Diagnosis of Metachromatic Leukodystrophy, Krabbe Disease, and Farber Disease after Uptake of Fatty Acid-labeled Cerebroside Sulfate into Cultured Skin Fibroblasts

Abstract

A B S T R A C T ['4C]Stearic acid-labeled cerebroside sulfate (CS) was presented to cultured skin fibroblasts in the media. After endocytosis into control cells 86% was readily metabolized to galactosylceramide, ceramide, and stearic acid, which was reutilized in the synthesis of the major lipids found in cultured fibroblasts. Uptake and metabolism of the ['4C]CS into cells from typical and atypical patients and carriers of metachromatic leukodystrophy (MLD), Krabbe disease, and Farber disease were observed. Cells from patients with late infantile MLD could not metabolize the CS at all, while cells from an adult MLD patient and from a variant MLD patient could metabolize -40 and 15%, respectively, of the CS taken up. These results are in contrast to the in vitro results that demonstrated a severe deficiency of arylsulfatase A in the late infantile and adult patient and a partial deficiency (21-27% of controls) in the variant MLD patient. Patients with Krabbe disease could metabolize nearly 40% of the galactosylceramide produced in the lysosomes from the CS. This is in contrast to the near zero activity for galactosylceramidase measured in vitro. Carriers of Krabbe disease with galactosylceramidase activity near half normal in vitro and those with under 10% of normal activity were found to metabolize galactosylceramide in cells significantly slower than controls. This provides a method for differentiating affected patients from carriers with low enzyme activity in vitro. Cells from patients with Farber disease could catabolize only '-15% of the ceramide produced from

An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity

“…This fact suggested a possible sap-B deficiency. To investigate this hypothesis, a sulphatide loading test 11 was performed on fibroblasts from patient PV, from a late infantile metachromatic leukodystrophy control, and from five normal controls. A high level of cerebroside sulphate was found compared with normal controls and low levels of galactosyl ceramide and of phosphatidylserine, -inositol, -choline gave additional evidence of a sap-B deficiency (Figure 1).…”

A simple fluorogenic method for determination of acid ceramidase activity and diagnosis of Farber disease

“…Although aCDase is a very well known enzyme, existing methods for determining its activity and for FD diagnosis still exhibit many disadvantages. The methods that have been used for FD diagnostic purposes can be classifi ed into three groups: i ) aCDase enzymatic assays ( 4-16 ), classically performed with radiolabeled substrates ( 4-13 ), which are rather water-insoluble and require at least one detergent (see Table 1 ) ; ii ) loading tests, consisting of the addition of exogenous radiolabeled sphingolipids, e.g., ceramide ( 17, 18 ), sulfatide ( 19,20 ), or sphingomyelin ( 21 ), on cultured living cells and the study of their metabolism; and iii ) determination of accumulated ceramide, either by sophisticated chromatographic methods ( 22,23 ) or through the use of the diacylglycerol kinase assay in the presence of ␥ [ Farber disease (FD) is a rare inherited lipid storage disorder, also known as lipogranulomatosis, which is characterized by accumulation of ceramide in the cells and tissues of patients ( 1, 2 ). This accumulation is the consequence of a defi cient intracellular activity of aCDase, a lysosomal …”

Metabolism of sphingomyelin in cultured skin fibroblasts from patients with different types of Niemann-Pick disease.

“…The disease is classified into several clinical types (Brady 1983;Elleder and Jirasek 1983). In types A and B of this disease, the specific activity of sphingomyelinase is markedly reduced in cultured skin fibroblasts, whereas in types C. D and E it is characteristically near normal.The in vitro measurement of lysosomal enzyme activities is not a good predictor of the clinical course (Porter et al 1971;Kudoh and Wenger 1982). Thus, the study of the uptake and metabolism of radiolabeled substrates by cultured skin fibroblasts has been suggested to more closely reflect the in vivo metabolism of such substrates (Kudoh et al 1981;Kudoh and Wenger 1982).…”

“…The in vitro measurement of lysosomal enzyme activities is not a good predictor of the clinical course (Porter et al 1971;Kudoh and Wenger 1982). Thus, the study of the uptake and metabolism of radiolabeled substrates by cultured skin fibroblasts has been suggested to more closely reflect the in vivo metabolism of such substrates (Kudoh et al 1981;Kudoh and Wenger 1982 …”

“…Thus, the study of the uptake and metabolism of radiolabeled substrates by cultured skin fibroblasts has been suggested to more closely reflect the in vivo metabolism of such substrates (Kudoh et al 1981;Kudoh and Wenger 1982 …”