The use of quantitative cytochemical analyses in rapid prenatal detection and somatic cell genetic studies of metabolic diseases

Synopsis The biochemical analysis of enzyme activities in cultured human cells has become an increasingly important tool in the (prenatal) diagnosis of inborn errors of metabolism and in fundamental studies of somatic cell genetics and metabolic interaction of normal and mutant human cells. In all these fields the use of microchemical analyses offers new possibilities in the assay of enzymes in small numbers or even single cultured cells.When cell cultivation is carried out in dishes with a thin plastic foil at the bottom, groups of a few hundred cells and single cells can be sampled after quick freezing and freeze-drying of the culture. Incubation of this material in (sub)microlitre volumes ofp-nitrophenyl or methylumbelliferyl substrates, followed by extinction or fluorescence measurements in microdroplets using a microspectrofluorometer enables lysosomal enzyme activities in the range of 10^–11–10^–14 mol/hr/cell to be detected.Application of these techniques in assays of -1,4-glucosidase and -galactosidase on groups of 100–300 freeze-dried cultured amniotic fluid cells have enabled the prenatal diagnosis of glycogenosis-II and Fabry's disease respectively within 10–12 days after amniocentesis in the 14–15th week of pregnancy. This is a considerable reduction in the time interval hitherto required, 4–6 weeks, for the prenatal diagnosis of metabolic diseases.Examples of application of enzyme assays on single cultured cells are presented for studies on the interaction between enzyme-deficient and normal cultured cells and for genetic complementation studies after hybridization of two human cell strains. By using Lowry's principle of NADP(H) cycling, glucose-6-phosphate dehydrogenase (G-6-PD) activity could be reliably measured in single binuclear hybrid cells after fusion of normal and G-6-PD deficient human cell strains. With the same technique we were also able to detect two cell populations (normal and G-6-PD-deficient) within one culture of a heterozygous carrier for G-6-PD deficiency without the need of elaborate cell cloning techniques.Finally, a microfluorometric assay procedure was developed to enable certain lysosomal enzyme activities to be measured in single (hybrid) human cells. An example of this application is presented for -galactosidase activity measurements in single cells from a mixed culture of normal fibroblasts and cells from a patient with GM1-gangliosidosis (-galactosidase-deficient). In none of our experiments was any evidence obtained for the metabolic correction of enzyme-deficient cells by the uptake of enzyme from normal cells.