Biophysical Characterisation of Amadori Modified Human Serum Albumin: A Prognostic Biomarker for Diabetic Complications

Abstract

Introduction: Glycation of biological macromolecules particularly protein leads to the generation of early and Advanced Glycation End (AGE) products. The interest in early glycation of protein is driven due to the fact of Amadori modified proteins having role in diabetic complications. Aim: To analyse the biophysical characterisation of Amadori modified human serum albumin as a prognostic biomarker for diabetic complications. Materials and Methods: This in-vitro experimental study was conducted at Department of Biochemistry, J.N. Medical College, Aligarh Muslim University, Uttar Pradesh, India, from May 2010 to December 2012. The structural characterisation of EGPs was generated by incubating Human Serum Albumin (HSA) with glucose for about a week. The generation of Early Glycation Products (EGPs) of HSA was quantitated as Hydroxy Methyl Furfural (HMF) by ThioBarbituric Acid (TBA) assay and authenticated by boronate affinity chromatography. Moreover, High Performance Liquid Chromatography (HPLC) and Electro Spray Ionisation/Mass Spectrometry (ESI/MS) was carried out to validate the presence of Amadori product formed. Additionally, Circular Dichroism (CD) and thermal denaturation studies were used to investigate the structural changes in Amadori albumin. Results: Glycated HSA was obtained as detected by the presence of HMF and chromatography peaks. On stratification, the structural perturbation was observed in Amadori HSA. Furthermore, the generation of furosine was also confirmed by obtaining a new peak in the HPLC profile of glycated HSA. The ESI/MS result also substantiated the presence of Amadori products. Conclusion: The therapeutic strategies that negate the Amadori modification of albumin might be a logical approach in the prevention of diabetic complications.

Keywords

• advanced glycation end product
• amadori adduct
• diabetes
• glycation
• hydroxy methyl furfural