hymecromone and Mucopolysaccharidoses

Conditions for assay of alpha-N-acetylglucosaminidase activity in human cultured fibroblasts, cultured amniotic fluid cells, leucocytes, serum, plasma and chorionic villi were studied using the fluorogenic substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside. The substrate was found to have advantage both in terms of sensitivity and ease of use over previously-used colorimetric substrates for assay of the enzyme in these tissues, and for diagnosis of Sanfilippo B disease and identification of carriers. It should have particular application in first trimester prenatal diagnosis using chorionic villus biopsies.

Topics: Acetylglucosamine; Acetylglucosaminidase; Amniotic Fluid; Chorionic Villi; Clinical Enzyme Tests; Female; Fibroblasts; Glucosamine; Hexosaminidases; Humans; Hydrogen-Ion Concentration; Hymecromone; Mucopolysaccharidoses; Mucopolysaccharidosis III; Pregnancy; Prenatal Diagnosis; Skin; Spectrometry, Fluorescence; Umbelliferones

Using 4-methylumbelliferyl-alpha-L-iduronide as a substrate, alpha-L-iduronidase activity was measured in leukocytes and in lymphoblastoid cells obtained from patients with alpha-L-iduronidase deficiency and from obligate heterozygotes for this disease. There was complete discrimination between alpha-L-iduronide in leukocytes and in lymphoblastoid cells from the patients and controls. However, overlap was observed between values of the activity in the obligate heterozygotes and those in the controls. 4-Methylumbelliferyl-alpha-L-iduronidase activity because of greater sensitivity, easier assay procedure and shorter incubation period.

Topics: Female; Fluorometry; Glycoside Hydrolases; Humans; Hymecromone; Iduronic Acid; Iduronidase; Male; Mucopolysaccharidoses; Mucopolysaccharidosis I; Umbelliferones

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

A fluorogenic substrate for alpha-L-iduronidase, 4-methylumbelliferyl alpha-L-iduronide, has been newly synthesized and the enzyme activity has been measured in urine samples obtained from normal persons and patients suffering from mucopolysaccharidosis. Urine samples derived from a patient with Scheie syndrome showed greatly reduced activity compared with a normal adult at a similar age. This patient exhibited a high level of urinary excretion of dermatan sulfate and heparan sulfate, which could be interpreted in terms of her low alpha-L-iduronidase activity. The use of the fluorogenic substrate has some advantages over existing methods because of the high sensitivity and the relative ease of handling, and it should be useful not only for diagnosis but also for following the purification process of the enzyme.

Topics: Adult; Child, Preschool; Dermatan Sulfate; Female; Fluorometry; Glycoside Hydrolases; Heparitin Sulfate; Humans; Hymecromone; Iduronic Acid; Iduronidase; Male; Middle Aged; Mucopolysaccharidoses; Mucopolysaccharidosis I

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