Absorption and passage through the dense layer of plasma membranes of rat hepatocytes and rabbit red blood cell ghosts, steroid hormones labelled with 3H were studied. The degree of absorption of steroid hormones by plasma membranes decreased in the following succession: progesterone greater than testosterone greater than oestrone greater than prednisolone greater than oestriol. The degree of the hormone absorption correlated with the solubility in lipids and with the effect of the enzyme release from the lysosomes. At the same time it is in inverse ratio to the speed of passage through the dense layer of the membranes. The nature of absorption of the steroid hormones is independent of the origin of plasma membranes.

A method for simultaneous quantitation of nine steroids in cord plasma is described which consists of Amberlite XAD-2-column chromatography at constant temperature of 45 degrees C, enzyme hydrolysis with beta-glucoronidase/aryl sulfatase, addition of five radioactive internal standards, ethyl acetate extraction, thin layer chromatography and quantitation by gas-liquid chromatography after trimethylsilyl ether derivative formation. Reliability criteria are established and the following steroid concentrations found: progesterone, 132.1 +/- 102.5 mug/100 ml; pregnenolone, 57.3 +/- 45.7 mug/100 ml; dehydroepiandrosterone, 46.5 +/- 29.4 mug/100 ml; pregnanediol, 67.5 +/- 46.6 mug/100 ml; 16-ketoandrostenediol 19.8 +/- 13.7 mug/100 ml; 16 alpha-hydroxydehydroepiandrosterone, 126.3 +/- 86.9 mug/100 ml; 16 alpha-hydroxypregnenolone, 78.2 +/- 56.5 mug/100 ml; androstenetriol, 22.2 +/- 17.5 mug/100 ml and oestriol, 127.7 +/- 116.9 mug/100.

Plasma oestradiol (E2), oestriol (E3), progesterone (P), human chorionic gonadotrophin (HCG) and human placental lactogen (HPL) levels, karyopyknotic index (KPI) and cervical mucus pattern were studied serially throughout pregnancy in 17 normal women. Hormonal patterns were similar to those reported by previous investigators. The P/E2 and P/E3 ratios were high in the first trimester but remained constant thereafter until term. There was no consistent change in E2, E3, and P levels and in P/E2 and P/E3 ratios in relation to onset of labour. With 2 exceptions, the cervical mucus and vaginal cytology were normal for pregnancy. The study provides a basis for a subsequent investigation into hormonal changes in threatened and recurrent abortions and the effect of treatment with HCG.

We compared two time-resolved fluoroimmunoassay systems for measuring free oestriol in human serum and progesterone in bovine milk. By reading the fluorescence of europium complex of 4,7-bis(chlorosulphophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid in solution, the measuring range is increased for both oestriol (10-50,000 ng/l instead of 25-50,000 ng/l) and for progesterone (10-50,000 ng/l instead of 25-10,000 ng/l). In addition, the interassay coefficients of variation were lowered from 9.5 to 5.7% for oestriol and from 7.5 to 5.4% for progesterone, at the smaller hormone concentrations detectable by each method.

After incubation of [4-14C]oestrone (E1) with kidney cortex slices of minipigs, [4-14C]oestradiol-17 beta (E2) and small amounts of a polar metabolite were detected in the ether-soluble fraction. E1, E2 and polar metabolites were found in the protein-bound fraction. The water-soluble fraction contained E1-3-glucuronide (80% of total glucuronides), E2-3-glucuronide and trace amounts of the 3-monoglucuronide of oestriol (E3). When E2 was used as substrate, the main product formed was E1; it was detected in both the ether-soluble and protein-bound fractions. E1-3-Glucuronide was the main metabolite in the water-soluble fraction, which also contained some E2-3-Glucuronide. In male minipigs, the mast, the rate of conversion of E1 and E2 as well as the formation of glucuronides were significantly greater in fertile females than in infertile females. Whereas no sex difference was observed in the metabolism of oestrogens in kidneys of infertile minipigs, the rate of oxidoreduction and glucuronidation was more pronounced in fertile female animals than in the corresponding males. The present results suggest that, in the kidneys of minipigs, the ratio of E1 to E2 is shifted towards the former; furthermore, by a comparatively rapid metabolism of the oestrogenic hormone, the renal tissue contributes to the maintenance of hormonal equilibrium.

The concentrations of mixed umbilical cord serum dehydroepiandrosterone sulphate (DHAS) and total oestriol, were determined by radioimmunoassay in 301 infants. Of 280 infants born between 36 and 42 weeks of gestation, 260 were born by vaginal delivery and 20 were delivered by Caesarean (C-section). We found that there was a significant elevation of serum total oestriol (P < 0.005) after 35 weeks with no concomitant gestational change of DHAS. Within the vaginal delivery group steroid values were similar among infants born following spontaneous, oxytocin augmented, and oxytocin induced labour. Within the C-section delivery group, steroid values were similar in infants who experienced spontaneous labour and those who did not. However, DHAS level was significant higher (P < 0.005) in the vaginal delivery group than in the C-section group. We postulate that the higher DHAS levels in the vaginal delivery group are the result of stress during the late first stage of labour or later.

We have developed an enzyme immunoassay for total oestrogens in pregnancy serum of plasma, using horseradish peroxidase (HRP) as the marker enzyme. A test combination consisting of an antiserum against oestriol-16/17-monosuccinyl-albumin and oestriol-16/17-monosuccinyl-HRP yielded a sensitive system, which reacted to approximately the same extent with oestrone, oestradiol, oestriol and their 16- and 17-conjugates. Samples had to be diluted 1 to 10 to avoid interference of plasma factors with the immune reaction. Bound/free separation was achieved with the double antibody solid phase (DASP) method. The HRP activity of the bound fraction was measured, after washing, to eliminate plasma factors disturbing the HRP reaction. The detection limit of the assay system was approx. 0.1 pmol/tube, while the index of precision lambda ranged from 0.02 to 0.06. To measure total oestrogens, including the 3-conjugated ones, we used an enzymatic hydrolysis with an extract of Helix pomatia. Hydrolysis was found to be optimal after 1 h at 50 degrees C and pH 5.0. The method was used on serum samples from normal pregnancies. The results showed a very good correlation (r=0.98) with those obtained by radioimmunoassay. Normal values for total oestrogens during pregnancy were determined in a multicentre clinical trial.

Novel chemiluminescent (CL) imaging microtiter plates with high-throughput, low-cost, and simple operation for detection of four biomarkers related to Down's syndrome screening were developed and evaluated. To enhance the sensitivity of CL immunosensing, soybean peroxidase (SBP) was used instead of horseradish peroxide (HRP) as a label enzyme. The microtiter plates were fabricated by simultaneously immobilizing four capture monoclonal antibodies, anti-inhibin-A, anti-unconjugated oestriol (anti-uE3), anti-alpha-fetoprotein (anti-AFP), and beta anti-HCG (anti-β-HCG), on nitrocellulose (NC) membrane to form immunosensing microtiter wells. Under a sandwiched immunoassay, the CL signals on each sensing site of the microtiter plates were collected by a charge-coupled device (CCD), presenting an array-based chemiluminescence imaging method for detection of four target antigens in a well at the same time. The linear response to the analyte concentration ranged from 0.1 to 40 ng/mL for inhibin-A, 0.075 to 40 ng/mL for uE3, 0.2 to 400 ng/mL for AFP, and 0.4 to 220 ng/mL for β-HCG. The proposed microtiter plates possessed high-throughput, good stability, and acceptable accuracy for detection of four antigens in clinical serum samples and demonstrated potential for practical applicability of the proposed method to Down's syndrome screening. Graphical Abstract Schematic evaluation of the microtiter plater for simultaneous detection of the four biomarkers.

The effect of 4 peroral oestrogen regimens without and with levonorgestrel (LNG) supplementation (250 micrograms/day) was studied in 7 post-menopausal women. The regimens were: A: 2.5 mg piperazine oestrone sulphate/day; B: 1.25 mg piperazine oestrone sulphate + 5.0 mg oestriol/day; C: 2.0 mg oestradiol valerate/day and D: 10.0 mg oestriol/day. A randomized complete cross-over design was employed and systemic blood levels of progesterone (P), oestrone (E1), oestradiol (E2), oestriol (E3), oestrone sulphate (E1S), oestradiol sulphate (E2S) and oestriol sulphate (E3S) were analyzed. All subjects were consistently post-menopausal, as indicated by low P, E2, E1S and E3S and high FSH and LH levels. The pretreatment and post-treatment levels of E2 and E2S as well as those of E3 and E3S were invariably below detection limit (60 pmol/l and 220 pmol/l, respectively). Treatment with regimens A, B and C resulted in consistently detectable levels of E2, E2S and E3S, but not of E3, and in grossly elevated E1 and E1S levels. Treatment with regimen D gave rise to very high E3S levels. The oestrogen profiles produced by regimens A and C were almost identical, with the exception of higher E1 and lower E3S levels following the administration of regimen A. Combination with LNG did not modify the levels of unconjugated oestrogens, but in certain cases it seemed to diminish those of oestrogen sulphates. A comparison with the results of a preceding study (Aedo A-R et al., Effect of orally administered oestrogens on gonadotrophins levels in post-menopausal women. Maturitas 1989; 11:) reveals that the gonadotrophin-suppressing effect of all 4 regimens still persisted in the post-treatment period at a time when the levels of all oestrogens analyzed returned to those of the pretreatment period.

The gas chromatography estimation using CHROM IV device enables a simultaneous investigation of oestrone, oestradiol and oestriol in urine. It is possible to use sorption on a solid sorbent PRESEP C 18 TESSEK for steroid extraction from biological material which shortens and simplifies the investigation method. The oestriol results gained by the described technique are compared with the values of total oestrogens estimated by the classical fluorometric method after Ittrich. The linear regression analysis gives the following result: Y = 0.7637x-2.7531, r = 0.9431, p less than 0.001 and confirms the correlation of both methods.

Estimates of the relative abundance of 16 alpha-hydroxy- and 16-deoxyoestrogens in late pregnancy urine lie between 13:1 and 5:1, yet the ratio of the concentrations of the major precursors 16 alpha-hydroxydehydroepiandrosterone sulphate and dehydroepiandrosterone sulphate in cord blood is about 2.5:1. This discrepancy might imply that 16 alpha-hydroxy-C19 steroids are used more efficiently for placental oestrogen biosynthesis than are the 16 alpha-deoxy-C19 steroids. On testing this hypothesis by incubation of placental microsomes with 16 alpha-hydroxy- and 16-deoxy- precursors together (concentration ratios 128:1 to 1:1), initial rates of oestrogen formation were highest from the 16-deoxy-C19 steroid. Additionally, whilst each substrate appeared to inhibit the aromatisation of the other, the 16-deoxy-C19 steroid was the more potent inhibitor. These findings were supported by an analogous experiment with placental slices. When each precursor was examined separately with microsomes from 4 placentae, aromatisation of the 16 alpha-hydroxy-C19 steroid (Michaelis constant, (Km) 0.75-1.24 mumol/l, maximum reaction velocity (Vmax) 28-69 pmol product/min/mg protein) was less efficient than that of the 16-deoxy-C19 steroid (Km 0.10-0.15 mumol/l, Vmax 71-145 pmol product/min/mg protein). To reconcile the disparity between the measured utilisation of precursors in vitro and expectations drawn from precursor availability and urinary excretion rates, sources of urinary 16 alpha-hydroxyoestrogens additional to placental aromatisation need to be considered. Hydroxylation of 16-deoxyoestrogens (the phenolic pathway) appears limited but aromatisation in fetal liver of 16 alpha-hydroxyandrostenedione not utilised by the placenta appears to be worth attention.

1. Gradient elution (sodium chloride concentration gradient) on DEAE-Sephadex columns is used to separate urinary oestrogen conjugates of pregnancy urine. Changes in the shape of the gradient alter the chromatograms in a predictable manner so that the dispersion of peaks can be modified at will. 2. Suitable choice of the gradient results in the separation from each other of oestrogen sulphates, oestrogen 16(or 17)-glucosiduronates, oestrogen 3-glucosiduronates and free oestrogens. 3. Evidence for the presence of oestriol 3-sulphate, oestrone 3-sulphate, 17beta-oestradiol 3-sulphate, 16-oxo-17beta-oestradiol 3-sulphate and oestriol 16(or 17)-sulphate in the sulphate fraction of DEAE-Sephadex chromatograms of pregnancy urine is provided.

The progressive rise in pregnancy of oestriol values and their significance in assessing fetal viability is well known, although the exact function of this increase is not understood. In considering the problem it was thought that further information of oestriol levels in mother, fetus and newborn could prove of value. The mean cord plasma oestriol was lower in small-for-dates than in normal cases, but in both groups it was ten times higher than in the relative maternal peripheral veins. In pooled capillary fetal scalp samples the mean oestriol was slightly higher in normal compared with small-for-dates cases. The use of gas chromatography--mass spectrometry was investigated and shown to give an accurate measurement of plasma oestriol levels. The expense of this procedure may be offset by its ability to determine simultaneously the nature and amount of other steroids present.

The absorption, metabolism and clearance of oestradiol, oestrone and oestriol from the peripheral circulation were investigated in five postmenopausal women after the oral administration of Hormonin (oestradiol 600 micrograms, oestrone 1400 micrograms, oestriol 270 micrograms) daily for five consecutive days. In addition the concentrations of plasma oestrone-3-sulphate, oestrone-3-glucuronide, LH and FSH were determined in the same plasma samples. The maximum concentration of each steroid in peripheral plasma and the time intervals during which the peak occurred after ingestion of the last tablet were: -oestrone (750 to 2116 pmol/l, 0.5 to 6.0 hours), oestradiol (246 to 813 pmol/l, 1 to 8 hours), oestriol (173 to 241 pmol/l, 5 to 12 hours), oestrone-3-glucuronide (32.2 to 78.8 nmol/l, 0.5 to 3.0 hours) and oestrone-3-sulphate (21.9 to 39.0 nmol/l, 1 to 7 hours). The mean factorial increases in the concentration of each compound during the first 12 hours post-treatment over the baseline values were: -oestrone (4.1); oestradiol (4.4); oestriol (1.7); oestrone-3-sulphate (2.6) and oestrone-3-glucuronide (8.1). After treatment, the concentration of plasma oestradiol remained significantly higher than the baseline values (p less than 0.025) for 24 hours and the values for each subject were within the normal range of premenopausal women. Moreover, there was a significant reduction (p less than 0.025) in the level of circulating LH. It is concluded that the simultaneous administration of oestrone, oestradiol and oestriol, by mouth, to postmenopausal women is a good approach to maintaining an appropriate concentration of oestradiol in peripheral plasma.

The capacity for removing wastewater-borne endocrine disrupting chemicals (EDCs) was investigated for two wastewater treatment plants (WWTPs) incorporating waste stabilisation ponds (WSPs) as the principal treatment technology. Samples were analysed for a number of steroidal oestrogens and androgens using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Removal efficiency for steroid androgens was high for both WWTPs (93-100%) but WSP treatment was observed to be less effective for removing steroid oestrogens, particularly oestriol.

Biochemical tests of fetal well-being are falling into disfavour. Difficulties arise because we are unable to ascribe specific functions to most test substances, and because they have a large normal range. Nevertheless, a critical survey of the evidence suggests biochemical tests have a part to play in antenatal monitoring, albeit a much reduced one. The main test substances are steroids and proteins produced by the trophoblast. These two classes of substances have a quite different metabolism and compartmental distribution, and the appropriate application of assays is different. A variety of progestogens and oestrogens are used as test substances, the most notable being oestriol. In view of their rapid metabolism, steroid assays serve best in pathological conditions of rapid onset. A dynamic test of the conversion of dehydroepiandrosterone to oestradiol has not fulfilled its initial promise. Among the placental proteins, human placental lactogen is most favoured, although some of the newly discovered proteins may have special applications in certain diseases. The use of biochemical tests in early pregnancy has not been sufficiently explored. The test substances may, in early pregnancy, play a more important role in fetal development and biochemical tests, particularly as a screening test to detect patients at risk of later overt disease, may be valuable.

The analytical and clinical advantages that would be expected to follow the adoption by clinical laboratories of a routine HPLC method for the partial oestriol conjugate profiling of human pregnancy urine are outlined in the Introduction. In order to ascertain if a candidate method for this assay has yet been devised, a complete survey of the published HPLC separations of oestrogen conjugate mixtures is presented, in tabular form, and discussed. From this survey it is concluded that a number of good separations of these steroids from synthetic mixtures have already been published. The third and final section of the paper contains the results of a detailed examination of those papers in which separation of oestriol conjugates present in pregnancy urine specimens have been reported. The paper is concluded with the recommendation that the method of Dixon, Lukha and Scott should be further investigated as a candidate method for adoption by clinical laboratories for the purpose of oestriol conjugate profiling.

After incubation of [4-14C]oestrone (E1) with liver slices from minipigs, the ether-soluble fraction contained [4-14C]oestradiol-17 beta (E2). In the protein-bound fractions, only polar metabolites were found, whereas in the water-soluble fraction the 3-monoglucuronide of oestriol (E1) was the preferred conjugate. When E2 was used as a substrate, E1 was present as main metabolite in the ether-soluble fraction. The radioactive metabolites in the protein-bound and water-soluble fractions were similar to those in the experiments with E1. The metabolism of E1 and E2 was dependent on age. Thus, the rate of conversion of oestrogens was greater in liver tissue of infertile male animals than in fertile males. In contrast, the two steroids were metabolised more rapidly in liver of fertile female minipigs than in infertile female animals. In fertile animals, the metabolic pattern of oestrogens in the ether-soluble, the protein-bound and the water-soluble fractions showed sex dependence: In females, E1 and E2 were metabolised to a greater extent by liver slices than in males. On the other hand, in experiments with male minipigs, E3-3-monoglucuronide was the only metabolite in the water-soluble fraction, whereas liver slices of female animals not only form E3-3-monoglucuronide, but also the 3-glucuronides of E1 and E2. The results described here show that, in liver tissue of minipigs, the oxidoreduction of E2 and E1 is the predominant reaction; in contrast to human liver, hydroxylation reactions play only a minor role. It may be concluded that there are differences in the metabolism of steroid hormones in man and minipig.

A novel liquid-phase immunoassay is described which involves: competitive binding of the analyte and labelled antigen to specific antibodies; enzymic transformation of the free ligand to a more hydrophobic derivative; and separation of the antibody-bound and free ligand by partition of the reactants into an organic and aqueous phase. The principles of the method, described as ligand differentiation immunoassay, have been used in conjunction with a tritiated antigen to establish a continuous-flow system for the measurement of oestriol-16 alpha-glucuronide in urine. Conventional autoanalyser (AAII) modules have been used, and the pump tubes and glass connections are standard accessories. The equipment is relatively inexpensive (pounds 1500 + detector) and the reagents are readily available. Each sample takes 55 minutes to be processed, and 30 results are obtained per hour.