Abstract
The advanced stage of the Maillard reaction that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it has been proposed that the intermediates contributing to AGE formation include dicarbonyl intermediates such as glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG). In the present study, we developed a novel, non-carboxymethyllysine (CML) anti-AGE antibody that recognizes serum proteins and peptides modified by 3-DG in vivo. AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with 3-DG or D-glucose. After immunization of rabbits, anti-AGE antisera were subjected to affinity chromatography on a Sepharose 4B column coupled with CML-BSA, or AGE-BSA created by incubation with 3-DG (AGE-6) or D-glucose (AGE-1). The AGE-Ab-6 and AGE-Ab-1 thus obtained was used to investigate AGEs in serum from diabetic patients on hemodialysis. Characterization of the novel AGE-Ab-6 obtained by immunoaffinity chromatography was performed with a competitive ELISA and immunoblot analysis. This antibody specifically cross-reacted with proteins modified by 3-DG. AGE-6 was detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD, while AGE-1 was detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD. This study provides new data on the pathways of AGE formation from 3-DG and methods for the immunochemical detection of AGEs. We also provide immunochemical evidence for the existence of six distinct AGEs in vivo among the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis.
Highlights
The modification, aggregation, and deposition of proteins is a prominent part of many pathological processes and can play a direct role in tissue damage
The formation of advanced glycation endproducts (AGEs) by rabbit serum albumin (AGE-RSA) during incubation with 3-DG for 2 weeks was monitored by AGE-specific fluorescence
There is general agreement that there are multiple sources and mechanisms involved in the formation of AGEs in vivo, including oxidative and nonoxidative processing of reducing sugars, Schiff bases, Amadori adducts, and metabolic intermediates such as glyceraldehyde and methylglyoxal (MGO) [17,25,26,29,30,31,32,33,34,35,36]
Summary
The modification, aggregation, and deposition of proteins is a prominent part of many pathological processes and can play a direct role in tissue damage. In the presence of hyperglycemia, glucose reacts with the free amino groups of amino acids, proteins, phospholipids, and nucleic acids This process begins with the conversion of reversible Schiff base adducts to more stable, covalently-bound Amadori rearrangement products. In the intermediate stage of the Maillard reaction, the Amadori products undergo multiple dehydration steps and rearrangements that produce highly reactive carbonyl compounds such as 3-deoxyglucosone (3-DG) [12,13,14,15] These reactive carbonyl compounds again react with free amino groups, leading to cross-linking and browning of proteins and the formation of AGEs in the late stage of the Maillard reaction [16]. Results: Characterization of the novel AGE-Ab-6 obtained by immunoaffinity chromatography was performed with a competitive ELISA and immunoblot analysis This antibody cross-reacted with proteins modified by 3-DG. We provide immunochemical evidence for the existence of six distinct AGEs in vivo among the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis
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