Background: Glycated proteins present new immunological epitopes on their surface against which autoantibodies are generated that have a possible role in immunopathogenesis in diabetic complications. Methods: In the present study, in vitro glycation- and reactive oxygen species (ROS)-modified human serum albumin (HSA) has been studied by different spectroscopic techniques (UV and fluorescence) and thermal denaturation profiles. The binding characteristics of circulating autoantibodies in diabetic patients and diabetic patients with secondary complications against native HSA (N-HSA) and ROS-modified glycated HSA (RG-HSA) were assessed by direct and competition enzyme-linked immunosorbent assay (ELISA). In another approach, antibodies against RG-HSA (RG-HSA-Abs) induced in experimental animals were used as an immunochemical probe for the detection of gluco-oxidative lesions in blood proteins of patients (n = 8) with diabetic retinopathy. Results: Modified RG-HSA showed marked structural changes. High recognition of RG-HSA was shown by diabetic serum autoantibodies. Diabetic patients with retinopathy, nephropathy and atherosclerosis showed significantly (p < 0.001) stronger binding to RG-HSA over N-HSA. Normal human sera exhibited negligible binding with either antigen. Competitive inhibition ELISA results show significantly high binding of RG-HSA-Abs to albumin, immunoglobulin G and red blood cell membrane isolated from diabetic retinopathic patients. Conclusion: In conclusion, these results suggest that hyperglycemia together with ROS may contribute to the immunopathogenesis of diabetes-associated complications.

Bennett PH: Definition, diagnosis and classification of diabetes mellitus and impaired glucose tolerance; in Kahn CR, Weir GC (eds): Joslins Diabetes Mellitus. Philadelphia, Lea & Febiger, 1994, pp 193–200.
Uribarri J, Cai W, Peppa M, Goodman S, Ferrucci L, Striker G, Vlassara H: Circulating glycotoxins and dietary advanced glycation endproducts: two links to inflammatory response, oxidative stress and aging. J Gerontol A Biol Sci Med Sci 2007;62:427–433.
Huijberts MS, Schaper NC, Schalkwijk CG: Advanced glycation end products and diabetic foot disease. Diabetes Metab Res Rev 2008;24:19–24.
Jakus V, Rietbrock N: Advance glycation end-products and the progress of diabetic vascular complications. Physiol Res 2004;53:131–142.
Makino H, Shikata K, Hironaka K, Kushiro M, Yamasaki Y, Sugimoto H, Ota Z, Araki N, Horiuchi S: Ultrastructure of nonenzymatically glycated mesangial matrix in diabetic nephropathy. Kidney Int 1995;48:517–526.
Kume S, Takeya M, Mori T, Araki N, Suzuki H, Horiuchi S, Kodama T, Miyauchi Y, Takahashi K: Immunohistochemical and ultrastructural detection of advanced glycation end products in atherosclerotic lesions of human aorta with a novel specific monoclonal antibody. Am J Pathol 1995;147:654–667.
Khan ZA, Chakrabarti S: Cellular signaling and potential new treatment targets in diabetic retinopathy. Exp Diabetes Res 2007;2007:3186–3187.
Shibayama R, Araki N, Nagai R, Horiuchi S: Autoantibody against Nε-(carboxymethyl)lysine: an advanced glycation end product of the Millard reaction. Diabetes 1999;48:1842–1849.
Giugliano D, Ceriello A, Paolisso G: Oxidative stress and diabetic vascular complications. Diabetes Care 1996;19:257–267.
Palm F, Cederberg J, Hansell P, Liss P, Carlsson OO: Reactive oxygen species cause diabetes-induced decrease in renal oxygen tension. Diabetologia 2003;46:1153–1160.
Rasheed Z, Khan MWA, Ali R: Hydroxyl radical modification of human serum albumin generated cross reactive antibodies. Autoimmunity 2006;39:479–488.
Khan MWA, Sherwani S, Khan WA, Ali R: Characterization of hydroxyl radical modified GAD65: a potential autoantigen in type 1 diabetes. Autoimmunity 2009;42:150–158.
Khan MWA, Rasheed Z, Khan WA, Ali R: Biochemical, biophysical and thermodynamic analysis of in vitro glycated human serum albumin. Biochemistry (Mosc) 2007;72:146–152.
Schalkwijk CG, Ligtvoet N, Twaalfhoven H, Jager A, Blaauwgeers HGT, Schlingemann RO, Tarnow L, Parving HH, Stehouwer CDA, Van Hinsbergh VWM: Amadori albumin in type 1 diabetic patients: correlation with markers of endothelial function, association with diabetic nephropathy, and localization in retinal capillaries. Diabetes 1999;48:2446–2453.
Khan WA, Habib S, Khan MWA, Alam K, Moinuddin: Enhanced binding of circulating SLE autoantibodies to cathecholestrogen-copper-modified DNA. Mol Cell Biochem 2008;315:143–150.
Goding JW: Use of staphylococcal protein A as immunological reagent. J Immunol Methods 1978;20:241–254.
Khan WA: Binding characteristics of SLE anti-DNA autoantibodies to Catecholestrogen-modified DNA. Scand J Immunol 2006;64:677–683.
Tayyab S, Qasim MA: Purification and properties of buffalo serum albumin. Biochem Int 1990;20:405–415.
Oakley BR, Kirsch DR, Morris ND: A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 1980;105:361–362.
Simpson AJ, Narita S, Gieseg S, Gebicki S, Gebicki MJ, Dean RT: Long-lived reactive species on free-radical-damaged proteins. Biochem J 1992;282:621–624.
Levine LR, Garland D, Oliver NC, Amici A, Climent I, Lenz AG, Ahn BW, Shaltiel S, Stadtman ER: Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 1990;186:464–478.
Miyazaki K, Nagai R, Horiuchi S: Creatine plays a direct role as a protein modifier in the formation of a novel advanced glycation end product. J Biochem 2002;132:543–550.
Bouma B, Kroon-Batenburg JML, Wu PY, Briinjes B, Posthuma G, Kranenburg O, De-Groot PG, Voest EE, Gebbink MFBG: Glycation induces formation of amyloid cross-β structure in albumin. J Biol Chem 2003;43:41810–41819.
Baynes JW, Thorpe SR: Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 1999;48:1–9.
Ahmed KA, Muniandy S, Ismail IS: Nε-(carboxymethyl)lysine and coronary atherosclerosis-associated low density lipoprotein abnormalities in type 2 diabetes: current status. J Clin Biochem Nutr 2009;44:14–27.
Boulanger E, Wautier JL, Dequiedt P, Schmidt AM: Glycation, glycoxidation and diabetes mellitus. Nephrol Ther 2006;2:6–16.
McNulty M, Mahmud A, Feely J: Advanced glycation end-products and arterial stiffness in hypertension. Am J Hypertens 2007;20:242–247.
Thornalley PJ, Langborg A, Minhas HS: Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose. Biochem J 1999;344:109–116.
Uesugi N, Sakata N, Nagai R, Jono T, Horiuchi S, Takebayashi S: Glucoxidative modification of AA amyloid deposits in renal tissue. Nephrol Dial Transplant 2000;15:355–365.
Turk Z, Ljubik S, Turk N, Benko B: Detection of autoantibodies against advanced glycation end products and AGE-immune complexes in serum of patients with diabetes mellitus. Clin Chim Acta 2001;303:105–115.
Schutt F, Bergmann M, Holz FG, Kopitz J: Proteins modified by malondialdehyde, 4-hydroxynonenal, or advanced glycation end products in lipofuscin of human retinal pigment epithelium. Invest Ophthalmol Vis Sci 2003;44:3663–3668.
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.