Published Research - Department of Chemistry


Date of this Version



Clin Chim Acta.2011 August 17; 412(17-18): 1606–1615; doi:10.1016/j.cca.2011.05.012. This archive contains the PubMed Central pdf version


Copyright 2011 Elsevier B.V. All rights reserved.Used by permission.


Background—The glycation of human serum albumin (HSA) during diabetes can affect the ability of this protein to bind drugs and small solutes in blood. This study describes the use of 16O/18O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to compare the levels of modification that occur throughout HSA under various glycation conditions in vitro. These quantitative studies build on a recent report that has identified the early and advanced glycation products that are formed on such samples of HSA.

Methods—Glycated HSA samples were prepared by incubating 42 g/l HSA with 0 to 15 mmol/l glucose at pH 7.4 and 37°C for up to 5 weeks. A control HSA sample was digested in 16Oenriched water and glycated HSA samples were digested in the presence of 18O-enriched water. These 2 types of samples were then mixed and the amounts of 16O- vs. 18O-labeled peptides were measured to determine the levels of modification that were occurring throughout HSA.

Results—The largest levels of modification occurred in residues 101-119, 1-10 or 42-51, 87-100, 360-372, 521-531, and 275-286 of HSA after 2 weeks of glycation, and in residues 21-41, 1-10 or 42-51, 521-531, 82-93, and 146-160 after 5 weeks of glycation. Some of these regions contained the N-terminus, K199, K439, and K525, which have been previously identified as major glycation sites on HSA. The glycation pattern of HSA was dominated by early glycation products (e.g., fructosyl-lysine) after a reaction period of 2 weeks for mildly glycated HSA, while advanced glycation end products became more prominent at longer reaction times.

Conclusions—The time course of the observed modifications indicated that the pattern of glycation products changed as HSA was incubated over longer periods of time with glucose. Several regions found to have significant levels of modification were at or near the major drug binding regions on HSA. These results explain why the interaction of some drugs with HSA has been observed to vary with the level of glycation for this protein.