This article describes a simple and selective procedure used for direct measurement of sulfated glycosaminoglycans (GAGs) in biological samples and its application to the determination of GAGs during tissue regeneration and myogenic differentiation. We describe a modified procedure of previous GAG assays that has improved specificity, reproducibility, and sensitivity. The assay is based on the ability of sulfated GAGs to bind the cationic dye 1,9-dimethylmethylene blue. We describe conditions that allow isolation of the GAG-dye complex. This complex was dissociated; the optical density measurement of the dissociated dye permitted quantification of GAGs in biological samples. Applied to the study of myogenic cell differentiation in vitro, muscle repair, and skin ulceration, this method revealed significant modifications in the patterns of expression of different sulfated GAGs in these tissues. In particular, application of the method after nitrous acid treatment revealed that heparan sulfate and chondroitin sulfate ratio changed during muscle regeneration process.

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Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies.

Glycosaminoglycans (GAGs) are measured in urine to screen for mucopolysaccharidoses. Other assay procedures are only qualitative (spot tests), can give false-negative results (spot tests, turbidity tests), or are relatively laborious (uronic acid-carbazole test). The present spectrophotometric procedure, based on the color reaction with dimethylmethylene blue (DMB), can be performed directly on untimed urine samples without prior precipitation. Reference values were age dependent. We tested urines of 27 patients with various mucopolysaccharidoses and compared results by three other procedures (cetylpyridinium chloride turbidity tests at pH 4.8 and at pH 7.0, and the uronic acid-carbazole test). In the DMB assay, GAGs were increased in 26 of the 27 patients. The exception was a Morquio A patient, whose activity of the defective enzyme was higher than in classical Morquio patients. Uronic acid, measured in precipitated GAG by the carbazole test, was increased in 23 of the 25 patients so tested. In the turbidity test at pH 7.0, values were increased in 24 of the 27 patients. In contrast, with the citrate-buffered (pH 4.8) turbidity measurement, GAG content was increased in only 19 of the 27 patients. This rapid and easy DMB method is a reliable screening procedure for mucopolysaccharidoses and compares well with procedures used hitherto.

Dimethylmethylene blue-based spectrophotometry of glycosaminoglycans in untreated urine: a rapid screening procedure for mucopolysaccharidoses.

Glycosaminoglycans detection methods: Applications of mass spectrometry.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) was first described by Dr. Charles Hunter in 1917. Since then, about one hundred years have passed and Hunter syndrome, although at first neglected for a few decades and afterwards mistaken for a long time for the similar disorder Hurler syndrome, has been clearly distinguished as a specific disease since 1978, when the distinct genetic causes of the two disorders were finally identified. MPS II is a rare genetic disorder, recently described as presenting an incidence rate ranging from 0.38 to 1.09 per 100,000 live male births, and it is the only X-linked-inherited mucopolysaccharidosis. The complex disease is due to a deficit of the lysosomal hydrolase iduronate 2-sulphatase, which is a crucial enzyme in the stepwise degradation of heparan and dermatan sulphate. This contributes to a heavy clinical phenotype involving most organ-systems, including the brain, in at least two-thirds of cases. In this review, we will summarize the history of the disease during this century through clinical and laboratory evaluations that allowed its definition, its correct diagnosis, a partial comprehension of its pathogenesis, and the proposition of therapeutic protocols. We will also highlight the main open issues related to the possible inclusion of MPS II in newborn screenings, the comprehension of brain pathogenesis, and treatment of the neurological compartment.

Mucopolysaccharidosis Type II: One Hundred Years of Research, Diagnosis, and Treatment.

Decreased urine glycosaminoglycan excretion in cats with interstitial cystitis

We describe a simple, rapid, precise, and sensitive spectrophotometric method for measuring urinary glycosaminoglycan (GAG) sulfate excretion. The GAG sulfates are precipitated with cetylpyridinium chloride, resuspended in water, and mixed with the basic dye 1,9-dimethylmethylene blue to produce a complex with the polyanionic molecule of sulfated GAGs. Absorbance is read at 535 nm. The standard curve for reaction was linear up to 12 micrograms of the different GAGs: dermatan sulfate, heparan sulfate, keratan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate. Within- and between-run precision (CV), measured at three different GAG concentrations (normal and pathological), varied from 1.6% to 2.5% and from 1.8% to 4.5%, respectively. Analytical recovery ranged from 71% to 107%. Urinary GAG excretion, measured by this procedure, correlates (r = 0.837; p less than 0.001) with the values obtained with the borate-carbazole reaction (Anal Biochem 1962;4:330-4).

Simple spectrophotometric quantification of urinary excretion of glycosaminoglycan sulfates.

The clinical application of a new method for the quantitative determination of urinary glycosaminoglycans (GAGs) is presented. 115 normal subjects and 44 patients affected by different types of mucopolysaccharidoses (MPSo-ses) have been studied by this method in comparison with the conventional borate-carbazole method. All patients showed an urinary GAG excretion well above the normal subjects. In particular, patients affected by Morquio syndrome showed values well beyond the normal range; on the contrary, with the borate-carbazole method, 6 out of 7 patients had borderline values of urinary GAGs. The new method is also more rapid and easier to perform than conventional methods.

Clinical application of a new simple method for the identification of mucopolysaccharidoses.

Metachromatic leukodystrophy (MLD) is an heterogeneous, neurodegenerative disorder, in which a deficient activity of the lysosomal hydrolase, arylsulfatase A, causes demyelination and accumulation of sulfatide in central and peripheral nervous tissues, as well as in other tissues and organs (Kolodny and Moser, 1983).

Biochemical and Ultrastructural Studies of a Fetus with Arylsulfatase A Deficiency

The basic dye: 1,9-dimethylmethylene blue (DMB) is one of the most sensitive thiazine compounds, giving a more marked metachromatic shift than other thiazine dyes when mixed with glycosaminoglycans (GAGs).

G. Panin

Papers

Simple spectrophotometric quantification of urinary excretion of glycosaminoglycan sulfates.

Clinical application of a new simple method for the identification of mucopolysaccharidoses.

Biochemical and Ultrastructural Studies of a Fetus with Arylsulfatase A Deficiency

A New Spot Test for the Screening of Pathological Urinary Sulfated Glycosaminoglycan Excretion