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Immunological Evidence that Non-carboxymethyllysine Advanced Glycation End-products Are Produced from Short Chain Sugars and Dicarbonyl Compounds in vivo

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Abstract

Background

The Maillard reaction that leads to the formation of advanced glycation end-products (AGE) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it was proposed that AGE were not only created by glucose, but also by dicarbonyl compounds derived from the Maillard reaction, autoxidation of sugars and other metabolic pathways of glucose. In this study, we developed four types of non-carboxymethyllysine (CML) anti-AGE antibodies that recognized proteins modified by incubation with short chain sugars and dicarbonyl compounds.

Materials and Methods

AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with glyceraldehyde, glycolaldehyde, methylglyoxal or glyoxal. After immunization of rabbits, four types of AGE-specific antisera were obtained that were specific for the AGE modification. To separate non-CML AGE antibodies (Ab) (non-CML AGE-Ab-2, -3, -4, and -5), these anti-AGE antisera were subjected to affinity chromatography on a matrix coupled with four kinds of AGE bovine serum albumin (BSA) or CML-BSA. These non-CML AGE antibodies were used to investigate the AGE content of serum obtained from diabetic patients on hemodialysis.

Results

Characterization of the four types of non-CML AGE antibodies obtained by immunoaffinity chromatography was performed by competitive ELISA and immunoblot analysis. Non-CML AGE-Ab-2 cross-reacted with the protein modified by glyceraldehyde or glycolaldehyde. Non-CML AGE-Ab-3 and -Ab-4 specifically cross-reacted with protein modified by glycolaldehyde and methylglyoxal, respectively. Non-CML AGE-Ab-5 cross-reacted with protein modified with glyoxal as well as methylglyoxal and glycolaldehyde. Three kinds of non-CML AGE (AGE-2, -4, and -5) were detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD; whereas, AGE-3 was detected as two peaks with apparent molecular weights of 200 and 0.85 kD.

Conclusion

We propose that various types of non-CML AGE are formed by the Maillard reaction, sugar autoxidation and sugar metabolism. These antibodies enable us to identify such compounds created by the Maillard reaction in vivo.

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Acknowledgments

We are grateful to Dr. Satoshi Miyata (Kobe University School of Medicine) for providing pyrraline-BSA and to Dr. Toshio Miyata (Tokai University School of Medicine) for providing pentosidine-BSA. These studies were supported in part by a Grant-in-Aid for Scientific Research (#09470204) to Zenji Makita from the Japanese Ministry of Education, Science, Sports, and Culture, and by a Health Science Research Grant (#H10-Chozyu-033) to Zenji Makita from the Japanese Ministry of Health and Welfare.

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Pharmaceutical Science, Hokuriku University, Kanazawa, Japan

    Masayoshi Takeuchi, Takako Suzuki & Yukihiko Kameda

  2. Department of Internal Medicine II, Hokkaido University School of Medicine, Sapporo, Japan

    Zenji Makita & Takao Koike

  3. Laboratory of Medical Biochemistry, The Picower Institute for Medical Research, Manhasset, New York, USA

    Richard Bucala

  4. Division of Endocrinology & Metabolism, Kurume University School of Medicine, 67 Asahi-machi, Kurume-city, Fukuoka, 830-0011, Japan

    Zenji Makita

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  1. Masayoshi Takeuchi
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  2. Zenji Makita
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  3. Richard Bucala
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  4. Takako Suzuki
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  5. Takao Koike
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  6. Yukihiko Kameda
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Correspondence to Zenji Makita.

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Communicated by R. Bucala.

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Takeuchi, M., Makita, Z., Bucala, R. et al. Immunological Evidence that Non-carboxymethyllysine Advanced Glycation End-products Are Produced from Short Chain Sugars and Dicarbonyl Compounds in vivo. Mol Med 6, 114–125 (2000). https://doi.org/10.1007/BF03401779

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  • DOI: https://doi.org/10.1007/BF03401779

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