Sex Differences in 11-Oxygenated Androgen Patterns Across Adulthood.

The gonads are the major source of sex steroids during reproductive ages. The gonadal function declines abruptly in women and gradually in men. The adrenals produce 11-oxygenated androgens (11-oxyandrogens), which start rising during adrenarche. Following menopause, 11-oxyandrogens levels remain similar to reproductive ages. To compare the circulating 11-oxyandrogen concentrations in men and women across adult ages. We used mass spectrometry to measure testosterone (T), androstenedione (A4), 11β-hydroxytestosterone (11OHT), 11-ketotestosterone (11KT), 11β-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), cortisol, and cortisone in morning sera obtained from adults in outpatient setting. We performed double immunofluorescence of 3β-hydroxysteroid dehydrogenase type 2 and cytochrome b5 in adrenal tissue from 19 men, age 23-78 years. We included 590 patients (319 men), aged 18 to 97 years, and 84% white. 11KT and 11KA4 were stable across ages in women, but they declined in men (0.21 and 0.06 ng/dL/year, respectively; P < 0.05). 11OHA4 and 11OHT increased modestly with age in women (0.6 and 0.09 ng/dL/year, respectively; P < 0.01), and both remained stable across ages in men. As body mass index (BMI) increased, 11KA4 decreased in women, and 11KT increased in men, both suggesting higher 17β-hydroxysteroid dehydrogenase activity in obese individuals. A4 and T declined with age and A4 with BMI in both sexes; T declined with BMI in men. Adrenal androgenic enzyme expressions in aging men were similar to those observed in women. In contrast with traditional androgens, the production of 11OHA4 and 11OHT is sustained with aging in both sexes. The bioactive androgen 11KT declines in aging men but not in women.

Davio A ，Woolcock H ，Nanba AT ，Rege J ，O'Day P ，Ren J ，Zhao L ，Ebina H ，Auchus R ，Rainey WE ，Turcu AF ... -  《-》

Profiling adrenal 11β-hydroxyandrostenedione metabolites in prostate cancer cells, tissue and plasma: UPC(2)-MS/MS quantification of 11β-hydroxytestosterone, 11keto-testosterone and 11keto-dihydrotestosterone.

Adrenal C19 steroids serve as precursors to active androgens in the prostate. Androstenedione (A4), 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT) are metabolised to potent androgen receptor (AR) agonists, dihydrotestosterone (DHT), 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). The identification of 11OHA4 metabolites, 11KT and 11KDHT, as active androgens has placed a new perspective on adrenal C11-oxy C19 steroids and their contribution to prostate cancer (PCa). We investigated adrenal androgen metabolism in normal epithelial prostate (PNT2) cells and in androgen-dependent prostate cancer (LNCaP) cells. We also analysed steroid profiles in PCa tissue and plasma, determining the presence of the C19 steroids and their derivatives using ultra-performance liquid chromatography (UHPLC)- and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). In PNT2 cells, sixty percent A4 (60%) was primarily metabolised to 5α-androstanedione (5αDIONE) (40%), testosterone (T) (10%), and androsterone (AST) (10%). T (30%) was primarily metabolised to DHT (10%) while low levels of A4, 5αDIONE and 3αADIOL (≈20%) were detected. Conjugated steroids were not detected and downstream products were present at <0.05μM. Only 20% of 11OHA4 and 11OHT were metabolised with the former yielding 11keto-androstenedione (11KA4), 11KDHT and 11β-hydroxy-5α-androstanedione (11OH-5αDIONE) and the latter yielding 11OHA4, 11KT and 11KDHT with downstream products <0.03μM. In LNCaP cells, A4 (90%) was metabolised to AST-glucuronide via the alternative pathway while T was detected as T-glucuronide with negligible conversion to downstream products. 11OHA4 (80%) and 11OHT (60%) were predominantly metabolised to 11KA4 and 11KT and in both assays more than 50% of 11KT was detected in the unconjugated form. In tissue, we detected C11-oxy C19 metabolites at significantly higher levels than the C19 steroids, with unconjugated 11KDHT, 11KT and 11OHA4 levels ranging between 13 and 37.5ng/g. Analyses of total steroid levels in plasma showed significant levels of 11OHA4 (≈230-440nM), 11KT (≈250-390nM) and 11KDHT (≈19nM). DHT levels (<0.14nM) were significantly lower. In summary, 11β-hydroxysteroid dehydrogenase type 2 activity in PNT2 cells was substantially lower than in LNCaP cells, reflected in the conversion of 11OHA4 and 11OHT. Enzyme substrate preferences suggest that the alternate pathway is dominant in normal prostate cells. Glucuronidation activity was not detected in PNT2 cells and while all T derivatives were efficiently conjugated in LNCaP cells, 11KT was not. Substantial 11KT levels were also detected in both PCa tissue and plasma. 11OHA4 therefore presents a significant androgen precursor and its downstream metabolism to 11KT and 11KDHT as well as its presence in PCa tissue and plasma substantiate the importance of this adrenal androgen.

du Toit T ，Bloem LM ，Quanson JL ，Ehlers R ，Serafin AM ，Swart AC ... -  《-》

11-Oxygenated C19 Steroids Do Not Decline With Age in Women.

The ovaries and adrenals are sources of androgens in women. Although dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and testosterone (T) all decline with age, these C19 steroids correlate poorly with parameters of androgen action in postmenopausal women. To comprehensively compare the androgen profiles of pre- and postmenopausal women. We quantified 19 steroids-including DHEA; DHEAS; T; androstenedione (A4); and the following adrenal-specific 11-oxygenated C19 steroids (11oxyandrogens): 11β-hydroxytestosterone (11OHT), 11-ketotestosterone (11KT), 11β-hydroxyandrostenedione (11OHA4), and 11-ketoandrostenedione (11KA4)-using liquid chromatography-tandem mass spectrometry in morning serum obtained from 100 premenopausal (age 20 to 40 years) and 100 postmenopausal (age ≥ 60 years) women. Double immunofluorescence of 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) with cytochrome b5 (CYB5A) or sulfotransferase 2A1 (SULT2A1) was performed in normal adrenal glands obtained from eight premenopausal and eight postmenopausal women. DHEA, DHEAS, A4, and T were significantly higher in pre- than in postmenopausal women (2.9, 2.8, 2.9, and 1.6-fold, respectively; P < 0.0001). In contrast, the 11-oxyandrogens did not decrease with aging, and the 11OHT/T and 11OHA4/A4 ratios showed strong positive correlations with age (r = 0.5 and 0.8, respectively; P < 0.0001). Double immunofluorescence analysis showed that with the involution of the zona reticularis in the old adrenals, the sharp zonal segregation of HSD3B2 and CYB5A becomes less distinct, and areas of HSD3B2 and CYB5A overlap are observed. Unlike DHEA, DHEAS, A4, and T, the 11oxyandrogens do not decline in aging women. Structural changes within the adrenal cortex might explain the evolution of androgen profiles in aging women.

Nanba AT ，Rege J ，Ren J ，Auchus RJ ，Rainey WE ，Turcu AF ... -  《-》

Circulating 11-oxygenated androgens across species.

The androgen precursors, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) are produced in high amounts by the adrenal cortex primarily in humans and a few other primates. The human adrenal also secretes 11-oxygenated androgens (11-oxyandrogens), including 11β-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), 11β-hydroxytestosterone (11OHT) and 11-ketotestosterone (11KT), of which 11OHT and 11KT are bioactive androgens. The 11-oxyandrogens, particularly 11KT, have been recognized as biologically important testicular androgens in teleost fishes for decades, but their physiological contribution in humans has only recently been established. Beyond fish and humans, however, the presence of 11-oxyandrogens in other species has not been investigated. This study provides a comprehensive analysis of a set of C19 steroids, including the traditional androgens and 11-oxyandrogens, across 18 animal species. As previously shown, serum DHEA and DHEAS were much higher in primates than all other species. Circulating 11-oxyandrogens, especially 11KT, were observed in notable amounts in male, but not in female trout, consistent with gonadal origin in fish. The circulating concentrations of 11-oxyandrogens ranged from 0.1 to 10 nM in pigs, guinea pigs and in all the primates studied (rhesus macaque, baboon, chimpanzee and human) but not in rats or mice, and 11OHA4 was consistently the most abundant. In contrast to fish, serum 11KT concentrations were similar in male and female primates for each species, despite significantly higher circulating testosterone in males, suggesting that 11KT production in these species is not testis-dependent and primarily originates from adrenal-derived 11-oxyandrogen precursors.

Rege J ，Garber S ，Conley AJ ，Elsey RM ，Turcu AF ，Auchus RJ ，Rainey WE ... -  《-》

11β-hydroxyandrostenedione, the product of androstenedione metabolism in the adrenal, is metabolized in LNCaP cells by 5α-reductase yielding 11β-hydroxy-5α-androstanedione.

11β-Hydroxyandrostenedione (11OHA4), which is unique to the adrenal, was first isolated from human adrenal tissue in the fifties. It was later shown in the sixties that 11β-hydroxytestosterone (11OHT) was also produced by the human adrenal. Attention has shifted back to these adrenal androgens once more, as improved analytical techniques have enabled more accurate detection of steroid hormones. In this paper, we investigated the origin of these metabolites as well as their subsequent metabolism and examined a possible physiological role for 11OHA4 in prostate cancer cells. In H295R cells treated with forskolin and trilostane, etomidate, a reported cytochrome P450 11β-hydroxylase (CYP11B1) inhibitor, blocked the production of corticosterone, cortisol, 11OHA4 and 11OHT. The metabolism of androstenedione and testosterone by CYP11B1 and aldosterone synthase (CYP11B2) was assayed. Androstenedione was converted by CYP11B1, while the conversion by CYP11B2 was negligible. Both enzymes readily converted testosterone. The metabolism of these 11β-hydroxylated metabolites by 11β-hydroxysteroid dehydrogenase (11βHSD) types 1 and 2 was subsequently investigated. 11βHSD2 catalyzed the conversion of both 11OHA4 and 11OHT to their respective keto-steroids, while 11βHSD1 catalyzed the conversion of 11-ketoandrostenedione and 11-ketotestosterone to their respective hydroxy-steroids in Chinese hamster ovary cells. Investigating a functional role, steroid 5α-reductase types 1 and 2 converted 11OHA4 to 11β-hydroxy-5α-androstanedione (11OH-5α-dione), identified by accurate mass detection. UPLC-MS/MS analyses of 11OHA4 metabolism in LNCaP androgen-dependent prostate cancer cells, identified the 5α-reduced metabolite as well as 11-ketoandrostenedione and 11-ketotestosterone, with the latter indicating conversion by 17β-hydroxysteroid dehydrogenase. Downstream metabolism by 11βHSD2 and by 5α-reductase may therefore indicate a physiological role for 11OHA4 and/or 11OH-5α-dione in normal and prostate cancer cells.

Swart AC ，Schloms L ，Storbeck KH ，Bloem LM ，Toit Td ，Quanson JL ，Rainey WE ，Swart P ... -  《-》