Circulating neuroactive C21- and C19-steroids in young men before and after ejaculation

Twelve neuroactive and neuroprotective steroids, androgens and androgen precursors i.e. 3alpha,17beta-dihydroxy-5alpha-androstane, 3alpha-hydroxy-5alpha-androstan-17-one, 3alpha-hydroxy-5beta-androstan-17-one, androst-5-ene-3beta,17beta-diol, 3beta,17alpha-dihydroxy-pregn-5-en-20-one (17alpha-hydroxy-pregnenolone), 3beta-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA), testosterone, androst-4-ene-3,17-dione (androstenedione), 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone), 3beta-hydroxy-pregn-5-en-20-one (pregnenolone), 7alpha-hydroxy-DHEA, and 7beta-hydroxy-DHEA were measured using the GC-MS system in young men before and after ejaculation provoked by masturbation. The circulating level of 17alpha-hydroxypregnenolone increased significantly, whereas the other circulating steroids were not changed at all. This fact speaks against the hypothesis that a drop in the level of neuroactive steroids, e.g. allopregnanolone may trigger the orgasm-related increase of oxytocin, reported by other authors.     

Bioconversion of C19- and C21-steroids with parent and mutant strains of Curvularia lunata

Regio- and stereospecificity of microbial hydroxylation was studied at the transformation of 3-keto-4-ene steroids of androstane and pregnane series by the filamentous fungus of Curvularia lunata VKM F-644. The products of the transformations were isolated by column chromatography and identified using HPLC, massspectrometry (MS) and proton nuclear magnetic resonance (¹H NMR) analyses. Androst-4-ene-3,17-dione (AD) and its 1(2)-dehydro- and 9α-hydroxylated (9-OH-AD) derivatives were hydroxylated by the fungus mainly in position 14α, while 6α-, 6β- and 7α-hydroxylated products were revealed in minor amounts. At the transformation of C₂₁-steroids (cortexolone and its acetylated derivatives) the presence of 17-acetyl group was shown to facilitate further selectivity of 11β-hydroxylation. Original procedures for protoplasts obtaining, mutagenesis and mutant strain selection have been developed. A stable mutant (M4) of C. lunata with high 11β-hydroxylase activity towards 21-acetate and 17α,21-diacetate of cortexolone was obtained. Yield of 11β-hydroxylated products reached about 90% at the transformation of 17α, 21-diacetate of cortexolone (1 g/l) using mutant strain M4.     

Evidence that 24- and 27-hydroxylation are not involved in the cholesterol-induced down-regulation of hydroxymethylglutaryl-CoA reductase in mouse liver.

Several authors have suggested that 27-hydroxycholesterol may be an important physiological regulator of cholesterol homeostasis. In the present study we investigated the possibility that 24- or 27-hydroxylation of cholesterol is of importance for the down-regulation of hydroxymethylglutaryl (HMG)-CoA reductase in mouse liver induced by dietary cholesterol. Using an accurate method based on isotope dilution-mass spectrometry with deuterated internal standards, we were able to detect significant levels of both 24- and 27-hydroxycholesterol in liver homogenates from normal mice. Feeding cholesterol, 2% for 4 days, increased the levels by 80 and 30%, respectively. No significant hepatic levels of 25-hydroxycholesterol could be demonstrated in untreated mice, and the level of this steroid in cholesterol-treated mice was just above the detection limit. Mouse liver mitochondria were able to catalyze 24- as well as 27-hydroxylation, but not 25-hydroxylation of cholesterol. There was no such conversion in liver microsomes. When using 24-2H2- or 23,23,24,24,25-2H5-labeled cholesterol as substrate a kinetic isotope effect of about 4.5 was observed for the mitochondrial 24-hydroxylation. When using 26,26,26,27,27,27-2H6-labeled cholesterol as substrate a kinetic isotope effect of about 2.5 was observed for the 27-hydroxylation. Use of those deuterium-labeled cholesterol species thus allowed a specific suppression of the rate of 24- and 27-hydroxylation. Feeding mice with 0.05% unlabeled pure cholesterol in the diet for 24 h inhibited the hepatic HMG-CoA reductase activity by about 50%. The same degree of suppression was obtained after feeding with 23,23,24,24,25-2H5- and 26,26,26,27,27,27-2H6-labeled cholesterol. Were mitochondrial 24- and 27-hydroxylation of importance, one would expect reduced suppression of HMG-CoA reductase when feeding deuterated cholesterol, due to the isotope effects. As this was not the case, it is concluded that neither 24-hydroxylation nor 27-hydroxylation are critical for the cholesterol-induced down-regulation of HMG-CoA reductase in mouse liver.     

Cyclic AMP-sensitive activation of hepatic sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We previously showed that preincubation of a 10,000 g supernatant (S(10)) from rat liver for 20 min at 37 degrees C dramatically increased the subsequent incorporation of [(14)C]acetate into sterols. No activation was seen with [(14)C]mevalonate as substrate. In the present studies we have examined the effect of preincubation on HMG CoA reductase. When microsomes were isolated from S(10) by calcium precipitation, preincubation of S(10) increased the specific activity of HMG CoA reductase threefold. No activation of HMG CoA reductase was observed in microsomes isolated by ultracentrifugation. Activation was cyclic AMP-sensitive. When cyclic AMP (0.001-1.0 mM) and MgATP (1 mM) were present during the preincubation period, there was little or no activation of HMG CoA reductase activity or of sterol synthesis from acetate. MgATP alone did not prevent activation. Neither cyclic AMP nor MgATP was inhibitory when present only during the assay of sterol synthesis. We propose that the in vitro activation represents the reversal of a physiologic cyclic AMP-mediated mechanism for the control of hepatic HMG CoA reductase. That a phosphoprotein phosphatase may catalyze the activation was supported by the observation that sodium fluoride, an inhibitor of phosphoprotein phosphatases, inhibited the activation. These results suggest that hormone-induced changes in the cellular level of cyclic AMP may regulate the activity of HMG CoA reductase and the rate of hepatic cholesterol synthesis.     

Accumulation of glycoprotein gonadotropin in the pituitary of juvenile rainbow trout in response to androgens and C21-steroids,including 11-steroids

Summary Effects of steroids on the accumulation of glycoprotein gonadotropin (GTH) in pituitaries of juvenile trout were investigated by means of scanning cytophotometry applied to immunocytochemical preparations, and with the use of a radioimmunoassay. Effects on other aspects of GTH-cell activity were analyzed by measuring the size of the gonadotrops and their nuclei.Progesterone added to aquarium water and methyltestosterone incorporated into the food showed a pronounced stimulatory effect on the accumulation of GTH. To a lesser extent, treatment with cortisol, cortisone, and desoxycorticosterone acetate administered to aquarium water, and 11-hydroxy-androstenedione added to the food resulted in an increase of the hypophysial content of GTH. Steroids stimulating the accumulation of GTH in the pituitary also exhibited a positive effect on GTH-cell activity as indicated by an increase in the size of gonadotropic cells. Progesterone incorporated into the food did not influence the GTH-content and the GTH-cell activity. It is suggested that the route of administration of an exogenous steroid is essential for its effect on GTH cells in trout.Comparison of GTH values reveals an excellent correlation between the data from the radioimmunoassay and those from the corresponding densitometric measurements. No correlation was observed between values of morphometrically determined GTH-cell activity and the densitometric values reflecting hypophysial GTH content.     

Inhibitors of sterol synthesis. Chemical syntheses, properties and effects of 4,4-dimethyl-15-oxygenated sterols on sterol synthesis and on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells

The chemical syntheses of a number of 4,4-dimethyl substituted 15-oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of the biosynthesis of cholesterol and other biological effects. Described herein are the first chemical syntheses of 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-3 beta-ol-15-one, 3 beta,15 alpha-diacetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene, 3 beta-acetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 beta-ol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 beta-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 alpha-ol-3-one, 3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene-7 alpha,15 alpha-diol, 7 alpha,15 alpha-diacetoxy-3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene, 4,4-dimethyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one and 3 beta,7 alpha,15 alpha-tri-o-bromobenzoyloxy-5 alpha-cholest-8(14)-ene. Also prepared for use in the biological experiments were 4,4-dimethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-5 alpha-cholest-8-ene-3 beta,15 alpha-diol and 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol. The effects of twelve 4,4-dimethyl substituted 15-oxygenated sterols and of four 4,4-dimethyl substituted 32-oxygenated sterols on sterol synthesis and on the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were evaluated in mouse L cells. With the exception of 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol, all of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-6) M and six of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-7) M. 4,4-Dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol caused a 50% decrease in sterol synthesis at 10(-8) M. The potencies of the 4,4-dimethyl substituted 15-oxygenated and C-32-oxygenated sterols with respect to inhibition of sterol synthesis and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity have been compared with those of the corresponding sterols lacking the 4,4-dimethyl substitution.     

Evidence for involvement of cytochrome P-450-linked oxygenase system in the conversion of C21-steroids to delta 16-C19-steroids catalyzed by pig testicular microsomes

Formation of androstadienone or androstadienol, a delta 16-C19-steroid, from progesterone or pregnenolone is catalyzed by the so-called delta 16-C19-steroid synthesizing enzyme in the pig testicular microsomes. The enzyme activity was also present in the testicular microsomes prepared from neonatal pig. The enzyme activity was considerably inhibited by CO, and such cytochrome P-450 inhibitors as SU 8000, SU 10603, and metyrapone. delta 16-C19-Steroid synthesizing enzyme activity was extracted from the testicular microsomes by sodium cholate in potassium phosphate buffer, pH 7.4, containing EDTA and dithiothreitol, and the solubilized enzyme activity was partially purified by DEAE-cellulose column chromatography. It was shown by reconstitution of the enzyme activity that delta 16-C19-steroid synthesizing enzyme is a cytochrome P-450-linked oxygenase system dependent on cytochrome P-450-reductase and cytochrome b5. In particular, cytochrome b5 was an essential component for the activity of delta 16-C19-steroid synthesizing enzyme.     

Lovastatin treatment inhibits sterol synthesis and induces HMG-CoA reductase activity in mononuclear leukocytes of normal subjects

The mechanism by which competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase decrease serum cholesterol is incompletely understood. The few available data in humans suggest that chronic administration of the competitive inhibitor, lovastatin, decreases serum cholesterol with little or no change in total body sterol synthesis. To further define the effect of lovastatin on cholesterol synthesis in normal subjects, we investigated the effect of a single oral dose of lovastatin and a 4-week treatment period of lovastatin on mononuclear leukocyte (ML) sterol synthesis as a reflection of total body sterol synthesis. In parallel, we measured serum lipid profiles and HMG-CoA reductase activity in ML microsomes that had been washed free of lovastatin. ML sterol synthesis did not significantly decrease (23 +/- 5%, mean +/- SEM) at 3 h after a single 40-mg dose of lovastatin. With a single oral 80-mg dose, ML sterol synthesis decreased by 57 +/- 10% (P less than 0.05) and remained low for the subsequent 6 h. With both doses, total HMG-CoA reductase enzyme activity in microsomes prepared from harvested mononuclear leukocytes was induced 4.8-fold (P less than 0.01) over baseline values. Both the 20-mg bid dose and the 40-mg bid dose of lovastatin administered for a 4-week period decreased serum cholesterol by 25-34%. Lovastatin at 20 mg bid decreased ML sterol synthesis by 23 +/- 6% (P less than 0.02) and increased ML HMG-CoA reductase 3.8 times (P less than 0.001) the baseline values. Twenty four hours after stopping lovastatin, ML sterol synthesis and HMG-CoA reductase enzyme activity had returned to the baseline values. The higher dose of lovastatin (40 mg bid) decreased ML sterol synthesis by 16 +/- 3% (P less than 0.05) and induced HMG-CoA reductase to 53.7 times (P less than 0.01) the baseline value at 4 weeks. Stopping this higher dose effected a rebound in ML sterol synthesis to 140 +/- 11% of baseline (P less than 0.01), while HMG-CoA reductase remained 12.5 times baseline (P less than 0.01) over the next 3 days. No rebound in serum cholesterol was observed. From these data we conclude that in normal subjects lovastatin lowers serum cholesterol with only a modest effect on sterol synthesis. The effect of lovastatin on sterol synthesis in mononuclear leukocytes is tempered by an induction of HMG-CoA reductase enzyme quantity, balancing the enzyme inhibition by lovastatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that changes in hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity are required partly to maintain a constant rate of sterol synthesis

The effects of insulin, glucagon, pyruvate, and lactate on the rate of sterol synthesis and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity were determined in hepatocytes obtained at different times of the day from rats maintained on a controlled lighting and feeding schedule. In hepatocytes from animals killed immediately before the start of the feeding period (D0 hepatocytes), the initially low activity of HMG-CoA reductase increased during incubation while that in hepatocytes prepared 6 h later (D6 hepatocytes) remained constantly high. The rates of sterol synthesis followed similar patterns of change. In both D0 and D6 cells, insulin stimulated HMG-CoA reductase but had little or no effect on the rates of sterol synthesis. In both types of cell preparation glucagon maximally suppressed HMG-CoA reductase activity at a concentration of 10(-7) M, but there was relatively little change in the rates of sterol synthesis. Both pyruvate and lactate mitigated the glucagon-mediated inhibition of HMG-CoA reductase. Each of these lipogenic precursors alone suppressed the rate of sterol synthesis in a dose-dependent manner. These changes were more apparent in the simultaneous presence of insulin and were greater in the D0 compared to the D6 hepatocytes. In the presence of lactate or pyruvate, the activity of HMG-CoA reductase was elevated, and the increase was greater when insulin was simultaneously present. In general, changes in the rate of fatty acid synthesis were positively correlated with changes in the activity of HMG-CoA reductase. These observations suggest that the latter changes are required to compensate for variations in the availability of simple precursors for sterol synthesis.     

Effects of mevinolin in rat liver: evidence for a lack of coupling between synthesis of hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase activity

The effect of treatment of rats with the hydroxymethylglutaryl-CoA reductase inhibitor, mevinolin, on 7 alpha-hydroxylation of cholesterol was studied. Treatment with 0.1% mevinolin in diet for 3 days was found to have an inhibitory effect on 7 alpha-hydroxylation of cholesterol (about 35%). Treatment with cholestyramine increased 7 alpha-hydroxylation of both exogenously added and endogenous microsomal cholesterol 3-4-fold. Combined treatment with both cholestyramine and mevinolin decreased this stimulation to 2-2.5-fold. Treatment with 2% cholesterol in diet increased 7 alpha-hydroxylation of exogenous cholesterol about 2-fold and 7 alpha-hydroxylation of endogenous cholesterol about 3.5-fold. The stimulatory effect of cholesterol was reduced or abolished when 0.1% mevinolin was added to the cholesterol-containing diet. With the exception of the experiments with cholesterol in the diet, all experiments including mevinolin gave a marked stimulation (up to 60-fold) of the hydroxymethylglutaryl-CoA reductase activity under the in vitro conditions employed. The concentration of free cholesterol in the liver microsomes was not significantly changed in any of these experiments. It is concluded that there is no coupling between induction of synthesis of hydroxymethylglutaryl-CoA reductase protein and cholesterol 7 alpha-hydroxylase activity. The inhibitory effect of mevinolin on cholesterol 7 alpha-hydroxylase activity under experimental conditions where most of the effect of mevinolin on hydroxymethylglutaryl-CoA reductase was abolished by treatment with cholesterol suggest that the effect of mevinolin on the cholesterol 7 alpha-hydroxylase may be independent of its effect on cholesterol synthesis. The over-all results do not favour the hypothesis that cholesterol synthesis and cholesterol availability are the most important determinants for the regulation of the cholesterol 7 alpha-hydroxylase.     

Inhibitory effects of phthalimide derivatives on the activity of the hepatic cytochrome P450 monooxygenases CYP2C9 and CYP2C19

Affecting hepatic cytochrome (CYP) activity is one of the major concerns in drug–drug interaction. Thus the testing of drug candidates on their impact on these enzymes is an essential step in early drug discovery. We tested a collection of 480 in-house phthalimide derivatives against different CYP450s using a high throughput inhibition assay. In initial tests with the isoform CYP2C19 about 57.5% of the tested phthalimide derivatives showed significantly enhanced inhibitory effects against this enzyme. In addition similar patterns of phthalimide inhibition for CYP2C9 and CYP2C19 were found, whereas the unrelated isoforms CYP2D6 and CYP3A4 were not specifically affected. Also less than 10% of randomly chosen substances inhibited CYP2C9. Analyses of structure-function relationships revealed that the substituent at the nitrogen atom in the isoindole ring is of crucial impact for the activity of CYP2C9/19.     

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and sterol synthesis by cholesterol sulfate in cultured fibroblasts

Although widely distributed throughout mammalian tissues, the biological function of cholesterol sulfate remains largely unknown. In these studies we have demonstrated that cholesterol sulfate suppresses de novo sterol synthesis in cultured human fibroblasts. It was further shown in these cultured cells that cholesterol sulfate is a potent inhibitor of the enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34), the rate-limiting enzyme in cholesterol biosynthesis and the site at which exogenous cholesterol suppresses endogenous cholesterol synthesis. Because cholesterol sulfate inhibited sterologenesis in steroid-sulfatase deficient fibroblasts derived from patients with recessive X-linked ichthyosis, it was inferred that cholesterol sulfate per se and not cholesterol liberated by intracellular desulfation was the inhibitor in these studies. Cholesterol sulfate may be an endogenous regulator of mammalian cholesterol biosynthesis.     

Inhibitors of sterol synthesis. Chemical synthesis of 5 beta-cholest-8-ene-3 beta,15 alpha-diol and its effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells

5 beta-Cholest-8-ene-3 beta,15 alpha-diol, prepared by hydroboration of 5 beta-cholesta-8,14-dien-3 beta-ol, was determined to have the 14 alpha-H,15 alpha-OH configuration by comparisons of observed and calculated lanthanide-induced shifts for the 3-tertbutyldimethylsilyl derivative. The 3 beta,15 alpha-diol was found to exist partially in a conformation in which ring B is a 5 beta, 6 alpha-half chair and the axial-equatorial orientation of ring A substituents is reversed. This conformation has been observed previously for 3 beta-(p-bromobenzoyloxy)-5 beta-cholesta-8,14-diene and for some cis-decalin derivatives. 5 beta-Cholest-8-ene-3 beta,15 alpha-diol was found to be highly active in the lowering of the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells and only slightly less active than the corresponding sterol (5 alpha-cholest-8-ene-3 beta,15 alpha-diol) with the trans A-B ring junction.     

Alternative pathways of sterol synthesis in yeast. Use of C(27) sterol tracers to study aberrant double-bond migrations and evaluate their relative importance

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the delta(8)-delta(7) isomerase (Erg2p) or delta(5) desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5 alpha-cholest-8-en-3beta-ol, 8-dehydrocholesterol (delta(5,8) sterol), or isodehydrocholesterol (delta(6,8) sterol), together with the corresponding 3 alpha-3H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven delta(22E) sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag(+)-HPLC) and identified by gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag(+)-HPLC. When delta(8)-delta(7) isomerization was blocked, exogenous delta(8) sterol underwent desaturation to delta(5,8), delta(6,8), and delta(8,14) sterols. Formation of delta(5,8) sterol was strongly favored over delta(6,8) sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The delta(5,8) sterol was metabolically almost inert except for delta(22) desaturation, whereas the delta(6,8) sterol was readily converted to delta(5,7), delta(5,7,9(11)), and delta(7,9(11)) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, delta(5,7) sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of delta(5,8) and delta(5,7,9(11)) sterols in wild-type yeast compared to Smith-Lemli-Opitz individuals.     

Age and sex differences in the metabolism of C19-delta5-steroids in human amniotic epithelium

In vitro transformation of [4-(14)C]-dehydroepiandrosterone and [4-(14)C]-5-androstenediol in human amniotic epithelium from the first and the second trimester was studied. Total conversion of both substrates used and formation of their "more polar" metabolites increased with advancing gestational age. The reduction of 17-oxo or the oxidation of 17 beta-hydroxy groups reached a maximum between weeks 17 and 20. A significant decrease in the conversion of delta 5-3 beta-hydroxy group from week 11 was evident. No significant sex differences were observed.