Formation and ways of detecting advanced glycation end-products in isolated human glomerular basement membrane and human serum albumin nonenzymatically glycated in vitro

• Published in: The Journal of diabetic complications Vol. 3; no. 2; pp. 88 - 91
• Main Authors: Sensi, Maurizio, Pricci, Flavia, De Rossi, Maria G., Bruno, Maria R., Morano, Susanna, Capuozzo, Elisabetta, Di Mario, Umberto
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.1989
• Subjects: Basement Membrane - analysis; Glucose - analysis; Glycoproteins - analysis; Glycosylation; Humans; Kidney Glomerulus - analysis; Protein Binding; Serum Albumin - analysis; Spectrometry, Fluorescence
• ISSN: 0891-6632
• DOI: 10.1016/0891-6632(89)90017-2

The formation of advanced glycation end-products (AGE) was investigated in samples of isolated human glomerular basement membrane (hGBM) and human serum albumin (hSA) which had been nonenzymatically glycated in vitro. In order to measure AGE, two methods which differ in principle—the standard spectrofluorescence technique and the spectrophotometric diazonium salt reaction—have been used and compared. Samples of finely homogenized hGBM and hSA were incubated for 10 days in buffer containing 500 mmol/L (9 × 10 3 mg%) and 100 mmol/L (1.8 × 10 3 mg%) D-glucose respectively. At the end of the incubation period, the ambient glucose was removed and the samples were incubated for a further 10 days in glucose-free buffer. During this time, loosely bound sugar was released into the buffer; at the end of the incubation, the emission fluorescence at 440 nm (following continuous excitation at 37- nm) and the absorbance at 492 nm of the glycated hGMB and hSA samples were measured and found to be significantly increased by comparison with native samples (1-way ANOVA: p < 0.05 with both techniques). Comparison of the two techniques used for AGE detection showed a positive linear correlation (Pearson's correlation coefficient r = 0.714; n = 8; p = 0.02). The released glucose probably originates from reversal of the Schiff base (the first and reversible step of the nonenzymatic glycation reaction), whereas fluorescence and photometric findings prove the presence of stable AGE on both hGBM and hSA. It is concluded that AGE can indeed be formed and detected by two different methods in hGBM (and hSA) subjected to nonenzymatic glycation in vitro. As the increased concentration of blood glucose in diabetes mellitus may lead to accelerated AGE formation, the in vitro model described may be of value in better understanding the pathophysiologic and biochemical mechanisms involved in the development of microangiopathy.