PROGRESS

IN ENDOCRINOLOGY

Heritable

AND METABOLISM

Catabolic and Anabolic of Lipid Metabolism

Disorders

Roscoe 0. Brady The principal manifestations and metabolic defects in ten heritable disorders of lipid metabolism are discussed. Facile procedures hove been developed for the diagnosis of patients with these conditions, the identification of heterozygour carriers, and the prenatal detection of any of these diseases. Enzyme replacement appears

promising for patients with Fabry’r disease and Gaucher’s disease who do not have central nervous system damage. The clinical and biochemical abnormalities that occur in patients with a novel inherited disorder of ganglioside anabolirm are described.

T

HE NATURE of the underlying metabolic defects in the group of heritable lipid storage diseases known as the sphingolipidoses was established just over a decade ago. The principle, elucidated in experiments on Gaucher’s disease, is quite straightforward: namely, a deficiency of specific catabolic, lipid-hydrolyzing enzyme. Although a defect of this type had been anticipated for some time,’ the great difficulty in synthesizing the appropriately labeled lipid for metabolic studies delayed this demonstration for more than 5 yr. In fact, even today, many of these naturally occurring lipids, found predominantly in cell membranes, are still completely refractory to chemical synthesis. Therefore, innovative approaches were required to obtain radioactive compounds required for the identification of enzymatic defects in many of these disorders. This article is designed as an overview of the field. It touches upon the practices that are now employed widely for the diagnosis of these patients, the detection of heterozygous carriers, and the monitoring of pregnancies at risk for such conditions. The results obtained in recent enzyme replacement trials which appear to offer promise for at least two of these disorders are summarized. We conclude with a brief clinical and metabolic summary of a newly documented heritable disorder caused by a deficiency of sphingolipid synthesis and an indication of the drastic consequences that attend a metabolic aberration of this type. GAUCHER’S

DISEASE

Signs and Symptoms Gaucher’s disease, as well as many other lipid storage diseases, bears the name of the clinician who first described the clinical manifestations in patients with the condition. It is an autosomal recessive disorder that has been subdivided into three clinical categories. The first, called type I, comprises patients From the Deveiopmentai and Metaboiic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Md. Receivedfor publication June 23, 1976. Reprint requests should be addressed to Dr. Roscoe 0. Brady, Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, NIH, Bethesda. Md. 20014. @ I977 by Grune & Stratton, Inc. Metabolism, Vol. 26, No. 3 (March),1977

329

330

ROSCOE

0.

BRADY

with the “adult” form of Gaucher’s disease. The symptoms include hepatosplenomegaly, a hemorrhagic diathesis, and extreme pain and pathologic fractures of the long bones, vertebrae, and pelvis due to the osteoporosis attending the infiltration of lipid-storing cells in the marrow. Patients with type II. the infantile form of Gaucher’s disease. have very early onset of the systemic manifestations that occur in patients with type I, along with severe mental retardation due to central nervous system involvement. Patients with type III. the juvenile form, have systemic signs and symptoms of type I: however, these individuals develop seizures and show gradual deterioration of the central nervous system that usually begins in their teens. Metabolic Defect An excessive quantity of the glycolipid known as glucocerebroside accumulates in the organs and tissues in all of the patients with this disorder. The hydrophobic portion of the lipid consists of the long-chain amino alcohol called sphingosine, to which a long-chain fatty acid is linked via an amide bond to the nitrogen atom on carbon 2 of sphingosine. This sphingosineefatty acid complex is called ceramide, and this moiety is common to all of the sphingolipids. In glucocerebroside, a single molecule of glucose is joined by a P-glycosidic bond to carbon atom 1 of the sphingosine portion of ceramide (Fig. 1). The accumulation of glucocerebroside in patients with Gaucher’s disease is caused by an insufficiency of the enzyme that catalyzes the cleavage of glucose from glucocerebroside:’ ceramide-glucose

+ H20

glucocerebrosidase

)

ceramide

+ glucose.

(1)

Patients with type II Gaucher’s disease have virtually no detectable glucocerebrosidase activity in their tissue. 3 Patients with type I always have some residual glucocerebrosidase activity which is usually greater than 22’55 and may be as high as 45% of that in normal individuals. Patients with type III Gaucher’s disease can have up to 20% of normal glucocerebrosidase activity, although it is generally somewhat less than this; however, it is always greater than in the type II patients. Sources of Accumulating Glucocerebroside Most of the glucocerebroside that accumulates in the reticuloendothelial cells of the liver, spleen, and bone marrow appears to be derived from senescent leukocytes and erythrocytes.4 The principal neutral glycolipid in leukocytes is ceramidelactoside (ceramide-glucose-galactose), and its catabolism is impaired due to the deficiency of glucocerebrosidase. Glucocerebroside is also derived

Fig. 1. structure cerebroside.

of

gluco-

CATABOLIC

AND ANABOLIC

331

DISORDERS

from globoside (ceramide-glucose-galactose-galactose-N-acetylgalactosamine), the principle neutral glycolipid in erythrocyte stroma. The central nervous system damage in patients with types II and III Gaucher’s disease is believed to be due at least in part to the turnover of acidic sphingoglycolipids called gangliosides. The major gangliosides in human brain are comprised of ceramide, 4 molecules of hexose, and l-4 molecules of N-acetylneuraminic acid linked to the internal and terminal molecules of galactose [ceramide-glucose(Ngalactose-(N-acetylneuraminic acid),_z -N-acetylgalactosamine-galactose acetylneuraminic acid)o_2]. Ganglioside turnover is very rapid in the neonatal period and thereafter declines to a fraction of this rate. It is assumed that the level of residual glucocerebrosidase activity in the brain of patients with type I Gaucher’s disease is sufficient to catabolize the glucocerebroside derived from ganglioside metabolism, and therefore there is no pathologic accumulation of this lipid in the nerve cells of these individuals. Diagnostic Tests

The diagnosis of homozygotes and the detection of heterozygous carriers of lipid storage diseases is readily available through measurement of lipid hydrolase activity in peripheral blood leukocytes or in extracts of cultured skin fibroblasts. Glucocerebroside labeled with 14C in the glucose portion of the molecule is the preferred substrate for identifying Gaucher’s disease patients5 and carriers.6 However, much emphasis is currently devoted to the development of reliable assays using artificial chromogenic and fluorogenic substrates for the detection of the sphingolipidoses. For example, monosaccharide derivatives of 4-methylumbelliferone do not fluoresce. When the hexose moiety is enzymatitally cleaved, the unsubstituted 4-methylumbelliferone is highly fluorescent (Fig. 2). The /3-glucoside derivative of 4-methylumbelliferone has been shown to be useful for the diagnosis of patients and carriers of Gaucher’s disease using cultured skin fibroblasts as the source of glucocerebrosidase.’ A similar claim has been made using leukocytes as the source of enzyme.* However, we and other investigators have found that the published procedure with leukocytes is not reliable. The test has been modified by separating homogenized leukocyte preparations into pellet and supernatant fractions. Assays of /Iglucosidase activity in the pellet have been found to be much more satisfactory for the diagnosis of Gaucher’s disease.70 The ability to diagnose patients and carriers through assays of enzymatic activity in extracts of cultured cells finds its maximum usefulness in the monitoring of pregnancies at risk for lipid storage diseases. Fetal cell cultures derived from amniotic fluid samples are generally obtained around the 14th gesta-

Fig. 2. Generic derivative of fluorogenic the diagnosis of lipid storage diseases.

oligosoccharides

used for

COLORLESS DERIVATIVE OF 4.METHYLUMBELLIFERONE

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332

0.

BRADY

tional week by transabdominal amniocentesis. When a sufficient quantity of cells have grown, they are harvested and the activity of the enzyme in question is assayed in extracts of these cells. This test has been shown to be reliable for the prenatal diagnosis of Gaucher’s disease’ and for the identification of Gaucher heterozygotes in utero.6 NIEMANN-PICK

DISEASE

Niemann-Pick disease is also transmitted as an autosomal recessive trait. Patients with this disorder have been divided into five categories on the basis of clinical findings. Type A is the severe infantile form with extensive neurologic involvement, emaciation, hepatosplenomegaly, and foam cells in the bone marrow. Type B patients have organomegaly, but are generally without central nervous system difficulties. Type C patients have both organomegaly and neurologic abnormalities, the latter appearing late in childhood or in the teens, in contrast with the early onset of mental retardation in patients with type A. Type D patients have organomegaly and central nervous system damage resembling that in type C, but the ancestry of these individuals is confined to the Nova Scotia area. In type E patients, central nervous system damage occurs in the form of cerebellar ataxia followed by epileptiform convulsions in the teens or early twenties. There is minimal if any hepatosplenomegaly in these patients, but they have lipid storage cells in the bone marrow and frequently have a cherry-red spot in the macular region of the eye. The enzymatic defect in Niemann-Pick disease is a deficiency of sphingomyelinase (Fig. 3, top), resulting in the accumulation of sphingomyelin in variis a ubiquitous component ous tissues throughout the body.” Sphingomyelin of cell membranes and subcellular particles, and therefore its accumulation is probably a consequence of normal cellular turnover. may be diagnosed using radioactive Homozygote2 and heterozygote@ sphingomyelin labeled in the choline portion of the molecule with either sonicated leukocyte preparations or extracts of cultured skin fibroblasts. A chromogenic substrate proposed several years ago6 (Fig. 3, bottom) has been synthesized. This substance has been found to be completely reliable for the diagnosis of Niemann-Pick patients and for the detection of heterozygotes.” More recently this chromogenic analogue of sphingomyelin has been shown to be useful for the prenatal diagnosis of Niemann-Pick disease (Table 1).

Table 1. Prenatal

Diagnosis of Niemann-Pick

Disease with the Chromogenic

Substrate 2-Hexadecanoylamino-4-Nitrophenyl-Phosphocholine Substrate Exp.

Source

NO.

1

Hydrolyzed

of Cultured

Amniotic

Cells

HNP

‘4C-Sphingomyelin

Control

130

55

Control

109

49

Type A Niemann-Pick homozygote 2

(HNP)

7.0

0.0

Control

163

81

Control

180

89

Type A Niemann-Pick heterozygote

55

24

Type A Niemann-Pick heterorygote

67

28

CATABOLIC

AND ANABOLIC

DISORDERS

333

ROSCOE

334

GLOBOID

LEUKODYSTROPHY

(KRABBE’S

0.

BRADY

DISEASE)

This autosomal recessive disorder is characterized by hyperirritability, hyperesthesia, and episodic fever that begins around 445 mo of age. These symptoms are followed by convulsions, mental retardation, hyperactivity, blindness, and deafness. There is no organomegaly or net accumulation of a specific lipid in the brain. The metabolic defect is a deficiency of the p-galactosidase that catalyzes the hydrolysis of galactocerebroside:” ceramide-galactose

+ Hz0

g”“~~~~~~~~~-‘-) ceramide

+ galactose.

(2)

For the past 6 yr, the use of galactocerebroside labeled with 14C or 3H has been required for the diagnosis of patients and the detection of carriers of this disorder.‘3,‘4 We recently synthesized a chromogenic analogue of galactocerebroside that is formally similar to the compound which is so useful for the diagnosis of Niemann-Pick disease. Here, galactose is linked by a @glycosidic bond to 2-hexadecanoylamino-4-nitrophenol instead of phosphocholine used for the diagnosis of Niemann-Pick disease. The chromogenic analogue of galactocerebroside has been found to be completely satisfactory for the diagnosis of Krabbe’s disease patients and the detection of heterozygotes.15 Furthermore, in contrast with the sphingomyelin analogue, where samples of tissue or cultured skin fibroblasts were required as sources of the enzyme, the galactocerebroside analogue can be used with peripheral blood leukocytes as well as for the diagnosis of Krabbe homozygotes and identification of heterozygotes. The availability of these new chromogenic analogues of sphingolipids makes the diagnosis of these patients possible in routine clinical chemistry laboratories, whereas elaborate research facilities were previously required for these procedures. METACHROMATIC

LEUKODYSTROPHY

Patients with the more common clinical form of this autosomal recessive disorder show progressive flaccidity and weakness of the arms and legs that begins at 12-18 mo of age. These signs are followed by loss of ability to stand and the onset of mental deterioration, which is initially manifested by a loss of speech. The disorder progresses to blindness and complete mental retardation. In other patients, many of the same signs and symptoms appear in the early teens and a slower clinical progression is observed. Nerve conduction velocity is decreased and sections of peripheral nerves show brownish-yellow metachromatic deposits when stained with cresyl violet dye. This disease is characterized by the accumulation of sulfatide, the 3-O-sulfate ester of galactocerebroside, because of a deficiency of the enzyme that catalyzes the following reaction:16 ceramide-galactose-sulfate

+ H,O - s”lfori”se ceramide-galactose

+ HzS04. (3)

The diagnosis of homozygotes and identification easily be accomplished with washed leukocytesi using nitrocatecholsulfate as substrate.”

of heterozygous carriers can or cultured skin fibroblasts’”

CATABOLIC

AND ANABOLIC

335

DISORDERS

In another important contribution from Austin’s laboratory, convincing evidence was presented for the presence of a protein in tissues from patients with metachromatic leukodystrophy which, though catalytically inactive, crossreacted with antibody against normal human arylsulfatase A.*’ This was the first documentation of the presence of an inactive enzyme in patients with lipid storage diseases. This demonstration provides strong support for the suggestion that structural mutations in the patient’s enzymes may be the principle pathogenetic mechanism in these disorders.2’ FABRY’S

DISEASE

Fabry’s disease is inherited as an X-linked recessive characteristic whose major clinical manifestations occur in men. Afflicted males have a reddishpurple maculopapular rash in the skin over the buttocks, inguinal regions, and scrotum. These patients experience excruciating peripheral neuralgia in their hands and feet which is worsened by hot weather. They are also unable to sweat. Males with this disorder usually experience renal failure in their late 40’s or early 50’s. Some have myocardial infarctions or cerebrovascular thromboses due to the extensive arteriosclerosis that results from lipid deposited in the blood vessels. Heterozygous females may exhibit some of the manifestations of the disease, including cornea1 opacification, signs of renal impairment, or EKG changes. The symptoms are usually milder in females, although several women have been reported with intense manifestations of the disease. There is a progressive accumulation of the glycolipid known as ceramidetrihexoside in the blood vessels, intestinal mucosa, and glomeruli of the kidneys due to a deficiency of the cY-galactosidase that catalyzes the following reaction:** ceramide-glucose-galactose-galactose

+ H20

ce”~O~~~~~~deW~* +

ceramide-glucose-galactose

+ galactose.

(4)

The principal source of the accumulating lipid is believed to be globoside (see above) and ceramidetrihexoside itself in erythrocyte stroma. Fabry’s disease patients have been identified by measuring ceramidetrihexosidase activity in tissues using the natural lipid labeled throughout the molecule23 or specifically in the terminal galactose moiety.24 The diagnosis of Fabry’s disease is greatly facilitated by determinations of a-galactosidase activity in leukocytes or cultured skin fibroblasts with chromogenic or fluorogenic a-galactosides2’ Heterozygotes may also be detected with these substrates, and a reliable procedure has been developed for the prenatal diagnosis of Fabry’s disease.26 TAY-SACHS

DISEASE

This lipid storage disease is transmitted as an autosomal recessive trait. There are several clinical as well as biochemically distinct forms of Tay-Sachs disease that are now known collectively as the G,,-gangliosidoses. The signs and symptoms of the classic infantile form of the disorder are restricted to the central nervous system. These patients appear quite normal for the first 5-6 mo and then fail to develop motor and mental capacities. Convulsions, apathy, and blindness follow. Death usually occurs in the third year. A cherry-red spot is

336

ROSCOE

0.

BRADY

present in the macular region of the eye. Other patients may have delayed onset of these manifestations until 18-21 mo of age. Here, the progression is slower, with death occurring at 5-6 yr of age. Occasionally a patient is seen with very early onset and rapid progression of the usual signs and symptoms along with some hepatomegaly. This is the Sandhoff-Jatzkewitz form of TaySachs disease. The metabolic defect in all of the different forms of Tay-Sachs disease is a varying degree of a deficiency of the hexosaminidase that catalyzes the catabolism of Tay-Sachs ganglioside (G M2) according to the following reaction:” “) ceramide-glucose-galactose-(N-acetylneuraminic N-acetylgalactosamine

acid)-

(GM*) + Hz0 m

galactose-N-acetylneuraminic

ceramide-glucose-

acid (GM,) + N-acetylgalactosamine.

(5)

This enzyme is normally found in lysosomes in brain and other tissues. Its absence in patients with Tay-Sachs disease results in the accumulation of lipid and protein in the form of membranous cytoplasmic bodies within the nerve cells of afflicted individuals. The diagnosis of most Tay-Sachs homozygotes and heterozygotes can be made through the use of the fluorogenic substrate 4-methylumbelliferyl-P_D-Nacetylglucosaminide.30-33 Patients with the most frequent form of Tay-Sachs disease have only one of two normally occurring hexosaminidase “isozymes,” which is called hexosaminidase A. Hexosaminidase B, the other isozyme, is greatly increased in activity in these patients over that in normal individuals. Patients with the Sandhoff-Jatzkewitz form of Tay-Sachs disease have virtually no hexosaminidase activity in their tissues. while patients with still another mutation have good activity with the fluorogenic substrate but cannot catabolize GMz. Because of these variations, much caution must be exercised concerning heterozygote and homozygote detection with artificial substrates. Furthermore, it has recently been shown that there are perfectly normal individuals without detectable hexosaminidase A activity who, in fact, can or fluorogenic substrate for precatabolize G M234 . If one used a chromogenic natal diagnosis as commonly practiced at this time, such a fetus would erroneously be classified as a Tay-Sachs homozygote. GENERALIZED

G,, GANGLIOSIDOSIS

This inherited disorder is also transmitted as an autosomal recessive trait. GM, gangliosidosis patients exhibit severe mental deterioration and frequently have a cherry-red spot in the retina. These patients also have hepatosplenomegaly, bony abnormalities, and foam cells in the marrow and viscera. At least two clinical forms have been described on the basis of the age of onset and rapidity of progression of the disease. The classic infantile type appears early in infancy and the juvenile form becomes manifest at a somewhat later time. G,, gangliosidosis is the result of the accumulation of ganglioside in various organs and tissues of afflicted individuals due to a deficiency of the P-galactosidase that catalyzes the following reaction:‘5

CATABOLIC

AND ANABOLIC

DISORDERS

337

ceramide-glucose-galactose-(N-acetylneuraminic

acid)-

N-acetylgalactosamine-galactose

8-pdacfosidase

+ Hz0

ceramide-glucose-galactose-(N-acetylneuraminic

acid)-

N-acetygalactosamine Mucopolysaccharides also accumulate in drastic reduction of total ,&galactosidase P-galactosidase activity in tissues of patients diagnosis of homozygotes and heterozygotes of chromogenic or throrogenic P-galactosides. able for carrier detectio’# and the prenatal

+ galactose

(6)

some of these patients due to the activity. The general reduction of with GM, gangliosidosis makes the readily available through the use Established techniques are availdiagnosis of this disorder.37*38

FUCOSIDOSIS

Fucosidosis is an autosomal recessive disorder in which patients have been divided into three clinical categories. The first is characterized by the onset of morbidity by 2-3 yr of age, manifested by coarse fecies, skeletal abnormalities resembling those in mucopolysaccharidoses, hepatosplenomegaly, cardiomegaly, thickening of the skin, respiratory difficulties, and mental retardation that progresses at a variable rate. A second group shows rapid onset of spastic quadriplegia, less dysmorphism than the first group, abnormalities in sweat electrolytes, loss of gall bladder function, and fibrotic degeneration of the pancreas. A third group shows slower progression of cerebral involvement, skeletal abnormalities, and angiokeratoma corporis diffusus. Although no satisfactory unification of the pathogenesis of the various forms of fucosidosis is available, there is a lack of cu-fucosidase activity in the organs and tissues of patients H-isoantigenic lipids and a dekasacin all three groups. 39 Fucose-containing charide accumulate in various tissues in these individuals40 due to the inability to catabolize the following substances: ceramide-glucose-galactose-N-acetylglucosamine-galactosefucose (H-isoantigen)

+ HZ0

s-jucosidpse+ceramide-glucose-

galactose-N-acetylglucosamine-galactose

+ fucose

(7)

(fucose-galactose-N-acetylglucosamine-mannose)~-mannoseN-acetylglucosamine

+ 2 HZ0

or~‘UcOSidaSe +

(galactose-N-acetylglucosamine-mannose),-mannoseN-acetylglucosamine Leukocyte preparations and the prenatal diagnosis

+ 2 fucose.

(8)

have been used for the detection of heterozygotes,41,42 of these patients seems feasible at this time. FARBER’S

DISEASE

The signs and symptoms of this rare disorder begin around 3-4 mo of age with the onset of hoarseness, aphonia, and a brownish desquamating dermatitis. Later there is an infiltration of foam cells in the bones and joints with striking deformations. A granulomatous reaction occurs in the lymph nodes, subcutan-

338

ROSCOE

0.

BRADY

eous tissues, heart, lungs, and kidneys. Central nervous system damage results in psychomotor retardation. The disorder is transmitted as an autosomal recessive characteristic. The metabolic lesion in this disease is a deficiency of ceramidase:43 ceramide

+ H20e

sphingosine

+ fatty acid.

Since ceramidase activity has been detected in cultured anticipated that genetic counseling will become available THERAPY

OF LIPID STORAGE

skin fibroblast? for this disorder.

it is

DISEASES

At the present time, the most direct approach to the treatment of the sphingolipidoses appears to be replacement of the deficient hydrolases with purified enzymes isolated from suitable human sources. Encouraging results have been obtained along this line in patients with Fabry’s disease and the adult form of Gaucher’s disease. Note that the clinical manifestations in these two disorders are confined to peripheral organs and tissues. Tay-Sachs Disease In an earlier investigation, purified hexosaminidase A was injected intravenously into an infant with Tay-Sachs disease. There was no indication that the exogenous enzyme crossed the blood-brain barrier and no clinical improvement was observed.45 However, several important observations were made during this investigation: (1) It was shown that an exogenous enzyme could be administered to humans without deleterious effects. (2) The injected hexosaminidase was rapidly cleared from the circulation with a half-time of about 8 min. (3) A 43% decrease in plasma globoside (see above) was observed 4 hr, after infusion of hexosaminidase A. (4) There was a surprising augmentation of hexosaminidase A activity in the liver of the recipient. Although only 487 x lo3 units of hexosaminidase A were injected, by 45 min after infusion, there was an increase of 2 x IO6 units of hexosaminidase A activity over the low basal level of this enzyme in the liver of the recipient. The significance of this unexpected finding was not fully appreciated until a similar augmentation of enzymatic activity was observed later in enzyme replacement trials in Fabry’s disease. Fabry’s Disease More hopeful findings were obtained in two patients with Fabry’s disease.46 These men received ceramidetrihexosidase that had been purified from human placental tissue. In each instance, there was a significant decrease in the level of circulating ceramidetrihexoside. The amount of lipid cleared from the blood was proportional to the quantity of enzyme injected. Since most of the ceramidetrihexoside that accumulates in the blood vessels and kidneys in these patients appears to be derived from globoside as a consequence of erythrocytorhexis in reticuloendothelial tissues such as the spleen and liver, reduction of the quantity of ceramidetrihexoside in the circulation might be expected to exert a beneficial effect on their vascular and renal problems. Several other important observations were made in the course of these experi-

CATABOLIC

AND ANABOLIC

339

DISORDERS

-

---

Fig. 4. Theoretical activation of mutated catalytically inactive ceramidetrihexoridare in Fabry’s disease patients by a monomer of functional placental enzyme. (From Brady et al.“)

FABRY

PLACENTA

‘“ZO--?

Q

+ -

GALACTOSE CERAMIDE LACTOSIOE

ments: (1) It appears likely that the injected enzyme exerted its catalytic effect after it was taken up by tissues such as the liver.” (2) Skin tests have been carried out on the recipients of placental ceramidetrihexosidase. There was no indication that they developed sensitivity to the placental enzyme. (3) There was an augmentation of liver cu-galactosidase activity similar to that observed in the Tay-Sachs patient who received hexosaminidase A. It was calculated that there was nearly 3.9 times more a-galactosidase activity than had actually been infused in the liver of one of the recipients of ceramidetrihexosidase 1 hr after the enzyme injection. Ceramidetrihexosidase is a tetramer of four polypeptide chains. We have postulated that a monomer of active placental ceramidetrihexosidase combined with subunits of the inactive enzyme in the patient’s liver and conferred catalytic activity to the patient’s mutated protein (Fig. 4). Gaucher’s Disease Purified human placental glucocerebrosidase has been infused into three patients with Gaucher’s disease. These injections caused a decrease in the quantity of accumulated glucocerebroside in the liver of each recipient (Table 2). Furthermore, the elevated glucocerebroside in the circulation, which is associated with erythrocytes, returned to normal by 72 hr after injection of enzyme in two of the three patients.48 This decrease persisted over a long period of time (Fig. 5). We believe the lack of reduction in circulating glucocerebroside in the third patient was due to the extraordinarily high level of glucocerebroside in her liver. In this patient we observed only an 8% decrease in liver glucocerebroside after infusion of glucocerebrosidase. The level of glucocerebroside in the blood appears to be a function of the amount of exchangeable glucocerebroside in tissues such as the liver.49 Several other observations made in the course of these investigation are noteworthy: (1) None of the patients had any fever, discomfort, or other unTable 2. Effect of Intravenous on Glucocerebroride

Injection of Purified

in the liver of Patients

Glucocerebroside

Amount of COZ

NO.

(rg/g

Enzyme Injected

Before

24hr

(nmolefhr)

Infusion

After Infusion

1’

1.5

106

702

2*

3.3 x lo6

1630

3

Glucocerebrosidare

with Gaucher’s Disease

9.3

*Summary

of data from

tf(vmbers

in parentheses

x

x

lo6

Brady

17900 et ~11.~

indicate

per cent change.

519 1210 16500

liver)

Change

183(26)1 420(26) 1400(S)

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340

3

-Lurr*-r .b- *--4wa-_,“.r+-++

*+.c*

0.

BRADY

NORMAL RANGE

2-

INJECTION

TIME

Fig. 5. long-term effect of human placental glucocerebrosidase glucocerebroside in two patients with Gaucher’s disease.

on

the

level

of

circulating

toward reaction to the enzyme injections. (2) The amount of glucocerebroside cleared from the liver of the recipients appeared to be equivalent to the quantity of lipid that had accumulated over a period of 4 yr in the first patient, 13.3 yr in the second, and 1.7 yr in the third.‘O Serum acid phosphatase activity in the third patient was 7.3 units before injection; it had decreased to 5.9 units by 1 mo after administration of the enzyme; 5 mo after infusion, her acid phosphatase level was 6.8 units. (4) There was a constant relationship between the amount of enzyme administered and the quantity of glucocerebroside cleared from the liver (Table 3). By obtaining needle biopsy specimens of the liver, we can determine the quantity of glucocerebroside that has accumulated. We propose to use this information to estimate how much enzyme will be required for the treatment of various patients with Gaucher’s diseases. Summary It is reasonable to conclude from these studies that enzyme replacement in noncerebral lipid storage diseases is feasible at this time and offers considerable hope for obtaining clinical improvement in Fabry’s disease and Gaucher’s disease. Dramatically novel approaches will have to be developed before amelioration of heritable disorders involving the central nervous system can be expected. Some potential avenues for the solution of these problems are discussed extensively elsewhereS’-53 and will not be reiterated here. Table 3. Catalytic Effectiveness

of Glucocerebroridare

Amount of CCW? NO.

x

enzyme)

0.35

3.3

1.2

0.36

9.3

4.0

0.43

3

cerebroside.

(nmole/unit

0.52

2

of glucocerebroside

on on esrlmoted

(nmoles x lo-6)t

lo-*)

1.5

*Nanomoles

Cleared From the Liver

Enzyme Injected (units’

1

tBased

Glucocerebroside

In Vivo

liver

hydrolyzed weight

of

per hour.

2200

g

and

an

average

molecular

weight

of

770

for

gluco-

CATABOLIC

AND ANABOLIC

HERITABLE

341

DISORDERS

DISORDER

OF GANGLIOSIDE

SYNTHESIS

Gangliosides are complex acidic glycolipids that are found in particularly high concentration in the brain. Thus, it is not at all surprising that metabolic disturbances in which their catabolism is impaired are manifested by severe brain dysfunction, as in Tay-Sachs disease and G,, (generalized) gangliosidosis. Investigators have long wondered what the consequence(s) of impaired ganglioside (and other lipid) synthesis would be. For many years, it was believed that such an anabolic defect would be lethal in utero. We have recently discovered a remarkable family in which this concept is convincingly refuted. The dramatic and tragic consequences of failure of ganglioside formation are well and in another male sibling born subillustrated in the index case (Fig. 6)54,55 sequently with identical clinical features. The presenting findings in the propositus, a male infant of European Jewish descent, were respiratory difficulties and seizures which began a few days after birth. By 3 mo of age, the patient was lethargic and exhibited poor physical and motor development. The facies were coarse and were accompanied by macroglossia and gingival hypertrophy. The skin was thickened, hirsute, and coarse. There were bilateral inguinal hernias and hepatosplenomegaly. The patient died at 3% mo of age. The central nervous system showed extensive vacuolization in the cerebral hemispheres, optic nerves, and spinal cord. There were large unmyelinated tracts and some small intracytoplasmic bodies in the astrocytess6 There was a strikingly abnormal pattern of brain gangliosides. Major normal gangliosides in this organ are comprised of ceramide, an oligosaccharide chain consisting of glucose-galactose-N-acetylgalactosamine-galactose, and 1-4 molecules of N-acetylneuraminic acid linked to the internal and terminal. molecules of galactose (see above). The ganglioside composition in the patient’s brain was greatly simplified and consisted of ceramide-glucose-galactose-Nacetylneuraminic acid (G& and a lesser amount of ceramide-glucose-galactoseN-acetylneuraminic acid-N-acetylneuraminic acid (Go)3).54 None of the higher ganglioside homologues were present. The metabolic defect was demonstrated to be a specific diminution of the activity of the aminosugar transferase that catalyzes the conversion of hematoside (G& to Tay-Sachs ganglioside (G,,):57

Fig. 6. Photograph of a patient with defective gonglioside biosynthesis. (From Macloren et 01.~~)

ROSCOE

342

ceramide-glucose-galactose-N-acetylneuramic

BRADY

acid (GM))

+ uridine-diphosphate-N-acetylgalactosamine

~~~?@~ +

ceramide-glucose-galactose-(N-acetylneuraminic N-acetylgalacto-samine

0.

(G,,)

+ uridine

acid)diphosphate

(10)

Since ganglioside formation occurs by the sequential addition of molecules of hexose and N-acetylneuraminic acid to the growing oligosaccharide chain, impairment of any one step precludes the formation of all higher homologues. In this patient, the synthetic defect was specific for the conversion of G,, to G,, since other enzymes involved in ganglioside formation were present in normal or slightly increased activity. Because a maternal uncle with the same outward features had died at 24 mo of age 30 yr previously, and lately a male sibling was born with identical clinical manifestations, we conclude that this condition is a heritable anabolic disorder. Perhaps the simplest explanation for the deficiency of the aminosugar transferase is a mutation in the primary structure of the enzyme rendering it less However, the situation in this anabolic discatalytically active than normal. 2o~58 order may not necessarily follow this pattern. For example, we have seen an identical block in ganglioside synthesis in cultured cells after tumorigenic transformation with the DNA viruses, Simian virus 40 and polyoma virus,59 and RNA virus. In the case of the murine sarcoma virus,6o which is a tumorigenic DNA virus transformants, we have shown that there is no alteration of the kinetic properties in the small residual activity of the deficient enzyme,6’ and we have concluded that the insertion of the tumorigenic virus genome into the genetic apparatus of the host cell results in diminished production of the aminosugar transferase. It might be speculated that such a tumorigenic virus might be carried on one of the X chromosomes in the females in this family. The absence of tumors in the propositus does not contradict this hypothesis since Simian virus 40 may not be tumorigenic in humans.6’ In sum, the multiplicity of deleterious effects of a block in ganglioside synthesis is dramatically demonstrated in this family. It was earlier predicted that hypomyelination might well be a consequence of an abnormality of ganglioside metabolism63; however, the widespread parenchymal tissue involvement was not anticipated. Emerging studies on the involvement of gangliosides in cell surface receptors for glycoprotein hormones such as thyrotropin,” chorionic and probably luteinizing hormone64 provide the first glimpses gonadotropin,@ into some of the biologic activities of these lipids. It may well be that one of the main functions of gangliosides is to mediate extrinsic metabolic signals impinging on cell membranes.66 CONCLUDING

REMARKS

Fundamental studies on the metabolism of sphingolipids have provided great benefit for the control of ten hereditary metabolic disorders. Current research emphasizes the solution of logistical problems for the delivery of exogenous enzymes as specific therapeutic agents for the treatment of patients with lipid storage diseases.These investigations have had important ramifications in

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DISORDERS

the understanding of abnormalities of lipid metabolism in developmental disorders, abnormalities of myelination, tumorigenic transformation of cells, and the site and mechanism of action of trophic hormones. It is difficult to predict how many other metabolic events will be shown to involve sphingolipids. There is already good evidence indicating that reactions of bacterial glycoprotein toxins67,68 and other substances such as interferon69 involve reactions with these lipids. The lessons we learned in the investigations into the sphingolipidoses have provided a solid point of departure for many hitherto unsuspected metabolic interrelationships. REFERENCES 1. Trams EC, Brady RO: Cerebroside synthesis in Gaucher’s disease. J Clin Invest 39 1546, 1960 2. Brady RO, Kanfer JN, Shapiro D: Metabolism of glucocerebrosides. II. Evidence of an enzymatic deficiency in Gaucher’s disease. Biochem Biophys Res Commun 18:22l, 1965 3. Brady RO, Kanfer JN, Bradley RM, et al: Demonstration of a deficiency of glucocerebroside-cleaving enzyme in Gaucher’s disease. J Clin Invest 45: 1112,1966 4. Kattlove HE, Williams JC, Gaynor E, et al: Gaucher cells in chronic myelocytic leukemia: An acquired abnormality. Blood 33~379, 1969 5. Kampine JP, Brady RO, Kanfer JN, et al: Diagnosis of Gaucher’s disease and NiemannPick disease with small samples of venous blood. Science 155:86, 1967 6. Brady RO, Johnson WC, Uhlendorf BW: Identification of heterozygous carriers of lipid storage diseases. Am J Med 51:423, 1971 7. Ho MW, Seek J, Schmidt D, et al: Adult Gaucher’s disease: Kindred studies and demonstration of a deficiency of acid @-glucosidase in cultured fibroblasts. Am J Hum Genet 24:37, 1972 8. Beutler E, Kuhl W: Diagnosis of the adult type of Gaucher’s disease and its carrier state by demonstration of a deficiency of &glucosidase activity in peripheral blood leukocytes. J Lab Clin Med 761747, 1970 9. Schneider EL, Ellis WC, Brady RO, et al: Infantile (type II) Gaucher’s disease: In utero diagnosis and fetal pathology. J Pediatr 81: 1134, 1972 IO. Brady RO, Kanfer JN, Mock MB, et al: The metabolism of sphingomyelin. II. Evidence of an enzymatic deficiency in Niemann-Pick disease. Proc Natl Acad Sci USA 55:366, 1966 I I. Gal AE, Brady RO, Hibbert SR, et al: A practical chromogenic procedure for the detection of homozygotes and heterozygous car-

riers of Niemann-Pick Disease. N Engl J Med 293:632, 1975 12. Suzuki K, Suzuki Y: Globoid cell leukodystrophy (Krabbe’s disease): Deficiency of Proc galactocerebroside beta-galactosidase. Nat1 Acad Sci USA 66:302, 1970 13. Suzuki Y, Suzuki K: Krabbe’s globoid leukodystrophy: Deficiency of galactocerebrosidase in serum, leukocytes, and fibroblasts. Science 171:73, 1971 14. Suzuki K, Schneider EL, Epstein CJ: In utero diagnosis of globoid cell leukodystrophy (Krabbe’s disease). Biochem Biophys Res Commun 45: 1363, 1972 15. Gal AE, Brady RO, Pentchev PG. et al: Synthesis and use of a chromogenic substrate for the detection of Krabbe’s disease. Fed Proc 35: 1726, 1976 16. Mehl E, Jatzkewitz H: Evidence for a genetic block in metachromatic leukodystrophy (ML). Biochem Biophys Res Commun 19~307, 1965 17. Percy AK, Brady RO: Metachromatic leukodystrophy: Diagnosis with samples of venous blood. Science 161:594, 1968 18. Kaback MM, Howell RR: Infantile metachromatic leukodystrophy: Heterozygote detection in skin fibroblasts and possible applications to intrauterine diagnosis. N Engl J Med 282: 1336, 1970 19. Austin J, Balasubramanian AS, Patabiraman TN. et al: A controlled study of enzymatic activities in three human disorders of glycolipid metabolism. J Neurochem 10: 805, 1963 20. Stumpf D, Neuwelt E, Austin J, et al: Metachromatic leukodystrophy (MLD). X. Immunological studies of the abnormal sulfatase A. Arch Neurol 25:427, 1971 2 1. Brady RO: The sphingolipidoses. N Engl J Med 275:312. 1966 22. Brady RO, Gal AE, Bradley RM, et al: Enzymatic defect in Fabry’s disease: Cera-

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midetrihexosidase deticiency. N Engl J Med 216: I 163, 1967 23. Brady RO, Gal AE, Bradley RM, et al: The metabolism of ceramidetrihexosides. 1. Purification and properties of an enzyme that cleaves the terminal galactose molecule of galactosylgalactosylglucosylceramide. J Biol Chem 242: 102 1, 1967 24. Radin NS. Hof L, Bradley RM. et al: Lactosylceramidase: Comparison with other sphingolipid hydrolases in developing rat brain. Brain Res 14:497. 1969 25. Kint JA: Fabry’s disease: Alpha-galactosidase deficiency. Science 167: 1268, 1970 26. Brady RO, Uhlendorf BW. Jacobson CB: Fabry’s disease: Antenatal detection. Science 172:174. 1971 27. Kolodny EH, Brady RO, Volk BW: Demonstration of an alteration of ganglioside metabolism in Tay-Sachs disease. Biochem Biophys Res Commun 37:526. 1969 28. Brady RO, Tallman JF, Johnson WC, et al: An investigation of the metabolism of TaySachs ganglioside specifically labeled in critical portions of the molecule. Adv Exp Med Biol 191277. 1972 29. Tallman JF, Johnson WC, Brady RO: The metabolism of Tay-Sachs ganglioside: Catabolic studies with lysosomal enzymes from normal and Tay-Sachs brain tissue. J Clin Invest 5 I :2339, 1972 30. Okada S, O’Brien JS: Tay-Sachs disease: Generalized absence of a beta-hexosaminidase component. Science 165:698, 1969 3 I O’Brien JS, Okada S, Chen A. et al: TaySachs disease: Detection of heterozygotes and homozygotes by serum hexosaminidase assay. N Engl J Med 283:15. 1970 32. Schneck L, Valenti C. Amsterdam D. et al: Prenatal diagnosis of Tay-Sachs disease. Lancet 1:582. 1970 33. O’Brien JS. Okada S, Fillerup DL. et al: Tay-Sachs disease: Prenatal diagnosis. Science 172:61. 1971 34. Tallman JF, Brady RO. Navon R, et al: Ganglioside catabolism in hexosaminidase A deficient adults. Nature 252:254, 1974 35. Okada S, O’Brien JS: Generalized gangliosidosis: Beta-galactosidase deficiency. Science 160:1002, 1968 36. Wolfe LS. Callahan J. Fawcett JS. et al: GM)-gangliosidosis without chondrodystrophy or visceromegaly. Neurology 20:23, 1970 37. Kaback MM, Sloan HR. Sonneborn M. et al: GM, gangliosidosis type I: In utero detection and fetal manifestations. J Pediatr 82: 1037. 1973

ROSCOE

0.

BRADY

38. Lowden JA. Cutz E. Conen PE, et al: Prenatal diagnosis of GM,-gangliosidosis. N Engl J Med 288:225. 1973 39. Van Hoof F. Hers HG: Mucopolysaccharidosis by absence of cu-fucosidase. Lancet l:I 198, 1968 40. Dawson G. Tsay GC: Fucosidosts, tn Volk BW. Schneck L (eds): Current Trends in Sphingolipidoses and Allied Disorders. New York. Plenum, 1976. p 187 41. Pate1 V. Zeman W: Variability of expressivity of a-fucosidase activity, in Volk BW. Schneck L (eds): Current Trends in Sphingolipidoses and Allied Disorders. New York. Plenum, 1976. p 167 42. Hirschhorn K. Beratis NC. Turner BM: Alpha-L-fucosidase in normal and deficient individuals. tn Volk BW, Schneck L (eds): Current Trends in Sphingolipidoses and Allied Disorders. New York. Plenum. 1976. p 205 43. Sugita M. Dulaney JT, Moser HW: Ceramidase deliciency in Farber’s disease (lipogranulomatosis). Science 178:1100, 1972 44. Dulaney J. Moser HW. Sidbury J. et al: The biochemical defect in Farber’s disease, in Volk BW. Schenck L (eds): Current Trends in Sphingolipidoses and Allied Disorders. New York, Plenum. 1976. p 403 45. Johnson WG. Desnick RJ. Long DM, et al: Intravenous injection of puritied hexosaminidase A into a patient with Tay-Sachs disease. in Desnick RJ, Bernlohr RW. Krivtt W (eds): Enzyme Therapy in Genetic Dtseases. New York. The National Foundation. 1973, p 120 46. Brady RO. Tallman JF. Johnson WC;. et al: Replacement therapy for inherited enzyme deticiency: Use of puritied ceramidetrihexosidase in Fabry‘s disease. N Engl J Med 7899. 1973 47. Brady RO. Pentchev PC, Gal AE: Investigations in enzyme replacement therapy in lipid storage diseases. Fed Proc 34:13 IO. 1975 48. Brady RO. Pentchev PG, Gal AE. et al: Replacement therapy for inherited enzyme deticiency: Use of puritied glucocerebrosidase in Gaucher’s disease. N Engl J Med 291:989. I974 49. Dawson G. Sweeley CC: In viva studtes on glycosphingolipid metabolism in porcine blood. J Biol Chem 245:410. 1970 50. Pentchev PG, Brady RO. Gal At, et al: Replacement therapy for inherited enzyme deficiency. Sustained clearance of accumulated glucocerebroside in Gaucher’s disease following infusion of purified glucocerebrosidase. J Mol Med 1:73. IY75

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AND ANABOLIC

DISORDERS

51. Brady RO: Hereditary diseases: Causes, cures and problems. Angew Chem (Engl) 12:1, 1973 52. Brady RO: The abnormal biochemistry of inherited disorders of lipid metabolism. Fed Proc 32: 1660. 1973 53. Brady RO: Inborn errors of lipid metabolism. Adv Enzymol 38:293, 1973 54. Max SR, Maclaren NK, Brady RO, et al: GM3 (hematoside) sphingolipodystrophy. N Engl Med 291:929, 1974 55. Maclaren NK, Max SR, Cornblath, et al: &a gangliosidosis: A novel human sphingolipodystrophy. Pediatrics 57: 106, 1976 56. Tanaka J. Garcia JH. Max SR, et al: Cerebral sponginess and GM~ gangliosidosis: Ultrastructure and probable pathogenesis. J Neuropathol Exp Neurol34:249, 1975 57. Fishman PH, Max SR, Tallman JF, et al: Deficient ganglioside biosynthesis: A novel human sphingolipidosis. Science 187:68. 1975 58. Meisler M, Rattazzi MC: Immunologicai studies of fl-galactosidase in normal human liver and in GM, gangliosidosis. Am J Hum Genet 26:683, 1974 59. Cumar FA, Brady RO, Kolodny EH, et al: Enzymatic block in the synthesis of gangliosides in DNA virus-transformed tumorigenic mouse cell lines. Proc Natl Acad Sci USA 67: 757, 1970 60. Mora PT. Fishman PH, Bassin RH, et al: Transformation of Swiss 3T3 cells by murine sarcoma virus is followed by decrease in a

345

glycolipid glycosyl-transferase. Nature (New Biol) 245:226, 1973 61. Fishman PH, Brady RO: Modification of membrane glycolipids by oncogenic agents, in Perkins EG, Witting LA (eds): Modification of Lipid Metabolism. New York, Academic, 1975, p 105 62. Fraumeni JR Jr. Stark CR. Gold E. et al: Simian virus 40 in polio vaccine: Follow up of newborn recipients, Science 167:59, 1970 63. Brady RO, Quarles RH: The enzymology of myelination. Mol Cell Biochem 2:23, 1973 64. Mullin BR, Fishman PH, Lee G. et al: Thyrotropin-ganglioside interactions and their relationship to the structure and function of thyrotropin receptors. Proc Natl Acad Sci USA 73~842, 1976 65. Lee G. Kohn LD, Fishman PH: unpublished observations 66. Fishman PH. Brady RO: The biosynthesis and function of gangliosides. Science (in press) 67. van Heyningen WE, Carpenter CCJ, Pierce NF, et al: Deactivation of cholera toxin by ganglioside. J Infect Dis 124:415, 1971 68. Moss J, Fishman PH. Manganiello VC, et al: Functional incorporation of gangliosides into intact cells: Induction of choleragen responsiveness. Proc Natl Acad Sci USA 73: 1034, 1976 69. Besancon F. Ankel H: Binding of interferon to gangliosides. Nature 252:478, 1974 70. Weinreb NJ, Pentchev PG. Brady RO: unpublished observations