The use of nonradiolabelled steroid infusions to investigate the origin of oestrone sulphate in postmenopausal women

Infusion of nonradiolabelled dehydroepiandrosterone sulphate (DHA-S) has been used to investigate the possible formation of oestrone sulphate via a sulphated conjugate of androstenedione. The metabolic clearance rate (MCR) of DHA-S also was measured and the mean value (25 1/24h) was similar to values reported using isotopic techniques. Although conversion of DHA-S to 5-androstenediol, a steroid with oestrogenic properties, was detected during infusion of DHA-S, there were no significant increases in plasma levels of conjugated androstenedione or oestrone sulphate. The MCR's oestrone sulphate measured using infusion of nonradiolabelled steroid in two menopausal women were 99 1/24h and 121 1/24h. For one woman, the production rate of oestrone sulphate, calculated from the conversion of oestrone and oestradiol to oestrone sulphate (151 nmol/day) was similar to the measured production rate of oestrone sulphate (144 nmol/day). It is concluded that in menopausal women, oestrone sulphate is derived from conversion of oestrone and oestradiol with no formation occurring via conjugated androstenedione.     

Studies on the metabolism of oestrone sulphate. Comparative perfusions of oestrone and oestrone sulphate through isolated rat livers

The metabolism of [4-(14)C]oestrone and of [6,7-(3)H(2)]oestrone sulphate was studied during cyclic perfusion and once-through perfusion of the isolated rat liver. The following results were obtained. 1. As shown by once-through perfusion, the two steroids are metabolized differently during the first passage through the organ. [4-(14)C]Oestrone was taken up by the liver and partly delivered as oestradiol-17beta and oestriol into the medium. After uptake of [6,7-(3)H(2)]oestrone sulphate, only oestrone, liberated by hydrolysis, was delivered into the medium; no oestradiol-17beta or oestriol could be detected in the medium after one passage through the organ. This indicates that intracellular oestrone, which was taken up as such, and oestrone, which derived from intracellular hydrolysis, may be metabolized in different compartments of the liver cell. 2. The results of the cyclic perfusion showed that intracellular oestrone is preferentially conjugated with glucuronic acid, and subsequently excreted into the bile. Intracellular oestrone sulphate is preferably reduced to oestradiol sulphate, thus indicating that oestrone sulphate is a better substrate for the 17beta-hydroxy steroid oxidoreductase than is oestrone. 3. Albumin-bound oestrone sulphate acts as a large reservoir, and in contrast with free oestrone is protected from enzyme attack by its strong binding to albumin. 4. Oestrone sulphate is partly converted into the hormonally active oestrone by liver tissue. This suggests that liver not only inactivates oestrogens, but also provides the organism with oestrone, which is subsequently readily taken up by other organs.     

Simulation of pregnancy levels of plasma oestrone sulphate by infusion in the non-pregnant mare: A preliminary study

Oestrone sulphate was infused intravenously in saline solution into two, non-pregnant pony mares in repeated trials to simulate levels of the oestrogen found in plasma after midpregnancy in mares. Rates of infusion of oestrone sulphate ranged from 32 to 231 mg of oestrone equivalent per hour. Blood samples were taken from the opposite jugular vein for measurement of oestrone sulphate by radioimmunoassay. Plasma levels rose rapidly from the start of each infusion of the oestrogen and remained elevated over the hour-long periods, with concentrations as oestrone ranging from about 175 to 700 ng/ml. During each subsequent infusion with saline alone the oestrogen levels in plasma fell within 1 h to about 25% of the preceding values. Results were similar for each animal and simulation of plasma levels of oestrone sulphate in pregnancy was achieved.     

Luteolytic effect of oestrone sulphate on cyclic beef heifers

The action of oestrone sulphate on luteal function was tested in cyclic beef heifers. Once daily injection of 28 or 56 mg oestrone sulphate in corn oil beginning on Day 10 of the cycle had a significant luteolytic effect as compared to corn oil-treated controls.     

A comparison between plasma oestrone sulphate concentration and doppler ultrasound as methods for pregnancy diagnosis in sows

Plasma oestrone sulphate (E(1)S) concentration and doppler ultrasound as methods of pregnancy diagnosis in sows were compared. Using either method, pregnancy was accurately detected (test sensitivity > 94% for pregnant sows). E(1)S was a better predictor of nonpregnant animals (test specificity 78 vs 66%, respectively; P < 0.01) and could be used at least 1 wk earlier than doppler ultrasound (24 to 30 d vs 35 d postservice, respectively). E(1)S concentration was not an accurate predictor of litter size.     

The appearance of oestrone sulphate in the peripheral plasma of the pig early in pregnancy

Detectable concentrations of oestrone sulphate were present in 50% of the plasma samples collected from pregnant animals by Day 17. No oestrone sulphate was detected in plasma from cyclic nonpregnant pigs.     

Intestinal absorption of oestrone, oestrone glucuronide and oestrone sulphate in the rat in situ--II. Studies with the Doluisio technique

The absorption of oestrone (E1), oestrone glucuronide (E1G) and oestrone sulphate (E1S) from the small intestine of anaesthetized rats has been evaluated using the Doluisio in situ technique. Luminal disappearance of E1 was biphasic, which is consistent with a 3-compartment model; t1/2 alpha (first phase) was less than 5 min and t1/2 beta (second phase) approx 27 min for each concentration of steroid studied (trace identical to 10 nM, 1 microM and 10 microM). In contrast, luminal disappearance of E1G and E1S was monoexponential; t1/2 for E1G was 159, 229 and 299 min (trace identical to 200 nM, 10 microM and 100 microM respectively) and for E1S, 215, 174 and 192 min (trace identical to 10 nM, 10 microM and 100 microM respectively). There was a good correlation between the luminal disappearance data and recovery of steroid in bile. Adsorption of E1S was estimated from the initial rapid fall in luminal content within the first 5 min after drug administration. The study provides further evidence that E1S can be absorbed intact. Since saccharolactone only caused a reduction in E1G absorption of 32% we also conclude that part of the administered E1G was absorbed intact.     

Intestinal absorption of oestrone, oestrone glucuronide and oestrone sulphate in the rat in situ--I. Importance of hydrolytic enzymes on conjugate absorption

The biliary excretion of steroid after administration of [3H]oestrone ([3H]E1), [3H]oestrone glucuronide ([3H]E1G) and [3H]oestrone sulphate ([3H]E1S) into the hepatic portal vein of anaesthetized rats was very rapid with more than 70% of E1S and greater than 80% of E1 and E1G excreted in the first 30 min. There was a lag period in the biliary excretion of E1S, this was less apparent with E1 and absent with E1G. Biliary excretion accurately reflects the amount of steroid in the portal circulation and was therefore used as an assessment of absorption from the gastrointestinal (GI) tract. Absorption (as judged by excretion in bile) was least after administration of each steroid into the stomach. The extent of absorption correlated well with the lipophilicity of the steroids as shown by their relative partition coefficients between n-octanol and pH 6.5 phosphate-buffered saline (E1 greater than or equal to E1S greater than or equal to E1G). There was no significant difference in excretion profile when the steroids were given into the caecum (at 5 h, E1, 46.3 +/- 9.1%; E1G, 42.2 +/- 14.5%; E1S, 39.9 +/- 7.1%). The similarity, despite marked differences in physicochemical properties, suggested conjugate hydrolysis to the parent steroid. In contrast, after administration into the small intestine, excretion of E1 was very rapid and was maximal at 1 h (72.5 +/- 8.0%); E1G showed a near-linear excretion rate (1 h, 14.4 +/- 3.0%; 5 h, 80.0 +/- 11.7%), whereas in comparison E1S excretion was low (1 h, 12.1 +/- 2.4%; 5 h, 36.9 +/- 2.7%). The involvement of hydrolytic enzymes in conjugate absorption was assessed. Ampicillin pretreatment (200 mg/kg/day for 2 days) reduced the absorption of E1G from both the proximal and distal small intestine (by approximately 50%) but had no effect on the absorption of E1S. There was, therefore, evidence that quantitative absorption of E1G requires prior hydrolysis (by mammalian and/or microbial enzymes) but intact absorption of E1S from this region of the tract was implicated. Ampicillin pretreatment reduced the absorption of both conjugates (greater with E1S) from the caecum; hydrolysis clearly precedes absorption from the caecum. The above findings were supported by an in vitro study which showed that ampicillin pretreatment abolished the hydrolysis of E1S by caecal contents but only partially reduced the hydrolysis of E1G. The presence of mammalian glucuronidase enzyme may account for this difference.     

Development and evaluation of a direct enzyme immunoassay for oestrone sulphate in urine as a tool for diagnosis of early pregnancy in swine

A competitive inhibition-type enzymeimmunoassay (EIA) has been developed using 3-hemisuccinate-oestrone-peroxidase as conjugate for direct measurement of the hormone in swine urine. The method has a minimum detection level at 0.3 ng ml(-1) and satisfactory specificity, recovery and reproducibility. In a field trial with a group of 387 sows (7 in oestrus, 16 non-pregnant and 364 pregnant sows at several stages post service), it was shown that the assay is potentially an accurate pregnancy test in assessing the viability of the fetoplacental unit from day 23 up to day 30 post service. The assay is well suited for routine testing, particularly as a swine early pregnancy diagnosis test since urine sampling is easier and does not disturb the animal, while in the present assay there is no restriction in the time of sampling and the sample storage conditions.     

A monoclonal antibody to scopolamine and its use for competitive enzyme-linked immunosorbent assay.

A hybridoma clone producing a monoclonal antibody (SC78.H81) against scopolamine was established. The monoclonal antibody was an IgG1 (k) antibody with high affinity (1.6 x 10(9) M-1 for methylscopolamine). The monoclonal antibody was cross-reactive with methylscopolamine and butylscopolamine, and showed weak cross-reactivity with 6 beta- and 7 beta-hydroxyhyoscyamine. The cross-reaction with L-hyoscyamine, atropine, scopine and DL-tropic acid was very weak. A competitive enzyme-linked immunosorbent assay using SC78.H81 was established to quantify scopolamine. The sensitivity of the assay allowed detection of 20 pg assay-1 (0.2 ng ml-1) of scopolamine. The assay was applied to the estimation of scopolamine content in hairy root cultures of a Duboisia hybrid.     

Determination of the plasma levels of metyrapone and its enantiomeric metyrapol metabolites by direct plasma injection and multidimensional achiral-chiral chromatography

The development and validation of a direct injection high-performance liquid chromatographic (HPLC) method, with column switching, for the determination of metyrapol enantiomers and metyrapone in human plasma is described. The system used in this work was composed of a restricted access media (RAM) bovine serum albumin (BSA) octyl column coupled to an amylose tris(3,5-dimethoxyphenylcarbamate) chiral column. Water was used as eluent for the first 5 min at a flow rate of 1.0 ml/min for the elution of the plasma proteins and then acetonitrile-water (30:70 v/v) for the transfer and analysis of metyrapol enantiomers and metyrapone, which were detected by UV at lambda = 260 nm. The total analysis time was about 32 min. The calibration curves for each enantiomer and for the metyrapone were linear in the ranges 0.075-0.75 microg/ml and 0.150-1.50 microg/ml, respectively. Recoveries, intra- and interday precision and accuracy were determined using three quality controls, one low (0.18 microg/ml), one medium (0.75 microg/ml), and one high (1.35 microg/ml) plasma concentration. Quantitative recoveries and good precision and accuracy were obtained. The limit of quantitation were 0.045 microg/ml for both enantiomers and for the metyrapone.     

Apolipoprotein E polymorphism in the Netherlands and its effect on plasma lipid and apolipoprotein levels

Summary By isoelectric focusing of delipidated sera followed by immunoblotting we studied the apolipoprotein (apo) E polymorphism in 2018 randomly selected 35-years-old males from three different areas in the Netherlands. Comparison of the APOE allele (E^*2, E^*3, and E^*4) frequencies estimated in this study with those reported for several other population samples showed that there are marked differences between the Dutch population and the populations of Japan, New Zealand, Finland, and the United States. These differences in APOE allele frequencies appeared to be mainly due to differences in frequencies of the E^*2 allele (decreased in Japan and Finland; increased in New Zealand) and the E^*4 allele (increased in Finland; decreased in Japan and the United States). No difference in APOE allele frequencies was found between the Dutch population and the populations of West Germany and Scotland. Measurements of plasma cholesterol and apo B and E concentrations showed that the E^*4 allele is associated with elevated plasma cholesterol and apo B levels and with decreased apo E concentrations, whereas the opposite is true for the E^*2 allele. In the Dutch population, the sum of average allelic effects of the common APOE alleles on plasma cholesterol and apo B levels is 6.8% and 14.2%, respectively, of the total population mean. The total average allelic effect on plasma apo E concentrations was more pronounced (50.1%), suggesting that the APOE alleles primarily affect apo E concentrations rather than plasma cholesterol and apo B levels. This hypothesis is sustained by the observation that for plasma apo E levels the genetic variance associated with the APOE gene locus contributed about 18% to the total phenotypic variance. For plasma cholesterol and apo B this contribution was only 1.4% and 2.3% and is relatively low as compared with that reported for other population samples.     

A luciferase reporter assay to investigate the differential selenium-dependent stability of selenoprotein mRNAs

The mechanisms regulating the differential selenium (Se)-dependent stability of selenoprotein mRNAs are partially characterized. To further study the Se-dependent regulation of selenoproteins, we developed a novel chemiluminescent reporter to monitor the steady-state mRNA level of an artificial selenoprotein. Our reporter is a fusion of the Renilla luciferase gene and of the β-globin gene, but contains features required for incorporation of selenocysteine (SEC), namely, a UGA-SEC codon and a 3' untranslated region RNA stem loop called a SEC incorporation sequence (SECIS). At various levels of Se, the activity of reporters containing GPX1 or GPX4 SECIS elements is proportional to the steady-state mRNA level of the reporter construct and reflects the level of the corresponding endogenous mRNA. In a reporter containing a UGA codon and a functional GPX1 SECIS, Se-dependent nonsense-mediated decay (NMD) occurred in the cytoplasm, as opposed to the more typical nuclear location. To validate the reporter system, we used genetic and pharmacologic approaches to inhibit or promote NMD. Modulation of UPF1 by siRNA, overexpression, or by inhibition of SMG1 altered NMD in this system. Our reporter is derived from a Renilla luciferase reporter gene fused to an intron containing B-globin gene and is subject to degradation by NMD when a stop codon is inserted before the second intron.