- Email: [email protected]
Leukocyte hydrolases in the Sanfilippo syndrome
Clin. Biochem. 4, 45-48 (1971)
NOTE LEUKOCYTE HYDROLASES IN THE SANFILIPPO SYNDROME* BRUCE A. GORDONt AND VERA FELEKI
Department of Biochemistry, Children's Psychiatric Research Institute and University of Western Ontario, London (Received October 18, 1970) EXAMINATION OF THE CIRCULATING LEUKOCYTE has been of considerable value in the diagnosis of the mucopolysaccharidoses since in these diseases the white blood cell has been shown to contain various characteristic metachromatic inclusions, e.g. Reilly's and Mittwoch's granules (1). Accumulating mucopolysaccharides in tissues such as the liver appear in abnormal organelles some of which resemble lysosomes (2-6). Those enzymes known to participate in mucopolysaccharide catabolism are localized within the lysosome (5). Assays of the activities of a series of lysosomal hydrolases from the skin (6-8), liver (9-12), kidney, brain, spleen (12), plasma (6, 7) or serum (11) from patients with mucopolysaccharidoses have demonstrated alterations in the activity of several. In view of the propensity of the leukocyte to accumulate mucopolysaccharides either in vivo (1, 18) or in culture systems (16) we have examined the activity of several acid hydrolases in leukocytes from patients with the Sanfilippo syndrome (type III mucopolysaccharidosis (16)). The level of these hydrolases in serum or liver from patients with type I (Hurler syndrome) or III mucopolysaccharidosis was shown to be altered in earlier studies (11). Leukocyte samples were collected from 12 patients with the Sanfilippo syndrome. Biochemical and clinical features of these patients are described in another publication (16). Control leukocyte samples were collected from 27 children admitted to this institute for assessment and short term care who presented with mental retardation and/or behavioral problems but without evidence of an inherited metabolic disease. The leukocyte suspension was prepared as outlined by Kampine et al. (17). After the final saline wash the leukocytes were suspended in 1.0 ml of isotonic saline and this suspension was diluted 1 in 10 for/%galactosidase and aryl sulfatase A assays, 1 in 20 for/~-glucuronidase, and/3-N-acetylglucosamindase assays and 1 in 40 for acid phosphatase assays. The various enzyme activities were measured as outlined previously (11) except aryl sulfatase A where the assay procedure of Percy and Brady (18) was utilized. The enzyme activities assayed in the leukocytes from the Sanfilippo patients are compared with normal control values in Table 1. The mean acid phosphatase, /3-galactosidase and /3-glucuronidase activities in the patients' cells were 730-/0, 740-/0 and 79% respectively of that in normal leukocytes. These differences were *Supported by grant MA 2326 from the Medical Research Council. tOntario Mental Health Foundation Research Scholar.
GORDON & FELEKI
46
TABLE 1 ACID HYDROLASESIN LEUKOCYTES'OFPATIENTSWITHSANFILIPPO SYNDROME Acid Phosphatase t,M/mg/min
#-N-acetylglucosaminidase /,M/mg/min
JMc IMc RD KH SH DM WL GL GR BL KW DR
0.039 0.048 0.035 0.026 0.044 0.040 0.078 0.037 0.047 0.019 0.036 0.062
0.012 0.017 0.013 0.025 0.032 0.020 0.010 0.014 0.015 0.022 0.026 0.015
0.30 0.30 0.27 0.50 0.47 0.43 0.46 0.37 0.48 0.46 0.48 0.47
Mean 4- s.e.m. n = 12
0.043 -4-0.0044
0.018 :t:0.0020
0.42 +0.023
0.059 4-0.0033 P < 0.01
0.021 :t:0.0010
0.53 4-0.024 P < 0.01
Patient
Control Mean 4- s.e.m. n = 27
#-glucuron- /3-galactos- Aryl Sulfatase idase idase /~ m/,M/mg/min mt~M/mg/min t,M/mg/min 0.191 0.200 0.112 0.152 0.147 0.114 0.142 0.095 0.078 0.163 0.200 0.122
0.300 0.423 0.561 0.738 0. 799 0.655 0.887 0.352 0.781 1.186 0.833 1.178
0.143 -4-0.011
0.724 4-0.082
0.194 4-0.0096 P < 0.01
0.772 :t:0.051
significant at the 1% level. The mean activities of/~-N-acetylglucosaminidase and aryl sulfatase A in patients' leukocytes although reduced were not significantly different from the normal. Reduced levels of/~-galactosidase activity have been described in the skin ((6-8), liver (9-12), brain, kidney and spleen (12) from patients with mucopolysaccharidoses while in plasma or serum the activity was increased (6, 7) or normal (11). The reduction observed in fl-galactosidase activity in leukocytes in the present study parallels the changes seen in other tissues in the mucopolysaccharidoses. MacBrinn and her associates have shown that the reduced ~-galactosidase activity apparent when assays are carried out with a synthetic substrate is also obvious if the natural substrates, ganglioside Gm, or a "keratan sulfate-like" mucopolysaccharide are employed (12). Hultberg and 0ckerman have studied the B-galactosidase isoenzymes in liver from control and mucopolysaccharidoses patients and in the liver from the latter have found a specific deficiency of the /~-galactosidase isoenzyme A (19, ~0). A number of other glycohydrolases particularly /~-glucuronidase and /%N-acetylglucosaminidase were increased in the tissues i.e. skin (6, 7), liver (9, 1~), brain, kidney and spleen (1~), and in plasma (6, 7) or serum (11) in the mucopolysaccharidoses. Reduced activity of the B-glucuronidase in the leukocyte in the Sanfilippo syndrome contrasts with the uniform elevations of this enzymic activity in other tissues in this and other mucopolysaccharidoses. Aryl sulfatase A activity was increased in both serum and liver in the type III mucopolysaccharidoses (11). Acid phosphatase activity
LEUKOCYTE HYDROLASES IN SANFILIPPO SYNDROME
47
was found to be reduced in the leukocytes b u t it was not altered significantly in either s e r u m or liver in the t y p e I I I disease although it was reduced in the s e r u m and increased in the liver in t y p e I disease (11). T h e reduced tissue O-galactosidase a c t i v i t y m i g h t be a p r i m a r y e v e n t in the etiology of these diseases however the variable although generally m o d e r a t e s e v e r i t y of the defect (6, 12), the occurrence of this single metabolic fault in t y p e s I and I I as well as t y p e I I I mucopolysaccharidosis (6, 12) where there is a genetically distinctive inheritance p a t t e r n (15), and the i m p e r f e c t correlation between the a c t i v i t y of this e n z y m e and the a c c u m u l a t i n g storage material are difficult to rationalize with this possibility. An a l t e r n a t i v e explanation m a y be t h a t the altered activities of the lysomal e n z y m e s are s e c o n d a r y events consequent to the a c c u m u l a t i o n of acid m u c o p o l y s a c c h a r i d e in the tissue. Some degree of tissue specificity could explain the reduced acid p h o s p h a t a s e a n d O-glucuronidase observed in the leukocyte in the Sanfilippo s y n d r o m e whereas in all o t h e r tissues in the mucopolysaccharidoses levels of this former e n z y m e are normal while t h a t of the latter are elevated. W h e t h e r the reduced tissue/3-galactosidase is the cause or an effect of the a c c u m u l a t i o n of acid m u c o p o l y s a c c h a r i d e in the mucopolysaccharidoses, d e m o n s t r a t i o n of this a b n o r m a l i t y in the leukocyte emphasizes the v a l i d i t y of e m p l o y i n g this readily available cell in the further studies necessary to define the underlying defect in these diseases. REFERENCES 1. M ITTWOCH, U. Abnormal lymphocytes in gargoylism. Brit. J. Haemat., 5, 365--368 (1959). 2. HERS, H.G. Inborn lysosomal diseases. Gastroenterol, 48, 625--633 (1965). 3. LAGUNOFF, D., & GRITZKA, T . L . The site of mucopolysaccharide accumulation in Hurler's syndrome. An electron microscopic and hlstochemical study. Lab. Investigation, 15, 1578-1588 (1966). 4. HAUST, M. D., ORIZAGA, M., BRYAN, A. M. & FRANK, H . F . The fine structure of liver in children with Hurler's syndrome. Exptl. Mol. Path., 1O, 141-161 (1969). 5. ARONSON, N. N. JR. & DE DUVE, C. Digestive activity of lysosomes. II. Digestion of macromolecular carbohydrates by extracts of rat liver lysosomes. J. Biol. Chem., 243, 45644573 (1968). 6. (~CKERMAN, P. A. Acid hydrolases in skin and plasma in gargoylism. Deficiency of 0-galactosidase in skin. Clin. Chim. Acta, 20, 1-6 (1968). 7. (~CKERMAN, P. A. & KOHLIN, P. Glycosidases in skin and plasma in Hunter's syndrome. Acta Paed. Scand., 57, 281-284 (1968). 8. GERICH, J.E. Hunter's syndrome Beta-galactosidase deficiency in skin. N. Eng. J. Med. 280, 799-802 (1969). 9. OCKERMAN, P . A . Lysosomal acid hydrolases in the liver in gargoylism. Deficiency of 4-metbylumbelliferyl-0-galactosidase. Scand. J. Clin. Lab. Invest., 22, 142-146 (1968). 10. VAN HOOF, F. & HERS, H.G. Abnormal lysosomal enzymes in mucopolysaccharidoses. European J. Biochem., 7, 34--44 (1968). 11. GORDON, B. A. & FELEKI, V. Acid hydrolases in the serum in mucopolysaccharidoses type I and III. Clin. Biochem., 3, 193-202 (1970). 12. MAcBRINN, M., OKADA, S., WOOLLACOTT, M., PATEL, V., HO, M. W., TAPPEL, A. L. & O'BRIEN, J . S . Beta-galactosidase deficiency in the Hurler syndrome. N. Eng. J. Med., 281,338-343 (1969). 13. ALBARRACIN, N. & HAUST, M.D. Unpublished observations. 14. FOLEY, K. M., DANES, B. S. & BEARN, A.G. White blood-cell cultures in genetic studies on human mucopolysaccharidoses. Science, 164, 424--426 (1969). 15. McCUSICK, V. A., KAPLAN, D., WISE, D., HANLEY, W. B., SUDDARTH, S. B., SEVICK, M. E. & MAUMANEE, A. E. The genetic mucopolysaccharidoses. Medicine, 44, 445-483 (1965).
48
GORDON & F E L E K I
16. GORDON, B. A. & HAUST, M . D . 17. 18. 19.
20.
The mucopolysaccharidoses type I, II and III. Urinary findings in 23 cases. Clin. Biochem., 3, 2.08-215 (1970). KAMPINE, J. P., BRADY, R. O., KANFER, J. N., FELD, M. & SHAPIRO, D. Diagnosis of Gaucher's disease and Niemann-Pick disease with small samples of venous blood. Science, 155, 86-88 (1966). PERCY, A. K. & BRADY, R . O . Metachromatic leukdystrophy: Diagnosis with samples of venous blood. Science, 161, 594-595 (1968). HULTBERG, B. & 0CKERMAN, P.A. Properties of 4-methylumbelliferyl-B-galactosidase activities in human liver. Scand. J. Clin. Lab. Invest., 23, 218-217 (1969). 0CKERMAN, P. A. & HULTBERG, B. Fractionation of 4-methylumbelliferyl-;~-galactosidase activities in liver in gargoylism. Scand. J. Clin. Lab. Invest. 22, 199 (1968).
NOTE LEUKOCYTE HYDROLASES IN THE SANFILIPPO SYNDROME* BRUCE A. GORDONt AND VERA FELEKI
Department of Biochemistry, Children's Psychiatric Research Institute and University of Western Ontario, London (Received October 18, 1970) EXAMINATION OF THE CIRCULATING LEUKOCYTE has been of considerable value in the diagnosis of the mucopolysaccharidoses since in these diseases the white blood cell has been shown to contain various characteristic metachromatic inclusions, e.g. Reilly's and Mittwoch's granules (1). Accumulating mucopolysaccharides in tissues such as the liver appear in abnormal organelles some of which resemble lysosomes (2-6). Those enzymes known to participate in mucopolysaccharide catabolism are localized within the lysosome (5). Assays of the activities of a series of lysosomal hydrolases from the skin (6-8), liver (9-12), kidney, brain, spleen (12), plasma (6, 7) or serum (11) from patients with mucopolysaccharidoses have demonstrated alterations in the activity of several. In view of the propensity of the leukocyte to accumulate mucopolysaccharides either in vivo (1, 18) or in culture systems (16) we have examined the activity of several acid hydrolases in leukocytes from patients with the Sanfilippo syndrome (type III mucopolysaccharidosis (16)). The level of these hydrolases in serum or liver from patients with type I (Hurler syndrome) or III mucopolysaccharidosis was shown to be altered in earlier studies (11). Leukocyte samples were collected from 12 patients with the Sanfilippo syndrome. Biochemical and clinical features of these patients are described in another publication (16). Control leukocyte samples were collected from 27 children admitted to this institute for assessment and short term care who presented with mental retardation and/or behavioral problems but without evidence of an inherited metabolic disease. The leukocyte suspension was prepared as outlined by Kampine et al. (17). After the final saline wash the leukocytes were suspended in 1.0 ml of isotonic saline and this suspension was diluted 1 in 10 for/%galactosidase and aryl sulfatase A assays, 1 in 20 for/~-glucuronidase, and/3-N-acetylglucosamindase assays and 1 in 40 for acid phosphatase assays. The various enzyme activities were measured as outlined previously (11) except aryl sulfatase A where the assay procedure of Percy and Brady (18) was utilized. The enzyme activities assayed in the leukocytes from the Sanfilippo patients are compared with normal control values in Table 1. The mean acid phosphatase, /3-galactosidase and /3-glucuronidase activities in the patients' cells were 730-/0, 740-/0 and 79% respectively of that in normal leukocytes. These differences were *Supported by grant MA 2326 from the Medical Research Council. tOntario Mental Health Foundation Research Scholar.
GORDON & FELEKI
46
TABLE 1 ACID HYDROLASESIN LEUKOCYTES'OFPATIENTSWITHSANFILIPPO SYNDROME Acid Phosphatase t,M/mg/min
#-N-acetylglucosaminidase /,M/mg/min
JMc IMc RD KH SH DM WL GL GR BL KW DR
0.039 0.048 0.035 0.026 0.044 0.040 0.078 0.037 0.047 0.019 0.036 0.062
0.012 0.017 0.013 0.025 0.032 0.020 0.010 0.014 0.015 0.022 0.026 0.015
0.30 0.30 0.27 0.50 0.47 0.43 0.46 0.37 0.48 0.46 0.48 0.47
Mean 4- s.e.m. n = 12
0.043 -4-0.0044
0.018 :t:0.0020
0.42 +0.023
0.059 4-0.0033 P < 0.01
0.021 :t:0.0010
0.53 4-0.024 P < 0.01
Patient
Control Mean 4- s.e.m. n = 27
#-glucuron- /3-galactos- Aryl Sulfatase idase idase /~ m/,M/mg/min mt~M/mg/min t,M/mg/min 0.191 0.200 0.112 0.152 0.147 0.114 0.142 0.095 0.078 0.163 0.200 0.122
0.300 0.423 0.561 0.738 0. 799 0.655 0.887 0.352 0.781 1.186 0.833 1.178
0.143 -4-0.011
0.724 4-0.082
0.194 4-0.0096 P < 0.01
0.772 :t:0.051
significant at the 1% level. The mean activities of/~-N-acetylglucosaminidase and aryl sulfatase A in patients' leukocytes although reduced were not significantly different from the normal. Reduced levels of/~-galactosidase activity have been described in the skin ((6-8), liver (9-12), brain, kidney and spleen (12) from patients with mucopolysaccharidoses while in plasma or serum the activity was increased (6, 7) or normal (11). The reduction observed in fl-galactosidase activity in leukocytes in the present study parallels the changes seen in other tissues in the mucopolysaccharidoses. MacBrinn and her associates have shown that the reduced ~-galactosidase activity apparent when assays are carried out with a synthetic substrate is also obvious if the natural substrates, ganglioside Gm, or a "keratan sulfate-like" mucopolysaccharide are employed (12). Hultberg and 0ckerman have studied the B-galactosidase isoenzymes in liver from control and mucopolysaccharidoses patients and in the liver from the latter have found a specific deficiency of the /~-galactosidase isoenzyme A (19, ~0). A number of other glycohydrolases particularly /~-glucuronidase and /%N-acetylglucosaminidase were increased in the tissues i.e. skin (6, 7), liver (9, 1~), brain, kidney and spleen (1~), and in plasma (6, 7) or serum (11) in the mucopolysaccharidoses. Reduced activity of the B-glucuronidase in the leukocyte in the Sanfilippo syndrome contrasts with the uniform elevations of this enzymic activity in other tissues in this and other mucopolysaccharidoses. Aryl sulfatase A activity was increased in both serum and liver in the type III mucopolysaccharidoses (11). Acid phosphatase activity
LEUKOCYTE HYDROLASES IN SANFILIPPO SYNDROME
47
was found to be reduced in the leukocytes b u t it was not altered significantly in either s e r u m or liver in the t y p e I I I disease although it was reduced in the s e r u m and increased in the liver in t y p e I disease (11). T h e reduced tissue O-galactosidase a c t i v i t y m i g h t be a p r i m a r y e v e n t in the etiology of these diseases however the variable although generally m o d e r a t e s e v e r i t y of the defect (6, 12), the occurrence of this single metabolic fault in t y p e s I and I I as well as t y p e I I I mucopolysaccharidosis (6, 12) where there is a genetically distinctive inheritance p a t t e r n (15), and the i m p e r f e c t correlation between the a c t i v i t y of this e n z y m e and the a c c u m u l a t i n g storage material are difficult to rationalize with this possibility. An a l t e r n a t i v e explanation m a y be t h a t the altered activities of the lysomal e n z y m e s are s e c o n d a r y events consequent to the a c c u m u l a t i o n of acid m u c o p o l y s a c c h a r i d e in the tissue. Some degree of tissue specificity could explain the reduced acid p h o s p h a t a s e a n d O-glucuronidase observed in the leukocyte in the Sanfilippo s y n d r o m e whereas in all o t h e r tissues in the mucopolysaccharidoses levels of this former e n z y m e are normal while t h a t of the latter are elevated. W h e t h e r the reduced tissue/3-galactosidase is the cause or an effect of the a c c u m u l a t i o n of acid m u c o p o l y s a c c h a r i d e in the mucopolysaccharidoses, d e m o n s t r a t i o n of this a b n o r m a l i t y in the leukocyte emphasizes the v a l i d i t y of e m p l o y i n g this readily available cell in the further studies necessary to define the underlying defect in these diseases. REFERENCES 1. M ITTWOCH, U. Abnormal lymphocytes in gargoylism. Brit. J. Haemat., 5, 365--368 (1959). 2. HERS, H.G. Inborn lysosomal diseases. Gastroenterol, 48, 625--633 (1965). 3. LAGUNOFF, D., & GRITZKA, T . L . The site of mucopolysaccharide accumulation in Hurler's syndrome. An electron microscopic and hlstochemical study. Lab. Investigation, 15, 1578-1588 (1966). 4. HAUST, M. D., ORIZAGA, M., BRYAN, A. M. & FRANK, H . F . The fine structure of liver in children with Hurler's syndrome. Exptl. Mol. Path., 1O, 141-161 (1969). 5. ARONSON, N. N. JR. & DE DUVE, C. Digestive activity of lysosomes. II. Digestion of macromolecular carbohydrates by extracts of rat liver lysosomes. J. Biol. Chem., 243, 45644573 (1968). 6. (~CKERMAN, P. A. Acid hydrolases in skin and plasma in gargoylism. Deficiency of 0-galactosidase in skin. Clin. Chim. Acta, 20, 1-6 (1968). 7. (~CKERMAN, P. A. & KOHLIN, P. Glycosidases in skin and plasma in Hunter's syndrome. Acta Paed. Scand., 57, 281-284 (1968). 8. GERICH, J.E. Hunter's syndrome Beta-galactosidase deficiency in skin. N. Eng. J. Med. 280, 799-802 (1969). 9. OCKERMAN, P . A . Lysosomal acid hydrolases in the liver in gargoylism. Deficiency of 4-metbylumbelliferyl-0-galactosidase. Scand. J. Clin. Lab. Invest., 22, 142-146 (1968). 10. VAN HOOF, F. & HERS, H.G. Abnormal lysosomal enzymes in mucopolysaccharidoses. European J. Biochem., 7, 34--44 (1968). 11. GORDON, B. A. & FELEKI, V. Acid hydrolases in the serum in mucopolysaccharidoses type I and III. Clin. Biochem., 3, 193-202 (1970). 12. MAcBRINN, M., OKADA, S., WOOLLACOTT, M., PATEL, V., HO, M. W., TAPPEL, A. L. & O'BRIEN, J . S . Beta-galactosidase deficiency in the Hurler syndrome. N. Eng. J. Med., 281,338-343 (1969). 13. ALBARRACIN, N. & HAUST, M.D. Unpublished observations. 14. FOLEY, K. M., DANES, B. S. & BEARN, A.G. White blood-cell cultures in genetic studies on human mucopolysaccharidoses. Science, 164, 424--426 (1969). 15. McCUSICK, V. A., KAPLAN, D., WISE, D., HANLEY, W. B., SUDDARTH, S. B., SEVICK, M. E. & MAUMANEE, A. E. The genetic mucopolysaccharidoses. Medicine, 44, 445-483 (1965).
48
GORDON & F E L E K I
16. GORDON, B. A. & HAUST, M . D . 17. 18. 19.
20.
The mucopolysaccharidoses type I, II and III. Urinary findings in 23 cases. Clin. Biochem., 3, 2.08-215 (1970). KAMPINE, J. P., BRADY, R. O., KANFER, J. N., FELD, M. & SHAPIRO, D. Diagnosis of Gaucher's disease and Niemann-Pick disease with small samples of venous blood. Science, 155, 86-88 (1966). PERCY, A. K. & BRADY, R . O . Metachromatic leukdystrophy: Diagnosis with samples of venous blood. Science, 161, 594-595 (1968). HULTBERG, B. & 0CKERMAN, P.A. Properties of 4-methylumbelliferyl-B-galactosidase activities in human liver. Scand. J. Clin. Lab. Invest., 23, 218-217 (1969). 0CKERMAN, P. A. & HULTBERG, B. Fractionation of 4-methylumbelliferyl-;~-galactosidase activities in liver in gargoylism. Scand. J. Clin. Lab. Invest. 22, 199 (1968).