Genetic engineering of Bacillus megaterium for high-yield production of the major teleost progestogens 17α,20ß-di- and 17α,20ß,21α-trihydroxy-4-pregnen-3-one.

17α,20ß-Dihydroxy-4-pregnen-3-one (17α,20ßDiOH-P) and 17α,20ß,21α-trihydroxy-4-pregnen-3-one (20ßOH-RSS) are the critical hormones required for oocyte maturation in fish. We utilized B. megaterium's endogenous 20ß-hydroxysteroid dehydrogenase (20ßHSD) for the efficient production of both progestogens after genetically modifying the microorganism to reduce side-product formation. First, the gene encoding the autologous cytochrome P450 CYP106A1 was deleted, resulting in a strain devoid of any steroid hydroxylation activity. Cultivation of this strain in the presence of 17α-hydroxyprogesterone (17αOH-P) led to the formation of 17α,20α-dihydroxy-4-pregnen-3-one (17α,20αDiOH-P) as a major and 17α,20ßDiOH-P as a minor product. Four enzymes were identified as 20αHSDs and their genes deleted to yield a strain with no 20αHSD activity. The 3-oxoacyl-(acyl-carrier-protein) reductase FabG was found to exhibit 20ßHSD-activity and overexpressed to create a biocatalyst yielding 0.22g/L 17α,20ßDiOH-P and 0.34g/L 20ßOH-RSS after 8h using shake-flask cultivation, thus obtaining products that are at least a thousand times more expensive than their substrates.

Bringing GC-MS profiling of steroids into clinical applications.

Abnormalities of steroid biosynthesis and excretion are responsible for the development and prevention of endocrine disorders, such as metabolic syndromes, cancers, and neurodegenerative diseases. Due to their biochemical roles in endocrine system, qualitative and quantitative analysis of steroid hormones in various biological specimens is needed to elucidate their altered expression. Mass spectrometry (MS)-based steroid profiling can reveal the states of metabolites in biological systems and provide comprehensive insights by allowing comparisons between metabolites present in cells, tissues, or organisms. In addition, the activities of many enzymes related to steroid metabolism often lead to hormonal imbalances that have serious consequences, and which are responsible for the progress of hormone-dependent diseases. In contrast to immunoaffinity-based enzyme assays, MS-based methods are more reproducible in quantification. In particular, high-resolution gas chromatographic (GC) separation of steroids with similar chemical structures can be achieved to provide rapid and reproducible results with excellent purification. GC-MS profiling therefore has been widely used for steroid analysis, and offers the basis for techniques that can be applied to large-scale clinical studies. Recent advances in analytical technologies combined with inter-disciplinary strategies, such as physiology and bioinformatics, will help in understanding the biochemical roles of steroid hormones. Therefore, comprehensive analytical protocols in steroid analysis for different research purposes may contribute to the elucidation of complex metabolic processes relevant to steroid function in many endocrine disorders, and in the identification of diagnostic biomarkers.

Preparation of a sustainable magnetic sorbent for the extraction and preconcentration of progestogens in natural water samples.

A film composed of agarose and graphene (G) and magnetic nanoparticles (G-MNPs) is proposed as a sorbent for the extraction and determination of medroxyprogesterone (MED), levonorgestrel (LEV), norethisterone (NOR) and progesterone (PRO) in natural water samples. Both the preparation of the film and the extraction procedure were optimized. The optimal extraction parameters were as follows: isopropyl alcohol as activation solvent, sample pH value of 3.0, extraction time of 30 min, 1.00 mL of acetonitrile as eluent, elution time of 5 min and sample volume of 100.00 mL. HPLC with photodiode array detector was used for the separation and determination. The method presented a linear range between 2.50 and 75.0 µg L-1 for all analytes, and the LODs were between 1.40 and 1.80 µg L-1. The method was applied to natural water samples, obtaining satisfactory recovery values (75-111 %). In conclusion, for the immobilization of the G-MNPs, agarose was used, which is a non-toxic, renewable and biodegradable material. The G-MNPs-agarose film was reused up to 70 times, without losing its extraction capacity significantly and presenting excellent sorbent properties, which allow the extraction and preconcentration of the progestogens under study.

Parallel derivatization strategy coupled with liquid chromatography-mass spectrometry for broad coverage of steroid hormones.

Steroid hormones are a type of crucial substances that mediate numerous vital physiological functions. The comprehensive detection of steroid hormones can help understand the physiopathologic mechanism of steroid hormone-related diseases. It is very difficult to determine steroid hormones in biological samples due to their low endogenous concentrations and poor ionization efficiency. In this study, an efficient and sensitive approach was developed for profiling steroid hormones by combining liquid-liquid extraction and parallel derivatization with liquid chromatography-tandem mass spectrometry. Methoxyamine and dansyl chloride were used to derivatize steroid hormones containing carbonyl and phenolic hydroxyl groups, respectively. Our established method achieved simultaneous analysis of carbonyl and phenolic hydroxyl-containing steroid hormones and could cover estrogens, androgens, corticoids and progestogens. Twenty-nine steroid hormones were detected at pg/mL levels with the sensitivity enhanced by three orders of magnitude after derivatization. The linearity (with linear range of 2-4 orders of magnitude), precision (less than 15%) and recovery (71.1-128.7%) were satisfactory for quantitative analysis of steroid hormones. Finally, the established method was successfully employed to the determination of steroid hormones in serum samples of healthy males and females as well as ovarian cancer patients. The results showed that this approach was suitable and reliable for routine test of steroid hormones containing carbonyl and phenolic hydroxyl groups.

The lecanindoles, nonsteroidal progestins from the terrestrial fungus Verticillium lecanii 6144.

Four new indolosesquiterpenes, lecanindoles A-D (1-4), were isolated from fermentations of the terrestrial fungus Verticillium lecanii 6144. The structures of compounds 1-4 were elucidated from analysis of spectroscopic data. Compound 2 was reduced to give 4 and its isomer 5. Compound 4 was found to be a potent and selective progesterone receptor agonist with an EC50 of 1.1 +/- 0.4 nM in a cell-based luciferase reporter assay.

Dimeric progestins from rhizomes of Ligusticum chuanxiong.

Five dimeric phthalides were isolated from rhizomes of Ligusticum chuanxiong and their structures deduced based on spectral data. All compounds and their parent extracts were assessed for progesterone-like activity using a progesterone receptor driven reporter-gene bioassay. Among all the compounds, riligustilide, displayed weak progesterone-like activity (EC50 approximately 81 microM), whereas, (3Z')-(3a'R,6'R,3R,6R,7R)-3,8-dihydro-6.6',7.3a'-diligustilide (Mr: 382, EC50 approximately 90 nM), was found to be a potent and specific activator of the progesterone receptor. Levistolide A, although having a very similar plenary structure, was inactive indicating the importance of stereochemistry of chiral centers and flexibility of butylidene side chain for progestogenic activity. These bioactive phthalides and their parent extracts (EC50 approximately 8 microg/ml) may have utility for treatment of conditions requiring progesterone action.

The development of a simple fecal immunoreactive progestin assay to monitor reproductive function in swine.

The objectives of the study were to (a) develop a simple fecal progestin extraction and radioimmunoassay method to measure immunoreactive progestin in porcine feces and (b) to characterize fecal progestin profiles during the estrous cycle and postpartum. A simple extraction method was developed in trial 1 and the mean (+/- SD) progestin recovery of the method was 84.3 +/- 3.5%. Progesterone levels measured at five different spiked concentrations (50, 100, 200, 400, and 500 ng/0.5 g feces) showed no systematic error. The sensitivity of the assay was 0.16 nmol/L of the extract. Trial 2 involved collecting fecal samples from six cycling sows every second or third day, beginning on the day of estrus (day 0) and continuing until day 22. The mean (+/- SD) fecal progestin concentrations of these sows determined by the above assay during days 0-5, days 6-10, days 11-15, and days 16-21 were 87.1 +/- 17.5, 262.6 +/- 102.1, 1188.2 +/- 454.1, and 897.3 +/- 274.1 x 10(-3) nmol/g feces, respectively. In trial 3, fecal samples from six postpartum sows were collected at weekly intervals beginning from day 7 after farrowing until day 50. The mean (+/- SD) fecal progestin concentrations were 111.0 +/- 61.1, 74.1 +/- 21.3, 66.5 +/- 26.1, 122.7 +/- 58.8 and 533.5 +/- 244.2 x 10(-3) nmol/g feces, during days 7-10, days 11-20, days 21-30, days 31-40, and days 41-50 postpartum, respectively. The results indicate that simple fecal progestin extraction and assay are feasible alternatives to the standard blood progesterone assays for monitoring reproductive function in swine.

Molecular screening of Chinese medicinal plants for progestogenic and anti-progestogenic activity.

Estrogen and progestins have adverse effects, and many of these adverse effects are caused by progestins. Due to this, many women choose to use botanical alternatives for hormone replacement therapy, which does not trigger steroidogenic properties. Therefore, it is necessary to screen these herbs for progestogenic and anti-progestogenic properties. Extract of 13 Chinese medicinal plants were analysed for progestogenic and anti-progestogenic activities by using progesterone response element-driven luciferase reporter gene bioassay. MTT assay was carried out to investigate the cytotoxic effect of herb extract on PAE cells. Among the 13 herbs, Dipsacus asperoides extract exhibited progestogenic activity, and 10 species - Cortex eucommiae, Folium artemisiae argyi, Glycyrrhiza uralensis, Angelica sinensis, Atractylodes macrocephala koidz, Scutellaria baicalensis, Cuscuta chinensis, Euscaphis japonica, Ailanthus altissima, and Dioscorea opposita - were recognized to have anti-progestogenic like activities. Extract of Dipsacus asperoides demonstrated dose-dependent progestogenic activity, and the progestogenic activity of 100 (mu)g/mL extracts was equivalent to 31.45 ng/mL progesterone activity. Herbs extracts that exhibited anti-progestogenic-like activity also inhibited the 314.46 ng/mL progesterone activity in a dose-response manner. None of the herb extracts shown significant toxic effect on PAE cells at 40-100 (mu)g/mL compared to control. This discovery will aid selection of suitable herbs for hormone replacement therapy.

Determination of endocrine disrupting compounds using temperature-dependent inclusion chromatography: I. Optimization of separation protocol.

In the present work we optimised the separation of battery of key UV non-transparent low-molecular-mass compounds having possible endocrine disrupting compounds (EDCs) activity or which may be used as the endocrine effect biomarkers. Simple optimization strategy was based on strong temperature effect that is driven by electrostatic interactions between macrocyclic mobile phase additives like cyclodextrins and eluted components of interest under C18 stationary phase and acetonitrile/water mobile phase conditions. Particularly, the effect of temperature involving native beta-cyclodextrin and its hydroxypropyl derivative to improve separation of number of natural (d-equilenin, equilin, estetrol, estriol, estrone, 17beta-estradiol, 17alpha-hydroxyprogesterone, 20alpha-hydroxyprogesterone, cortisol, cortisone, progesterone, testosterone, tetrahydrocortisol and tetrahydrocortisone) and artificial steroids (ethynylestradiol, norgestrel isomers, medroxyprogesterone, mestranol, methyltestosterone, norethindrone, 17alpha-estradiol) as well as non-steroidal compounds (diethylstilbesterol, bisphenol A, 4-tert-butylphenol, dimethyl phthalate, dibutyl phthalate and dioctyl phthalate) was investigated. It has been found that successful isocratic separation of 27 chemicals can be achieved using acetonitrile/water eluents modified with beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin at concentration of 10 mM and temperature of 47 degrees C. Separation protocol is simple, reliable, direct and non-radioactive and may be easily adapted for rapid separation and quantification of wide range of given steroids and related EDCs in environmental samples, particularly those that are characterised by unstable biological matrix and components of interest load.

Optimization of the reversed-phase high-performance liquid chromatographic separation of synthetic estrogenic and progestogenic steroids using the multi-criteria decision making method.

The optimization of the reversed-phase high-performance liquid chromatographic separation of a mixture of ethynylestradiol, desogestrel and three related compounds is described. A procedure is used that allows the prediction of the capacity factors of each individual synthetic steroid, depending on the mobile phase composition. Therefore, complete chromatograms can be predicted and evaluated with the multi-criteria decision making method.

[Structure-mobility relationships in thin-layer chromatography. Applications to some progestogens (author's transl)]. / Relations structure-mobilité en chromatographie sur couches minces. Applications à quelques progestatifs

A study has been made on the quantitative relationship between the chromatographic mobility and the structure of progestogenic steroids. The coefficient K, corresponding to the fundamental skeleton, and the parameters of different substituents were calculated from the RM values. The results obtained with eighteen steroids tested in two solvent systems of low dielectric constant and low polarity have been improved satisfactorily.

[Determination of naturally occurring sex steroid hormones (androgens and progestagens) in beef]. / Bestimmung natürlich vorkommender steroidaler Sexualhormone (Androgene und Gestagene) in Rindfleisch.

The content of 10 naturally occurring steroidal sex hormones (androgens and progestogens), their biosynthetic precursors and metabolites was determined in 62 samples of beef (bulls, steers, heifers). After enzymatic hydrolysis of their conjugates, the steroids were extracted from the tissue by liquid-liquid extraction and purified by solid phase extraction. The identification and quantification was carried out by GC-MS of the trimethylsilyl ethers. The progestogens progesterone and pregnenolone were quantitatively dominant (43.7 and 6.5 micrograms/kg respectively). The highest steroid concentrations were determined in female cattle.

High-pressure liquid chromatographic separation of a mixture of corticosteroids, androgens, and progestins.

Fifteen steroids including corticosteroids, androgens, progestins, and their derivatives were completely separated by reverse-phase high-pressure liquid chromatography on a ChemcoPak 7 ODS-H column in 50 min. The elution procedures were first with water:methanol:acetonitrile:isopropanol 55:32:6.5:7.5 (v/v) for 15 min and followed with a linear gradient elution for 35 min from 0 to 80% of water:methanol:n-butanol 40:40:20 (v/v). The applicability of this method was successfully demonstrated in the analyses of the biological samples of carp plasma, testis, and head kidney.

Transformations of C21 and C19 steroids in porcine ovarian preparations and the chromatographic separation of the metabolites.

The separation of several C21 steroids, such as 17-hydroxyprogesterone, and androgens, such as testosterone, from the non-polar steroids like 16-androstenes has been achieved on one thin-layer chromatographic plate using a two-dimensional technique. This method has been further developed to include a separation of some oestrogens from C19 steroids. The thin-layer chromatographic development was then utilised to separate the metabolites of porcine ovarian incubations. Homogenised preparations of corpora lutea (5-14 days post ovulation) were incubated separately with [4-14C] testosterone, [4-14C] progesterone and [4-14C] pregnenolone, using NADPH as cofactor. After two-dimensional thin-layer chromatography the metabolites were identified by establishing their radiochemical purity either by repeated thin-layer chromatography or by gas-fraction collection. Pregnenolone was converted to small yields of 4,16-androstadien-3-one (0.13-0.28%) and 5 alpha-androst-16-en-3-one (less than 0.1%) and also to 17-hydroxypregnenolone and 17-hydroxyprogesterone (0.8 and 0.37% respectively). Increased activity of 5-ene-3 beta-hydroxysteroid dehydrogenase/4,5-isomerase was shown by the high yields (73-83%) of progesterone obtained from pregnenolone. This was associated with decreased C-17.20 lyase activity as reflected in the relatively small amounts of 4-androstenedione obtained from both pregnenolone and progesterone. None of the well-known oestrogens or 16-androstenes was formed from progesterone or testosterone, but the latter was converted into 4-androstenedione, in small yield. Both pregnenolone and progesterone gave rise to a metabolite in 1-2% yield (of similar polarity to the 16-androstenes but separated from them by thin-layer chromatography on AgNO3-impregnated plates) which has been tentatively identified as 5 alpha-pregnane-3,20-dione. Incubations of porcine follicular fluid and tissue with labelled pregnenolone or progesterone did not result in the biosynthesis of labelled 16-androstenes.