Determination of membrane protein glycation in diabetic tissue
Journal Title: The AAPS Journal - Year 1999, Vol 1, Issue 4
Abstract
Diabetes-associated hyperglycemia causes glycation of proteins at reactive amino groups, which can adversely affect protein function Although the effects of glycation on soluble proteins are well characterized, there is no information regarding membrane-associated proteins, mainly because of the lack of reproducible methods to determine protein glycation in vivo. The current study was conducted to establish such a method and to compare the glycation levels of membrane-associated proteins derived from normal and diabetic tissue. We present a detailed sample preparation protocol based on the borohydride-periodate assay, modified to allow manipulation of animal tissue. Assay noise associated with extraction protocols and nonproteinaceous buffer components was eliminated by the using 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) as a membrane detergent, applying desalting columns, and including a protein precipitation step. The glycation level of membrane proteins from diabetic rats is elevated to 4.89 nmol/mg protein (standard deviation [SD] 0.48) compared with normoglycemic control tissue (2.23 nmol/mg protein, SD 0.64). This result is consistent with and correlated to the total glycated hemoglobin levels in diabetic and normoglycemic rats. Using<100 μg protein, the described methods allow further study of protein glycation effects on the function of individual transporter proteins and the role of these modifications in diabetes.
Authors and Affiliations
Eric Y. Zhang, Peter W. Swaan
Keywords
Related Articles
- EP ID EP682126
- DOI 10.1208/ps010420
- Views 76
- Downloads 0
Bioequivalence studies for levothyroxine
The Food and Drug Administration (FDA) Guidance for Bioavailability and Bioequivalence Studies for Levothyroxine has been challenged by companies that manufacture brand-name products. Their contention is that the current...
Effect of Traumatic Brain Injury, Erythropoietin, and Anakinra on Hepatic Metabolizing Enzymes and Transporters in an Experimental Rat Model
The online version of this article (doi:10.1208/s12248-015-9792-y) contains supplementary material, which is available to authorized users.
Comparison of Methods for Handling Missing Covariate Data
Missing covariate data is a common problem in nonlinear mixed effects modelling of clinical data. The aim of this study was to implement and compare methods for handling missing covariate data in nonlinear mixed effects...
In Vitro Evaluation of Reversible and Irreversible Cytochrome P450 Inhibition: Current Status on Methodologies and their Utility for Predicting Drug–Drug Interactions
It is widely accepted that today’s practice of polypharmacy inevitably increases the incidence of drug–drug interactions (DDIs). Serious DDI is a major liability for any new chemical entity (NCE) entering...
Alteration in P-glycoprotein Functionality Affects Intrabrain Distribution of Quinidine More Than Brain Entry—A Study in Rats Subjected to Status Epilepticus by Kainate
This study aimed to investigate the use of quinidine microdialysis to study potential changes in brain P-glycoprotein functionality after induction of status epilepticus (SE) by kainate. Rats were infused with 10 or 20&#...