI will facilitate the clinical assessment of pathologically important AGEs and potentially aid in the design of more specific, pharmacological inhibitors of AGE formation. ACKNOWLEDGMENT

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