A new approach to the synthesis of 3 beta, 23-diacetoxy-24-nor-5-cholene from 3 beta, 21-diacetoxy-5-pregnen-20-one

Synthesis of 3 beta, 23-diacetoxy-24-nor-5-cholene in six steps from 3 beta, 21-diacetoxy-5-pregnen-20-one has been achieved by a new approach. The method would allow to label the side chain of a bile acid at carbon-atom 21.     

Engineering steroid 5 beta-reductase activity into rat liver 3 alpha-hydroxysteroid dehydrogenase

Delta 4-3-Ketosteroid-5 beta-reductase (5 beta-reductase) precedes 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) in steroid hormone metabolism. Both enzymes are members of the aldo-keto reductase (AKR) superfamily and possess catalytic tetrads differing by a single amino acid. In 3 alpha-HSD, the tetrad consists of Tyr55, Lys84, Asp50, and His117, but a glutamic acid replaces His117 in 5 beta-reductase. By introducing the H117E point mutation into 3 alpha-HSD, we engineered 5 beta-reductase activity into the dehydrogenase. Homogeneous H117E 3 alpha-HSD reduced the double bond in testosterone to form 5 beta-dihydrotestosterone with kcat = 0.25 min-1 and Km = 19.0 microM and reduced the double bond in progesterone to generate 5 beta-dihydroprogesterone with kcat = 0.97 min-1 and Km = 33.0 microM. These kinetic parameters were similar to those reported for homogeneous rat liver 5 beta-reductase [Okuda, A., and Okuda, R. (1984) J. Biol. Chem. 259, 7519-7524]. The H117E mutant also reduced 5beta-dihydrosteroids to 5 beta, 3 alpha-tetrahydrosteroids with a 600-1000-fold decrease in kcat/Km versus wild-type 3 alpha-HSD. The ratio of 5 beta-reductase:3 alpha-HSD activity in the H117E mutant was approximately 1:1. Although the H117A mutant reduced Delta 4-3-ketosteroids, the 3 alpha-HSD activity predominated because the 5 beta-dihydrosteroids were rapidly converted to the 5 beta,3 alpha-tetrahydrosteroids. The pH-rate profiles for carbon-carbon double-bond and ketone reduction catalyzed by the H117E mutant were superimposable, suggesting a common titratable group (pKb = 6.3) for both reactions. In wild-type 3 alpha-HSD, the titratable group responsible for 3-ketosteroid reduction has a pKb = 6.9 and is assignable to Tyr55. The pH-rate profiles for 3-ketosteroid reduction by the H117A mutant were pH-independent. Our data indicate that Tyr55 functions as a general acid for both 3 alpha-HSD and 5 beta-reductase activities. We suggest that a protonated Glu117 increases the acidity of Tyr55 to promote acid-catalyzed enolization of the Delta 4-3-ketosteroid substrate. Further, the identity of amino acid 117 determines whether an AKR can function as a 5 beta-reductase by reorienting the substrate relative to the nicotinamide cofactor. This study provides functional evidence that utilization of modified catalytic residues on an identical protein scaffold is important for evolution of enzymatic activities within the same metabolic pathway.     

Effect of total gamma-irradiation on the rate of steroid hormone hydroxylation by rat liver microsomes

gamma-irradiation in doses of over 600 r proved to decrease the rate of androgen hydroxylation significantly; the rate of hydroxylation of the estrogens under study altered but little. Under the mentioned irradiation conditions the content of cytochrome P-450 in rat hepatic microsomes diminished. A reduction of the hydroxylation rate of steroid hormones by rat hepatic microsomes under the effect of total gamma-irradiation apparently depended on the qualitative changes of cytochrome P-450.     

Identification of a specific binding site for the anabolic steroid stanozolol in male rat liver microsomes

Male rat liver microsomes contain a [3H]dexamethasone binding site, capable of binding glucocorticoids and progesterone. We have shown previously that the 17 alpha-alkylated androgen, stanozolol, can inhibit the [3H]dexamethasone binding to microsomes through a negative allosteric mechanism, which gives rise to the possibility of its interaction with a different binding site. In this study, the existence of a single-saturating binding site, capable of binding the radioactive steroid with a maximum number of the specific binding site of 49 +/- 2 pmol/mg of protein and a Kd of 37 +/- 1.3 nM was demonstrated by using [3H]stanozolol. In competition experiments, only stanozolol and danazol were able to compete with [3H]stanozolol for its binding to microsomes, among more than 60 steroids and other compounds tested. The binding of [3H]stanozolol was depressed after protease treatment of the microsomes, or after the administration of cycloheximide to adult male rats for 24 hr, which suggest its proteic nature. The [3H]stanozolol binding site was detected in many tissues of the rat, with the highest concentrations being found in the liver. It was detected from birth, increasing afterward in concentration and reaching a peak at 2 to 3 months of age. This is the first experimental verification of the existence in liver microsomes of a specific binding site for some 17 alpha-alkylate androgens, such as stanozolol and danazol, different from the androgen receptor or the [3H]dexamethasone binding site.     

17 alpha,20 alpha-Dihydroxy-4-pregnen-3-one is the naturally occurring spermiation-inducing hormone in the testis of a frog, Rana nigromaculata

Spermiation, the process in which vertebrate spermatozoa are detached from investing Sertoli cells into the lumen of the seminiferous tubule, is a prerequisite for the successful fertilization. Using an in vitro Rana nigromaculata spermiation bioassay, we have shown that gonadotropin initiates spermiation by inducing the synthesis of delta 4-steroids by testis fragments. Among all of the delta 4-steroid metabolites produced by R. nigromaculata testis fragments, spermiation-inducing activity was confined to only one metabolite; this metabolite was identified as 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17 alpha,20 alpha-DP). Induction of spermiation by gonadotropin in vitro was accompanied by marked elevations in 17 alpha,20 alpha-DP concentrations in incubation media. These findings provide evidence that 17 alpha,20 alpha-DP is the nautrally occurring spermiation-inducing hormone in R. nigromaculata.     

Sterol synthesis. Syntheses of 15-oxygenated 5alpha, 14beta-cholest-7-en-3beta-ol derivatives

Treatment of 3beta-benzoyloxy-14alpha, 15alpha-epoxy-5alpha-cholest-7-ene with boron trifluoride-etherate gave, in 43% yield, 3alpha-benzoyloxy-5alpha, 14beta-cholest-7-en-15-one with the unnatural C-D ring juncture. Reduction of the latter compound with lithium aluminum hydride gave 15alpha, 14beta-cholest-7-en-3beta, 15alpha-diol and 5alpha, 14beta-cholest-7-en-3beta, 15beta-diol in 9% and 81% yields, respectively.     

Conversion of ectoderm into a neural fate by ATH-3, a vertebrate basic helix-loop-helix gene homologous to Drosophila proneural gene atonal

We have isolated a novel basic helix-loop-helix (bHLH) gene homologous to the Drosophila proneural gene atonal, termed ATH-3, from Xenopus and mouse. ATH-3 is expressed in the developing nervous system, with high levels of expression in the brain, retina and cranial ganglions. Injection of ATH-3 RNA into Xenopus embryos dramatically expands the neural tube and induces ectopic neural tissues in the epidermis but inhibits non-neural development. This ATH-3-induced neural hyperplasia does not require cell division, indicating that surrounding cells which are normally non-neural types adopt a neural fate. In a Xenopus animal cap assay, ATH-3 is able to convert ectodermal cells into neurons expressing anterior markers without inducing mesoderm. Interestingly, a single amino acid change from Ser to Asp in the basic region, which mimics phosphorylation of Ser, severely impairs the anterior marker-inducing ability without affecting general neurogenic activities. These results provide evidence that ATH-3 can directly convert non-neural or undetermined cells into a neural fate, and suggest that the Ser residue in the basic region may be critical for the regulation of ATH-3 activity by phosphorylation.     

Formation of a novel 20-hydroxylated metabolite of lipoxin A4 by human neutrophil microsomes

Lipoxin A4 (LXA4) is a biologically active compound produced from arachidonic acid via interactions of lipoxygenases. Incubation of LXA4 either with human neutrophils or with the neutrophil microsomes leads to formation of a polar compound on a reverse-phase high-performance liquid chromatography. We have identified the metabolite as 20-hydroxy-LXA4, a novel metabolite of arachidonic acid, on the basis of ultraviolet spectrometry and gas chromatography-mass spectrometry. The LXA4 omega-hydroxylation requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide, by antibodies raised against NADPH-cytochrome P-450 reductase, or competitively by leukotriene B4 (LTB4) and LTB5, substrates of LTB4 omega-hydroxylase. These findings indicate that the formation of 20-hydroxy-LXA4 is catalyzed by a neutrophil cytochrome P-450, the LTB4 omega-hydroxylase.     

Suppression in gonadotropes of gonadotropin-releasing hormone-stimulated follicle-stimulating hormone release by the gonadal- and neurosteroid 3 alpha-hydroxy-4-pregnen-20-one involves cytosolic calcium

The gonadal- and neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) suppresses FSH release in cultures of anterior pituitary cells. In a previous report, we showed that this suppression is achieved at least in part by an interaction at the plasma membrane level. We undertook to examine the possible interaction of 3 alpha HP at the level of intracellular Ca2+. Anterior pituitary cells from adult randomly cycling female rats were treated for 4 h with 10 nM GnRH and 0.1 nM 3 alpha HP with or without protein kinase C activator (SC10), antagonist (H-7), intracellular Ca2+ chelator (TMB-8), and intracellular Ca2+ mobilizer (glutamate), and with or without EGTA and Ca2+ in the medium. FSH content in media and cells was determined by RIA. The protein kinase C (PKC) activator, SC10, increased basal levels of secreted FSH. 3 alpha HP suppressed (P < 0.05) SC10-stimulated basal FSH release. The PKC inhibitor, H7, decreased GnRH-induced FSH release; FSH was further suppressed (P < 0.05) by 3 alpha HP in the presence of H7. These results were interpreted to indicate that 3 alpha HP may act in part at the level of PKC and also at another site(s). The intracellular Ca2+ chelator, TMB-8, suppressed released and cellular GnRH-stimulated FSH to the same extent as 3 alpha HP; FSH was not further decreased by 3 alpha HP in the presence of TMB-8. 3 alpha HP suppressed glutamate-stimulated FSH release in Ca(2+)-free medium (P < 0.01). Moreover, GnRH-induced release of FSH was suppressed to the same degree by 10(-10) M 3 alpha HP as by 10(-4) M EGTA. In pituitary cell suspensions, the GnRH-induced [Ca2+]i elevations were significantly (P < 0.05) attenuated by 3 alpha HP. From these and previous results, a model is proposed for the action of 3 alpha HP. The model suggests that 3 alpha HP may interact with gonadotropes at the level of the PKC cell signaling pathway and intracellular Ca2+ mobilization, in addition to the plasma membrane/calcium channel. The interaction effects a decrease in intracellular Ca2+, leading to decreases in FSH release from those pituitary gonadotropes that are responsible for FSH. The consistent decrease in total FSH (released plus cellular content) by 3 alpha HP suggests that this neurosteroid may also suppress FSH synthesis.     

Effects of chemotherapy-induced testicular damage on inhibin, gonadotropin, and testosterone secretion: a prospective longitudinal study

To investigate the role of inhibin in the control of follicle-stimulating hormone (FSH) secretion, we have measured levels of immunoreactive inhibin (ir-inhibin), inhibin B, Pro-alpha C containing inhibins, FSH, luteinizing hormone (LH), and testosterone in twelve men with hematological malignancies before, during, and after chemotherapy. Inhibin B levels fell significantly by 1 month from a mean +/- SE baseline level of 273.2 +/- 32.8 pg/mL, reaching a nadir of 52.6 +/- 15.3 pg/mL at 4 months (P < 0.0001). FSH levels increased within the first month from a baseline level of 3.9 +/- 0.6 IU/L, reaching a peak level of 22.4 +/- 3.3 IU/L at 4 months (P < 0.0001). FSH and inhibin B were significantly and inversely correlated (r = 0.69, P < 0.0001). Pro-alpha C containing inhibin levels increased significantly (P < 0.05) at 3 months and were significantly and positively correlated with FSH (r = 0.38, P = 0.002). LH levels increased significantly but to a much lesser extent than FSH, the increase becoming evident only 4 months after treatment commenced (P < 0.03). Levels of ir-inhibin and testosterone remained unchanged throughout the study. These data provide strong support to the hypothesis that inhibin B is the physiologically important form of inhibin in men, negatively regulating FSH secretion at the pituitary. Furthermore, they suggest that FSH stimulates inhibin alpha-subunit secretion by the testis.     

Glucuronidation of entacapone, nitecapone, tolcapone, and some other nitrocatechols by rat liver microsomes

PURPOSE: Nitrocatechol COMT inhibitors are a new class of bioactive compounds, for which glucuronidation is the most important metabolic pathway. The objective was to characterize the enzyme kinetics of nitrocatechol glucuronidation to improve the understanding and predicting of the pharmacokinetic behavior of this class of compounds. METHODS: The glucuronidation kinetics of seven nitrocatechols and 4-nitrophenol, the reference substrate for phenol UDP-glucuronosyltransferase activity, was measured in liver microsomes from creosote-treated rats and determined by non-linear fitting of the experimental data to the Michaelis-Menten equation. A new method that combined densitometric and radioactivity measurement of the glucuronides separated by HPTLC was developed for the quantification. RESULTS: Apparent K(m) values for the nitrocatechols varied greatly depending on substitution pattern being comparable with 4-nitrophenol (0.11 mM) only in the case of 4-nitrocatechol (0.19 mM). Simple nitrocatechols showed two-fold Vmax values compared with 4-nitrophenol (68.6 nmol min-1 mg-1), while all disubstituted catechols exhibited much lower glucuronidation rate. Vmax/K(m) values were about 10 times higher for monosubstituted catechols compared to disubstituted ones. The kinetic parameters for COMT inhibitors were in the following order: K(m) nitecapone > > entacapone > tolcapone; Vmax nitecapone > entacapone > tolcapone; Vmax/K(m) tolcapone > nitecapone > entacapone. CONCLUSIONS: Nitrocatechols can in principle be good substrates of UGTs. However, substituents may have a remarkable effect on the enzyme kinetic parameters. The different behaviour of nitecapone compared to the other COMT inhibitors may be due to its hydrophilic 5-substituent. The longer elimination half-life of tolcapone in vivo compared to entacapone could not be explained by glucuronidation kinetics in vitro.     

Uptake of 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate into isolated rat hepatocytes by three transport systems

Uptake of norcholansulfonate (3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate), an isogeometric analogue of cholate into isolated rat liver hepatocytes occurs only by saturable transport. In order to identify the transport systems involved, uptake of norcholansulfonate was studied using 7 beta-NBD-NCT ({N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl})-2'-aminoethanesulfonate) as a competing substrate. For transport of both bile salt derivatives, which mutually inhibit their mediated transport competitively, the existence of at least three transport systems must be assumed. Uptake studies using the cloned hepatic Na+/cholyltaurine cotransporting polypeptide stably expressed in CHO cells (Chinese hamster ovary cells) showed that both bile salt derivatives were transported and furnished the definite KT values of this single transport system and the ratio of the maximal uptake velocities. On the basis of these data, uptake of both bile salt derivatives into rat hepatocytes and their mutual competitive inhibition could be analyzed for three transport systems. The maximal flux rates J2 and the half-saturation constants KT2 in the presence of Na+ (143 mM) are for norcholansulfonate: J1(Na+ 143) = 1.0 +/- 0.2 nmol/(min . mg protein), KT1(Na+ 143) = 15 +/- 4 microM, J2(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT2(Na+ 143) = 15 +/- 2 microM, J3(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT3(Na+ 143) = 60 +/- 15 microM, and for 7 beta-NBD-NCT J1(Na+ 143) = 0.14 +/- 0.04 nmol/(min.mg protein), KT1(Na+ 143) = 3.1 +/- 0.5 microM, J2(Na+ 143) = 0.014 +/- 0.005 nmol/(min.mg protein), KT2(Na+ 143) = 21 +/- 2 microM, J3(Na+ 143) = 1.0 +/- 0.1 nmol/(min.mg protein), KT3(Na+ 143) = 190 +/- 25 microM. The kinetic parameters are in accordance with the assumptions that the cloned Na+/cholyltaurine cotransporting polypeptide represents transport system 2 and that the kinetically identified additional transport system 1 is either strictly or partially Na(+)-dependent.     

The delta 5-3 beta-hydroxy steroid acyl transferase activities in tissues of the male rat and sheep

The present study reports on the results of a follow-up examination of patient material, 5 years after the installation of the fixed supraconstruction. The patient group comprised 11 individuals. Briefly, a split-mouth technique of treatment was used. In the right side of the mandible the traditional 2-step surgical approach for implant installation was used. In the left jaw quadrant a 1-step surgical procedure was used. A clinical and radiographical examination was performed 5 years after the initial connection of the supraconstruction to the implants. At the 5-year follow-up examination all 61 implants examined at the 18-month follow-up were still in service and found to be clinically stable irrespective of the surgical procedure used. Furthermore, the results of the present clinical and radiographical follow-up study demonstrate that the marginal bone level at implants placed anteriorly in the edentulous mandible and supporting fixed supraconstructions is stable between 18 and 60 months irrespective of whether placed according to a 1-step or 2-step surgical procedure.     

Metabolism of N,N-diethylbenzamide and N,N-diethyl-alpha,alpha'-13C-benzamide by rat liver microsomes

The O2 and NADPH-dependent metabolism of N,N-diethylbenzamide (DEB), a topically applied insect repellent, has been investigated in liver microsomal suspensions from phenobarbital-pretreated male Wistar rats. Incubation conditions for the enzymatic production of N-ethylbenzamide (EB) from DEB (0.2 mM) were studied by use of HPLC with UV detection. With the microsomal enzymes suspended in 10 mM phosphate buffer (pH 7.4) and the substrate delivered in acetone (10 microliter), the yield of EB was 81.9 +/- 2.9% in five replicated experiments with NADPH (2 mM) and MgCl2 (4 mM). Conversion of DEB to EB was strongly inhibited by carbon monoxide, a cytochrome P-450 inhibitor. A sample of N,N-diethyl-alpha,alpha'-13C-benzamide was synthesized and was used, in conjunction with high-resolution 13C NMR spectroscopy, to identify the metabolites arising from oxidation of the ethyl group. Using the distortionless enhancement by polarization transfer pulse sequences and a substrate concentration of 0.4 mM, 13C-labeled acetaldehyde and glycolaldehyde, both detected as the hydrates, were found in the microsomal incubates. N-Ethyl-N-(alpha-hydroxyethyl)benzamide, the presumed metabolic progenitor of acetaldehyde, was not detected in these metabolic experiments. A pathway is proposed to account for the metabolic generation of glycolaldehyde by hydroxylation of both carbons of the N-ethyl group.     

Relationship between symptom severity and steroid variation in women with premenstrual syndrome: study on serum pregnenolone, pregnenolone sulfate, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one

The relationship between symptoms of premenstrual syndrome (PMS) and serum levels of pregnenolone (Pe), pregnenolone sulfate (PS), 5 alpha-pregnane-3,20-dione (5 alpha-DHP), 3 alpha-hydroxy-5 alpha- pregnan-20-one (5 alpha-THP), LH, 17 beta-estradiol (E2), and progesterone (P) was investigated during 2 consecutive menstrual cycles in 12 patients using daily measurements. Corresponding hormones were also measured during 1 cycle in 8 control women. Pe, PS, 5 alpha-DHP, and 5 alpha-THP showed a significant cyclicity within menstrual cycles and a high rate of correlation with P variation in both PMS patients and controls. No significant difference was found between PMS patients and controls in average serum concentrations of Pe, PS, 5 alpha-DHP, 5 alpha-THP, and LH during the luteal phase, whereas a significantly higher level of E2 and a lower level of P were observed in PMS patients. The variation in symptom scores was compared with that in hormone levels within each woman. The symptom peak showed a delay of 3-4 days after the serum P, Pe, 5 alpha-DHP, and 5 alpha-THP peaks. However, the plasma PS peak appeared on the same day or only 1 day before the symptom peak in PMS patients. When comparing the 2 cycles studied, more negative symptoms occurred in cycles with higher luteal phase E2, Pe, and PS concentrations, whereas higher luteal phase 5 alpha-DHP and 5 alpha-THP concentrations were associated with improved symptom ratings in PMS patients. These results suggest that the mentioned steroids are related to the severity of distressing symptoms in PMS patients.     

Fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, scavenges free radicals and inhibits lipid peroxidation in rat liver microsomes

A total 17,121 patients with pancreatic cancer have been collected by the Pancreatic Cancer Registration Committee of the Japan Pancreas Society. Significant differences in the postoperative prognosis were observed between patients with tumor limited to the pancreas and with tumor extending to surrounding tissues or adjacent organs. The lymph node metastases and distant metastases were definitive factors on the prognosis after resection. It might was possible for the subdivision of regional lymph nodes as Japanese classification to provide the Stage classification. Further development of the TNM system is necessary for assessing the outcome of most advanced cancers.