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Two cases of mucopolysaccharidosis Type III (Sanfilippo)
Journal of the Neurological Sciences, 1979, 40:77-86 © Elsevier/North-HollandBiomedicalPress
77
TWO CASES OF MUCOPOLYSACCHARIDOSIS TYPE III (SANFILIPPO). A Biochemical Study
G. VAN DESSEL, A. LAGROU, J. J. MARTIN, C. CEUTERICK and W. DIERICK UIA-Laboratory for Pathological Biochemistry and RUCA-Laboratory for Human Biochemistry; Department of Neuropathology, Born-Bunge Foundation and UIA-Department of Medicine, University of Antwerp, Antwerp (Belgium)
(Received 19 April, 1978) (Accepted 20 September, 1978)
SUMMARY The mucopolysaccharide and lipid composition of human nervous tissue and viscera from one case of Sanfilippo disease type A and one case of Sanfilippo disease type C, were investigated. In the brain a moderate increase of acid glycosaminoglycans occurred. This phenomenon was much more pronounced in the viscera, especially in the liver. In all tissues this increase was mainly due to an accumulation of heparan sulphate. Changes in lipid composition were noted, but can be regarded as secondary effects. The biochemical results reported also suggest some general conclusions. (a) A G A G and lipid analyses do not permit differentiation between the subtypes of Sanfilippo disease. (b) The differences in lipid composition can probably be considered as consequences of variation in secondary effects. (c) The severe demyelination in brain correlates well with the biochemical lipid analysis. However, in other instances it remains difficult to bridge the gap still existing between some morphological and biochemical data.
INTRODUCTION Mucopolysaccharidoses (MPS) are a group of genetic diseases characterized by storage of incompletely degraded acid glycosaminoglycans (AGAG). All the classified mucopolysaccharidoses except for the Morquio syndrome (MPS IV) and flglucuronidase deficiency (MPS VII) show a disturbance in the lysosomal catabolism Reprint requests to: Dr. G. Van Dessel, UIA-Laboratory for Pathological Biochemistry, Groenenborgerlaan 171, B-2020 Antwerp (Belgium).
78 of two polymeric substances, dermatan sulphate (DS) and heparan sulphate (HS), singly or in combination (Neufeld 1974). Few studies deal with the A G A G (Constantopoulos, M c C o m b and Dekaban 1976; Shimamura, Hakozaki, Takahashi, Kimura, Fujino, Suzuki and N a k a m u r a 1976; Kindler, Klein and von Figura 1977) and glycolipid (Dekaban and Patton 1973; Kint, Dacremont, Carton, Orye and Hooft 1973) composition of brain and viscera from Sanfilippo patients. No information is available with regard to changes in neutral and phospholipids except for one report concerning cholesterol and phospholipid content of a Sanfilippo brain (Dekaban and Patton 1973). This paper describes the lipid and mucopolysaccharide composition of brain and viscera of two patients showing the clinical features of Sanfilippo disease. CASE REPORTS Case 1 is an 11-year-old white male patient with clinical features of MPS III proven to belong to type III A by assaying heparan sulphate sulphamidase on cultured fibroblasts (Kresse and von Figura, personal communication). Light microscopy of autopsy material shows a diffuse neuronal storage in brain and dilated perivascular spaces filled with ballooned bistiocytes in the white matter. Signs of visceral storage are found in liver, spleen, kidney, lymph nodes and cartilage. Heart valves and arterial walls are also infiltrated by foam cells. Electron microscopy shows zebra bodies, membranous cytoplasmic bodies and membrano-granulo-vacuolar inclusions in neurons, glial cells, Schwann cells and myocardial fibres. Large electron-lucent vacuoles are found in liver, spleen, kidney and fibroblasts. Case 2 is a 13-year-old white male patient with clinical features of MPS III. He belongs to type III C as demonstrated by assaying a-glucosaminidase on cultured fibroblasts (von Figura, personal communication). Light- and electron-microscopic features of storage are found both in the nervous system and visceral organs. They are essentially similar to those described in Case 1. It was not possible to discover any significant morphological difference between our types IIIA and III C cases. MATERIAL Fresh brain and viscera from a control and from the two patients were obtained at autopsy. The tissues were stored at - - 2 0 °C. The enzymes papain and pronase were purchased from Sigma. The standard mucopolysaccharides hyaluronic acid (HA), chondroitin-4-sulphate (C-4S), dermatan sulphate (DS), chondroitin-6-sulphate (C6S) and heparin (Hep) as well as the enzymes chondroitinase ABC and chondroitinase AC were obtained from Miles Laboratories. The cellulose polyac~tate strips (Sepraphore III) were from Gelman Instruments. All chemicals were analytical grade and used without further purification. METHODS
Mucopolysaccharide analysis (i) Isolation ofglycosaminoglycans. The tissues were defatted through successive homogenization with 20 volumina chloroform:methanol 2/1 (v/v), 10 volumina 1/1 (v/v) and 10 volumina 1/2 (v/v). After each step the mixture was filtered through sintered glass of medium porosity and the extracts were combined. These chloroformmethanol extracts were used for lipid analysis. The defatted tissue was subjected to proteolytic digestion with papain and pronase (25 mg enzyme/g delipidated tissue;
79 0.1 M acetate buffer pH 5.5; 60 °C; overnight). At the end of incubation the mixture was centrifuged at 10,000 x g for 15 min and the supernatant collected. The sediment was washed with 2 ml acetate buffer and the washing combined with the first supernatant. The AGAG were isolated by precipitation with 5 ~ cetylpyridiniumchloride (CPC) (4 °C, overnight). The resulting AGAG-CPC complexes were spun down (10,000 × g for 10 min) and treated with 20 ml 10 Yopotassium acetate in 95 ~o ethanol. After standing for 1 hr and centrifugation (10,000 x g for 10 min) the sediment was washed successively with 20 ml ethanol and 20 ml diethylether and finally dried at room temperature. The precipitate was dissolved in 0.05 M NaOH and allowed to stand for at least 1 hr at 4 °C. After centrifugation (I0,000 x g for 10 min) the supernatant yields the AGAG extract used for further analysis. CPC-non precipitable AGAG degradation products were also quantitated in the supernatant obtained after CPC addition. (ii) Constituent analysis. Uronic acid was determined by the carbazole reaction as modified by Bitter and Muir (1962) using glucuronolactone as a standard. For DS identification the carbazole reaction was carried out with and without borate since iduronic acid gives a very low colour yield without borate. DS was also assayed by the naphthoresorcinol method (Teller 1967). For the analysis of N-substituted hexosamines the AGAG fractions were hydrolysed in aqueous 4 N HCI for 3 hr at 110 °C and the hydrolysates dried in vacuo over PeO5 and NaOH. The residue was dissolved in citrate buffer pH 2.2. The hexosamine content and ratio glucosamine/galactosamine were determined on an amino acid analyser with L-a-aminofl-guanido-propionic acid as internal standard using Aminex A5 cation exchanger (1.5 x 14 cm) (Van Dessel, Lagrou, Hilderson, Dierick and Dacremont 1977). It must be stressed that considerable losses of hexosamine occur during the hydrolysis procedure. Therefore a correction has been applied. (iii) Estimation of AGAG composition. After enzymic depolymerization with the eliminases chondroitinase AC and chondroitinase ABC, the susceptibility of the AGAG to the two enzymes was examined by measuring the uronic acid content of the released disaccharides. (Constantopoulos et al. 1976). Chondroitinase AC specifically digests chondroitin, HA, C-4S and C-6S to the unsaturated nonsulphated, 4-sulphated and 6-sulphated disaccharides, respectively, but does not attack DS (Saito, Yamagata and Suzuki 1968). The chondroitinase ABC digests in supplement to the AGAG mentioned above, also DS to the unsaturated 4-sulphate disaccharide. The uronic acid not susceptible to the two enzymes represents HS. The AGAG mixtures (50/~g AGAG-uronic acid) were subjected to enzymic degradation with chondroitinase AC (0.05 I.U.) and chondroitinase ABC (0.1 I.U.) in enriched Tris buffer, pH 8.0 (total volume of 200/~1) (Saito et al. 1968). The time of incubation was 1 hr at 37 °C for the enzymic digestion with chondroitinase AC and 4 hr at 37 °C for the other incubations. The reactions were stopped by boiling for 2 min, whereafter the mixtures were cooled and 100/~1 of 3.0 M NaC1 and 1.4 ml of 95 ~ ethanol added. After standing 1 hr at 4 °C the tubes were centrifuged (10,000 × g for 10 min) and uronic acid content of the released disaccharides measured in the supernatants by the carbazole-borate method (Saito et al. 1968).
80
(iv) Electrophoresis. Electrophoresis on Sepraphore membrane was carried out following Wessler's method (Wessler 1968, 1971) in 0.05 M barium acetate [75 min; potential gradient (8V/cm)] and in 0.1 M HC1 (37 min applying the same voltage). Lipid analysis (i) Lipid extraction and fractionation. The crude lipid extracts, prepared as previously described (Martin, Van Dessel, Lagrou, De Barsy and Dierick 1974) were separated in a neutral, a glycolipid and a phospholipid fraction by column chromatography on Biosil A (200--400 mesh) (Van Dessel et al. 1977).
Analytical methods Total lipids were estimated by the phosphovanillin colorimetric assay (Knight, Anderson and Rawle 1972). Free and esterified cholesterol, total phospholipids and phospholipid patterns were assayed as reported earlier (Martin et al. 1974). Glycolipids. Following silicic acid chromatography and alkaline saponification (Van Dessel et al. 1977) ceramide hexosides were separated by TLC on silicagel G using C :M :H20 65/25/4 (v/v/v) as eluent. The amount of glycolipid was determined by GLC of the trimethylsilylether derivatives of the methylglycosides (Van Dessel et al. 1977). Sulphatides and neutral glycolipids were separated on DEAE-Sephadex (Yu and Ledeen 1972). Gangliosides. Total lipid-bound sialic acid in the Folch upper phase was determined by the thiobarbituric acid assay (Van Dessel et al. 1977). Ganglioside patterns were determined after separation on silicagel G with C :M :NH3 2.5 N 60:35:8 (v/v/v) as eluent. Quantitative determination of the individual gangliosides was performed applying Suzuki's method as modified by McMillan and Wherrett (1969). RESULTS
Acid glycosaminoglycans The A G A G concentrations in brain, liver, spleen and kidney are represented in Table 1. In comparison with normal tissue the A G A G content in the organs of the TABLE 1 A G A G CONTENT IN BRAIN A N D VISCERA
Brain (l)
Control 0.5 Case 1 1.1 Case2 0.5
Liver
Spleen
(2)
(1)
(2)
0.5 1.3 0.5
0.9 0.1 27.5 25.4 44.1 24.9
(1)
Kidney (2)
1.9 0.2 9.2 6.1 11.2 5.0
(1)
(2)
2.7 0.7 10.7 N.D. 7.5 2.1
(1) AGAG expressed as mg uronic acid/g defatted tissue. (2) Total N-substituted hexosamines in mg/g defatted tissue. N.D. ~ not determined because of lack of material.
81 TABLE 2 COMPOSITION a OF AGAG IN BRAIN AND VISCERA Brain
Liver
Spleen
Kidney
(a)
(b)
(c)
(a)
(b)
(c)
(a)
(b)
(c)
(a)
(b)
(c)
Control 74 Case 1 44 Case 2 42
14 7 5
12 49 53
20 9 2
25 4 7
55 87 91
15 2 4
36 29 23
49 69 73
47 27 20
16 9 13
37 64 67
(a) (b) (c) a
Hyaluronic acid ÷ chondroitin-4-sulphate ÷ chondroitin-6-sulphate. Dermatan sulphate. Heparan sulphate. Expressed as ~o of total uronic acid.
patients was much higher, especially in the liver, where a 30-50-fold elevation was noted. The other organs contained 2-12 times as much as the controls. In the brain of one patient the A G A G concentration was nearly normal. The A G A G composition of brain and viscera as derived from their susceptibility to chondroitinases AC and ABC is shown in Table 2. In normal brain predominance of HA, C-4S and C-6S is evident. By contrast in the brain of the two Sanfilippo patients a fifty-fifty distribution is found for this group vs. HS. This is also substantiated by the higher intensity of the spot in the electropherogram migrating just ahead of the Hep standard. It is interesting to note that although there is no marked difference in the total A G A G content in the brain, there is a significant change in the procentual distribution: in normal brain HA, C-4S and C-6S comprise 74 ~o, HS 12 % and DS 14 % of the total A G A G ; in the affected brains a 4fold increase of the HS content was observed, mainly reflected in the concomitant decrease of HA, C-4S and C-6S. In both patients 85 % of AGAG-hexosamines was identified as glucosamine, confirming the major brain A G A G (4- 50 % of total) to be HS. Compared to the normal values the A G A G in affected liver were increased 30-50-fold (Table 1). Table 2 shows that even in normal liver HS is the main A G A G (55 %). The HS content for both patients, however, amounted to 87-91%. This agrees with the determination of the N-substituted hexosamines (Table 1). The galactosamine content was beyond the detection limit indicating a drastic decrease of C-4S, C-6S and DS containing this hexosamine. The electrophoretic pattern of liver A G A G is characterized by the presence of a very intense spot having the same migration rate as HS. The total A G A G content was elevated 5-6 times in the spleen of both patients. HA, C-4S and C-6S content ( A G A G susceptible to chondroitinase AC) is drastically reduced (Table 2). A 40-50% increase in HS was observed. The results are in agreement with the electrophoretic pattern obtained for spleen total AGAG. Chemical analyses of the A G A G isolated from the normal and phatological human kidneys are tabulated in Tables 1 and 2. The predominant hexosamine in kidneys was glucosamine. The enzymatic degradation of the A G A G showed that
82 TABLE 3 CPC-NONPRECIPITABLE AGAG DEGRADATION PRODUCTS a IN BRAIN AND VISCERA
Control Case 1 Case 2
Brain (whole) Liver
Spleen
Kidney
275 371 312
72 600 984
48 1020 972
48 1092 780
a Expressed as uronic acid (/~g/g wet weight).
chondroitinase AC hydrolysed substantial amounts of the A G A G from normal kidney (47 ~). From pathological kidneys only a minor fraction of the A G A G (20-27 ~ ) was hydrolysed with chondroitinase AC. The results indicate that HS is the major A G A G of normal and pathological kidney, but is more elevated in the affected organs. Again the results were confirmed by the electrophoresis experiments. Liver, spleen and kidney of our patients contained large amounts of CPCnonprecipitable A G A G degradation products (Table 3), while in brain only a slight increase was apparent.
Lipids No uronic acid was found in the lipid extracts. The lipid composition of white matter is summarized in Table 4. A marked decrease in total lipids and phospholipid was noted. Cholesterol was the main neutral lipid component with no increase of cholesterol esters. Total glycolipid was within the normal range. Nevertheless the ratio cerebrosides/sulphatides for Case 1 was raised up to 8.5. The presence of minor quantities of lactosylceramide could be demonstrated, but was also observed in the control tissue. The phospholipid patterns looked nearly normal in both cases with the exception of a decrease for phosphatidylinositol (PI) and phosphatidylserine (PS). The content of lipid-bound sialic acid was normal for Case 1 and increased for Case 2. The results of the chemical analyses of grey matter are also represented in Table 4. The same general tendency was noted as in white matter. However, in grey matter, an increase in PI and PS occurred while decrease in total glycolipids was noted only for Case 2. The ganglioside content was nearly normal, although in Case 1 relatively high concentrations of less polar gangliosides were found (Table 4). The lipid content of liver is given in Table 4. These analyses revealed for both patients a decrease of total lipid, cholesterol, glycolipids and phospholipids. The phospholipid pattern was normal. Like in normal liver on thin layer chromatography (TLC) only a ganglioside with the same R/-value as GMa could be detected. Spleen and kidney showed no differences in lipid composition from the controls. DISCUSSION In all tissues of the patients reported here (except for the brain of Case 2) an absolute increase of A G A G content was observed being most pronounced in the liver.
83 TABLE 4 B I O C H E M I C A L ANALYSIS O F LIPIDS IN B R A I N A N D LIVER All values are expressed in mg/g wet weight except lipid-bound sialic acid (#g/g wet weight). For liver the concentration of lower phase glycolipids was expressed as/~moles/g wet weight.
Total lipids Cholesterol Cholesterol esters Triglycerides Lower phase glycolipids Glucosylceramide Galactosylceramide Sulphatide Dihexosylceramide Trihexosylceramide Tetrahexosylceramide Phospholipids total phospholipids: PE a PC b SPH e PS + PI a unidentified Lipid-bound sialic acid lipid bound sialic acid: GMa GM~ GM1 GDla GDlb GT1
White matter
Grey matter
Liver
control case 1 case 2
control case 1 case 2
control case 1 case 2
107.2 31.3 . . . . 29.0 N.D. 22.1 5.5 1.4 N.D. N.D. 55.7
67.4 74.1 36.3 27.2 33.2 9.5 . . . . . . . . 28.7 28.2 10.3 N.D.N.D. N.D. 24.2 23.0 7.5 2.8 4.3 1.4 1.7 0.9 1.4 N.D.N.D. N.D. N.D.N.D. N.D. 36.0 37.5 20.8
40.1 26.6 12.0 21.9
50.9 41.9 50.3 24.7 29.6 28.9 13.9 16.5 14.1 10.5 12.1 7.0 . . . . 296 550 869
. 332
-------
.
-------
-------
N.D. N.D. 18.9 28.3 29.7 23.0
24.8 8.7
25.8 7.6
10.1 6.5 N.D.N.D. 7.6 4.6 1.9 1.2 0.6 0.6 N.D.N.D. N.D.N.D. 14.7 15.7 42.4 25.4 17.9 14.2 766
50.6 23.5 15.7 10.2 623
20.1 N.D. 12.3 N.D. 16.9 29.9 19.6 26.9 15.9 23.3 15.0 19.7
30.0 3.9 0.5 1.4 -0.04 N.D. N.D. 0.07 0.09 0.04
12.7
13.4 2.2 0.3 1.3
11.5 2.0 0.2 1.6
0.01 0.01 N.D.N.D. N.D. N.D. 0.04 0.03 0.02 0.02 0.02 0.02
8.0
5.3
22.3 52.1 8.5 15.1 1.9 120
25.5 24.9 47.8 41.6 8.4 15.4 13.9 19.6 4.3 8.5 110 110
÷ N.D. N.D. N.D. N.D. N.D.
+ ÷ N.D.N.D. N.D.N.D. N.D.N.D. N.D.N.D. N.D.N.D.
Phosphatidylethanolamine; b Phosphatidylcholine; e Sphingomyelin; d Phosphatidylserine + phosphatidylinositol. N.D. = not detectable; - - = not done; + = major ganglioside, ganglioside nomenclature according to Svennerholm (1970).
The AGAG
p a t t e r n w a s , i n all a f f e c t e d o r g a n s , c h a r a c t e r i z e d b y t h e p r e s e n c e o f
elevated HS. The biochemical analyses of the two cases are consistent. Th•AGAG
content was nearly normal in the brain. However, the distribution
of the different components was not. HS was the major AGAG
accumulating in the
brains of both patients. These results are in good agreement with the results obtained b y o t h e r i n v e s t i g a t o r s ( G e o r g e a n d B a c h h a w a t 1970; C o n s t a n t o p o u l o s
e t al. 1976). O n
the other hand in the viscera of the patients drastic changes occur both in total amount (2-49-fold increase of total AGAG) 49-91 ~ of total AGAG). o c c u r r e d as g l u c o s a m i n e .
and
in AGAG
composition
(HS represents
I n v i s c e r a o f b o t h c a s e s n e a r l y all A G A G
hexosamine
84 Although in normal liver, HS is already the major A G A G (Table 2), a pronounced increase for this compound is noted in our patients. Similar findings are reported in the literature (Shimamura et al. 1976; Constantopoulos et al. 1976). Gordon and Haust (1971) found that 93 ~o of the total hexosamine present in the liver of MPS III patients was glucosamine. This is confirmed by our hexosamine analysis but in contrast with Kindler's data (Kindler et al. 1977). In the kidney of our patients a 5-10-fold increase of HS occurred (Table 1 and Table 2). A similar value was reported by Kindler et al. (1977). However, for our patients no cortex vs. medulla study was performed to detect eventual concentration differences for those regions (Inoue, Sawada, Fukunaga and Yoshikawa 1970; Constantopoulos, Louie and Dekaban 1973; Murata 1975). In the spleen of both patients, a 5-fold increase in total A G A G was observed. This value is lower than the 10-20-fold higher concentrations found by Kindler et al. (1977) and Constantopoulos et al. (1976). In the procentual distributions of A G A G HS represented about 70 ~ but substantial amounts of DS were still present (Table 1 and Table 2). Constantopoulos et al. (1976) found 90 ~o of spleen A G A G to be HS with only 8 ~ DS and 2 ~o HA q- C-4S q- C-6S. Besides the drastic changes in A G A G content and composition of brain and viscera the results obtained for the CPC-nonprecipitable uronic acid suggest that the A G A G polymers are much smaller. As can be seen from Table 3 in viscera a 10-20fold increase in CPC-nonprecipitable uronic acid occurs. However, this phenomenon was not observed for brain tissue. In brain tissue the lower value for total lipid, mainly due to a 25-30 ~o decrease of total phospholipids, is in accordance with the neuronal depletion and the severe accompanying demyelination. The changes in lipid content were much more pronounced than reported by Dekaban and Patton (1973), who in white matter even found an increase of total phospholipids. In white matter of both patients a simultaneous decrease of the acidic lipids PIq-PS and sulphatides was observed, while the opposite phenomenon was noted in grey matter. No self evident explanation for this finding can be offered. The neutral glycolipid fraction was characterized by the presence of moderate concentrations of lactosylceramide. This moderate elevation of lactosylceramide can be explained by the reduced fl-galactosidase activity in the tissues. The lactosylceramide content in brain seems to vary, making it difficult to decide whether or not the increase in affected brain can really be regarded as a specific finding for the disease (Suzuki and Chen 1967; Kint et al. 1973). As Shimamura et al. (1976) we were not able to confirm the marked increase of polyhexosylceramides reported by Dekaban and Patton (1973). Lipid-bound sialic acid was nearly normal in the brain with the exception of the white matter in Case 2. Similar increases were found in Sanfilippo white matter by other authors (George and Bachhawat 1970; Shimamura et al. 1976). The higher percentage of monosialogangliosides (GM3 and GM2 in Case 1 and GM1 in Case 2) present in grey matter, is in agreement with data reported by Shimamura et al. (1976).
85 These less polar gangliosides are probably not fully degraded catabolites stored in the lysosomes. In the liver of our Sanfilippo patients the total lipid composition was depleted by about 30 ~ . Both cholesterol and phospholipids were decreased, while the triglyceride content was not changed. Shimamura et al. (1976) reported a 7-fold increase of triglycerides in a MPS III B liver. However, it must be stressed that the triglyceride composition of liver can vary considerably even in normal subjects (Kwiterovich, Sloan and Fredrickson 1970). In agreement with Rushton and Dawson (1977) liver from our patients with Sanfilippo disease showed normal levels of glycosphingolipids in contrast to patients with Hurler's disease where in the livers an overall 2-3-fold nonspecific elevation of all glycosphingolipids was found. Data from spleen and kidney lipids from patients suffering from MPS I I I A and MPS III C are, to our knowledge, not present in the literature and therefore comparison is impossible. From our results, it is clear that in both organs no striking changes in the lipid composition occur. For kidney from a MPS III B patient almost normal values were recorded for lipid analysis (Shimamura et al. 1976). ACKNOWLEDGEMENTS We are indebted to Prof. Dr. J. Leroy for making the clinical diagnosis. Dr. G. Dierick put the pathological material to our disposal. Prof. Dr. H. Kresse and Prof. Dr. K. von Figura performed the enzymatic assays. The skilful technical assistance of M. Van Acker is gratefully acknowledged.
REFERENCES Bitter, T. and H. Muir (1962) A modified uronic acid carbazole reaction, Anal. Biochem., 4: 330-334. Constantopoulos, G., M. Louie and A. Dekaban (1973) Acid mucopolysaccharides (glycosaminoglycans) in normal human kidneys and in kidneys of patients with mucopolysaccharidoses, Biochem. Med., 7: 376-388. Constantopoulos, G., R. McComb and A. Dekaban (1976) Neurochemistry of the mucopolysaccharidoses - - Brain glycosaminoglycansin normals and four types of mucopolysaccharidoses, J. Neurochem., 26: 901-908. Dekaban, A. and V. Patton (1973) Anatomo-pathologie et ~tude des lipides dans les mucopolysaccharidoses de type Hurler et Sanfilippo, Nouv. Presse todd., 2: 415-417. George, E. and B. Bachhawat (1970) Brain glycosaminoglycansand glycosaminoglycansulfotransferase in Sanfilippo syndrome, Clin. chim. Acta, 30: 317-324. Gordon, B. and M. Haust (1971) Hepatic acid mucopolysaccharides in the mucopolysaccharidoses type I, II and III, Clin. Biochem., 4: 147-155. Inoue, G., T. Sawada, Y. Fukunaga and M. Yoshikawa (1970) Levels of acid mucopolysaccharidesin aging human kidneys, Gerontologia, 16: 261-269. Kindler, A., U. Klein and K. yon Figura (1977) Characterization of glycosaminoglycansstored in mucopolysaccharidosisIII A--Evidence for a generally occurring degradation of heparan sulfate by endoglycosidases, Hoppe Seyler 's Z. physiol. Chem., 358 : 1431-1438. Kint, J., G. Dacremont, D. Carton, E. Orye and C. Hooft (1973)Mucopolysaccharidosis-- Secondarily induced abnormal distribution of lysosomal isoenzymes, Science, 181 : 352-354. Knight, J., S. Anderson and J. Rawle (1972) Chemical basis of the sulpho-phospho-vanillin reaction for estimating total serum lipids, Clin. Chem., 18 : 199-202. Kwiterovich, P., H. Sloan and D. Fredrickson (1970) Glycolipids and other constituents of normal human liver, J. Lipid Res., 11 : 322-330.
86 McMillan, V. and J. Wherrett (1969) Modified procedure for the analysis of mixtures of tissue gangliosides, J. Neurochem., 16: 1621-1624. Martin, J. J., G. Van Dessel, A. Lagrou, A. M. De Barsy and W. Dierick (1974) Multiple system atrophies - - A neuropathological and neurochemical study, J. neurol. Sci., 21 : 251-272. Murata, K. (1975) Acidic glycosaminoglycans in human kidney tissue, Clin. chim. Acta, 63 : 157-169. Neufeld, E. (1974) The biochemical basis for mucopolysaccharidoses and mucolipidoses. In: A. Steinberg and A. Beam (Eds.), Progress in Medical Genetics, Vol. 10, Grune and Stratton, New York, NY. Rushton, A. R. and G. Dawson (1977) The effect of glycosaminoglycans on the in vitro activity of human skin fibroblast glycosphingolipid fl-galactosidases and neuraminidases, Clin. chim. Acta., 80: 133-139. Saito, H., T. Yamagata and S. Suzuki (1968) Enzymatic methods for the determination of small quantities of isomeric chondroitin sulphates, J. bioL Chem., 243 : 1536-1542. Shimamura, K., H. Hakozaki, K. Takahashi, A. Kimura, J. Fujino, Y. Suzuki and N. Nakamura (1976) Sanfilippo B syndrome - - A case report, A cta path. jap., 26: 739-764. Suzuki, K. and G. Chen (1967) Brain ceramide hexosides in Tay-Sach's disease and generalized gangliosidosis (GMI gangliosidosis), J. Lipid Res., 8:105-113. Svennerholm, L. (1970) Ganglioside metabolism. In: M. Florkin and E. Stotz (Eds.), Comprehensive Biochemistry, Vol. 18, Elsevier, Amsterdam. Teller, W. (1967) Kolorimetrischer Nachweis von Chondroitinsulfat B im Gemisch mit anderen sauren Mucopolysacchariden, Z. klin. Chim. klin. Biochem., 5 : 29-32. Van Dessel, G., A. Lagrou, H. J. Hilderson, W. Dierick and G. Dacremont (1977) Quantitative determination of the neutral glycosyl ceramides in bovine thyroid gland, Biochimie, 59: 839-848. Wessler, E. (1968) Analytical and preparative separation of acidic glycosaminoglycans by electrophoresis in barium acetate, Anal. Biochem., 26: 439-444. Wessler, E. (1971) Electrophoresis of acidic glycosaminoglycans in hydrochloric acid - - A micro method for sulphate determination, Anal Biochem., 41 : 67-69. Yu, R. and R. Ledeen (1972)Gangliosides of human, bovine and rabbit plasma, J. Lipid. Res., 13: 680-686.
77
TWO CASES OF MUCOPOLYSACCHARIDOSIS TYPE III (SANFILIPPO). A Biochemical Study
G. VAN DESSEL, A. LAGROU, J. J. MARTIN, C. CEUTERICK and W. DIERICK UIA-Laboratory for Pathological Biochemistry and RUCA-Laboratory for Human Biochemistry; Department of Neuropathology, Born-Bunge Foundation and UIA-Department of Medicine, University of Antwerp, Antwerp (Belgium)
(Received 19 April, 1978) (Accepted 20 September, 1978)
SUMMARY The mucopolysaccharide and lipid composition of human nervous tissue and viscera from one case of Sanfilippo disease type A and one case of Sanfilippo disease type C, were investigated. In the brain a moderate increase of acid glycosaminoglycans occurred. This phenomenon was much more pronounced in the viscera, especially in the liver. In all tissues this increase was mainly due to an accumulation of heparan sulphate. Changes in lipid composition were noted, but can be regarded as secondary effects. The biochemical results reported also suggest some general conclusions. (a) A G A G and lipid analyses do not permit differentiation between the subtypes of Sanfilippo disease. (b) The differences in lipid composition can probably be considered as consequences of variation in secondary effects. (c) The severe demyelination in brain correlates well with the biochemical lipid analysis. However, in other instances it remains difficult to bridge the gap still existing between some morphological and biochemical data.
INTRODUCTION Mucopolysaccharidoses (MPS) are a group of genetic diseases characterized by storage of incompletely degraded acid glycosaminoglycans (AGAG). All the classified mucopolysaccharidoses except for the Morquio syndrome (MPS IV) and flglucuronidase deficiency (MPS VII) show a disturbance in the lysosomal catabolism Reprint requests to: Dr. G. Van Dessel, UIA-Laboratory for Pathological Biochemistry, Groenenborgerlaan 171, B-2020 Antwerp (Belgium).
78 of two polymeric substances, dermatan sulphate (DS) and heparan sulphate (HS), singly or in combination (Neufeld 1974). Few studies deal with the A G A G (Constantopoulos, M c C o m b and Dekaban 1976; Shimamura, Hakozaki, Takahashi, Kimura, Fujino, Suzuki and N a k a m u r a 1976; Kindler, Klein and von Figura 1977) and glycolipid (Dekaban and Patton 1973; Kint, Dacremont, Carton, Orye and Hooft 1973) composition of brain and viscera from Sanfilippo patients. No information is available with regard to changes in neutral and phospholipids except for one report concerning cholesterol and phospholipid content of a Sanfilippo brain (Dekaban and Patton 1973). This paper describes the lipid and mucopolysaccharide composition of brain and viscera of two patients showing the clinical features of Sanfilippo disease. CASE REPORTS Case 1 is an 11-year-old white male patient with clinical features of MPS III proven to belong to type III A by assaying heparan sulphate sulphamidase on cultured fibroblasts (Kresse and von Figura, personal communication). Light microscopy of autopsy material shows a diffuse neuronal storage in brain and dilated perivascular spaces filled with ballooned bistiocytes in the white matter. Signs of visceral storage are found in liver, spleen, kidney, lymph nodes and cartilage. Heart valves and arterial walls are also infiltrated by foam cells. Electron microscopy shows zebra bodies, membranous cytoplasmic bodies and membrano-granulo-vacuolar inclusions in neurons, glial cells, Schwann cells and myocardial fibres. Large electron-lucent vacuoles are found in liver, spleen, kidney and fibroblasts. Case 2 is a 13-year-old white male patient with clinical features of MPS III. He belongs to type III C as demonstrated by assaying a-glucosaminidase on cultured fibroblasts (von Figura, personal communication). Light- and electron-microscopic features of storage are found both in the nervous system and visceral organs. They are essentially similar to those described in Case 1. It was not possible to discover any significant morphological difference between our types IIIA and III C cases. MATERIAL Fresh brain and viscera from a control and from the two patients were obtained at autopsy. The tissues were stored at - - 2 0 °C. The enzymes papain and pronase were purchased from Sigma. The standard mucopolysaccharides hyaluronic acid (HA), chondroitin-4-sulphate (C-4S), dermatan sulphate (DS), chondroitin-6-sulphate (C6S) and heparin (Hep) as well as the enzymes chondroitinase ABC and chondroitinase AC were obtained from Miles Laboratories. The cellulose polyac~tate strips (Sepraphore III) were from Gelman Instruments. All chemicals were analytical grade and used without further purification. METHODS
Mucopolysaccharide analysis (i) Isolation ofglycosaminoglycans. The tissues were defatted through successive homogenization with 20 volumina chloroform:methanol 2/1 (v/v), 10 volumina 1/1 (v/v) and 10 volumina 1/2 (v/v). After each step the mixture was filtered through sintered glass of medium porosity and the extracts were combined. These chloroformmethanol extracts were used for lipid analysis. The defatted tissue was subjected to proteolytic digestion with papain and pronase (25 mg enzyme/g delipidated tissue;
79 0.1 M acetate buffer pH 5.5; 60 °C; overnight). At the end of incubation the mixture was centrifuged at 10,000 x g for 15 min and the supernatant collected. The sediment was washed with 2 ml acetate buffer and the washing combined with the first supernatant. The AGAG were isolated by precipitation with 5 ~ cetylpyridiniumchloride (CPC) (4 °C, overnight). The resulting AGAG-CPC complexes were spun down (10,000 × g for 10 min) and treated with 20 ml 10 Yopotassium acetate in 95 ~o ethanol. After standing for 1 hr and centrifugation (10,000 x g for 10 min) the sediment was washed successively with 20 ml ethanol and 20 ml diethylether and finally dried at room temperature. The precipitate was dissolved in 0.05 M NaOH and allowed to stand for at least 1 hr at 4 °C. After centrifugation (I0,000 x g for 10 min) the supernatant yields the AGAG extract used for further analysis. CPC-non precipitable AGAG degradation products were also quantitated in the supernatant obtained after CPC addition. (ii) Constituent analysis. Uronic acid was determined by the carbazole reaction as modified by Bitter and Muir (1962) using glucuronolactone as a standard. For DS identification the carbazole reaction was carried out with and without borate since iduronic acid gives a very low colour yield without borate. DS was also assayed by the naphthoresorcinol method (Teller 1967). For the analysis of N-substituted hexosamines the AGAG fractions were hydrolysed in aqueous 4 N HCI for 3 hr at 110 °C and the hydrolysates dried in vacuo over PeO5 and NaOH. The residue was dissolved in citrate buffer pH 2.2. The hexosamine content and ratio glucosamine/galactosamine were determined on an amino acid analyser with L-a-aminofl-guanido-propionic acid as internal standard using Aminex A5 cation exchanger (1.5 x 14 cm) (Van Dessel, Lagrou, Hilderson, Dierick and Dacremont 1977). It must be stressed that considerable losses of hexosamine occur during the hydrolysis procedure. Therefore a correction has been applied. (iii) Estimation of AGAG composition. After enzymic depolymerization with the eliminases chondroitinase AC and chondroitinase ABC, the susceptibility of the AGAG to the two enzymes was examined by measuring the uronic acid content of the released disaccharides. (Constantopoulos et al. 1976). Chondroitinase AC specifically digests chondroitin, HA, C-4S and C-6S to the unsaturated nonsulphated, 4-sulphated and 6-sulphated disaccharides, respectively, but does not attack DS (Saito, Yamagata and Suzuki 1968). The chondroitinase ABC digests in supplement to the AGAG mentioned above, also DS to the unsaturated 4-sulphate disaccharide. The uronic acid not susceptible to the two enzymes represents HS. The AGAG mixtures (50/~g AGAG-uronic acid) were subjected to enzymic degradation with chondroitinase AC (0.05 I.U.) and chondroitinase ABC (0.1 I.U.) in enriched Tris buffer, pH 8.0 (total volume of 200/~1) (Saito et al. 1968). The time of incubation was 1 hr at 37 °C for the enzymic digestion with chondroitinase AC and 4 hr at 37 °C for the other incubations. The reactions were stopped by boiling for 2 min, whereafter the mixtures were cooled and 100/~1 of 3.0 M NaC1 and 1.4 ml of 95 ~ ethanol added. After standing 1 hr at 4 °C the tubes were centrifuged (10,000 × g for 10 min) and uronic acid content of the released disaccharides measured in the supernatants by the carbazole-borate method (Saito et al. 1968).
80
(iv) Electrophoresis. Electrophoresis on Sepraphore membrane was carried out following Wessler's method (Wessler 1968, 1971) in 0.05 M barium acetate [75 min; potential gradient (8V/cm)] and in 0.1 M HC1 (37 min applying the same voltage). Lipid analysis (i) Lipid extraction and fractionation. The crude lipid extracts, prepared as previously described (Martin, Van Dessel, Lagrou, De Barsy and Dierick 1974) were separated in a neutral, a glycolipid and a phospholipid fraction by column chromatography on Biosil A (200--400 mesh) (Van Dessel et al. 1977).
Analytical methods Total lipids were estimated by the phosphovanillin colorimetric assay (Knight, Anderson and Rawle 1972). Free and esterified cholesterol, total phospholipids and phospholipid patterns were assayed as reported earlier (Martin et al. 1974). Glycolipids. Following silicic acid chromatography and alkaline saponification (Van Dessel et al. 1977) ceramide hexosides were separated by TLC on silicagel G using C :M :H20 65/25/4 (v/v/v) as eluent. The amount of glycolipid was determined by GLC of the trimethylsilylether derivatives of the methylglycosides (Van Dessel et al. 1977). Sulphatides and neutral glycolipids were separated on DEAE-Sephadex (Yu and Ledeen 1972). Gangliosides. Total lipid-bound sialic acid in the Folch upper phase was determined by the thiobarbituric acid assay (Van Dessel et al. 1977). Ganglioside patterns were determined after separation on silicagel G with C :M :NH3 2.5 N 60:35:8 (v/v/v) as eluent. Quantitative determination of the individual gangliosides was performed applying Suzuki's method as modified by McMillan and Wherrett (1969). RESULTS
Acid glycosaminoglycans The A G A G concentrations in brain, liver, spleen and kidney are represented in Table 1. In comparison with normal tissue the A G A G content in the organs of the TABLE 1 A G A G CONTENT IN BRAIN A N D VISCERA
Brain (l)
Control 0.5 Case 1 1.1 Case2 0.5
Liver
Spleen
(2)
(1)
(2)
0.5 1.3 0.5
0.9 0.1 27.5 25.4 44.1 24.9
(1)
Kidney (2)
1.9 0.2 9.2 6.1 11.2 5.0
(1)
(2)
2.7 0.7 10.7 N.D. 7.5 2.1
(1) AGAG expressed as mg uronic acid/g defatted tissue. (2) Total N-substituted hexosamines in mg/g defatted tissue. N.D. ~ not determined because of lack of material.
81 TABLE 2 COMPOSITION a OF AGAG IN BRAIN AND VISCERA Brain
Liver
Spleen
Kidney
(a)
(b)
(c)
(a)
(b)
(c)
(a)
(b)
(c)
(a)
(b)
(c)
Control 74 Case 1 44 Case 2 42
14 7 5
12 49 53
20 9 2
25 4 7
55 87 91
15 2 4
36 29 23
49 69 73
47 27 20
16 9 13
37 64 67
(a) (b) (c) a
Hyaluronic acid ÷ chondroitin-4-sulphate ÷ chondroitin-6-sulphate. Dermatan sulphate. Heparan sulphate. Expressed as ~o of total uronic acid.
patients was much higher, especially in the liver, where a 30-50-fold elevation was noted. The other organs contained 2-12 times as much as the controls. In the brain of one patient the A G A G concentration was nearly normal. The A G A G composition of brain and viscera as derived from their susceptibility to chondroitinases AC and ABC is shown in Table 2. In normal brain predominance of HA, C-4S and C-6S is evident. By contrast in the brain of the two Sanfilippo patients a fifty-fifty distribution is found for this group vs. HS. This is also substantiated by the higher intensity of the spot in the electropherogram migrating just ahead of the Hep standard. It is interesting to note that although there is no marked difference in the total A G A G content in the brain, there is a significant change in the procentual distribution: in normal brain HA, C-4S and C-6S comprise 74 ~o, HS 12 % and DS 14 % of the total A G A G ; in the affected brains a 4fold increase of the HS content was observed, mainly reflected in the concomitant decrease of HA, C-4S and C-6S. In both patients 85 % of AGAG-hexosamines was identified as glucosamine, confirming the major brain A G A G (4- 50 % of total) to be HS. Compared to the normal values the A G A G in affected liver were increased 30-50-fold (Table 1). Table 2 shows that even in normal liver HS is the main A G A G (55 %). The HS content for both patients, however, amounted to 87-91%. This agrees with the determination of the N-substituted hexosamines (Table 1). The galactosamine content was beyond the detection limit indicating a drastic decrease of C-4S, C-6S and DS containing this hexosamine. The electrophoretic pattern of liver A G A G is characterized by the presence of a very intense spot having the same migration rate as HS. The total A G A G content was elevated 5-6 times in the spleen of both patients. HA, C-4S and C-6S content ( A G A G susceptible to chondroitinase AC) is drastically reduced (Table 2). A 40-50% increase in HS was observed. The results are in agreement with the electrophoretic pattern obtained for spleen total AGAG. Chemical analyses of the A G A G isolated from the normal and phatological human kidneys are tabulated in Tables 1 and 2. The predominant hexosamine in kidneys was glucosamine. The enzymatic degradation of the A G A G showed that
82 TABLE 3 CPC-NONPRECIPITABLE AGAG DEGRADATION PRODUCTS a IN BRAIN AND VISCERA
Control Case 1 Case 2
Brain (whole) Liver
Spleen
Kidney
275 371 312
72 600 984
48 1020 972
48 1092 780
a Expressed as uronic acid (/~g/g wet weight).
chondroitinase AC hydrolysed substantial amounts of the A G A G from normal kidney (47 ~). From pathological kidneys only a minor fraction of the A G A G (20-27 ~ ) was hydrolysed with chondroitinase AC. The results indicate that HS is the major A G A G of normal and pathological kidney, but is more elevated in the affected organs. Again the results were confirmed by the electrophoresis experiments. Liver, spleen and kidney of our patients contained large amounts of CPCnonprecipitable A G A G degradation products (Table 3), while in brain only a slight increase was apparent.
Lipids No uronic acid was found in the lipid extracts. The lipid composition of white matter is summarized in Table 4. A marked decrease in total lipids and phospholipid was noted. Cholesterol was the main neutral lipid component with no increase of cholesterol esters. Total glycolipid was within the normal range. Nevertheless the ratio cerebrosides/sulphatides for Case 1 was raised up to 8.5. The presence of minor quantities of lactosylceramide could be demonstrated, but was also observed in the control tissue. The phospholipid patterns looked nearly normal in both cases with the exception of a decrease for phosphatidylinositol (PI) and phosphatidylserine (PS). The content of lipid-bound sialic acid was normal for Case 1 and increased for Case 2. The results of the chemical analyses of grey matter are also represented in Table 4. The same general tendency was noted as in white matter. However, in grey matter, an increase in PI and PS occurred while decrease in total glycolipids was noted only for Case 2. The ganglioside content was nearly normal, although in Case 1 relatively high concentrations of less polar gangliosides were found (Table 4). The lipid content of liver is given in Table 4. These analyses revealed for both patients a decrease of total lipid, cholesterol, glycolipids and phospholipids. The phospholipid pattern was normal. Like in normal liver on thin layer chromatography (TLC) only a ganglioside with the same R/-value as GMa could be detected. Spleen and kidney showed no differences in lipid composition from the controls. DISCUSSION In all tissues of the patients reported here (except for the brain of Case 2) an absolute increase of A G A G content was observed being most pronounced in the liver.
83 TABLE 4 B I O C H E M I C A L ANALYSIS O F LIPIDS IN B R A I N A N D LIVER All values are expressed in mg/g wet weight except lipid-bound sialic acid (#g/g wet weight). For liver the concentration of lower phase glycolipids was expressed as/~moles/g wet weight.
Total lipids Cholesterol Cholesterol esters Triglycerides Lower phase glycolipids Glucosylceramide Galactosylceramide Sulphatide Dihexosylceramide Trihexosylceramide Tetrahexosylceramide Phospholipids total phospholipids: PE a PC b SPH e PS + PI a unidentified Lipid-bound sialic acid lipid bound sialic acid: GMa GM~ GM1 GDla GDlb GT1
White matter
Grey matter
Liver
control case 1 case 2
control case 1 case 2
control case 1 case 2
107.2 31.3 . . . . 29.0 N.D. 22.1 5.5 1.4 N.D. N.D. 55.7
67.4 74.1 36.3 27.2 33.2 9.5 . . . . . . . . 28.7 28.2 10.3 N.D.N.D. N.D. 24.2 23.0 7.5 2.8 4.3 1.4 1.7 0.9 1.4 N.D.N.D. N.D. N.D.N.D. N.D. 36.0 37.5 20.8
40.1 26.6 12.0 21.9
50.9 41.9 50.3 24.7 29.6 28.9 13.9 16.5 14.1 10.5 12.1 7.0 . . . . 296 550 869
. 332
-------
.
-------
-------
N.D. N.D. 18.9 28.3 29.7 23.0
24.8 8.7
25.8 7.6
10.1 6.5 N.D.N.D. 7.6 4.6 1.9 1.2 0.6 0.6 N.D.N.D. N.D.N.D. 14.7 15.7 42.4 25.4 17.9 14.2 766
50.6 23.5 15.7 10.2 623
20.1 N.D. 12.3 N.D. 16.9 29.9 19.6 26.9 15.9 23.3 15.0 19.7
30.0 3.9 0.5 1.4 -0.04 N.D. N.D. 0.07 0.09 0.04
12.7
13.4 2.2 0.3 1.3
11.5 2.0 0.2 1.6
0.01 0.01 N.D.N.D. N.D. N.D. 0.04 0.03 0.02 0.02 0.02 0.02
8.0
5.3
22.3 52.1 8.5 15.1 1.9 120
25.5 24.9 47.8 41.6 8.4 15.4 13.9 19.6 4.3 8.5 110 110
÷ N.D. N.D. N.D. N.D. N.D.
+ ÷ N.D.N.D. N.D.N.D. N.D.N.D. N.D.N.D. N.D.N.D.
Phosphatidylethanolamine; b Phosphatidylcholine; e Sphingomyelin; d Phosphatidylserine + phosphatidylinositol. N.D. = not detectable; - - = not done; + = major ganglioside, ganglioside nomenclature according to Svennerholm (1970).
The AGAG
p a t t e r n w a s , i n all a f f e c t e d o r g a n s , c h a r a c t e r i z e d b y t h e p r e s e n c e o f
elevated HS. The biochemical analyses of the two cases are consistent. Th•AGAG
content was nearly normal in the brain. However, the distribution
of the different components was not. HS was the major AGAG
accumulating in the
brains of both patients. These results are in good agreement with the results obtained b y o t h e r i n v e s t i g a t o r s ( G e o r g e a n d B a c h h a w a t 1970; C o n s t a n t o p o u l o s
e t al. 1976). O n
the other hand in the viscera of the patients drastic changes occur both in total amount (2-49-fold increase of total AGAG) 49-91 ~ of total AGAG). o c c u r r e d as g l u c o s a m i n e .
and
in AGAG
composition
(HS represents
I n v i s c e r a o f b o t h c a s e s n e a r l y all A G A G
hexosamine
84 Although in normal liver, HS is already the major A G A G (Table 2), a pronounced increase for this compound is noted in our patients. Similar findings are reported in the literature (Shimamura et al. 1976; Constantopoulos et al. 1976). Gordon and Haust (1971) found that 93 ~o of the total hexosamine present in the liver of MPS III patients was glucosamine. This is confirmed by our hexosamine analysis but in contrast with Kindler's data (Kindler et al. 1977). In the kidney of our patients a 5-10-fold increase of HS occurred (Table 1 and Table 2). A similar value was reported by Kindler et al. (1977). However, for our patients no cortex vs. medulla study was performed to detect eventual concentration differences for those regions (Inoue, Sawada, Fukunaga and Yoshikawa 1970; Constantopoulos, Louie and Dekaban 1973; Murata 1975). In the spleen of both patients, a 5-fold increase in total A G A G was observed. This value is lower than the 10-20-fold higher concentrations found by Kindler et al. (1977) and Constantopoulos et al. (1976). In the procentual distributions of A G A G HS represented about 70 ~ but substantial amounts of DS were still present (Table 1 and Table 2). Constantopoulos et al. (1976) found 90 ~o of spleen A G A G to be HS with only 8 ~ DS and 2 ~o HA q- C-4S q- C-6S. Besides the drastic changes in A G A G content and composition of brain and viscera the results obtained for the CPC-nonprecipitable uronic acid suggest that the A G A G polymers are much smaller. As can be seen from Table 3 in viscera a 10-20fold increase in CPC-nonprecipitable uronic acid occurs. However, this phenomenon was not observed for brain tissue. In brain tissue the lower value for total lipid, mainly due to a 25-30 ~o decrease of total phospholipids, is in accordance with the neuronal depletion and the severe accompanying demyelination. The changes in lipid content were much more pronounced than reported by Dekaban and Patton (1973), who in white matter even found an increase of total phospholipids. In white matter of both patients a simultaneous decrease of the acidic lipids PIq-PS and sulphatides was observed, while the opposite phenomenon was noted in grey matter. No self evident explanation for this finding can be offered. The neutral glycolipid fraction was characterized by the presence of moderate concentrations of lactosylceramide. This moderate elevation of lactosylceramide can be explained by the reduced fl-galactosidase activity in the tissues. The lactosylceramide content in brain seems to vary, making it difficult to decide whether or not the increase in affected brain can really be regarded as a specific finding for the disease (Suzuki and Chen 1967; Kint et al. 1973). As Shimamura et al. (1976) we were not able to confirm the marked increase of polyhexosylceramides reported by Dekaban and Patton (1973). Lipid-bound sialic acid was nearly normal in the brain with the exception of the white matter in Case 2. Similar increases were found in Sanfilippo white matter by other authors (George and Bachhawat 1970; Shimamura et al. 1976). The higher percentage of monosialogangliosides (GM3 and GM2 in Case 1 and GM1 in Case 2) present in grey matter, is in agreement with data reported by Shimamura et al. (1976).
85 These less polar gangliosides are probably not fully degraded catabolites stored in the lysosomes. In the liver of our Sanfilippo patients the total lipid composition was depleted by about 30 ~ . Both cholesterol and phospholipids were decreased, while the triglyceride content was not changed. Shimamura et al. (1976) reported a 7-fold increase of triglycerides in a MPS III B liver. However, it must be stressed that the triglyceride composition of liver can vary considerably even in normal subjects (Kwiterovich, Sloan and Fredrickson 1970). In agreement with Rushton and Dawson (1977) liver from our patients with Sanfilippo disease showed normal levels of glycosphingolipids in contrast to patients with Hurler's disease where in the livers an overall 2-3-fold nonspecific elevation of all glycosphingolipids was found. Data from spleen and kidney lipids from patients suffering from MPS I I I A and MPS III C are, to our knowledge, not present in the literature and therefore comparison is impossible. From our results, it is clear that in both organs no striking changes in the lipid composition occur. For kidney from a MPS III B patient almost normal values were recorded for lipid analysis (Shimamura et al. 1976). ACKNOWLEDGEMENTS We are indebted to Prof. Dr. J. Leroy for making the clinical diagnosis. Dr. G. Dierick put the pathological material to our disposal. Prof. Dr. H. Kresse and Prof. Dr. K. von Figura performed the enzymatic assays. The skilful technical assistance of M. Van Acker is gratefully acknowledged.
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