Steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities in mouse tissues

The metabolism of estrone sulfate and dehydroisoandrosterone sulfate to the free, unconjugated steroids, estrone and dehydroisoandrosterone, was demonstrated in more than thirty different tissues from male and female BALB/c mice. The activity of steroid sulfatase, when expressed per mg tissue, was greatest in both the pituitary gland and the adrenal glands. The pituitary gland, however, had the lowest capacity for hydrolysis of steroid sulfates while the liver had the greatest capacity. 17 beta-Hydroxysteroid oxidoreductase activity also was demonstrated in all mouse tissues by the formation of estradiol-17 beta when using estrone sulfate as the substrate. The highest apparent activity for 17 beta-hydroxysteroid oxidoreductase was found in lung tissue, and the greatest capacity to form estradiol-17 beta from estrone sulfate was found in liver, lungs, kidneys and testes. This study demonstrates that the majority of mouse tissues have steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities.     

Origin of deoxycorticosterone and deoxycorticosterone sulfate in human pregnancy: absence of steroid 21-sulfatase activity in sulfatase-deficient placenta

The activity of steroid 21-sulfatase, the enzyme that catalyzes the hydrolysis of deoxycorticosterone sulfate (DOC-SO4) is demonstrable in human placenta. Thus, it is possible that this placental enzyme, by way of the hydrolysis of either DOC-SO4 or 21-hydroxypregnenolone mono- or di-sulfate of fetal origin, may be important in the biosynthesis of DOC, which is present in the plasma of pregnant women in high concentration. To investigate this issue further, we evaluated steroid 21-sulfatase activity in microsomal preparations of a sulfatase-deficient placenta. Immediately after delivery, at term, of a living male fetus with sulfatase deficiency, a microsome-enriched fraction of placental tissue was prepared; sulfatase activity was evaluated by use of three substrates, viz. dehydroisoandrosterone sulfate (DS), estrone sulfate (E1-SO4), and DOC-SO4, in various concentrations. Similar incubations were conducted with aliquots of a microsome-enriched fraction prepared from placental tissue of a normal fetus that was delivered, at term, within minutes of the time of delivery of the infant with sulfatase deficiency. In microsomal fractions from the normal placenta, each of the steroid sulfates was hydrolyzed. In the absence of microsomes, and in the presence of microsomal fractions from the sulfatase-deficient placenta, the hydrolysis of DOC-SO4 and DS was not detected. Moreover, in microsomes prepared from the sulfatase-deficient placenta, E1-SO4 was hydrolyzed at a rate that was only 10% of that in incubations with microsomal preparations of the normal placenta. We conclude that with sulfatase deficiency, the placenta is deficient not only in sulfatase activity for steroid-3-sulfates but for steroid 21-sulfates, e.g. DOC-SO4, as well.     

Immunohistochemical analysis of steroid sulfatase in human tissues

Steroid sulfatase (EC 3.1.6.2) is an enzyme that removes the sulfate group from 3β-hydroxysteroid sulfates. This enzyme is best known for its role in estrogen production via the fetal adrenal–placental pathway during pregnancy; however, it also has important functions in other physiological and pathological steroid pathways. The objective of this study was to examine the distribution of steroid sulfatase in normal human tissues and in breast cancers using immunohistochemistry, employing a newly developed steroid sulfatase antibody. A rabbit polyclonal antiserum was generated against a peptide representing a conserved region of the steroid sulfatase protein. In Western blotting experiments using human placental microsomes, this antiserum crossreacted with a 65 kDa protein, the reported size of steroid sulfatase. The antiserum also crossreacted with single protein bands in Western blots of microsomes from two human breast cancer cell lines (MDA-MB-231 and MCF-7) and from rat liver; however, there were some size differences in the immunoreactive bands among tissues. The steroid sulfatase antibody was used in immunohistochemical analyses of individual human tissue slides as well as a human tissue microarray. For single tissues, human placenta and liver showed strong positive staining against the steroid sulfatase antibody. ER+/PR+ breast cancers also showed relatively strong levels of steroid sulfatase immunoreactivity. Normal human breast showed moderate levels of steroid sulfatase immunoreactivity, while ER−/PR− breast cancer showed weak immunoreactivity. This confirms previous reports that steroid sulfatase is higher in hormone-dependent breast cancers. For the tissue microarray, most tissues showed some detectable level of steroid sulfatase immunoreactivity, but there were considerable differences among tissues, with skin, liver and lymph nodes having the highest immunoreactivity and brain tissues having the lowest. These data reveal the utility of immunohistochemistry in evaluation of steroid sulfatase activity among tissues. The newly developed antibody should be useful in studies of both humans and rats.     

Simultaneous azo-coupling method for steroid acetate hydrolyzing enzyme

A simultaneous azo-coupling method for histochemical localization of steroid acetate hydrolyzing enzyme is described. It is based on the observation that d-equilenin, a natural oestrogenic steroid hormone, forms deeply coloured insoluble reaction products with diazonium salts under reaction conditions suitable for histochemical purposes. An acetate at position 3 of d-equilenin is rapidly hydrolysed by tissue esterase and the liberated d-equilenin couples with a diazonium salt to form a coloured precipitate. Steroid acetate hydrolyzing enzyme activity was observed in various tissues of the rat; a comparison with nonspecific esterase activity using alpha-naphthyl acetate as substrate suggested that steroid acetate hydrolyzing enzyme activity represents the activity of one or several isozymes of classical nonspecific esterase. This conclusion has also been drawn previously from biochemical studies using esters of other steroids.     

Lack of effect of insulin or insulin-like growth factor I on the steroid sulfatase activity of human placental cytotrophoblasts

Hyperinsulinemia is known to reduce serum dehydroepiandrosterone sulfate (DHEA-S) levels in normal females. A possible mechanism for this phenomenon would be an insulin-mediated increase in steroid sulfatase activity, with insulin acting either via activation of the insulin receptor or via cross-reaction with the insulin-like growth factor I (IGF-I) receptor. Using a well characterized human cytotrophoblast system, the presence of steroid sulfatase activity in isolated cytotrophoblasts was documented. Half maximal cellular hydrolysis of DHEA-S was observed at a substrate concentration of 9.6-14.5 microM, and maximal hydrolysis at a concentration of 75-100 microM. The hypothesis that insulin increases steroid sulfatase activity was examined by exposing cytotrophoblasts to supraphysiological concentrations of either insulin (2 micrograms/ml) or IGF-I (20 ng/ml) for 24 h and then measuring the rate of DHEA-S hydrolysis. Insulin failed to affect cytotrophoblastic steroid sulfatase activity, irrespective of whether the substrate concentration was 20 microM or 100 microM. IGF-I also exerted no effect on steroid sulfatase activity. These data indicate that neither insulin nor IGF-I affect the steroid sulfatase activity of human cytotrophoblasts. An effect of insulin or IGF-I on the steroid-sulfatase activity of other tissues has not been excluded. These observations suggest that the decline in serum DHEA-S levels during hyperinsulinemia is not mediated via an insulin-induced increase in steroid sulfatase activity.     

Estrogen sulfatase and steroid sulfatase activities in intrauterine tissues of the pregnant guinea pig

The possible role of intrauterine estrogen sulfatase and steroid sulfatase around the time of parturition in the guinea pig was investigated. [3H]Estrone sulfate or [3H]pregnenolone sulfate was incubated with intrauterine tissues. Estrogen sulfatase was found in placenta, endometrium, decidua basalis, amnion and chorion. The presence of steroid sulfatase was established in endometrium and decidua basalis but not in placenta or the fetal membranes. Examination of activities in early (days 32-35), mid (days 44-46) and late (within 5 days of parturition) gestation revealed no significant change in estrogen sulfatase specific activity in decidua basalis. However, in chorion and endometrium this activity was seen to increase approx. 12-fold (P less than 0.001) and 2.8-fold (P less than 0.001), respectively, from early to late gestation. In placenta, estrogen sulfatase activity appeared to increase 2.4-fold (P less than 0.001) and in amnion it decreased 2.8-fold (P less than 0.002). Steroid sulfatase activity in decidua basalis did not change during gestation, while activity in endometrium was found to increase by a factor of 5.3 (P less than 0.001), from early to late gestation. The increases, both in estrogen sulfatase activity in chorion, endometrium and placenta and in steroid sulfatase activity in endometrium, occurred primarily within the final 3 weeks of gestation. In contrast, the decrease in estrogen sulfatase activity in amnion occurred principally between the fifth and sixth weeks of gestation. Analysis of radiolabelled metabolites indicated that estradiol and progesterone could be produced via estrogen sulfatase and steroid sulfatase activities in certain tissues. Subcellular fractionation of tissues revealed that the greatest specific activity and total activity, in all cases, was associated with the 105,000 g pellet. Significant activity was also detected in the 750 and 10,000 g pellets but not in the 105,000 g supernatant. Radioimmunoassay of endogenous estradiol-17 beta (estradiol) in chorion extracts revealed a 6.3-fold increase in the hormone from mid to late gestation. Estradiol levels in endometrium and myometrium did not appear to change during this time. It was concluded that increased estrogen sulfatase activity in guinea pig chorion in late gestation occurs along with elevated levels of the hormone estradiol which may be important for parturition in this species.     

Steroid sulphatase activity in the human ovarian corpus luteum, stroma, and follicle: comparison to activity in other tissues and the placenta

Steroid sulfatase activity was measured in 89 human samples, using dehydroepiandrosterone sulfate (DHEAS) as substrate. The lowest activity was that of follicular fluid which was significantly lower than that of other tissues tested (each P less than 0.01). The steroid sulfatase activity of ovarian tissue taken collectively (corpus luteum, stroma, and follicles) was higher than that of other tissues taken collectively (abdominal skin, uterus, and fallopian tube) (P less than 0.001), and the steroid sulfatase activity of either the follicle (P less than 0.01) or the stroma (P less than 0.05) was significantly greater than that of the corpus luteum. The geometric mean steroid sulfatase activity of the placenta was significantly higher than other tissues tested (each P less than 0.01) and was 22-fold higher than that of the follicle, the tissue with the next highest activity. These data indicate that the human ovary (particularly the stroma and follicle) is capable of utilizing DHEAS, an adrenal product, as a substrate for production of other androgens such as dehydroepiandrosterone (DHEA), androstenedione, and testosterone.     

Steroid sulfatase activities in human leukocytes: biochemical and clinical aspects.

Steroid sulfatase is a membrane-bound microsomal enzyme, present in various tissues. In this report, data on sulfatase activity in peripheral blood leukocytes isolated from normal women and the characterization of its enzyme are studied. In addition, sulfatase activities in placental sulfatase deficiency (PSD) and ichthyosis patients including ichthyosis vulgaris (IV) and recessive X-linked ichthyosis (RXLI) were analysed and were compared with normal subjects. Steroid sulfatase activity was measured by using tritium labeled steroid sulfate as the reaction substrate. It is demonstrated that human leukocytes contain a sulfatase activity for pregnenolone sulfate (P5-S), dehydroepiandrosterone sulfate (DHA-S) and estrone sulfate (E1-S) respectively. This enzyme has a greatest affinity for P5-S, but the activity for E1-S was the highest among the three substrates. The steroid sulfatase activity in female leukocytes is significantly stronger than that in normal males (p less than 0.001) as determined by the cleavage of DHA-S. Sulfatase in leukocytes obtained from the PSD babies and RXLI patients had lower sensitivity. In the case of the mother affected with PSD, the activity was less than half of that in normal men (p less than 0.001) and the levels did not overlap with that in normal women. In patients with IV, the activities were in the normal ranges for both males and females. The measurement of leukocyte sulfatase activity would be a clinically useful tool for the diagnosis of PSD carriers and pedigree analysis.     

Steroid sulfatase and sulfuryl transferase activity in monkey brain tissue

Dehydroepiandrosterone and its sulfated form are commonly known as modulators of gamma-aminobutyrate A and N-methyl-D-aspartate receptors. In spite of poor permeability of the blood-brain barrier for sulfated steroids, high concentrations of dehydroepiandrosterone and also its sulfate have been found in brain tissue. Physiological concentrations of these neuromodulators are maintained by two enzymes present in the blood and many peripheral tissues, including the brain, namely, steroid sulfatase and neurosteroid sulfuryl transferase (NSST). This prompted us to investigate activities of these enzymes in primate brain tissue. Rather low neurosteroid sulfuryl transferase activity was detectable in in vitro incubations of cytosol fractions from male and female Macaca mulatta brains, dissected to cerebral cortex, subcortex, and cerebellum. In male monkeys, the highest activity was found in the cerebellum followed by cortex and subcortex. On the other hand, in female monkeys, the highest activity was determined in the cortex followed by subcortex and cerebellum. Steroid sulfatase activity was determined in in vitro microsomal samples from each of the above-mentioned brain regions. Specific activities in female cerebral regions declined in the order: cerebellum, cortex, and subcortex. In male monkeys, no significant difference among the studied regions was observed. Using dehydroepiandrosterone sulfate as a substrate, the apparent kinetic characteristics of steroid sulfatase were determined as follows: K(M) 36.10 +/- 8.33 microM, V(max) 8.38 +/- 1.68 nmol/h/mg protein. These results will serve as a basis for further studies concerning the pathophysiology of human brain tumors.     

Characterization of arylsulfatase C isozymes from human liver and placenta

Arylsulfatase C and steroid sulfatase were thought to be identical enzymes. However, recent evidence showed that human arylsulfatase C consists of two isozymes, s and f. In this study, the biochemical properties of the s form partially purified from human placenta were compared with those of the f form from human liver. Only the placental s form has steroid sulfatase activity and hydrolyses estrone sulfate, dehydroepiandrosterone sulfate and cholesterol sulfate. The liver f form has barely detectable activity towards these sterol sulfates. With the artificial substrate, 4-methylumbelliferyl sulfate, both forms demonstrated a similar KM but the liver enzyme has a pH optimum of 6.9 while the placental form displayed two optima at 7.3 and 5.5. The molecular weight of the native enzyme determined with gel filtration was 183,000 for the s form and 200,000 for the f form and their pI's were also similar at 6.5. However, the T50, temperature at which half of the enzyme activity was lost, was 49.5 degrees C for the f form and 56.8 degrees C for the s form. Polyclonal antibodies raised against the placental form reacted specifically against the s and not the f form. They immuno-precipitated concomitantly greater than 80% of the total placental arylsulfatase C and steroid sulfatase activities while less than 20% of the liver enzyme was immuno-precipitable. In conclusion, the two isozymes s and f of arylsulfatase C in humans purified from placenta and liver, respectively, have similar KM, pI' and native molecular weight. However, they are distinct proteins with different substrate specificity, pH optima, heat-lability and antigenic properties. Only the s form is confirmed to be steroid sulfatase.     

Human placental steroid sulfatase: purification and properties

Steroid sulfatase is recovered quantitatively from the 105,000 g h supernatant of human placental microsomes extracted with Triton X-100. The solubilized enzyme has been purified using conventional techniques. Throughout the purification procedure, steroid sulfatase appears to be heterogeneous as evidenced by certain, but not all, criteria. Following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the final preparation exhibits a major component and varying amounts of two minor ones. Antibodies raised in rabbits with the heterogeneous immunogen give rise to a single precipitation line when the native enzyme is analyzed by double immunodiffusion or by immunoelectrophoresis. In addition, using aged preparations of microsomes and immunoaffinity techniques, steroid sulfatase activity was found to be associated with the fastest migrating minor component. This finding would suggest that the apparent heterogeneity of purified steroid sulfatase is linked to degradation processes occurring within the microsomal preparations. Steroid sulfatase has a Stokes radius of 56 A, a sedimentation coefficient of 4.85 +/- 0.15S (in Triton-containing buffers) and binds 1.3 g of Triton X-100-per g of protein. The molecular weight of the Triton-protein complex was calculated to be 166,000 in which the glycoprotein portion contribution is about 43% (72,000). In contrast, the apparent molecular weight of the major polypeptide determined on calibrated SDS-gels is 62,000. The purified enzyme exhibits two pH optima with cholesterol sulfate as substrate, an acidic one at pH 5.0 and a second one at pH 7.5. The Km values for cholesterol sulfate, dehydroandrosterone sulfate and p-nitrophenylsulfate were 5.26, 14 and 1,320 microM, respectively.     

STEROID SULFATASE IN BRAIN: COMPARISON OF SULFOHYDROLASE ACTIVITIES FOR VARIOUS STEROID SULFATES IN NORMAL AND PATHOLOGICAL BRAINS, INCLUDING THE VARIOUS FORMS OF METACHROMATIC LEUKODYSTROPHY

Abstract– The enzymatic hydrolysis by brain homogenate of the sulfate esters of estrone, pregnenolone, dehydroepiandrosterone, testosterone, cholesterol and p-nitrophenol was studied. With homogenate of young rat brain, the pH optima of estrone sulfatase ⁴ 4 The term steroid sulfatase is used as a general name for the enzyme(s) which hydrolyzes the sulfate ester of a steroid. Simplified terms, such as estrone sulfatase, instead of the more formal terms, such as estrone sulfate sulfohydrolase, have been used throughout.
and arysulfatase C (p-nitrophenyl sulfate as substrate) were 8.2 and all other steroid sulfatases had pH optima at 6.6. Apparent K_ms for these steroid sulfates were widely different. The highest K_m value was 32.2 μm for estrone sulfate and the lowest was 0.66 μm for testosterone sulfate; the K_m for p-nitrophenyl sulfate was 30 fold higher than for estrone sulfate. Specific activity was also highest with estrone sulfatase and lowest with testosterone sulfatase; specific activity with aryl sulfatase C was over 3 fold higher than with estrone sulfatase. Estrone sulfatase activity was inhibited noncompetitively by sulfate esters of dehydroepiandrosterone, pregnenolone, and cholesterol; on the other hand, other steroid sulfatases were inhibited by these latter three sulfates competitively. Developmental changes of these sulfohydrolase activities in rat brain were almost identical with the exception of testosterone sulfatase activity; the latter sulfatase had a peak activity at 30 days old, while all other sulfatase had a peak at 20 days old. Thermal stability of all these activities was identical. Testosterone sulfatase activity in neurological mouse mutants, jimpy, msd, and quaking mice, was less than one half of littermate controls, while other steroid sulfatase levels in these mutants' brain were normal. All sulfatase activities were diminished in the brain of a metachromatic leukodystrophy patient with multiple sulfatase deficiency. The brains of classical metachromatic leukodystrophy patients contained normal levels of all steroid sulfatases and arylsulfatase C, with the single exception of testosterone sulfatase which level was less than 50% of control.     

Histochemistry of estrogen sulfatases in human breast diseases

Two estrogen sulfatases, arylsulfatase C-estrone sulfatase (ASC-ES) and d-equilenin sulfatase (EqS) were demonstrated histochemically in the normal human female breast, in benign breast diseases and in infiltrating mammary ductal carcinomas to study their significance in the pathogenesis of epithelial proliferations. By hydrolyzing estrone sulfate, the amount of which in female blood is about ten times greater than that of estradiol or estrone, estrogen sulfatases can produce a high local concentration of estrogens. A simultaneous azo-coupling method for histochemical demonstration of ASC-ES is described in the present study; EqS was demonstrated by a previously described method. Estrogen sulfatases were not found in the normal female breast. Both estrogen sulfatases were found in epithelial cells in some examples of mastopathic disease and in fibroadenomas, while ASC-ES was found in periductal fibroblasts. In some cases of infiltrating ductal carcinomas, estrogen sulfatases were present in carcinoma cells. In most of these tumors ASC-ES activity was observed in fibroblasts around infiltrative cell cords. There was no correlation between the presence of estrogen sulfatases and of hormone receptors in carcinomas. It is concluded that estrogen sulfatases play no role in the early stages of benign or malignant epithelial proliferations. However, the induction of estrogen sulfatases may promote epithelial proliferation in some cases if estrogen receptors are present in epithelial cells.     

Purification and properties of steroid sulfatase from human placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.     

Effects of estrogen and growth hormone on steroid sulfatase activity and estrogen binding in rat liver

It is now well established that the activity of certain liver enzymes displays sex differences and that administration of human growth hormone to male rats alters the liver metabolism in a "female" direction. In this work we studied steroid sulfatase activity and binding of estradiol-17 beta in livers from intact rats and found a sex difference, with considerably higher enzyme activity in male as compared to female liver tissue. Continuous infusion of native and recombinant human growth hormone and estradiol-17 beta to male rats reduced sulfatase activity to "female" levels. A specific binding of estradiol-17 beta with receptor properties was found in the rat livers, but the concentration of binding sites did not change after administration of growth hormone or estradiol in this group of intact animals. Our data confirm previous reports that continuous administration of human growth hormone "feminize" liver metabolism, and since estradiol was found to have an identical effect on sulfatase activity it is suggested that the effect of estradiol-17 beta in this respect may be indirect, mediated via an altered secretory pattern of rat growth hormone.     

An estimate of unique DNA sequence heterozygosity in the human genome

Summary Patients with recessive X-linked ichthyosis Patients with recessive X-linked ichthyosis (RXLI), one hereditary form of scaly skin, lack activity of the enzyme steroid sulfatase in all tissues studied. To investigate the molecular defect underlying the lack of enzyme activity, we prepared antisera against normal enzyme by injecting normal placental microsomal suspensions or partially purified steroid sulfatase into rabbits. Antibody activity was assessed by immunoprecipitation of detergent solubilized steroid sulfatase. In addition, we prepared rabbit antisera against RXLI placental microsomal suspensions. To detect immunologically cross-reactive material in patients' placentas, extracts were studied by immunoblot techniques and by competition with normal enzyme for antibody binding. Patients' extracts did not contain immunoreactive material co-migrating on electrophoresis with purified enzyme nor did they inhibit immunoprecipitation of normal enzyme. Sera from rabbits immunized with RXLI placental microsomes contain no antibodies to normal steroid sulfatase, as judged by their failure to immunoprecipitate normal enzyme or to react with normal steroid sulfatase on immunoblot. Thus the mutation in RXLI appears to reduce steroid sulfatase enzyme protein as well as enzyme activity. Portions of this material have appeared in abstract form in Clinical Research 31:564A, 1983 and 32:138A, 1984     

Roles of steroid sulfatase in brain and other tissues

Steroid sulfatase (EC 3.1.6.2) is an important enzyme involved in steroid hormone metabolism. It catalyzes the hydrolysis of steroid sulfates into their unconjugated forms. This action rapidly changes their physiological and biochemical properties, especially in brain and neural tissue. As a result, any imbalance in steroid sulfatase activity may remarkably influence physiological levels of active steroid hormones with serious consequences. Despite that the structure of the enzyme has been completely resolved there is still not enough information about the regulation of its expression and action in various tissues. In the past few years research into the enzyme properties and regulations has been strongly driven by the discovery of its putative role in the indirect stimulation of the growth of hormone-dependent tumors of the breast and prostate.     

New assay for steroid sulfatase (EC 3.1.6.2) and its application for studies of human placental and skin sulfatase

A new, simple, fast and highly practicable sulfatase assay and its application is described. Sterol sulfatase sulfohydrolase (EC 3.1.6.2) activity is determined by a two-phase scintillation technique separating the unreacted [4-14C]dehydroepiandrosterone sulfate from carbon-14-labeled products. The principle of the separation relies on the limited emulsifying capacity of the dioxane-based scintillation solution for water and the different partition of dehydroepiandrosterone sulfate and sulfate-free steroid products between the scintillation fluid and the aqueous phase as recently applied for determination of aromatase activity [1]. [7-3H]Dehydroepiandrosterone sulfate can also be used as a substrate for this assay. This test was applied to studies of microsomal sulfatase prepared from human term placenta and to the detection of sulfatase activity in human skin biopsies. Using placental microsomes, the Km of dehydroepiandrosterone sulfate was determined to be 5.0 X 10(7)M. Sulfatase activity in frozen scrotal skin was found to be 2-3 fold than with vaginal skin. Using an incubation time of 24h/skin sulfatase can be detected in biopsies as small as 2.5 mm2. The sulfatase assay can be applied for routine detection of human placental sulfatase deficiency and, furthermore, the application of this assay has to be demonstrated for the analysis of sulfatase activity in patients with congenital ichthyosis (X-chromosomal, recessive type).