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PloS One 2015Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of...
Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing.
Topics: Cell Line; Chromatography, Liquid; Endometrium; Female; Humans; Metabolome; Metabolomics; Pregnancy; Progesterone; Reproducibility of Results; Tandem Mass Spectrometry
PubMed: 25680188
DOI: 10.1371/journal.pone.0117984 -
Clinical Endocrinology Feb 2021Increasing magnesium intake might reduce the risk of cardiovascular disease (CVD). Whether potential effects on cortisol contribute to these beneficial effects on... (Randomized Controlled Trial)
Randomized Controlled Trial
OBJECTIVE
Increasing magnesium intake might reduce the risk of cardiovascular disease (CVD). Whether potential effects on cortisol contribute to these beneficial effects on cardiovascular health remains unclear. We therefore studied effects of long-term oral magnesium supplementation on glucocorticoid metabolism, specifically on the excretion of urinary cortisol, cortisone and their metabolites, as well as on the ratios reflecting enzymatic activity of 11β-hydroxysteroid dehydrogenases (11β-HSDs) and A-ring reductases.
DESIGN
A post-hoc analysis of a randomized trial with allocation to a magnesium supplement (350 mg/day) or a placebo for 24-week.
PATIENTS
Forty-nine overweight men and women, aged between 45 and 70 years.
MEASUREMENTS
Cortisol, cortisone and their metabolites (tetrahydrocortisol [THF], allo-tetrahydrocortisol [allo-THF] and tetrahydrocortisone [THE]) were measured in 24-h urine samples. Enzymatic activities of 11β-HSD overall and of 11β-HSD type 2 were estimated as the urinary (THF + allo-THF [THFs])/THE and cortisol/cortisone ratios, respectively. A-ring reductase activity was assessed by ratios of THF/allo-THF, allo-THF/cortisol, THF/cortisol and THE/cortisone.
RESULTS
After 24-week, urinary cortisol excretion was decreased in the magnesium group as compared with the placebo group (-32 nmol/24-h, 95% CI: -59; -5 nmol/24-h, p = .021). Ratios of THFs/THE and cortisol/cortisone were decreased following magnesium supplementation by 0.09 (95% CI: 0.02; 0.17, p = .018) and 0.10 (95% CI: 0.03; 0.17, p = .005), respectively. No effects were observed on A-ring reductase activity.
CONCLUSIONS
We observed a beneficial effect of magnesium supplementation towards a lower 24-h urinary cortisol excretion together with an increased activity of 11β-HSD type 2. Our findings may provide another potential mechanism by which increased magnesium intake lowers CVD risk (ClinicalTrials.gov identifier: NCT02235805).
Topics: Aged; Cortisone; Dietary Supplements; Female; Glucocorticoids; Humans; Hydrocortisone; Magnesium; Male; Middle Aged; Tetrahydrocortisone
PubMed: 33030273
DOI: 10.1111/cen.14350 -
Journal of Cellular Physiology May 2013A group of bioactive steroidal glycosides (pregnanes) with anorectic activity in animals was isolated from several genera of milkweeds including Hoodia and Asclepias. In...
A group of bioactive steroidal glycosides (pregnanes) with anorectic activity in animals was isolated from several genera of milkweeds including Hoodia and Asclepias. In this study, we investigated the effects, structure-activity relationships, and mechanism of action of pregnane glycosides on steroidogenesis in human adrenocortical H295R cells. Administration of pregnane glycosides for 24 h suppressed the basal and forskolin-stimulated release of androstenedione, corticosterone, and cortisone from H295R cells. The conversion of progesterone to 11-deoxycorticosterone and 17-hydroxyprogesterone to either androstenedione or 11-deoxycortisol was most strongly affected, with 12-cinnamoyl-, benzoyl-, and tigloyl-containing pregnanes showing the highest activity. Incubation of pregnane glycosides for 24 h had no effect on mRNA transcripts of CYP11A1, CYP21A1, CYP11B1 cytochrome enzymes and steroidogenic acute regulatory protein (StaR) protein, yet resulted in twofold decrease in HSD3B1 mRNA levels. At the same time, pregnane glycosides had no effect on the CYP1, 2, or 3 drug and steroid metabolism enzymes and showed weak Na(+) /K(+) ATPase and glucocorticoid receptor binding. Taken together, these data suggest that pregnane glycosides specifically suppress steroidogenesis through strong inhibition of 11β-hydroxylase and steroid 17-alpha-monooxygenase, and weak inhibition of cytochrome P450 side chain cleavage enzyme and 21β-hydroxylase, but not 3β-hydroxysteroid dehydrogenase/isomerase.
Topics: 3-Hydroxysteroid Dehydrogenases; Adrenal Cortex Hormones; Androstenedione; Animals; Cell Line, Tumor; Cholesterol Side-Chain Cleavage Enzyme; Corticosterone; Cortisone; Glycosides; Humans; Pregnanes; Progesterone; Steroid 11-beta-Hydroxylase; Steroid 17-alpha-Hydroxylase; Structure-Activity Relationship
PubMed: 23065845
DOI: 10.1002/jcp.24262 -
Clinical Chemistry and Laboratory... Apr 2022Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far,...
OBJECTIVES
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration.
METHODS
Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials.
RESULTS
Using CAL1, intra-laboratory CVs ranged between 2.8-7.4%, 4.4-18.0% and 5.2-22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p<0.001), respectively. Using CAL1, median bias vs. all laboratory-medians ranged from -6.6 to 6.9%, -17.2 to 7.8% and -12.0 to 16.8% for cortisol, 17OH-progesterone and aldosterone, respectively. Regression lines significantly deviated from the best fit for most laboratories. Using CAL3 improved cortisol and 17OH-progesterone between-method bias and correlation.
CONCLUSIONS
Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements.
Topics: Aldosterone; Calibration; Chromatography, Liquid; Humans; Hydrocortisone; Progesterone; Reproducibility of Results; Tandem Mass Spectrometry
PubMed: 35172417
DOI: 10.1515/cclm-2021-1028 -
Psychopharmacology Sep 2014Given that the pregnane neurosteroid, 5α-pregnan-3α-ol-20-one (3α,5α-THP), is increased following behavioral challenges (e.g., mating), and that there is...
Involvement of pregnane xenobiotic receptor in mating-induced allopregnanolone formation in the midbrain and hippocampus and brain-derived neurotrophic factor in the hippocampus among female rats.
RATIONALE
Given that the pregnane neurosteroid, 5α-pregnan-3α-ol-20-one (3α,5α-THP), is increased following behavioral challenges (e.g., mating), and that there is behavioral-induced biosynthesis of 3α,5α-THP in midbrain and mesocorticolimbic structures, 3α,5α-THP likely has a role in homeostasis and motivated reproduction and reproduction-related behaviors (e.g., affect, affiliation). The role of pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism, for these effects is of continued interest.
OBJECTIVES
We hypothesized that there would be differences in brain levels of 3α,5α-THP following varied behavioral experiences, an effect abrogated by knockdown of PXR in the midbrain.
METHODS
Proestrous rats were infused with PXR antisense oligonucleotides (AS-ODNs) or vehicle to the ventral tegmental area before different behavioral manipulations and assessments. Endpoints were expression levels of PXR in the midbrain, 3α,5α-THP, and ovarian steroids (estradiol, progesterone, dihydroprogesterone) in the midbrain, striatum, hippocampus, hypothalamus, prefrontal cortex, and plasma.
RESULTS
Across experiments, knocking down PXR reduced PXR expression and 3α,5α-THP levels in the midbrain and hippocampus. There were differences in terms of the behavioral manipulations, such that paced mating had the most robust effects to increase 3α,5α-THP levels and reduce open field exploration and social interaction. An additional question that was addressed is whether brain-derived neurotrophic factor (BDNF) is a downstream factor for regulating effects of behavioral-induced 3α,5α-THP biosynthesis. Rats infused with PXR AS-ODNs had lower levels of BDNF in the hippocampus.
CONCLUSION
Thus, PXR may be a regulator of mating-induced 3α,5α-THP formation and behavioral changes and neural plasticity, such as BDNF.
Topics: Animals; Brain-Derived Neurotrophic Factor; Dehydroepiandrosterone; Estrous Cycle; Female; Hippocampus; Mesencephalon; Oligonucleotides, Antisense; Pregnane X Receptor; Pregnanolone; Progesterone; Rats; Rats, Long-Evans; Receptors, Steroid; Sexual Behavior, Animal; Ventral Tegmental Area
PubMed: 24781516
DOI: 10.1007/s00213-014-3569-3 -
International Journal of Molecular... Jun 2022Progesterone biotransformation is worth studying because of the high industrial value of its derivatives. This study investigated the catalytic ability of the...
Progesterone biotransformation is worth studying because of the high industrial value of its derivatives. This study investigated the catalytic ability of the entomopathogenic filamentous fungus strain KCh KW1.1 to transform progesterone derivatives: 11α-hydroxyprogesterone, 17α-hydroxyprogesterone, 16α,17α-epoxyprogesterone and pregnenolone. In the culture of KCh KW1.1, 11α-hydroxyprogesterone was effectively transformed into only one product: 6β,11α-dihydroxyprogesterone. Transformation of 17α-hydroxyprogesterone gave three hydroxy derivatives: 6β,17α-dihydroxyprogesterone, 12β,17α-dihydroxyprogesterone and 6β,12β,17α-trihydroxyprogesterone. Two products: 6β-hydroxy-16α,17α-epoxyprogesterone and 6β,11α-dihydroxy-16α,17α-epoxyprogesterone, were obtained from the 16α,17α-epoxyprogesterone transformation. We isolated two compounds from the biotransformation medium with pregnenolone: 11α-hydroxy-7-oxopregnenolone and 5α,6α-epoxy-3β,11α-dihydroxypregnan-7,20-dione. In this study, we observed only mono- and dihydroxy derivatives of the tested substrates, and the number of obtained products for each biotransformation did not exceed three.
Topics: Algestone; Biotransformation; Cordyceps; Hydroxylation; Hydroxyprogesterones; Pregnenolone; Progesterone
PubMed: 35806021
DOI: 10.3390/ijms23137015 -
Steroids Sep 2017Five new pregnane-type steroidal glycosides, named menarandrosides A-E (1-2, 5-7) were isolated from the aerial parts of Cynanchum menarandrense, together with three...
Five new pregnane-type steroidal glycosides, named menarandrosides A-E (1-2, 5-7) were isolated from the aerial parts of Cynanchum menarandrense, together with three known compounds, carumbelloside I (3), carumbelloside II (4), and pregnenolone-3-O-gentiobioside (8). Their structures were determined on the basis of spectroscopic analyses including NMR and mass spectrometry, reporting C-21 steroids glycosylated only by one or two glucose moieties. Compounds were then investigated for their potential to stimulate glucagon-like peptide-1 (GLP-1) secretion in intestinal cells; although none of the pure compounds had any influence, the fraction enriched in pregnanes exhibited a significant activity, suggesting a possible synergistic effect. Furthermore, none of the purified compounds affected cell viability.
Topics: Cell Line; Cell Survival; Cynanchum; Glucagon-Like Peptide 1; Glycosides; Intestines; Pregnanes
PubMed: 28636871
DOI: 10.1016/j.steroids.2017.06.005 -
The Journal of Pharmacology and... Apr 2016Synaptic GABAA receptors are primary mediators of rapid inhibition in the brain and play a key role in the pathophysiology of epilepsy and other neurologic disorders....
Synaptic GABAA receptors are primary mediators of rapid inhibition in the brain and play a key role in the pathophysiology of epilepsy and other neurologic disorders. The δ-subunit GABAA receptors are expressed extrasynaptically in the dentate gyrus and contribute to tonic inhibition, promoting network shunting as well as reducing seizure susceptibility. However, the neurosteroid structure-function relationship at δGABA(A) receptors within the native hippocampus neurons remains unclear. Here we report a structure-activity relationship for neurosteroid modulation of extrasynaptic GABAA receptor-mediated tonic inhibition in the murine dentate gyrus granule cells. We recorded neurosteroid allosteric potentiation of GABA as well as direct activation of tonic currents using a wide array of natural and synthetic neurosteroids. Our results shows that, for all neurosteroids, the C3α-OH group remains obligatory for extrasynaptic receptor functional activity, as C3β-OH epimers were inactive in activating tonic currents. Allopregnanolone and related pregnane analogs exhibited the highest potency and maximal efficacy in promoting tonic currents. Alterations at the C17 or C20 region of the neurosteroid molecule drastically altered the transduction kinetics of tonic current activation. The androstane analogs had the weakest modulatory response among the analogs tested. Neurosteroid potentiation of tonic currents was completely (approximately 95%) diminished in granule cells from δ-knockout mice, suggesting that δ-subunit receptors are essential for neurosteroid activity. The neurosteroid sensitivity of δGABA(A) receptors was confirmed at the systems level using a 6-Hz seizure test. A consensus neurosteroid pharmacophore model at extrasynaptic δGABA(A) receptors is proposed based on a structure-activity relationship for activation of tonic current and seizure protection.
Topics: Animals; Anticonvulsants; Dentate Gyrus; GABA Agonists; GABA Modulators; In Vitro Techniques; Interneurons; Mice; Mice, Inbred C57BL; Mice, Knockout; Midazolam; Neurons; Neurotransmitter Agents; Pregnanes; Pregnanolone; Receptors, GABA-A; Seizures; Structure-Activity Relationship
PubMed: 26857959
DOI: 10.1124/jpet.115.229302 -
Molecules (Basel, Switzerland) Mar 2023The ethnobotanical plant has been used for hundreds of years for Dai people in Yunnan Province, China. Previously, chemical investigations on this plant have revealed...
The ethnobotanical plant has been used for hundreds of years for Dai people in Yunnan Province, China. Previously, chemical investigations on this plant have revealed that pregnane glycosides were the main biological constituents. Nine new pregnane glycosides, marsdeosides A-I (-), were isolated from cultivated dried stems of the medicinal plant in this study. The structures were analyzed by extensive spectroscopic analysis, including 1D, 2D NMR, HRESIMS, and IR spectroscopic analysis. The absolute configurations of the sugar moieties were identified by comparing the values and specific optical rotations with those of the commercially available standard samples and the data reported in the literature. Marsdeosides A () featured an unusual 8,14--pregnane skeleton. Compounds , , and showed activity against nitric oxide production in lipopolysaccharide-activated macrophage RAW264.7, with IC values of 37.5, 38.8, and 42.8 μM (L-NMMA was used as a positive control, IC 39.3 μM), respectively. This study puts the knowledge of the chemical profile of the botanical plant one step forward and, thereby, promotes the sustainable utilization of the resources of traditional folk medicinal plants.
Topics: Humans; Plants, Medicinal; Marsdenia; China; Pregnanes; Glycosides
PubMed: 36985677
DOI: 10.3390/molecules28062705 -
Biomolecules Apr 2023: Allopregnanolone is one of the most studied neuroactive steroids; yet, despite its relevance to neuropsychiatric research, it is not known how it, as well as its ratio...
: Allopregnanolone is one of the most studied neuroactive steroids; yet, despite its relevance to neuropsychiatric research, it is not known how it, as well as its ratio to progesterone, varies across all six subphases of the menstrual cycle. Two enzymes-5α-dihydroprogesterone and 5α-reductase-convert progesterone to allopregnanolone, and, based on immunohistochemical studies in rodents, the activity of 5α-reductase is considered the rate-limiting step in the formation of allopregnanolone. It is not clear, however, whether the same phenomenon is observed across to the menstrual cycle, and, if so, at what point this takes place. : Thirty-seven women completed the study during which they attended eight clinic visits across one menstrual cycle. We analyzed their allopregnanolone and progesterone serum concentrations using ultraperformance liquid chromatography-tandem mass spectrometry, and we implemented a validated method to realign the data from the original eight clinic study visits, following which we imputed the missing data. Hence, we characterized allopregnanolone concentrations, and the ratio of allopregnanolone:progesterone at six menstrual cycle subphases: (1) early follicular, (2) mid-follicular, (3) periovulatory, (4) early luteal, (5) mid-luteal, and (6) late luteal. : There were significant differences in allopregnanolone levels between (1) early follicular and early luteal, (2) early follicular and mid-luteal, (3) mid-follicular and mid-luteal, (4) periovulatory and mid-luteal, and (5) mid-luteal and late luteal. We detected a sharp drop in allopregnanolone:progesterone ratio in the early luteal subphase. Within the luteal subphase, the ratio was the lowest in the mid-luteal subphase. : Allopregnanolone concentrations are the most distinct, relative to the other subphases, in the mid-luteal subphase. The shape of the allopregnanolone trajectory across the cycle is similar to that of progesterone; however, the proportion of the two neuroactive steroid hormones is drastically different due to enzymatic saturation, which takes place at the start of the early luteal subphase, but continuing through, and peaking, in the mid-luteal subphase. Hence, the estimated activity of 5α-reductase decreases, but does not cease, at any point across the menstrual cycle.
Topics: Female; Animals; Progesterone; Pregnanolone; Menstrual Cycle
PubMed: 37189398
DOI: 10.3390/biom13040652