Urinary Glycosaminoglycan Electrophoresis With Optimized Keratan Sulfate Separation Using Peltier System for the Screening of Mucopolysaccharidoses

Mihriban Tijen Tanyalcin

doi:10.1177/2326409815613805

Abstract

The purpose of this communication is to indicate a simple and rapid method with a small volume of urine sample to detect urine glycosaminoglycan (GAG) and serve as a screening procedure for mucopolysaccharidoses (MPSs). Total GAG measurement for patients with MPS disorders is considered to be the first step in diagnosis of those heterogeneous group of lysosomal storage disorders presenting clinical phenotype. In this study, modified 9-dimethylmethylene blue method is used for total GAG measurement. Following GAG quantitation, the procedure described here allows GAG isolation from a very a small volume of urine sample and subjected to high-resolution GAG electrophoresis, which can be easily performed in routine clinical diagnostic laboratories. Glycosaminoglycan precipitation is a modified method based on total GAG concentration in the urine. For optimized isolation of total GAG for electrophoresis, instead of considering the urine creatinine concentration, 300 μg/mL GAG containing urine is considered to be the target concentration for the best precipitation with 1000 μL cetylpyridinium chloride (CPC)/citrate buffer. Glycosaminoglycan concentration-based precipitation of urine with CP Ca llows the laboratory to be able to work with as mall volume of urine sample by keeping the precipitating ratio with CPC constant for samples that contain GAG less than 300 μg/mL. Based on the effect of cold buffer using low voltage, GAGs high-resolution electrophoresis banding patterns described here enable a clear separation of keratan sulfate from chondroitin sulfate as well as dermatan sulfate (DS1 and DS2) and heparan sulfate. By this procedure, GAG patterns are more clear, easily identified, and provide a guide for the enzyme analysis deficient in the MPS disorders.

Highlights

Cellulose acetate (CA) membrane has been used for many years as the supporting medium for glycosaminoglycans (GAGs)
The value observed for total GAG as mg/L is corrected with regard to the amount of urine creatinine value obtained by the automated dry chemistry analyzer Vitros 350 (OrthoClinical Diagnostic NY, USA)
There is a clear separation of keratan sulfate in Morquio case (MPS IV A) which is from ERNDIM MPS QA Scheme sample (No:[15]; Figure 1)

Summary

Introduction

Cellulose acetate (CA) membrane has been used for many years as the supporting medium for glycosaminoglycans (GAGs). Almost all the experimental techniques in urine GAG electrophoresis (GAGE) involve the use of buffers with different pH and voltage to optimize GAG separation and to make an accurate interpretation in order to facilitate further enzymatic or molecular analysis.[1,2,3] The major GAG excreted by the healthy individuals is chondroitin-6sulfate, whereas heparan sulfate (HS), dermatan sulfate (DS), and keratan sulfate (KS) are excreted in excess in patients with mucopolysaccharidosis (MPS). In well-optimized electrophoretic assay condition, chondroitin-4-sulfate separation appears to be justified. The GAGs can migrate as compact, clear bands without tailing. The method outlined here is simple, requires less volume of urine sample, less time consumption, and can be performed in any hospital or clinic diagnostic laboratory

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Conclusion