New 19-oxygenated and 4-methylated steroids from the Formosan soft coral Nephthea chabroli

Chemical investigation of the Formosan soft coral Nephthea chabroli resulted in the isolation of four new 19-oxygenated steroids, nebrosteroids I-L (1-4), together with a new 4α-methylated steroid, nebrosteroid M (5). The molecular structures of these isolated metabolites were elucidated on the basis of extensive spectroscopic analysis and by comparison of the data with those of related metabolites. Compounds 1-5 were evaluated for anti-inflammatory activity using RAW 264.7 macrophages.     

Studies on the metabolism of steroids in the foetus. Biosynthesis of 6α-hydroxytestosterone in the human foetal liver

After incubation of testosterone with 105000g microsomes of human foetal liver, 6alpha-hydroxytestosterone was isolated and identified by t.l.c. and g.l.c.-mass spectrometry. This is the first example of 6alpha-hydroxylation of C(19) steroids in the human liver, and the finding is discussed in relation to earlier reports of 6-oxygenated C(19) and C(18) steroids in pregnant women.     

A time-dependent inactivation of aromatase by 19-oxygenated androst-4-ene-3,6,17-triones

19-Hydroxyandrost-4-ene-3,6,17-trione (19-OHAT), its 19-oxo derivative (19-oxo AT) and 4β,5β-epoxyandrostane-3,6,17-trione (5) were synthesized as possible intermediates involved in a mechanism-based inactivation of aromatase caused by androst-4-ene-3,6,17-trione (AT). These compounds inhibited the enzyme in a competitive manner with K_i's of 0.61, 7.5 and 5.1 μM for 19-OHAT, 19-oxo AT, and compound 5. The two 19-oxygenated steroids showed a time-dependent, pseudo-first order rate of inactivation of aromatase with k_inact's of 0.222 and 0.076 min⁻¹ for 19-OHAT and 19-oxo AT, respectively, while compound 5 did not. NADPH and oxygen were required for the inactivation. Androstenedione blocked the inactivation, while -cycteine partially prevented that of 19-OHAT and almost completely that of 19-oxo AT. When the 19-oxygenated steroids were separately subjected to reaction with , these rapidly disappeared from the reaction mixture with of 25 min (19-OHAT) and 20 s (19-oxo AT). This finding indicates that -cysteine prevents inactivation by a chemical dependent elimination of the inhibitors from the incubate. These results suggest that the 19-oxygenation rather than the 4,5-epoxidation may be involved in the time-dependent inactivation by AT.     

Probing the active site of aromatase with 2-methyl-substituted androstenedione analogs

To gain insight into the spatial nature of the androstenedione (AD) binding (active) site of aromatase in relation to the catalytic function of the enzyme, we synthesized 2,2-dimethylAD (4), 2beta- and 2alpha-methylADs (5 and 6), 19-oxygenated derivatives of compounds 4 and 6, and 2-methyleneAD (17), and we then tested their inhibitory activity as well as their aromatase reaction (aromatization for 2-methyl and 2-methylene analogs or 19-oxygenation for 2,2-dimethyl steroids) with human placental aromatase. 2-Methyl and 2-methylene steroids 5, 6, and 17 were good competitive inhibitors of aromatase (K(i)=22-68nM), but less effective compared to the 2,2-dimethyl analog 4 (K(i)=8.8nM), indicating that a combination of 2beta- and 2alpha-methyl moieties is essential for the formation of a thermodynamically stable inhibitor-aromatase complex. A series of 2alpha-methyl steroids were good substrates for aromatase, whereas 2beta-methyl steroid 5 was an extremely poor substrate, and a series of 2,2-dimethyl steroids did not serve as substrate, suggesting that a 2beta-methyl moiety of the 2,2-dimethyl and 2beta-methyl steroids would prevent the aromatase reaction probably due to steric hindrance in each case. The 2-methylene compound 17 was also aromatized to produce 2-methylestrogen with a low conversion rate where the 1,4-diene structure may have been created before the C(10)-C(19) bond cleavage. Kinetic analysis of the aromatization of androgens revealed that a good substrate was not essentially a good inhibitor for aromatase.     

Seasonal changes in serum steroid hormones in a protandrous teleost, the sobaity (Sparidentex hasta Valenciennes)

Serum concentrations of 11-ketotestosterone, 11β-hydroxytestosterone, testosterone, testoster-one glucuronide, oestradiol and 17,20β -dihydroxy-4-pregnen-3-one (17,20β -P) were measured in the sobaity at monthly intervals through their second breeding season. Concentrations of the 11-oxygenated androgens in the males and of oestradiol in the females peaked during the spawning season in January-February, while maximum levels of testosterone were found in the summer when these steroids were low. Testosterone glucuronide showed two peaks, one in the post-spawning period as oestradiol and the 11-oxygenated androgens were falling and the other coincident with the summer peak of testosterone. 17,20β -P was detectable in only one male and one female fish in February. Serum concentrations of 11-oxygenated androgens are more reliable than those of oestradiol for determining the sex of sobaity, and may also be used as indicators of the occurrence of sex reversal. The seasonal pattern of serum steroids correlated well with the changes of sexual status of the gonads during regression and recrudescence observed histologically and suggests that oestradiol may be involved in the sex inversion of this species.     

Disulphates of 16-oxygenated ketonic c19 steroids as biliary metabolites of androsterone sulphate in female rats

The chemical synthesis of 16β-hydroxyandrosterone was described preparatory to studies of the disulphates of the 16-oxygenated ketonic C₁₉ steroids present in the bile of female rats dosed with [³H]androsterone sulphate. The biliary metabolites were separated by chromatography on Sephadex LH-20 to afford monosulphate and dicon jugate fractions. After solvolysis of the diconjugate fraction, the liberated steroids were separated by partition chromatography on Celite 545 and analyzed by gas chromatography-mass spectrometry. In addition to 3α, 17β-dihydroxy-5α-androstan-16-one isolated previously, 16β-hydroxyandrosterone was identified as a disulphate.     

Effect of testosterone-estradiol-binding globulin in the enzymic oxidoreduction of 17-oxygenated c19 steroids.

The effect of both testosterone-estradiol-binding globulin (TeBG) and albumin on enzymic oxidoreduction of four 17-oxygenated C19 steroids by bacterial 17beta-hydroxysteroid:NAD oxidoreductase from Pseudomonas testosteroni was investigated. The decreased yields of products under presence of TeBG were found in both directions of reversible enzymic reaction. This finding was unexpected in the case of enzymic reduction in which the opposite effect could be assumed with respect of high affinity of the product but not the substrate to TeBG. Kinetically, the competition between enzyme and binding protein for the substrate occurs at enzymic oxidation, whereas the mechanism resembling non-competitive inhibition operates in the enzymic reduction.     

Simultaneous determination of dehydroepiandrosterone and its 7-oxygenated metabolites in human serum by high-resolution gas chromatography--mass spectrometry

A highly sensitive and specific method has been developed for the simultaneous measurement of free (unconjugated) or sulfate-conjugated forms of dehydroepiandrosterone (DHEA), 7alpha-hydroxy-DHEA (7alpha-OH-DHEA), 7beta-hydroxy-DHEA (7beta-OH-DHEA), and 7-oxo-DHEA (7-oxo-DHEA) in human serum. This method is based upon a stable isotope-dilution technique by gas chromatography-selected-ion monitoring mass spectrometry. Free steroids were extracted from serum with an organic solvent and the sulfate-conjugated steroids remained in aqueous phase. Free steroids were purified by solid-phase extraction, while sulfate-conjugated steroids were hydrolyzed by sulfatase and deconjugated steroids were purified by solid-phase extractions. The extracts were treated with O-methylhydroxylamine hydrochloride and were subsequently dimethylisopropylsilylated. The resulting methyloxime-dimethylisopropylsilyl (MO-DMIPS) ether derivatives were quantified by gas chromatography-selected-ion monitoring mass spectrometry in a high-resolution mode. The detection limits of MO-DMIPS ether derivatives of DHEA, 7alpha-OH-DHEA, 7beta-OH-DHEA and 7-oxo-DHEA were 1.0, 0.5, 0.5 and 2.0pg, respectively. Coefficients of variation between samples ranged from 10.6 to 22.9% for free 7-oxygenated DHEA to less than 10% for DHEA and sulfate-conjugated 7-oxygenated DHEA. The concentrations of these steroids were measured in 18 sera samples from healthy volunteers (9 males and 9 females; aged 23-78 years). Free DHEA, 7alpha-OH-DHEA, 7beta-OH-DHEA and 7-oxo-DHEA levels ranged between 0.21-3.55, 0.001-0.194, 0.003-0.481, and 0.000-0.077ng/ml, respectively, and the sulfate-conjugated steroid levels of these metabolites ranged between 253-4681, 0.082-3.001, 0.008-0.903, and 0.107-0.803ng/ml, respectively. The free DHEA-related steroid concentrations were much lower than those previously measured by RIA and low-resolution GC-MS. The present method made it possible to determine simultaneously serum DHEA-related steroid levels with sufficient sensitivity and accuracy.     

High levels of 15-oxygenated steroids in circulation of patients with multiple sclerosis: fact or fiction?

15-Oxygenated cholesterol species such as 5α-cholest-8(14)ene-3β,15α-diol (15HC) and 3β-hydroxy-5α-cholest-8(14)-en-15-one (15KC) are commercially available synthetic products unlikely to occur in biological systems. Surprisingly, Farez et al. recently reported that these two steroids occur in human circulation at levels considerably higher than those of any other endogenous oxysterol [Farez, M. et al. 2009. Toll-like receptor 2 and poly(ADP-ribose) polymerase 1 promote central nervous system neuroinflammation in progressive EAE. Nat. Immunol. 10: 958-964]. The levels were reported to be increased in patients with multiple sclerosis in a progressive phase and the authors suggested that this could be utilized diagnostically. Based on extensive in vitro experiments exposing cells to the same high levels of 15HC as found in vivo (1000 ng/ml) the authors concluded that 15HC may be an important pathogenetic factor in multiple sclerosis. Using combined gas chromatography-mass spectrometry we fail to detect significant plasma levels of 15HC either in healthy controls or in patients with multiple sclerosis (levels < 2 ng/ml). If 15KC is present in these plasma samples, the concentration of it must be <10 ng/ml. Our failure to detect significant levels of the above steroids could not be due to loss during hydrolysis and work-up because recovery of the added two oxysterols was close to 100%. Autoxidation of lipoprotein-bound cholesterol resulted in extensive conversion of cholesterol into 7-oxygenated but not 15-oxygenated sterols. We conclude that if present there are trace amounts only of the above 15-oxygenated steroids in human circulation and that the role of such oxysterols as pathogenetic factors and biomarkers must be reconsidered.     

Steroid and sterol 7-hydroxylation: ancient pathways

B-ring hydroxylation is a major metabolic pathway for cholesterols and some steroids. In liver, 7 alpha-hydroxylation of cholesterols, mediated by CYP7A and CYP39A1, is the rate-limiting step of bile acid synthesis and metabolic elimination. In brain and other tissues, both sterols and some steroids including dehydroepiandrosterone (DHEA) are prominently 7 alpha-hydroxylated by CYP7B. The function of extra-hepatic steroid and sterol 7-hydroxylation is unknown. Nevertheless, 7-oxygenated cholesterols are potent regulators of cell proliferation and apoptosis; 7-oxygenated derivatives of DHEA, pregnenolone, and androstenediol can have major effects in the brain and in the immune system. The receptor targets involved remain obscure. It is argued that B-ring modification predated steroid evolution: non-enzymatic oxidation of membrane sterols primarily results in 7-oxygenation. Such molecules may have provided early growth and stress signals; a relic may be found in hydroxylation at the symmetrical 11-position of glucocorticoids. Early receptor targets probably included intracellular sterol sites, some modern steroids may continue to act at these targets. 7-Hydroxylation of DHEA may reflect conservation of an early signaling pathway.     

Aromatization of androstenedione and 16alpha-hydroxyandrostenedione in human placental microsomes. Kinetic analysis of inhibition by the 19-oxygenated and 3-deoxy analogs

Inhibition of aromatase activity in human placental microsomes with androstenedione (AD) (1a) and its 19-oxygenated derivatives 1b and 1c, their 16alpha-hydroxy compounds 2 and 3, and 3-deoxyandrost-4-ene compounds 5 and 6 was studied using [1beta-(3)H]AD as a substrate and compared to that with [1beta-(3)H]16alpha-hydroxyandrostenedione (16-OHAD). AD series of steroids, compounds 1, inhibited competitively [1beta-(3)H]AD aromatization whereas other 16alpha-hydroxy steroids 2, 3, 5, and 6 inhibited AD aromatization in a non-competitive manner. On the other hand, all of 16-OHAD series, compounds 2, blocked the [1beta-(3)H]16-OHAD aromatization in a competitive manner whereas the AD series steroids 1 as well as the 3-deoxy-16alpha-hydroxy-17-one steroids 5 and 3-deoxy-16alpha,17beta-diol steroids 6 inhibited 16-OHAD aromatization non-competitively. 3-Carbonyl and 16alpha-hydroxy functions of 16-OHAD play a critical role of selection of the 16-OHAD binding site. The results suggest that the AD derivatives 1 are kinetically aromatized at a different site from the 16-OHAD derivatives 2. Physical and/or chemical environments around the aromatase protein in the microsomal membrane may play a significant role in the expression of the substrate specificity, and the present results do not exclude the idea that the placental microsomes have a single binding site.     

Aldosterone synthase activity in the Y-1 adrenal cell line

The Y-1 adrenal cell line was shown to produce 20-dihydroaldosterone from deoxycorticosterone. This compound was identified by GC-MS by comparison with the previously synthesized reference compound. Two other 18-hydroxylated metabolites were identified as 11β,18-dihydroxy-20-dihydroprogesterone from endogenous cholesterol and 18-hydroxy-20-dihydro-11-dehydrocorti-costerone from DOC. The conditions necessary for the synthesis of these compounds are culturing in 20% serum-supplemented medium and repeated incubations with the substrate. The production of 11β-hydroxylated steroids and that of 18-oxygenated steroids is stimulated differently by ACTH and angiotensin II suggesting the expression of two different enzymes, cytochrome P-450_11β and cytochrome P-450_aldo The Y-1 cell line can secrete either 11β-hydroxylated steroids characteristic of the glucocorticoid pathway or 18-oxygenated steroids characteristic of the mineralocorticoid pathway, which in vivo are generally produced in two different zones of the adrenal cortex. This cell line should be an interesting model for the study of the molecular mechanisms regulating the expression of these two enzymes involved in the final steps of the steroidogenic pathways.     

C20 steroids: The metabolism of 3-hydroxy-19-nor[21-14C]pregna-1,3,5(10)-trien-20-one in the rabbit

1. The metabolism of 3-hydroxy-19-norpregna-1,3,5(10)-trien-20-one, a possible product of the aromatization of progesterone or pregnenolone, has been studied. 2. After oral administration of this C(20) steroid as the 21-(14)C-labelled compound to two groups of rabbits, the excretion pattern of metabolites in the urine was examined. 3. At 14 days after administration, 3.3-6.5% of the radioactivity had appeared in the urine, 71-79% in the faeces and approx. 10% remained in the gut. 4. A metabolite, isolated from urine mainly as the unconjugated steroid, was identified as 19-norpregna-1,3,5(10)-triene-3,20alpha-diol and constituted 18.5-22% of the total urinary radioactivity. 5. A minor component of the urinary unconjugated steroids was identified as 19-norpregna-1,3,5(10)-triene-3,17alpha,20alpha-triol. 6. A further 2-7.5% of the total urinary radioactivity, isolated only from the urinary sulphate fraction, was tentatively identified as an 18-oxygenated derivative of the administered steroid.     

6-Oxygenation of 3-oxo-4-ene-steroids in high yields after 3-imine-formation

3-Imine formation between primary amines and 3-oxo-4-ene-steroids, followed by hydrolysis of the imines (either spontaneously during work up or induced by acetic acid) has been shown to cause 6-oxygenation of the steroids tested (17 beta-hydroxy-4-androsten-3-one, 4-androstene-3,17-dione, 4-pregnene-3,20-dione and 4-cholesten-3-one). The main products are the 6 beta-hydroxy- and the 6-oxo-derivatives of the respective steroid. These derivatives were identified by chromatographic mobilities and by gas chromatography-mass spectrometry. The formation of 6 beta-hydroperoxy-derivatives is suggested and these derivatives were tentatively identified. The highest yields of 6-oxygenated products (30-50%) were found when cadaverine and spermine were reacted with the steroids. The addition of reduced glutathione during hydrolysis of the steroid 3-imines of cadaverine, hexylamine and ethanolamine as well as addition of ascorbic acid during the hydrolysis of the steroid 3-imines of cadaverine substantially reduced the 6-oxygenation. Steroid 3-imine formation and hydrolysis which yields 6-oxygenated derivatives has also been shown to occur during work up (evaporation) of organic solvent extracts of rat liver microsomes (105,000 g sediments) to which 17 beta-hydroxy-4-androsten-3-one, 4-androstene-3,17-dione, 4-pregnene-3,20-dione or 4-cholesten-3-one respectively had been added. It is concluded that there is a risk that these organic reactions are mistaken for enzymatic conversions during in vitro investigations of 3-oxo-4-ene-steroids.     

Chemical synthesis of 4,4'-dimethyl-7-oxygenated sterols. Inhibitors of 3-hydroxy-3-methylglutaryl reductase

The chemical syntheses of 4,4'-dimethylcholest-5-en-3 beta-ol-7-one, 4,4'-dimethylcholest-5-ene-3 beta, 7 beta-diol and 4,4'-dimethylcholest-5-ene-3 beta, 7 alpha-diol are described. All of these compounds were found to be potent inhibitors of 3-hydroxy-3-methylglutaryl (HMG-CoA) reductase activity in cultured mouse L cells. The synthetic scheme developed in this study utilizes commercial cholesterol as the starting material and provides a simplified method for the preparation of 4,4'-dimethyl-7-oxygenated steroids.     

Human placental estrogen synthetase (aromatase). Effect of environment on the kinetics of protein-protein and substrate-protein interactions and the production of 19-oxygenated androgen intermediates in the purified reconstituted cytochrome P450 enzyme system

Estrogen synthetase (aromatase) catalyzes the conversion of androgen into estrogen via two hydroxylations at C19 and a subsequent C19-10 lyase reaction. We report here the results of a reconstitution study using a highly purified aromatase cytochrome P450 monooxygenase enzyme system, with both protein components (cytochrome P450 and NADPH-cytochrome P450 reductase) obtained from human term placental microsomes. By varying one of the components (amounts of cytochrome P450, NADPH-cytochrome P450 reductase, or androgen substrate) as the other two were held constant in four different environments (phospholipid, non-ionic detergent, mixture of phospholipid and non-ionic detergent and buffer alone), we obtained evidence supporting the following conclusions. The reconstituted enzyme is more active and the protein components exhibit much lower apparent Km values in the detergent and/or lipid environment compared with buffer alone. Although the apparent Km and Vmax values for each aromatase protein component differ significantly in most cases with the particular limiting component and environment, the catalytic efficiency (Kcat/Km) was independent of the limiting protein component and varied with the environment only (highest in the lipid-detergent mixture and lowest in lipid alone). When the concentration of androgen substrate (androstenedione or testosterone) was varied at constant amounts of the aromatase protein components (NADPH-cytochrome P450 reductase saturating), the Km was lower and the Vmax was higher for adrostenedione. The specificity constant (Vmax/Km) was a function of the reconstitution environment (highest in lipid alone and lowest in detergent alone) and was, on average, about 4-fold higher for androstenedione in a particular environment. The extent of production of 19-oxygenated androgen intermediates (19-hydroxy and 19-oxo androstenedione) was examined at three different levels of aromatase cytochrome P450 (subsaturating, saturating, super-saturating) relative to the NADPH-cytochrome P450 reductase component in the three different hydrophobic environments using androstenedione as substrate. Both 19-oxygenated androgens, each made in comparable amounts relative to control, were isolatable in greatest amounts under cytochrome P450 super-saturating conditions in the detergent-lipid mixed environment, and in least amounts under cytochrome P450 subsaturating conditions in the lipid-only environment. Based on these data, we propose that 19-oxygenated androgen intermediates are biosynthesized sequentially in a step-wise fashion as the cytochrome P450 and NADPH-cytochrome P450 reductase form transient complexes, and that the amount of isolatable 19-oxygenated androgen is proportional to the amount of excess cytochrome P450 component.     

LC-NMR and LC-MS analysis of 2,3,10,11-oxygenated protoberberine metabolites in Corydalis cell cultures

The metabolism of 2,3,10,11-oxygenated protoberberine alkaloids was studied in cell cultures of Corydalis species. Without prior isolation, the structures of the metabolites were determined by LC-MS and LC-NMR analyses. Tetrahydropseudocoptisine alpha-N-metho salt, pseudoprotopine, and pseudomuramine were identified for the first time, and preliminary evidence for metabolic pathways to the formation of these alkaloids were obtained.     

Steroid metabolism in primates. XV. Excretion of C19 and C21 steroids in the urine of baboons (Papio hamadryas) after long-term treatment with a combination of mestranol-chlormadinone acetate

The urinary excretion of C21- and C19-steroids was investigated in female babons (Papio hamadryas) treated with the ovulation inhibitor Ovosiston (mestranol + chlormadinone-acetate), in comparison with an untreated control Group Urinary C21-steroid excretion was not significantly altered by Ovosiston. 17-Ketosteroids were decreased, predominantly 11-oxygenated compounds.     

Ergosteroids. VI. Metabolism of dehydroepiandrosterone by rat liver in vitro: a liquid chromatographic-mass spectrometric study

Because relatively large amounts of dehydroepiandrosterone (DHEA) are required to demonstrate its diverse metabolic effects, it is postulated that this steroid may be converted to more active molecules. To search for the possible receptor-recognized hormones. DHEA was incubated with whole rat liver homogenate and metabolite appearances were studied by LC-MS as a function of time to predict the sequence of their formation. An array of metabolites has been resolved, identified and characterized by highly specific and accurate technique of LC-MS, and several of these steroids were analyzed quantitatively. Their identities were established by comparison with pure chemically synthesized compounds and by chemical degradation of isolated fractions. In the present study, we have reasonably established that DHEA was converted to 7alpha-OH-DHEA, 7-oxo-DHEA, and 7beta-OH-DHEA in sequence. These metabolites were further reduced at position 7 and/or 17 to form their respective diols and triols, which were also sulfated at 3beta-position. DHEA and its 7-oxygenated derivatives were also converted to their respective 3beta-sulfate esters. Several of these steroids are being reported for the first time. 16Alpha-hydroxy-DHEA, androst-5-ene-3beta,16alpha,17beta-triol, androst-4-ene-3,17-dione, 11-hydroxy-androst-4-ene-3,17-dione, androst-5-ene-3,17-diol and testosterone were also identified and characterized. In all, 19 metabolites of DHEA are being reported in this extensive study. We have also detected the formation of 12 additional metabolites including several conjugates, which are the subject of current investigation.     

3-Keto steroids from the marine organisms Dendrophyllia cornigera and Cymodocea nodosa

The new (20R)-22E-cholesta-4,22-diene-3,6-dione (1), along with three known 3-keto steroids were isolated from the deep-water Mediterranean scleractinian coral Dendrophyllia cornigera (2-4). Moreover, four known related 3-keto steroids were isolated from the sea grass Cymodocea nodosa (5-8). The structure elucidation of steroid 1 and the full NMR resonance assignments of all isolated metabolites were based on interpretation of their spectral data. All compounds are reported for the first time as metabolites of the investigated organisms. Compounds 2 and 3 showed significant cytotoxicity against lung cancer NSCLC-N6 cell line.