hymecromone and Mucopolysaccharidosis-III

A new fluorogenic substrate, 4-methylumbelliferyl beta-D-glucosaminide, was used for the assay of acetyl CoA:glucosaminide N-acetyltransferase in chorionic villi, cultured villus cells, and amniocytes. Optimal conditions for the assay and the ranges of enzyme activity were established for the various types of fetal cells. This simple fluorometric assay provides a reliable method for early prenatal diagnosis of Sanfilippo disease type C which is more convenient than current methods using radiolabelled substrates. The method was applied to amniotic fluid cells and fetal fibroblasts from an at-risk pregnancy in which an affected fetus was diagnosed by two-dimensional electrophoresis of glycosaminoglycans in the amniotic fluid.

Topics: Acetylglucosamine; Acetyltransferases; Amniotic Fluid; Cells, Cultured; Chorionic Villi; Female; Fibroblasts; Fluorescent Dyes; Humans; Hymecromone; Mucopolysaccharidosis III; Pregnancy; Prenatal Diagnosis

4-Methylumbelliferyl-alpha-N-acetylglucosamine 6-sulphate was synthesized and shown to be a substrate for the lysosomal N-acetylglucosamine-6-sulphate sulphatase (GlcNAc-6S sulphatase). Fibroblasts and leukocytes from 3 different Sanfilippo D patients showed < 1% of mean normal GlcNAc-6S sulphatase activity. The enzymatic liberation of the fluorochrome from 4-methyl-umbelliferyl-alpha-N-acetylglucosamine 6-sulphate requires the sequential action of the GlcNAc-6S sulphatase and alpha-N-acetylglucosaminidase. A normal level of alpha-N-acetylglucosaminidase activity was insufficient to complete the hydrolysis of the reaction intermediate 4-methylumbelliferyl-alpha-N-acetylglucosaminide formed by the GlcNAc-6S sulphatase. A second incubation in the presence of excess alpha-N-acetylglucosaminidase is needed to avoid underestimation of the GlcNAc-6S sulphatase activity.

Topics: Acetylglucosamine; Fibroblasts; Fluorometry; Humans; Hymecromone; Leukocytes; Lysosomes; Mucopolysaccharidosis III; Organometallic Compounds; Substrate Specificity; Sulfatases

Both the alpha- and beta-anomers of 4-methylumbelliferyl-D-glucosaminide were synthesized and shown to be substrates for the lysosomal acetyl-CoA:glucosaminide N-acetyltransferase. Using the beta-anomer, fibroblasts and leukocytes from 11 different Sanfilippo C patients showed < 1% of mean normal N-acetyltransferase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl-beta-D-glucosaminide requires the sequential action of the N-acetyltransferase and beta-hexosaminidase. Normal beta-hexosaminidase activity caused complete hydrolysis of the reaction intermediate 4-methylumbelliferyl-beta-D-N-acetylglucosaminide formed by the N-acetyltransferase. In cell extracts with a beta-hexosaminidase deficiency, however, a second incubation in the presence of excess beta-hexosaminidase is needed to avoid underestimation of the N-acetyltransferase activity.

Topics: Acetyltransferases; Cells, Cultured; Fibroblasts; Fluorometry; Heterozygote; Humans; Hymecromone; Leukocytes; Mucopolysaccharidosis III; Reference Values

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