ent-Steroids: Novel tools for studies of signaling pathways

Membrane receptors are often modulated by steroids and it is necessary to distinguish the effects of steroids at these receptors from effects occurring at nuclear receptors. Additionally, it may also be mechanistically important to distinguish between direct effects caused by binding of steroids to membrane receptors and indirect effects on membrane receptor function caused by steroid perturbation of the membrane containing the receptor. In this regard, ent-steroids, the mirror images of naturally occurring steroids, are novel tools for distinguishing between these various actions of steroids. The review provides a background for understanding the different actions that can be expected of steroids and ent-steroids in biological systems, references for the preparation of ent-steroids, a short discussion about relevant forms of stereoisomerism and the requirements that need to be fulfilled for the interaction between two molecules to be enantioselective. The review then summarizes results of biophysical, biochemical and pharmacological studies published since 1992 in which ent-steroids have been used to investigate the actions of steroids in membranes and/or receptor-mediated signaling pathways.     

Novel soft steroids: effects on cell growth in vitro and on wound healing in the mouse.

Evaluation of three "soft" steroids is described. The test compounds were compared with the standard anti-inflammatory steroids betamethasone and prednicarbate in two studies. Soft steroids are designed based on the "inactive metabolite approach" to be rapidly inactivated by predictable metabolism after performing their therapeutic function. Consequently, lower circulating (peripheral) levels of potentially harmful steroids result, and undesirable systemic and local side effects are minimized. The soft and standard steroids behaved similarly in an in vitro cell culture model, whereas in a whole animal study the advantages of the soft steroids were evident.     

Metabolism and excretion of anabolic steroids in doping control--new steroids and new insights

The use of anabolic steroids in sports is prohibited by the World Anti-Doping Agency. Until the 1990s, anabolic steroids were solely manufactured by pharmaceutical companies, albeit sometimes on demand from national sports agencies as part of their doping program. Recently the list of prohibited anabolic steroids in sports has grown due to the addition of numerous steroids that have been introduced on the market by non-pharmaceutical companies. Moreover, several designer steroids, specifically developed to circumvent doping control, have also been detected. Because anabolic steroids are most often intensively subjected to phase I metabolism and seldom excreted unchanged, excretion studies need to be performed in order to detect their misuse.

This review attempts to summarise the results of excretion studies of recent additions to the list of prohibited steroids in sports. Additionally an update and insight on new aspects for “older” steroids with respect to doping control is given.     

On the origin of vertebrate-type steroids present in Locusta migratoria: do they originate from the food?

1. This study investigates the origin of vertebrate-type steroids which were reported to be present in Locusta migratoria: are the steroids synthesized by the locust or are they derived from the diet, i.e. grass and rolled oats? 2. It is unlikely that the steroids are synthesized by locust tissues. In vitro incubations of eleven different tissues with labeled pregnenolone or androstenedione did not result in androgen or estrogen synthesis respectively. 3. Steroid synthesis was also not detected when tissues were incubated in the presence of the early precursors cholesterol and isopentenyl pyrophosphate. 4. It is unlikely that the steroids are derived from the diet. Feeding experiments indicate that only low amounts of steroids are capable of crossing the gut-body barrier. 5. Injection of testosterone in the hemolymph also resulted in rapid excretion, instead of storage in tissues. 6. Moreover, radioimmunological measurements indicate that vertebrate-type steroids are absent in the food of locusts. 7. Specificity studies indicate that substances other than vertebrate-type steroids are detected by radioimmunoassay in locust tissue extracts. 8. Because vertebrate-type steroids are absent in locust tissues, it can be concluded that vertebrate-type steroids do not have a physiological function in Locusta.     

The identification and simultaneous quantification of neuroactive androstane steroids and their polar conjugates in the serum of adult men, using gas chromatography-mass spectrometry

Certain androstane steroids (AS) modulate ionotropic receptors, as do the pregnane steroids. Whereas women produce significant amounts of neuroactive progesterone metabolites, the steroid neuromodulators in men originate mainly from the 3-oxo-4-ene C(19)-steroids, which are converted to their 3alpha- and 3beta-hydroxy-5alpha/5beta-reduced metabolites. The neuromodulating effects of AS prompted us to monitor circulating levels of the steroids to estimate metabolic pathways in the periphery that may influence brain concentrations of AS. Hence, the serum levels of 20 steroids and 16 steroid polar conjugates including 17-oxo- and 17beta-hydroxy-derivatives of 5alpha/beta-androstane-3alpha/beta-hydroxy-androstane steroids were quantified in 15 men (16-62 years of age) using GC-MS. The conjugated AS for the most part reached micromolar concentrations, these being two or three orders of magnitude higher than those of the free steroids. The ratios of conjugates to free steroids were one to two orders of magnitude higher than the values for the corresponding pregnane steroids. This data suggested that conjugation may considerably restrain the transport of free AS from the periphery into the central nervous system.     

Neuropsychopharmacological properties of neuroactive steroids.

In addition to the well-known genomic effects of steroid molecules via intracellular steroid receptors, certain steroids rapidly alter neuronal excitability through interaction with neurotransmitter-gated ion channels. Several of these steroids accumulate in the brain after local synthesis or after metabolism of adrenal steroids. The 3alpha-hydroxy ring A-reduced pregnane steroids allopregnanolone and tetrahydrodeoxycorticosterone have been thought not to interact with intracellular receptors, but enhance gamma-aminobutyric acid (GABA)-mediated chloride currents, whereas pregnenolone sulfate and dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABA(A) receptors. We demonstrated that these neuroactive steroids can regulate also gene expression via the progesterone receptor after intracellular oxidation. Thus, in physiological concentrations these neuroactive steroids regulate neuronal function through their concurrent influence on transmitter-gated ion channels and gene expression. When administered in animal studies, memory-enhancing effects have been shown for pregnenolone sulfate and DHEA. The 3alpha-hydroxy ring A-reduced neuroactive steroids predominantly display anxiolytic, anticonvulsant, and hypnotic activities. Sleep studies evaluating the effects of progesterone as a precursor molecule for these neuroactive steroids revealed a sleep electroencephalogram pattern similar to that obtained by the administration of benzodiazepines. These findings extend the concept of a "cross-talk" between membrane and nuclear hormone effects and provide a new role for the therapeutic application of these steroids in neurology and psychiatry.     

Ketosteroids and hydroxyketosteroids, minor metabolites of sugarcane wax

Besides beta-sitosterol and stigmasterol, the major steroids of sugarcane, the following minor steroids have been isolated and identified from sugarcane wax: 3,6-diketosteroids, Delta(4)-3-keto steroids, and Delta(4)-6-hydroxy-3-keto steroids. Their structures were established by spectroscopic techniques and chemical correlations.     

Fractionation of fecal neutral steroids by high performance liquid chromatography

Fecal neutral steroids were fractionated by high performance liquid chromatography (HPLC) into three major fractions: 5 beta-H, 3-keto steroids; 5 beta-H, 3 beta-hydroxy steroids; and 5 alpha-H and delta 5-3 beta-hydroxy steroids. This separation was achieved in about 10 minutes, with greater than 97% recovery of standards in each fraction. Gas-liquid chromatographic quantitation of fecal steroids fractionated by either HPLC or thin-layer chromatography gave nearly identical results. A method using both C18 reverse phase and silica HPLC to purify radiolabeled sterols is also described.     

Steroid modulation of the GABA/benzodiazepine receptor-linked chloride lonophore

Recent findings suggest that steroids with sedative-hypnotic properties interact specifically with the gamma-aminobutyric acidA/benzodiazepine receptor-chloride ionophore complex (GBRC). They show positive heterotropic cooperativity by allosterically enhancing the binding of GABA agonists and the clinically useful benzodiazepines (BZs) to their respective recognition sites. These steroids have stringent structural requirements for activity at the GBRC, with the essential requirements for high potency being a 3 alpha-hydroxyl group and a 5 alpha-reduced A-ring. Some of these steroids are naturally occurring metabolites of progesterone and deoxycorticosterone and have nanomolar potencies as potentiators of chloride channel conductance. These 3 alpha-hydroxylated, 5 alpha-reduced steroids do not act through any known sites on the GBRC. Thus, the exact site and mechanism of action remain to be determined. Together with the observation that physiological levels of these metabolites are sufficient to influence the function of the GBRC, the evidence clearly suggests a role for these steroids in the normal regulation of brain excitability by potentiating the postsynaptic effects of gamma-aminobutyric acid (GABA). Pharmacological studies of the GBRC-active steroids show that they possess anxiolytic and anticonvulsant activities. The potential therapeutic application of these steroids in the treatment of mood disorders and catamenial exacerbation of seizures associated with the menstrual cycle is discussed. Collectively, the evidence from the studies of these steroids imply that another mechanism by which the endocrine system influences brain function has been identified. Its characterization will provide important insight into how steroids modulate brain excitability under normal and pathophysiological states.     

Rapid and efficient method for extraction and separation of glucocorticosteroids and sex steroids from urines

The influence of different pH values on the recoveries of glucocorticosteroids and sex steroids from Kieselguhrfilled minicolumns has been investigated. While the recoveries of all steroids tested were similar if samples had acidic or neutral pH values, sex steroids could effectively be separated from glucocorticosteroids by increasing the pH value of 13.7: recoveries were 1.7% for glucocorticosteroids and 56–76% for sex steroids. For the determination of sex steroids in biological samples it is recommended to adjust samples to a strong alkaline pH before extraction; this holds especially true for samples with very high glucocorticosteroid levels.     

Identification of 5 beta-pregnane and 5 beta-androstane derivatives in adrenal venous and peripheral blood plasma of the female possum (Trichosurus vulpecula)

In the possum a marked sex difference has been found in the steroids in adrenal venous plasma. Four 5 beta-pregnane and four 5 alpha (beta) androstane derivatives together with ten 4-ene-3-keto steroids were isolated from the adrenal venous plasma of the female and definitively identified by gas chromatography-mass spectrometry. The major reduced steroids were: 5 beta-pregnane-3 alpha,17 alpha-diol-20-one and 5 beta-pregnane-3 alpha,17 alpha,20 alpha-triol, at concentrations of 52 +/- 12 micrograms/100 ml and 44 +/- 8 micrograms/100 ml mean +/- SEM respectively. The concentration of cortisol was 198 +/- 47 micrograms/100 ml. The concentration of the 2 reduced steroids in peripheral plasma were approx. 100 times less. In contrast the adrenal venous plasma of a male contained 14 steroids of which only three, found in trace amounts, were reduced. The results confirm previous in vitro observations that reduced steroids are produced by the adrenocortical special zone, which is only present in the female. The physiological significance of the presence of reduced steroids of adrenocortical origin in the circulation of the female possum is discussed.     

Production and secretion of C-19 steroids by rat and guinea pig adrenals

The concentrations of C-19 steroids were measured in guinea pig and rat adrenals before and after castration as well as after stimulation with adrenocorticotropin hormone (ACTH). Characterization of adrenal C-19 steroids was also carried out by isolation with high-performance liquid chromatography and gas chromatography/mass spectrometry (GC/MS). From radioimmunoassay (RIA) data, androstenedione (4-DIONE) and 11 beta hydroxyandrostenedione (11 beta-DIONE) were the major C-19 steroids found in guinea pig adrenals, and castration induced a decrease of 4-DIONE levels only while all other C-19 steroids remained unchanged. In rat adrenals, the major C-19 steroids were 4-DIONE and testosterone, and they were also markedly inhibited after castration. With the exception of 11 beta-DIONE, all other C-19 steroids in circulation were eliminated after castration in both animals species. After ACTH administration in the guinea pig, adrenal 4-DIONE and 11 beta-DIONE levels were markedly stimulated, while an increase of only 11 beta-DIONE was observed in plasma. In the rat, ACTH had a small stimulatory effect on adrenal 52-androstane-3 alpha, 17 beta-diol (3 alpha-DIOL) and plasma 11 beta-DIONE levels. Analysis of guinea pig adrenal steroids by GC/MS confirmed the presence of C-19 steroids in adrenals (namely, 4-DIONE and 11 beta-DIONE) while, in the rat, this could not be confirmed. Our data indicate that production of C-19 steroids occurs in guinea pig adrenals, and 11 beta-DIONE is the major C-19 steroid as well as the only C-19 steroid secreted into the circulation. In the rat, the production of C-19 steroids detected by RIA is not supported by GC/MS data.     

Relationships between unconjugated and sulphated steroids in porcine primary Leydig cell culture

Steroidogenesis in immature porcine Leydig cells was investigated in primary culture at 48-84 h under basal conditions and in the presence of hCG. The basal accumulation of unconjugated steroids was close to linear only during the first 4 h of study, whereas the sulphate-conjugated steroids accumulated essentially linearly over the 36 h experimental period. At the last time point, 95% of the steroids measured were sulphated. Stimulation with hCG (1 ng/ml) led to a still more pronounced sulphate conjugation, and approx 99% of the steroids measured were sulphated at 36 h. Under maximal stimulation with hCG (100 ng/ml) the sulphates accounted for 74% of the total steroids measured at 36 h. Testosterone, androstenedione, dehydroepiandrosterone, 5-androstene-3 beta, 17 beta-diol and estrone were usually quantitatively the most important unconjugated steroids, and sulphated dehydroepiandrosterone, estrone, testosterone and 5-androstene-3 beta, 17 beta-diol were the most important steroid sulphates, especially following maximal stimulation of the cultures. These data emphasize the importance of steroid sulphates in porcine testicular steroid metabolism. Under stimulation with hCG, there was a rapid response in testicular steroidogenesis, initially seen as a rapid increase in the secretion of unconjugated and sulphated steroids. At approx 4-12 h, the rate of sulphate conjugation appeared to reach or even to exceed that of steroid biosynthesis, which lead to stabilisation or a decrease in the concentrations of unconjugated steroids. Only high doses of hCG, 10-100 ng/ml, were then able to lead to a net accumulation of unconjugated steroids, at 24-36 h of incubation with hCG.     

Steroid-induced conformational changes of FITC-labelled sarcoplasmic reticulum Ca2+-ATPase

Interactions between transmembrane and cytoplasmic domains of Ca2+-ATPase from sarcoplasmic reticulum (SR) have been studied. To affect the hydrophobic transmembrane domain, we used four amphiphilic steroids - esters of a dibasic acid and 20-oxypregnene. All four steroids contained cholesterol-like nuclei and differed by the structure of side chains. Steroids with carboxyl groups in the side chains inhibited the rates of ATP hydrolysis and Ca2+ transport, whereas a steroid without the carboxyl group did not appreciably affect Ca2+-ATPase function. Fluorimetric titration of FITC-labelled Ca2+-ATPase in SR vesicles by Nd3+ showed that steroids increased the apparent dissociation constant for Nd3+ bound to the hydrolytic site, the potency order of the steroids being the same as for the sterol-induced inhibition of the hydrolytic activity of Ca2+-ATPase. These results suggest structural changes in the active site. Ca2+ transport was inhibited more efficiently by steroids than the hydrolytic activity of the enzyme. This could be partially due to the increase of the membrane passive permeability induced by steroids, which, in turn, reflected the efficiency of the interaction of the steroids with lipid bilayers. The effects of the steroids were largely dependent on their amphiphilicity (the availability of polar groups in regions A and D), the structure of the side chains, and, possibly, on the distance between the molecular polar groups. We suggest that the inhibition of hydrolytic and transport functions of Ca2+-ATPase in the SR membrane is due to the interaction of the steroids with the transmembrane alpha-helical segments.     

Analysis of profiles of conjugated steroids in urine by ion-exchange separation and gas chromatography—mass spectrometry

A simplified, flexible method for the analysis of metabolic profiles of steroids in urine is described. Solid extraction with Amberlite XAD-2 or Sep-Pak C_{1 g} cartridges is followed by group fractionation of unconjugated neutral and phenolic steroids, monoglucuronides, monosulphates and disulphates on the lipophilic strong anion exchanger triethylaminohydroxyproply Sephadex LH-20 (TEAP-LH-20). Following brief enzymatic hydrolysis or solvolysis the steroids are purified on TEAP-LH-20. O-Methyloxime and trimethylsilyl ether derivatives are prepared and purified by filtration through Lipidex 5000, and are then analyzed by glass capillary column gas—liquid chromatography and gas chromatography—mass spectrometry.Between 2 and 5 ml of urine are used for a comprehensive analysis. Unconjugated neutral and phenolic steroids are isolated in half a day, corresponding steroids in the conjugate fractions in two days. No fraction containing steroids is discarded, but the analysis can be limited to a selected fraction.     

类固醇激素非基因组作用的机制及意义

现已证明各种类型的类固醇激素均能通过非基因组作用快速改变生理过程。不同的激素,或同一激素对不同的细胞,其非基因组作用的机制各不相同,多种多样,并且不时有新的机制诞生。本文将迄今为止类固醇激素快速非基因组作用的可能机制作一综述,并初步阐述其可能的意义。     

Partial filling affinity capillary electrophoresis with cationic poly(vinylpyrrolidone)-based copolymer coatings for studies on human lipoprotein-steroid interactions

Human plasma lipoproteins have strong hydrophobic interactions with steroids and their fatty acyl derivatives such as estradiol fatty acyl esters. In this work, affinity capillary electrophoresis with the partial filling technique was applied to study the hydrophobic interactions between lipoproteins, which are nanometer-sized particles, and nonconjugated steroids. The capillaries were first rinsed with one of two novel poly(vinylpyrrolidone) (PVP)-based cationic copolymers that were strongly adsorbed onto the fused-silica surface via electrostatic interactions. This surface treatment greatly suppressed the adsorption of lipoproteins. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles were then employed in the coated capillaries as pseudostationary phase in the partial filling mode. The changes in corrected migration times of steroids increased linearly with the filling time of the lipoproteins. The affinity constants between the steroids and lipoproteins were calculated, and the most hydrophobic steroid studied, progesterone, had stronger affinity than testosterone or androstenedione toward both LDL and HDL. Affinity between steroids and LDL was stronger than that between steroids and HDL. Interactions between the steroids and lipoproteins were mainly nonspecific with particle lipid components, whereas some were site specific with the apolipoproteins. The developed technique has great potential for determination of the affinity of various compounds toward lipoproteins.     

Antitumor and hepatoprotective activity of natural and synthetic neo steroids

In this review, steroids with a tertiary butyl group, which are usually called neo steroids, are a small group of natural lipids isolated from higher plants, fungi, marine sponges, and yeast. In addition, steroids with a tertiary butyl group have been synthesized in some laboratories in Canada, USA, Europe, and Japan and their biological activity was studied. Some natural neo steroids demonstrate antitumor or hepatoprotective activities. In addition, synthetic neo steroids exhibit anticancer and neuroprotective properties. However, to confirm the above data, both practical and clinical experimental studies are necessary. Nevertheless, the results may be useful for pharmacologists, chemists, biochemists, and the pharmaceutical industry.     

Steroid interference in iron-cholesterol reactions: A comparative study

Interference from various physiological and non-physiological steroids in the spectrophotometric determination of cholesterol by the Zak method (ferric chloride) and the method of Parekh and Jung (ferric acetate) was quantitatively measured. Contribution of the steroids at the specific absorption maxima of the cholesterol assays was determined by employing the steroids (40 mg/dl) alone, or added to a serum pool of known cholesterol content. The results show that the method of Parekh and Jung is less influenced by the presence of steroids than the Zak method. Observations on the structural specificity of the iron-cholesterol reaction are discussed.