Department of Chemistry

 

Development of Affinity Microcolumns for Drug–Protein Binding Studies in Personalized Medicine: Interactions of Sulfonylurea Drugs with in vivo Glycated Human Serum Albumin

Jeanethe Anguizola, University of Nebraska-Lincoln
K. S. Joseph, University of Nebraska–Lincoln
Omar S. Barnaby, University of Nebraska-Lincoln
Ryan Matsuda, University of Nebraska-Lincoln
Guadalupe Alvarado, University of Nebraska–Lincoln
William Clarke, Johns Hopkins University School of Medicine
Ronald Cerny, University of Nebraska-Lincoln
David S. Hage, University of Nebraska - Lincoln

Document Type Article

Copyright © 2013 American Chemical Society. Used by permission.

Abstract

This report used high-performance affinity microcolumns to examine the changes in binding by sulfonylurea drugs to in vivo glycated HSA that had been isolated from individual patients with diabetes. An immunoextraction approach was developed to isolate HSA and glycated HSA from clinical samples, using only 20 μL of plasma or serum and 6–12 nmol of protein to prepare each affinity microcolumn. It was found that the affinity microcolumns could be used in either frontal analysis or zonal elution studies, which typically required only 4–8 min per run. The microcolumns had good stability and allowed data to be obtained for multiple drugs and experimental conditions over hundreds of sample application cycles. Both the overall binding, as measured by frontal analysis, and site-specific interactions, as examined by zonal elution, showed good agreement with previous data that had been obtained for in vitro glycated HSA with similar levels of modification. It was also possible to directly compare the changes in site-specific binding that occurred between sulfonylurea drugs or as the level of HSA glycation was varied. This method is not limited to clinical samples of glycated HSA but could be adapted for work with other modified proteins of interest in personalized medicine.