Lipid abnormalities in a variant of the Hurler syndrome.

Numerous studies have established that the excretion in urine and the deposition in tissues and in cultured fibroblasts of acid mucopolysaccharides (AMPS) are markedly increased in Hurler's disease.1-8 Previous reports have indicated an increase in ganglioside content in brain and visceral organs of patients with the Hurler syndrome.9-13 The relationship of the increase in glycolipids to that of AMPS is not clear. In the course of a study of two brothers with clinical evidence of Hurler's syndrome, fibroblasts obtained from one of the brothers were strikingly abnormal but did not show typical metachromasia. However, after the specimens were treated with chloroform: methanol (2:1, v/v), metachromatic granules could be demonstrated, thus suggesting that excess lipids were present which interfered with staining. It is the purpose of this communication to report details regarding the occurrence of abnormal quantities of certain lipids in fibroblasts of this patient as compared with the fibroblasts of a patient with typical Hurler's disease and those of a normal child. Materials and Methods.-Carrier-free H2S'504 was obtained from New England Nuclear Corporation. Sodium acetate-C'4 (53 mc/mmole) was obtained from Nuclear Chicago. Monosialogangliosides, GM1, GM2, and disialoganglioside GD1 (nomenclature of Svennerholm14) were a gift from Drs. B. Kaufman and S. Roseman, Johns Hopkins University. Tissue culture: Fibroblast cultures were established from skin biopsies of a previously studied Hurler patient (B. H.), a normal child, and the proband J. S. and his parents. Cells were grown in 100-mm plastic Petri dishes as previously described.7 Isolation of lipids: Cells were removed from the Petri dishes by using a rubber policeman, homogenized, and lyophilized, and lipids were extracted 4 times with chloroform: methanol (2:1, v/v) with the addition of KCl.15 Isolation of AMPS: Following digestion of the cells with crystalline papain, the solution was dialyzed and AMPS were precipitated with cetylpyridinium chloride (CPC) as previously described.16 In the case of J. S., a copious precipitate was obtained from CPC which was found to include lipids in addition to AMPS. Addition of 2.0 M LiCl, which dissolves AMPS-CPC complexes, did not clear the solution. However, the subsequent addition of 3 vol of ethanol resulted in a precipitate containing AMPS and a clear supernatant solution containing the lipids. Fractionation of AMPS: Separation of AMPS was accomplished by chromatography on Dowex 1-X2, Clcolumns utilizing stepwise sodium-chloride elution.17 Thin-layer chromatography: Thin-layer chromatography (TLC) was carried out on silica-gel plates. Plates 1 mm thick were used for preparative studies. For analysis of the crude lipid fraction, the solvent was chloroform: methanol: H20 (16: 6: 1, v/v) and the stain was iodine vapor. In this solvent, gangliosides remain close to the origin, neutral fats migrate with the solvent front, and other components including phospholipids display an intermediate mobility. For further study, the gangliosides were eluted from the silica gel with chloroform:methanol (1:1, v/v) and rechromatographed on silica-gel