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Toxics Nov 2022This paper describes a methodology for simultaneous determination of 19 steroid hormones, viz. estrone, estradiol, estriol, testosterone, 5α-dihydrotestosterone,...
This paper describes a methodology for simultaneous determination of 19 steroid hormones, viz. estrone, estradiol, estriol, testosterone, 5α-dihydrotestosterone, androstenedione, androstenediol, dehydroepiandrosterone, progesterone, pregnenolone, 17α-OH-progesterone, 17α-OH-pregnenolone, cortisone, cortisol, 11-deoxycortisol, 11-deoxycorticosterone, 11-dehydrocorticosterone, aldosterone, and corticosterone, in 500-µL of urine or serum/plasma. The method was optimized using isotopically labeled internal standards and liquid-liquid extraction followed by detection using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS). Dansylation of estrogens significantly improved their sensitivities (~11- to 23-fold) and chromatographic separation. The respective limit of detection (LOD) and limit of quantification (LOQ) of all analytes were 0.04−0.28 and 0.14−0.92 ng/mL in human urine, and 0.11−0.35 and 0.38−1.18 ng/mL in human serum/plasma. Recoveries of all analytes (except for progesterone) fortified at 10, 20, and 200 ng/mL in urine and serum were 80−120%, with standard deviations ranging from 0 to 17.3%. Repeated analysis of similarly fortified urine and serum samples yielded intra-day and inter-day variations of 0−21.7% and 0.16−11.5%, respectively. All analytes except cortisone exhibited weak matrix effects in urine and serum (−13.9−18.2%). The method was further validated through the analysis of the National Institute of Standards and Technology (NIST) plasma Standard Reference Material (SRM1950) with certified concentrations for cortisol, progesterone, and testosterone (coefficient of variation: 3−11%). The developed method was applied in the analysis of urine samples from 20 volunteers, which revealed the occurrence of 16 analytes with detection frequencies (DFs) > 80%. Furthermore, 15 analytes were found in plasma SRM1950, indicating the feasibility of our method in the analysis of steroid hormones in urine and serum/plasma. This method will facilitate analysis of steroid hormones in population-based biomonitoring studies.
PubMed: 36422894
DOI: 10.3390/toxics10110687 -
Andrology Jan 2021Previous studies on gonadal steroidogenesis have not compared metabolic pathways between fetal and adult mouse testes to date. (Comparative Study)
Comparative Study
BACKGROUND
Previous studies on gonadal steroidogenesis have not compared metabolic pathways between fetal and adult mouse testes to date.
OBJECTIVES
To evaluate comparative metabolic signatures of testicular steroids between fetus and adult mice using gas chromatography-mass spectrometry (GC-MS)-based steroid profiling.
MATERIALS AND METHODS
GC-MS with molecular-specific scan modes was optimized for selective and sensitive detection of 23 androgens, 7 estrogens, 14 progestogens, and 13 corticoids from mouse testes with a quantification limit of 0.1-5.0 ng/mL and reproducibility (coefficient of variation: 0.3%-19.9%). Based on 26 steroids quantitatively detected in testes, comparative steroid signatures were analyzed for mouse testes of 8 fetuses on embryonic day 16.5 and 8 adults on postnatal days 56-60.
RESULTS
In contrast to large amounts of steroids in adult testes (P < .0002), all testicular levels per weight unit of protein were significantly increased in fetal testes (P < .002, except 6β-hydroxytestosterone of P = .065). Both 11β-hydroxyandrostenedione and 7α-hydroxytestosterone were only measurable in fetal testes, and metabolic ratios of testosterone to androstenediol and androstenedione were also increased in fetal testes (P < .05 for both).
DISCUSSION AND CONCLUSION
Testicular steroid signatures showed that both steroidogenic Δ and Δ pathways in the production of testosterone were activated more during prenatal development. Both 7α- and 11β-hydroxylations were predominant, while hydroxylations at C-6, C-15, and C-16 of testosterone and androstenedione were decreased in the fetus. The present GC-MS-based steroid profiling may facilitate understanding of the development of testicular steroidogenesis.
Topics: Animals; Fetus; Gas Chromatography-Mass Spectrometry; Gonadal Steroid Hormones; Male; Mice; Testis
PubMed: 32810374
DOI: 10.1111/andr.12893 -
Journal of Bone and Mineral Research :... Aug 2021Bone metastasis is a complication of prostate cancer in up to 90% of men afflicted with advanced disease. Therapies that reduce androgen exposure remain at the forefront...
Bone metastasis is a complication of prostate cancer in up to 90% of men afflicted with advanced disease. Therapies that reduce androgen exposure remain at the forefront of treatment. However, most prostate cancers transition to a state whereby reducing testicular androgen action becomes ineffective. A common mechanism of this transition is intratumoral production of testosterone (T) using the adrenal androgen precursor dehydroepiandrosterone (DHEA) through enzymatic conversion by 3β- and 17β-hydroxysteroid dehydrogenases (3βHSD and 17βHSD). Given the ability of prostate cancer to form blastic metastases in bone, we hypothesized that osteoblasts might be a source of androgen synthesis. RNA expression analyses of murine osteoblasts and human bone confirmed that at least one 3βHSD and 17βHSD enzyme isoform was expressed, suggesting that osteoblasts are capable of generating androgens from adrenal DHEA. Murine osteoblasts were treated with 100 nM and 1 μM DHEA or vehicle control. Conditioned media from these osteoblasts were assayed for intermediate and active androgens by liquid chromatography-tandem mass spectrometry. As DHEA was consumed, the androgen intermediates androstenediol and androstenedione were generated and subsequently converted to T. Conditioned media of DHEA-treated osteoblasts increased androgen receptor (AR) signaling, prostate-specific antigen (PSA) production, and cell numbers of the androgen-sensitive prostate cancer cell lines C4-2B and LNCaP. DHEA did not induce AR signaling in osteoblasts despite AR expression in this cell type. We describe an unreported function of osteoblasts as a source of T that is especially relevant during androgen-responsive metastatic prostate cancer invasion into bone. © 2021 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.
Topics: Androgens; Animals; Cell Line, Tumor; Dehydroepiandrosterone; Humans; Male; Mice; Osteoblasts; Prostatic Neoplasms; Receptors, Androgen; Testosterone
PubMed: 33900658
DOI: 10.1002/jbmr.4313 -
PloS One 2021Animal experiments have consistently shown that estrogen receptor β (ERβ)-selective ligands have antidepressant and anxiolytic effects. In humans, endogenous ligands...
Animal experiments have consistently shown that estrogen receptor β (ERβ)-selective ligands have antidepressant and anxiolytic effects. In humans, endogenous ligands for ERβ include 5α-androstane-3β, 17β-diol (3βAdiol) and androstenediol (Δ5-diol). We determined, for the first time, the exact serum levels of 3βAdiol and Δ5-diol in young healthy volunteers using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We investigated the effect of the menstrual cycle on the levels of these steroids in women; then, we performed a gender comparison. Blood samples were collected from 48 subjects: 23 women (mean age = 28.4±7.8 years) and 25 men (mean age = 31.4±7.8 years). We collected the blood samples of women at three time-points in the menstrual cycle: the early follicular phase, ovulatory or mid-cycle phase, and mid-luteal phase. A total of 92 blood samples were analyzed using LC-MS/MS. The levels of two well-studied steroids, namely dehydroepiandrosterone (DHEA) and 17β-estradiol (E2), were simultaneously measured. Depression rating scale (Hamilton Rating Scale for Depression, Beck Depression Inventory-II and Quick Inventory of Depressive Symptomatology) scores were also recorded at the time of blood sampling. Significant differences in the levels of 3βAdiol and E2 and in the depression rating scale scores were observed over the duration of the menstrual cycle of the women. The levels of 3βAdiol and Δ5-diol were significantly lower in women than in men. E2 levels were higher in women than in men, and DHEA levels did not differ significantly between men and women. Further, women had higher scores than men on the Hamilton Rating Scale for Depression. Sex differences in depressive symptoms can be explained by 3βAdiol and Δ5-diol levels, and the effect of the menstrual cycle on mood can be explained by 3βAdiol and E2 levels, not by Δ5-diol level.
Topics: Adult; Androstenediol; Chromatography, Liquid; Dehydroepiandrosterone; Estradiol; Female; Humans; Male; Menstrual Cycle; Sex Characteristics; Tandem Mass Spectrometry; Young Adult
PubMed: 34910781
DOI: 10.1371/journal.pone.0261440 -
Communications Biology Apr 2022Identifying the genetic determinants of inter-individual variation in lipid species (lipidome) may provide deeper understanding and additional insight into the...
Identifying the genetic determinants of inter-individual variation in lipid species (lipidome) may provide deeper understanding and additional insight into the mechanistic effect of complex lipidomic pathways in CVD risk and progression beyond simple traditional lipids. Previous studies have been largely population based and thus only powered to discover associations with common genetic variants. Founder populations represent a powerful resource to accelerate discovery of previously unknown biology associated with rare population alleles that have risen to higher frequency due to genetic drift. We performed a genome-wide association scan of 355 lipid species in 650 individuals from the Amish founder population including 127 lipid species not previously tested. To the best of our knowledge, we report for the first time the lipid species associated with two rare-population but Amish-enriched lipid variants: APOB_rs5742904 and APOC3_rs76353203. We also identified novel associations for 3 rare-population Amish-enriched loci with several sphingolipids and with proposed potential functional/causal variant in each locus including GLTPD2_rs536055318, CERS5_rs771033566, and AKNA_rs531892793. We replicated 7 previously known common loci including novel associations with two sterols: androstenediol with UGT locus and estriol with SLC22A8/A24 locus. Our results show the double power of founder populations and detailed lipidome to discover novel trait-associated variants.
Topics: Amish; DNA-Binding Proteins; Founder Effect; Genetics, Population; Genome-Wide Association Study; Humans; Lipidomics; Lipids; Nuclear Proteins; Transcription Factors
PubMed: 35393526
DOI: 10.1038/s42003-022-03291-2 -
The Journal of Steroid Biochemistry and... May 2023The canonical androgen synthesis in Leydig cells involves Δ5 and Δ4 steroids. Besides, the backdoor pathway, eompassing 5α and 5α,3α steroids, is gaining interest...
The canonical androgen synthesis in Leydig cells involves Δ5 and Δ4 steroids. Besides, the backdoor pathway, eompassing 5α and 5α,3α steroids, is gaining interest in fetal and adult pathophysiology. Moreover, the role of androgen epimers and progesterone metabolites is still unknown. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 20 steroids and used it to investigate the steroid secretion induced by human chorionic gonadotropin (hCG) in the mouse Leydig tumor cell line 1 (mLTC1). Steroids were extracted from 500 µL supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100 × 3 mm, 3 µm column, with 100 µM NH4F and methanol as mobile phases, and analyzed by positive electrospray ionization and multiple reaction monitoring. Sensitivity ranged within 0.012-38.0 nmol/L. Intra-assay and inter-assay imprecision were < 9.1% and 10.0%, respectively. Trueness, recovery and matrix factor were within 93.4-122.0, 55.6-104.1 and 76.4-106.3%, respectively. Levels of 16OH-progesterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, 17OH-progesterone, androstenedione, epitestosterone, dihydrotestosterone, progesterone, androsterone and 17OH-allopregnanolone were effectively measured. Traces of 17OH-dihydroprogesterone, androstanediol and dihydroprogesterone were found, whereas androstenediol, 17OH-pregnenolone, dehydroepiandrosterone, pregnenolone and allopregnanolone showed no peak. hCG induced an increase of 80.2-102.5 folds in 16OH-progesterone, androstenedione and testosterone, 16.6 in dihydrotestosterone, 12.2-27.5 in epitestosterone, progesterone and metabolites, 8.1 in 17OH-allopregnanolone and ≤ 3.3 in 5α and 5α,3α steroids. In conclusion, our LC-MS/MS method allows exploring the Leydig steroidogenesis flow according to multiple pathways. Beside the expected stimulation of the canonical pathway, hCG increased progesterone metabolism and, to a low extent, the backdoor route.
Topics: Humans; Chorionic Gonadotropin; Animals; Mice; Cell Line, Tumor; Leydig Cells; Male; Chromatography, Liquid; Tandem Mass Spectrometry; Gonadal Steroid Hormones
PubMed: 36764496
DOI: 10.1016/j.jsbmb.2023.106270 -
American Journal of Obstetrics and... Jul 2022Risk-reducing salpingo-oophorectomy is an effective ovarian cancer risk reduction strategy. However, bilateral oophorectomy has also been associated with increased...
BACKGROUND
Risk-reducing salpingo-oophorectomy is an effective ovarian cancer risk reduction strategy. However, bilateral oophorectomy has also been associated with increased long-term nonneoplastic sequelae, effects suggested to be mediated through reductions in systemic sex steroid hormone levels. Currently, it is unclear whether the postmenopausal ovary contributes to the systemic hormonal milieu or whether postmenopausal ovarian volume or other factors, such as body mass index and age, affect systemic hormone levels.
OBJECTIVE
We examined the impact of oophorectomy on sex steroid hormone levels in postmenopausal women. Furthermore, we explored how well ovarian volume measured by transvaginal ultrasound correlated with direct ovarian measures obtained during surgical pathology evaluation and investigated the association between hormone levels and ovarian volumes.
STUDY DESIGN
Postmenopausal women who underwent risk-reducing salpingo-oophorectomy (180 cases) or ovarian cancer screening (38 controls) enrolled in an international, prospective study of risk-reducing salpingo-oophorectomy and risk of ovarian cancer algorithm-based screening among women at increased risk of ovarian cancer (Gynecologic Oncology Group-0199) were included in this analysis. Controls were frequency matched to the cases on age at menopause, age at study entry, and time interval between blood draws. Ovarian volume was calculated using measurements obtained from transvaginal ultrasound in both cases and controls and measurements recorded in surgical pathology reports from cases. Serum hormone levels of testosterone, androstenedione, androstenediol, dihydrotestosterone, androsterone, dehydroepiandrosterone, estrone, estradiol, and sex hormone-binding globulin were measured at baseline and follow-up. Spearman correlation coefficients were used to compare ovarian volumes as measured on transvaginal ultrasound and pathology examinations. Correlations between ovarian volumes by transvaginal ultrasound and measured hormone levels were examined using linear regression models. All models were adjusted for age. Paired t tests were performed to evaluate individual differences in hormone levels before and after risk-reducing salpingo-oophorectomy.
RESULTS
Ovarian volumes measured by transvaginal ultrasound were only moderately correlated with those reported on pathology reports (Spearman rho [ρ]=0.42). The median time interval between risk-reducing salpingo-oophorectomy and follow-up for the cases was 13.3 months (range, 6.0-19.3), and the median time interval between baseline and follow-up for the controls was 12.7 months (range, 8.7-13.4). Sex steroid levels decreased with age but were not correlated with transvaginal ultrasound ovarian volume, body mass index, or time since menopause. Estradiol levels were significantly lower after risk-reducing salpingo-oophorectomy (percentage change, -61.9 post-risk-reducing salpingo-oophorectomy vs +15.2 in controls; P=.02), but no significant differences were seen for the other hormones.
CONCLUSION
Ovarian volumes measured by transvaginal ultrasound were moderately correlated with volumes directly measured on pathology specimens and were not correlated with sex steroid hormone levels in postmenopausal women. Estradiol was the only hormone that declined significantly after risk-reducing salpingo-oophorectomy. Thus, it remains unclear whether the limited post-risk-reducing salpingo-oophorectomy changes in sex steroid hormones among postmenopausal women impact long-term adverse outcomes.
Topics: Estradiol; Female; Gonadal Steroid Hormones; Humans; Ovarian Neoplasms; Postmenopause; Prospective Studies; Salpingo-oophorectomy
PubMed: 35216968
DOI: 10.1016/j.ajog.2022.02.022 -
Molecules (Basel, Switzerland) Jan 2020Steroidal glycosides are important sources of innovative drugs. The increased diversification of steroidal glycosides will expand the probability of discovering active...
Steroidal glycosides are important sources of innovative drugs. The increased diversification of steroidal glycosides will expand the probability of discovering active molecules. It is an efficient approach to diversify steroidal glycosides by using steroidal glycosyltransferases. OcUGT1, a uridine diphosphate-d-glucose (UDP-Glc)-dependent glycosyltransferase from , is a multifunctional enzyme, and its glycodiversification potential towards steroids has never been fully explored. Herein, the glycodiversification capability of OcUGT1 towards 25 steroids through glucosylation and transglucosylation reactions were explored. Firstly, each of 25 compounds was glucosylated with UDP-Glc. Under the action of OcUGT1, five steroids (testosterone, deoxycorticosterone, hydrocortisone, estradiol, and 4-androstenediol) were glucosylated to form corresponding mono-glucosides and biosides. Next, OcUGT1-mediated transglucosylation activity of these compounds with another sugar donor -nitrophenyl-β-d-glucopyranoside (NPGlc) was investigated. Results revealed that the same five steroids could be glucosylated to generate mono-glucosides and biosides by OcUGT1 through transglucosylation reactions. These data indicated that OcUGT1-assisted glycodiversification of steroids could be achieved through glucosylation and transglucosylation reactions. These results provide a way to diversify steroidal glycosides, which lays the foundation for the increase of the probability of obtaining active lead compounds.
Topics: Glucosides; Glycosides; Glycosylation; Glycosyltransferases; Ornithogalum; Steroids
PubMed: 31979165
DOI: 10.3390/molecules25030475 -
Diseases (Basel, Switzerland) Mar 2020Evidence of altered cholesterol and steroid hormones in autism is increasing. However, as boys are more often affected, evidence mainly relates to autistic males,...
Evidence of altered cholesterol and steroid hormones in autism is increasing. However, as boys are more often affected, evidence mainly relates to autistic males, whereas evidence for affected autistic girls is sparse. Therefore, a comprehensive gas chromatography mass spectrometry-based steroid hormone metabolite analysis was conducted from autistic girls. Results show increased levels of several steroid hormones, especially in the class of androgens in autistic girls such as testosterone or androstenediol. The increase of the majority of steroid hormones in autistic girls is probably best explained multifactorially by a higher substrate provision in line with the previously developed cholesterol hypothesis of autism.
PubMed: 32183287
DOI: 10.3390/diseases8010006 -
Molecules (Basel, Switzerland) Jul 2022Multiple biological functions of extract were evaluated in the current work. Phytochemical components of the extract were detected by Gas Chromatography-Mass...
Molecular Interaction Studies and Phytochemical Characterization of L. Constituents with Multiple Biological Utilities as Antioxidant, Antimicrobial, Anticancer and Anti-Hemolytic Agents.
Multiple biological functions of extract were evaluated in the current work. Phytochemical components of the extract were detected by Gas Chromatography-Mass Spectrometry (GC-MS) and High-performance liquid chromatography (HPLC). Moreover, extract was estimated for antioxidant potential by 2,2-Diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical scavenging, antimicrobial activity by well diffusion, and anticoagulant activity via prothrombin time (PT) and activated partial thromboplastin time (APTT). GC-MS analysis detected compounds including cholesterol margarate, stigmast-5-en-3-ol, 19-nor-4-androstenediol, androstan-17-one, pulegone-1,2-epoxide, isochiapin B, dotriacontane, hexadecanoic acid and neophytadiene. Chrysoeriol (15.36 µg/mL) was followed by kaempferol (11.14 µg/mL) and 7-OH flavone (10.14 µg/mL), catechin (4.11 µg/mL), hisperdin (3.05 µg/mL), and luteolin (2.36 µg/mL) were detected by HPLC as flavonoids, in addition to ferulic (13.19 µg/mL), cinnamic (12.69 µg/mL), caffeic (11.45 µg/mL), pyrogallol (9.36 µg/mL), -coumaric (5.06 µg/mL) and salicylic (4.17 µg/mL) as phenolics. Antioxidant activity was detected with IC 18 µg/mL, hemolysis inhibition was recorded as 79.8% at 1000 μg/mL, and PT and APTT were at 21.5 s and 49.5 s, respectively, at 50 μg/mL of extract. The acute toxicity of extract was recorded against PC3 (IC 97.99 µg/mL) and MCF7 (IC 80.21 µg/mL). Antimicrobial activity of extract was documented against , , , , , but not against black fungus . Molecular docking was applied using MOE (Molecular Operating Environment) to explain the biological activity of neophytadiene, luteolin, chrysoeriol and kaempferol. These compounds could be suitable for the development of novel pharmacological agents for treatment of cancer and bacterial infections.
Topics: Anti-Bacterial Agents; Anti-Infective Agents; Antioxidants; Hemolysis; Hemolytic Agents; Kaempferols; Luteolin; Mentha pulegium; Molecular Docking Simulation; Phytochemicals; Plant Extracts
PubMed: 35956775
DOI: 10.3390/molecules27154824