Andrew E. Gal

Replacement Therapy for Inherited Enzyme Deficiency: Use of Purified Ceramidetrihexosidase in Fabry's Disease

Abstract The effect of intravenous administration of glucocerebrosidase isolated from human placenta was investigated in two patients with Gauchers disease who are deficient in this enzyme. The first received one injection of 1.5 x 106 units of glucocerebrosidase, and the second an injection of 1.65 x 106 units on two successive days. Liver biopsies were obtained before and 24 hours after injection of enzyme. Glucocerebroside in the liver of the first patient decreased from 702 to 519 μg per gram wet weight and from 1634 to 1214 μg per gram in the second after infusion of glucocerebrosidase. The quantity of glucocerebroside in erythrocytes of the two patients before infusion was 7.4 and 6.2 μg per milliliter of cells respectively and 2.9 and 2.6 μg per milliliter of cells 72 hours afterward. These findings indicate that exogenous glucocerebrosidase causes definite decreases in the quantity of accumulated lipid in patients with Gauchers disease. (N Engl J Med 291:989–993, 1974)

A lysosomal storage disorder in mice characterized by a dual deficiency of sphingomyelinase and glucocerebrosidase

Lipid and lysosomal enzyme levels in the tissues of a strain of mice afflicted with an autosomal rescessive neuroviscereal storage disorder were examined. Sphingomyelinase and glucocerebrosidase activities were consistently diminished in a wide variety of tissues obtained from the affected mice. The activities of these enzymes were clearly attenuated in new-born mice, which at this age, were otherwise indistinguishable from littermates and age-matched controls. The deficiency of sphingomyelinase was more pronounced than glucocerebrosidase. There was progressive accumulation of sphingomyelin, glucocerebroside, lactosylceramide and unesterified cholesterol in the tissues of these mice in the postnatal period. Gangliosides GM2 and GM3 accumulated in the brain of the animals, and GM3 and asialo-GM2 were stored in the liver. Furthermore, there was a large increase in the quantity of hepatic bis(monoacylglycero)phosphate. The accumulation of lipids was parallelled by a progressive elevation in the activity of several lysosomal hydrolases in various tissues. Heterozygous mice were biochemically indistinguishable from normal controls. The phenotypic manifestations in these metabolically mutated animals are compared with those in Niemann-Pick disease and Gauchers disease in humans.

A practical chromogenic procedure for the detection of homozygotes and heterozygous carriers of Niemann-Pick disease.

Niemann-Pick disease is caused by a deficiency of sphingomyelinase in organs and tissues. Determinations of sphingomyelinase activity had required the use of sphingomyelin labeled with radiocarbon or radiohydrogen. These materials are expensive, and their use is restricted to laboratories with radioactive counting facilities. An analogue of sphingomyelin, 2-hexadecanoylamino-4-nitrophenylphosphorylcholine, was synthesized. This substance is hydrolyzed by highly purified sphingomyelinase, and by sphingomyelinease in extracts of human liver tissue, cultured skin fibroblasts, cultured amniotic cells and washed leukocyte preparations. Extracts of tissues and cells from patients with Niemann-Pick disease Type A do not hydrolyze this compound, whereas heterozygotes and patients with Niemann-Pick disease Type C have an intermediate level of hydrolytic activity. Thus, the analogue is a reliable chromogenic reagent for the diagnosis of patients with Niemann-Pick disease and the detection of heterozygous carriers of the Niemann-Pick trait.

Separation and identification of monosaccharides from biological materials by thin-layer chromatography

Abstract Thin-layer chromatographic methods are described for the separation of the following monosaccharides: glucose, galactose, glucosamine, galactosamine, N-acetylglucosamine, N-acetylgalactosamine and neuraminic acids. Neutral solvent systems and unaltered silica gel G plates were used. The monosaccharides are separated sharply, which allows these methods to be used for the determination of these compounds. The preparation and use is discussed of seven facile spray reagents that afford further specificity for detection and evaluation of the sugars at the microgram level. The feasibility of these procedures was demonstrated with a hydrolyzate of human erythrocyte globoside.

The isolation and characterization of sphingomyelinase from human placental tissue

Human placental sphingomyelinase activity was eluted as a single symmetrical peak from Sephadex G-200 with a molecular weight of 290000; however, the enzyme behaved heterogeneously on ion exchange chromatography. A specific species of sphingomyelinase was purified approx. 10 000-fold to a constant specific activity of 274 000 nanomol of sphingomyelin hydrolyzed per mg protein per h. When the purified enzyme was examined on sodium dodecyl sulfate disc gel electrophoresis, two distinct protein bands in approximately equal proportions with molecular weights of 36 800 and 28 300 were found. The specificity of the enzyme is directed towards both the hydrophilic phosphocholine and the hydrophobic ceramide moieties of sphingomyelin. Possible interrelationships between the heterogenous forms of placental sphingomyelinases are discussed.

A practical chromogenic procedure for the diagnosis of Krabbe's disease.

Krabbes disease is caused by a deficiency of galactocerebrosidase in organs and tissues. Determinations of galactocerebrosidase activity had required the use of galactocerebroside labeled with radiocarbon or radiohydrogen. These materials are expensive and their use is restricted to laboratories with radioactive counting facilities. An analogue of galactocerebroside, 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside, was synthesized. The hydrolysis of this analogue by extracts of tissues and cells from patients with Krabbes disease is greatly reduced from normal levels. Cultured skin fibroblasts preparations derived from heterozygous carriers of Krabbes disease have an intermediate level of hydrolytic activity. Thus, the analogue is a reliable chromogenic reagent for the diagnosis of patients with Krabbes disease and for the detection of heterozygous carriers of the Krabbe trait.

Glucocerebrosidase deficiency and lysosomal storage of glucocerebroside induced in cultured macrophages

A cell culture model stimulating the genetic deficiency of glucocerebrosidase has been developed, utilizing macrophages and conduritol B epoxide (CBE), the specific irreversible inhibitor of the enzyme. Rat peritoneal macrophage glucocerebrosidase was completely inhibited when cells were treated with 10 microM CBE for 16 h or 100 microM CBE for 2 h. The t1/2 of inactivation was 30 min at 10 microM concentration. When cells were washed free of CBE, the enzyme activity reappeared linearly with time, reaching 50% of control activity 48 h after removal of the inhibitor. CBE-treated macrophages have normal phagocytic activity toward [3H]glycine-coupled latex beads and a normal number of mannose receptors. CBE was found to have no effect on other lysosomal enzymes. When [14C]glucocerebroside, encapsulated in multilamellar liposomes with alpha-D-mannopyranoside covalently coupled to the surface, was fed to glucocerebrosidase-depleted macrophages, the radiolabelled glycolipid accumulated and was undegraded. Subcellular fractionation on a Percoll density gradient demonstrated that the stored glucocerebroside in the CBE-treated macrophages was localized in lysosomes.

Synthesis of 2-n-(hexadecanoyl)-amino-4-nitrophenyl phosphorylcholine-hydroxide, a chromogenic substrate for assaying sphingomyelinase activity.

2-N-(Hexadecanoyl)-amino-4-nitrophenyl phosphorylcholine-hydroxide a compound resembling sphingomyelin is synthesized. It is cleaved by sphingomyelinase to the chromogenic N-acylaminonitrophenyl moiety. Phospholipase C preparations do not hydrolyze this compound. The starting material is 2-amino-4-nitrophenol which when acylated with palmitoyl chloride yields the hexadecananilide. Reaction with beta-bromoethylphosphoryldichloride gives the phosphate which is quaternized with trimethylamine to give the title compound.

Response of sphingolipid hydrolases in spleen and liver to increased erythrocytorhexis

Abstract The injection of phenylhydrazine, zymosan, and human erythrocyte stroma caused an increase in the specific activities of various hydrolytic enzymes in rat spleen and liver tissues. The activity of spleen glucocerebrosidase and sphingomyelinase was particularly augmented by the injection of phenylhydrazine whereas the increases obtained with zymosan were not statistically significant. The injection of red-cell stroma brought about a significant increase in the activity of these enzymes; the injection of lipid-extracted stroma had no effect. The injection of partially purified stromal sphingolipids consisting mainly of globoside and sphingomyelin caused an increase in the activity of glucocerebrosidase and sphingomyelinase in liver but not in spleen.

Biliary excretion of glycolipid in induced or inherited glucosylceramide lipidosis

Metabolically inert L-[1-14C]glucosylceramide is stored predominantly in the liver after intravenous administration to mice. The half-time of this glycolipid analogue in the liver is 3.5 days and its clearance occurs predominantly via the bile. Within the limited number of Gaucher specimens available for examination very high levels of glucosylceramide were found in the bile of one patient and in the liver of two patients with biliary obstruction. The question of a possible relationship between biliary excretion of glycolipid and the pathogenesis of Gauchers disease will require further studies.