Intestinal absorption of oestrogen: the effect of altering transit-time.

OBJECTIVE: The mechanism by which a high fibre diet may reduce serum oestrogens is unknown. We hypothesized that time is a rate-limiting factor in oestrogen absorption from the colon so that changes in colonic transit-rate affect the proportion of oestrogen that is deconjugated and/or absorbed. AIM: To determine if alteration of intestinal transit rate would influence the absorption of an oral dose of oestradiol glucuronide. PARTICIPANTS: Twenty healthy postmenopausal women recruited by advertisement. SETTING: Department of Medicine, Bristol Royal Infirmary. METHODS: Volunteers consumed, in turn, wheat bran, senna, loperamide and bran shaped plastic flakes, each for 10 days with a minimum 2 week washout period between study periods, dietary intake being unchanged. Before and in the last 4 days of each intervention whole-gut transit-time, defecation frequency, stool form, stool beta-glucuronidase activity, stool pH and the absorption of a 1.5 mg dose of oestradiol glucuronide were measured. RESULTS: Wheat bran, senna and plastic flakes led to the intended reduction in whole-gut transit-time, increase in defecatory frequency and increase in stool form score. Loperamide caused the opposite effect. The length of time the absorbed oestrogen was detectable in the serum fell with wheat bran and senna, although this was only significant for oestradiol. Oestrone, but not oestradiol, was detectable for a longer time with loperamide. Plastic flakes had no effect on either oestrogen. Areas under the curve did not change significantly but tended to fall with the three transit-accelerating agents and to rise with loperamide. CONCLUSION: Our data indicate there is likely to be an effect of intestinal transit on the absorption of oestrogens but more refined techniques are needed to characterize this properly.

Lower serum oestrogen concentrations associated with faster intestinal transit.

Increased fibre intake has been shown to reduce serum oestrogen concentrations. We hypothesized that fibre exerts this effect by decreasing the time available for reabsorption of oestrogens in the colon. We tested this in volunteers by measuring changes in serum oestrogen levels in response to manipulation of intestinal transit times with senna and loperamide, then comparing the results with changes caused by wheat bran. Forty healthy premenopausal volunteers were placed at random into one of three groups. The first group took senna for two menstrual cycles then, after a washout period, took wheat bran, again for two menstrual cycles. The second group did the reverse. The third group took loperamide for two menstrual cycles. At the beginning and end of each intervention a 4-day dietary record was kept and whole-gut transit time was measured; stools were taken for measurement of pH and beta-glucuronidase activity and blood for measurement of oestrone and oestradiol and their non-protein-bound fractions and of oestrone sulphate. Senna and loperamide caused the intended alterations in intestinal transit, whereas on wheat bran supplements there was a trend towards faster transit. Serum oestrone sulphate fell with wheat bran (mean intake 19.8 g day(-1)) and with senna; total- and non-protein-bound oestrone fell with senna. No significant changes in serum oestrogens were seen with loperamide. No significant changes were seen in faecal beta-glucuronidase activity. Stool pH changed only with senna, in which case it fell. In conclusion, speeding up intestinal transit can lower serum oestrogen concentrations.

Serum cortisol binding capacity measured with Concanavalin A-Sepharose in patients with a recent inflammatory response.

It has been suggested that elastase released from activated neutrophils degrades cortisol binding globulin. A novel assay for serum cortisol binding capacity was therefore devised and applied to assess whether such degradation was evident in patients showing a recent inflammatory response as indicated by a raised serum C-reactive protein. In 49 patients with evidence (C-reactive protein > 50 mg/l) of a recent inflammatory response, mean serum cortisol binding capacity (288 nmol/l, S.D. = 82.9) was significantly lower (P < 0.05, t test) than in 48 patients (320 +/- 75.8 nmol/l) whose response was quiescent (C-reactive protein < 6 mg/l) or in 49 healthy controls (335 +/- 72.4 nmol/l). Four patients with septic shock had markedly reduced values (167 +/- 49.9 nmol/l) but low values were not restricted to this condition. It is concluded that a population experiencing a recent inflammatory response exhibits reduced serum cortisol binding capacity but a role for elastase in this process remains to be defined.

Steroid sulphatase deficiency: identification of heterozygotes using hydrolysis of dehydroepiandrosterone sulphate by peripheral leucocytes.

Diagnosis of X-linked recessive ichthyosis, which is expressed only in males, can readily be made by measurement of leucocyte steroid sulphatase activity. However, because the gene for steroid sulphatase activity partly escapes from the process of X-chromosome inactivation associated with gene dosage compensation, identification of heterozygotes (females) is more difficult. We have measured the steroid sulphatase (by hydrolysis of dehydroepiandrosterone sulphate) and beta-glucuronidase (by hydrolysis of methylumbelliferyl glucuronide) activities in leucocytes from 18 heterozygotes, 100 normal females, 100 normal males and 11 affected subjects. When the ratio of the activities of steroid sulphatase and beta-glucuronidase in mixed leucocytes was plotted as a function of the steroid sulphatase activity, 85% heterozygotes were distinguished from normal females. Measurement of steroid sulphatase activity alone with these cells enabled identification of 78% heterozygotes. Measurements on mononuclear leucocytes were much less effective. Thrombocytes showed 1% of the steroid sulphatase activity of leucocytes. In females, leucocyte steroid sulphatase activity was independent of the stage of the ovarian cycle at which the cells were collected.

Steroids in human intrauterine fluids of early pregnancy.

OBJECTIVE: Little is known of the hormone environment of the developing early human embryo. We have therefore measured selected steroids in the intrauterine fluids of early pregnancy. DESIGN: Measurement of progesterone, 17 alpha-hydroxyprogesterone, oestradiol-17 beta, testosterone, androstenedione, cortisol and dehydroepiandrosterone sulphate in matched samples of coelomic fluid, amniotic fluid and maternal serum collected before pregnancy termination from 12 women between 8 and 12 weeks gestation. RESULTS: Mean concentrations of progesterone, oestradiol-17 beta and 17 alpha-hydroxyprogesterone in coelomic fluid were respectively 20, 6 and 2 times greater than in maternal serum and 8, 13 and 2.6 times those in amniotic fluid. Concentrations of testosterone and androstenedione were highest in maternal serum and lowest in amniotic fluid. Cortisol and dehydroepiandrosterone sulphate were found in intrauterine fluids only at the limit of detection but in normal concentrations in maternal serum. CONCLUSIONS: Coelomic fluid contains relatively high concentrations of progesterone, oestradiol-17 beta and 17 alpha-hydroxyprogesterone which may be synthesized locally. Amniotic fluid contains lower concentrations of steroids (other than progesterone) than are found in coelomic fluid or maternal serum. Free diffusion of steroids across the amnion appears limited. This may constitute a mechanism to protect the embryo from unwanted exposure to biologically active steroids.

Why so much oestriol? A comparison of the aromatisation of androstenedione and 16 alpha-hydroxyandrostenedione when incubated alone or together with human placental microsomes.

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.

Urinary multiple marker screening for Down's syndrome.

We have examined the possibility of using multiple markers in maternal urine rather than serum in order to screen for Down's syndrome. Urine samples were available from 36 cases (24 Down's syndrome, five Edwards' syndrome, three Turner's syndrome, one Klinefelter's syndrome, one triploidy, one triple-X, one twin discordant for Down's syndrome) and 294 controls, including three twins. Three markers were tested: the beta-core fragment of human chorionic gonadotrophin (hCG), total oestrogen (tE) and the free alpha subunit of hCG. Levels were corrected for creatinine excretion and expressed as multiples of the gestation-specific median (MOM) level from the singleton controls. The median value for the singleton Down's syndrome cases was 6.02, 0.74, and 1.08 MOM for beta-core-hCG, tE, and alpha-hCG, respectively. The increases in beta-core-hCG and the reduction in tE levels were highly significant (P < 0.0001 and 0.005, respectively; Wilcoxon rank sum test) but the increase in free alpha-hCG was not (P = 0.40). On the basis of a mathematical model, the expected detection rate for a 5 per cent false-positive rate was 79.6 per cent for beta-core-hCG alone, which increased to 82.3 per cent when combined with tE. Aneuploidies other than Down's syndrome were characterized by low levels of tE and either low or high beta-core-hCG.

A method for the determination of sex hormone binding globulin using Concanavalin A-sepharose.

A binding assay for sex hormone binding globulin (SHBG) has been developed in which SHBG is saturated with tritiated dihydrotestosterone (DHT). Separation of bound and free DHT is achieved by using Concanavalin A-Sepharose as a solid phase matrix. The method is described and its performance, including linearity, imprecision and comparison with other methods, is assessed. The assay is simple and robust and is suitable for analysis of samples of plasma or serum for clinical or research use.

The effects of fasting on plasma corticosterone kinetics in rats.

Plasma corticosterone clearance in anaesthetized rats was measured from the disappearance of radioactivity after a bolus injection of [3H]corticosterone. Mean fractional clearance rates were significantly (P less than 0.05) reduced after a 48 h fast, by 32 and 22% for males and females respectively. Plasma corticosterone concentrations were increased by fasting in both sexes. Corticosterone secretion rates, calculated as the product of fractional clearance and plasma corticosterone concentration, did not differ between fed and fasted groups in either sex. The mean activity (U/liver) of the rate-limiting enzyme for corticosterone degradation, hepatic 4,5-dihydrocorticosterone:NADP+ delta 4-oxidoreductase, was significantly reduced by 51 and 78% after fasting in males and females respectively. This was due to changes in both the soluble and microsomal forms of the enzyme. The binding capacity of corticosterone-binding globulin in plasma was significantly reduced by fasting in females (P less than 0.001), but was not altered in males. The results suggest that reduced hormone clearance is the dominant cause of fasting hypercorticosteronaemia in the rat.

Evidence for separate sites for aromatisation of androstenedione and 16 alpha-hydroxyandrostenedione in human placental microsomes.

Much greater quantities of 16 alpha-hydroxyoestrogens (e.g. oestriol) than of 16-deoxyoestrogens (e.g. oestradiol-17 beta) are formed in human pregnancy than might be expected from the relative availability to the placenta of the 16 alpha-hydroxy- and 16-deoxy-C19 precursors. To investigate this further, 16 alpha-hydroxyandrostenedione (16 alpha-OH-A4) and androstenedione (A4) were tested in vitro as substrates and mutual inhibitors of human placental aromatase. It was found that the Km for aromatisation of A4 (mean = 0.26 mumol/l) was very similar to Ki (0.30, 0.35 mumol/l) for the inhibition by A4 of the aromatisation of 16 alpha-OH-A4. Similarly, Km for aromatisation of 16 alpha-OH-A4 (mean = 1.21 mumol/l) had the same value as the Ki (1.0, 1.2 mumol/l) for the inhibition by 16 alpha-OH-A4 of the aromatisation of A4. From graphical analysis of Lineweaver-Burk plots, both inhibitions were characterised as noncompetitive. Hence, it was concluded that the two 16-deoxy- and 16-hydroxy-C19 substrates bind at separate, but interactive, sites and that each substrate on binding inhibits the aromatisation of the other. Additional evidence for the separate but interactive substrate binding sites for the 16-deoxy- and 16-hydroxy-C19 steroids was obtained by use of the suicide inhibitor 4-hydroxyandrostenedione (4-OH-A4), which is recognised as binding to the aromatisation site for A4. Aromatisation of 16 alpha-OH-A4 was found to be inhibited by pre-incubation of the microsomes with 4-OH-A4 (0.1 mumol/l). The presence of A4 (4.6 mumol/l), but not of 16 alpha-OH-A4 (4.0 mumol/l) during the pre-incubation successfully protected the subsequent aromatisation of 16 alpha-OH-A4 from this inhibition. In addition, the Km values, reported here, suggest also that the 16-deoxyandrogens are preferred to the 16 alpha-hydroxyandrogens as oestrogen precursors. In consequence, factors other than substrate affinity and plasma concentrations must be presumed to be involved in the overwhelming production of 16 alpha-hydroxyoestrogens in human pregnancy.

In-vivo and in-vitro endocrine investigation of pure gonadal dysgenesis.

Diagnosis of XY pure gonadal dysgenesis was established in a patient of female phenotype, with female internal genitalia, but with a chromosomal constitution of 46 XY. Streak gonads had undergone neoplastic transformation--gonadoblastoma and dysgerminoma. Before operation the concentrations of gonadotrophins in plasma were high and of oestradiol was low. Administration of oestradiol benzoate initially suppressed and then stimulated an increase in the plasma concentration of LH. These changes were not accompanied by changes in blood levels of endogenous sex steroids. A single injection of hCG failed to stimulate steroid secretion. The activities in vitro of steroid-metabolizing enzymes in the dysgenetic gonadal tissue more closely resembled those of ovarian tissue from a premenopausal and from a postmenopausal women than those in testes from two androgen-insensitive patients. However, aromatase activity was higher in the dysgenetic gonads than in the pre or post-menopausal ovaries. Examination of enzymes in genital skin fibroblasts demonstrated normal activities of 3 alpha/beta-beta-hydroxysteroid dehydrogenase and 17 beta-hydroxysteroid dehydrogenase (oxidative and reductive directions). However, 5 alpha-reductase activity was low in minces and fibroblasts of genital skin from the patient. Androgen binding was within the range for male controls.

Steroid metabolism in testes of patients with incomplete masculinization due to androgen insensitivity or 17 beta-hydroxysteroid dehydrogenase deficiency and normally differentiated males.

For purposes of establishing suitable controls in studies of patients with a suspected enzyme deficiency, activities of enzymes involved in the biosynthesis of testosterone were compared in testes of patients with androgen insensitivity syndrome (AIS) and normally differentiated males with carcinoma of the prostate (Ca prostate) or testis (Ca testis). Activities of 17,20-desmolase and of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) were higher in the testes of pre-, peri- or postpubertal patients with AIS than in elderly men (58-80 yr) with Ca prostate. Activities of 17 beta-HSD (reductive direction) and 3 beta-HSD tended to be higher in peri- or postpubertal than in prepubertal patients with AIS. Activity of 3 beta-HSD was low in the patient with Ca testis. In a peripubertal (12 yr) patient with incomplete masculinization due to a severe deficiency of 17 beta-HSD, reductive activity of 17 beta-HSD was very low compared with that of patients with Ca prostate, Ca testis or AIS. In contrast, in testes from the younger sibling (4 yr), in whom the deficiency of 17 beta-HSD was less severe, 17 beta-HSD reduction of dehydroepiandrosterone was as high as that of men with Ca prostate, yet deficient in comparison with that of more closely age-matched patients with AIS. This emphasizes the desirability of using age-matched tissue for control purposes in enzyme studies.

Oestriol binding to plasma proteins.

Simple diffusion experiments indicated that oestriol was retained by human pregnancy plasma more effectively than by albumin solutions of a corresponding concentration. Oestriol bound (Ka = 6 X 10(6) l/mol at 4 degrees C) to a glycoprotein which had been isolated from plasma by adsorption to Concanavalin A. The free energy of binding at 37 degrees C was -38 kJ/mol. Competition experiments indicated that the oestriol binding glycoprotein had properties expected of sex hormone binding globulin. The distribution of oestriol among the protein fractions of human pregnancy plasma--glycoprotein bound 7.8%, albumin bound 78.6%, unbound 13.6%--suggests that this glycoprotein plays little part in the transport of oestriol.

Evidence for secondary 5 alpha-reductase deficiency in genital and supra-pubic skin of subjects with androgen insensitivity syndrome.

The activity of 5 alpha-reductase in genital and supra-pubic skin (homogenate or fibroblasts) from subjects with complete or incomplete androgen insensitivity syndrome was low compared with mean activity in samples from normally differentiated male controls. Also, in two subjects with incomplete androgen insensitivity syndrome the ratio of the concentration of testosterone to that of 5 alpha-dihydrotestosterone in plasma was raised after hCG stimulation but normal under basal conditions. In three subjects with complete androgen insensitivity syndrome there was no evidence of raised ratios of testosterone to 5 alpha-dihydrotestosterone in plasma under basal or hCG-stimulated conditions. The activities of other steroid metabolizing enzymes, e.g. 17 beta-hydroxysteroid dehydrogenase, 3 alpha/beta-hydroxysteroid dehydrogenase, were not decreased. The low 5 alpha-reductase activity of androgen insensitive subjects reported here, and by others, may imply that this enzyme in genital skin is in some way androgen dependent, or responsive to other factors associated with androgen insensitivity syndrome.

Antenatal detection of placental steroid sulphatase deficiency by measurement of urinary 16 alpha-hydroxydehydroepiandrosterone sulphate.

A simple gas chromatographic technique for the measurement of 16 alpha-hydroxydehydroepiandrosterone sulphate in urine from pregnant women is described. An assessment was made of the effectiveness of the measurement of this oestriol precursor for the antenatal diagnosis of placental steroid sulphatase deficiency. Twenty-two patients whose pregnancies were complicated by subnormal oestrogen excretion for gestation were studied. In nine of these, where placental steroid sulphatase activity was found subsequently in vitro to be normal, the excretion of 16 alpha-hydroxydehydroepiandrosterone sulphate was less than 27 mumol/24 h. In the remaining 13 patients, in whom postnatal in vitro assay demonstrated absence of placental steroid sulphatase activity, urinary excretion of 16 alpha-hydroxydehydroepiandrosterone sulphate was 59-360 mumol/24 h. The excretion of this metabolite was below the limit of detection (20 mumol/24 h) in 30 uncomplicated pregnancies. It is concluded that urinary excretion of 16 alpha-hydroxydehydroepiandrosterone sulphate greater than 50 mumol/day or a ratio of urinary 16 alpha-hydroxydehydroepiandrosterone sulphate to urinary oestrogen greater than 2.0 correctly identifies, before delivery, those pregnancies in which fetus and placenta are deficient in steroid sulphatase activity.

Cloning of a cDNA for steroid sulfatase: frequent occurrence of gene deletions in patients with recessive X chromosome-linked ichthyosis.

A human steroid sulfatase (steryl-sulfatase; steryl-sulfate sulfohydrolase, EC 3.1.6.2) cDNA 2.4 kilobases long was isolated from a human placental lambda gt11 cDNA expression library. The library was screened with monospecific rabbit antibodies elicited by injection of steroid sulfatase protein purified from human placentas. Hybridization of the cDNA with EcoRI-digested genomic DNA indicated that patients from 14 of 15 apparently unrelated families have gross deletions of the gene for steroid sulfatase. One patient had genomic DNA fragments that were identical to those from normal individuals, indicating the absence of any major deletions as the cause of his lack of steroid sulfatase enzyme activity.

Oestriol and non-protein-bound oestriol concentrations in human peripheral plasma before labour and delivery.

The concentration of oestriol and the proportion of this hormone not bound to plasma protein were measured using radioimmunoassay and centrifugal ultrafiltration respectively, in 55 samples of plasma obtained from 12 women in the last 2 to 7 weeks of uncomplicated pregnancy. Among individuals, the mean plasma concentration of oestriol varied from 25.8 +/- 94.8 nmol/l; in nine subjects, there was a tendency for oestriol concentrations to increase as delivery approached. The mean proportion of oestriol not bound to plasma protein in the different subjects varied from 13.1 to 18.9%, but values from any individual subject remained essentially constant during the periods of study. These measured values were used to calculate, for each sample, the apparent concentration of oestriol not bound to plasma protein. The results were combined with analogous values for oestradiol and progesterone obtained from the same plasma samples and described in a previous study. It was found that the mean ratio of the concentration of oestriol and oestradiol was 0.75, the mean concentration of non-protein-bound oestriol was 8.7 times that of non-protein-bound oestradiol, and in individual subjects, there was no consistent trend as delivery approached in the ratio of the concentration of progesterone to that of oestriol in either the total or non-protein-bound form.

Hydrolysis of deoxycorticosterone-21-yl sulphate and dehydroepiandrosterone sulphate by microsomal preparations of human placentae: evidence for a common enzyme.

The human placenta can hydrolyse both dehydroepiandrosterone-3 beta-yl sulphate (DHASO4-) and deoxycorticosterone-21-yl sulphate (DOCSO4-). There is some uncertainty as to whether the same or different enzymes are responsible for hydrolysis of these substrates. As a fresh approach to this problem we have compared the quantities of DHASO4- and DOCSO4- hydrolysed by microsomal preparations of placentae obtained from 14 normal pregnancies and from 14 pregnancies complicated by steroid sulphatase deficiency. Under the conditions used, and standardizing the results to unit time and quantity of protein, 1380-8830 fmol DHASO4- were hydrolysed by 14 normal placentae whereas less than 1000 fmol DHASO4- were hydrolysed by the other 14 placentae, thereby designated as steroid sulphatase deficient. Net hydrolysis of DOCSO4- by the preparations of normal tissue was 9-52 fmol; hydrolysis of this substrate by steroid sulphatase-deficient tissues was indistinguishable from that by boiled tissue (less than 29 fmol). Thus preparations of placentae which hydrolysed DHASO4- also hydrolysed DOCSO4-; tissues which did not hydrolyse DHASO4- also failed to hydrolyse DOCSO4-. The quantities of DHASO4- and DOCSO4- hydrolysed by the 28 individual placentae showed a positive correlation (r = 0.91, P less than 0.001). The apparent Michaelis constants for hydrolysis of DHASO4- and DOCSO4- were 38 and 274 mumol/l respectively. These results are consistent with the proposal that these substrates are hydrolysed by a common enzyme.

Non-protein-bound oestradiol and progesterone in human peripheral plasma before labour and delivery.

Plasma samples were obtained at weekly intervals from the peripheral circulation of 12 women in the last 2-7 weeks of pregnancy. The concentrations of oestradiol and progesterone (isolated by chromatography) were measured by radioimmunoassay; the proportion of each hormone which was not bound to protein was measured by steady-state gel filtration. From these, the apparent concentration of the non-protein-bound form of each hormone was calculated. The mean proportion of oestradiol not bound to protein varied from 0.84 to 2.71% in the different subjects, but within each subject variation was within experimental error. For progesterone, the mean proportion not bound to protein in the different subjects varied from 1.76 to 2.77%; within individuals the proportion remained essentially constant. There was no consistent, recognizable trend as labour approached in the concentration of oestradiol; the concentration of progesterone; the concentrations of non-protein-bound oestradiol or non-protein-bound progesterone; the ratio of the concentrations of progesterone and oestradiol; the ratio of the concentrations of non-protein-bound progesterone and oestradiol. In nine out of 12 subjects, the ratio of the concentration of non-protein-bound progesterone to that of non-protein-bound oestradiol was greater than the corresponding ratio based on total hormone concentrations. These results therefore provide no support for the hypothesis that human labour is preceded by alteration in the progesterone to oestradiol ratio which can be detected by measurement of these hormones in peripheral blood.