Steroid Assays Research Articles

Isolation and characterization of a novel uronic acid-containing acidic glycosphingolipid from the ascidian Halocynthia roretzi

A novel uronic acid-containing glycosphingolipid (UGL-1) was isolated from the ascidian Halocynthia roretzi. UGL-1 was prepared from chloroform-methanol extracts and purified by the use of successive column chromatography on DEAE-Sephadex, Florisil, and Iatrobeads. Chemical structural analysis was performed using methylation analysis, gas chromatography, gas chromatography-mass spectrometry, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, and 1H-NMR spectra. The chemical structure of UGL-1 was determined to be a glucuronic acid-containing glycosphingolipid, Galbeta1-4(Fucalpha1-3)GlcAbeta1-1Cer. The ceramide component was composed of C16:0 and C18:0 acids and C16-, C17-, and C18-phytosphingosines as major components.

Comparison of Bayer Advia Centaur® immunoassay results obtained on samples collected in four different Becton Dickinson Vacutainer® tubes

Medicines and Healthcare products Regulatory Agency's (MHRA's) Medical Device Alert MDA/2004/048 described bias in some endocrine test results obtained on a few immunoassay platforms, particularly the Bayer Advia Centaur instrument, when using blood specimens collected into Becton Dickinson (BD) Vacutainer SSTII Advance tubes. As users of BD tubes and the Advia Centaur instrument, we addressed our concerns about the quality of the results that we had previously reported by undertaking an independent study. We compared the results of 15 immunoassays performed on Bayer Advia Centaur using blood specimens collected into four different BD Vacutainer tubes (plain, old and newly released BD SSTII Advance, and BD PSTII). Compared with plain tubes, old SSTII Advance tube results showed no bias for testosterone, CA15-3, follicle-stimulating hormone and folate assays, but gave a positive bias for cortisol and a negative bias for vitamin-B12. Compared with plain tubes, BD PSTII tubes gave no significant bias for thyroid function tests, prolactin, parathyroid hormone, and CA125, but gave a negative bias for steroid assays, and a positive bias for gonadotrophins. The results obtained using new BD SSTII Advance tubes were generally comparable with those on plain tubes. Only for cortisol did our findings support the bias described by MHRA. Based on our results, apart from vitamin-B12 and possibly cortisol, there may have been no significant influence on clinical decisions as a result of using the old BD SSTII Advance specimen tubes. New BD SSTII Advance tubes and plain tubes give generally comparable results. BD PSTII tubes should not be used for steroid hormone measurements on the Bayer Advia Centaur instrument.

Estrus behavior and fecal steroid profiles in the Asiatic lion ( Panthera leo persica) during natural and gonadotropin-induced estrus

In this paper the behavior of the Asiatic lion was studied during natural and gonadotropin-induced estrus in relation to fecal estradiol and progesterone concentration. The average length of estrus was 5.4 days and no significant difference was observed between natural and gonadotropin-induced estrus. Vocalization and rolling were the major estrus behavioral activities of Asiatic lions and the frequency of these activities were similar in both natural and gonadotropin-induced estrus and treatment with exogenous gonadotropin did not alter estrus behavioral activities. A significant positive correlation was observed between fecal estradiol and frequency of estrus behavior during natural and gonadotropin-induced estrus. Following gonadotropin treatment estrus could be induced in 69% of animals and these induced animals ovulated following hCG treatment. This study reports for the first time the successful use of the non-invasive fecal steroid assay for monitoring the induction of estrus and ovulation in the Asiatic lion.

Establishment and Application of Enzyme Immunoassay for Saliva Cortisol in Taiwanese Context

Saliva steroid assay is an upcoming area of research, with much potential for growth and progress. Expensive, varying results with commercial kits and the disadvantages of radioimmunoassay have forced researchers to develop their own system of enzyme immunoassay (EIA). A modification from our established EIA system was used to develop a saliva cortisol (F) assay system. The system sensitivity (>90 pg/mL) was checked by various experiments, including comparison of data with a commercial kit obtained from Salimetrics®. The assay system was employed to investigate the saliva F level in a young Taiwanese population, and compared with the total and free serum levels of F.

Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry

Background Serum steroid assays play an important role in the clinical evaluation of a number of common endocrine disorders. Among various assays, tandem mass spectrometry (MS/MS) has being increasingly applied in clinical laboratories for its high sensitivity, specificity, and simultaneous multi-analyte quantitation capability. Our first generation isotope dilution steroid profile assay by HPLC-tandem MS/MS with a C-18 column allowed for the measurement of 9 steroids in 18 min employing a sample volume of 760 ul serum. We describe our second generation steroid profile assay which allows for the quantitation of 12 steroids simultaneously employing HPLC-MS/MS and isotope dilution tandem MS in 11 min. This method requires a sample volume of 200 μl. Methods An API-5000 triple-quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with the PhotoSpray source and Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia, MD) was used employing isotope dilution with deuterium labeled internal standard (IS) for each analyte. Two hundred microliters of serum were deproteinized by adding 300 μl of acetonitrile containing internal standards. After centrifugation, 450 μl of supernatant were diluted with 900 μl of water and 1000 μl aliquot were injected onto a C-8 column. After a 3 min wash the valve was activated to initiate the gradient elution program which eluted the steroids. Quantitation by MRM analysis was performed both in positive ion mode for 11 analytes and in negative ion mode for aldosterone. Within-day and between-day precision, reliability and accuracy of this method were assessed by correlation with other MS/MS and immunoassay methods and by recovery study. Results Within-day CVs were < 11.5% for all analytes tested and between-day CVs ranged from 3.5% to 12.2%. The results of the comparison study yield r values ranging between 0.908 and 0.999. Recovery ranged from 90% to 110%. Conclusions This method can simultaneously measure 12 steroids in serum within 11 min with minimal sample preparation. It can be routinely employed in a clinical environment and is attractive because of its simplicity in sample processing and high throughput.

The influence of spacer-containing enzyme conjugate on the sensitivity and specificity of enzyme immunoassays for hapten

Background In hapten enzyme immunoassays (EIA), there is an increase or decrease of labeled hapten recognition by antibody that affects sensitivity of the assay. We incorporated a spacer between a hapten derivative and enzyme to test its influence on the sensitivity and specificity of enzyme immunoassays. Method Antibodies were generated against cortisol-3- O-carboxymethyl-oxime-bovine serum albumin (cortisol-3- O-CMO-BSA) and cortisol-21-hemisuccinate-bovine serum albumin (cortisol-21-HS-BSA) as an immunogen. Four cortisol horseradish peroxidase (HRP) enzyme conjugates were prepared using 2 cortisol derivatives (cortisol-3- O-CMO and cortisol-21-HS) with and without adipic acid dihydrazide (ADH) as a spacer. Eight combinations of homologous and heterologous assays were evaluated. Result The incorporation of ADH spacer in cortisol-enzyme conjugate improved the sensitivity in heterologous (bridge and site plus bridge) EIA systems. In heterologous assays (site plus bridge), the presence of spacer in enzyme conjugate reduced the cross-reactivity with cross-reacting steroids. Conclusion Spacer in the enzyme conjugate for hapten ELISA can improve the sensitivity of heterologous assay of hapten-like steroids. It may also reduce the cross-reactivity for some assays.

A Case-Control Study Evaluating the Association of Purposeful Physical Activity, Body Fat Distribution, and Steroid Hormones on Premenopausal Breast Cancer Risk

The objective of this case-control study was to investigate the relationship between purposeful physical activity, body fat distribution, body mass index, and steroid hormones. These factors are known to be implicated in modulating breast cancer risk in premenopausal women. A total of 112 newly diagnosed, premenopausal breast cancer patients and 106 age-matched premenopausal disease-free controls were admitted to the study. Information regarding personal, medical, hormonal, and reproductive history, smoking and alcohol use, physical activity history, and anthropometric measurements was obtained. Serum samples for steroid hormone assays were collected and analyzed. Disease-free premenopausal controls had a significantly higher physical activity index (PAI) (p</=0.05), however, significantly higher weight (p</=0.05), body mass index (BMI) (p=0.01), waist (p</=0.005) and hip (p</=0.05) circumferences, waist:hip ratios (p</=0.05), and serum total estradiol levels (p<0.0005) were observed in cancer cases. The final model using stepwise logistic regression analysis indicates that the variables that significantly predicted breast cancer risk were waist:hip ratio (odds ratio [OR]=1.11, p</=0.005) and serum total estradiol levels (OR=1.03, p</=0.0001). Our study provides some evidence that purposeful physical activity may reduce upper body fat distribution associated with adult weight gain. This may be a result of alterations in the steroid hormone pathway, such as reduced estradiol levels. This demonstrates the potential mechanism through which increased physical activity can reduce the risk for breast cancer in premenopausal women.

Collision/Composite Tumors of the Adrenal Gland: A Pitfall of Scintigraphy Imaging and Hormone Assays in the Detection of Adrenal Metastasis

In patients with a history of extraadrenal tumor, incidental discovery of an adrenal mass necessitates excluding the possibility of metastatic malignancy. Detection of the malignant tissue is a difficult challenge when metastasis occurs in an adrenal adenoma, forming a collision/composite tumor. We report two patients with adrenal collision/composite tumors referred to two French university hospitals. Two patients with histories of mammary and sigmoid carcinomas, respectively, presented with adrenal mass discovered 8 and 3 yr after surgical removal of the primary tumor. In the two cases, computerized tomographic scan showed that the adrenal tumor contained two components with low and high attenuation values, respectively. Uptake of iodocholesterol by the adrenal tumor in case 1 and elevated plasma ACTH-stimulated 17-hydroxyprogesterone values in case 2 strongly argued for the diagnosis of primary adrenocortical tumors. Enlargement of the adrenal mass during follow-up in case 1 and association of the adrenal lesion with a hepatic mass in case 2 led to adrenalectomy. In both cases, histological examination of the tumor demonstrated the presence of metastatic carcinoma tissue in an adrenocortical adenoma, allowing classification of the neoplasia as a collision/composite tumor. These observations show that collision/composite tumors of the adrenal gland formed by carcinoma metastasis in benign adenomas are a pitfall of iodocholesterol scintigraphy and/or plasma steroid assays to exclude the diagnosis of adrenal metastasis. Conversely, computerized tomographic scan is a useful tool for the distinction between the benign and malignant tissues in adrenal collision/composite tumors.

Dosagem de cortisol e cortisona livres urinários empregando cromatografia líquida associada a espectrometria de massa em tandem (LC-MS/MS)

Free urinary cortisol (UFF) measurement is one of the most useful screening tests for Cushing's syndrome. Immunoassays employed today by most clinical laboratories present limitations, specially concerning specificity. These limitations restrain a widespread application of the method, as well as the comparison of results obtained by the use of different methods. We present the development and characterization of a UFF and cortisone method based on liquid chromatography and tandem mass spectrometry (LC-MS/MS). A 200 microL aliquot from a 24 h urine sample is mixed with a solution containing a known quantity of deuterated cortisol and on-line extracted in solid phase (C18). The eluate is transferred to a second C18 column (Phenomenex Luna, 3 micro, 50 x 2 mm) and the isocratic mode elution profile is directly applied to a tandem mass spectrometer model Quattro Micro operating in positive mode atmospheric pressure chemical ionization (APCI). All process is automated and the quantification is performed by isotopic dilution, based on the analyte and the deuterated internal standard peak area ratios. The specificity study showed that all the steroids tested presented cross reactivity of <1% for cortisol and cortisone. Functional sensitivity is <1 microg/L for both steroids, and the interassay CV <8%. Recovery and linearity studies were satisfactory and comparison of results obtained using a RIA for UFF and the present method in 98 routine samples showed a correlation of r= 0.838, with the results obtained with LC-MS/MS significantly lower (medians of 22.0 vs. 49.4 microg/24 h for RIA) (P<0.0001). Reference values for cortisol were defined as values between 11 and 43 microg/24 h, compatible to those recently described for similar methods. The concomitant measurement of UF cortisone allows the study of the activity of the enzyme 11beta-HSD2 and the diagnosis of the apparent mineralocorticoid excess syndrome. The method represents the first steroid assay of a new generation, based on automated preparative methods and tandem mass spectrometry, described in our country.

Assessing reproductive status of right whales ( Eubalaena glacialis) using fecal hormone metabolites

Long-term studies of the endangered North Atlantic right whale, Eubalaena glacialis, have revealed declining reproductive parameters over the past two decades, threatening recovery of this small population if current trends continue. Little is known about right whale reproductive physiology, and investigating this reproductive decline has been limited by a lack of non-lethal methods for assessing reproductive status (e.g., sexual maturation, ovarian activity, pregnancy, lactation, and reproductive senescence) in free-swimming whales. This paper describes validation of existing radioimmunoassay techniques to study reproduction in right whales by measuring estrogens, progestins, androgens, and their related metabolites in fecal samples. Over the past decade fecal steroid hormone assays have been used to assess reproductive status and function in a wide range of terrestrial wildlife species, but this is the first application of this methodology in wild cetaceans. Analysis of fecal hormone metabolite levels in combination with life history data from photographically identified whales shows that this non-invasive method can be used to determine gender, detect pregnancy and lactation, and to assess age at sexual maturity in right whales and potentially other endangered whale populations.

Manganese superoxide dismutase activity in the rat adrenal

In the light of studies suggesting that transcription of the gene coding for manganese superoxide dismutase (MnSOD) is induced by ACTH in the rat adrenal gland, northern blot analysis was used to determine its mRNA distribution. It was found that mRNA coding for MnSOD is primarily present in the inner zones of the rat adrenal cortex, and not the glomerulosa. To investigate the functional relationships between MnSOD activity and expression and adrenocortical function, adrenals and blood were taken from animals pretreated with corticotrophin or betamethasone (Betnesol), or subjected to a low-sodium diet. MnSOD activity in inner zone mitochondrial fractions was enhanced by corticotrophin and by a low-sodium diet, but suppressed by betamethasone. Apparent cytosolic MnSOD activity, total cytosolic MnSOD and CuZnMn-SOD, and glomerulosa mitochondrial MnSOD all were unaffected. Steroid assays showed a clear correlation between circulating corticosterone and inner zone mitochondrial MnSOD, but none between aldosterone and glomerulosa MnSOD. Immunoblot analysis of MnSOD showed two apparent isoforms, at approximately 25 kDa and 75 kDa. There was a partial relationship between expression of the 75 kDa isoform and MnSOD activity, in that it was induced by corticotrophin. However, there was also a slight induction with betamethasone, and a low-sodium diet had no effect. The 25 kDa MnSOD isoform was unaffected by the treatments. The results suggest that MnSOD may have a specific role in the steroidogenic function of the fasciculata/reticularis of the rat adrenal, but not in that of the glomerulosa.

Analysis of anabolic steroids by partial filling micellar electrokinetic capillary chromatography and electrospray mass spectrometry

A partial filling micellar electrokinetic capillary chromatography (PF-MEKC) separation of six anabolic androgenic steroids (androstenedione, metandienone, fluoxymesterone, methyltestosterone, 17-epimetandienone and testosterone) is introduced. The method utilises a mixed micellar solution consisting of sodium dodecyl sulphate (SDS) and sodium taurocholate. The analytes are detected with a photodiode array detector at 247 nm wavelength. Methyltestosterone is used as internal standard. The detection limits were 39 μg/L for androstenedione, 40 μg/L for testosterone, 45 μg/L for fluoxymesterone, 45–90 μg/L for 17-epimetandienone, 59 μg/L for methyltestosterone and 90 μg/L for metandienone. Linear correlation between concentration (0.1–5.0 mg/L) and detector response was obtained with r2 of 0.994 for fluoxymesterone, 0.998 for 17-epimetandienone and 0.999 for androstenedione, metandienone and testosterone. In addition, ionisation of the investigated compounds in electrospray mass spectrometry (ESI-MS) was studied in positive ion mode. The most intense signal (100%) was the protonated molecular ion [M+H]+, except for 17-epimetandienone, which gave its strongest signal at m/z corresponding to [M−H2O+H]+. Finally, separation and identification of fluoxymesterone, androstenedione and testosterone by PF-MEKC–ESI-MS is described. This is the first use of PF-MEKC and PF-MEKC–ESI-MS assays for anabolic androgenic steroids.

Analysis of anabolic steroids by partial filling micellar electrokinetic capillary chromatography and electrospray mass spectrometry

A partial filling micellar electrokinetic capillary chromatography (PF-MEKC) separation of six anabolic androgenic steroids (androstenedione, metandienone, fluoxymesterone, methyltestosterone, 17-epimetandienone and testosterone) is introduced. The method utilises a mixed micellar solution consisting of sodium dodecyl sulphate (SDS) and sodium taurocholate. The analytes are detected with a photodiode array detector at 247 nm wavelength. Methyltestosterone is used as internal standard. The detection limits were 39 μg/L for androstenedione, 40 μg/L for testosterone, 45 μg/L for fluoxymesterone, 45–90 μg/L for 17-epimetandienone, 59 μg/L for methyltestosterone and 90 μg/L for metandienone. Linear correlation between concentration (0.1–5.0 mg/L) and detector response was obtained with r 2 of 0.994 for fluoxymesterone, 0.998 for 17-epimetandienone and 0.999 for androstenedione, metandienone and testosterone. In addition, ionisation of the investigated compounds in electrospray mass spectrometry (ESI-MS) was studied in positive ion mode. The most intense signal (100%) was the protonated molecular ion [ M+H] +, except for 17-epimetandienone, which gave its strongest signal at m/ z corresponding to [ M−H 2O+H] +. Finally, separation and identification of fluoxymesterone, androstenedione and testosterone by PF-MEKC–ESI-MS is described. This is the first use of PF-MEKC and PF-MEKC–ESI-MS assays for anabolic androgenic steroids.

Circadian rhythm of salivary cortisol, 17alpha-hydroxyprogesterone, and progesterone in healthy children.

Very few reference intervals for salivary steroids in children have been established to date (1). Even the manufacturers of salivary steroid assays do not provide sufficient reference data for their products. This lack of information is surprising because the measurement of salivary steroids has been accepted as being noninvasive and stress-free (2)(3). In particular, psychiatric and neuro-endocrinologic experiments are frequently designed with saliva as the medium of choice for steroid analysis (4)(5)(6). A large variety of stressors can rapidly affect the adrenal cortex, causing increased adrenal steroid concentrations. For example, hypoglycemia (7) or physical exercise (8) are potent physiologic stressors, whereas fear (9), feelings of inferiority (10)(11), or experiences at school (12)(13) can affect the adrenal cortex activity as psychologic stressors. The taking of blood can also influence adrenal steroid concentrations in children; saliva collection, however, is almost stress-free (14). The use of saliva for steroid analysis in children is therefore an excellent alternative to blood. The aim of our study was to establish age-dependent reference values for salivary cortisol, 17α-hydroxyprogesterone (17OHP), and progesterone in a large cohort of healthy children. The availability of such reference intervals will improve the applicability of saliva analysis as a diagnostic tool in pediatric endocrinology. We collected 252 saliva profiles from healthy children and adolescents (125 boys; age range, 4 days to 15 years; 127 girls; age range, 6 days to 13 years) with normal body length/height and weight. None of the girls had developed a regular menstrual cycle. The parents of the children gave informed consent. Saliva was collected either with the Salivette®, using polyester swabs (Sarstedt), from children >1 year of age or with modified medical pacifiers (Buttner-Frank; see Fig. 1⇓ in the Data Supplement that accompanies the …

Friedel-Crafts acylation as a quality control assay for steroids.

The rapid Friedel-Crafts chromogenic acylation of alkene groups at ambient temperatures using a 25:1 mixture of 98% acetyl chloride and 70% perchloric acid is shown to have all the properties needed to serve as a potential quality control reagent that can be used to routinely discriminate among steroid analogs. Although ostensibly a non-selective reagent, from these and prior applications in terpenes and polyunsaturated acid esters, it is seen that the reaction is capable of discriminating bewteen geometric isomers and even enantiomers. The selectivity towards acylation of the alpha- over the beta-position at C-17 makes the method adaptable to screening for anabolic steroids. Reactions at that position produce the more unusual results, including a positive color reaction for alpha-methyltestosterone even though there is no alkene functional group in the vicinity of C-17. For molecules with more than one alkene, concurrent acylations are independent one from the other and, in the absence of any interferences, their spectral properties are found to be additive.

Analysis of biological samples by gas chromatography–mass spectrometry without a reference standard: measurement of urinary 18-hydroxytetrahydro-11-dehydrocorticosterone excretion rate in human subjects

Reference standards for some minor urinary steroid metabolites are sometimes unavailable. We describe a novel procedure to quantitate a urinary steroid metabolite of known structure and mass spectrum, using as a standard a compound which produces ions in common with it and has a similar retention time in gas chromatography–mass spectrometry. The steroid of interest was 18-hydroxy-11-dehydrotetrahydrocorticosterone (18-OH-THA), the major urinary metabolite of 18-hydroxycorticosterone (18-OH-B), a putative intermediate in the conversion of 11-deoxycorticosterone to aldosterone. The steroid used as an alternative to the authentic 18-OH-THA standard was β-cortol which, like 18-OH-THA, produces a fragmentation ion at m/ z 457. Allo-tetrahydrodeoxycorticosterone (5α-THDOC) was used as the internal standard. β-Cortolone also has the fragmentation ion at m/ z 449 (in common with β-cortol) and an authentic standard is available commercially. To validate the procedure, we quantitated β-cortolone urinary excretion rate against this alternative standard and also against authentic β-cortolone standards. Both methods produced similar results (adjusted R 2: 0.998, P<0.001). The method was then used to measure urinary excretion of 18-OH-THA rate in healthy volunteers. The reference range obtained was 20–204 μg/24 h ( n=32). This is similar to the few results available by conventional assay. Method performance was also similar to other assays of urinary steroids. This procedure could be generally applicable for assays when authentic standards are not available but mass spectra are known or can be predicted.

Detection of illegal growth promoters in biological samples using receptor binding assays

In the European Union (EU), the use of growth-promoting substances in meat production is banned. The control of growth promoters, especially steroid hormones, is presently based on expensive and time-consuming chromatographic methods of analysis or, sometimes, for screening purposes, on radio- or enzyme-immunoassays, all of which are often too specific to allow effective multi-analyte control. In order to develop rapid and inexpensive multi-analyte detection tests, we proposed the use of hormonal receptors as detection tools. The system described here (radio-receptor assays) is based on a direct binding assay of steroid hormones to their respective receptors. Human receptors to estrogens (hERα), androgens (hAR), progestagens (hPR) and glucocorticoids (hGR) have been produced by genetic engineering in bacteria or in eucaryotic cells. Binding analyses revealed that the obtained receptor proteins retained a high affinity for their corresponding native ligand. In addition, competition studies confirmed that each of the four receptors displays a specificity profile for a series of analogs in agreement with the literature. Finally, the stability of these recombinant receptors is sufficient to allow their use in test kits.

Faecal progesterone metabolites and behavioural observations for the non-invasive assessment of oestrous cycles in the common wombat ( Vombatus ursinus) and the southern hairy-nosed wombat ( Lasiorhinus latifrons)

Wombats belong to Australia’s unique marsupial species. Two of the three remaining species, the common wombat ( Vombatus ursinus) and the southern hairy-nosed wombat ( Lasiorhinus latifrons) are abundant. The third species, the northern hairy-nosed wombat ( Lasiorhinus krefftii) has only about 115 individuals left in the wild. This study aimed to gain further insight into the basic reproductive biology of wombat species and evaluate the value of faecal progesterone metabolites and behavioural patterns as a means for non-invasive monitoring of the oestrous cycle in common and the southern hairy-nosed wombats. In an initial study, three different faecal steroid assays showed that 20α-OH-pregnanes were the main progesterone metabolites. These metabolites were examined in captive female common wombats ( n=5) and southern hairy-nosed wombats ( n=2). In one female common wombat and in one male common wombat oestrous related behavioural data were obtained. Individual cycling females exhibited a significant relationship between plasma progesterone and faecal pregnanes. In the common wombat, the values for faecal pregnanes showed an oestrous cycle length of 55.1±11.7 days with a follicular phase of 25.6±6.3 days and a luteal phase of 28.2±12.7 days. The data for faecal pregnanes obtained in the southern hairy-nosed wombat during the breeding season gave an oestrous cycle length of 41.1±12.8 days with a follicular phase of 27.9±12.3 days and a short luteal phase of 13.3±1.1 days. The behavioural data show that the faecal sniffing behaviour of the male, tended to increase around the time that oestrous was found. In conclusion, monitoring of 20α-OH-pregnanes in wombat faeces could be a useful methodology to monitor reproductive cycles in the wombat, and can possibly be applied to monitor the endangered northern hairy-nosed wombat.

Excretion and measurement of estradiol and progesterone metabolites in the feces and urine of female squirrel monkeys (Saimiri sciureus).

The first objective of the present study was to determine the metabolic form and rate of excretion of ovarian hormone metabolites in the urine and feces of female squirrel monkeys injected with radiolabeled progesterone (Po) and estradiol. The major portion of the urinary metabolites of both hormones was excreted within 16-24 hr post-injection. Estrogen and Po isotopes in feces exhibited an excretion peak at 16 hr post-injection. The majority of recovered radiolabel of both hormones was excreted in feces. Chromatographic separation of fecal extractions indicated that the major estrogen metabolites in feces are in the free as opposed to the conjugated form. The radioactivity and immunoreactivity for estrone and estradiol (E(1) and E(2), respectively) in eluates of fecal samples subjected to celite co-chromatography indicated that both free E(1) and E(2) exist as excretion products in the feces of female squirrel monkeys. The major radioactive peaks for Po metabolites showed peaks in the elution profile at or very near the Po standard, and corresponded with the celite co-chromatography elution profile of Po standard when subjected to enzyme immunoassay (EIA). The second objective was to validate the application of EIA systems to measure fecal metabolites. Reproductive events of one female squirrel monkey across one annual reproductive cycle are described using the endocrine profile generated from fecal steroid assays. Examination of this profile confirmed that longitudinal fecal sampling and steroid hormone metabolite measurement in feces was not only feasible and practical, but accurately detected known reproductive events as well.

Human granulosa cells are a site of sulphatase activity and are able to utilize dehydroepiandrosterone sulphate as a precursor for oestradiol production.

Dehydroepiandrosterone sulphate (DHEAS) is the most abundant androgen in the circulation and in ovarian follicular fluid. A steroid sulphatase accepting DHEAS as a substrate has been identified in the follicle, but the cellular location has not been determined. As DHEAS is also a potential source of oestrogen for endocrine-dependent tumours, a potent steroid sulphatase inhibitor oestrone-3-O-sulphamate (EMATE) has been developed which inhibits this activity in rat liver and mammary tumour. The aim of this study was to investigate human granulosa cells as a site of steroid sulphatase activity, to determine whether DHEAS can be utilized as a precursor for oestrogen synthesis and to investigate the inhibitory capacity of EMATE in these cells. Conversion of DHEAS to DHEA was assessed in luteinized granulosa cells by tritiated steroid assay following incubation with or without LH or insulin and steroid accumulation in the medium measured by RIA. The effects of EMATE were assessed by addition of a range of doses during the measurement of conversion of DHEAS to DHEA. Cells from three sizes of small follicles from an unstimulated ovary were also assessed for their ability to produce oestradiol from DHEAS. Sulphatase enzyme activity was present in all cells; the mean conversion of tritiated DHEAS to DHEA was 50% (range 4-65%). LH and EMATE inhibited and insulin stimulated this activity. Addition of DHEAS to granulosa cells caused a dose-dependent increase in oestradiol and androstenedione production with no change in progesterone concentration. LH increased the accumulation of oestradiol in the medium. DHEAS also stimulated oestradiol production by granulosa cells from small follicles. This is the first demonstration that granulosa cells are a site of sulphatase activity and that DHEAS can be utilized as a substrate for androstenedione and oestrogen production. This may be of physiological importance for both normal folliculogenesis and oestrogen-dependent tumour growth.