hymecromone and Mucopolysaccharidosis-VI

A new technique is introduced for the differential assay of arylsulphatases A and B in centrifuged homogenates of cultured human skin fibroblasts, using 4-methylumbelliferyl-sulphate as a substrate and AgNO3 as a selective inhibitor of arylsulphatase A. The method can be applied in the diagnosis of metachromatic leucodystrophy, mucopolysaccharidosis type VI and mucosulphatidosis. Normal arylsulphatase activities were found in fibroblasts derived from patients with mucopolysaccharidoses types II, III-A and IV, known to be caused by deficiencies of various other sulphatases.

Topics: Cerebroside-Sulfatase; Chondro-4-Sulfatase; Diagnosis, Differential; Diploidy; Fibroblasts; Humans; Hymecromone; Leukodystrophy, Metachromatic; Mucopolysaccharidoses; Mucopolysaccharidosis VI; Silver Nitrate; Skin; Sulfatases

Metachromatic leukodystrophy and Maroteaux-Lamy syndrome can be diagnosed by assay of leukocyte or fibroblast arylsulfatase A and B activity with the fluorogenic substrate 4-methylumbelliferyl sulfate. The arylsulfatases are extracted into a 27000 x g supernatant by sonication in 0.9% sodium chloride and then separated with CM-32 on columns or in test tubes. In 0.05 M sodium acetate pH 6.0, arylsulfatase A is not absorbed while arylsulfatase B is retained by the resin. The arylsulfatase B is then eluted from the resin with 0.3 M sodium chloride. The arylsulfatase A activity obtained from normal leukocytes and fibroblasts is linear for the initial 10 minutes of the reaction, is stimulated 3-fold by 6 mM lead acetate and inhibited 80% by 0.24 mM silver nitrate. After separation with CM-32, the arylsulfatase B activity is stimulated 3-fold by Triton X-100 (0.1%). Arylsulfatase A but not arylsulfatase B is destroyed by heat (60 degrees). Both leukocyte and fibroblast arylsulfatase A activity was reduced to 11% of control values in metachromatic leukodystrophy. Essentially no arylsulfatase B activity was detected in cells from patients with Maroteaux-Lamy syndrome. Metachromatic leukodystrophy heterozygotes but not Maroteaux-Lamy syndrome heterozygotes can also be distinguished by this method. A heat inactivation technique utilizing the differential thermal stabilities of the two enzymes for diagnosis of patients with Marotezux-Lamy syndrome is also described. The advantages of these 4-methylumbelliferyl sulfate assay procedures over the p-nitrocatechol sulfate method of assay are greater sensitivity, selectivity for the desired enzyme and potential for use in large scale testing.

Topics: Arylsulfatases; Cerebroside-Sulfatase; Chondro-4-Sulfatase; Drug Stability; Electrophoresis, Cellulose Acetate; Fibroblasts; Humans; Hydrogen-Ion Concentration; Hymecromone; Isoenzymes; Kinetics; Leukocytes; Leukodystrophy, Metachromatic; Methods; Mucopolysaccharidoses; Mucopolysaccharidosis VI; Skin; Sulfatases