Neurodegenerative course in ceramidase deficiency (Farber disease) correlates with the residual lysosomal ceramide turnover in cultured living patient cells

JO”RN*L

OF

THE

NEUROLOGICAL SCIENCES ELSEVIER

Journal of the Neurological Sciences 134 (1995) 108-114

Neurodegenerative course in ceramidase deficiency ( Farber disease) correlates with the residual lysosomal ceramide turnover in cultured living patient cells Thierry Levade a** , Hugo W. Moser b, Anthony H. Fensom ‘, Klaus Harzer d, Ann B. Moser b, Robert Salvayre a a Laboratoire

de Biochimie, “Maladies M&aboliques”, CJF INSERM 9206, Institut Louis Bugnard, C.H.U. Rangueil, F-31054 Toulouse, France b Departments ofNeurology and Pediatrics, Kennedy Krieger Institute, Johns Hopkins University, Baltimore, MD, USA ’ Division of Medical and Molecular Genetics, Supraregional Laboratory for Genetic Enzyme Defects, Guy’s Ho.gpital, London, UK ’ Neurochemical Laboratory, Institut fir Hirnforschung, Uniuersiry of Tiibingen, Tiibingen, Germany

Received 18 April 1995; revised 16 June 1995; accepted 25 June 1995

Abstract Farber’s lipogranulomatosis is an inborn lipid storage disease characterized by tissue accumulation of ceramide due to deficient activity of lysosomal ceramidase. Symptoms include painful swelling of joints, subcutaneous nodules, a hoarse cry, hepatosplenomegaly and nervous system dysfunction of markedly variable degree. In most cases the neural dysfunction rather than the general dystrophy, seems to limit the duration of Farber disease. We examined whether the severity can be shown as a function of ceramide turnover by lysosomal ceramidase. The lysosomal degradation of sphingomyelin-derived ceramide was studied in situ in patient skin fibroblasts and lymphoid cells loaded with LDL-associated radioactive sphingomyelin. We could show for the first time a significant correlation between the ceramide accumulated in situ and the severity of Farber disease. Our method provides an alternative means for determining ceramide degradation by lysosomal ceramidase, but in intact c-ells. The relatively simple method is at least of the same diagnostic use for Farber disease as the in vitro assay of acid ceramidase using cell homogenates and may also have some prognostic use. Keywords:

Farber disease; Ceramide; Ceramidase; Sphingomyelin; Lysosomes; Lysosomal storage disease

1. Introduction

Farber disease(lipogranulomatosis) is a rare, inherited lipid storage disorder characterized by accumulation of ceramide (IV-acylsphingosine) in the tissues of patients (Moser et al., 1989). Clinical features include painful swelling of joints, subcutaneous nodules, a hoarse cry, hepatosplenomegalyand nervous system dysfunction. Clinical subtypes have been distinguishedbased on the age of onset, mean age of death and the degree of neurologic involvement (psychomotor deterioration). Storage material has been reported in the nervous system of nearly all

Abbreviations: SPM, sphingomyelin; LDL, low density lipoprotein; TLC, thin-layer chromatography * Corresponding author. Tel.: (+33-61) 32 29 84; Fax: (+33-61) 32 29 53.

0022-510X/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved 0022-510X(95)00231-6

SSDI

autopsiedcases(Moser et al., 1989). The pathophysiological basis for the clinical heterogeneity of Farber disease remains unknown. Diagnosis of Farber diseaseis confirmed by demonstration of a deficient

activity

of lysosomal

ceramidase

(IV-

acylsphingosine deacylase, EC 3.5.1.23) (Sugita et al., 1972; Dulaney et al., 1976; Fensomet al., 1979). Although this enzyme has been known for 30 years (Gatt, 1963), its in vitro assay remains extremely complex and tedious. As the hydrolysis of ceramide can be catalyzed by three ceramidaseswith different pH optima, tissue distributions and subcellular localizations (Gatt, 1963, 1966; Yavin and Gatt, 1969; Nilsson, 1969; Sugita et al., 1975; Stoffel and Melzner, 1980; Momoi et al., 1982; Spenceet al., 1986; Al et al., 19891, attempts to specifically assay the lysosomal enzyme in vitro for the enzymatic diagnosis of Farber diseasewere successfulin a very limited number of specialized laboratories (Sugita et al., 1975; Momoi et al.,

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1982). An alternative approach to the diagnosis of Farber disease used radiolabelled ceramide (Chen et al., 1981; Sutrina and Chen, 1982) or cerebroside sulfate (sulfatide) (Kudoh and Wenger, 1982; Inui et al., 1987) loading tests on intact, cultured skin fibroblasts. However, the sulfatide loading test cannot be employed on lymphoid cells due to the existence in these cells of a non-lysosomal sulfatidedegrading pathway (Tempesta et al., 1994; Levade et al., 1995a). Moreover, none of the previously reported assays of lysosomal ceramidase has provided a differentiation between the various clinical Farber phenotypes, the resid-

Table 1 Clinical features, in vitro acid ceramidase activity disease after 24 hours loading with LDL-associated

and in situ accumulation Iceramide-3HlSPM.

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ual activities in vitro being similar in the mild and severe cases (Moser et al., 1989). In the present study, the usefulness of a loading test with sphingomyelin @PM), which is a direct precursor of ceramide, for determining lysosomal ceramide turnover has been investigated in normal and Farber fibroblasts and Epstein-Barr virus-transformed lymphoid cells. Our work on a dozen of Farber patients reports for the first time a coherent metabolic study of this rare disease, allowing to differentiate between cases with different residual ceramide turnover rates.

of ceramide

by intact cells from

patients

affected

with Farber

(or Farber-like)

Patient Moh

Ste

Sch

GM 5748 GM 5752

Moz

Cal

Sex Age at onset Age at death Skin nodules Joint contractures Hoarseness Organomegaly Neurological features

F in utero 3 days

F 6mo 3 years +

M birth 4mo

F 3 mo 6 mo +

F 4mo 10.5 mo + + +

M 6mo 3 years + + +

Psychomotor retardation Areflexia Amyotrophy Increased CSF protein

Ataxia Nystagmus Myoclonus Tremor

Other

Hydrops

0.5 71.3

0.5 67.2

signs

In vitro acid ceramidase Radiolabelled ceramide

Sex Age at onset Age at death Skin nodules Joint contractures Hoarseness Organomegaly Neurological features

Other signs In vitro acid ceramidase Radiolabelled ceramide

+ + Seizures

9.0 79.9

15.0 78.8

Lung infiltrates Lymphadenopathy Hypercalcemia 0 7;.8,76.0

GM 2314

Alb

Ben

Dou

Fra

GM 2315

F 6mo 5.9 years + + +

F 3 mo 9.5 mo + + +

F 18 mo 3.5 years + + +

F 11 mo 4 years + + + -

F 10 mo 11 years + + + -

F 20 mo 30 years + + + -

Psychomotor retardation Hypotonia Decreased NCV Increased CSF protein

Psychomotor retardation Dementia Myoclonus Mild myopathy

Psychomotor retardation Hypotonia Myoclonus

Psychomotor retardation Amyotrophy

Mental retardation Muscle weakness

n.d. 63.0

4.0 56.2

8.0 54.7

a n.d. b 98.0

a 8.0 b 64.5

fetalis

Psychomotor retardation Ataxia Tremor Loss of motor function Decreased NCV Cherry-red spot

+ + Poor head control

+ Hypotonia Seizures

Prosaposin

deficiency

Cachexia 6.0 50.0

10.0 48.9

L In vitro ceramidase activity was assayed as described by Dulaney and Moser (19771 and is expressed as percentage of controls (nd., not determined). Cells were incubated for 24 h in a medium supplemented with 2% Ultroser HY and LDL-associated [ceramide-3H]SPM (about 50 pg apolipoprotein B/ml; 12500 dpm/pg). Then, the cell-associated lipids were extracted, separated on TLC and their distribution analyzed by radiochromatoscanning as described in Methods. The amount of undegraded ceramide is expressed as the percentage of the total radiolabelled metabolic products of SPM. For each Farber cell line, up to 4 separate determinations were performed. In control fibroblasts (n = 12) and lymphoid cells (n = 51, the ceramide averaged 5.9 f 2.6 and 6.2 f l.S%, respectively.

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2. Materials

of the Neurological

and methods

2.1. Chemicals

and radiolabelled

lipids

SPM was prepared from bovine brain or purchased from Sigma. Silica gel 60 TLC plates were from Merck (Darmstadt, Germany). All solvents obtained from Merck or SDS (Peypin, France) were of analytical grade. [ceramide- 3H]Sphingomyelin (400 Ci/mol) was obtained from C.E.A. (Gif-stir-Yvette, France) by catalytic tritiation of bovine brain SPM, and repurified by TLC, using chloroform/methanol/water (100:42:6, by vol.) as developing solvent. Acidic hydrolysis (Gaver and Sweeley, 1965) of [ ceramide-3H]SPM showed that the radiolabel was on both the sphingoid base and fatty acid moieties. Standard radiolabelled ceramide was synthesized by condensing sphingosine and [9,10-3H]oleic acid (10 Ci/mmol; Du Pont N.E.N., Les Ulis, France) according to previously described procedures (Dulaney and Moser, 1977; Hammarstrom, 1971). Alternatively, radioactive ceramide was obtained by hydrolysis of [ceramide-3H]SPM with Bacillus cereus sphingomyelinase(Sigma). RPM1 1640 medium, penicillin, streptomycin, L-glutamine, trypsin-EDTA and fetal calf serum were from Gibco BRL (Cergy-Pontoise, France). Ultroser HY, a serumsubstitute devoid of lipoproteins, was from IBF (Villeneuve-la-Garenne, France). 2.2. Cell cultures

Human skin fibroblasts were derived from 12 normal individuals (children and adults, malesand females), from affected patients (see Table 1; lines Alb, Ben, Cal, Dou, Fra, Moh, Ste, GM 2314, GM 2315 and GM 5752) and a heterozygous relative (line Iko) with Farber diseaseand from a patient with prosaposin deficiency (line Sch). Fibroblasts were studied between passages4 and 15. Longterm human lymphoid cell lines were establishedby Epstein-Barr virus-transformation of peripheral blood B lymphocytes (Neitzel, 1986; Levade et al., 1991) from normal individuals and from patients affected with Farber disease (lines Moz and GM 5748). The fibroblast cell line GM 5752 and the lymphoid cell line GM 5748 were derived from the same patient with typical Farber disease (Antonarakis et al., 1984). The Farber cell lines Fra and GM 2315 were kindly provided by Drs. R. Gatti and P. Durand (Istituto G. Gaslini, Genova, Italy) and Dr. A. Fiumara (Catania, Italy), and derive from membersof the same family (Pavone et al., 1980; Fiumara et al., 1993). Reports concerning casesCal, Dou and Sch have also been published (Colamaria et al., 1992; Palcoux et al., 1985; Harzer et al., 1989). The GM cell lines were from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ), the other cell lines originate from the authors’ laboratories. Materials from patients Dou, Moz and Sch were provided by Dr. J.B. Palcoux (Hotel-Dieu, Clermont-Ferrand, France), Dr.

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J.M. Richardet (Hopita Trousseau,Paris, France) and Drs. E. Kattner, H. Lietz and P. Meinecke (Altonaer Kinderkrankenhaus, Hamburg, Germany), respectively. The cells were routinely grown in a humidified 5% CO, atmosphere at 37°C in RPM1 1640 medium containing L-glutamine (2 mmol/l), penicillin (100 U/ml), streptomycin (100 pg/ml) and heat-inactivated fetal calf serum (10%) as previously reported (Levade et al., 1991). 2.3. Preparation

of LDL-associated

[ceramide-3H]SPM

LDL-associated [ ceramide-3H]SPM was prepared as previously described (Graber et al., 1994). Briefly, an ethanolic solution of [ ceramide-3H]SPM (500-700 X 10” dpm) was mixed with 10 ml of fresh human sera and incubated overnight at 37°C. The subsequently labelled LDL was isolated by discontinuous density gradient ultracentrifugation, dialyzed and filtered through a 0.2-pm pore diameter membranefilter. The incorporation of SPM into LDL ranged from 8500 to 25000 dpm/pg apolipoprotein B. More than 95% of the radioactive SPM was associated with LDL as measuredafter agarosegel (Hydragel, Sebia) electrophoresis. 2.4. Incubation

of intact cells with [ceramide-3H]SPM

Before the experiments were initiated, the cells (for fibroblasts, when confluent) were grown for 2-3 days in RPM1 1640 medium containing L-glutamine, antibiotics and 2% Ultroser HY, a serum substitute. Cells were then incubated with medium containing 2% Ultroser HY and LDL-associated [ ceramide- 3H]SPM (the final concentration of LDL in the incubation medium approximated 50-70 pg apolipoprotein B/ml). In pulse-chaseexperiments, after either 3 or 24 h pulse, the medium was removed and the cells were washed twice (lymphoid cells were sedimented by slow-speed centrifugation) in RPM1 medium containing 10% fetal calf serum. Fresh medium supplemented with 10% fetal calf serum was added to the cells and incubation continued for the indicated times. At the end of incubation, cells were washed 3 times with PBS containing bovine serum albumin (2 mg/ml) and then twice with PBS alone (Levade et al., 1991). The cell pellets were stored at - 20°C until use. 2.5. Lipid

extraction

and analyses

Cell pellets were suspendedin 0.5 ml distilled water and sonicated for 3 X 15 s (Soniprep MSE sonicator). An aliquot was taken for protein determination (Smith et al., 1985) another for estimating the total cell-associatedradioactivity by liquid scintillation counting (Packard Tricarb 4530 spectrometer), and the remainder was extracted with 2.5 ml of chloroform/methanol (2:1, by vol.), vortex mixed and centrifuged at 1000 X g for 15 min (Folch et al., 1957). The lower phasewas evaporated under nitrogen

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and the lipids were resolved by analytical TLC developed in chloroform/methanol/water (100:42:6, by vol.) up to the 2/3 of the plate and then in chloroform/methanol/acetic acid (94:5:1, by vol.)(Levade et al., 1993). The distribution of the radioactivity on the plate was analyzed using a Berthold LB 2832 radiochromatoscan. Unlabelled and radioactive lipid standards were used to identify the various [ceramide-3H]SPM metabolic products.

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The approach we used for evaluating the ceramide turnover by lysosomal ceramidase was to administer to the cultured cells a purified preparation of LDL-associated [ ceramide- 3H]SPM which was selectively hydrolyzed by the lysosomal sphingomyelinase &evade et al., 1991, 1993; Graber et al., 1994). Fig. 1 shows the TLC patterns of the lipids extracted from normal and Farber cells incubated with LDL-associated [ cerumide- 3H]SPM. While normal cells actively metabolized the SPM and the subsequently released ceramide, Farber fibroblasts and lymphoid cells were almost devoid of ceramide metabolic products, suggesting the selective action of lysosomal ceramidase in the degradation of ceramide. Fig. 2 gives the rates of degradation of the ceramide produced from LDL-associated [ cerumide- 3H]SPM in normal and Farber cells after various incubation times. The degradation of ceramide was calculated (from the respective radioactivities) as the percentage of undegraded ceramide from the total lipid products of SPM hydrolysis, that is by neglecting the final SPM radioactivity (and, therefore, possibly some resyntheLymphoid

Fibrobiasts 500

5001 NOAYAL

DISTANCE

(cm)

cells

NORMAL

DISTANCE

(cm)

Fig. 1. TLC patterns of the radiolabelled lipids extracted from normal and Farber cells incubated with LDL-associated [ ceramide- 3 H]SPM. Skin fibroblasts and lymphoid cells from normal subjects and a patient with Farber disease (GM 5752 and GM 5748 cell lines) were incubated in medium supplemented with 2% Ultroser and LDL-associated [ ceramide3H]SPM (53 fig apolipoprotein B/ml; 12500 dpm/ pg). After 3 h pulse, cells were chased for 21 h in a medium supplemented with serum. Then, the cell-associated lipids were extracted, separated by TLC and radiochromatoscanned. Approximate running distances were as follows: SPM, 5 cm; phosphatidylcholine, 7 cm; phosphatidylethanolamine, 10.5 cm; ceramide (arrow), 15 cm; neutral lipids, 17-19 cm.

14

111

Fibroblasts

Lymphoid

cells

3

%ih

z

I SPWLDL T

I

loo

q NORNALS n FARBER

so

E 3. Results

108-I

P3CO

P3C21

P24CO

P3CO

P3CZl

PZ4CO

Fig. 2. Ceramide after degradation of LDL-associated [ceramide-‘H]SPM by intact normal and Farber cells. Cultured skin fibroblasts (left) and lymphoid cells (right) from normal subjects and a patient with Farber disease were incubated with LDL-associated [ ceramide- 3 H]SPM (as in Fig. 1). After 3 h pulse, incubation was stopped (P3CO) or continued for additional 21 h in a chase medium supplemented with serum (P3C21). In another set of experiments, cells were pulsed with LDL-associated [ceramide3H]SPM for 24 h (P24CO). The amount of undegraded ceramide is expressed as the percentage of the total radiolabelled metabolic products of SPM. The data correspond to the mean of 2 to 8 separate experiments. Up to 5 and 4 different cell lines of normal fibroblasts and lymphoid cells, respectively, were tested; for Farber disease, GM 5752 and GM 5748 cell lines (same patient) were used.

sized SPM) but considering the ceramide as the substrate. Already after 3 h incubation, the examination of the undegraded ceramide levels permitted differentiation between skin fibroblasts or lymphoid cells from normal subjects and Farber patients. This difference became much more striking after a 3-h pulse followed by a 21-h chase (Fig. 2). While in normal cells the undegraded ceramide represented less than 6% of the total SPM metabolites, it now accounted for 90% in GM 5748/GM 5752 Farber cells. Similar data were obtained after one day pulse, the ceramide levels in Farber cells being 11-13-fold higher than in normal cells. Using the latter method, a series of normal and pathologic skin fibroblasts were studied. Table 1, which highlights the marked variability of phenotypic expression of Farber disease in 12 patients examined, reports their respective residual lysosomal ceramide turnover. While in normal cells the undegraded ceramide averaged 5.9% of SPM metabolic products (irrespective of age, sex, passage level), it amounted from 49 to 98% in the various Farber cell lines. The high ceramide accumulation found in the prosaposin-deficient cells (line Sch), in which there is a low in vitro ceramidase activity although ceramidase is not the primary defect, confirms previous findings (Harzer et al., 1989; Paton et al., 1992). A normal amount of ceramide (7%) was found in cells from a heterozygous male (data not shown). We found that there is significant inverse correlation between the ceramide accumulated in situ and the decimal logarithm of the age at death of patients who died essentially from their neurologic involvement (Fig. 3). We have omitted the data of patient Sch from our statistical analy-

112

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y = 84.7 - 13.7 log(x)

AGE

OF DEATH

of the Neurological

(r = - 0.874)

(months)

Fig. 3. In situ ceramide accumulation as an antiproportional measure residual lysosomal ceramide turnover correlates with age at death Farber patients. The amounts of ceramide correspond to those given Table I (the Farber-like cell line Sch has been omitted). Logarithmic at death of patients correlates with ceramide accumulation: n= r = - 0.874 (99% confidence limits - 0.412 to - 0.979).

of of in age 11,

sis, since he died from Farber-like prosaposin-deficiency rather than pure Farber disease. Patient Moh, who had the most severe symptoms (including organomegaly, hydrops fetalis and vegetative insufficiency ending with death 3 days after birth) had the highest ceramide accumulation and, therefore, the lowest (if any) inferable ceramidase activity. Conversely, cases GM 2315 and Fra with a very prolonged survival had the highest activity. Interestingly, we also found that the age of onset of symptoms, another clinical feature, inversely correlated with the ceramide accumulated in situ (y = 101.6 - 16.7 log(x), with r = - 0.864, n = 11).

4. Discussion Ceramide is a key intermediate in the synthesis and degradation of all sphingolipids, i.e., membrane, lipoprotein and myelin constituents (Morel1 and Braun, 1972; Barenholz and Thompson, 1980). Ceramide and its metabolic products have recently been suggested to be very potent biological effecters (Hannun and Bell, 1989; Merrill, 1991; Kolesnick, 1992; Hannun and Linardic, 1993; Hannun, 1994). Ceramide accumulates in the lysosomes of Farber cells due to deficient activity of lysosomal ceramidase (Moser et al., 1989). The level of lysosomal ceramidase activity appears crucial both for studying its role in the generation/degradation of lipid mediators and for diagnosing Farber disease. However, the determination of lysosomal ceramidase activity remains one of the most complex assays of lysosomal enzymes and is therefore restricted to a very small number of laboratories (Moser et al., 1989; Wenger and Williams, 1991). Problems are encountered in both the in vitro and in situ assay of lysosomal ceramidase. In vitro, the measurement of acid ceramidase activity is difficult for its low specific activity, and because non-lysosomal ceramidases can hydrolyze the substrate (Nilsson, 1969; Sugita et al., 1975; Stoffel and Melzner, 1980; Momoi et al., 1982; Spence et al., 1986).

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As to the in situ assay, few data only for cultured human skin fibroblasts are available (Chen et al., 1981; Sutrina and Chen, 1982; Kudoh and Wenger, 1982; Inui et al., 1987). The degradation of the ceramide itself (Chen et al., 1981; Sutrina and Chen, 1982) or that derived from sulfatide hydrolysis (Kudoh and Wenger, 1982; Inui et al., 1987) was followed. The fate of the ceramide produced from SPM has also been studied and its accumulation reported in fibroblasts derived from patients with prosaposin deficiency (Harzer et al., 1989) or combined Sandhoff and Farber disease (Fusch et al., 1989). However, in the above mentioned studies, the turnover of ceramide by lysosomal ceramidase could not be correctly assessed because part of the ceramide remained in subcellular compartments other than lysosomes (Sutrina and Chen, 1982; Inui et al., 1987) or was degraded by non-lysosomal ceramidase(s) (Chen et al., 1981; Sutrina and Chen, 1982). We therefore reexamined the use of sphingolipid loading tests to measure the lysosomal ceramide turnover in intact normal and Farber cells. SPM loading test was selected because SPM, an immediate precursor of ceramide and a naturally occurring constituent of LDL (Barenholz and Thompson, 1980), can be associated with LDL @wade and Gatt, 1987; Levade et al., 1991) and therefore targetted to lysosomes (Graber et al., 1994). The ceramide released by the action of lysosomal sphingomyelinase is subsequently hydrolyzed by lysosomal ceramidase. Thus, incubation of cells in the presence of lipoproteins permitted the degradation of SPM-derived ceramide to be followed accurately, and allowed the selective determination of lysosomal ceramide turnover. The specificity of this determination could be achieved by using a purified [ cerumide-3H]SPM-labelled LDL preparation. The fact that, in our study, SPM was targeted to lysosomes via LDL explains why our data markedly differed from those of Sutrina and Chen (Sutrina and Chen, 1984). After loading fibroblasts with SPM liposomes, these authors did not find any ceramide accumulation in Farber cells compared to normal cells, most probably because the majority of SPM did not enter the lysosomes. Whereas the residual lysosomal ceramidase activity determined in vitro does not correlate with the severity of clinical symptoms (see Table l), our in situ data show significant inverse correlation between ceramide accumulation and logarithmic age at death of the patient (Fig. 3). This suggests that in Farber disease the individual residual ceramide turnover to an appreciable degree defines the duration of the disease. In fact, at least 8 of the 12 analyzed Farber patients have died as a consequence of nervous system involvement according to clinical data. More generally, the severity of Farber disease seems to be the higher the lower is the in situ residual ceramide turnover. Such a correlation between the effective residual activity of a sphingolipid hydrolase and the severity of the ensuing lysosomal storage disorder has recently been demonstrated for GM,-gangliosidosis, metachromatic

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leukodystrophy, Niemann-Pick and Gaucher diseases (Leinekugel et al., 1992; Graber et al., 1994; Meivar-Levy et al., 1994). Hence, our present observations provide an additional support to the theoretical model of Conzelmann and Sandhoff (1983/84) on the pathogenesis of sphingolipidoses, which links the effective lysosomal hydrolase activity, the degree of substrate accumulation and the severity of the ensuing disorder to each other. The usefulness of our loading method for prenatal diagnosis of Farber disease on amniotic fluid cells has just recently been demonstrated &evade et al., 1995b). Furthermore, our biochemical data, beyond their diagnostic specificity, may have some course-predictive value in living Farber patients.

Acknowledgements The technical assistance of J.P. Basile and M.A. Berges is acknowledged. This work was supported by grants from INSERM (CJF 92061, UniversitC Paul Sabatier (Toulouse, France, JE DRED 174) and the association “Vaincre les Maladies Lysosomales”.

References Al, B.J.M., C.W. Tiffany, D.S. Gomes de Mesquita, H.W. Moser, J.M. Tager and A.W. Schram (1989) Properties of acid ceramidase from human spleen. Biochim. Biophys. Acta, 1004: 245-251. Antonarakis, S.E., D. Valle, H.W. Moser, A. Moser, S.J. Qualman and W.H. Zinkham (1984) Phenotypic variability in siblings with Farber disease. J. Pediatr., 104: 406-409. Barenholz, Y. and T.E. Thompson (1980) Sphingomyelins in bilayers and biological membranes. Biochim. Biophys. Acta, 604: 129-158. Chen, W.W., A.B. Moser and H.W. Moser (1981) Role of lysosomal acid ceramidase in the metabolism of ceramide in human skin fibroblasts. Arch. Biochem. Biophys., 208: 444-455. Colamaria, V., L. Giardina, M. Simeone, A. Salviati, A.H. Fensom and B. Dalla Bernardina (1992) Neurologic progressive form (type 5) of Farber’s lipogranulomatosis (ceramidase deficiency) in monozygotic twins. Meeting of the European Neurological Society (Abstract). Conzelmann, E. and K. Sandhoff (1983/84) Partial enzyme deficiencies: residual activities and the development of neurologic disorders. Dev. Neurosci., 6: 58-71. Dulaney, J.T., A. Milunsky, J.B. Sidbury, N. Hobolth and H.W. Moser (1976) Diagnosis of lipogranulomatosis (Farber disease) by use of cultured fibroblasts. J. Pediatr., 89: 59-61. Dulaney, J.T. and H.W. Moser (1977) Farber disease (lipogranulomatosis). In: Glew, R.H. and Peters, S.P. (Eds.), Practical Enzymology of the Sphingolipidoses, Alan R. Liss, Inc., New York, pp. 283-296. Fensom, A.H., P.F. Benson, B.R.G. Neville, H.W. Moser, A.E. Moser and J.T. Dulaney (1979) Prenatal diagnosis of Farber’s disease. Lancet, 2: 990-992. Fiumara, A., F. Nigro, L. Pavone and H.W. Moser (1993) Farber disease with prolonged survival. J. Inherit. Metab. Dis., 16: 915-916. Folch, J., M. Lees and G.H. Sloane Stanley (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226: 497-509. Fusch, C., R. Huenges, H.W. Moser, A.C. Sewell, W. Roggendorf, B. Kustermann-Kuhn, A. Poulos, W.F. Carey and K. Harzer (1989) A

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