ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 7, No. 2 Copyright (£) 1977, Institute for Clinical Science

Enzymological Approaches to the Lipidoses

ROSCOE O. BRADY, M.D.

Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, MD 20014

ABSTRACT

There are now ten known heritable disorders of metabolism for which the nature of the underlying enzymological defect is conclusively established. In addition to devising procedures for successful enzyme re­ placement therapy, much current work deals with the development of con­ venient, effective methods for the rapid diagnosis of patients with these disorders, the detection of heterozygous carriers of these diseases, and the monitoring of pregnancies at risk for these conditions. Clinical en- zymologists are required to assume increasing responsibilities in the per­ formance of these tests, and the present contribution describes the applica­ tion of fundamental principles and discusses recent developments along this line. In particular, the development of facile chromogenic reagents for the diagnosis of patients with Niemann-Pick disease and Krabbe’s disease is delineated. Previously, the diagnosis of such patients required the use of radioactivity labeled compounds and such testing was limited to relatively few research laboratories. The novelty of these reagents and their applica­ tion comprise the major aspect of this presentation.

Introduction der, and eventually for all of the nine known other lipid storage diseases, that The role and contributions of clinical these conditions are due to an insuffi­ enzymologists are becoming increasingly ciency of hydrolytic enzymes required important for the management of heredit­ for the catabolism of the accumulating ary metabolic diseases. The translation of substances. basic research discoveries to practical benefits requires further extensive en­ This discovery permitted the rapid de­ gagement of clinical scientists. Nowhere velopment of sensitive, facile procedures has this concept been more evident than for the diagnosis of patients with these in the control of the lipid storage dis­ conditions using readily available mate­ eases. Just over a decade ago, the pivotal rials such as washed peripheral blood advance in this area was provided leukocytes27 (figure 1), cultured skin fib­ through the elucidation of the enzymatic roblasts25 (figure 2) and, in some disor­ defect in Gaucher’s disease.12, Is It was ders, serum samples.32 Initially, most of specifically demonstrated for this disor­ these tests required the use of radioactiv- 105 1 06 BRADY

Fig u re 1. Determina­ 't> ~ (D • - (pi tion of enzyme activity in O ) Separate red and white blood cells. A com­ ^ * white blood cells. monly used procedure for © the diagnosis of metabolic disorders.

-white cells 3 ^ Assay enzyme activity in white -red cells © blood cells.

ity labeled that accumulated in the of this article. Only a minimum of clinical various disorders. However, it soon be­ manifestations of these disorders will be came apparent that chromogenic or cited since they are described exten­ fluorogenic artificial substrates could be sively in previous contributions.5,6,7,9 used in a number of these conditions. Some very recent developments will Gaucher’s Disease be cited in this communication that now permit the detection of all except one of This disorder and, in fact, all of the the known lipid storage diseases through lipid storage diseases are characterized the use of artificial substrates. These rea­ by the accumulation of derivatives of the gents have distinct advantages for day to long chain amino alcohol called sphin- day application in clinical chemistry gosine [CH3-(CH2)12-CH = CH- laboratories where the lack of radioactive CH(OH)-CH(NH2)-CH2OH] to which a counting equipment precludes the utili­ long chain fatty acid is linked via an zation of labeled compounds. Therefore, amide bond to the nitrogen atom on car­ the use of chromogenic and fluorogenic bon 2. This combination of substances for the diagnosis of lipid stor­ and fatty acid is called , and it is age diseases, the detection of heterozyg­ shared by all of the accumulating lipids. ous carriers of these disorders and the Various hexoses, oligosaccharides or monitoring of pregnancies at risk for phosphorylcholine are linked to carbon 1 these conditions will be the main theme of the sphingosine moiety of ceramide.

^ 1 Obtain small skin biopsy. Grow skin cells in tissue culture carriers © (3-4 weeks). deficient normal Et Fig u re 2. Determina­ tion of enzyme activity in cultured skin cells. An alternative procedure for the diagnosis of metabolic disorders. © Harvest. Assay enzyme activity in the cultured skin © cells. ENZYMOLOGICAL APPROACHES TO THE LIPIDOSES 1 0 7

Thus, the lipid that accumulates in Gaucher s disease is called glucocereb- roside, and it consists of a single mole­ cule of joined by a /3-glycosidic bond to ceramide. Glucocerebroside labeled in the glucose portion of the mol­ ecule with radiocarbon-14 was synthe­ sized and this labeled compound was used to demonstrate the metabolic lesion F i g u r e 3. Structure of 4-methylumbelliferyl-/3- in Gaucher’s disease. Here there is a de­ D-glucopyranoside used for the diagnosis of ficiency of the enzyme that catalyzes the Gaucher’s disease. hydrolytic cleavage of the glucose por­ tion of glucocerebroside according to Gaucher’s disease.23 However, assays Reaction 1. with this substrate are somewhat less sensitive than those with 14 C- 1. Glucocerebroside + H20 glucocerebroside or 4-methylumbelli- glucocerebroside-/3- feryl-/3-D-glucopyranoside and may not g lucosidase be practical when leukocytes are used as the source of tissue to be analyzed. ceramide + glucose Niemann-Pick Disease Radioactive glucocerebroside has been used extensively for the diagnosis of pa­ The metabolic defect in Niemann-Pick tients with Gaucher’s disease,27 the de­ disease was identified a decade ago as a tection of carriers11 and the prenatal deficiency of sphingomyelinase13 (Reac­ diagnosis of fetuses with this condition.42 tion 2). The fluorescent substrate, 4-methyl- umbelliferyl-/3-D-glucopyranoside (fig­ 2. + H20 ure 3), has been used for a number of sphingomyelinase ------» years to diagnose patients with Gaucher’s disease and the detection of carriers with ceramide + phosphorylcholine extracts of cultured skin fibroblasts under rigidly specified conditions.25 An early at­ For nine of these years, the use of tempt to use this fluorogenic compound labeled sphingomyelin was required for for patient and carrier detection with leukocyte preparations4 has not provided the reliability required for general use. It has recently been found that this sub­ strate may be used with confidence if leukocytes are homogenized and sub­ jected to centrifugation. /3-Glucosidase activity in the pellet obtained by this pro­ c = o cedure is then determined with the fluorogenic glucoside. This procedure {CH2)i4 has been found to be reliable for diagnos­ I ing patients with Gaucher’s disease.51 c h 3 Recently, a chromogenic analogue of F i g u r e 4. 2-Hexadecanoylamino-4-nitrophenyl- /3-D-glucopyranoside, a chromogenic analogue of glucocerebroside (figure 4) has been glucocerebroside for diagnosing patients with used successfully for the diagnosis of Gaucher’s disease. 10 8 BRADY

O diagnosis of patients and the detection of II carriers of this disorder. However, a o 2 n - O —P —o —c h 2—c h 2 - N(CH3 )3o h chromogenic analogue has been synth­ \\ // OH esized of galactocerebroside similar to N-H that used for the diagnosis of Gaucher’s c=oI disease and Niemann-Pick disease ex­ I cept that galactose is linked by a (CH,)2 '1 4 /3-glycosidic bond to 2-N-hexadeca-

CH, noylamino-4-nitrophenol instead of glucose or phosphorylcholine. This F i g u r e 5. 2-Hexadecanoylamino-4-nitrophenyl- galactoside derivative has been found phosphorylcholine, a chromogenic analogue of sphingomyelin used for the diagnosis of patients to be useful for the diagnosis of pa­ with Niemann-Pick disease. tients with Krabbe’s disease using sam­ ples of brain or liver tissue obtained from the diagnosis of patients and the detec­ patients with this metabolic disorder.21 tion of carriers of Niemann-Pick disease. Assay conditions have now been estab­ In 1975, a major diagnostic breakthrough lished which permit the use of the galac­ occurred through the synthesis of a useful tocerebroside analogue for the diagnosis chromogenic analogue of sphingomyelin of patients and carriers of Krabbe’s dis­ that has proven to be completely reli­ ease using extracts of cultured skin fib­ able for the enzymatic detection of roblasts.22 Niemann-Pick disease and the carriers of this trait20,23 and the monitoring of pre­ Metachromatic Leukodystrophy gnancies at risk for this disorder9 (figure Patients with this hereditary disorder 5). This compound may be used for the lack arylsulfatase A activity in their or­ diagnosis of Niemann-Pick disease using gans and tissues. This enzyme catalyzes tissue specimens or extracts of cultured the cleavage of sulfuric acid from sul- skin fibroblasts as source of enzyme. At fatide, the 3-0'-sulfate ester of galac­ the moment, radioactive sphingomyelin tocerebroside31 (Reaction 4). is preferred when leukocytes are used since it provides a slightly more sensitive 4. + H20 test system than the chromogenic sulfatidase analogue. (arylsulfatase A) Krabbe’s Disease galactocerebroside + H2S04 Here there is a deficiency of the en­ zyme that catalyzes the cleavage of the Even before this defect had been dem­ molecule of galactose from galactocereb­ onstrated with the natural lipid substrate, roside44 (Reaction 3). Austin and co-workers had clearly shown that there was a decrease in sulfatase ac­ 3. Galactocerebroside + H20 tivity in tissues of patients with this dis­ galactocerebroside-(}- order through the use of nitrocatechol galactosidase sulfate as substrate.1 These observations have stood the test of time, and this ceramide + galactose chromogenic substrate is still widely used for the diagnosis of patients with Until recently, the use of radioactive metachromatic leukodystrophy3" and the galactocerebroside was mandatory for the detection of heterozygotes.3,49 ENZYMOLOGICAL APPROACHES TO THE LIPIDOSES 1 0 9

Fabry’s Disease Patients with this disorder may be In contrast with all of the other known readily diagnosed by measuring hexo­ saminidase activity in tissues and blood lipid storage diseases which are au­ with the fluorogenic substrate 4-meth- tosomal recessive disorders, Fabry’s dis­ ylumbelliferyl-/3-D-N-acetylglucosam- ease is transmitted as an X-chromosome inide.32,35 There are two major hexo­ linked recessive trait. In this disorder, saminidase isozyes in mammalian cells39 ceramidetrihexoside accumulates be­ and only one of them, called Hex-A, is cause of an insufficiency of the galac- lacking in most patients with Tay-Sachs tosidase that catalyzes the cleavage of the terminal molecule of galactose from disease. The activity of the other hexo­ saminidase isozyme, called Hex-B, is ceramidetrihexoside10 (Reaction 5). greatly increased. Therefore, it is neces­ sary to differentiate between these ac­ 5. Ceramide-glucose-galactose- tivities in order to carry out assays for galactose + H20 homozygotes and heterozygous carriers ceramidetrihexoside-a------—»• of Tay-Sachs disease. g alactosidase ceramide-glucose-galactose + galactose The most widely used procedure for this differentiation is the selective heat 3H-labeled ceramidetrihexoside has inactivation of Hex-A.8’32*33 Total tissue been used to detect patients and carriers hexosaminidase activity is measured in of this disorder. However, most current an aliquot of the sample under investiga­ diagnostic work is based on the demon­ tion. Another aliquot is heated for four stration by Kint that patients with Fabry’s hours at 50°. Hex A activity is destroyed disease have decreased a-galactosidase by this procedure and the residual activity in their tissues.28 Either 4-methyl- hexosaminidase activity represents the umbelliferyl-a-D-galactopyranoside or Hex B component. The amount of Hex A activity is determined by subtracting Hex p-nitrophenyl-a-D-galactopyranoside may be used for this assay. Heterozygotes and B activity from total hexosaminidase ac­ afflicted fetuses may readily be detected tivity. When performed carefully, this with these substrates.17 test permits reliable identification of pa­ tients with Tay-Sachs disease and the de­ tection of carriers. Other techniques for Tay-Sachs Disease determining Hex A and Hex B activity This inherited metabolic disorder is include separation of the isozymes by gel caused by a deficiency of the enzyme that electrophoresis,39 inactivation of Hex A catalyzes the cleavage of the molecule of under acidic conditions37 and separation N-acetylgalactosamine from the acidic of isozymes by small ion-exchange col­ called Tay-Sachs ganglio- umns.19 side29,47 (Reaction 6). In contrast with most Tay-Sachs pa­ 6. Ceramide-glucose-galactose- tients, some individuals, currently re­ (N-acetylneuraminic acid)-N- ferred to as patients with Sandhoff s dis­ acetylgalactosamine + H20 ease, have very little total hexosamin­ hexosaminidase idase activity in their tissues.40 The diag­ ------—» nosis of these patients and the detection ceramide-glucose-galactose- of carriers is also feasible using 4-methyl- N-acetylneuraminic acid + umbelliferyl-/3-D-N-acetylglucosaminide N-acetylgalactosamine as substrate.5 110 BRADY

Clinical chemists should be aware of umbelliferyl-/3-D-galactopyranoside or two other aspects of Tay-Sachs disease p-nitrophenyl-j8-D-galactopyranoside.16 which might prove troublesome. A few The prenatal diagnosis of fetuses with patients with classic manifestations of this condition has also been demon­ Tay-Sachs disease have been described strated through the use of these sub­ where Hex A and B activities are per­ strates.26,30 fectly normal when measured with arti­ ficial substrates such as 4-methylum- Fucosidosis belliferyl-/3-D-N-acetylglucosaminide or p-nitrophenyl - ¡5- D - N - acetylgalactos- Patients with this disorder have a aminide.41 In contrast, the catabolism of coarseness of the skin, bony changes, or­ the itself is severely com­ ganomegaly and mental retardation. promised. Fucose-containing H-isoantigenic lipids Furthermore, it has recently been dis­ and a dekasaccharide accumulate in vari­ covered that there are perfectly normal ous tissues of these patients18 because of individuals without detectable Hex A ac­ a lack of a-fucosidase activity.50 This en­ tivity in their tissues who, in fact, can zymatic defect is readily detected using catabolize Tay-Sachs ganglioside.46 If one 4-methylumbelliferyl-a-L-fucopyranoside were to use a chromogenic or fluorogenic as substrate. substrate for prenatal detection as com­ Farber’s Disease monly practiced at the present time,33 such a fetus would be classified as a Patients with this rare inherited disor­ Tay-Sachs homozygote. Here again, one der of disseminated lipogranulomatosis must rely on the use of Tay-Sachs have hoarseness of the voice, a brownish ganglioside specifically labeled in the dermatitis and mental retardation that N-acetylgalactosaminyl portion of the begins between the third and fourth molecule for correct diagnosis. Proce­ month. The metabolic lesion in this con­ dures for the preparation of this substrate dition is a deficiency of the enzyme that have been published.2'45,48 catalyzes the cleavage of ceramide into its component parts, sphingosine and a long chain fatty acid.43 So far, radioac- Generalized tively labeled ceramide has been re­ This disorder is caused by a lack of the quired to measure this reaction, although enzyme that catalyzes the cleavage of the it is conceivable that a useful chromo­ terminal molecule of galactose from genic substrate can be developed along ganglioside GM134 (Reaction 7). the lines which are so helpful for the diagnosis of Niemann-Pick and Krabbe’s 7. Ceramide-glucose-galactose- disease. (N-acetylneuraminic acid)-N-acetyl- galactosamine-galactose (GM1) + HzO Conclusion /3-galactosidase It is apparent that clinical chemists will eeramide-glucose-galactose- be required to become increasingly in­ (N-acetylneuraminic acid)-N- volved in the use of enzyme assays for the acetylgalactosamine + galactose diagnosis of heritable metabolic disor­ ders, the detection of symptom-free Patients and carriers may be readily heterozygous carriers of these deleteri­ identified by measuring tissue /3-ga­ ous traits and the monitoring of pregnan­ lactosidase activity with 4-methyl- cies at risk for these conditions. There is a ENZYMOLOGICAL APPROACHES TO THE LIPIDOSES 111

clear movement towards the rapid appli­ 10. B r a d y , R. O ., G a l , A. E., B r a d l e y , R. M., cation of these tests to medical practice. M a r t e n s s o n , E ., W a r s h a w , A. L ., and L a s - t e r , L.: Enzymatic defect in Fabry’s disease: Since weighty decisions are based on the ceramidetrihexosidase deficiency. New Eng. J. results of these determinations, it is im­ M ed. 276:1163-1167, 1967. perative that the clinical scientist 11. B r a d y , R. O., J o h n s o n , W. G., and U h l e n - familiarize himself with the concepts and DORF, B. W.: Identification of heterozygous carriers of lipid storage diseases. Amer. J. Med. techniques used in these analyses. At the 51:423-431, 1971. present time, considerations involving 12. B r a d y , R. O., Ka n f e r , J. N., B r a d l e y , R. 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Lan­ 37. PERLE, G. and Sa i f e r , A.: Serum hex A cet 2:1198, 1968. analysis with the pH-inactivation method for 51. W e i n r e d , N. J., Pe n t c h e v , P. G., and B r a d y , the detection of TSD heterozygotes and R. O.: (Submitted for publication).