In diabetes, the non-enzymatic modification of human serum albumin (HSA) by glucose (a process known as glycation) is believed to effect the transport, displacement and non-bound protein concentrations of drugs. HSA is a blood protein known to interact with a wide range of drugs and other solutes. It has been shown in the past that high-performance affinity chromatography (HPAC) using immobilized HSA columns is a fast, precise tool for studying the thermodynamics and stoichiometries of these drug-protein interactions. However, no previous studies have used this approach with glycated HSA. One challenge in such work is the fact that glycated HSA is a heterogeneous protein population, in which the number and types of glycation sites vary with the levels of glucose in blood. In addition, little is known about how this glycation affects the binding of HSA at specific regions on this protein or how this modification compares with the other changes in blood during diabetes, such as increased levels of fatty acids, that can alter drug interactions with HSA. The overall goal of this project is to develop and test new approaches for the analysis of glycated HSA and other heterogeneous ligand systems by HPAC. This will be accomplished through three sets of studies. The first set will consider the use of entrapment or site-selective immobilization for HSA/glycated HSA to eliminate or minimize immobilization-induced heterogeneity when these proteins are used in HPAC columns for studying glycated HSA's inherent heterogeneity; this work will use both glycated HSA isolated from diabetic patient samples and in vitro glycated HSA. The second set of studies will use chromatographic theory to develop procedures by which HPAC can detect and characterize the binding heterogeneity of ligands. The third set of studies will use these tools to examine the changes in drug interactions that occur upon the glycation of HSA and to compare these effects with those produced by increased fatty acid levels in diabetes. The tools developed in this project should also be useful in studying the binding heterogeneity of other complex ligands, such as phosphorylated or glycosylated proteins. This research is relevant to public health since it will provide a better understanding of how diabetes affects the ability of HSA to bind drugs and other compounds. This, in turn, should allow an improved description of how the transport, metabolism, and effective dose of a drug will differ between a diabetic patient and an individual without diabetes. ? ? ?
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