Principle Of Method:
Hydrolysis of the synthetic substrate 4-methylumbelliferyl-a-D-glucopyranoside at acid pH is followed by measuring the fluorescence of the liberated 4-methylumbelliferone in alkaline solution.
Uses And Limitations Of The Method:
Deficiency of acid a-glucosidase (also called acid maltase or acid a1,4-glucosidase) is the primary defect in glycogen storage disease type II (GSD II). In all types there is a severe deficiency of a-glucosidase, as measured by this method using cultured fibroblasts, where residual activity correlates to some extent with severity of disease. Deficiency can also be demonstrated in muscle biopsy (or PM). White blood cells are not suitable for the diagnosis using the standard assay because of the presence of a neutral a-glucosidase in these cells which interferes with determination of the acid enzyme. A modified method in which an initial precipitation step is carried out to remove most of the neutral enzyme is under development but definitive diagnosis is only offered by use of the standard assay using fibroblasts.
The standard method is suitable for prenatal diagnosis of GSD II using chorionic villi directly or cultured amniotic cells, and this usually presents no problems. Heterozygote detection is not reliable, although we have found that some obligate heterozygotes have reduced a-glucosidase activity in fibroblasts when this is expressed relative to a-galactosidase.
Fibroblasts cultured from a skin biopsy are needed. Biopsy material should be collected aseptically into a sterile bottle containing tissue culture medium (available from the laboratory), and sent at room temperature to arrive at the laboratory within 24 hours. Biopsies for tissue culture should not be frozen. Fibroblast cultures established in other laboratories should be sent in plastic 25 cm2 flasks filled with medium.
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