Leukocyte Sulfatidase for the Reliable Diagnosis of Metachromatic Leukodystrophy

Abstract

A simple assay technique for the determination of sulfatidase activity in leukocytes has been developed for the reliable diagnosis of metachromatic leukodystrophy (MLD). Sulfatide is tritiated in sphingosine and fatty acid by reduction with [3H]sodium borohydride in alkali in the presence of palladium chloride. This labeled natural substrate for aryl sulfatase A (AsA) is hydrolyzed by normal human leukocytes in 25 mM-acetate buffer, pH 5.0, in the presence of 0.3% sodium taurodeoxycholate. The enzyme activity is greatly improved after dialysis, exhibiting better linearity with protein concentration. It is stimulated maximally by 5 mM-MnCl2 with an apparent Km of 0.17 mM for the substrate. Patients with MLD exhibited virtually no detectable sulfatidase activity although they had residual AsA activity that was measured with the synthetic substrate, p-nitrocatechol sulfate (NCS). Potential heterozygotes could be identified by the sulfatidase assay in instances where the NCS assay for AsA was inconclusive. Several individuals with levels of AsA activity characteristic of MLD, including a few healthy carriers and certain patients with unknown neurological diseases, were shown not to have MLD by the presence of measurable levels of sulfatidase in their leukocytes.

Deficiency of lysosomal hydrolases in apparently healthy individuals

“…Eleven of these were found in a screening for MLD heterozygotes among healthy adults from a small Jewish community (the Habbanite community) in which a very high incidence of MLD had been found (1175 live births) [Zlotogora et al, 1980;Schaap et al, 19811. There are also reports of persons with very low ASA activity who are not affected with MLD but had various neurological manifestations and are sometimes referred to as atypical MLD variants (Table 11) [Dubois et al, 1980;Raghavan et al, 1981;Butterworth et al, 1978;Weiter et al, 19801. In some of these individuals low activity of arylsulfatase B was noted in addition to the low ASA activity [Raghavan, 19811.…”

Arylsulfatase A (ASA) defect and psychiatric illness

“…substrate, nitrocatechol sulfate (ASA-NCS). The assay method using the natural substrate, cerebroside sulfate (ASA-CS), has been shown to have a distinct advantage over the one that utilizes ASA-NCS in determining ASA activity in the presence of arylsulfatase B (ASB), and for the identification of heterozygotes (Raghavan et al 1981). A second study was therefore undertaken, in which a large number of psychiatric patients were screened using less stringent selection criteria.…”

“…This discrepancy necessitates more extensive screening studies with more rigorously standardized assay systems. Our study, for example, has clearly indicated a poor correlation between the two assay systems, using natural substrate (sulfatide) and synthetic substrate (nitrocatechol sulfate) (Shah et al, 1985); and it has also been indicated that, for determining heterozygosity, assay systems using natural substrate appears to provide more reliable results than assay using synthetic sulfate (Raghavan et al, 1981). Differences in populations may constitute yet another source of variation, since variations in gene frequency for the pseudodeficiency allele (ASAp) among different population groups have been noted (e.g., 13.7 and 12.1% for the Arab population, reported by Bach's group Schaap et al, 1981, Herz andBach, 1984, vs 7% for the German population, reported by Propping (1986).…”

Arylsulfatase A and β-galactosidase activities in leukocytes and lymphocytes from normal and psychiatric subjects

“…Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (Krabbe disease) are associated with a deficiency in the lysosomal hydrolases arylsulfatase A (ASA) and galactocerebroside 3-galactosidase (cerebrosidase), respectively. Assays of ASA and cerebrosidase activities in leukocytes have been routinely used as a diagnostic tool for determining the homozygous or heterozygous states for MLD (Percy and Brady, 1968;Pilz, 1972;Raghavan et al, 1981;Shah et at., 1985) or Krabbe disease (Suzuki et al, 1972;Poulos and Pollard, 1976). Leukocyte fractions contain mixed cell types that contain different hydrolase activity.…”