5alphaDH-DOC (5alpha-dihydro-deoxycorticosterone) activates androgen receptor in castration-resistant prostate cancer.

Prostate cancer often relapses during androgen-depletion therapy, even under the castration condition in which circulating androgens are drastically reduced. High expressions of androgen receptor (AR) and genes involved in androgen metabolism indicate a continued role for AR in castration-resistant prostate cancers (CRPCs). There is increasing evidence that some amounts of 5alpha-dihydrotestosterone (DHT) and other androgens are present sufficiently to activate AR within CRPC tissues, and enzymes involved in the androgen and steroid metabolism, such as 5alpha-steroid reductases, are activated in CRPCs. In this report, we screened eight natural 5alphaDH-steroids to search for novel products of 5alpha-steroid reductases, and identified 11-deoxycorticosterone (DOC) as a novel substrate for 5alpha-steroid reductases in CRPCs. 11-Deoxycorticosterone (DOC) and 5alpha-dihydro-deoxycorticosterone (5alphaDH-DOC) could promote prostate cancer cell proliferation through AR activation, and type 1 5alpha-steroid reductase (SRD5A1) could convert from DOC to 5alphaDH-DOC. Sensitive liquid chromatography-tandem mass spectrometric analysis detected 5alphaDH-DOC in some clinical CRPC tissues. These findings implicated that under an extremely low level of DHT, 5alphaDH-DOC and other products of 5alpha-steroid reductases within CRPC tissues might activate the AR pathway for prostate cancer cell proliferation and survival under castration.

The use of high-resolution solid-state NMR spectroscopy and differential scanning calorimetry to study interactions of anaesthetic steroids with membrane.

We have used a combination of high-resolution solid-state 13C-NMR and DSC (differential scanning calorimetry) to study the distinctively different thermotropic and dynamic properties of the anaesthetic steroid alphaxalone and its inactive congener delta16-alphaxalone in dipalmitoylphosphatidylcholine (DPPC) model membranes. In the solid-state 13C-NMR, the techniques included cross polarization (CP) and/or magic angle spinning (MAS). The observed data revealed the following important results. (a) DSC as a bulk method showed that the active steroid lowers the main phase transition temperature and broadens the pretransition more significantly than the inactive congener. The 13C-CP/MAS experiments allowed us to detect the pretransition temperature in the alphaxalone-containing preparation, which was not discernible in DSC. (b) The chemical shift values varied with temperature, indicating different degrees of trans-gauche isomerization in the lipid acyl chains when the bilayer is in the liquid crystalline phase. (c) Only specific additional peaks appeared in the 13C-CP/MAS spectra when each of the steroids was present in the preparation. delta16-alphaxalone gives rise to more additional peaks than alphaxalone, indicating a different mobility of the corresponding molecular moiety in the phospholipid bilayer environment. (d) The relative intensities of these peaks also confirmed that alphaxalone is fully incorporated in the bilayer, whereas delta16-alphaxalone is only partially so. These results suggest that the differential effects of these two analogues in the membrane may, at least in part, explain the reason for their different biological activities.

Gas chromatographic-mass fragmentographic quantitation of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and its precursors in blood and brain of adrenalectomized and castrated rats.

Coupling high performance liquid chromatography with gas chromatography-mass fragmentography has made it possible to simultaneously measure subpicomolar concentrations of allopregnanolone and its precursors, pregnenolone, progesterone and 5 alpha-dihydroprogesterone (5 alpha-DHP) in various brain areas. Allopregnanolone was measured in the brain of adrenalectomized/castrated (ADX/CX) rats in nanomolar concentrations long after peripheral sources of allopregnanolone were removed. A partial decrease (approximately 30%) in the content of allopregnanolone was found in the brains of ADX/CX rats compared to sham-operated rats. Moreover, the content of allopregnanolone in brains of sham-operated as well as ADX/CX rats was nonuniformly distributed (olfactory bulb > striatum > cortex > hippocampus) and was one to two orders of magnitude higher than in plasma or liver. Infusion of pregnenolone sulfate in ADX/CX rats elicited a fourfold increase in 5 alpha-DHP and progesterone content and a seven- to eightfold increase in the content of allopregnanolone in brain but not in liver or plasma. Furthermore, the content of allopregnanolone in brain increased to the same extent in both sham-operated and ADX/CX rats following pregnenolone sulfate infusion. The 5 alpha-reductase inhibitor, (17 beta)17[[bis(1-methylethyl)amino]carbonyl] androstane-3,5-diene-3-carboxylic acid (SKF 105111), reduced the brain content of allopregnanolone and blocked the increased formation of allopregnanolone in brain following pregnenolone sulfate infusion. The results clearly demonstrate that the synthesis of allopregnanolone from 5 alpha-DHP and progesterone occurs in the brain and that a significant amount of allopregnanolone is synthesized locally in brain from its precursors. These experiments suggest that the brain, like adrenals and gonads, is a steroidogenic organ which produces allopregnanolone as one of its own most important physiologically relevant steroids.

Progestin content and biosynthetic potential of the corpus luteum of the African elephant (Loxodonta africana).

The aim of this study was to examine the progestin content and biosynthetic potential of the corpus luteum of the African elephant (Loxodonta africana). Luteal tissue was collected from nonpregnant and early, mid- and late pregnant elephants (n = 2 per group) shot in the Kruger National Park. Pieces of individual corpora lutea (2-3 per animal; 23 in total) were stored directly in ethanol before hormone analysis. Matching tissue pieces were incubated for 2 h with [3H]pregnenolone (2 x 10(5) c.p.m.), after which tissue plus medium were also stored in ethanol. Progesterone and 17 alpha-hydroxyprogesterone immunoreactivity in tissue extracts were determined by enzymeimmunoassay and radioimmunoassay, respectively, before and after reverse phase HPLC. Progesterone immunoreactivity predominated over that of 17 alpha-hydroxyprogesterone in all corpora lutea examined but concentrations of both hormones were very low (73-374 ng g-1 and 3-93 ng g-1, respectively after HPLC). There were no obvious differences in hormone concentrations in corpora lutea from animals at different reproductive stages. Progesterone and 17 alpha-hydroxyprogesterone immunoreactivity assayed before HPLC was 10-30 times higher than that measured after chromatographic separation. HPLC consistently revealed two large immunoreactive peaks associated with relatively nonpolar compounds, which together accounted for most (at least 75%) of all progesterone immunoreactivity measured. Large amounts of radioactivity with the same retention times as these peaks were also detected after HPLC in samples incubated with [3H]pregnenolone. Analysis of conversion products from four corpus luteum incubations indicated that between 52% and 84% of [3H]pregnenolone had been converted; 19-33% was accounted for by progesterone, and 12-50% by the two substances represented by the unidentified peaks. Subsequent GCMS analysis identified the two immunoreactive peaks as 5 alpha-pregnane-3 alpha-ol-20-one and 5 alpha-pregnane-3,20-dione (5 alpha-dihydroprogesterone). These results indicate that the major progestins contained within and biosynthesized by corpora lutea of African elephants are 5 alpha-reduced metabolites, and that progesterone and 17 alpha-hydroxyprogesterone are quantitatively of minor importance.

Regional distribution of progesterone and 5 alpha-pregnane-3,20-dione in rat brain during progesterone-induced "anesthesia".

Progesterone and 5 alpha-pregnane-3,20-dione (5 alpha-DHP) concentrations were measured in seven brain areas and in plasma during "anesthesia" induced by progesterone (1-2 mg IV) in female rats. The highest levels of progesterone were detected in the striatum and hypothalamus (23.3 +/- 5.27 and 22.7 +/- 4.30 micrograms/g +/- SEM, respectively); these concentrations were approximately 1000 times higher than those during the post-ovulatory phase. Highest levels of 5 alpha-DHP were detected in the striatum and hippocampus (11.5 +/- 1.74 and 10.4 +/- 3.15 micrograms/g +/- SEM, respectively). The ratio of 5 alpha-DHP to progesterone was approximately 100 times higher in brain tissue than in plasma. We conclude that a conversion of progesterone to 5 alpha-DHP occurs in the brain during the course of progesterone-induced "anesthesia". This metabolic step may be an important contributory factor to the anesthetic potency of progesterone.

Low polarity ligands of sex hormone-binding globulin in pregnancy. Part II--Identification.

Certain previously unrecognized ligands of SHBG of low polarity in pregnancy were identified. They include two weakly bound compounds: 5 alpha-pregnane-3,20-dione and progesterone; and two strongly bound substances, 2-methoxyestrone and a new steroid, estradienolone (17 beta-hydroxy-1,5-estradiene-3-one). The identification of the first three peaks was based on chromatographic elution patterns, binding characteristics and gas chromatography-mass spectrometry. The identification of the fourth peak, the new steroid, was based on similar kinds of evidence and, in addition, solubility characteristics and ultraviolet absorption spectrum.

Radioimmunoassay of 5 alpha-pregnane-3,20-dione. A metabolite of placental progesterone.

A radioimmunoassay (RIA) procedure has been developed for the measurement of 5 alpha-pregnane-3,20-dione (DHP) in human plasma after ether extraction of the plasma-samples, followed by column chromatography. The antiserum was generated in rabbits with 6 alpha-carboxyethylmer-captoprogesterone-BSA conjugate. The high affinity (Ka = 3.72 X 10(9) l/mol) antiserum binds 40% of 40 picograms of tritiated 5 alpha-pregnane-3,20-dione at working dilutions of 1 : 2800. Negligible cross-reactivity of the antiserum was detected with 5 beta-pregnane-3,20-dione (1.8%). Other hydroxylated pregnanes showed minor cross-reactivity (5-40%). The cross-reacting steroids were all separated from pregnanedione by one chromatographic step. The plasma levels of 5 alpha-pregnane-3,20-dione were measured from 22nd to 42nd week of normal pregnancy and compared to published data.

[Analysis of the steroid hormones in pig blastocysts]. / Analiz steroidnykh gormonov v blastotsistakh svinei.

The steroid hormonal activity was established in the pig blastocysts by means of histochemical methods. This is supported by the presence of hormones (pregnenolon, pregnandion), activity of 3beta-ol steroid dehydrogenase, high content of lipids (sterols) in the trophoblast. The results of thin-layer chromatography on silicagel suggest that the qualitative composition of lipids of the early embryos and the yellow bodies of the ovaries at the corresponding stage of pregnancy is the same.

Physico-chemical and analytical studies on Halopredone acetate.

Physico-chemical, spectroscopic (UV, CD, ORD, IR-1H-DMR, 19F-NMR, PFT13C-NMR, MS), and chromatographic (TLC, HPLC) properties of 17,21-bis(acetyloxy)-2-bromo-6beta,9-difluoro-11beta-hydroxypregna-1,4-diene-3,20-dione (halopredone acetate; Topicon) are reported. Densitometric and high-pressure liquid chromatographic quantitative analytical methods are described. The usual descriptive data, purity tests, potential impurities arising from the synthesis and stability tests are given.

Effect of oestradiol-17beta and progesterone on the metabolism of [1,2(-3)H] progesterone by the rabbit endometrium and myometrium.

The metabolism of [3H]progesterone in the rabbit endometrium and myometrium was studied in vitro. The major metabolities identified were 5alpha-pregnane-3,20-dione, 20alpha-hydroxypregn-4-en-3-one, 3beta-hydroxy-5alpha-preganan-20-one and 5alpha-pregnane-3beta,20alpha-diol. Other minor metabolites tentatively identified were 3alpha-hydroxy-5beta-pregnan-20-one,20alpha-hydroxy-5beta-pregnan-3-one and 5beta-pregnane-3alpha,20alpha-diol. The ability of the endometrium to metabolize progesterone on a unit weight bais was about 2.7 times that of the myometrium. The metabolism of [3H]progesterone in the rabbit uterus under the influnce of oestradiol-17beta and progesterone was studied. The ability of the oestradiol-treated rabbit uterus to metabolize progesterone was increased to 3.47 times that of the overiectomized control uterus, whereas the oestradiol-progesterone-treated rabbit uterus metabolized only 1.86 times that of the control. Study of the metabolism of progesterone with uterine subcellular preparations revealed that the 5alpha-reductase enzyme was present mainly in the nuclear fraction; 20alpha-hydroxysteroid dehydrogenase was found in the cytosol fraction and 3beta-hydroxysteroid dehydrogenase in the particulate fraction of the uterus. The metabolic pathways of progesterone in the rabbit uterine tissue are discussed.