The accumulation of cerebroside sulfates by fibroblasts in culture from patients with late infantile metachromatic leukodystrophy

Porter, Myna T.; Fluharty, Arvan L.; Harris, Sandra E.; Kihara, Hayato

doi:10.1016/0003-9861(70)90392-9

Cited by 53 publications

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“…Under similar conditions normal fibroblasts ingest the sulfatides and degrade them, as evidenced by release of inorganic sulfate into the medium (4). We now find that the manifestations of the genetic disorder in the tissue culture model can be corrected 18 JUNE 197112.…”

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confidence: 75%

“…Procedures for initiation of primary cultures from skin biopsies, culture of outgrowths, enzyme assays, preparation of 35S-labeled sulfatides, and determination of [35S]sulfatide uptake and degradation have been described (3,4). Cultures for the present study were derived from two patients with late infantile MLD, an obligate heterozygous carrier (a parent of one of the patients), and a normal adult.…”

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confidence: 99%

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Correction of Abnormal Cerebroside Sulfate Metabolism in Cultured Metachromatic Leukodystrophy Fibroblasts

Porter¹,

Fluharty²,

Kihara³

1971

Science

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Cultured fibroblasts derived from patients with late infantile metachromatic leukodystrophy incorporated arylsulfatase A from the growth medium. Upon exposure to cerebroside sulfate, they exhibited patterns of uptake and hydrolysis indistinguishable from cells derived from control subjects. Furthermore, inclusion granules formed in the metachromatic leukodystrophy fibroblasts upon exposure to sulfatides were cleared by subsequent supplementation of the growth medium with arylsulfatase A.

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confidence: 75%

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confidence: 99%

Correction of Abnormal Cerebroside Sulfate Metabolism in Cultured Metachromatic Leukodystrophy Fibroblasts

Porter¹,

Fluharty²,

Kihara³

1971

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“…The substrate is of very high radiopurity (99.6%) and is relatively simple to prepare according to a published procedure (23). Previous studies have demonstrated that CS could be incorporated into cultured skin fibroblasts, and that it could induce the formation of metachromatic inclusions in cells from MLD patients (24). Ultrastructural studies revealed that these inclusions consisted of undigested CS in the lysosomes which lacked CS sulfatase activity (1).…”

Section: Discussionmentioning

confidence: 99%

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

Kudoh¹,

Wenger²

1982

J. Clin. Invest.

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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

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“…The test has been modified somewhat from that originally described (19). With fibroblasts,,the Eagle's minimal essential medium of confluent cultures was replaced with medium 199 and cultures were incubated in a 5% COz:atmosphere 2 to 4 days before subculture.…”

Section: Cerebroside Sulfate Loading Testmentioning

confidence: 99%

“…Previous cerebroside sulfate loading tests had been camed out on monolayer fibroblast cultures (8,19,20). and it was uncertain whether the test would be effective in cultured amniotic fluid cells.…”

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Prenatal Diagnosis of Metachromatic Leukodystrophy in a Family with Pseudo Arylsulfatase A Deficiency by the Cerebroside Sulfate Loading Test

Kihara

Fluharty

et al. 1980

Pediatr Res

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SummaryPrenatal diagnosis was requested by a family at risk for metachromatic leukodystrophy (MLD). An examination of the family leukocyte arylsulfatase A profile revealed that the mother had pseudo arylsulfatase A deficiency. Cultured amniotic fluid cells were deficient in arylsdfatase A, so two possibilities were indicated. the fetus was affected with MLD or had the pseudodeficiency phenotype. The only known biochemical test to differentiate the two enzyme deficient phenotypes is cerebroside sulfate loading of growing fibroblasts. The pseudodeficient cells hydrolyze the incorporated sulfatide as efficiently as control cells, whereas MLD cells show no hydrolysis. Application of this test to the at risk cultured amniotic fluid cells resulted in appreciable uptake of the sulfolipid, but no hydrolysis. Control amniotic fluid cell cultures hydrolyzed 82 to 95% of the incorporated sulfatide. Therefore, an affected fetus was indicated. Fibroblasts derived from the aborted fetus sbowed a deficiency of arylsulfatase A and a similar inability to hydrolyze cerebroside sulfate i n the loading test. The loading technique allowed the prenatal diagnosis of MLD when the arylsulfatase A analysis was equivocal. Speculation I n metachromatic leukodystrophy families with pseudo arylsulfatase A deficiency, the usual enzyme assays on cultured amniotic fluid cell extracts fail to differentiate between the fetus with the affected phenotype and the fetus with the pseudodeficiency phenotype. The cerebroside sulfate loading test in growing cultured amniotic fluid cells allowed this discrimination. I t is important to examine the family enzyme profile for the peudodeficiency phenotype as a prerequisite in the prenatal diagnosis of metachromatic leukodystrophy to avoid the erroneous identification of a pseudodeficient fetus as a metachromatic leukodystrophy fetus.In metachromatic leukodystrophy (MLD), the profound deficiency of arylsulfatase A (ajlsuifaie ~ulfohydrola&, EC 3.1.6.1) leads to the accumulation of cerebroside sulfate in neural tissue ~ resulting in progressive neurological degeneration (5). Based on the age of onset of symptoms, three classical types of MLD are recognized: late infantile, juvenile, and adult. Each type appears to be an independent autosomal recessive disorder, so allelism is implied. Although no treatment is available, the disorder can be prevented in at risk families because MLD is amenable to prenatal diagnosis. In affected pregnancies, cultured amniotic fluid cells are deficient in arylsulfatase A (10,14,17,(21)(22)(23)(24).Dubois el al. (3, 4). Lott el al. (15). and Fluharty el al. (8) described four MLD families in which one of the parents and some of the unaffected children had very low arylsulfatase A activities which overlapped the range of probands. These individuals showed no neurologic dysfunction and were healthy, so it would be appropriate to iypifi the apparent enzyme deficikncy as a ~seudodeficiencv. The attenuated enzyme activity was observed in' leukocyte anda fibroblast extracts khether thk ...

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The accumulation of cerebroside sulfates by fibroblasts in culture from patients with late infantile metachromatic leukodystrophy

Cited by 53 publications

References 15 publications

Correction of Abnormal Cerebroside Sulfate Metabolism in Cultured Metachromatic Leukodystrophy Fibroblasts

Correction of Abnormal Cerebroside Sulfate Metabolism in Cultured Metachromatic Leukodystrophy Fibroblasts

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

Prenatal Diagnosis of Metachromatic Leukodystrophy in a Family with Pseudo Arylsulfatase A Deficiency by the Cerebroside Sulfate Loading Test

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