Archives of
Deermatological
Arch. Dermatol. Res. 266, 95 97 (1979)
search
9 Springer-Verlag 1979
Determination of Arylsulfatase C in Hair Follicles J. Ch. Meyer, H.-P. Grundmann, and U. W. Schnyder Department of Dermatology, University Hospital Ztirich (Director: Prof. Dr. U. W. Schnyder), CH-8091 Zttrich, Switzerland
The use of hair follicles for the determination of genetically defined enzyme defects was described by several authors in the last ten years, especially for the detection of heterozygotes in X-linked disorders [3, 4, 6 - 8 , 15, 20] or autosomal recessive inherited disease [11]. But in some cases it may be of great interest to detect a certain enzyme defect in the patient itself in order to ensure the clinical diagnosis. Steroid sulfatase and arylsulfatase C deficiencies have recently been shown to be associated with X-linked ichthyosis [12, 13, 17], a skin disorder which is difficult to distinguish clinically and histologically from other forms of ichthyosis [10], whereas marked ultrastructural differences can be detected [1, 2]. Having these facts in mind and in order to have a simple and fast assay in hand it was obvious to try to estimate sulfatase in hair follicles. From each individual 2 0 - 3 0 scalp hairs were collected and microscopically differentiated. Only anagen hairs proved to be useful for the assay. In most cases hairs were tested immediately after plucking but it was possible to store them for at least 3 - 4 days at room temperature without loss of activity. All selected anagen hairs were cut about 3 - 4 mm above the hair bulbs as described by others [3] and placed separately in 50 pl of 0.1 M phosphate buffer p H 8.0. Before we had shown that the use of 0.1 M phosphate prevents the simultaneous estimation of the activities of the arylsulfatases A and B because these enzymes are inhibited by phosphate [5, 14]. The buffer solutions with the single hairs were then subjected to l 0 cycles of freezing and thawing using liquid nitrogen and a waterbath of 37 ~C to make the cells of the hair follicles penetrable for the substrate. Because the arylsulfatase C is tightly bound to microsomal membranes [5] the substrate has to penetrate into the cell. To the 50 ~1 of phosphate buffer containing the hair follicle 100 pl of 0.4 mM 4-methylumbelliferyl sulfate potassium salt [9, 18, 19] in 0.1 M phosphate buffer were added and this solution together with the hair follicle was incubated for 5 h in a waterbath at 37 ~C. The reaction was then stopped by the addition of 1 ml of 0.1 M glycine-carbonate buffer p H 10.3. The fluorescence which had developed by the release of free methylumbelliferone was measured with the Offprint requests to." Dr. J. Ch. Meyer (address see above)
0340-3696/79/0266/0095/$1.00
96
J. Ch. Meyer et al.
2s
CONTROLS
X-LINKED ICHTYOSIS
E
E o
o
,, : T o ..
-.-
?
: T
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i
:
9
."
9
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.
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.*
Fig. 1. Arylsulfatase C in individual hair follicles of controls and subjects with X-linked ichthyosis. Enzyme activity is expressed as micromoles methylumbelliferone liberated per hair follicle in 5 h
Zeiss spectrophotometer DMR21 (Fa. Carl Zeiss AG, Zfirich) equipped with a mercury vapour lamp and a 365nm excitating filter. The emission of the fluorescence intensity was estimated at 455 nm. Preliminary experiments had shown that photometric methods with acetylphenyl sulfate or p-nitrophenyl sulfate as substrates [5, 14, 16] were too insensitive for the detection of arylsulfatase C in hair follicles. Enzyme activity was expressed as micromoles of methylumbelliferone liberated per hair in 5h. For this purpose there were five concentrations of methylumbelliferone included in each assay run. Concentrations were then calculated from a linear regression using a programmable HP-97 calculator (Fa. Hewlett-Packard, CH-8952 Schlieren, Switzerland). Results obtained from laboratory personal and from patients of our clinic suffering from other than ichthyotic disorders revealed an enormous variation of the arylsulfatase C activity (Fig. 1) among individual hairs of the same person and between different subjects. In addition, in opposition to others measuring arylsulfataseC histologically in skin biopsies [12,13], we could also detect arylsulfatase C in hair follicles of male patients with X-linked ichthyosis.This fact and the great variation of the activity in control persons made it impossible to distinguish significantly between controls and subjects with X-linked ichthyosis, using this assay method without further modifications. But nevertheless there seemed to exist a trend toward a quantitative difference between controls and Xlinked ichthyosis. Because this great variation of arylsulfatase C activity apparently depends on the qualitiy of the hair follicles (adhering tissue, developmental state, cosmetic history, etc.) it will be necessary to include another parameter in the assay which controls this qualitative properties, for instance a reference enzyme
Arylsulfatase C in Hair Follicles
97
[3, 4, 7, 15]. The detection of arylsulfatase C in hair follicles of X-linked ichthyosis could be explained by the existance of more than one isoenzyme of arylsulfatase C which cannot be demonstrated in skin fibroblasts and with histological techniques or by an arylsulfatase C inhibitor, which is not present in hair follicles.
References 1. Anton-Lamprecht, I.: Zur Ultrastruktur hereditfirer Verhornungsst6rungen. III. Autosomaldominante Ichthyosis vulgaris. Arch. Dermatol. Forsch. 248, 149-172 (1973) 2. Anton-Lamprecht, I. : Zur Ultrastruktur hereditfirer Verhornungsst6rungen. IV. X-chromosomalrezessive Ichthyosis. Arch. Dermatol. Forsch. 248, 3 6 1 - 378 (1974) 3. Beaudet, A. L., Caskey, C. T.: Detection of Fabry's disease heterozygotes by hair root analysis. Clin. Genet. 13, 251-258 (1978) 4. De Bruyn, C. H. M. M., Oei, T: L., Ter Haar, B. G. A. : Studies on hair roots for carrier detection in hypoxanthine-guanine phosphoribosyl transferase deficiency. Clin. Genet. 5, 449-456 (1974) 5. Dodgson, K. S., Spencer, B., Wynn, C. H.: Studies on sulphatases. 12. The arylsulphatases of human tissues. Biochem. J. 62, 500-507 (1956) 6. Gartler, S. M., Gandini, E., Angioni, G., Argiolas, N.: Glucose-6-phosphate dehydrogenase mosaicism: utilization as a tracer in the study of the development of hair root cells. Ann. Hum. Genet. 33, i71 - 176 (1969) 7. Gartler, S. M., Scott, R. S., Goldstein, J. L., Campbell, B. : Lesch-Nyhan syndrome : rapid detection of heterozygotes by use of hair follicles. Science 172, 572-573 (1971) 8. Grimm, T., Wienker, T. F., Ropers, H.-H. : Fabry's disease: Heterozygote detection by hair root analysis. Hum. Genet. 32, 329-334 (1976) 9. Hameister, H., Wolff, G., Lauritzen, C. H., Lehmann, W. O., Hauser, A., Ropers, H. H. : Clinical and biochemical investigations on patients with partial deficiency of placental steroid sulfatase. Hum. Genet. 46, 199-207 (1979) 10. Hofbauer, M., Schnyder, U. W.: Zur Differentialdiagnose yon autosomal-dominanter Ichthyosis vulgaris und X-chromosomaler Ichthyose. Hautarzt 25, 319-325 (1974) 11. King, R. A., Witkop, C. J.: Detection of heterozygotes for tyrosinase-negative oculocutaneous albinism by hairbulb tyrosinase assay. Am. J. Hum. Genet. 29, 164- 168 (1977) 12. Koppe, J. G., Marinkovi6-Ilson, A., Rijken, Y., De Groot, W. P., J6bsis, A. C. : X-linked ichthyosis. A sulphatase deficiency. Arch. Dis. Childh. 53, 803-806 (1978) 13. Marinkovi6-Ilsen, A., Koppe, J. G., J6bsis, A. C., De Groot, W. P. : Enzymatic basis of typical X-linked ichthyosis. Lancet 18, 1097 (1978) 14. Milsom, D. W., Rose, F. A., Dodgson, K. S. : The specific assay of arylsulphatase C, a rat liver microsomal marker enzyme. Biochem. J. 128, 3 3 1 - 336 (1972) 15. Romeo, G., Rinaldi, A., Urbano, F., Filippi, G. : Hair root versus red cell individial phenotype in sardinian heterozygotes for G6PD deficiency (mediterranean type). Am. J. Hum. Genet. 28, 5 0 6 513 (1967) 16. Shapiro, L. J., Cousins, L., Fluharty, A. L., Stevens, R. L., Kihara, H.: Steroid sulphatase deficiency. Pediat. Res. 11, 894-897 (1977) 17. Shapiro, L. J., Weiss, R., Buxman, M. M., Vidgof~ J., Dimond, R. L., Roller, J. A., Wells, R. S. : Enzymatic basis of typical X-linked ichthyosis. Lancet 7, 756 (1978) 18. Sherman, W. R., Stanfield, I. F.: Measurement of arylsulphatase of Patella vulgata with 4methylumbelliferone sulphate. Biochem. J. 102, 905-909 (1967) 19. Yoshikatsu, E., Wiesmann, U. N., Carson, J. H., Herschkowitz, N. N.: Multiple sulphatase deficiencies in cultured skin fibroblasts. Occurrence in patients with a variant form of metachromatic leukodystrophy. Arch. Neurol. 30, 153 - 156 (1974) 20. Yutaka, T., Fluharty, A. L., Stevens, R. L., Kihara, H. : Iduronate sulphatase analysis of hair roots for identification of hunter syndrome heterozygotes. Am. J. Hum. Genet. 30, 575-582 (1978)
Received June 18, 1979
Deermatological
Arch. Dermatol. Res. 266, 95 97 (1979)
search
9 Springer-Verlag 1979
Determination of Arylsulfatase C in Hair Follicles J. Ch. Meyer, H.-P. Grundmann, and U. W. Schnyder Department of Dermatology, University Hospital Ztirich (Director: Prof. Dr. U. W. Schnyder), CH-8091 Zttrich, Switzerland
The use of hair follicles for the determination of genetically defined enzyme defects was described by several authors in the last ten years, especially for the detection of heterozygotes in X-linked disorders [3, 4, 6 - 8 , 15, 20] or autosomal recessive inherited disease [11]. But in some cases it may be of great interest to detect a certain enzyme defect in the patient itself in order to ensure the clinical diagnosis. Steroid sulfatase and arylsulfatase C deficiencies have recently been shown to be associated with X-linked ichthyosis [12, 13, 17], a skin disorder which is difficult to distinguish clinically and histologically from other forms of ichthyosis [10], whereas marked ultrastructural differences can be detected [1, 2]. Having these facts in mind and in order to have a simple and fast assay in hand it was obvious to try to estimate sulfatase in hair follicles. From each individual 2 0 - 3 0 scalp hairs were collected and microscopically differentiated. Only anagen hairs proved to be useful for the assay. In most cases hairs were tested immediately after plucking but it was possible to store them for at least 3 - 4 days at room temperature without loss of activity. All selected anagen hairs were cut about 3 - 4 mm above the hair bulbs as described by others [3] and placed separately in 50 pl of 0.1 M phosphate buffer p H 8.0. Before we had shown that the use of 0.1 M phosphate prevents the simultaneous estimation of the activities of the arylsulfatases A and B because these enzymes are inhibited by phosphate [5, 14]. The buffer solutions with the single hairs were then subjected to l 0 cycles of freezing and thawing using liquid nitrogen and a waterbath of 37 ~C to make the cells of the hair follicles penetrable for the substrate. Because the arylsulfatase C is tightly bound to microsomal membranes [5] the substrate has to penetrate into the cell. To the 50 ~1 of phosphate buffer containing the hair follicle 100 pl of 0.4 mM 4-methylumbelliferyl sulfate potassium salt [9, 18, 19] in 0.1 M phosphate buffer were added and this solution together with the hair follicle was incubated for 5 h in a waterbath at 37 ~C. The reaction was then stopped by the addition of 1 ml of 0.1 M glycine-carbonate buffer p H 10.3. The fluorescence which had developed by the release of free methylumbelliferone was measured with the Offprint requests to." Dr. J. Ch. Meyer (address see above)
0340-3696/79/0266/0095/$1.00
96
J. Ch. Meyer et al.
2s
CONTROLS
X-LINKED ICHTYOSIS
E
E o
o
,, : T o ..
-.-
?
: T
"-o
i
:
9
."
9
:
9
..:_,
44e,. "7"
.
0,
:
.*
Fig. 1. Arylsulfatase C in individual hair follicles of controls and subjects with X-linked ichthyosis. Enzyme activity is expressed as micromoles methylumbelliferone liberated per hair follicle in 5 h
Zeiss spectrophotometer DMR21 (Fa. Carl Zeiss AG, Zfirich) equipped with a mercury vapour lamp and a 365nm excitating filter. The emission of the fluorescence intensity was estimated at 455 nm. Preliminary experiments had shown that photometric methods with acetylphenyl sulfate or p-nitrophenyl sulfate as substrates [5, 14, 16] were too insensitive for the detection of arylsulfatase C in hair follicles. Enzyme activity was expressed as micromoles of methylumbelliferone liberated per hair in 5h. For this purpose there were five concentrations of methylumbelliferone included in each assay run. Concentrations were then calculated from a linear regression using a programmable HP-97 calculator (Fa. Hewlett-Packard, CH-8952 Schlieren, Switzerland). Results obtained from laboratory personal and from patients of our clinic suffering from other than ichthyotic disorders revealed an enormous variation of the arylsulfatase C activity (Fig. 1) among individual hairs of the same person and between different subjects. In addition, in opposition to others measuring arylsulfataseC histologically in skin biopsies [12,13], we could also detect arylsulfatase C in hair follicles of male patients with X-linked ichthyosis.This fact and the great variation of the activity in control persons made it impossible to distinguish significantly between controls and subjects with X-linked ichthyosis, using this assay method without further modifications. But nevertheless there seemed to exist a trend toward a quantitative difference between controls and Xlinked ichthyosis. Because this great variation of arylsulfatase C activity apparently depends on the qualitiy of the hair follicles (adhering tissue, developmental state, cosmetic history, etc.) it will be necessary to include another parameter in the assay which controls this qualitative properties, for instance a reference enzyme
Arylsulfatase C in Hair Follicles
97
[3, 4, 7, 15]. The detection of arylsulfatase C in hair follicles of X-linked ichthyosis could be explained by the existance of more than one isoenzyme of arylsulfatase C which cannot be demonstrated in skin fibroblasts and with histological techniques or by an arylsulfatase C inhibitor, which is not present in hair follicles.
References 1. Anton-Lamprecht, I.: Zur Ultrastruktur hereditfirer Verhornungsst6rungen. III. Autosomaldominante Ichthyosis vulgaris. Arch. Dermatol. Forsch. 248, 149-172 (1973) 2. Anton-Lamprecht, I. : Zur Ultrastruktur hereditfirer Verhornungsst6rungen. IV. X-chromosomalrezessive Ichthyosis. Arch. Dermatol. Forsch. 248, 3 6 1 - 378 (1974) 3. Beaudet, A. L., Caskey, C. T.: Detection of Fabry's disease heterozygotes by hair root analysis. Clin. Genet. 13, 251-258 (1978) 4. De Bruyn, C. H. M. M., Oei, T: L., Ter Haar, B. G. A. : Studies on hair roots for carrier detection in hypoxanthine-guanine phosphoribosyl transferase deficiency. Clin. Genet. 5, 449-456 (1974) 5. Dodgson, K. S., Spencer, B., Wynn, C. H.: Studies on sulphatases. 12. The arylsulphatases of human tissues. Biochem. J. 62, 500-507 (1956) 6. Gartler, S. M., Gandini, E., Angioni, G., Argiolas, N.: Glucose-6-phosphate dehydrogenase mosaicism: utilization as a tracer in the study of the development of hair root cells. Ann. Hum. Genet. 33, i71 - 176 (1969) 7. Gartler, S. M., Scott, R. S., Goldstein, J. L., Campbell, B. : Lesch-Nyhan syndrome : rapid detection of heterozygotes by use of hair follicles. Science 172, 572-573 (1971) 8. Grimm, T., Wienker, T. F., Ropers, H.-H. : Fabry's disease: Heterozygote detection by hair root analysis. Hum. Genet. 32, 329-334 (1976) 9. Hameister, H., Wolff, G., Lauritzen, C. H., Lehmann, W. O., Hauser, A., Ropers, H. H. : Clinical and biochemical investigations on patients with partial deficiency of placental steroid sulfatase. Hum. Genet. 46, 199-207 (1979) 10. Hofbauer, M., Schnyder, U. W.: Zur Differentialdiagnose yon autosomal-dominanter Ichthyosis vulgaris und X-chromosomaler Ichthyose. Hautarzt 25, 319-325 (1974) 11. King, R. A., Witkop, C. J.: Detection of heterozygotes for tyrosinase-negative oculocutaneous albinism by hairbulb tyrosinase assay. Am. J. Hum. Genet. 29, 164- 168 (1977) 12. Koppe, J. G., Marinkovi6-Ilson, A., Rijken, Y., De Groot, W. P., J6bsis, A. C. : X-linked ichthyosis. A sulphatase deficiency. Arch. Dis. Childh. 53, 803-806 (1978) 13. Marinkovi6-Ilsen, A., Koppe, J. G., J6bsis, A. C., De Groot, W. P. : Enzymatic basis of typical X-linked ichthyosis. Lancet 18, 1097 (1978) 14. Milsom, D. W., Rose, F. A., Dodgson, K. S. : The specific assay of arylsulphatase C, a rat liver microsomal marker enzyme. Biochem. J. 128, 3 3 1 - 336 (1972) 15. Romeo, G., Rinaldi, A., Urbano, F., Filippi, G. : Hair root versus red cell individial phenotype in sardinian heterozygotes for G6PD deficiency (mediterranean type). Am. J. Hum. Genet. 28, 5 0 6 513 (1967) 16. Shapiro, L. J., Cousins, L., Fluharty, A. L., Stevens, R. L., Kihara, H.: Steroid sulphatase deficiency. Pediat. Res. 11, 894-897 (1977) 17. Shapiro, L. J., Weiss, R., Buxman, M. M., Vidgof~ J., Dimond, R. L., Roller, J. A., Wells, R. S. : Enzymatic basis of typical X-linked ichthyosis. Lancet 7, 756 (1978) 18. Sherman, W. R., Stanfield, I. F.: Measurement of arylsulphatase of Patella vulgata with 4methylumbelliferone sulphate. Biochem. J. 102, 905-909 (1967) 19. Yoshikatsu, E., Wiesmann, U. N., Carson, J. H., Herschkowitz, N. N.: Multiple sulphatase deficiencies in cultured skin fibroblasts. Occurrence in patients with a variant form of metachromatic leukodystrophy. Arch. Neurol. 30, 153 - 156 (1974) 20. Yutaka, T., Fluharty, A. L., Stevens, R. L., Kihara, H. : Iduronate sulphatase analysis of hair roots for identification of hunter syndrome heterozygotes. Am. J. Hum. Genet. 30, 575-582 (1978)
Received June 18, 1979
