339
Clinica Chimica Acta, 68 (1976) 339-341 @ Elsevier Scientific Publishing Company,
SHORT COMMUNICATIONS &__
Amsterdam
-
Printed
in The Netherlands
-__
CCA 7631
RAPID METHOD FOR MEASURING ARYLSULFATASE A AND B IN LEUCOCYTES AS A DIAGNOSIS FOR SULFATIDOSIS, LMUCOSULFATIDOSIS AND MUCOPOLYSACCHARIDOSIS VI
R. HUMBEL State Pediatric (Received
Clinic,
October
Luxembourg
(G.D.L.)
24, 1975)
Summary A simple and rapid method for the separation and measurement of arylsulfatase A and B is presented which is suitable for the diagnosis of sulfatidosis, mucosulfatidosis and mucopolysaccharidosis VI.
Introduction In recent years, determination of arylsulfatase A and B has been utilized more frequently for the diagnosis of lysosomal storage diseases. Three diseases are directly concerned with a deficiency of arylsulfatase: sulfatidosis due to an absence of arylsulfatase A, mucosulfatidosis in which there is a multiple sulfatase deficiency, and mucopolysaccharidosis VI with a deficiency of arylsulfatase B. Arylsulfatase activity is usually measured with p-nitrocatechol sulfate as substrate. Assay systems for the independent assay of arylsulfatase A and B were devised by Baum et al. [l]. However, in spite of the differences exhibited by the two enzymes, differential assay of either enzyme in the presence of the other is somewhat problematic. Quite good separation of arylsulfatase A and B could be obtained by chromatography on DEAE-cellulose [ 21: at neutral pH, the cationic arylsulfatase B is not absorbed by the ion exchanger, while the anionic arylsulfatase A is retained and is only eluted at relatively high NaCl concentrations. The present paper describes a simple micro-chromatographic method for the differential measurement of arylsulfatase A and B. Results obtained in cases of sulfatidosis, mucosulfatidosis and mucopolysaccharidosis VI confirmed the validity of this procedure.
340
Materials and methods DEAE-cellulose was suspended in water, cleared of fine particles and washed with Tris buffer 0.05 M, pH 7.55. Micro-columns of 3 mm X 50 mm were packed with the ion-exchanger. Substrates for the measurement of arylsulfatase A and B were prepared according to Baum [ 11. Reagents for the determination of proteins were prepared according to Hartree [ 31. Human-protein standard (DADE) was utilized for standardization. Blood leucocytes, from normal controls and patients [4] were separated by sedimentation in Dextran [ 51. The leucocyte-pellet of 10 ml blood was suspended in 1 ml of 0.1% Triton Xl00 and frozen and thawed 6 times. The clear supernatant was immediately used for the chromatography. All of the following operations were performed at 4” C. 0.5 ml of the leucocyte extract was placed on a column of DEAE-cellulose, and followed by 0.5 ml of Tris buffer 0.050 M, pH 7.55 to wash down the resin. The two effluents were mixed and used for the determination of arylsulfatase B. Two portions of 0.5 ml, 0.5 M NaCl in Tris buffer were then passed through the column to elute arylsulfatase A. The protein content in each fraction was determined with 0.2 ml effluent by the method of Hartree [ 31. Arylsulfatase B was measured by mixing 0.2 ml of effluent 1 with 0.2 ml of the substrate B of Baum [ 11. Arylsulfat~e A was measured in the same way by mixing 0.2 ml of effluent 2 with 0.2 ml of the substrate A of Baum [ l] . After an incubation time of 1 h, 0.1 ml of 10% NaOH was added and the red coloration measured at 530 nm. A blank, as well as standards of p-nitrocatechol (lO100 nM) were prepared in the same manner. Results Results are summarized in Table I. It can be seen clearly that ~ylsulfa~e is strongly reduced in the two cases of sulfatidosis, whereas only arylsulfatase
TABLE I ARYLSULFATASE
A AND B IN LEUCOCYTES
OF CONTROLS
AND PATIENTS
The activities are expressed as nmol hydrolyzed per mg protein per h.
Controls (38) Sulfatidosis
1)
2) Mucosulfatidosis 1) 2)
Arylsulfatase A
Arylsulfatase B
330.08 f 76.72 (264.62 - 416.7) 19.10 7.18 0 0
418.90 + 75.87 (261.23-684.67) 292.56 345.80 25.00 12.00
Mucopolysaccharidosis VI 1) 2)
280.9i 352.38
13.71 31.27
A B
341
is deficient in mucopolysaccharidosis VI. The two arylsulfatases A and B are reduced so as to be undetectable in mucosulfatidosis. Thus, the method described here allows an easy and rapid way of diagnosing these three diseases. References 1 2 3 4 5
Baum. H., Dogson. K.S. and Spencer, B. (1959) Clin. Chim. Acta 4,453 Fluharty. A.L., Stevens. R.L., Sanders. D.L. and Kimara. H. (1974) Biochim. Biophys. Acta 59,455 Hartree, E.F. (1972) Anal. Biochem. 48. 422 Humbel. R. (1975) Helv. Paediatr. Acta 30, 191 Perky. A.K. and Brady, R.O. (1969) Science 161. 594
Clinica Chimica Acta, 68 (1976) 339-341 @ Elsevier Scientific Publishing Company,
SHORT COMMUNICATIONS &__
Amsterdam
-
Printed
in The Netherlands
-__
CCA 7631
RAPID METHOD FOR MEASURING ARYLSULFATASE A AND B IN LEUCOCYTES AS A DIAGNOSIS FOR SULFATIDOSIS, LMUCOSULFATIDOSIS AND MUCOPOLYSACCHARIDOSIS VI
R. HUMBEL State Pediatric (Received
Clinic,
October
Luxembourg
(G.D.L.)
24, 1975)
Summary A simple and rapid method for the separation and measurement of arylsulfatase A and B is presented which is suitable for the diagnosis of sulfatidosis, mucosulfatidosis and mucopolysaccharidosis VI.
Introduction In recent years, determination of arylsulfatase A and B has been utilized more frequently for the diagnosis of lysosomal storage diseases. Three diseases are directly concerned with a deficiency of arylsulfatase: sulfatidosis due to an absence of arylsulfatase A, mucosulfatidosis in which there is a multiple sulfatase deficiency, and mucopolysaccharidosis VI with a deficiency of arylsulfatase B. Arylsulfatase activity is usually measured with p-nitrocatechol sulfate as substrate. Assay systems for the independent assay of arylsulfatase A and B were devised by Baum et al. [l]. However, in spite of the differences exhibited by the two enzymes, differential assay of either enzyme in the presence of the other is somewhat problematic. Quite good separation of arylsulfatase A and B could be obtained by chromatography on DEAE-cellulose [ 21: at neutral pH, the cationic arylsulfatase B is not absorbed by the ion exchanger, while the anionic arylsulfatase A is retained and is only eluted at relatively high NaCl concentrations. The present paper describes a simple micro-chromatographic method for the differential measurement of arylsulfatase A and B. Results obtained in cases of sulfatidosis, mucosulfatidosis and mucopolysaccharidosis VI confirmed the validity of this procedure.
340
Materials and methods DEAE-cellulose was suspended in water, cleared of fine particles and washed with Tris buffer 0.05 M, pH 7.55. Micro-columns of 3 mm X 50 mm were packed with the ion-exchanger. Substrates for the measurement of arylsulfatase A and B were prepared according to Baum [ 11. Reagents for the determination of proteins were prepared according to Hartree [ 31. Human-protein standard (DADE) was utilized for standardization. Blood leucocytes, from normal controls and patients [4] were separated by sedimentation in Dextran [ 51. The leucocyte-pellet of 10 ml blood was suspended in 1 ml of 0.1% Triton Xl00 and frozen and thawed 6 times. The clear supernatant was immediately used for the chromatography. All of the following operations were performed at 4” C. 0.5 ml of the leucocyte extract was placed on a column of DEAE-cellulose, and followed by 0.5 ml of Tris buffer 0.050 M, pH 7.55 to wash down the resin. The two effluents were mixed and used for the determination of arylsulfatase B. Two portions of 0.5 ml, 0.5 M NaCl in Tris buffer were then passed through the column to elute arylsulfatase A. The protein content in each fraction was determined with 0.2 ml effluent by the method of Hartree [ 31. Arylsulfatase B was measured by mixing 0.2 ml of effluent 1 with 0.2 ml of the substrate B of Baum [ 11. Arylsulfat~e A was measured in the same way by mixing 0.2 ml of effluent 2 with 0.2 ml of the substrate A of Baum [ l] . After an incubation time of 1 h, 0.1 ml of 10% NaOH was added and the red coloration measured at 530 nm. A blank, as well as standards of p-nitrocatechol (lO100 nM) were prepared in the same manner. Results Results are summarized in Table I. It can be seen clearly that ~ylsulfa~e is strongly reduced in the two cases of sulfatidosis, whereas only arylsulfatase
TABLE I ARYLSULFATASE
A AND B IN LEUCOCYTES
OF CONTROLS
AND PATIENTS
The activities are expressed as nmol hydrolyzed per mg protein per h.
Controls (38) Sulfatidosis
1)
2) Mucosulfatidosis 1) 2)
Arylsulfatase A
Arylsulfatase B
330.08 f 76.72 (264.62 - 416.7) 19.10 7.18 0 0
418.90 + 75.87 (261.23-684.67) 292.56 345.80 25.00 12.00
Mucopolysaccharidosis VI 1) 2)
280.9i 352.38
13.71 31.27
A B
341
is deficient in mucopolysaccharidosis VI. The two arylsulfatases A and B are reduced so as to be undetectable in mucosulfatidosis. Thus, the method described here allows an easy and rapid way of diagnosing these three diseases. References 1 2 3 4 5
Baum. H., Dogson. K.S. and Spencer, B. (1959) Clin. Chim. Acta 4,453 Fluharty. A.L., Stevens. R.L., Sanders. D.L. and Kimara. H. (1974) Biochim. Biophys. Acta 59,455 Hartree, E.F. (1972) Anal. Biochem. 48. 422 Humbel. R. (1975) Helv. Paediatr. Acta 30, 191 Perky. A.K. and Brady, R.O. (1969) Science 161. 594
