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Immunological Detection of a Novel Advanced Glycation End-Product

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Abstract

Background

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.

Materials and Methods

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.

Results

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.

Conclusion

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.

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Acknowledgments

These studies were supported in part by a Grant-in-Aid for Scientific Research (#13470197) to Masayoshi Takeuchi from the Japanese Ministry of Education, Science, Sports, and Culture, by a research grant to Masayoshi Takeuchi from Japan Foundation for Aging and Health, by a research grant to Masayoshi Takeuchi from the Hokuriku University, by a research foundation (#12HIAC124) to Masayoshi Takeuchi from the Hokuriku Industrial Advancement Center, and by the diabetes research foundation (#1-2000-263) to Soroku Yagihashi from Juvenile Diabetes Foundation International.

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Pharmaceutical Science, Hokuriku University, 3-Ho Kanagawa-machi, Kanazawa-city, Ishikawa, 920-1181, Japan

    Masayoshi Takeuchi, Yukiko Yanase & Yukihiko Kameda

  2. Biotechnology Research Center, Toyama Prefectural University, Toyama, Japan

    Nobuyasu Matsuura

  3. Division of Endocrinology and Metabolism, Department of Medicine, Kurume University School of Medicine, Kurume, Japan

    Sho-ichi Yamagishi & Zenji Makita

  4. The Picower Institute for Medical Research, Manhasset, New York, USA

    Richard Bucala

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  1. Masayoshi Takeuchi
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  2. Yukiko Yanase
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  3. Nobuyasu Matsuura
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  4. Sho-ichi Yamagishi
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  5. Yukihiko Kameda
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  6. Richard Bucala
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  7. Zenji Makita
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Correspondence to Masayoshi Takeuchi.

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Contributed by R. Bucala.

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Takeuchi, M., Yanase, Y., Matsuura, N. et al. Immunological Detection of a Novel Advanced Glycation End-Product. Mol Med 7, 783–791 (2001). https://doi.org/10.1007/BF03401969

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