Nephrotic syndrome and renal failure associated with a novel glycolipid storage disorder.

A 57-year-old Caucasian male presented with severe nephrotic syndrome and diffuse organomegaly; he subsequently developed renal failure and died. Intracellular, crystalloid material was identified by light and electron microscopy in bone marrow, liver, spleen, mesenteric lymph nodes, and kidneys. Tissue extraction analysis identified the material as glucocerebroside and its immediate precursor, ceramide lactoside. Although Gaucher's disease cannot be completely excluded, glycolipid profiles do not conform to those of known storage disorders. Additionally, electron-microscopic studies indicate that the structural features of the glycolipid deposits are different from those of previously described storage diseases. These findings suggest a unique crystalloid deposition as the probable cause of a multisystem process, which was associated with renal insufficiency and death.

Studies of the pathogenesis of Gaucher's disease: tissue distribution and biliary excretion of [14C]L-glucosylceramide in rats.

The time course of the clearance from the blood and the tissue localization of [14C]L-glucosylceramide, a nonmetabolizable enantiomorph of D-glucosylceramide that accumulates in Gaucher's disease, has been determined. 14C-labeled L-glucosylceramide injected intravenously in the form of micelles or liposomes is rapidly removed from the circulation. Most of this lipid is taken up by the liver where it is found in both hepatocytes and nonparenchymal cells. This sphingolipid analog is promptly cleared from hepatocytes and a significant portion is recovered in the bile. The clearance of [14C]L-glucosylceramide from Kupffer cells is greatly prolonged in comparison with its brief residence in hepatocytes. These findings have significant implications regarding the pathogenesis and treatment of Gaucher's disease.

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.

Animal and cellular models of sphingolipid storage disorders of humans.

The synthesis of L-galactosylceramide is described. Data are presented indicating that this enantiomorph of D-galactocerebroside is not cleaved by galactocerebroside-beta-galactosidase obtained from mammalian tissues. The synthesis of L-glucosylceramide and beta-D-glucothiocerebroside are outlined. These compounds are also refractory to catabolism by glycosidases in mammalian tissues that catalyze the hydrolysis of naturally occurring cerebrosides. L-Hexosyl- and thioanalogs of cerebrosides and perhaps psychosines as well may be helpful for investigating the pathogenesis of Krabbe's disease and Gaucher's disease.

Normolipemic subcutaneous xanthomatosis.

A patient with diabetes mellitus is described in whom an unusual xanthomatosis developed involving large areas of the subcutaneous tissue and vocal cords. Few lesions were present on the skin. Plasma lipid, lipoprotein, apolipoprotein, and cholestanol levels revealed normal patterns. Electron microscopy showed macrophages with vacuolar and crystal lipid inclusions. Results of lipid and enzyme analysis of the subcutaneous xanthoma were similar to those of xanthomas derived from a patient with diabetes mellitus and type V hyperlipidemia. The mechanism of this xanthomatosis remains unknown.

Amygdalin (Laetrile) and prunasin beta-glucosidases: distribution in germ-free rat and in human tumor tissue.

Amygdalin, the gentiobioside derivative of mandelonitrile commonly referred to as Laetrile, is presently under intensive investigation as a potential cancer chemotherapeutic agent. Because of this interest, we investigated the activity of beta-glucosidases that cleave glucose from amygdalin and from prunasin (mandelonitrile monoglucoside) in tissues from germ-free rats and in normal and neoplastic human tissues. Rat and human small intestinal mucosa contain high levels of activity of glucosidases that act on both of these cyanogenic glucosides. Release of glucose from these compounds was not detected in any of the human neoplastic tissues examined in the present study. These observations are consistent with reports of cyanide toxicity through the oral use of amygdalin or prunasin and pose serious questions concerning the alleged tumoricidal effect of amygdalin.

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 Gaucher's disease will require further studies.

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 Gaucher's disease in humans.

Diagnosis of adult Gaucher disease: use of a new chromogenic substrate, 2-hexadecanoylamino-4-nitrophenyl-beta-D-glucopyranoside, in cultured skin fibroblasts.

Gaucher disease is a group of lipid storage diseases in which the glycosphingolipid glucocerebroside accumulates in tissues because of deficiency of the enzyme glucocerebrosidase. Radioactively labelled glucocerebroside and the artificial fluorogenic substrate 4-methylumbelliferyl-beta-D-glucopyranoside are commonly used for its diagnosis. We studied the use of a new chromogenic substrate, 2-hexadecanoylamino-4-nitrophenyl-beta-D-glucopyranoside in cultured skin fibroblasts. The amount of reaction product, 2-hexadecanoylamino-4-nitrophenol, increased linearly with incubation time for at least 4 h and was proportional to the amount of fibroblast protein added up to 150 micrograms per incubation. The pH optimum was 4.8. The Km was 0.19 mmol/l. The mean activity of control cultured skin fibroblasts was 22.9 +/- 5.4 nmol of product formed per mg fibroblast protein per hour under standard conditions. Cultured skin fibroblasts from patients with adult non-neuropathic Gaucher disease had reduced activity, 6.4 +/- 2.4 nmol/mg/h or 28% of control activity. This compared well with mean enzyme activity in the same patients determined using the natural substrate, [14C]glucocerebroside: 28% of control activity. Heterozygotes had reduced activity with the new substrate.

Status of enzyme replacement therapy for Gaucher disease.

At this point in time, enzyme replacement therapy for Gaucher disease appears both biochemically effective (patients 1-3, and 10) as well as clinically promising (patients 4 and 5), although these salutary responses have not been obtained consistently (patients 6-9). The discrepancy may be due to the method employed for the isolation of the enzyme. One current opinion is that glucocerebrosidase prepared by conventional fractionation and ion exchange chromatographic procedures may have been more effective in vivo than enzyme prepared by butanol extraction and hydrophobic column chromatography, which we developed because of the difficulty in obtaining large quantities of glucocerebrosidase by the conventional method. It should be remembered that the enzyme prepared by the latter procedure was fully active on glucocerebroside in liver biopsy specimens in vitro. Furthermore, glucocerebrosidase prepared by the second method exerted a positive effect in 2 young patients, who received relatively large amounts of enzyme, and in an adult who was treated with corticosteroid prior to infusion of the enzyme. At this moment, a possible explanation is that a necessary associated factor, perhaps a lipid, may have been removed by the large-scale isolation procedure. We are attempting to improve the biochemical and clinical responses to enzyme infusion by investigating the effects of lysosomal modifying agents and by enhancing the uptake and localization of the infused enzyme in lysosomes of cells that contain the stored glucocerebroside.

A lysosomal storage disorder in mice characterized by the accumulation of several sphingolipids.

A strain of BALB/c mice with an autosomal recessive neurologic disorder has been reported previously [1, 2]. The tissues of affected animals have been further examined and the activities of varius lysosomal hydrolases and levels of sphingolipids were compared to those in control mice. There was a substantial diminution of sphingomyelinase and glucocerebrosidase activities in liver, spleen, lung, thymus, and kidney of affected mice. There was a corresponding accumulation of sphingomyelin and glucocerebroside in these tissues. The activity of several other lysosomal hydrolases was elevated. Heterozygotes did not show any of the enzymatic alterations. The brain of affected animals showed substantial accumulation of the gangliosides GM3 and GM2.

L-glucosylceramide: synthesis, properties, and resistance to catabolism by glucocerebrosidase in vitro.

Procedures for the synthesis and radioactive labeling of L-glucosylceramide are described. This compound is a stereoisomeric analogue of D-glucosylceramide which occurs in nature and accumulates in pathological quantity in the organs and tissues of patients with Gaucher disease. The properties of L-glucosylceramide that have been examined so far have been found to be indistinguishable from those of the naturally occurring glycolipid. However, L-glucosylceramide is completely refractory to enzymatic hydrolysis by purified placental glucocerebrosidase and enzyme(s) present in whole tissue extracts. It is anticipated that L-glucosylceramide will be a uniquely useful substance for exploring pathogenetic processes in animal analogues of Gaucher disease.

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.

Krabbe's 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 Krabbe's disease is greatly reduced from normal levels. Cultured skin fibroblasts preparations derived from heterozygous carriers of Krabbe's disease have an intermediate level of hydrolytic activity. Thus, the analogue is a reliable chromogenic reagent for the diagnosis of patients with Krabbe's disease and for the detection of heterozygous carriers of the Krabbe trait.

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.