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Studies in the morphology, histochemistry, and ultrastructure of the cerebellum, with special reference to the Purkinje cell dendrites, have been undertaken in eight cases of gargoylism. The results suggest that the demonstration of ovoid swellings of the Purkinje cell dendrites by the Cajal method for the cerebellum, together with certain histochemical findings, may enable a diagnosis of mucopolysaccharidosis to be made. Possible mechanisms for the formation of these swellings are briefly discussed.

The sugars present in hydrolyzed extracts of human liver and brain were analyzed by gasliquid chromatography after conversion to their alditol acetates. The samples analyzed were obtained from control subjects, patients with gargoylism, and patients with a few other kinds of storage disorders. Accumulation of galactose was demonstrated in the liver and the brain of two patients with gargoylism, and in the liver samples, high levels of mannose were found too. We also studied the hydrolysis of a number of galactosides by homogenates from different tissues in the control subjects and in the patients. Separation methods and kinetic studies demonstrated the presence in normal human tissues of two different beta-galactosidases, which we call enzyme A and enzyme B, respectively. Enzyme A hydrolyzed all the beta-galactosides tested. Enzyme B hydrolyzed the synthetic substrates tested (4-methylumbelliferyl-, p-nitrophenyl-, o-nitrophenyl-, and phenyl-beta-galactoside) but not the natural substrates tested (ceramide-beta-galactoside, ceramide lactoside, transferrin glycopeptide, and keratan sulfate). Enzyme B also exerted beta-glucosidase activity. In various tissues from patients with gargoylism, deficiency of beta-galactosidase A could be demonstrated. It is suggested that the high level of galactose found in the hydrolyzed extracts of tissues from gargoylism patients is due to storage of galactose-rich glycosaminoglycans and glycopeptides, and that this storage is a result of the deficiency of beta-galactosidase A. The high level of mannose in the liver from gargoylism patients seems to indicate storage of glycopeptide, adding a new group of substances to those known to be stored in gargoylism.

Catabolism of sulfated mucopolysaccharide by normal human fibroblasts in culture is progressively inhibited as the pH of the growth medium is raised from 6.8 to 8.0. The final cell density increases with the change in pH. The capacity to degrade mucopolysaccharide is rapidly restored by lowering the pH, and this reactivation does not require protein synthesis. Such pH dependence is not observed in cells from patients with genetic impairment of mucopolysaccharide degradation, such as the Hurler or Hunter syndromes. These results may have relevance not only to studies of mucopolysaccharide metabolism in cell culture, but also to the use of metachromasia as a genetic marker and to the observation that normal fibroblasts are released from contact inhibition of growth as the pH of the growth medium is raised.

Cultured fibroblasts from the genetic mucopolysaccharidoses store higher than normal amounts of the polyanionic glycosaminoglycans (mucopolysaccharides); histochemical staining with the cationic dyes toluidine blue O and Alcian blue detects such intracellular accumulation. With these stains as phenotypic markers, correction of the cellular abnormality by fibroblasts derived from different patients and their heterozygous parents has been observed among several genetic types of mucopolysaccharidoses. In this way, syndromes previously considered clinically homogeneous have been separated into distinct groups.

Increased amounts of hyaluronic acid accumulate in fibroblasts cultured from patients with Marfan's disease, an autosomal dominant disorder. In the recessive Hurler's disease, the storage of glycosaminoglycan (GAG) is due to impaired degradation. This study examines the kinetics of GAG accumulation in Marfan's disease in order to determine whether the mechanism of accumulation differs from that in Hurler's disease.Marfan-derived fibroblasts incorporated [(14)C]acetate or [(14)C]glucosamine into GAG to a level 4-6 times greater than control fibroblasts. Sugar analyses, electrophoretic mobility, and enzyme susceptibility studies showed that the isolated material was hyaluronic acid. There were no differences in activity of a variety of glycosidases between Marfan and control fibroblasts, nor were there differences in the ability to degrade prelabeled hyaluronate by cell-free extracts. Finally, chase experiments showed parallel rates of loss of labeled GAG from control fibroblasts and fibroblasts from Marfan patients. It appears that hyaluronic acid was accumulating in greater amounts in the fibroblasts from patients with Marfan's disease because of a greater rate of synthesis as opposed to a decreased rate of breakdown.

The molar ratios of N-acetyl neuraminic acid, hexose, hexosamine, and sphingosine have been determined for the abnormal ganglioside in Tay-Sachs disease that was previously detected as a fast-moving band in thin-layer chromatography, and in two abnormal fast-moving bands of gangliosides from the cortex and white matter of the brain in cases of gargoylism and Niemann-Pick's disease. The fastest-moving ganglioside band in these two conditions contains neither hexosamine nor glucose.