Detection of 5α-androst-2-en-17-one and variants: Identification of main urinary metabolites in human urine samples by GC-MS and NMR.

Two steroids were identified in a supplement named D-2 following the detection of unknown compounds during the routine testing of an athlete's sample. The main glucuroconjugated metabolites were isolated from this urine by high performance liquid chromatography (HPLC) following enzymatic hydrolysis and identified by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analyses as being 2α-hydroxy-5α-androst-3-en-17-one (M1) and 2ß,3α-dihydroxy-5α-androstan-17-one (M2). A third metabolite, 3α,4ß-dihydroxy-5α-androstan-17-one (M3) was also detected, however in lower amounts. The precursor steroids, 5α-androst-2-en-17-one (1) and 5α-androst-3-en-17-one (2) were present in the first D-2 products offered on the Internet. Later, the corresponding 17-hydroxyl compounds were offered as such or as esters (acetate, cypionate) in different relative ratios. Both M2 and M3 were synthesized from the trans-diaxial hydrolysis of the corresponding 2α,3α- and 3α,4α-epoxides (3). These were excreted in the hours following the controlled administration of the commercial product called D-2 R to a male volunteer and were also produced from the incubation of 1 and 2 with S9 liver fractions. Some preparations contain predominantly the alkene in C-2 and, therefore, an efficient detection method must include both primary metabolites M1 and M2. The latter was found equally in the fractions extracted following the enzymatic hydrolysis with ß-glucuronidase and the chemical solvolysis, which may ease its identification. Copyright © 2016 John Wiley & Sons, Ltd.

Studies on neurosteroids XX. Liquid chromatography-tandem mass spectrometric method for simultaneous determination of testosterone and 5alpha-dihydrotestosterone in rat brain and serum.

Testosterone (T) and 5alpha-dihydrotestosterone (DHT) are now referred to not only as androgenic steroid hormones, but also as neuroactive steroids, because they elicit anesthetic and anxiolytic effects. Methods using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) coupled with derivatization are developed and validated to examine rat brain and serum levels of T and DHT and their stress-induced changes. The steroids are extracted with methanol-acetic acid from the brain tissue or serum, purified using solid-phase extraction cartridges, derivatized with a permanently charged reagent, 2-hydrazino-1-methylpyridine, and subjected to LC-MS-MS. [19,19,19-(2)H3]-T is used as the internal standard. The intra- and inter-assay coefficients of variation are below 10.0%, and the analytical recoveries are 98.1-103.0%. The developed methods are applied to the animal study and it was found that a fair amount of DHT is continuously and locally synthesized in the brain, and its level is not changed by the immobilization stress and depends on the brain T level.

A one-step enzymatic assay for the measurement of 17 beta-hydroxy- and 17-oxo-steroid profiles in biological samples.

We describe a simple enzymatic method for the sensitive and specific detection and quantitation of families of hydroxy- and oxo-steroids in biological mixtures. Analysis of the profiles of individual steroids may be achieved following their chromatographic separation. The objectives of this analytical system are, therefore, different from conventional methods which are designed to measure single steroids with a high degree of specificity. The method employs highly purified and active bacterial hydroxysteroid dehydrogenases (HSD) which promote stereospecific, nicotinamide nucleotide-dependent oxidations and reductions at specified positions of steroids. In the presence of catalytic quantities of steroids these enzymes promote the transfer of hydrogen (transhydrogenation) between NADH and NAD analogues. A recently purified 17 beta-HSD from an Alcaligenes species (D. W. Payne and P. Talalay, J. biol. Chem., 260, 13468-13655, 1985) shows almost complete specificity for the 17 beta-hydroxy- and 17-oxo-groups of both C18 and C19 steroids. This enzyme catalyzes steroid-dependent transhydrogenation between NADH and the thionicotinamide analogue of NAD (S-NAD). When these components are incubated at pH 8.5 in the presence of minute quantities of steroid substrates, S-NADH (measured at 398 nm where NADH does not absorb) accumulates at a constant rate which is proportional to the concentrations of steroid and enzyme. The linear increase in absorbance with time is a measure of the total concentration of 17 beta-hydroxy- and 17-oxo-steroids, and can be used to detect subpicomol quantities of steroids. The method is illustrated by the detection and identification of free and conjugated androgens in human serum following their separation by high pressure liquid chromatography. The specificity of the transhydrogenase assay is completely dependent on the specificity of the enzyme and is thus applicable to the detection of other hydroxy- and oxo-steroids by making use of HSDs with appropriate specificities (e.g. 3 alpha-HSD for the measurement of 3 alpha-hydroxy- and 3-oxo-steroids). The simple one-step reaction lends itself to automation, needs no auxiliary detection systems, and requires only an inexpensive colorimeter.

Gynecomasty: estrogen and androgen receptors. A clinical-pathological investigation.

The histology, estrogen (ER)- and androgen (AR)- receptor content, s-FSH, s-LH, s-17-beta-estradiol, U-17-ketosteroid and U-17-ketogenic steroid patterns were measured in a consecutive series of 20 patients operated on for gynecomasty. Two out of 22 specimens were ER-positive, and 4 out of 22 were AR-positive. No relationship was demonstrated between the histopathologic features, ie. active-intermediate-inactive gynecomasty, and the steroid receptor content in the present limited number of cases. In addition, there was no marked relationship between the histopathologic features and the hormone values.

Evidence for adrenal and/or ovarian dysfunction as a possible etiology of idiopathic hirsutism.

Thirty-one cases of idiopathic hirsutism, characterized biochemically in the basal state by increased levels of urinary 3 alpha-androstane-5 alpha, 17 beta-diol and normal levels of the main androgens, were studied. In order to determine a possible etiologic heterogeneity of idiopathic hirsutism, pituitary gonadotropin responses to synthetic luteinizing-releasing hormone (LRH) and adrenal steroid responses to adrenocorticotropic hormone (ACTH) stimulation were evaluated and the results were compared to those in six normal women. On the basis of the results obtained in each hirsute patient after LRH and ACTH tests, two groups were identified. The majority, 23 of 31 hirsute patients (group I), had results similar to those in the control group. In the other eight patients (group II), biologic abnormalities were disclosed and suggested a partial adrenal 11 beta-hydroxylase defect in two patients, an incomplete form of adrenal 3 beta-ol deficiency in one patient, an adrenal hyperreactivity without evident cause in two patients, and polycystic ovary syndrome in association with an adrenal hyperreactivity in three patients. As a group, the eight patients showed ACTH-stimulated increments in testosterone, delta 4-androstenedione, dehydroepiandrosterone, and 17-ketosteroids that were significantly greater (p less than 0.01) than the mean responses in the control group. The conclusion is that some women who previously were designated as having "idiopathic" hirsutism had an adrenal and/or ovarian component to their hyperandrogenism which could be shown only by appropriate dynamic tests.

Simultaneous azo-coupling method for an estrogen sulfatase in human tissues.

A simultaneous azo-coupling method for the histochemical localization of d-equilenin sulfatase is described. d-Equilenin is a natural estrogenic steroid hormone, and its sulfuric acid ester was synthesized. It was found that the d-equilenin liberated during hydrolysis of d-equilenin sulfate by tissue sulfatase could be coupled with a diazonium salt to produce a purple precipitate indicating enzyme activity. d-Equilenin sulfatase was found in human tissues, but not in tissues of the rat. The optimum substrate concentration was 0.8 mM, activity was demonstrable over the wide pH range 5.0-8.0. Enzyme activity localized diffusely in the cytoplasm in optimally fixed specimens. Enzyme activity was also fairly well demonstrable in unfixed cryostat sections. Enzyme activity was completely inhibited by 0.1 M phosphate, 1 mM sodium tetraborate, 1 mM p-nitrophenyl sulfate and by 2 mM p-nitrocatechol sulfate. Estrone sulfate at concentration 0.8 mM had no effect, but at 4 mM caused marked inhibition of the reaction. At the same concentrations dehydroepiandrosterone sulfate did not inhibit the reaction. The chemical properties and tissue localizations of d-equilenin sulfatase differed from the properties of arylsulfatases A, B and C and other steroid sulfatases reported previously in the literature.

Determination of 17-oxosteroid glucuronides and sulfates in urine and serum by fluorescence high-performance liquid chromatography using dansyl hydrazine as a prelabeling reagent.

A fluorescence high-performance liquid chromatographic method is described for the direct determination of conjugated 17-oxosteroids in biological fluids without hydrolysis. Conjugated 17-oxosteroids are extracted with Sep-Pak C18 cartridge, labeled with dansyl hydrazine in trichloroacetic acid--benzene solution and then separated by high-performance liquid chromatography on reversed-phase muBondapak C18 column using 0.01 M sodium acetate in methanol-water-acetic acid (65:35:1, v/v) as the mobile phase. The eluate is monitored by a fluorophotometer at 365 nm (excitation) and 520 nm (emission). Linearities of fluorescence intensities (peak heights) with the amounts of various conjugated 17-oxosteroids were obtained between 10 pmol and 100 pmol. This method is sensitive, reliable and useful for the simultaneous determination of conjugated 17-oxosteroids in urine and serum.

[Prenatal sex determination from the maternal saliva (author's transl)]. / Zur Geschlechtsbestimmung des Fetus aus dem Speichel der Mutter.

A modified Zimmermann reaction (detection of 17-keto-steroids) has been tested for its applicability to fetal sex diagnosis after 20 weeks of pregnancy. In 90 pregnant women the overall reliability of prediction was only 52%. It increased, however, to 67% by analyzing non-morning samples of saliva, while it decreased to 40% if the first saliva was collected. The sensitivity of the method was found to be unequivocally too low to measure salivary androgens. While this procedure in its present form is not suited to antenatal sex determination, further studies of the still unknown nature of the colour reaction might lead to a new understanding of the changes in salivary composition during pregnancy.

A decade's experience with an individualized clomiphene treatment regimen including its effect on the postcoital test.

During a 10-year period, 428 women received clomiphene citrate according to a graduated therapeutic regimen in which the dose of clomiphene and the laboratory studies were individualized according to each patient's history, examination and response. Of the 428 patients, 85.3% ovulated and 42.8% conceived. The great majority of those who conceived did so during the first three ovulatory cycles. There was no evidence that clomiphene therapy was associated with the induction of another cause of infertility. Overall, 88.2% of those with no other causes for infertility who ovulated also conceived. However, only 7.8% of those who had one or more factors in addition to anovulation became pregnant. There was no evidence that clomiphene adversely affected the postcoital test, as only 15% of the patients had poor cervical mucus. The low rate of complications of this treatment, 5.1% cyst formation as well as the 14% abortion rate and the 2.6% congenital anomaly rate and the excellent gestational outcome in those who conceived support the use of this treatment regimen.

[Endocrinologic and ophthalmologic findings before and after transnasal surgery in so-called chromophobe pituitary adenomas]. / Endokrinologische und ophthalmologische Befunde vor und nach transnasaler Operation bei sogenannten chromophoben Hypophysenadenomen.

Pre- and postoperative visual and endocrine functions were examined in 54 patients with so-called chromophobe pituitary adenomas operated on by the transnasal approach. The preoperative examination showed visual disturbances in 41 of 48 patients. A loss of gonadal function was present in 93% of the women and 69% of the men. Impairment of adrenal function was found in 37% and of thyroid function in 18% of patients. Hyperprolactinemia was present in 16 of 29 patients examined. The visual findings remained unchanged, normal or improved in 75% of patients. Impairment occurred in 3%. Eight patients experienced improvement of gonadal function and 3 of adrenal function, whereas no postoperative improvement of the thyroid function was observed. Deterioration of gonadal function occurred in 4, of adrenal function in 7, and of thyroid function in 9 patients. Hyperprolactinemia was reduced in all cases.

[Clinical aspects of the anti-androgens]. / Kliniese aspekte van die anti-androgene.

The ongoing development and gradual availability of the new anti-androgens hold exciting clinical implications for the future. The biosynthesis and interchangeability of the sex steroids, the roles of the ovaries and adrenals and the value and interpretation of biochemical assays in clinical practice are briefly discussed. Because the anti-androgens are used primarily for seborrhoea/acne/hirsutism/oligomenorrhoea, the pathophysiological basis of this socially debilitating syndrome is discussed. The classification of the anti-androgens, their indications, side-effects, dosage schemes and results of treatment are reviewed. Finally, a summary of a possible clinical management regimen is presented.

[Gas chromatographic determination of urinary 17-ketosteroids in normal adults of various ages]. / Dosaggio gascromatografico dei 17-chetosteroidi urinari nell'adulto sano di diverse età.

The excretion of individual urinary 17-ketosteroids, pregnanediol and pregnanetriol, as measured in 98 healthy adults subdivided into groups according to age and sex, by means of a gas chromatographic analysis method based on enzymatic hydrolysis, extraction with ethyl ether, and conversion of the extracted steroids into trimethylsilyl ethers. The results showed that age and sex substantially influence the steroid pattern. Metabolites with 5-alpha configuration are predominant in young subjects; those with 5-beta configuration are pre-eminent in the more advanced ages, particularly in women. Metabolites with definite androgenous significance (C19O2-17KS) decline rapidly with advancing age, while the C19O3-17KS undergo a lesser decrease.

Androgen producing adrenocortical carcinoma.

Two cases of androgen secreting adrenocortical carcinoma have been described by light and electron microscopy. The histological and ultrastructural features of the tumour cells were similar to those of compact cells of zona reticularis and to those described in virilizing adenomas. They possess numerous mitochondria with lamellar and tubular cristae, abundant smooth endoplasmic reticulum, lipofuscin bodies and scanty lipid. Irregularly shaped, crenated mitochondria, with outpouchings of the outer limiting membrane have also been observed. The clusters of neoplastic cells were surrounded by basement membrane which demonstrated a focal discontinuity, probably reflecting malignancy of the tumours. Hyperplasia of smooth endoplasmic reticulum and the presence of outpouchings of the mitochondrial outer limiting membrane might be the morphological manifestation of endocrine activity of the tumours.