Dimethylmethylene blue: a tool for MPS screening

Author(s): Sanjeev Kumar Pandey*, Sunil Kumar Polipalli, Seema Kapoor

Abstract

Mucopolysaccharidoses are a group of lysosomal diseases characterized by storage of glycosaminoglycans (GAGs) in tissue and as a consequence in urine. GAGs consist of N- and /or O-sulfate groups and are covalently linked to core proteins to form proteoglycans. During catabolism, GAGs are internalized in lysosome and degraded into their monomolecular constituents by lysosomal endoglycosidases, exoglycosidases, and exosulfatases. In Mucopolysaccharidoses (MPS) one of the hydrolytic lysosomal enzymes is deficient, which results in the accumulation of GAGs in the lysosomes and the resulting syndrome depends on which enzyme is deficient. The present spectrophotometric procedure based on the color reaction with Dimethyl Methylene Blue can be performed directly on untimed urine sample without prior precipitation. We have tested urine samples without preservative of 21 patients with various Mucopolysaccharidoses (eight hunters, four Hurler, two morquio, two scheie, three sanfilippo A and two sanfilippo C) through Dimethyl Methylene Blue assay and compared the result with Cetypyridinium Chloride (CPC) turbidity assay at pH: 4.8 (Citrate buffered) and at pH: 7.0 (Tris buffered). A total 72 normal urine samples were also tested with the CPC turbidity assay and the DMB assays to establish a reference range. GAG content is measured at absorbance 520 nm. Condroitin-4- sulfate standard were used to prepare calibration curves. In Normal subjects the GAG content in the younger age groups was more than the older one. Glycosaminoglycans level in the DMB assay were increased in 21 of 21 patients; whereas GAG’s level in the turbidity test at pH 7.0, value were increased in 19 of the 21 patients and at pH 4.0, GAG’s content was increased in only 15 of the 21 patients. The Dimethyl Methylene Blue assay procedure is a rapid, sensitive and reliable screening procedure to determine concentration of GAG’s for various MPS.

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