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Two siblings with Sandhoff's disease manifested clinical and laboratory evidence of significant cardiomyopathy. Postmortem examination of the hearts of both patients revealed anatomic, biochemical and ultrastructural abnormalities resulting from deficient activities of the enzymes hexosaminidase A and B. A study of the biochemical and ultrastructural changes in the hearts revealed the accumulation of the glycosphingolipid globoside in all cardiac tissues. The necropsy findings indicate that Sandhoff's disease should be considered in the differential diagnosis in infants with cardiomyopathy or mitral insufficiency.

Summary A mammalian model system was developed to determine the in vivo fate of intravenously administered bovine β-glucuronidase in β-glucuronidase deficient mice. Sensitive and reliable discrimination of bovine liver β-glucuronidase activity from residual murine tissue activities was achieved by selective heat inactivation of the bovine activity, providing the ability to monitor the fate of administered enzyme. Following injection, the bovine activity was rapidly cleared from the circulation and recovered almost exclusively in the liver. Subcellular fractionation of hepatic tissue obtained 2 hours post injection localized 72% of recovered activity in the lysosomally-enriched fraction. This mammalian system provides an in vivo model to serially evaluate and maximize methods for the protection and delivery of an exogenous enzyme, prior to trials of enzyme replacement in patients with inherited enzymatic deficiency diseases.

Methods were investigated for the stabilization of human splenic α-galactosidase A (α- d -galactoside galactohydrolase, EC 3.2.1.22). Anti-α-galactosidase A antiserum was produced in a goat by repeated immunization with highly purified α-galactosidase A. When this antiserum was incubated at 37°C with α-galactosidase A in varying enzyme: antiserum ratios, a significant increase in the thermal (50 °C) stability and in the resistance to protease digestion of these mixtures was observed compared to appropriate controls. The enzyme was also treated with the bifunctional cross-linking reagent, hexamethylene diisocyanate, and the thermal stability and protease resistance of the cross-linked derivative were increased compared to the native enzyme treated with the monofunctional reagent, butyl isocyanate.

Recently, an inborn error of glycosphingolipid metabolism was described in which activities of beta-N-acetylhexosaminidase A and B were totally deficient. This resulted in the accumulation of two neutral glycosphingolipids, globoside and asialo G M2 ganglioside in the brain. The systemic storage of a particular type of glycosphingolipid results in specific anatomic and pathophysiologic manifestations. Our patient had cardiovascular symptoms prior to the development of signs of central nervous system degeneration. Multiple lamellar cytoplasmic inclusion bodies were visualized under electron microscopy in every tissue examined. The diagnosis was confirmed by extensive biochemical analysis of tissue glycosphingolipids. Biochemical diagnosis during life can be accomplished by the demonstration of elevated levels of globoside ∗ ∗The use of the term globoside indicates the presence of galactosamine (GalNAc), galactose (Gal), glucose (Glc), sphingosine base and fatty acid in molar ratio of 1:2:1:1:1, and assignment of the galNAc to the terminal position, but does not indicate the stereochemistry and linkages of the respective sugar moieties. in plasma and urinary sediment as well as deficient activities of hexosaminidase A and B.

Neutral and acidic glycosphingolipids were isolated and quantitated from fresh, washed human blood platelets. Enzymatic assays for α-galactosidase, β-galactosidase, and N-acetyl-β-hexosaminidase A and B showed significant activity of these glycosyl hydrolases in whole platelet homogenates. Only minimal β-glucosidase activity was found. Activity levels for normal controls and patients with two glycosphingolipidoses, Fabry's and Sandhoff's diseases, were determined and the diagnostic value of platelet enzyme assay for such glycosphingolipidoses established.

A method is described for the isolation and quantitation of six neutral glycosyl ceramides from human urinary sediment. Total lipids were extracted from sediments of 24-hr urine collections, and the glycosyl ceramides were isolated by silicic acid column chromatography followed by thin-layer chromatography. Methanolysis of the individual glycosyl ceramides yielded methyl glycosides which were quantitated as the trimethylsilyl ethers by gas-liquid chromatography. By this technique, the submicromolar concentrations of six glycosyl ceramides in normal subjects and in individuals with Fabry's disease, an hereditary glycosphingolipid storage disease, were determined. Trihexosyl ceramide (galactosyl-galactosylglucosyl ceramide) and a digalactosyl ceramide accumulated in the urinary sediment of patients with Fabry's disease.

The first in utero diagnosis of Sandhoff's disease was made in an at-risk fetus by the demonstration of deficient β-N-acetyl-hexosaminidase A and B activities in amniotic fluid components the day of amniocentesis. These enzymatic deficiencies were determined by enzyme assay and electrophoresis using 4-methylumbelliferyl-β-N-acetyl-glucosaminide as substrate. The concentrations of the neutral glycosphingolipids were quantified in amniotic fluid; the level of the glycosphingolipid substrate, globoside, was markedly increased in amniotic fluid from the at-risk fetus compared to that of fetal controls. In addition, ultrastructural examination demonstrated pathologic glycosphingolipid accumulation in uncultured amniotic cells. These enzymatic, chemical and ultrastructural procedures provided the rapid and accurate in utero diagnosis Sandhoff's disease within three days of amniocentesis. The in utero diagnosis was confirmed by the marked deficiencies of β-N-acetyl-hexosaminidase A and B in plasma and various tissues from the aborted fetus. These findings indicated that maternal hexosaminidases do not cross the fetal-placental barrier.

Publisher Summary This chapter focuses on Fabry's disease. Fabry's disease is an X-linked inborn error of glycosphingolipid catabolism. Transplantation of enzymatically active renal tissue into patients with Fabry's disease has resulted in decrease to normal of the accumulated glycosphingolipid substrate in plasma. The diagnosis of hemizygotes and heterozygotes for Fabry's disease can be confirmed by the deficient activity of ceramide trihexosidase in plasma, urine, leukocytes, cultured fibroblasts, or biopsied tissue. The chapter also discusses clinical characteristics of Fabry's disease. The clinical manifestations of the disease result from the generalized visceral deposition of the Fabry glycosphingolipid, particularly in the cardiovascular-renal system. Three separate approaches to enzyme replacement have been utilized in Fabry's disease. These are: (1) the plasma infusion, (2) renal transplantation, and (3) direct enzyme infusion.

Six neutral glycosphingolipids were identified in dental pulp from normal individuals and hemizygotes and heterozygotes for Fabry's disease by biochemical analysis. Histochemical stains were selected to demonstrate the presence of glycosphingolipid. Only small amounts of glycosphingolipid material could be detected in normal pulp or in pulp from heterozygotes for Fabry's disease by histochemical or biochemical techniques. However, the pulp from a hemizygote demonstrated a marked deposition of PAS positive material and a specific increase of trihexosyl ceramide, the glycosphingolipid which accumulates in this disorder. These procedures provide a reliaable method for the diagnostic confirmation of hemizygotes with Fabry's disease.

Two patients with Fabry's disease were infused with normal plasma to provide active enzyme (ceramide trihexosidase) for hydrolysis of the plasma substrate, galactosylgalactosylglucosylceramide. Maximum ceramide trihexosidase activity occurred 6 hours after infusion of the plasma, attaining a level approximately 150 percent of that in normal plasma; enzymatic activity was detectable for 7 days. The amount of accumulated substrate in the plasma of these recipients decreased about 50 percent on day 10 after infusion. Thus, periodic replacement of ceramide trihexosidase activity in the plasma of patients with Fabry's disease might lead to consistently lower amounts of substrate in the plasma and a decrease in its rate of accumulation in tissues.

Fabry's disease,1 heretofore a rare and esoteric diagnostic rubric, has become an important focus for research by biochemists, enzymologists, geneticists, clinical investigators and transplant surgeons.2 As noted by Clarke and his colleagues in this issue of the Journal, several centers have transplanted normal kidneys into patients with renal failure secondary to Fabry's disease. Of the nine patients throughout the world who have received transplants, four have had normal renal function for periods of six months to over three years.3 , 4 These successful transplants have had documented clinical improvement, with subjective relief of their frequent excruciating pain, return of sweating and . . .

Fabry’s disease is a systemic metabolic disorder of glycosphingolipid metabolism (Sweeley and Klionsky, 1963). It can be recognized clinically in childhood or early adolescence by cutaneous vascular lesions (angiokeratoma) and periodic episodes of fever and pains in the extremities (Sweeley and Klionsky, 1966). With increasing age, these symptoms are usually accompanied by proteinuria and gradual development of renal dysfunction. A significant mortality occurs in the fourth and fifth decades from renal failure or cardiovascular complications. Pedigree studies (Opitz et al., 1965) and linkage data (Johnston, Warland, and Weller, 1966; Johnston et al.,1969) indicate that the metabolic defect in Fabry’s disease is transmitted by an X-linked gene. The most severely affected individuals are, therefore, hemizygous males, although some heterozygous females also have significant clinical manifestations.

Sandhoff’s disease results from the deficient activity of both N-Acetyl-β-hexosaminidase A and B (14,17,19–22) and is characterized by the neural and visceral deposition of Gm2 ganglioside, its asialo derivative (NAcgal-gal-glc-cer) and globoside (NAcgal-gal-gal-glc-cer) in affected individuals (Figure 1) (3,6,9,10,14,19–22,24,25).

Fabry's diease was originally conceived diagnostically as a dermatological disorder (angiokeratoma). The systemic manifestations due to blood vessel involvement of renal, cardiac, and neural tissues widened the spectruk of clinical disease. The earliest manifestations occur in childhood and are a confusing diagnostic problem. The scope of the disease lias been enlarged and manifested by the female heterozygote and by the several “forme fruste” genetic variants in males without skin lesions. Recent studies have provided explicit knoweledge about the metabolic abnormality. Increased levels of galactosyl-galactosyl-glycosyl ceramide (GL-3) in blood, urine sediment, and most tissues have been consistently noted (J. Lipid Res., 10: 188, 1969 and 11: 31, 1970). These are correlated with the absence of a specific galactosyl hydrolase in hemizygotes. Heterozygous females have intermediate levels of the lipid and partial deficiency of the enzyme. Recently, the same galactosyl hydrolase which is absent in patients with Fabry's disease has been found in normal blood plasma. This suggested the possibility the possibility of direct enzyme therapy by plasma transfer. Six hours after the plasma infusion an “enhanced” or “induced” level of enzyme was noted. This level was 20-fold greater in the recipient than could be accounted for by the amount from the donor. Therapy with plasma infusion is under active investigation at present.