Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with Canavan disease

American Journal of Medical Genetics 29463471 (1988)

ASPARTOACYLASE DEFICIENCY AND N-ACETYLASPARTIC ACIDURIA IN PATIENTS WITH CA” DISEASE R. Matalon, K. Michals, D. Sebesta, M. Deanching P. Gashkoff, and J. Casanova

The De-ts of Pediatrics, and Nutrition and Medical Dietetics, University of Illinois at Chicago; Chicago, Illinois

An increased amount of N-acetylaspartic acid was found in urine and plasna of Wee patients, frcm ism families, with the diagnosis of cerebral spongy degeneration (Caravan disease). Asgarbacylase was assayed in cultured skin fibroblasts frcm one patient of each family and a profound deficiency of this enzyme was found. Although the function of N-acetylaspartic acid is not undersm, it is knmn to occur in high cancentration in human brain. The finding of a defect in the metablisn of N-acetylaspartic acid causing progressive spongy degeneration of the brain may lead to a better understanding of the function of t h i s amino acid derivative. The asparbacylase assay affords a new tool for determining the diagnosis of Canavan disease. Since aspartoacylase activity w a s present in cultured amniotic cells and chorionic villi, it is likely that the assay for t h i s enzyme can be used for the prenatal diagnosis of Canavan disease.

Keymrds: aspartoacylase, N-acetylaspartic aciduria, Canavan disease, ~ n g degeneration y of brain, autosanal recessive inheritance Address reprint requests to Reuben Matalon, University of Illinois at Chicago, Deparhnent of Pediatrics m/c 856, 840 S. Wood Street, Chicago, Illinois 60612. 0 1988 Alan R. Liss, Inc.

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N-Acetylaspartic acid, discovered by Tallan e t al. (1956), is found i n high Concentration i n the brain of most vertebrates. The concentration in the human brain is very high, 6-7 pmle/gm tissue, w i t h the highest concentration in the cerebral cortex and the lawest i n the medulla (Miyake e t a l , 1982). The concentration of N-acetylaspartic acid is second only to glutamic acid i n the free amino acid pool of the brain and is higher in concentratian than gaMna mimbutyric acid (GABA). The level of N-acetylaspartic acid has also been reported to be increased i n the developing brain (Miyake e t a1,1982; Jacobson,1957). In spite of its abundance i n the brain the function of N-acetylaspartic acid is not w e l l understood. Increased N-acetylaspartic acid Concentration in blood, urine and cerebrospinal fluid w a s recently reported by Kvittirigen e t al. (1986) in a mentally retarded patient. Hagenfeldt et al. (1987) reported another case of N-acetylaspartic aciduria i n a patient with leukodystrophy. Aspartoacylase (EC.3.5.1.15) w a s assayed in both cases; normal activity w a s mted i n the first report (Kvittingen e t a l , 1986) while aspartoacylase deficiency w a s found i n the second (Hagenfeldt e t al, 1987). Neither of these reports linked N-acetylaspartic aciduria or aspartoacylase deficiency w i t h a neurological disorder that results i n spongy degeneration of the brain, Canavan disease. Canavan disease is a form of leukodystmplq inherited as an autoscmal recessive disorder (van Bogaert and Bertrand, 1967; Buchanan and Davis, 1965). It has been found to be mre ccrmyln m n g Ashkenazi Jews (warand Goodman, 1983). The neurological findings usually appear i n the f i r s t few mths of l i f e and are due to demyelinization and leukodystmphy w i t h associated megalencephaly, blindness and spasticity. The patkalcgic changes involve astrocytic swelling w i t h nonnal neurons. Electron micsoscopy shaws vacuoles i n the myelin sheaths, swelling of astrocytes and elongated mitochondria. Various themies have been proposed t o explain the pathogenesis of the spongy degeneration of the brain including disturbance i n water and electrolyte s h i f t wiL& a possible defect i n the ionic pmp. However, to date no biochemical marker or enzyme defect has been found i n this disease (Adomato e t al, 1972; Torack e t a l , 1960; Adachi e t a l , 1972). Three patients w i t h Canavan disease are the subject of this study. These patients were found to have

aspartoacylase deficiency and increased levels of N-acetylaspartic acid i n blood and urine.

Canavan Disease

CLINICAL REWRTS One patient with Canavan disease was diagnosed in a Boston Medical Center on the basis of clinical features of leukcdystrophy, megalenceghaly, aptic atrophy, and canputed t m c g r a m ((2) of the head. Brain biopsy shawed spongy degeneration of the brain which confirmed carxivan disease. The other 2 patients were sibliqs whose diagmses w e r e made in a medical center in Chicago on the basis of clinical manifestations of leukodystrophy, megalocephaly, optic atrophy and CT scan of the head. Brain biopsy was m t performed on the .two siblings. The 2 families are unrelated and both are of Ashkenazi Jewish descent. METHODS

cell cultures Cells frcm the three Canavan disease patients and four controls were cultured. In addition, there was one anmiocyte sample and one chorionic villi sample. These were cultivated in Matalon's modified Eagle's medium (Gibco, Grand Island, N.Y.) according to the method described by Matalon and D o r m (1966).

Assay for Aspartoacylase Asparbacylase activity w a s determined similar to the method of Hagenfeldt et al. (1987). Fibroblasts were rinsed, scraped and suspended in cold water then sonicated by three 10 second bursts using Heat Systems M x k l W 185 F Sonifier. The sonicates were then centrifuged at 10,OOO x g for 20 minutes and the supernatant aliquots were used for asparbacylase assay. Each incubation mixture contained 400 pg of protein (Lawry et al, 1951). Duplicate aliquots of the supernatant were incubated with and without the substrate, 1.7 m l / L of N-acetyl-L-aspartic acid (Sigma Biochemicals, St. Louis, PO), in 0.05M Tris HC1 buffer pH 8.0. We found that asparbacylase activity increased 2 fold when Tris buffer was used instead of phosphate buffer. Samples were incubated 8 hours and the reaction was terminated by boiling for 3 minutes. At the termination of the aspartoacylase reaction 2~xcglutarate(1.4 mnol/L), NADH (0.148 ml/L), malate dehydrogenase and aspartate aminotransferase were added to each assay tube. Aspartic acid was detennined spectrophotanekically by measuring the decrease in absorbance at 340 nm by the conversion of NADH to NAD. The aspartate content, as indicated bj the difference in the formation of NAD between the control and the assay tube, was a measure of N-acetylaspartate

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hydrolysis. A m t h e r method which was utilized for measuring the hydrolysis of N-acetylaspartic acid w a s direct analysis of aspartic acid by an a m i m acid analyzer (Beclanan System 7300 Amino Acid Analyzer, Palo Alto, CA). Aspartic acid peaks were integrated and quantified at 570 m. N-Acetylaspartic Acid in Urine and Plasma Urine and plasma were solvent extracted and silylated according to the m W of Gmdman and Markey (1981). Urine and plasma extracts were analyzed by gas chrclmatography-mass spectroscopy (Hewlett-Packard mdel 5993 B, Palo Alto, CA) using OV 22 capillary column 25 meters long. Hydrolysis of urinary N-acetyla5partic acid w a s performed similar to the method of Kvittingen et al. (1986) and the hydrolyzed aspartic acid w a s quantified on a Beckman amino acid analyzer. RESULTS

The 3 patients with Canavan disease were found to excrete excessive amtKlIlts of N-acetylaspartic acid in the urine. Their plasma also contained elevated levels of N-acetylaspartic acid. Urine frun 31 individuals who served as Controls contained only trace amounts of N-acetylaspartic acid. m n g these were three age matched children with other leukodlystmphies and 6 obligate carriers for Canavan disease. N m e of these had elevated urine or blood levels of N-acetylaspartic acid (Table I).

Table I. N-Acetylaspartic Acid in Urine and Plasma of Canavan Disease Patients Urine creatinine

pl/ml

Plasna

Pwfl

Patient A 1299.5 0.92 Patient B 1626.8 1.37 Patient C 3680.9 1-00 Controls a ( n=31) 10.0 52.1 n.d. Tadxols include 3 patients of similar age with other leukodystrophies and 6 obligate carriers for Canavan disease. n.d. - m t detected The identity of N-acetylaspartic acid was confirmed by mass spectroscopy. The win& cl-mma-am f m one patient with peak X and the ion masses of that peak are shown in Figure 1. This peak and the ion masses match authentic

Canavan Disease

"

I

Fig. 1. The urine chrcmatogram f m one patient with Peak X and the ion masses of that peak. A = malonic (internal standard), B = phosphoric, X = N-acetylaspartic, D = citric, E = 3(m-OH-phenyl)-3-OH-propionic, F = hippuric, C-24 (external standard).

A

Fig. 2. The c m t c g r a m of authentic N-acetylaspartic acid w i t h the same standards and the ion masses.

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N-acetylaspartic acid (Fig. 2). In the mass spectrum of the patient's urine there are sa-e ion masses of aconitic acid, a m m l urine ccmponent.

In order to further document excessive quantities of N-acetylaspartic acid in urine of patients with -van disease, urine fran the patients and normal controls was subjected to hydrolysis using bmgem'zedextracts of normal cultured skin fibroblasts as an enzyme source (Kvittingen et al, 1986). Upon hydrolysis with the fibroblast extracts the 3 patients with Canavan disease had a sharp increase in aspartic acid as canpared to controls. These data are presented in Table 11. Table 11. Aspartic Acid in Urine After Hydrolysis with Skin Fibroblasts in Canavan Disease Patients and Control Subjects

Patient A Patient B Patient C controls (n=6)

Aspartic Acid p-ml/ml creatinine 98.9 104.8 95.6 6.7~1.1

The assay for asparbacylase was performed on cultured skin fibroblasts on one patient fm each family, Control

fibroblasts, cultured amniocytes and a cultured chorionic villi sample. No fibroblasts available fran obligate carriers. Aspartoacylase activity was deficient in the fibroblast extracts f m the two patients with Canavan disease (Table 111). Asparbacylase activity was also present in normal cultured anmiocytes and cultured &orionic villi. Table 111. Aspartoacylase Activity in Skin Fibroblasts of 2 Patients with Canavan Disease and in Other Cultured Cells n

m*

Asartic Acid** protein 0.00

u/mg protein &l/mg

Patient B 0.04 Patient C 0.01 0.00 controls 4 2.38-4.00 6.15-7.40 1 0.70 1.75 cultured anmiccytes 0.50 0.89 cultured chorionic villi 1 *Aspartoacylase activity was determined spectrcmetrically by the conversion of NADH to NAD. **Aspartic acid was determined by amino acid analyzer.

Canavan Disease

DISCUSSION Synthesis of N-acetylaqarkic acid has not been found in any organ other than the central nervous system (Benuck and D'Adamo, 1968). The function of this ccmpound is m t mder&md. McIntosh and Cooper (1968) suggested that N-acetylaspartic acid is metabolized slowly in the mature brain. ShigematsU et al. (1983) have suggested that N-acetylaspartic acid is an essential ccmponent in a series of reactions required for the ccanrersion of ligmceric acid to cerebronic acid and the formation of glutamic acid. Since cerebnic acid is a precursor for the formation of ceramide in the brain, which is a cunpnent of myelin, it is possible that a defect in the metabolian of N-acetylaspartic acid is responsible for the abnormal myelinization process and spongy degeneration in this disease. 0x1the other hand, since aspartic acid is a neurotransmitter (Bradford and Thanas, 1968), the high concentration of N-acetylaspartic acid in the brain may serve for chemical ccmparhnentation of aspartic acid. Thus, a defect in the hydrolysis of the acetyl group could result in a deficiency of free aspartic acid at the neurcMal junction, which may lead to neurological disturbances. Another mcharn' s n for the pathogenesis of Canavan disease is that the accumulation of N-acetylaspartic in brain tissue may result in myelin damage and spongy degeneration. Whether any or all of these mchanisns are operative in the patkcgenesis of Canavan W e work is needed to test these disease is unkrmm. hypotheses.

Our findings indicate a specific defect in aspartoacylase activity in the cases of Canavan disease studied in our laboratory. Increased levels of plasma and urinary N-acetylaspartic acid is a biochemical marker for this disease. It is likely that the cases of Kvittingen et al. (1986) and Hagenfeldt et al. (1987) have the same disease and Canavan disease may not be that rare, altbough it is not understood the finding of normal aspartoacylase activity in the case of Kvittingen et al. ( 1986). Our data indicate that it should be possible to diagnose cases of Canavan disease prenatally by examination of cultured anmiocytes or chorionic villi samples for aspartoacylase activity. Further studies of other cases of Canavan disease for aspartoacylase deficiency is clearly indicated. Casual and pathcgmetic heterogeneity of Canavan disease is not excluded by OUT studies.

We would like to thank the National T a y Sachs and Allied Diseases Association for monetary and human

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resaurces.

The fibroblasts fran the Boston patient were kindly sent to us by Dr. A. Johnson and Dr. H. Nitowsky, A l b e r t Einstein College of Medicine, N.Y. The fibroblasts

fran the Chicago patients w e r e kindly given to us by Dr. A. Horwitz, University of Chicago and Dr. J. charrow, Children's Memorial Hospital, Chicago.

Adachi M, Torii J, Schneck L, Volk BW (1972): Electron microsoopic and enzyme histocherm'cal studies of the cerebellum in spongy degeneration (van Bogaert and Bertrand type). Acta Neuropath 20:22-31. Adomato BT, O'Brien JS, L a m p e r t FW, Roe TF, Neustein HB (1972): Cerebral spangy degeneration of infancy: a biochemical and ultrastructural study of affected t w i n s . Neurology 22:202-210. Benuck M, D'Adamo AF Jr (1968): AcetyltranspOrt mecham'sn: metabolism of N-acetylaSpartic acid in mn-nenmus tissue of rat. Bicchem Biophys Acta 152:611-618. van Eegaert L, l3ertrand I ( 1967): Spongy degeneration of brain in infancy. Amsterdam: North Holland Publishing CO. p ~ 3-132. . Bradford HF, Thunas AJ ( 1969): Metabolism of glucose and glutamate by synaptoms fran mamnalian cerebral cortex. J Neurochem 16:1495-1504. Buchanan DS, Davis RG (1965): Spongy dqeneratim of the nervous system: a reprt of 4 cases with a review of the literature. Neurology 15:207-222. Goodman SI, Markey SP (1981): "Diagnosis of organic acidmias by gas chranatmgra~-massspectrunetq." New York: Alan R. Liss, Inc, pp.3-24. Hagenfeldt L, Bollgren I, Venizelos N (1967): N-Ace-laspartic aciduria due to aspartoacylase deficiency - a new etiology of childhood leukodystrophy. J Inher Metab Dis 10:135-141. Jacobson KB (1957): Studies on the role of N-acetylaspartic acid in mamnalian brain. J Gen Physiol 43:323-333. K v i t t i n g e n EA, Guldal G, Borsting S, Skalpe 10, Stokke 0, Jellum E (1986): N-Acetylaspartic aciduria in a child with a progressive cerebral atrophy. Clin Chim Acta 158:217-227. Lowry OH, Rosenbraugh NJ, Farr At, Randall RJ (1951): protein measurement with the folin phenol reagent. J Biol Chem 193:265-275. Matalon R, Dorfman A (1966): Hurler's syndrane: Biosynthesis of acid rrrum~lysaccharidesin tissue culture. Pro Nat Acad Sci USA 56:1310-1316. McIntosch JM, Cooper JR (1965): Studies on the function of N-acetylaspartic acid in the brain. J Neurochem 12:825-835.

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Miyake M, Pbrim H, Mizobuchi M, Kakimoto Y (1982):

N-Acetylaspartic acid, N-acetyl-alpha-L-aspartyl-L-glutamic acid and beta-citryl-L-glutamic acid in human urine. Clin Chim Ada 120:119-126. S h i g m t s u H, Okamura N, Shimen0 H, Kishimoto Y, Kan L, Fenselau C (1983): Purification and characterization of the heat stable factors essential for conversion of ligmceric acid to cerebronic acid and glutamic acid: identificatim of N-acetyl-L-aspartic acid. J Neurochem 40:814-820. Tallan HH, mre. S, Stein WH (1956): N-acetyl-L-aspartic acid in brain. J Biol chem 219:257-264. Torack FW, Terry RP, Z i r m w m a n HM (1960): The fine structure of cerebral fluid accumulation and swelling produced by triethyltin p0iscmi.q and its canparism with that in the human brain. Amer J Path 36:273-287. w a r M, Goodman FW (1983): Spongy degeneration of the brain in Israel: A retrospective study. Clin Genet 23:23-29. ADDENDUM After the suhnissim of t h i s manuscript, fibroblasts and a sample of f r o m brain tissue frun a patient with Canavan disease, confinxed by brain biopsy, were examined

for aspartoacylase activity. Both the cultured skin fibroblasts and the brain tissue were deficient for asparbacylase while normal mtrol brain tissue showed an activity of 3.30 U/mg protein. The Sample w a s kindly sent to us by Professor David M. Danks, The Murdoch Institute, Royal Children's Hospital, Parksville, Australia.

Edited by John M. Opitz and James F. Reynolds Received for publication November 12, 1987; revision received December 9, 1987.