hymecromone and Leukodystrophy--Globoid-Cell

Globoid cell leukodystrophy is an inherited metabolic disorder of the central nervous system caused by deficiency of the lysosomal enzyme galactocerebrosidase. Haematopoietic stem cell transplantation is the only available effective treatment. The engraftment from normal donors provides competent cells able to correct the metabolic defect. Umbilical cord blood cells have proved to significantly decrease complications and improve engraftment rate compared to adult marrow cells in haematopoietic stem cell transplantation. Umbilical cord blood cells must be of sufficient activity to provide central nervous system recovery after engraftment is obtained. Galactocerebrosidase activity is known to be affected by two polymorphic alleles found at nucleotides 502 and 1637 of the cDNA for this gene. This enzyme activity and the polymorphic alleles noted above were analysed in 83 random samples of umbilical cord blood. The activity, assayed with the fluorogenic substrate 6-hexadecanoylamino-4-methylumbelliferyl-beta-galactopyranoside, in those with neither polymorphic allele was 4.6 +/- 1.7 units (nmol/h per mg protein). This optimal choice of cord blood was found in only 24% of specimens. Homozygotes for 1637T > C with activity of only 1.5 +/- 0.4 units represented 16% of the samples. Those heterozygous for 1637T > C with slightly better activity (2.3 +/- 0.7 units) represented 52% of the samples. Choice of umbilical cord blood for haematopoietic stem cell transplantation, therefore, requires consideration not only of cell quantity and HLA compatibility but also selection for normal alleles to obtain maximal enzymatic activity for central nervous system correction.

Topics: Alleles; Central Nervous System; Cryopreservation; DNA, Complementary; Fetal Blood; Galactosides; Galactosylceramidase; Hematopoietic Stem Cell Transplantation; Heterozygote; HLA Antigens; Homozygote; Humans; Hymecromone; Leukodystrophy, Globoid Cell; Lysosomes; Mutation; Polymorphism, Genetic

6-Hexadecanoylamino-4-methylumbelliferyl-beta-D-galactopyranoside (HMGal) has been shown to be a specific fluorogenic substrate of galactocerebrosidase and to facilitate the simple enzymatic diagnosis of Krabbe disease in human patients and in twitcher mice. HMGal hydrolysis at pH 4.5 is optimally stimulated by sodium taurocholate (0.25%) and oleic acid (0.05%) with a Km of 0.150, 0.04 and 0.03 mM, respectively for control mouse kidney, human fibroblasts and leukocytes. In control samples, the specific activity (nmol/mg prot./h) for HMGal is higher than for the natural substrate, galactocerebroside, and is severely deficient in the twitcher mouse and in patients with Krabbe disease. Comparative investigation of galactocerebrosidase activity in fibroblasts, leukocytes and brain with radioactive and fluorogenic substrates reveals a good agreement between the results of the two methods. Galactocerebroside (Gal-Cer) is a competitive inhibitor of HMGal hydrolysis in mouse kidney homogenates while GM1-ganglioside has no inhibitory effect in the same assay system. The sensitivity and specificity of this fluorogenic substrate for galactocerebrosidase provides a simple and rapid method for the diagnosis of Krabbe disease, and for the purification of this enzyme from normal tissues.

Topics: Animals; Brain; Carbohydrate Sequence; Cells, Cultured; Clinical Enzyme Tests; Dose-Response Relationship, Drug; Female; Fibroblasts; Fluorescent Dyes; Galactosides; Galactosylceramidase; Galactosylceramides; Humans; Hydrolysis; Hymecromone; Leukocytes; Leukodystrophy, Globoid Cell; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Oleic Acid; Oleic Acids; Substrate Specificity; Taurocholic Acid

Topics: Cells, Cultured; Fibroblasts; Fluorescent Dyes; Galactosides; Galactosylceramidase; Humans; Hymecromone; Leukodystrophy, Globoid Cell

The properties of beta-galactocerebrosidase from human chorionic villi, cultured chorionic villi and cultured skin fibroblasts were compared, using 6-hexadecanoylamino-4-methylumbelliferyl-beta-D-galactopyranoside (HMGaL) as substrate. The effects of bile salt and Triton X-100 on beta-galactocerebrosidase were examined. It was shown that optimization of the HMGaL assay system requires the presence of pure sodium taurocholate and Triton X-100 at concentrations of 4.5 mM and 0.28 mM, respectively. The optimal pH value was found to be equal to 4.5-5.0; Km for the substrate was 0.03 mM. A comparison of beta-galactocerebrosidase from chorionic villi and cultured chorionic villi with the enzyme from skin fibroblasts revealed the similarity of some properties of these enzymes. The experimental results suggest that HMGaL can be used as a substrate for the identification of chorionic villi beta-galactocerebrosidase in an early prenatal diagnosis of Krabbe's disease.

Topics: Cells, Cultured; Chorion; Fibroblasts; Fluorescent Dyes; Galactosidases; Galactosides; Galactosylceramidase; Glycosides; Humans; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Leukodystrophy, Globoid Cell; Substrate Specificity; Umbelliferones