Spectrophotometric and fluorimetric assays of galactocerebrosidase activity, their use in the diagnosis of Krabbe's disease

Derivatives of galactocerebroside were prepared containing coloured (ω-2,4,6-trinitrophenylaminolauric acid) or fluorescent (11-(9-anthroyloxy) undecanoic acid) fatty acid moieties. These cerebrosides were used as substrates for galactocerebrosidase activity. By overcoming problems associated with the radioactively labelled substrates normally used, yet retaining good enzyme-substrate specificity, these derivatives provided useful and reliable alternative substrates for galactocerebrosidase activity. Enzyme activities in whole extracts of brain, liver, fibroblasts and cultured amniotic fluid cells were compared, using as substrates the novel cerebrosides as well as [³H] galactocerebroside. Good correlation of activities was obtained. In extracts derived from patients with Krabbe's disease marked deficiency of galactocerebrosidase activity was observed with each substrate, whereas extracts from heterozygous carriers exhibited a partial reduction in enzyme activity. The results show that these coloured and fluorescent galactocerebrosides may be used with confidence in the diagnosis and carrier detection of Krabbe's disease.