Humangenetik 29, 299 307 (1975) © by Springer-Verlag 1975

Suliatide Excreting Heterozygous Carrier of Juvenile Metachromatic Leukodystrophy or Asymptomatie Patient of Adult Metaehromatie Leukodystrophy K. Harzer and A. S. l%eeke Institut fiir tIirnforschung der Universit~t T/ibingen und Neuropsychiatrische Klinik der Universit~t Mainz Received~Iay 15,1975 Summary. In a family with juvenile metachromatic leukodystrophy (sulfatide lipidosis) 2 patients showed residual arylsulfatase A activities of 5--6O/o. The patients' healthy father was characterized biochemicMly by a 39% normal activity of leukoeyte plus plasma arylsulfatase A. The father was further characterized by a high sulfatide excretion (0.2--0.5 mg/l urine) and, paradoxically, by a normal sulfatide degrading enzyme activity in vitro. This special carrier is suspeeted to be heterozygous for a) arylsulfatase A deficiency and b) arylsulfatase A (sulfatidase) lability. This presumed additional genetic defect could be the cause of the sulfatide excretion which, in turn, would be a sign of the preclinical stage of an exceptional form of adult metaehromatie leukodystrophy. The normal sulfatidase activity seems to be due to an in vitro effect.

Metachromatic leukodystrophy (MLD, sulfatide lipidosis, for review see Moser, 1972) is a fatal disorder with progressive central and peripheral nervous system involvement and excessive sulfatide accumulation in the affected tissues. I n fact, high amounts of snlfatide are stored not only in the brain but also in the kidney and are exereted in the urine. The metabolic basis of MLD is a generalized and profound deficiency of arylsulfatase A (sulfatidase, sulfatide degrading enzyme) activity (Austin, 1963; Mehl and Jatzkewitz, 1965). The genetic heterogeneity of the sulfatide lipidosis finds expression in at least three clinical types of the disease. The late infantile, juvenile and adult type (Moser, 1972) of MLD are also biochemically different (Stumpf and Austin, 1971). The more delayed is the onset of the clinical symptomatology and the higher is the residual sulfatidase activity found in cultivated skin fibroblasts (Porter et al., 1971). The genetic heterogeneity of the lipidosis is further substantiated by the recent finding of a family with juvenile MLD in which only "one component of arylsulfatase A" was missing (Suzuki and Mizuno, 1974). Our studies of another family with juvenile MLD may add a further aspect to said heterogeneity. The MLD family the biochemical data of which are reported below, comprises the following members: 1. The proband, M. S., is a 12-year-old girl. She shows the clinical picture of juvenile MLD (Moser, 1972), excretes sulfatide in the urine and has about 6% of the normal arylsulfatase A level of activity in urine and blood. 2. P. S. is a 9-year-old sib who appears to be clinically healthy expect for legasthenia and "exhaustible elonus of the right foot". In addition, he excretes large

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a m o u n t s of sulf~tide a n d his arylsulfatuse A is reduced to 5 % of the n o r m a l level. 3. The p r o b a n d ' s second brother, S.S., aged 6 years, is h e a l t h y a n d has ~lmost n o r m a l arylsulfatase A levels. 4. The p r o b a n d ' s 40-year-old father, F. S., has no clinical s y m p t o m s b u t excretes appreciable a m o u n t s of sulfatide in the urine ~nd his arylsulfatase A is reduced to a 39% level of n o r m a l activity. However, his urine enzyme a c t i v i t y readily degrades sulfatide in vitro. 5. The p r o b a n d ' s h e a l t h y mother, It. S., ~ged 38, is biochemically characterized as a MLD carrier b y her a b o u t 4 6 % reduced level of arylsulfatase A a c t i v i t y . - - U r i n e arylsulfatase A from F. S. shows ~ slightly decreased physical stability ~s compared to t h a t o f H . S.

Methods Sul[atide Determination in Urine. Qualitative testing of urine sulfatide was carried out with pseudoisocyanin (Serva, Heidelberg, Cat. No. 33 800) as described elsewhere (Harzer and Benz, 1973). The sulfatide dependent pseudoisocyanin metachromasia was demonstrated as a peak at 570--580 nm in a partial absorption spectrum (540--630 nm) of the sulfatidepseudoisocyanin complex (ttarzer and Benz, 1973). For the quantitative assay of sulfatide a thin layer chromatographic-densitometrie system was used starting fl'om the hydrophobic phase of a 24-hr sample of urine. The hydrophobic phase was prepared by mixing 10 ml of vigorously shaken urine with 80 ml chloroform/methanol (1:1). After 3 subsequent phase partitioning steps with each 20 ml water, the last hydrophobic (lower) phase was collected, transferred to a polypropylene (glass is unsuitable) vessel of about 5 cm diameter and dried under nitrogen at about 50°C. The residue was extracted twice with 2 ml of chloroform/ methanol (1:1), boiling solvent being used in the second extraction. Volume of combined extracts was reduced and applied to thin layer plates. The upper phases were discarded, since sulfatide losses with these phases were lower than 10~o as studied with radioactive sulfatide (see below) added to urine. Precoated silica gel plates (Merck, Darmstadt, art. No. 5729, not additionally activated) with the applied samples were run twice in chloroform/methanol/25°/o aqueous ammonia 92:46:3, the first run being 3, and the second 15 cm from start. Co-ehromatographed reference sulfatide was a product of Applied Science/Serva. Spraying reagent and densitometric evaluation were those described elsewhere (ttarzer et al., 1968). Arylsul/atase A was assayed in leukocyte containing plasma supernatant of venous blood. 10 ml venous blood were mixed with 1.8 ml of a dextran and heparin containing solution as described (Kampine et al., 1967), and allowed to stand at room temperature for 1 hr. The supernatant that had been formed was collected, frozen and thawed 3 times and centrifuged at 30000 g for 15 rain. The clear supe1~atant containing plasma and leukocyte enzyme was used for sulfatase assay. For the A enzyme one part was mixed with two parts of substrate solution A according to Baum et al. (1959), incubated for 5 hrs at 37°C and alealized with 3 parts of 1 IN sodium hydroxide. Absorption at 515 nm, corrected with appropriate blanks but not with the very low (< 5~o) percentage of arylsulfatase B that is not inhibited by substrate solution A, was taken as a measure of arylsulfatase A activity. Arylsul/atase B in leukocyte containing plasma supernatant was assayed by mixing 1 part of supernate (see above) with 2 parts of substrate "solution T" (total arylsulfatase; l0 mM p-nitrocatechol sulfate, Sigma, St. Louis, in 2.0 IV[ sodium acetate buffer pH 6.0); further proceeding as for arylsulfatase A. Arylsulfatase B activity was calculated from the absorption value at 515 nm by subtracting 30~o of the value obtained for arylsulfatase A, since with "solution T" isoelectrically focused arylsulfatase A was found to produce about 30~o of the As15 value obtained with solution A (see Fig. 4). For lability o] urine enzymes see Note added in proof. Sul]atidase was assayed in urine by adjusting 1 ml of dialyzed (3 hrs) urine to the following concentrations of assay compounds: a) 0.4 mM sulfatide (commercial product as indicated above, purified by florisil column chromatography) which had been catalytically hydrated with tritium at the sphingosine and fatty acid double bounds (Amersham Bucks. England tritiating service, code TR 3). Final specific radioactivity after dilution with unlabeled sul-

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fatide was about 50 gCi/izmole, b) 0.4% crude sodium tauroeholate (Merck-Schuehardt, Munich, Cat. No. 12353). e) 0.05 M sodium acetate buffer pH 4.8 (optimum under the used conditions), d) 0.004~o ampicillin.--Ineubationperiod was 20 hrs. 50 fzl of assay were applied to precoated silica gel plates (see above) and run in chloroform/methanol/water 14:6:1. Galaetosyl ceramide as the final product of enzymatic desulfation of snlfatide was localized and measured by radioscanning (Harzer et al., 1973). Isoelectric Focusing of arylsulfatase A and B was performed according to a previously described method (Itarzer et al., 1973) with the variation that 4--8 ml of dialyzed (3 hrs) urine were used as starting enzyme sample. As a re/erence system for sulfatase activities served a mean relative reference enzyme factor R (see below). Practically, comparability of equal volumes of leukocyte containing plasma supernatants and of urine samples was achieved by adjusting all samples of one series to the mean relative reference enzyme activity found for the sample with the lowest activity. Adjustment was done by appropriate dilution with water. The mean relative reference enzyme activity R i of a sample i out of a series of samples 1 . . . n was calculated with the aid of the following formula

Ri

A.~ Bi Ci Z A 1 . . . n +. X B t. .. n + . ZC1 .

n ~-

A, B and C are the absolute activities of 3 reference enzymes. Each absolute activity of a sample i is divided by the sum of all corresponding absolute activities of the complete series. /~l being the lowest mean relative reference enzyme activity found in one series, the dilution factor for each sample i of this series was calculated as Ri/IRt. As reference enzymes were determined: Total fi-hexosaminidase, acid phosphatase and fl-glucuronidase, using 2 mM solutions of the respective p-nitrophenyl snbstrates (Serva) either in 0.1 M sodium citrate buffer pH 4.5 (total fl-hexosaminidase) or in the same buffer pH 5.0 (acid phosphatase) or in 0.5 N sodium acetate buffer pH 5.0 (fi-glncuronidase). For the reference enzyme assays, 1 volume of sample was mixed with either 10 volumes of substrate solution and incubated for 1 hr (total fi-hexosaminidase, acid phosphatase) or 1 volume of substrate solution and incubated 5 hrs (fi-glueuronidase). Absorption a~ 405 nm after alcalizing to pit 10.3 was used a measure proportional to absolute activity and taken as A, /?, C for the formula.

Results The m e m b e r s of the MLD family are identified b y their initials as given in the first section of this paper. These are used below. The s u l f a t i d e - d e p e n d e n t metaehromasia with pseudoisoeyanin (absorption m a x i m u m at a b o u t 575 rim) in n o r m a l a n d 3/ILD family urine is shown in Fig. 1. The sulfatide c o n t e n t of the u r i n e as d e t e r m i n e d b y t h i n layer c h r o m a t o g r a p h y is listed i n Table 1. A corresponding c h r o m a t o g r a m a n d densitogram are shown i n Fig. 2 a a n d b. Arylsulfatase A a n d B activities in the blood of the m e m b e r s are given in Table 2. The i n t a c t arylsulfatase B in the 2 p a t i e n t s with MLD excludes the m u l t i p l e sulfatase deficiency, A u s t i n v a r i a n t of MLD (Austin, 1973). The residual arylsulfatase A a c t i v i t y ( 5 - - 6 % of controls) in the p a t i e n t s with juvenile m e t a e h r o m a t i c l e u k o d y s t r o p h y is higher ( S t u m p f a n d Austin, 1971) t h a n in p a t i e n t s with the late infantile form of the disorder (about 1--4Yo). Sulfatidase a c t i v i t y in the urine of 3/[LD family m e m b e r s is listed in Table 3 a n d a n example of the m e t h o d used for its d e t e r m i n a t i o n is shown in Fig. 3. p H - d e p e n d e n e y of urine arylsulfatase A from F. S., the sulfatide excreting father ("special carrier") of the 2 p a t i e n t s was found to be n o t different from t h a t of control enzymes ( o p t i m u m at p H 4.9).

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AlcmA 2D 1.0 s, o

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Fig. 1. Partial absorption spectrum of metaehromatie pseudoisocyanin-sulfatide complex obtained with 10 ml urine samples from MLD patient (P. S., upper curve), MLD special carrier (F. S., middle) and control (lower). Intensity of "polymeric band" (Appel and Seheibe, 1958) with its maximum absorption at about 575 nm roughly parallels urine sulfatide content (see Table 1) Table 1. Sulfatide content in the urine from the family with juvenile metaehromatie leukodystrophy; milligram per liter urine collected during 24 hrs (2 determinations) Controls (n = 5) Patients M.S. Patient P . S . Patients' father F . S . (special carrier) Patients' mother I~. S. Healthy brother S.S.

< 0.05 0.4/0.8 0.6/0.7 0.2/0.5 < 0.05 < 0.05

Table 2. Arylsulfatase A and B activities in leukocyte containing plasma supematants (see Methods) from the family with juvenile metachromatic leukodystrophy. Activities as nmoles p-nitrocatechol sulfate cleaved per hour and per milliliter of supernatant at 37°C. Mean of 3 determinations Controls (n = 5)

Arylsulfatase A Arylsulfatase B 30~42 32--60 (mean 36) (mean 50)

Patient M.S. Patient P . S . Patients' father F . S . Patients' mother H . S . Patients' brother S.S.

2.4 1.7 14 20 33

60 32 50 66 40

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Fig. 2. (a) Detection of urine sulfatide by t h i n layer chromatography. Precoated silica gel plates were r u n in chloroform/methanol/25~o aqueous ammonia 92:46:3, sprayed with 2~o sulfuric acid/0.5~o anisaldehyde in acetic acid and heated for 10 min at 120°C. Lane 1 : 1 0 gg of brain lipid extract from a p a t i e n t with infantile metachromatic leukodystrophy. Lane 2: hydrophobic phase of 10 ml urine from patient M. S. of the family with juvenile metachromatic leukodystrophy. Lane 3 : 9 ,~g of reference sulfatide. S sulfatide (with slightly depressed rf value in lane 2). (b) Thin layer densitogram of the hydrophobic phase of 10 ml urine from the special carrier (F. S.) of juvenile metachromatic leukodystrophy (upper trace). Lower trace: 9 ~g of reference sulfatide. Shadowed areas correspond to sulfatide spots. Starting position of chrom a t o g r a m was exactly at the righthand end of the traces. Normal urine shows no sulfs~ide spots

Table 3. Sulfatide degrading enzyme activity in the urine from the family with juvenile metachromatic leukodystrophy. Activity is expressed as nmoles sulfatide desulfated per hour and of milliliter urine at 37 ° C. For comparability of urine see Methods Controls (n = 5) P a t i e n t M.S. Patient P.S. Patients' father F.S. excreting sulfatide

3--10 < 0.05 < 0.05 4

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Fig. 3. Sulfatide degrading enzyme activity (sulfatidase) in the urine of patient P. S. with juvenile metachromatic leukodystrophy and of his father F. S. excreting sulfatide. Thin layer radioscan of 50 fzl of sulfatidase assay chromatographed in chloroform/methanol/water 14:6:1 (start at the bottom). Lane 1 = non-incubated assay, lane 2 = assay with urine from 1). S., incubated for 70 hrs, lane 3 = assay with urine from F. S., incubated for 70 hrs. S tritium-labeled sulfa~ide; C cerebroside (galactosylceramide) produced by enzymatic desulfation of sulfatide in the presence of crude taurocholate

Sulfatide excretion in t h e presence of active sulfatidase a n d a r y l s u l f a t a s e A has been f o u n d in t h r e e out of four different urine samples o b t a i n e d from F. S. in t h e course of a 15 m o n t h s period. F o r urine e n z y m e l a b i l i t y see N o t e a d d e d in proof.

Discussion H e t e r o z y g o u s carriers of one of t h e recessively i n h e r i t e d sphingolipidoses are n o t k n o w n to a c c u m u l a t e or excrete p a t h o l o g i c a l a m o u n t s of sphingolipids. I t is c o m m o n knowledge though, t h a t affected persons w i t h a t o t a l or n e a r l y t o t a l deficient a c t i v i t y of a sphingolipid d e g r a d i n g e n z y m e a c c u m u l a t e t h e corresponding sphingolipid in t h e i r tissues or b o d y fluids or both. I n t h e r e p o r t e d f a m i l y w i t h juvenile m e t a c h r o m a t i c l e u k o d y s t r o p h y (MLD) t h e heterozygous f a t h e r - - c h a r a c t e r i z e d b y a 39% l e u k o c y t e / p l a s m a a r y l s n l f a t a s e A a c t i v i t y (Table 2 ) - - e x c r e t e s high a m o u n t s of snlfatide in his urine (Fig. 1).

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Fig. 4. Isoeleetrie focusing of urine arylsulfatases from a control female aged 30 (upper panel), the su]fatide excreting father F. S. (middle pane]) and the patient M. S. (lower panel) of the family with juvenile metaehromatie leudodystrophy. 4 ml of dialyzed urine were used for a 12 ml eleetrofoeusing eolumn. Fractions were incubated for 2 hrs with "solution T" (broken line; see Methods) or solution A (solid line). Lefthand small peaks of broken line curves correspond to arylsulfatase B, high peaks of solid line eurves to arylsulfatase A. The double line in the lower panel shows activities of N-acetyl-/3-glueosaminidasesB and A, respectively, which reference enzymes were assayed as described for total fl-hexosaminidase D e s p i t e this finding, t h e e n z y m e a c t i v i t y in t h e urine from t h e f a t h e r (hereafter d e s i g n a t e d as "special carrier") degrades sulfatide in vitro a t a b o u t t h e same r a t e as control e n z y m e a c t i v i t y (Table 3). The question arises how to e x p l a i n these p a r a d o x i c a l findings. T h e following possibilities m a y be considered. 1. P h y s i c a l l a b i l i t y of a r y l s u l f a t a s e A (and therefore also of sulfatidase) from t h e "special carrier" (Note a d d e d in proof) is t h e cause of his defective sulfatide c a t a b o l i s m in vivo which is d e t e c t a b l e b y t h e urine sulfatide excretion. I t seems

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possible t h a t in vivo the pathologic enzyme is i n a c t i v a t e d so r a p i d l y t h a t only a p a r t of the n o r m a l e n z y m a t i c sulfatide degrading p o t e n t i a l is available. This p a r t m a y be lower in vivo t h a n in vitro, where sulfatide d e g r a d a t i o n (desulration) is n o t impaired (Table 3, Fig. 3) as long as the fresh urine e n z y m e is stabilized or a c t i v a t e d b y one or a n o t h e r c o n s t i t u e n t of the e n z y m e assay, especially b y sodium tauroeholate, a well k n o w n activator of sulfatidase a n d of other sphingolipid degrading hydrolases. 2. I f there would exist a specific activator of sulfatidase (see J a t z k e w i t z a n d Stinshoff, 1973), one could speculate t h a t a gene selectively responsible for this activator could be defective i n the special carrier a n d t h e r e b y could be the cause of the impaired sulfatide catabolism. A t a n y rate it seems difficult to explain the observed lability of arylsulfatase A in terms of a deficient sulfatidase activator. The special carrier, who excretes sulfatide i n his urine, m a y be i n the preclinical stage of a late a d u l t onset form of MLD. Clinical studies are p l a n n e d to search for discrete s y m p t o m s of this disorder, for example, for a slightly reduced nerve c o n d u c t i o n velocity. I f the special carrier would manifest s y m p t o m s of late a d u l t MLD i n the future, one would have to consider t h a t even high residual activities of arylsulfatase A do n o t exclude exceptional forms of MLD. I t was one aim of the present work to develop a sufficiently exact direct m e t h o d for sulfatide assay i n the urine. The methods available from the literature were n o t completely satisfactory in our hands. F u r t h e r m o r e , we feel t h a t the herein described m e t h o d for d e t e r m i n a t i o n of leukocyte/plasma arylsulfatase A which starts from leukocyte c o n t a i n i n g plasma s u p e r n a t a n t has some a d v a n t a g e s also for heterozygote identification (see Table 2). The technical assistance of Mrs. I. Schuster is thankfully acknowledged. Note Added in Proo/. An additional clinical finding in the patient P. S. (see Introduction) is the deficient blocking of alpha-rhythm in EEG upon eye-opening. --Additional biochemical findings are: Blood leukocyte sulfatidase from patients' father, the special carrier F. S. as well as from the mother It. S. is at ("heterozygous") 55~o normal levels; about 4:5 nmoles sulfatide are cleaved per hour and mg leukocyte protein.--The physical stability of urine arylsulfatase A from F. S. appears to be reduced: When testing the effect of heat (30--180 min, 50°C), 15fold concentration, storage of undialyzed urine (pH 6.0, --20°C, 6 weeks) on the enzyme, the activity of F. S. is lost to a higher rate than that of H. S. The relative differences between both persons are about 10, 50 and 800/o for the three respective methods of testing enzyme stability. The strong effect of the storage period on the activity of the F. S. enzyme and the weak effect on the H. S. enzyme are accompanied by an intermediate or weak effect on control enzymes. Other lysosomal urine enzymes from F, S. too are sensitive to storage period. Acid B-galactosidase is as labile as, $-hexosaminidase is somewhat less labile than arylsulf~tase A. If the effect of storage period is not simply due to different urine composition, one could discuss that for genetic reasons F. S.' enzymes studied could be labile against physical alterations.~In a recent paper the activator of sulfatidase mentioned in Discussion has now been extensively studied and characterized as a protein or glycoprotein (G. Fischer and H. Jatzkewitz, Hoppe-Seylers Z. physiol. Chem. 856, 605--613, (1975)).

References Austin, J. It. : Studies in metachromatic leukodystrophy XII. Multiple sulfatase deficiency. Arch. Neurol. 28, 258--264 (1973) Austin, J. H., Balasubramian, A. S., Pattabiraman, T. N., Saraswathi, S., Basu, D. K., Bachhawat, B. K. : A controlled study of enzymic activities in three human disorders of glycolipid metabolism. J. Neurochem. 10, 805--816 (1963)

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Appel, W., Scheibe, G. : Uber die Bildung reversibler Polymerisate des Pseudoisocyanins durch polare, kettenfSrmige Hochpolymere (Heparin) I. Z. Naturforsch. 13b, 359--364 (1958) Baum, tt., Dodgson, K , Spencer, I%. : The assay of arylsulfa.tase A and B in human urine. Clin. chim. Acta 4, 453 455 (1959) Harzer, K., Benz, H. U. : Quantitative ~etachromasie mit Pseudoisocyanin. Eine neue Merhode zur Bestimmung von Sulfatiden sowie ihre Anwendung bei der Diagnose der metachromatischen Leukodystrophie (Sulfatid-Lipidose). Z. klin. Chem. Biochem. 11, 471--475 (1973) ttarzer, K., Stinshoff, K., Mraz, W., Jatzkewitz, H. : The patterns of arylsulphatases A and B in human normal and metachromatic leukodystrophy tissues and their relationship to the cerebroside sulphatase activity. J. Neurochem. 20, 279--287 (1973) Harzer, K., Wiissle, W., Sandhoff, K., Jatzkewitz, H. : Densitometrische Mikrobestimmung yon Lipiden nach Diinnschiehtchromatographie des Gesamtlipidextrakts. Z. analyt. Chem. 243, 527--536 (1968) Jatzkewitz, H., Stinshoff, K.: An activator of eerebroside sulphatase in human normal liver and in cases of congenital metaehromatic leukodystrophy. F E B S Letters 32, 129--131 (1973) Kampine, J. R., :Brady, 1% 0., Kanfer~ J. N. : Diagnosis of Gaueher's disease and NiemannPick disease with small samples of venous blood. Science 155, 86--88 (1967) ~Iehl, E., Jatzkewitz, I-I. : Evidence for the geneLic block in metaehromatic leukodystrophy (ML). Biochem. biophys. Res. Commun. 19, 4~07 411 (1965) Moser, H. W. : Sulfatide lipidosis : Metaehromatic leukodystrophy. In : The metabolic basis of inherited disease (eds. J. :B. Stanbury, J. B. Wyngaarden, D. S. Fredriekson), pp. 688--729. New York: McGraw-Hill, :Blakiston Div. 1972 Porter, M. T., Fluharty, A. L., Trammel, J., Kihara, H.: A correlation of intracellular cerebroside sulfatase activity in fibroblasts with latency in metaehromatic leukodystrophy. Biochem. biophys. Res. Commun. 44, 660--666 (1971) Stumpf, D., Austin, J. tI. : Metachromatic leukodystrophy (M:LD) IX. Qualitative and quantitative differences in urinary arylsulfat~se A in different forms of MLD. Arch. Neurol. 24, 117--124 (1971) Suzuki, Y., Mizuno, Y.: Juvenile metachromatic leukodystrophy: Deficiency of an arylsulfatase A component. J. Pediat. 85, 823--825 (1974) Dr. K. Harzer Institut fiir Hirnforschung Korbinian-Brodmann-Itaus D-7400 Ttibingen, BelthlestraBe 15 Federal l~epublic of Germany