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

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 CPC allows the laboratory to be able to work with a small 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.

Clinical Presentation of Mucopolysaccharidosis Type II (Hunter’s Syndrome)

“…The presences of excess heparin and dermatan sulphates in the urine is evidence of MPS type I, MPS type II or MPS type VII [9,10]. In our case urinary electrophoresis reveals excess of glycosaminoglycans.…”

Mucopolysaccharidoses Differential Diagnosis by Mass Spectrometry-Based Analysis of Urine Free Glycosaminoglycans—A Diagnostic Prediction Model

“…Quantitative assays are usually colorimetric using 1,9-dimethylene blue (DMB); however, this method, although rapid and cheap, may give false positive or false negative results due to the presence of interfering substances in the samples [ 7 , 8 , 9 ]. Next, qualitative electrophoretic analysis can help with differentiating MPS subtypes based on the pattern of bands of the four main GAGs (CS, DS, HS and KS) [ 10 ], except for MPS I and MPS II, which remain undistinguishable, as well as the different forms of MPS III. However, in the last decade, an increasing number of laboratories have switched to assays based on mass spectrometry (MS) for differential diagnosis, given its superior accuracy, speed, specificity and sensitivity [ 11 ].…”

Comparative Study of Urinary N-Acetyl-Beta-D-Glucosaminidase Levels, Urinary Glycosaminoglycans and Plasma Glycosaminoglycans Levels in Cats With Feline Idiopathic Cystitis

“…In human, the damage of urothelial GAGs barrier layers were postulated to be the underlie of pathogenesis in chronic bladder pathologies (Bassi et al, 2011). The changes in urinary GAGs excretion might be a tool for detecting and monitoring the pathogenesis of bladder cancer (Hennessey et al, 1981) and Mucopolysaccharidoses (Tanyalcin, 2015a). As a result of defective GAGs layers, many researchers try to investigate the intravesical exogenous GAGs into the damaged bladder of mouse (Kyker et al, 2005) and also in human IC (Davis et al, 2008).…”

“…In human, urinary GAGs excretion was relatively high in childhood, decreased in adults and relatively increased again in old age (Manley et al, 1968). Several study suggested that urinary GAGs excretion can be a biomarker for screening many diseases such as Mucopolysaccharidoses (Tanyalcin, 2015b) and bladder carcinoma (Hennessey et al, 1981). As previously stated, The decreased urinary GAGs excretion has been demonstrated in patients with lower urinary tract diseases such as PBS/IC (Lucon et al, 2014), idiopathic detrusor overactivity (Siracusano et al, 2009) and also in cats with FIC (Buffington et al, 1996) but the mechanism was not precisely described (Lucon et al, 2014).…”